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Molecular characterization and differential expression of a invertase gene family in maize

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Molecular characterization and differential expression of a invertase gene family in maize
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Xu, Jian, 1963-
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x, 127 leaves : ill., photos ; 29 cm.

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Amino acids ( jstor )
Complementary DNA ( jstor )
Corn ( jstor )
Cytokinins ( jstor )
Enzymes ( jstor )
Messenger RNA ( jstor )
Plants ( jstor )
RNA ( jstor )
Root tips ( jstor )
Sugars ( jstor )
Dissertations, Academic -- Plant Molecular and Cellular Biology -- UF ( lcsh )
Plant Molecular and Cellular Biology thesis Ph. D ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis (Ph. D.)--University of Florida, 1994.
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Includes bibliographical references (leaves 110-126).
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Typescript.
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Vita.
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by Jian Xu.

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MOLECULAR CHARACTERIZATION AND DIFFERENTIAL EXPRESSION OF
A INVERTASE GENE FAMILY IN MAIZE
















By

JIAN XU


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA 1994














ACKNOWLEDGEMENTS


My sincerest appreciation is extended to the members of my committee, Dr. Karen Koch, Dr. Alice Harmon, Dr. Ken Boote and Dr. Don McCarty, for their support and guidance during the completion of this degree. I am also truly grateful to the other faculty, staff and graduate students for their help and encouragement during my time here, especially Kurt Nolte, Ed Duke, Wayne Avigne, Don Merhaut, Gwendolyn Pemberton, Betsy Bihn, Summer Osterman and Aiyu Li.

Finally, I extend my deepest thanks to my wife Naidong Shao, who was always there whenever I needed her and also to my parents and my brother in China. They have all given me support and encouragement throughout my education here, and I can never thank them enough.














TABLE OF CONTENTS


ACKNOWLEDGEMENTS .............

LIST OF FIGURES ..................

ABSTRACT .......................

CHAPTERS


I INTRODUCTION ..................................


2 LITERATURE ERVIEW ..


Invertase and its Functions ............
Regulation of Invertase Gene Expression ..

3 ISOLATION AND CHARACTERIZATION
INVERTASE GENES ...............

Introduction ......................
Materials and Methods ...............
R esults ..........................
D iscussion .......................


OF MAIZE
. ., . . . . . .


4 DEVELOPMENTAL EXPRESSION AND CARBOHYDRATE
RESPONSIVENESS OF TWO MAIZE INVERTASE GENE
SUBFAM ILIES ...................................


Introduction ...............................
Materials and Methods ........................
R esults . . . . . . . . . . . . . . . . . .
D iscussion ................................


5 CYTOKININ MIMICS AND SUPERSEDES THE SUGARINDUCIBILITY OF MAIZE INVERTASE FAMILY MEMBERS


. . . . . . . . . . . . . ii

. . . . . . . . . . . . . V

. . . . . . . . . . . . viii








AND FACILITATES THEIR DIFFERENTIAL RESPONSIVENESS TO ABSCISIC ACID ................................. 85


Introduction ....... Materials and Methods Results ...........
Discussion ........


6 SUMMARY AND CONCLUSIONS .................... 105

REFERENCE LIST ............................................ 109

BIOGRAPHICAL SKETCH ..................................... 126


. .. . . . . .. . . .. . . .
. . . . . . . . . . . . . . .














LIST OF FIGURES


Figure 3-1 Restriction maps of Ivr clones for maize soluble acid invertases . 26

Figure 3-2 Schematic diagram of the genomic organization of the IvrlG .... 28

Figure 3-3 The deduced amino acid sequence for maize invertase 1 gene ..... 30

Figure 3-4 The hydropathy and fold values of the deduced polypeptide for
m aize invertase gene 1 .................................... 32

Figure 3-5 Conserved regions within derived amino acid sequences of higher
plant invertases ......................................... 34

Figure 3-6 Conserved regions within derived amino acid sequences of the
IvrlG for maize soluble acid invertase and either other soluble
invertases or insoluble invertases from higher plant ................ 36

Figure 3-7 DNA gel blot analysis of cross-reactivity between Ivrl, lvr2,
Ivr2C-1 and Ivr2C-2 ..................................... 38

Figure 4-1 Abundance of mRNA from the Ivr] and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize
invertases in root tips, a sink leaf, a source leaf, a prop root, anthers,
silk and kernels ........................................ 55

Figure 4-2 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize
invertases during kernel development ........................... 57

Figure 4-3 Abundance of mRNA from the Ivr] and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize
invertases in pedicel, middle and top portions of kernels at 8, 10, 12
DAP .................................................. 59

Figure 4-4 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize








invertases in kernels sampled daily from 2 days before to 2 days after
pollination ............................................. 61

Figure 4-5 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize invertases during the final 3 days of anther development and in mature
pollen .................................................. 63

Figure 4-6 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble and insoluble maize invertases
in silk sampled daily from 2 days before to 2 days after pollination ... 65

Figure 4-7 Abundance of mRNA from the Ivr] and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble and insoluble maize invertases in tip, mid and low portions of silk sampled at pollination, 3hr later, 6hr
later, or 24hr later .. ...................................... 67

Figure 4-8 Abundance of mRNA from the Ivr] and Ivr2 subfamilies of soluble
acid invertase in maize root tips incubated for 24hr in white's basal salts
medium supplemented with glucose or sucrose ................... 69

Figure 4-9 Abundance of mRNA from the Ivr] and Ivr2 subfamilies of soluble
acid invertase during starvation of maize root tips ................. 71

Figure 4-10 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase during post- starvation recovery of maize root
tips .................................................. 73

Figure 4-11 Abundance of mRNA from the Ivr] and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble acid invertases in maize root tips incubated for 24hr in White's basal salts medium supplemented with either 2.0% glucose, fructose, sucrose, L-glucose or
m annitol .. ............................................. 75

Figure 5-1 Abundance of mRNA from the Ivr] and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble acid invertases in maize root tips incubated for 24hr in White's basal salts medium supplemented either with (+G) or without (-G) 0.5% glucose and either with (+K) or
without (-K) 5 ptM Kinetin ................................ 95

Figure 5-2 Abundance of mRNA from the IvrJ and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble acid invertases in maize root tips incubated for 24hr in White's basal salts medium supplemented with








0.5% glucose, either with (+K) or without (-K) 5 piM Kinetin, either with
(+ABA) or without (-ABA) abscisic acid (50 pM) ................ 97

Figure 5-3 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble acid invertases in maize root tips incubated for 24hr in White's basal salts medium supplemented with
0.5% glucose, alone (+0) or with either GA or IAA ............... 99














Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

MOLECULAR CHARACTERIZATION AND DIFFERENTIAL EXPRESSION OF A INVERTASE GENE FAMILY IN MAIZE By

Jian Xu

December, 1994

Chairperson: Dr. Karen E. Koch
Major Department: Plant Molecular and Cellular Biology Program

A family of soluble invertase genes in maize (Zea mays L.) were cloned and characterized to test several hypotheses regarding their potential significance in specific instances of developmental and/or environmental adjustment. The responses of two invertase gene subfamilies were examined at the level of both gene expression and overall enzyme activity.

Five maize cDNA clones (Ivrl, Ivr2, Ivr2C-1, IvrC-2 and IvrC-3) and one genomic clone (IvrlG) were isolated and found to encode probable isozymes of soluble invertase. The deduced amino acid sequences show significant identities, especially to previously characterized soluble acid invertases of higher plants, and are particularly strong in key regions conserved among these enzymes. One of the most strongly conserved regions among all invertase sequences (NDPNG) was found








to be carried on an unusually small 9 nucleotide exon identified in the maize genomic DNA.

Two subfamilies of maize soluble invertases (each cross-reactive with either Ivr] or Ivr2 [Ivr2C-1 + Ivr2C-2]) were differentially expressed in an array of tissues. A comparison between message and enzyme activity was consistent with both subgroups encoding soluble acid invertases. The spatial and temporal patterns of expression for the two invertase classes, as well as the contrast between them implicate their potential involvement in several stages of development. Data support the hypothesis that invertase could be especially important during stages requiring expansion of specific cells, such as during pollination and early kernel development.

Maize root tips were used to further test the extent to which expression of the two subfamilies for soluble invertase isozymes may have been regulated by sugar levels or specific developmental signals.

The mRNA levels from both subgroups were elevated in the presence of

exogenous sugar supplies as long as these were readily metabolizable, however, the extent of this response differed. The Ivr2 group of genes showed a greater sensitivity to carbohydrate deprivation. The differential responsiveness of invertase gene subfamilies to carbohydrate availability provides a potential mechanism for different isozyme genes to predominate in various tissues developmental stages, and/or altered environmental conditions.

Data also indicated that specific developmental cues could affect expression of both invertase subgroups as well as soluble activity of acid enzymes. Cytokinin








signals (typically produced by dividing cell, endosperm, root tips and symbionts) could alone apparently replace and supersede the carbohydrate upregulation of invertase transcript levels by sugars. Both Ivrl and Ivr2 type mRNA abundance was upregulated by exogenous ABA (elevated in developing seeds and in response to some stresses). However, simultaneous presence of cytokinin appeared to be required before the ABA-induced changes at the message-level could be transduced at the level of enzyme activity. The differential response of invertase isozyme genes to sugar levels and specific plant hormones suggests that integration of these types of signals may mediate developmental responses, symbiosis, and/or adaptation to stresses.










CHAPTER 1
INTRODUCTION


Sucrose is the most abundant long-distance transport carbohydrate in the plant kingdom. As such, it plays a central and vital role in plant growth and development. In vascular plants there are two known enzymatic reactions that can breakdown this sucrose. These are catalyzed by invertase and sucrose synthase.

Invertase is often considered an essential enzyme for carbohydrate

metabolism and partitioning because of the nearly ubiquitous role of sucrose in photoassimilate translocation (Avigad, 1982; Hawker, 1985; Turgeon, 1989). This is supported by the observation that invertase deficient kernels of the miniature-] maize mutant develop abnormally in addition to their reduced size (Miller and Chourey, 1992). Further, primary root tips of another invertase deficient maize mutant, OH43, can not grow normally on sucrose agar (Robins, 1958; Duke et al., 1991).

Invertase activity is widely distributed within and among vascular plants. Several isoforms of invertase often can be present simultaneously in a given plant and/or organ. However, the roles of the individual isoforms are not well understood.

Early work with maize kernels (Shannon, 1972; Shannon and Dougherty, 1972; Doehlert and Felker, 1987; Doehlert et al., 1988) indicated that imported sucrose moved from phloem into the extracellular space where it was hydrolyzed by a cell-wall-bound, acid invertase. This was presumed to contribute to a sucrose










concentration gradient between the phloem and apoplast which facilitated translocation of sucrose into sugar-utilizing tissues. Although more recent studies have largely been consistent with this scenario, they also indicate that the entire story may not be as simple (Shannon et al., 1993).

Rapidly expanding tissues require either invertase or sucrose synthase to convert sucrose to substrates necessary for respiratory and synthetic processes (Giaquinta, 1979; Avigad, 1982; Hawker, 1985). Invertase can be especially important to cell expansion through generation of hexoses and their associated osmotic potential (Kaufman et al., 1973; Schmalstig and Cosgrove, 1988; 1990).

Genes involved in metabolic pathways are often regulated by levels of

metabolites (Carlson, 1987; Schuster, 1989; Maas et al., 1990; Koch et al., 1992; Sadka et al., 1994). In vascular plants, sugar-responsive genes have been primarily characterized in storage organs (Rocha-Sosa et al., 1989; Salanoubat and Belliard, 1989; Muller-Rober et al., 1990; DeWald et al., 1994; Sadka et al., 1994). However, carbohydrate-induced changes in gene expression have also been identified for enzymes involved in photosynthesis (Sheen, 1990) and other metabolic pathways (Maas et al., 1990; Koch et al., 1992; Graham et al., 1994). Data at the enzyme level have suggested that a similar sugar-regulated gene expression may underlay responses of invertase to carbohydrate availability (Sacher et al., 1963; Glasziou et al, 1966; Ricardo et al., 1972; Kaufman et al., 1973). Together, sugar-sensitivity of these sucrose-metabolizing genes may comprise a system for sensing and transducing signals of whole plant carbohydrate status.










Plant growth regulators often have pleiotropic effects on plant growth and development. When an organism becomes more complex during its development, communication between its different part requires an appropriate signaling system. In vascular plants, a complex hormonal system is largely responsible for signaling such a communication system (Libbenga and Mennes, 1987). Invertase activity can be upregulated by abscisic acid, auxin, cytokinins and/or gibberellic acid in an array of vascular plants (Sacher et al., 1963; Glasziou et al., 1966; Kaufman et al., 1973; Howard and Withan, 1983; Ackerson, 1985; Weil and Rausch, 1990; Miyamota et al., 1993; Wu et al., 1993).

The purpose of the present research was to test the hypothesis that regulation of two subfamilies of soluble invertase isozymes in maize could be consistent with a proposed role for these isozymes in specific aspects of cell expansion during development and/or environmental adjustment. Specific objectives were as follows.

1. Clone and characterize maize invertase genes.

2. Test the effects of altered carbohydrate availability on soluble invertase activity and abundance of mRNA from the two invertase subfamilies.

3. Determine the extent to which changes in mRNA abundance and enzyme activity correspond to those proposed for given physiological roles of these isozymes in maize development.

4. Characterize the responsiveness of genes for invertase isozymes to developmental signals at both message and enzyme activity levels.










CHAPTER 2

LITERATURE REVIEW


Invertase and Its Functions



Invertase catalyzes one of the only two enzyme reactions known to

breakdown sucrose into its constituent monosaccharides, glucose and fructose, in vascular plants. Lack of this enzyme will result in abnormal growth and development of plants, which is evident in root tips of the Oh43 maize mutant (Robbins, 1958; Duke et al., 1991) and kernels of the miniature-] (Miller and Chourey, 1992). Overexpression of invertase in leaves strongly influences growth and phenotype of transgenic plants (von Schaewen et al., 1990; Dickinson et al., 1991). Expression of an invertase gene also controls sugar composition in tomato fruit (Klann et al., 1993).

Invertase may be ubiquitous among multicellular plant species. Acid and neutral forms can be distinguished based on pH optima, and/or invertases can be classified as soluble vs cell-wall-bound forms (Avigad, 1982). An "alkaline" (neutral) invertase has recently been purified from sprouting soybean (Chen and Black, 1992). However, acidic invertases are generally more common and have been studied from an array of plants (Hanft and Jones, 1986a; Sturm and Chrispeels, 1990; Arai et al., 1991; Klann et al., 1992; Elliott et al., 1993; Ramloch-Lorenz et al., 1993; Schwebel-Dugue et al., 1994; Unger et al., 1994).










The physiological significance of invertase action has been debated over a

considerable period of time. Work with sugarcane stems (Hawker and Hatch, 1965; Glasziou and Gayler, 1972) and corn kernels (Shannon, 1968; Shannon, 1972; Shannon and Dougherty, 1972) indicated that imported sucrose moved from phloem into the extracellular space where it was hydrolyzed by a cell-wall-bound, acid invertase. This was presumed to contribute to a sucrose concentration gradient between the phloem and apoplast, enhancing the rate of sucrose transfer into sucrose utilizing tissues. More recent evidence is also consistent with the initial hypothesis (Lin et al., 1984; Doehlert, 1986; Doehlert and Felker, 1987; Doehlert et al., 1988; Doehlert, 1990 ). Nonetheless, work done by other groups indicates that sucrose can be transported into Zea mays L. endosperm without invertase hydrolysis (Hitz et al., 1985; Cobb and Hannah, 1986; Schmalstig and Hitz, 1987; Cobb and Hannah, 1988). It is possible that sucrose can move into sucrose-utilizing tissues of maize kernels by both mechanisms.

Soluble acid invertase activity is closely correlated with leaf expansion in

bean (Phaseolus vulgaris L.) and Citrus whereas sucrose synthase activity is minimal and fairly constant (Morris and Arthur, 1984; Schaffer et al., 1987). The authors suggest that soluble acid invertase activity is the primary enzyme responsible for sucrose catabolism in the expanding bean and citrus leaves. Its activity is considered to be the primary determinant of sink potential in these systems.

Invertase also predominates over sucrose synthase (which is barely

detectable) in the earliest stages of development for maize kernels (Tsai et al., 1970;










Chourey and Nelson, 1976; Chourey, 1981) and snap bean pods (Sung et al., 1994). In addition, maize kernels induced to abort by high temperature have a much reduced activity of pedicel soluble invertase than do nonaborting kernels (Hanft and Jones, 1986a). The rapid expansion characteristic of this early development requires both osmotic constituents and substrates for respiratory and synthetic processes. Soluble invertase can be especially important to cell enlargement through generation of hexoses and their associated osmotic potential (Kaufman, 1973; Avigad, 1982; Schmalstig and Cosgrove, 1988; 1990). Either invertase or sucrose synthase can provide an avenue for carbohydrate entry into respiratory and biosynthetic processes (Giaquinta, 1979; Avigad, 1982; Morris and Arthur, 1984b; Hawker, 1985; Schaffer et al., 1987).

Soluble invertase activity is also closely associated with other phases of

reproductive development (Tsai et al., 1970; Jaynes and Nelson, 1971a; Shannon and Dougherty, 1972; Singh and Knox, 1984; Hubbard et al., 1989; Miron and Schaffer, 1991; Klann et al., 1992). Pryke and Berneir (1978) have found that increased content of sugar and activity of soluble acid invertase in the apices consistently appear to accompany the transition to flowering in Sinapis alba. Invertase also appears to be involved in pollen function. The sucrose content from pollen grains of Camellia japonica decreases rapidly during growth of the tube. Soluble invertase activity also increases during germination of cultured pollen and a high constant activity is found during the later stages of pollen tube growth (Nakamura et al., 1980). Further, invertase is demonstrated to contribute to an in vitro chemotropism










of pearl millet pollen tubes toward stigmatic tissue through its production of glucose (Reger et al., 1992a; 1992b; 1993).

A number of different genes may be involved in these processes in vascular plants (Sturm and Chrispeels, 1990; Arai et al., 1992; Klann et al., 1992; Elliott et al., 1993; Ramloch-Lorenz et al., 1993; Schwebel-Dugue et al., 1994; Unger et al., 1994). In yeast, one gene encodes both cell-wall-bound and soluble invertases through differential splicing (Carlson and Botstein, 1982). However, in carrot at least seven different invertase genes have been distinguished (A. Sturm, personal communication).



Regulation of Invertase Expression



Invertase and Its Endogenous Inhibitors



Proteinaceous invertase inhibitors are found in an array of vascular plants (Pressey, 1966; 1967; 1968; Jaynes and Nelson, 1971b; Matsushita and Uritani, 1976; Bracho and Whitaker, 1990a; 1990b; Isla et al., 1992; Weil et al., 1994). In Solanum tuberosum they are located in the vacuole (Bracho and Whitaker, 1990a; 1990b; Isla et al., 1992), whereas in Nicotiana tabacum they are in the extracellular space (Weil et al., 1994). These inhibitors bind tightly and specifically to acid invertase and have molecular weights ranging from 17 to 23 KDa (Bracho and Whitaker, 1990b; Weil et al., 1994). However, it remains unclear by what










mechanism the endogenous inhibitors may regulate invertase activities either spatially or temporally.



Plant Growth Regulators and Invertase



Invertase activity appears to be upregulated by abscisic acid, auxin,

cytokinins and/or gibberellic acid depending on the system and tissues involved (Sacher et al., 1963; Glasziou et al., 1966; Gayler and Glasziou, 1969; Kaufman et al., 1973; Howard and Witham, 1983; Morris and Arthur, 1984; Ackerson, 1985; Schaffer et al., 1987; Weil and Rausch, 1990; Miyamoto et al., 1993; Wu et al., 1993).

Both auxin and gibberellic acid stimulate cell enlargement, cell elongation and possibly phototropism and gravitropism (Davies, 1987; Kaufman and Song, 1987; Kim et al., 1993; Wu et al., 1993a; 1993b). The concentration of GA (gibbellic acid), which promotes growth, closely parallels that which increases invertase activity in Avena stem segments (Kaufman et al., 1973). The increased rate of hydrolysis of sucrose to hexose following the stimulation of acid invertase activity by GA is considered one means of generating an elevated level of osmotic constituents in the growing region of the stem (Morris and Arhtur, 1985). The stimulation of both invertase activity and stem growth by auxin is consistent with the finding that invertases are especially active in tissues undergoing rapid cell enlargement, such as regions near shoot and root apices (Avigad, 1982). The










mechanism by which IAA leads to an increase in acid invertase activity, however, remains obscure. It is not yet clear whether the observed increase in activity is a cause or a consequence of auxin-induced growth (Morris and Arthur, 1984a).

Both abscisic acid and cytokinins are reported to stimulate assimilate

translocation from source to sink (Gersani and Kender, 1982; Howard and Witham, 1983; Hein et al, 1984; Schussler et al., 1984; Ackerson, 1985; Jones et al., 1986; Brokovec and Prochazka, 1992; Jones et al., 1992). Such enhancement may involve the capacity of invertase to hydrolyze sucrose to hexoses and thus increase sink potential. This in turn could stimulate the translocation of sugar to seeds (Shannon, 1968; Shannon, 1972; Shannon and Dougherty, 1972; Lin et al., 1984; Doehlert, 1986; Doehlert and Felker, 1987).

The mechanisms originally proposed to explain the effects of plant hormones on invertase have been questioned (Sacher et al., 1963; Glasziou et al., 1966; Chrispeels and Varner, 1967; Gayler and Glasziou, 1969; Hagen et al., 1984). Gayler et al. (1969) suggested that auxin and gibberellic acid may have aided stabilization of the mRNA for invertase. They further suggested that the mechanism of abscisic acid (ABA) action in this instance involved processes subsequent to formation of invertase-mRNA and prior to invertase destruction. In contrast, Chripeels et al. (1967) suggested that the gibberellic acid effect required synthesis of enzymespecific RNA molecules. They also proposed that abscisin exerted its action either by inhibiting the synthesis of these enzyme-specific RNA molecules or by preventing their incorporation into an active enzyme-synthesizing unit.










Most recent work at the molecular level, however, indicates that there are

ABA-responsive elements and GA-responsive sequences located on promoter regions in a number of structural genes (Jacobsen and Beach, 1985; Zwar and Hooley, 1986; Libbenga and Mennes, 1987; Marcott et al., 1989; Mundy et al., 1990; Salmenkallio et al., 1990; Jacobsen and Close, 1991; Skriver et al., 1991; Lanahan et al., 1992). An auxin-responsive promoter appears to be differentially induced by auxin gradients during tropisms (Li et al., 1991). It is more likely that the effects of abscisic acid, auxin, cytokinin and/or gibberellic acid on invertase are mediated by their respective influence on transcription, but these may well occur by different mechanisms.



Wounding and Invertase



Wounding typically stimulates expression of invertase genes (Matsushita and Urttani, 1974; Sturm and Chrispeels, 1990). A general increase in the respiratory activity in response to wounding in various plant storage tissues is well documented (Matsushita and Urttani, 1974). In root tissue of sweet potato, respiratory activity doubles within 20 hours after wounding. The increased respiratory activity is paralleled by increases in RNA content and the de novo synthesis of enzymes (Shirras and Northcote, 1984). Invertase may well be one of these and would be advantageous in its enhancement of capacity to initiate sucrose breakdown.










Sugar Levels and Invertase



A number of recent reports demonstrate that various genes involved in metabolic pathways are either induced or repressed by sugars (Carlson, 1987; Schuster, 1989; Maas et al., 1990; Sheen, 1990; Koch et al., 1992; Sadka et al., 1994). Studies of carbohydrate assimilation in potato tubers have revealed that genes encoding patatin (Rocha-Sosa et al., 1989), sucrose synthase (Salanoubat and Belliard, 1989) and ADP-glucose pyrophosphorylase (Muller-Rober et al., 1990) can all be induced by elevated levels of sucrose. Similarly, the tuberous root storage protein genes of sweet potato (Hattori et al., 1990) and the vegetative storage protein genes (vegetative storage proteins, Sadka et al., 1994) are upregulated by sugars. In Arabidopsis, sucrose mimics the light induction of nitrate reductase gene transcription (Cheng et al., 1992). In maize, elevated carbohydrate levels regulate the sucrose synthase genes differentially such that Sus] is stimulated whereas Shl is repressed (Maas et al., 1990; Koch et al., 1992).

Repression of gene expression by sugars has also been shown for other plant genes. In maize mesophyll protoplasts, seven photosynthetic genes are downregulated by photosynthetic end products sucrose and glucose and by the exogenous carbon source acetate (Sheen, 1990). In tobacco, the glutamate dehydrogenase gene is suppressed by feeding glucose (Maestri et al., 1992), and in cucumber, genes encoding enzymes of the glyoxylate cycle (malate synthase and isocitrate lyase) are repressed by carbon catabolites (Graham et al., 1994). Together,










these mechanisms may comprise a means for sensing and transducing signals of whole plant carbohydrate status, and subsequently altering plant metabolism and/or development.

Particularly important in such a possibility are the genes encoding those enzymes which can break down sucrose, sucrose synthase and invertase. Sugar responsiveness of the former has been characterized (Salanoubat and Belliard, 1989; Maas et al., 1990; Koch et al., 1992). Although invertase clones have not been previously characterized, levels of these enzymes appear to show a long-term, carbohydrate responsiveness (Sacher et al., 1963; Glasziou et al., 1966; Ricardo et al., 1972; Kaufman et al., 1973; Sarokin and Carlson, 1984).

Kaufman et al. (1973) demonstrated that increased levels of invertase were correlated with the sustained growth of Avena stem segments in the presence of sucrose. Their data further indicated that the presence of sucrose greatly enhanced the GA effect on elevation of invertase activity. They suggested that substrate may stabilize the enzyme and/or aid its induction. Their studies also support the view that gibberellic acid, as well as substrate (sucrose) and end products (glucose and fructose), play a significant role in regulating invertase levels in Avena stem tissue. Moreover, such regulation could provide a mechanism for increasing the level of soluble saccharides needed for gibberellic acid-promoted growth.

However, Morris and Arthur (1984a) documented a drop in concentration of hexose sugars in internodal segments of Phaseolus vulgaris during incubation in the presence of auxin. The greatest decline in hexose concentrations occurred when










segments were treated with compounds which stimulated the most growth. They also suggested that by reducing sucrose concentrations in the apoplast and/or symplast of sink tissues, the acid invertases located in these respective compartments may contribute significantly to maintenance of source-to-sink gradients in sucrose concentration and hydrostatic pressure which drives phloem transport.



Fungi, Bacteria, and Invertase



Increased invertase activities have been reported in tissues of several plants infected by biotrophic fungi and/or bacteria (Callow and Ling, 1973; Long et al., 1975; Billett et al., 1977; Billett and Burnett, 1978; Callow et al., 1980; Krishnan and Pueppke, 1988; Sturm and Chrispeels, 1990;). In addition, a common feature of biotrophic fungal infections of vascular plants is an increased translocation of photosynthetic assimilates into infected plant parts, which is typically accompanied by accumulation of one or more host carbohydrates (Callow and Ling, 1973; Long et al., 1975; Billett et al., 1977; Billett and Burnett, 1978; Callow et al., 1980).

Billett et al. (1977) have shown that infection of maize by the corn smut,

Ustilago maydis, stimulates assimilate movement into, and accumulation of soluble sugars, and starch, in tissues. Smut in maize kernels results in rapid growth, cell division, and elevated rates of respiration. Enhancement of maize invertase activity in these regions could facilitate competition with other sinks for the sugars needed to support these processes. Sucrose import and unloading from phloem could be








14

accelerated by a greater capacity for invertase to remove this sugar from the terminal end of the transport path.

Little research, however, has been directed toward understanding the

mechanisms by which invertase activity is elevated in response to pathogens (Callow and Ling, 1973; Long et al., 1975; Billett et al., 1977; Billett and Burnett, 1978; Callow et al., 1980; Heidecker and Messing, 1986; Collinge and Slusarenko, 1987; Sheridan, 1988; Sturm and Chrispeels, 1990). The origin of the induced invertase protein (fungal vs host) has remained controversial (Billett et al., 1977; Callow et al., 1980).

Agrobacterium tumefaciens and Pseudomonas syringae pv Savastanoi,

however, contain genes that specify the biosynthesis of cytokinin and indoleacetic acid (Morris, 1986; Morris, 1987; Ishikawa et al., 1988; Weil and Rausch, 1990). Cytokinins and/or cytokinin-like substances are also reported to be synthesized in mycorrhizal fungi (Miller, 1967; Crafts and Miller, 1974; Ng et al., 1982) and Bradyrhizeobium japonicum (Sturtevant and Taller, 1989). Allen et al. (1980) found that cytokinin levels increase in the host plant following infection by vesiculararbuscular mycorrhizae. Elevated levels of IAA and/or cytokinin have also been implicated in maize tissues infected by Ustilago maydis (Turian and Hamilton, 1960; Billett et al., 1977; Billett and Burnett, 1978).

Upregulation of invertase expression by fungal infection could facilitate enhancement and/or establishment of a symbiosis by providing hexoses for those fungi unable to metabolize sucrose. A resulting question is whether or not plant








15

hormones, such as IAA and/or GA, act as signals to target the elevation of invertase in plant parts infected by certain biotrophic fungi and/or bacteria (Heidecker and Messing, 1986; Morris, 1986; Collings and Slusarenko, 1987; Davies, 1987; Libbenga and Mennes, 1987; Morris, 1987; Ishikawa et al., 1988; Sheriden, 1988; Weil and Rausch, 1990)?

Sturm and Chrispeels (1990) imply that the homology between extracellular carrot P-fructosidase and the levan hydrolyzing enzyme, levanase, may allow carrot 1-fructosidase (invertase) to hydrolyze the bacterial slime coat. In this way, invertase action could inhibit bacterial growth directly or make the pathogen susceptible to further defense reactions. In this scenario, invertase would function in a positive, protective role as a new and unrecognized pathogenesis-related protein.












CHAPTER 3

ISOLATION AND CHARACTERIZATION OF MAIZE INVERTASE GENES




Introduction



Only two avenues known for enzymatic breakdown of sucrose exist in

vascular plants. One is catalyzed by sucrose synthase, the other by invertase. Two distinct types of invertase activities are found in plants (Avigad, 1982). One class has an optimum pH of 4.5 to 5.0, and includes acid invertases. The second class hydrolyzes sucrose at a maximal rate at pH 7.5 to 8.0, and is designed as the alkaline invertases. The existence of these two types of 0-fructosidase is evident in many plants and/or organs (Avigad, 1982). Acid invertases are located either inside the vacuole (soluble form) or in the extracellular space (varying degrees of soluble and cell-wall-bound forms). In contrast, alkaline invertases are compartmentalized in cytoplasm (Hawker, 1985).

Invertase genes encoding cell-wall and vacuolar (soluble) acid invertases have been characterized from carrot (Sturm and Chrispeels., 1990; Ramloch-Lorenz et al., 1993; Unger et al., 1994), tomato (Klann et al., 1992; Elliott et al., 1993), mung bean (Arai et al., 1992), and Arabidopisis (Schwebel-Dugue et al., 1994).










In the present study, a tomato invertase clone (Klann et al., 1992) was used to isolate an invertase cDNA from maize, and this, in turn, was used to obtain additional maize clones. These findings provide the tools for further investigation along two lines. The first of these will be aimed at combining an analysis of sugarresponsiveness of these genes with that of the sucrose synthases, to define carbohydrate regulation of two different avenues for sucrose breakdown. The second will be to further clarify the potential functional significance for soluble invertase isozymes in development and/or environmental adjustment in maize.



Materials and Methods



Probe for cDNA Library Screening



A 0.45 Kb fragment from the 5'-end of a cDNA encoding a soluble acid invertase in tomato (Klann et al., 1992) was isolated from tomato clone and subcloned into a pUC19 vector. The recombinant plasmid was amplified in E. coli cells, purified, and used to screen a maize cDNA library (Sambrook et al., 1989).



cDNA Library Screening



A maize root tip cDNA library (Xgt 10, Clontech, Palo Alto, CA) was

screened with the 0.,+5 kb tomato invertase cDNA fragment. One positive clone










containing a 1.2 kb cDNA was obtained. This maize fragment was subcloned and used to probe for further cDNAs from the same library. Twelve positive clones ranging from 0.5 to 2.2 kb were identified.



Genomic Library Screening



A maize genomic fragment containing 8 kb DNA was identified by screening a genomic library (EMBL 3, Clontech, Palo Alto, CA) with the 1.2 kb maize invertase cDNA clone.



Hybridization with DNA probe



Procedures for library plating and production of filter replicas were conducted as recommended by Clontech (Palo Alto, CA). Plaques or colonies were blotted to nylon membranes, and DNA was denatured in situ with NaOH (0.5 M), neutralized with Tris buffer (1.0 M, pH 7.5), and fixed by baking (80 'C, 0.5-2 hr) (Sambrook et al., 1989). Filters were hybridized at either 50 C (low stringency) or at 65 C (high stringency) in a solution with the selected cDNA, 7% SDS, 250 mM Na2HPO4 (pH 7.2) and 1% BSA (Church and Gilbert, 1984). Tomato and/or maize invertase cDNA fragments were radiolabed by random primer (BRL, Gaithersberg, MD). Blots were washed as described by Church and Gilbert (1984), and exposed to X-ray film with intensifying screens at -80 'C.










DNA Sequencing



Selected cDNA and genomic DNA fragments were subcloned into a pUC 19 vector. The recombinant plasmids were amplified in E. coli cells and purified through CsC12 ultracentrifigation and/or with the use of QIAGEN-tip (QIAGEN Inc., Chatsworth, CA). Both strands of each cDNA and genomic DNA were sequenced by the Sequence Core Lab of ICBR (Interdiciplinary Center for Biotechnology Research) located at the University of Florida.



Analysis of DNA and Protein Sequences



Computer-assisted analyses of DNA and protein sequences were carried out with Geneworks (Release 2.2, IntelliGenetics, Inc., Mountain View, CA).










Results



One positive clone containing a 1.2 kb maize cDNA (Ivrl) was obtained (Figure 3-1) when a cDNA fragment encoding a soluble acid invertase in tomato (Klann et al., 1992) was used to screen a maize root tip cDNA library (Xgt 10, Clontech, Palo Alto, CA).

This 1.2 kb maize fragment was used to rescreen the same library. Twelve

positive clones ranging from 0.5 to 2.2 kb were identified. Sequences obtained from the longest of these indicated that none of them included a full-length cDNA clone. For this reason, a HindIII-EcoRI fragment from the longest clone (2.2 kb, Figure 31) was used to rescreen the library a second time. Seven positive clones were identified.

From the total of twenty clones, five were selected for full length sequencing based on their sizes, hybridization characteristics, and location of restriction sites (Figure 3-1). Sequence was provided by the Sequence Core Lab of the ICBR at the University of Florida. They were designated IvrJ through Ivr2C-3 (Figure 3-1). Ivr2C-2 was identical to Ivr2, and Ivr2C-3 contained the same but shorter sequence as Ivr2C-].

Further information was sought in the corresponding genomic sequence. A maize seedling genomic DNA library (EMBL 3, Clontech, Palo Alto, CA) was screened with a I kb KpnI-EcoRI fragment from the Ivrl cDNA Figure 3-1, 3-7). One positive genomic clone was isolated and characterized. This clone consisted of








21

ca 8 kb DNA. Digestion with BamHI and KpnI generated three fragments, each of which was subcloned and sequenced.

The invertase coding region was deduced according to the information from cDNAs, recognition sites for intron splicing (Goodall and Filipowicz, 1989; 1991) and/or comparision with other invertases from vascular plants (Figure 3-5, 3-6). The gene for maize invertase 1 (IvrlG) was organized into seven exons and six introns, as diagrammed in Figure 3-2A. The second exon was only 9 nucleotides long (Figure 3-4B), and has also been reported in tomato fruit vacuolar invertase (Elliott et al., 1993). The amino acids encoded by this 9 bp exon are located in a highly conserved domain found in all invertases cloned thus far (NDPNG, the 3fructosidase motif, Sturm and Chrispeels, 1990, Figure 3-2B).

The genomic DNA (IvrlG) is almost identical to the Ivrl cDNA clone at the level of amino acid sequence, except for a few amino acid replacements. Genomic and cDNA clones are from different maize lines, lvriG being isolated from a B73 genomic library, and the Ivrl cDNA from a 'Merit' root tip libary.

The deduced amino acid sequence from lvriG consisted of 670 residues

(Figure 3-3) which predicted a molecular weight of 71,942 and an isoelectric point of 7.5. This protein also included five potential glycosylation sites (N-X-S/T): N65, N275, N518, N595 and N639 (Figure 3-3). The amino-terminal sequence of the IvriG protein indicated a hydrophobic region between basic N and polar C terminals (Figure 3-3, 3-4) and other characteristics typical of a signal peptide ([-3,-1] rule,von Heijne, 1986; K. Cline, personal communication). The predicted excision site for










the signal sequence according to the (-3, -1)-method of von Heijne (1986) was between A73 and G74.

A comparison between the invertase genes isolated from maize in the present work and other invertases from vascular plants (Table 3-1; Figure 3-6), IvrlG shared an approximate 60% amino acid identity with soluble invertases (Arai et al., 1992; Klann et al., 1992; Elliott et al., 1993; Unger et al., 1994) and 40% with insoluble forms (Sturm and Chrispeels, 1990; Ramloch-Lorenz et al., 1993); moreover the conserved key domains (NDPND [D-fructosidase motif, Sturm and Chrispeels, 1990], as well as FRDPTTA, TGMWEC and YASKTF) (Figure 3-5). In addition, the maize invertase gene has a significantly greater amino acid identity to the soluble isoforms of invertase than to the cell-wall-bound ones found in other vascular plants, especially at the C-terminus of this protein (Figure 3-6).

Restriction maps of invertase cDNA and genomic clones from maize are shown in Figure 3-1. Ivr2 was found to share a 53% sequence similarity at the amino acid level to IvrlG (Table 3-1), especially have extensive sequence similarity located at conserved domains (data not shown). The Ivrl probe (1 kb KpnI-EcoRI fragment) did not cross-hybridize with the Ivr2 or Ivr2C-1 cDNAs at high stringency (Figure 3-7B). The Ivr2 probe (200 bp PstI-PstI fragment) cross-reacted with the Ivr2C-1 cDNA but not that of Ivrl (Figure 3-7C).

Ivrl was missing its 3'end and contained one 5' unspliced (putative) intron (according to the sizes of 5'RACE products, E. Bihn, unpublished data). Ivr2









23

contained an unusual 5'end, missing the NDPNG (sequence indicates possible incomplete intron splicing). Ivr2C-1 was lacking the 3'end of its coding sequence.




















Table 3-1. Percentage comparative sequence similarity shared at the amino acid level between genomic and cDNA clones for maize soluble invertases (Ivr]G and Ivr2) and those of other invertases from vascular plants

Amino Acid Identity (%)


aTomato soluble invertase (Klann et al., 1992). bS represents soluble isoform for invertase. cMung bean soluble invertase (Arai et al., 1992). dCarrot soluble invertases (Unger et al., 1994) eCarrot insoluble invertases (Sturm and Chrispeels, 1990; Sturm, unpublished data). fCW represents cell-wall-bound (insoluble) invertase.


Ivr1G Ivr2
Tomato' (S)b 61 48 Mung Bean' (S) 59 56 Carrotd (SI) 59 49 Carrotd (SII) 59 49 Carrot' (CWI)' 42 32 Carrote(CWII)f 45 32 IvrlG 100 53 Ivr2 53 100































Figure 3-1. Restriction maps of Ivr clones for maize soluble acid invertases.
Restriction maps of maize soluble invertases (IvrlG, Ivrl, Ivr2 and Ivr2C-1).
Sites on the restriction maps are as follows: B, BamH I; H, Hind III; K, Kpn
I; P, Pst I; S, Sma I.





















H K S BP H I I I I I



K




III I I I


Ivr2C-1


:1 Kb


Ivrl G Ivrl Ivr2


, |























Figure 3-2. Schematic diagram of the genomic organization of the IvrlG gene for soluble invertase from Zea mays L..
A, The entire IvrlG gene for soluble invertase and bordering regions is depicted with exons as solid boxes. The
locations of a putative CAAT box, TATA box, translation start (ATG), translation stop (TGA) are designated
with arrows. B, Enlarged area from A, which encodes the most strongly conserved region among all invertase
sequences (NDPNG, the P-fructosidase motif) (Sturm and Chrispeels, 1990).
















E. 4
14 4 ______________


V

Area enlarged
below


.


- m -


-m U


I Kb


fl-fructosidase motif































Figure 3-3. The deduced amino acid sequence for maize invertase I gene. The
arrow indicates the cleavage site for potential signal peptide. The box represents P3-fructosidase motif (NDPNG, Sturm and Chrispeels, 1990).
Underlined sequences are for putitive glycosylation sites (N-X-S/T).























.4 ~. ~ is ~ .~ a a~ o ~ ~.. a ~' 4 .4 xs .3 z 3 ... .~s ~r .




t.1 ko V n "4 .4 N xR~ .. a 0 a x -5 0 a )I~ 3
r. IHN of j1 t. A . a j r r 3'. a x zOg


au to Pa a z .4 w4 < -C is -C a t. x x t a s~ s ~ ~ .


is 3. r4 v a x ag -Cst Na~ 0~~ 34 ~ at si ~KNE .4 a' 3









.4 ) .4 is is 0 4 0 j N .4 N i x a 4 3 .4 a a us . .4 ft 4 0 .4 A a r a' 4 .4 4 c0 n 0 x 114 A1 a. w Ri w a a R .C R a .0 2 'e a 9 A K 04 1 4 0 -0 g4 t, a s ii 0 a v R40 a R )0 1 0 R r R0 x 9 9 .4 m .t































Figure 3-4. The hydropathy and folding values of the deduced polypeptide for
maize invertase gene 1. A, hydropathy. B, folding structure. The dashed
lines indicate the putative signal peptide cleavage site.















A. Hydropathy


.k.1 ,i 4 I J~iid,~


phil
0 134 268 402 536


B. Structure


A M


J.A,. A AA ^A y%-


AAIAL. AAAAL A A AAAA.AA AA d


. A 1hA LkW A,. -A,.-


A I ,,' -v'v -v "' A! 'AA'


IV I.. v rw I I' Y v yy-, v 'v 1 'FYI


711


Helix


Sheet



Turn



Coil


1. 111 1hil i. J I


. . .. . . .. .. I 1 1 4 Pq P 4 I . .


A


I


0 134


268 402


536 670























Figure 3-5. Conserved regions within derived amino acid sequences of higher plant invertases, shown here for the
Ivr]G for maize soluble acid invertase gene 1, a mung bean soluble invertase (Arai et al., 1992), a tomato
soluble invertase (Klann et al., 1992), two carrot soluble invertases (Unger et al., 1994), and two carrot insoluble
invertases (Sturm and Chrispeels, 1990; Sturm, unpublished data). Boxes represent highly conserved regions.































_t-.7A MA& 5 PM-44. L~-......

TL593.18 SI.U)35A4 9*MWL 815)38 SI3 VL
-0I----5 -U,1T5555 ----5-1555 3 ..... 1,104 -3
9('1+1354315*8S 44KJF7ITS 5*555I3*-- -953.4.*553ll.


*........ .......... ..... *..... ......... ..... 011.1,I

-----. Srffl .......... .... LAIlf .. .......... ..... F..--I-D
*-n5AA 115l--L58iW ON,45 805 111A ------ -9 51914 5 LIL MS .1,1r ,LLYWNVW wtoa"tmo "m I* ++x illa i&--VWiiYiMtl SO IAS nwo XnA.Vllal v 1 Al8939W~ L-ISISSO- 3. 5V35WM VE 5WVA l.811.".831 9.ow55550 155555554(5 thsS88 .s4. ...... IV .......... 9)ITL5I5 .............. 3 4

..-.................. ... ....... .......V. .. ..


?SC W. .-505*v5155)*8.8




555551*94.3 :0104aw 55586MM"5
n9XV.1638. .5040583- --vw5--- a
5.1 ......... ..3.83 @ .


C..t I41I3)





1-t(. III) C.... %41611 C.,." 0tl














C.... 35
Ils. ISO C.,..t 4*43 94**5 St (54l












C... 4313S ttet 41534| ...., 3 IS












.-.. 403
(an I 4833il







CI... ISO C ... tt I
31.4I5 11




C :. .al C.. r, Ill3


Sf35 lll



*lot (4.533I
5.5....I

5.54. II5 33 ?.4. 45)


* I.5l 4







31.55q P.. 38)
*ll5 ll C...I 4rl 3


I


B. 3' end


C."S.. 455133 Cir ttt 11111





latti 11 o C...* ll


C.n.4 Call



C ... 4 5IS 5.*..t 45333 I 08.8 *. (81






A .:"r I'll


C..." I 333




C*,."l fil A i-it ll C ...1 ASCSIl








C .... t "M53


0.. 41.1
0-0r 1- IS1 Ca :." IS 1,




C-... 53

5S.. ttL Can
5.5.55l (5l34









" Im .is:




C...333
tWo OIvll l
1i ,t. ISOl



C-_ INS C .., ,Lill



.I5ri
?55 35) Il






Clrit 5333 1.1I5 35l t0535 535)Il 855... 4il3 50(5. 453
*I .s(0 al 505151 31i433

5*551III


F~I55 fS 53344 5-5 4~I4~0





M ,~4~4 'y" 9 i584.5 l.. 4'
p5I(W'ol 055344 3 O55541454.

~fi~ 3 5o5ni 5
3 555 .11 008 1

So 5 5

3atii 3955t 15
5 AO~ 58511 TCMS- t
If VUM -O -5*875t A7 558 r u~rc-o -t
SI'I E uwvTc "- 1






3 80 I9531* 4 .. a?
II 9,o"
LAM SV4(3.350

U=M5155581 3.55

rim 55xiU jjSVS.N I fa IL









35)5 1 .5-ZL 55155 NO 38
NO A 185394? 535 5155"
595...50 SO O --LIPS ~ ~ ~ V.-&J A I.V...


A. 5' end


85M 4 95~~~o.3 u 4 53






II 59 ..30 .5 J Im





4934955 9118uZ85. 44. SO=---j5$ I morn.LM if ~ ~ ~ ~ ~ rm- 3i!! bITSA8L .594IfmJl
v5. 49913 ~ 5 555*0 513 lo" 9555M 304358 4 454





















Figure 3-6. Conserved regions within derived amino acid sequences of the IvrlG for maize soluble acid invertase gene
1 and either A, other soluble invertases or B, insoluble invertases from other vascular plants. A, Derived amino acid sequence similarities shared between the IvrlG cDNA clone for maize soluble invertase, mung bean soluble invertase (Arai et al., 1992), tomato soluble invertase (Klann et al., 1992), and carrot soluble invertases (Unger et al., 1994). B, Derived amino acid sequence similarities shared between the IvrlG and the insoluble invertases of
carrot (Sturm and Chrispeels, 1990; Sturm, unpublished data). Boxes in A and B represent the most highly
conserved regions and underlined sequences are those shared among soluble but not insoluble invertases.
























RTIW p---T DP ISUtvMM- --ora IOMlS ...---- |loir
HM---- 9 PIJSBSNAA -8 5-T IUD L.LVLC---. -OLLFJS-*L
PITISH 1PL100 1MP SL11I1MWAI 8 2LT-- -852,8883IL.
_FAVAOP f7 A.Y=A588 I8P 880 AMAZQ8-o -88,A ?878VL ........L....S.. .. ........ .. ..

LO-VILLLSV A *'--SLM 92904 1a0 SPA--;;; ...- 0a LSZILSVV 2 U.l IVN QFQM WVIaI sI S )og m103 35 88 S M VA--vooTyA SOVPIAHLJLS 8gO s V, I M...0IIN L-.. ..- =I!!VV8E =I!!VA-4....
TAVVSAVLLL VLVAVTVAI 2g4VDitV F8AOZDXWZ VAAsfIA .8.......... .. ..... 0 .............. SI
- VT- CmNVIR

O0 Ia8I58 IakTAK8S
VSUSSNLLV AOIOAS3Af
VSP8S PA L-UP-NJIeFAw11
vsWn ...... -0.. 50 0I


Tumato 45) Caret SM1| Hong Dean (So Cgcgot 4S5|


linen u5 Tomato (so Coret |Sil 1lung Dean ISO carrot (oil

Carrot 481s3




Carter lo ll Slyrloi




160sto IS

Carrot 4l1ll

vs|O o"Sonous

Tomato is) Carrot ISII 1wng Dean IS)
C~rcot (SI)



C I O)
Conasnsus


Toato 0ISO Carrot toll $lung e.n IS)
0.110| 484)



C Isl.I
C...I0u.





TMW*4to p
Cit 4Mi3| 80flg Uran 453



Cerst 4563 38.55 aa.48 Cerot 3823~


I Il l I


A. Soluble 5' end


U


B. Insoluble 5' end







Carrot 40l) | ....... S.-L--------- ---------- ----------- s



* srI0| SAVLLWVA V, 6V -pJ1ov8 AVw PGVAV8r 1s8 Caro 'C, r "+F-I1--------...... -- ... 4r...".t ICH ., -H-.. im_3 .......... ----1 ..... 3
Consensus -5.2...LV..TI ---U----- -----. iA.U.V ....... ice





Corsot .e ) OV r 0 Carrot 40411)-------3UIAVWX LIV-1 pa sI Noi 7 Consensus-----.....L.A M..L V-M m a ISO


carot IIT


Carrot 0C413 9 IS Consensus. 20






"* t 0 NILV88aza 28
Carrot0c 4 3 t1 t 1 !1 Carrot (CI413 I 1 ConsnusV ..F=2504




Carrot 4041) 8 27




Carrot4011 I )F(8daSP3 18300ir 4 Conseonsus 'vW... a.. 0




ca rrot 1CMI) 9)dO


Consonsus 2A7




continued


iU -180." ."I8



838pAul I o? SC 1 ,92 CS 8







continued






















Figure 3-7. DNA gel blot analysis of A, Approximate size of the Ivrl, Ivr2, Ivr2C-1 and Ivr2C-2 cDNA clones and B,
Extent of cross-reactivity between them. A, EtBr stained DNA gel blot analysis of approximate length for Ivrl, Ivr2, Ivr2C-1 and Ivr2C-2. B and C, DNA gel blots with equal amounts (1 p.g) of recombinant DNA from each
cDNA (Released from pUC 19 vector by digestion with EcoRI) and probed with 32p-labeled fragments from
either A, Ivrl (Kpn I-EcoR I, 1 kb fragment) or B, Ivr2 (Pst I-Pst I, 200 bp fragment). Both blots were exposed
to X-ray film for 1 hr. Sites on the restriction maps are K, Kpn I; P, Pst I; R, EcoR I.













MW
Ivrl Ivr2 Ivr2C-1 Ivr2C-2


AA A


Ivrl Ivr2 Ivr2C-1 Ivr2C-2


Ivrl Ivr2 Ivr2C-1 Ivr2C-2


A A
Ad -.1


ii


.-0
0
0r (D
CL












Discussion



Soluble invertase genes were cloned and characterized for two reasons. The first of these was to characterize the extent of their carbohydrate-responsiveness relative to that of genes for sucrose synthase, ultimately to provide a more complete picture of how sugars influence the capacity for their own metabolism at the transcriptional level. The second planned use for the invertase clones was to clarify the potential significance of these gene family members during development and/or environmental adjustment by maize tissue and organs.

Three lines of evidence support the designation of these genes not only as maize invertases but also as soluble ones. First, the full length sequence of the putative maize invertase clone (IvrlG) has extensive sequence similarity to other invertases found in vascular plants (Table 3-1), and shares the conserved key domains identified in other invertases (NDPNG [P-fructosidase motif, Sturm and Chrispeels, 19901, plus FRDPTTA, TGMWEC and YASKTF) (Figure 3-5). Second, the maize invertase gene examined here has a considerably greater amino acid identity to the soluble isoforms of invertase than to the cell-wall-bound ones found in other vascular plants (Table 3-1; Figure 3-6). The underlined areas in figure 3-6 are those highly conserved regions which are shared among soluble invertases but not insoluble ones. In particular, the amino acid sequence at the C-terminus of the IvrlG protein is significantly more similar to that of soluble vs. insoluble forms.










Targeting signals for vacuolar proteins are frequently present in this region as Cterminal propeptides (Bednarek et al., 1990; Chrispeels, 1991; Bednarek and Raikhel, 1992). Third, message abundance of Ivrl and Ivr2 correlates well with total soluble invertase activities in an array of maize tissues and/or developmental stages (see Chapter 4).

Invertases of maize and other vascular plants are presumably encoded by different genes, although in yeast, variable splicing allows a single gene to encode both cell-wall-bound and soluble invertases (Carlson and Botstein, 1982). There are at least two Ivrl-like genes in the maize genome, and the Ivrl and Ivr2 subfamilies have been tentatively mapped to two and four different loci respectively (data not shown, collaboration with Scott Wright, Genetic Linkages, Salt Lake, Utah).

The genomic clone of maize invertase has typical CAAT and TATA boxes located in the upstream untranslated region (Figure 3-2). The second exon is unusually small (9 bp) in maize Ivrl invertase (Figure 3-2) and tomato soluble invertase genes (Elliott et al., 1993). The amino acids encoded by this 9 bp exon are located in a highly conserved domain found in all invertase clones (NDPNG, the 3fructosidase motif, Sturm and Chrispeels, 1990). This represents one of the smallest exons currently known to function in the plant genome (M. Schuler, personal communication).

In the Ivrl maize invertase genomic gene, several introns (number 1, 3, 4 and 5) are also found to contain one or more copies of an RY sequence motif (CATGCATG, data not shown), which thus far has been implicated in seed-specific










gene expression (Dickinson et al., 1988; Baumlein et al., 1992; Lelievre et al., 1992). This suggestion is also supported by the preferential expression of the IvrJ subfamily genes in reproductive tissues (see Chapter 4).

The polypeptide encoded by the Ivr]G invertase gene has 670 residues with a molecular weight of 71,942 (Figure 3-3). The calculated isoelectric point is 7.5, which is intermediate between that of carrot soluble invertases (SI: 3.8; SI1: 5.7, Unger et al, 1994) and insoluble invertases (carrot CW: 9.9, Sturm and Chrispeels, 1990; Arabidopsis CW: 9.1, Schwebel-Dugue et al., 1994). This protein also contains five putative glycosylation sites (N-X-S/T) and a potential peptide signal from M1 to A73 (Figure 3-3).














CHAPTER 4
DEVELOPMENTAL EXPRESSION AND CARBOHYDRATE
RESPONSIVENESS OF TWO MAIZE INVERTASE GENE SUBFAMILIES


Introduction



Invertases (13-fructosidase, EC 3.2.1.26) play a key role in sugar metabolism. In vascular plants, different isoforms are located in different cellular compartments (Avigad, 1982; Hawker, 1985). Isoforms with an acidic pH optimum are found in the vacuole and/or apoplasm whereas isoforms with a neutral pH optimum are located in the cytoplasm. Work with sugar cane stems (Hawker and Hatch, 1965; Glasziou and Gayler, 1972) and corn kernels (Shannon, 1968; Shannon, 1972; Shannon and Doughty, 1972; Shannon et al, 1993) has indicated that imported sucrose moves from phloem into the extracellular space where it is hydrolyzed by a cell-wall-bound, acid invertase. This is presumed to contribute to a sucrose concentration gradient between the phloem and apoplast, facilitating transfer of sucrose into importing tissues (Lin et al., 1984; Doehlert, 1986; Doehlert and Felker, 1987; Doehlert et al., 1988; Turgon, 1989). Soluble invertase has been found in the vacuoles of sucrose-storing cells (Avigad, 1982). Thus, soluble invertases with acidic pH optima are often thought to be localized in the cell vacuoles of other tissues as well, where they can mobilize sucrose temporarily stored in this compartment.










Rapidly expanding tissues require either invertase or sucrose synthase to convert sucrose into substrates necessary for respiratory and synthetic processes (Giaquinta, 1979; Avigad, 1982; Morris and Arthur, 1984b; Hawker, 1985; Schaffer et al., 1987). Invertase can be especially important to cell expansion through generation of hexoses and their associated osmotic potential (Kaufman, 1973; Avigad, 1982; Schmalstig and Cosgrove, 1988; 1990). Invertase activity is also associated with reproductive organs (Jaynes and Nelson, 1971a; Shannon and Dougherty, 1972; Singh and Knox, 1984; Hubbard et al., 1989; Miron and Schaffer, 1991; Klann et al., 1992; Reger et al., 1992). Invertase can aid competition for sink capacity for reproductive growth. Soluble invertase is the predominant enzyme for sucrose breakdown during the early developmental stage of maize kernel (Tsai et al., 1970) and snap bean seed (Sung et al., 1994).

Genes regulated by carbohydrate were first studied in microorganisms

(Carlson, 1987; Schuster, 1989). Those genes are usually involved in metabolic pathways. In vascular plants, sugar-responsive genes have been primarily characterized in storage organs (Rocha-Sosa et al., 1989; Salanoubat and Belliard, 1989; Muller-Rober et al., 1990; DeWald et al., 1994; Sadka et al., 1994). Those genes generally encode storage proteins such as patatin from potato (Rocha-Sosa et al., 1989), tuberous root storage protein genes from sweet potato (Hattori et al., 1990), vegetative storage proteins from soybean (Sadka et al., 1994). In addition, carbohydrate-induced changes in gene expression have also focused on metabolic pathways, especially those involved in sugar metabolism such as sucrose synthase








44

(Maas et al., 1990; Koch et al., 1992), malate synthase, isocitrate lyase (Graham et al., 1994) and/or photosynthetic pathway (Sheen, 1990) and are considered critical mechanisms for sensing environmental and developmental signals.

Invertase is one of the only two enzymes known for sucrose breakdown in vascular plants and has shown a relatively long-term responsiveness to carbohydrate availability at the enzyme level (Sacher et al., 1963; Glasziou et al., 1966; Ricardo et al., 1972; Kaufman et al., 1973).

Previous research indicated that invertase was vital at both the specific organ level and at the whole plant level. Robbins (1958) found that OH43 primary roots could not grow on sucrose arga, and Duke et al. (1991) showed these roots to be invertase deficient. Miller and Chourey (1992) also found that the abnormal development of miniature kernels was associated with an invertase deficiency. The present study utilizes two acid invertase gene-probes to determine the effects of developmental processes and altered carbohydrate availability on expression of the Ivr] and Ivr2 classes for soluble acid invertase genes. The report presented here also demonstrates the extent of developmental differences and carbohydrate responsiveness in two subfamilies of maize genes for acid invertase (probably soluble). These findings indicate that there may be specific roles for soluble invertases during development and that these could differentially contribute to adjustment of sucrose import, cellular volume, and possibly metabolism in vascular plants.










Materials and Methods



Plant Material



The Zea mays hybrid NK 508 was used for all experiments. For analyses of developmental changes, plants were grown under greenhouse or field conditions. Samples harvested included leaves, anthers, silk, cobs, pollen, prop roots, and kernels at different developmental stages.

For experiments with root tips, seeds were first emersed in 20% Clorox for 30 min, followed by 30 min of continuous rinsing with water. Germination took place in the dark at 18 'C on two layers of moist 3 MM paper (Whatman, Inc., Clifton, NJ) in 17 x 26 cm glass pans. Air flowed continuously at 1 liter min' through each pan for the 6-day period, with 40% 02 supplied during the final 24 hr before root tip excision. The moisture level was adjusted daily by applying mist and draining excess water. The terminal 1 cm was cut from root tips (at ca. 3 to 6 cm total length) under a sterile transfer hood.



Experimental Conditions



Experimental treatments were as described by Koch et al. (1992).

Approximately 100 root tips (- 500 mg) were used for each experimental treatment. Excised root tips were incubated in the dark at 18 C for 6 to 48 hr in Whites'








46

medium, either with or without an array of supplemental sugars. Each group of root tips was agitated at 120 cycles per minute in a 125-ml side-arm Erlenmeyer flask with 50 ml of sterile media. Airflow (40% 02, make sure to supply enough 02) through air stones in each flask was maintained at 250 ml min' throughout the incubations.



RNA Isolation and Gel Blot Analysis



Root tip samples were rinsed twice in sterile water, blotted dry, weighed, and frozen in liquid N2. Other samples (as mentioned in the previous text) were harvested from greenhouse and/or field-grown plants, weighed, and frozen immediately in liquid N2. Samples were ground into fine power in liquid N2 and total RNA was extracted (McCarty, 1986). RNA was quantified spectrophotometrically (Sambrook et al., 1989). Total RNA (10 jig) was separated by electrophoresis in I % agarose gels containing formaldehyde (Thomas, 1980), blotted to nylon membranes, and fixed by baking and/or UV treatment (Sambrook et al., 1989). Filters were hybridized at 65 OC in a solution with 7 % SDS, 250 mM Na2HPO4 (pH 7.2) and I % BSA (Church and Gilbert, 1984). Maize Ivr 1 and Ivr 2 invertase cDNA clones were radiolabeled by random primer. No cross-reactivity was observed between the Ivr 1 and Ivr 2 gene probes when hybridizations were conducted at high stringency (data not shown). Blots were washed as described by








47

Church and Gilbert (1984), and exposed against X-ray film with intensifying screens at -80 0C.



Enzyme Extraction



Soluble invertase was extracted as per Duke et al. (1991). Frozen tissue samples were ground to a fine powder in liquid N2 using a mortar and pestle. Frozen powder was transferred to a second mortar containing ice-cold 200 mM HEPES buffer (pH 7.5) with 1 mM DTT, 5mM MgCI2, 1 mM EGTA and 10%(w/w) PVPP. One ml of extraction buffer was used for each 100 mg of tissue fresh weight. Buffered extract was centrifuged at 15,000 x g for 10 min to sediment particulate matter. Pellets were saved for salt-solubilized particulate invertase extraction. Supernatant was dialyzed (50,000 MW cutoff) at 4 C for 24 hr against extraction buffer diluted 1:40. Buffer was changed twice. Soluble dialyzed extract was centrifuged again at 15,000 x g for 10 min and supernatant assayed for soluble invertase activity as described below.

Insoluble invertase was extracted as described by Doehlert and Felker (1987). Pellets remaining from extractions of soluble invertase were washed three times by sequentially resuspending each in 5 to 10 ml extraction buffer and centrifuging at 15,000 x g for 10 min. Salt-solubilized particulate invertase was extracted by resuspending the pellet in extraction buffer containing 1 M NaC1. Solubilized particulate invertase was recovered in supernatant following centrifugation at 15,000










x g for 10 min. Pooled supematant fractions were assayed for insoluble invertase assay as described below.



Enzme Assay



Both soluble and salt-solubilized invertase activities were assayed for 15 to 30 min at 37 C in an assay medium with 100 mM Na-acetate (pH 4.5) and 100 mM sucrose in a final volume of 500 p1. Activity was determined by measuring reducing sugars as described by Nelson (1944) and Somogyi (1951).










Results



Developmental and organ-level differences were evident in expression of the two classes of invertase genes (Figure 4-1 A). Message levels for the Ivr] group were markedly higher in reproductive structures than vegetative tissues, whereas those of the Ivr2 type transcripts were abundant in essentially all of the sucroseimporting structures examined (loading same amount of total RNA). Message from both classes of invertase were present in sink leaves, dropping below detectable levels during sink-to-source transition. Transcript levels of both types were also evident in those tissues undergoing rapid growth and/or cell division, such as root tips, anthers, pollen and silk (styles). As observed for relative mRNA abundance, activity of this enzyme fraction also predominated in the most rapidly elongating tissues (such as root tips and silk) regardless of whether data were expressed per unit protein or fresh weight (data not shown). Activity was also generally elevated in instances of enhanced sucrose import. The greater ratio of RNA/protein recovered from root tip extracts vs those from other tissues suggests that if changes in total RNA encoding invertase messages are viewed relative to protein levels, then invertase mRNA levels in root tips are greater relative to enzyme activity than is evident in Figure 4-1. The greater values for RNA/protein recovery from root tip extracts may possibly be due to the extensive meristematic activity in these organs.

Shifts in region of localization were evident during kernel development for the two subfamilies of maize invertase (Figure 4-2 A and Figure 4-3 A). Message










(Days After Pollination), dropping below detection within 16 DAP. However, the Ivr2 type mRNA was abundant in the pedicel region and barely detectable in the middle and top portions of the kernels (Figure 4-3A). In contrast, levels of Ivrlrelated messages in the pedicel region were similar or less than those in the middle and top sections of kernels at the same developmental stage. In addition, developmental differences in timing were evident, with a narrow peak in Ivrl transcript abundance at 8 DAP in the upper kernel, vs a broader elevation in Ivr2 message in the pedicel between 8 and 12 days after pollination. Transcript levels of the Ivrl subgroup were approximately similar in pericarp and endosperm at 10 DAP, whereas the Ivr2 mRNAs were considerably more abundant in the pericarp (data not shown).

Figure 4-3 B showed that total soluble acid invertase activity, like that of Ivrl and Ivr2 mRNA was highest in the pedicel region and lowest in the top area of the same kernels when expressed per unit total soluble protein (similar results were observed when data were calculated per unit fresh weight [data not shown] expcept that peak activity was elevated for two days longer). Total activity of soluble acid invertase was maximal in extracts of kernels sampled 12 days after pollination, dropping gradually to below detection in those from between 20 and 24 DAP (Figure 4-2C). In contrast, salt-solubilized particulate invertase activity (insoluble) increased gradually in developing kernels, but did so most rapidly between 2 and 6 DAP. Peak activity was observed at ca 16 DAP, and decreased slowly thereafter. Activity remained detectable at 32 DAP (well past maturity under local growing










Activity remained detectable at 32 DAP (well past maturity under local growing conditions). This salt-solubilized activity was also maximal in the pedicel area and lowest in the top portion of the same kernels when expressed per unit total saltsolubilized protein. If decreases in mRNA levels encoding IvrJ and Ivr2 are viewed relative to protein levels, then the drop in message abundance is more pronounced than pictured due to the onset of enhanced protein storage in kernels between 10 and 12 DAP.

During the earliest stages of kernel development, message levels for both Ivr] and Ivr2 subfamilies and total soluble invertase activity increased markedly (Figure 4-4). Soluble invertase activity from kernels two days after pollination was twice as high as that of unpollinated ones (Figure 4-4B) and insoluble activity from the same kernels (Figure 4-4C).

During anther development, transcript levels of both Ivrl and Ivr2 classes of invertase increased gradually through anthesis (Figure 4-5A). Both message types were also abundant in RNA extracted from pollen. This was probably not the basis for localization in young anthers, because shedding anthers had greater apparent levels of both classes of mRNA than did pollen itself.

Both the IvrJ and Ivr2 types of mRNA were abundant in silk if tissue was sampled before or immediately after pollination (Figure 4-6A). A gradient in relative message levels for these gene classes was also evident along the length of the silk, with lowest levels in the top (distal) 1/3 and greatest abundance in the 1/3 closest to the ovary (proximal) (Figure 4-7A). A rapid response to pollination was








52

also observed in a progressive decline of message levels for both classes of invertase transcripts. The longitudinal gradient of invertase mRNA levels from tip to base of silks was reduced during this decrease by the rapid decline in message abundance observed in the basal region of the style. At the enzyme level, temporal and spatial changes in total soluble activity were consistent with those of the Ivr] and Ivr2 message levels (Figure 4-6B, 4-7B). Salt-solubilized invertase was relatively constant before and/or after pollination, and no activity gradation was evident along the length of silk (Figure 4-6C, 4-7C). The drop in mRNA abundance of invertase is still more pronounced than pictured if considered relative to protein levels.

Differential responses of the Ivrl and Ivr2 class genes to sugar supplies

became apparent when excised root tips were supplemented with a range of glucose and/or sucrose concentrations and incubated for 24 hr (Figure 4-8). Ivrl class message levels were maximal with ca 0.5% exogenous glucose (Figure 4-8A) and ca

0.2% sucrose (Figure 4-8B), whereas those of Ivr2 remained relatively constant when media glucose and/or sucrose levels were between 0.2 and 4.0%. In addition, levels of the Ivrl subfamily of transcripts appeared to drop less markedly during a 24 hr period without exogenous carbohydrate than did those of Ivr2 (Figure 4-8). In excised maize root tips, soluble sugars reportedly drop to minimal levels within 10 hr if no supplemental sugars are provided (Saglio and Pradet, 1980). Differential responses of the Ivrl and Ivr2 classes of invertase to carbohydrate deprivation were further explored by an analysis of their progression over time in excised root tips (Figure 4-9). Levels of the Ivrl type mRNAs decreased less rapidly than did those








53
of the Ivr2 subgroup and persisted for considerably longer. Relatively little change was evident during 24 hr of starvation, and message remained readily apparent for at least 48 hr. In contrast, levels of the Ivr2 class of mRNA dropped below detection after between 12 and 18 hr of carbohydrate deprivation (Figure 4-9). Although Ivrl message abundance appeared to be relatively insensitive to an 18-hr starvation period or subsequent additions of sugar to media, levels of mRNA for the Ivr2 subfamily were sensitive to both (Figure 4-10). Glucose replacement after 18 hr of C-depravation appeared to counter initial decreases in levels of message for the Ivr2 subfamily. These returned to pre-starvation levels after 18 hr incubation in 0.5 % glucose (Figure 4-10).

The responses of the Ivr] and Ivr2 class genes to different types of sugars

(Figure 4-1 IA) also showed that expression of both appeared to require a supply of metabolizable sugars. Transcripts remained abundant in the presence of 2% Dglucose, fructose, or sucrose in the exogenous media, but dropped when these were replaced by either L-glucose or mannitol.


























Figure 4-1. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in root
tips, a sink leaf, a source leaf, a prop root, anthers, silk and kernels (2 DAP).
A, RNA gel blots with equal amounts (10 ptg) of total RNA from above
tissues were probed with 32p-labeled Ivrl or Ivr2 representing the two
subfamilies of maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12 hr, respectively. B, Total soluble acid invertase activity
from the above tissues. C, Insoluble acid invertase activity from the above tissues. Values for RNA/protein recovery were ca 0.04 (+ 0.02) for tissues other than root tips and did not otherwise differ significantly between tissue
types. Root tip values were greater (0.15 + 0.04) possibly due to more
extensive meristematic activity.

















Invertase activity
(ptmol glucose mg-1 protein hr"1)

0>


T


iii


ZH ZZJH IIIIIIIIZI

IN


Probe


root tips sink leaf source leaf prop root anthers silk

kernel (2DAP)


root tips sink leaf source leaf prop root anthers

silk

kernel (2DAP)


If II







IN


7)

0
C,

-



























Figure 4-2. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases during
kernel development. A and B, RNA gel blots with equal amounts (10 tg) of
total RNA from kernels between 6 and 32 DAP (full maturity at ca 30 DAP
under local conditions) were probed with 32P-labeled Ivrl or Ivr2 representing
the two subfamilies of maize soluble acid invertase. Blots were exposed to X-ray film for two days. Relative abundance of mRNA was quantified by
phosphor image quantifications. C, Total soluble acid invertase activity from the above tissues. D, Insoluble acid invertase activity from the above tissues.
Values for RNA/protein recovery from this set of kernels were ca 0.04
(0.02), except at 10 and 12 DAP (0.08 + 0.04) (consistent with changes in
cell division and protein levels during early kernel development).































o 0 a

B. Ivr2 mRNA

E
x
m
E 100







so






C. Soluble activity


0
CL
















10 -rSO-
50





















0 1 -H













6 8 10 12 16 20 24 28 32 DAP



























Figure 4-3. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in
pedicel, middle and top portions of kernels at 8, 10 and 12 DAP. A, RNA gel blots with equal amounts (10 rig) of total RNA from above tissues were
probed with 32P-labeled Ivrl or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for two days.
B, Total soluble acid invertase activity from the above tissues. C, Insoluble
acid invertase activity from the above tissues. Values for RNA/protein
recovery were ca 0.03 (+ 0.01) with variability independent of tissue gradient
from kernel top to pedicel. Values for RNA/protein recovery dropped from
ca 0.04 ( 0.02) to 0.02 (_ 0.01) past 10 DAP, and is consistent with
elevated protein storage in kernels at this stage.









A. mRNA
8 DAP 10 DAP 12 DAP
top mid ped top mid ped top mid ped


It@as


10.


Sf


B. Soluble activity ,


rFl


FE1r]


S


C. Insoluble activity


300 -


200 k


rr~7i


[471


~=,fT1


Ivrl Ivr2


4


W E 4 0 0)
E


- LI


top mid ped top mid ped top mid ped
8 DAP 10 DAP 12 DAP


Fz i i-



























Figure 4-4. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in
kernels (ovules) sampled daily from 2 days before to 2 days after pollination.
A, RNA gel blots with equal amounts (10 jtg) of total RNA from above
tissues were probed with 32P-labeled Ivr] or Ivr2 representing the two
subfamilies of maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12 hr, respectively. B, Total soluble acid invertase activity
from the above tissues. C, Insoluble acid invertase activity from the above
tissues. Values for RNA/protein recovery were ca 0.03 (. 0.01) with
variability independent of development.











A. mRNA

Days -/+ pollination (fert)
-2 -1 0 +1 +2
(-3) (-2) (-1) (0) (+1)


-'U


B. Soluble activity


C. Insoluble activity

4 T





0


-2 -1
(-3) (-2)


0
(-1)


+1 +2
(0) (+1)


Days -/+ pollination (fert)


Ivrl Ivr2 [


'.


4
I
0



E cca

*-6c



























Figure 4-5. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases during the final 3 days of anther development and in mature pollen. A, RNA gel
blots with equal amounts (10 ptg) of total RNA from above tissues were probed with 32P-labeled Ivr] or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively. B, Total soluble acid invertase activity from the above
tissues (*, not assayed). C, Insoluble acid invertase activity from the above
tissues (*, not assayed). Values for RNA/protein recovery were ca 0.03 (0.01) for mature and shedding anthers. Values from extracts of young
anthers were greater (0.07 + 0.03), possibly due to more extensive
meristematic activity.













































































3 1 0 0
anther pollen Days to anthesis


0
.
0
CL






















I.
>,_=

0



4- (



o E :=L


























Figure 4-6. Abundance of mRNA from the Ivr] and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in silk sampled daily from 2 days before to 2 days after pollination. A, RNA gel
blots with equal amounts (10 gg) of total RNA from above tissues were probed with 32p-labeled Ivrl or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively. B, Total soluble acid invertase activity from the above
tissues. C, Insoluble acid invertase activity from the above tissues. Values
for RNA/protein recovery were ca 0.06 (_ 0.02) before pollination and
dropped to 0.02 (L 0.01) after pollination. Transcription may be markedly reduced by pollination and/or message longevity may largely determine the
extent of change in types of mRNA predominating.










A. mRNA Days -/+ pollination (fert)


-2 -1 0
(-3) (-2) (-1)


+1 +2
(0) (1)


Ivrl 1-


"V9'U


B. Soluble activity


-I -L


C. Insoluble activity






-2 -1 0 +1 +2
(-3) (-2) (-1) (0) (+1)

Days -/+ pollination (fert)


60 T


40 F


E I=.



" O
=a,



0

E :::

























Figure 4-7. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in tip,
mid, and low portions of silk (portions of silk [ca 4 cm total length] relative
to ovary) sampled at pollination, 3 hr later, 6 hr later, or after 24 hr. A,
RNA gel blots with equal amounts (10 p.g) of total RNA from above tissues
were probed with 32P-labeled Ivrl or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively. B, Total soluble acid invertase activity from the above
tissues. C, Insoluble acid invertase activity from the above tissues. Values
for RNA/protein recovery were ca 0.06 (. 0.02) before pollination and
dropped to 0.04 (. 0.02) within 3hr after pollination, then to 0.02 (. 0.01) at
6hr and 24hr after pollination. Transcription may be markedly reduced by
pollination and/or message longevity may largely determine the extent of
change in types of mRNA predominating.











A. mRNA


.0 hr AP +3 hr AP +6 hr AP +24 hr AP

tip mid low tip mid low tip mid low tip mid low


00S.


S.




S


B. Soluble activity


100 I-


1J1 iL1 "m-


C. Insoluble activity Lifl-1iT-F


tip mid low tip mid low tip mid low tip mid low +0 hr AP +3 hr AP +6 hr AP +24 hr AP


Ivrl


Ivr2


.
>1 S



C, 0)
E L.0 > -

E
I


mm II II I


P. 0 0 4





























Figure 4-8. Abundance of mRNA from the Ivr] and Ivr2 subfamilies of soluble acid
invertase in maize root tips incubated for 24 hr in White's basal salts medium supplemented with either 0, 0.2, 0.5, 2.0, 4.0% glucose or sucrose. RNA gel
blots with equal amounts (10 pig) of total RNA from above tissues were probed with 32p-labeled Ivr] or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively.











glucose (%)

0 0.2 0.5 2.0 4.0


Ivrl 00-


Ivr2 loo


sucrose (%)

0 0.2 0.5 2.0 4.0


Ivrl Pop-


Ivr2 ]o


A





























Figure 4-9. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase in maize root tips depleted of carbohydrates for either 6, 12, 18, 24,
36, or 48 hr, respectively, in White's basal salts medium without an
exogenous sugar supply. RNA gel blots with equal amounts (10 jig) of total
RNA from above tissues were probed with 32P-labeled Ivrl or Ivr2
representing the two subfamilies of maize soluble acid invertase. Blots were
exposed to X-ray film for 24 or 12 hr, respectively.













starvation (hr)


6 12 18 24 36 48


V


Ivrl No


Ivr2 P





























Figure 4-10. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase during post-starvation recovery of maize root tips. Sugar depletion in White's basal salts without sugars (18 hr) was followed by
incubation for various periods of time (6-18 hr) in media with 0.5% glucose supplements. RNA gel blots with equal amounts (10 [tg) of total RNA from above tissues were probed with 32P-labeled Ivrl or Ivr2 representing the two
subfamilies of maize soluble acid invertase. Blots were exposed to X-ray
film for 24 or 12 hr, respectively.










post-starvation recovery (hr)


0 6


12 18


Ivr 0w

Ivr2 ,


























Figure 4-11. Abundance of mRNA from the Ivr] and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble acid invertase in maize root tips
incubated for 24 hr in White's basal salts medium supplemented with either
2.0% glucose, fructose, sucrose, L-glucose or mannitol respectively. A, RNA
gel blots with equal amounts (10 gg) of total RNA from above tissues were
probed with 32P-labeled Ivr 1 or Ivr 2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively. B, Total soluble acid invertase activity from the above
samples. Insoluble invertase activity (not shown) was consistently ca 10-fold
less than that in the soluble fraction of maize root tips. Values for
RNA/protein recovery were ca 0.14 ( 0.05) with variability independent of
presence or absence of metabolizable C-source.


















Ivrl


Ivr2


No +G


+F +S +L-G Type of sugar










Discussion



The significance of findings presented here extends from implications of special roles for soluble invertases during development (especially pollination and early kernel development) to broader possible contributions to adjustment of sucrose import, cell volume, and metabolism in a multi-celled higher plant. The spatial and temporal patterns of expression for the two invertase subfamilies, as well as the contrast between them suggest involvement in specific developmental processes. The availability of these clones has also allowed the hypothesis to be tested that regulation of transcript level by photosynthate availability could contribute to adjustment of both avenues for sucrose breakdown in a cell (invertase as well as sucrose synthase). Moreover, a surprising similarity in differential carbohydrate responsiveness was evident between the two invertase subfamilies and the two sucrose synthase genes. In both instances, the more broadly distributed of the two (Ivr2 or Susl) was found to be readily induced by enhanced carbohydrate availability, whereas the form which was upregulated during more specific developmental and environmental signals (Ivrl or Shl) was less sensitive to sugar supplies (Koch et al., 1992).

The present work indicates that each subfamily of the invertase genes is expressed differentially depending on developmental stage and the tissue/organ involved. Although invertase activity was detected in extracts of almost every sucrose-importing tissue examined, the Ivr] type message was preferentially










associated with reproductive organs (Figure 4-1 A). Data shown here for general association between soluble invertase activity and rapid growth/cell division were consistent with previous suggestions for the role of this enzyme relative to sucrose import. Rapidly expanding tissues require either invertase or sucrose synthase to convert sucrose to substrates necessary for respiratory and synthetic processes (Glasziou and Gayler, 1972; Giaquinta, 1979; Avigad, 1982; Morris and Arthur, 1984b; Hawker, 1985; Schaffer, 1986; Schaffer et al, 1987). Invertase in particular can be important to cell expansion through generation of hexoses and their associated osmotic potential. Changes in both message and activity in the present study were also consistent with the gradual sink-to-source transition in leaves (Ho, 1988; Turgeon, 1989; Nguyen-Quoc et al., 1990).

Data presented here indicate that soluble invertases may be especially

important during the early stages of maize kernel development. This is consistent with a hypothesis advanced on the basis of previous work (Hanft and Jones, 1986a; 1986b; Reed and Singletary, 1989), which suggests that the soluble forms of these enzymes in the pedicel may be critical to initiation of normal kernel development. The expression pattern of both the Ivrl and Ivr2 classes of invertase, as well as total soluble activity (Figure 4-2 and Figure 4-4), are also in agreement with this possibility.

Past research on invertase and kernel development has tended to focus on the insoluble "cell-wall-bound" form of this enzyme primarily because of its apparent importance during later stages of kernel fill. Shannon and coworkers proposed that








78

the driving force for assimilate movement into normally developing kernels was the sucrose-gradient between the leaves and the pedicel apoplasm combined with the monosaccharide-gradient between the pedicel apoplasm and the starchy endosperm cells (Shannon, 1968; Shannon, 1972; Shannon and Dougherty, 1972). Both gradients are presumably maintained by the activity of an apoplastic pedicel invertase (Shannon et al. 1993).

Early phases of kernel growth may differ from previous hypothesis, despite the apparent importance of insoluble invertase and sucrose synthase activity in later development (ca 22 DAP) (Tsai et al., 1970; Chourey and Nelson, 1976; Chourey, 1981). Sucrose synthase activity is not detectable prior to 12 DAP (Tsai et al.,1970; Chourey and Nelson, 1976; Chourey, 1981). Instead, activity of soluble invertase as well as mRNA for both classes of soluble invertase peak at 8 to 12 DAP (Tsai et al., 1970; Figure 4-2). Soluble acid invertase may also be important to initial establishment and maintenance of sink strength.

The potential role of soluble invertase genes in early kernel development may be related to the difference between tissues in which message classes were most strongly expressed. Ivrl mRNA levels were greater in endosperm and the upper kernel whereas Ivr2 message was most abundant in the pedicel region (Figure 4-3A). This distinction could be important for two reasons. The first of these is the suggestion that the miniature phenotype results initially from reduced endosperm invertase and its subsequent effect on pedicel invertase (both presumably insoluble, Miller and Chourey, 1992). The second is the hypothesis advanced by Hanft and










Jones (1986a; 1986b) which tentatively attributes kernel abortion under water and heat stresses to reduced activity of soluble invertase in the pedicel. The following scenario represents one possible explanation for the combination of data on the greater sensitivity of the Ivr2 genes to carbohydrate deprivation and the abundance of their transcripts in the pedicel. Any early limitation of assimilate flux into the endosperm would be expected to reduce soluble sugar concentration in the pedicel within a relatively short time (Hanft and Jones, 1986a). The depletion of pedicel sugars could in turn result in decreased levels of the carbohydrate-responsive Ivr2 gene products and a subsequent decrease in soluble invertase activity in this region. This is consistent with the observation that it is the soluble rather than insoluble acid invertase activity which is most markedly affected in pedicel of kernels that have been induced to abort vs. nonaborting kernels (Hanft and Jones, 1986a).

The role of soluble invertases during anther and pollen development is probably twofold. First, there are no plasmodesmatal connections between developing pollen grains and the surrounding tapetum layer (Kesselback, 1949). The tapetum thus lies at the terminal end of the maternal transport path. Any invertase or sucrose synthase present in these cells could theoretically enhance sugar transport to pollen grains by creating a sucrose gradient between phloem and the secretory surface, much as hypothesized for developing kernels (Shannon, 1972; Shannon and Dougherty, 1972; Lin et al., 1984). Presumably, enhanced hydrolysis could also benefit the probable elevation in respiratory and biosynthetic demands. Our results










indicated that significant amounts of both Ivr] and Ivr2 type message were present in the anther tissues collectively (Figure 4-5).

Second, invertase message in pollen may be important for subsequent

germination, to facilitate use of endogenous as well as exogenous sucrose. Sucrose represents the major soluble sugar present in the majority of angiosperm pollen grains, including maize. Mature pollen grains from diverse plants contain sucrose but not starch as reserve carbohydrate (Portnoi and Horovitz, 1977; Nakamura et al., 1980). Germinating pollen grains show an extremely high rate of growth and thus have a high demand for carbon skeletons required for pollen tube wall synthesis as well as substrates for respiration (Hoekstra, 1983; Singh and Knox, 1984). The sucrose content from pollen grains of Camellia japonica decreases rapidly during pollen growth and the activity of soluble invertase increases during culturing and a high constant activity is found at the later stages of pollen tube growth (Nakamura et al., 1980). Our data are consistent with the possibility that invertase has multiple functions during anther development and pollen grain maturation (Figure 4-5). Further localization of invertase at the message and/or protein level in situ could help clarify the functions of invertase in reproductive processes.

The association between high levels of both invertase activity and message levels with rapidly elongating styles (silk) in maize may have a twofold biological implication. First, invertase, as one of the important constituents of sink strength (Morris and Arthur, 1984; Schaffer et al., 1987), can provide an important avenue for sucrose cleavage. Resulting hexoses can either be subsequently metabolized in










support of high respiratory rates and/or compartmentalized in vacuoles to maintain turgor for cell expansion. Second, invertase is demonstrated to contribute to an in vitro chemotropism of pearl millet pollen tubes toward stigmatic tissue through its production of glucose (Reger et al., 1992a; 1992b; 1993). Although other factors, such as calcium and an ovarian protein, are also important for the chemotropic response of pearl millet pollen tubes, our results were consistent with an invertase role in forming gradients of hexoses for pollen growth in maize. The message gradient of Ivr] and Jvr2 abundance along the length of the silk (Figure 4-6) further supported the concept that glucose produced by invertase may be at least one key factor underlying the chemotropic response of pollen tubes, and their pathway towards the ovule.

Genes encoding carbohydrate metabolizing enzymes are regulated at the

transcriptional level by sugar availability in yeast and vascular plants (Carlson, 1987; Schuster, 1989; Maas et al., 1990; Koch et al., 1992). Invertase, one of the only two enzymes known with a capacity to breakdown sucrose in vascular plants (Avigad, 1982), is also shown here to be regulated at the message level by sugar availability. Further, invertase gene subfamilies are found to be differentially expressed even within the same organ. The potential exists for these isozyme gene subfamilies to confer particular biological advantage through their presence in specific tissue at various stages of development and/or altered environmental conditions.

Photosynthesis and carbohydrate availability are often greatly reduced under environmental stresses, such as drought, flooding, severe cold and/or insect attack.










Because the Ivr2 subfamily of genes are extremely sensitively to carbohydrate deprivation (Figures 4-8, 4-9), the invertases encoded by these messages would be expected to contribute less to physiological process of importing cells under these circumstances.

However, specific, high-priority developmental processes, such as pollination and/or reproductive growth, once initiated, should ideally be less sensitive to changes in carbohydrate availability than is vegetative growth. If the associated enzymes for sucrose metabolism are less sensitive to de novo down-regulation during sugar deprivation, then this could provide a mechanism for giving reproductive and other essential tissues "import priority" during stresses. The Ivr] and Ivr2 subgroup of genes could play contrasting roles in these instances much like those of Shl and Sus]. Ivrl related gene expression is strongly associated with reproductive structures (Figure 4-1) and is less markedly affected by carbohydrate deprivation (Figure 4-9). In contrast, the Ivr2 subfamily of messages are widely distributed and clearly downregulated by in the absence of sugar supply. The altered pattern of gene expression for Ivrl and Ivr2 classes in response to carbohydrate deprivation may be an important adaptive strategy during different stresses, in which plant survival and/or reproduction could depend on the preservation of vital organs and/or tissues at the expense of others.

This differential regulation of the two invertase subfamilies in response to sugar suggests that these genes and their respective enzymes may also have an important function in carbohydrate partitioning between sink and source tissues.










Under source-limited conditions, invertase involved in certain physiological processes could act to increase sink activity and stimulate assimilate translocation to these sinks to compete with others. Under normal growth conditions, assimilate levels are plentiful. Thus, the Ivr2 class of genes tend to be widely expressed in sink tissues and their gene products are abundant. This is especially evident in rapidly growing tissues, which is consistent with the concept that high activity of "soluble" invertase is usually associated with rapid tissue expansion (Glasziou and Gayler, 1972; Giaquinta, 1979; Avigad, 1982; Hawker, 1985; Schaffer et al., 1987). Invertase is considered to facilitate assimilate transportation from the site of phloem unloading to sink tissues by steepening the gradient of sucrose between source and sink (Shannon, 1968; Shannon et al, 1972; Shannon and Dougherty, 1972; Shannon et al., 1993). However, soluble invertase can also promote cell elongation and/or rapid growth by hydrolyzing sucrose to hexoses, thereby providing osmotically active solutes and the osmotic pressure necessary to support growth (Kaufman et al., 1973; Schmalstig and Cosgrove, 1988; 1990).

Gene responses to sugars in vascular plants have been known for some time (Rocha-Sosa et al., 1989; Salanoubat and Belliard, 1989; Muller-Rober et al., 1990; Maas et al., 1990; Koch et al., 1992). However, the mechanism, by which the sugar signal is sensed by plant genes, is not clear. Our results (Figure 4-12) indicated that naturally occurring, metabolizable sugars, such as sucrose, D-glucose and fructose, meet the requirement for invertase responsiveness, although data shown here can not rule out other possibilities for certain non-metabolizable sugars.








84

Sadka et al. (1994) propose that sugar modulates transcription of the soybean vegetative storage proteins and other sugar-inducible genes by using phosphate as a signal. In their model, phosphate acts as a negative factor to those sugar-responsive genes. Carbohydrate activates those genes by accumulation of sugar-phosphates and concomitant reduction of cellular phosphate levels. High phosphate levels relative to those of sugars are also found in starved sycamore cells (Rebeille et al., 1985).

Graham et al. (1994), on the other hand, propose that not metabolism per se, but the phosphorylation by hexokinase per si maybe signaling intracellular sugarresponsiveness of gene expression. In their experiments, they demonstrate that 2deoxyglucose and mannose, like glucose and fructose (which are phosphorylated by hexokinase but not further metabolized) specifically repress cucumber malate synthase and isocitrate lyase gene expression. However, 3-methylglucose, an analog of glucose that is not phosphorylated, does not result in repression of either malate synthase or isocitrate lyase.

Many of the genes involved in metabolic pathways are subject to regulation by the fluctuation of internal and external metabolites in multicellular vascular plants (Maas et al., 1990; Sheen, 1990; Koch et al., 1992; Graham et al., 1994; Sadka et al., 1994;). The metabolic regulation of gene expression should play a role of fundamental importance in maintaining an economical balance of the supply and demand of biomolecules in different organs of vascular plants. Metabolic control of specific gene expression now appears to be a widespread phenomenon, although the mechanism of signal transduction and response for different genes will not








85

necessarily be the same. Environmental and developmental signals may also have contrasting influences, and depending on the role of the gene product, the sensitivity and degree of the response may also vary.














CHAPTER 5
CYTOKININ MIMICS AND SUPERSEDES THE SUGAR-INDUCIBILITY OF
MAIZE INVERTASE FAMILY MEMBERS AND FACILITATES THEIR
DIFFERENTIAL RESPONSIVENESS TO ABSCISIC ACID



Introduction


Plant growth regulators often affect many different aspects of plant growth

and development. As an organism becomes more complex, communication between its different parts requires a signaling system with a progressively greater capacity to integrate distant messages. Hormonal responses belong to such a communication system (Libbenga and Mennes, 1987). Much of the signalling in vascular plants is dependent upon a relatively complex array of hormonal signals.

Sucrose, as the major form in which photoassimilates are transported, and as such plays a central and vital role in plant life (Avigad, 1982; Hawker, 1985). Invertase is one of the two known enzymes which can initiate breakdown of this sucrose for further metabolism in vascular plants. It is thus considered a key enzyme for carbohydrate partitioning and utilization (Robbins, 1958; Glasziou and Gayler, 1972; Avigad, 1982; Turgeon, 1989; Sturm and Chrispeels, 1990; Duke et al, 1991; Miller and Chourey, 1992). Early studies of its activity showed that upregulation of capacity could be observed in response to abscisic acid, auxin, cytokinins, and/or gibberellic acid depending on species and conditions. The 86










diversity of systems examined included sugarcane stem segments (Sacher et al., 1963; Glasziou et al., 1966; Gayler and Glasziou, 1969), Avena stem segments (Kaufman et al., 1973), radish cotyledons (Howard and Witham, 1983), Phaseolus vulgaris seeds (Morris and Arthur, 1985), soybean seeds (Ackerson, 1985), Citrus leaves (Schaffer et al., 1987), tobacco crown gall cells (Weil and Rausch, 1990), etiolated Pisum sativum seedlings (Miyamoto et al., 1993), and shoots of dwarf pea (Pisum sativum) (Wu et al., 1993).

The present research was motivated by studies which showed that high

invertase activity is usually associated with rapid growth. Action of this enzyme can provide tissues with not only substrates for their respiratory and synthetic demands (Morris and Arthur, 1984b; Schaffer et al., 1987), but also elevate turgor for cell expansion (Avigad, 1982; Schmalstig and Cosgrove, 1988; 1990).

Both cytokinins and ABA are reported to stimulate assimilate translocation

from source to sink (Gersani and Kender, 1982; Howard and Witham, 1983; Hein et al., 1984; Schussler et al., 1984; Ackerson, 1985; Jones et al., 1986; Brokovec and Prochazka, 1992; Jones et al., 1992). ABA has been called a stress hormone, since it accumulates during an array of stresses (Chen et al., 1983; Chen and Gusta, 1983; LaRosa et al., 1985; LaRosa et al., 1987; Davies and Zhang, 1991; Thomas et al., 1992). Both auxin and gibberellins stimulate cell enlargement, cell elongation and possibly phototropism and gravitropism (Davies, 1987; Kaufman and Song, 1987; Kim et al., 1993; Wu et al., 1993a; 1993b).










The hypotheses tested here are as follows. Invertase gene expression could be responsive to ABA (aiding osmoregulation), gibberellins and auxin (aiding gravitropism and phototropism), and/or cytokinins (aiding sink potential and/or symbiosis).

In this report, we demonstrate that in maize root tips, both Ivr] and Ivr2 expression for soluble acid invertase genes includes an unexpected, differential responsiveness to specific hormonal signals. These findings indicate that different invertase isozymes may have specialized functions in a diverse set of developmental and/or environmental processes.



Materials and Methods



Plant Material



Zea mays hybrid NK 508 was used for all experiments. Seeds were first

emersed in 20 % Clorox for 30 min. followed by 30 min. of continuous rinsing with water. Germination took place in the dark at 18 'C on two layers of moist 3 MM paper (Whatman, Inc., Clifton, NJ) in 17 x 26 cm glass pans. Air flowed continuously at 1 liter min-' through each pan for the 6-day period, with 40% 02 supplied during the final 24 hr before root tip excision. The moisture level was adjusted daily by applying mist and draining excess water. Root tips (ca 1 cm each) were excised under a sterile transfer hood.












Experimental Conditions



Experimental treatments were as described by Koch et al. (1992).

Approximately 100 root tips (- 500 mg) were used for each experimental treatment. Excised root tips were incubated in the dark at 18 'C for 6 to 48 hr in Whites' medium, plus 0.5% glucose, either with or without specific supplemental plant growth regulators (ABA, A1049; GA, G7645; Kinetin, K0753; IAA, 12886; all from Sigma). Each group of root tips was agitated at 120 cycles per minute in a 125-ml side-arm Erlenmeyer flask with 50 ml of sterile media. Airflow (40% 02) through air stones in each flask was maintained at 250 ml min' throughout the incubations.



RNA Isolation and Blot Analysis



Root tip samples were rinsed twice in sterile water, blotted dry, weighed, and frozen in liquid N2. Samples were ground into fine power in liquid N2 and total RNA was extracted as per McCarty (1986). RNA was quantified spectrophotometrically (Sambrook et al., 1989).

Total RNA was separated by electrophoresis in I % agarose gels containing formaldehyde (Thomas, 1980), blotted to a nylon membrane, and fixed by baking and/or UV treatment (Sambrook et al., 1989). Filters were hybridized at 65 'C in a solution containing 7 % SDS, 250 mM Na2HPO4, pH 7.2, 1 % BSA (Church and










Gilbert, 1984). Maize Ivr] and Ivr2 invertase cDNA clones were radiolabeled by random primer. No cross-reactivity between Ivr] and Ivr2 gene probes was observed when hybridizations were conducted at high stringency (data not shown). Blots were washed as described by Church and Gilbert (1984), exposed to X-ray film with intensifying screens at -80 C.



Enzyme Extraction



Soluble invertase was extracted as per Duke et al. (1991). Frozen tissue samples were ground to a fine powder in liquid N2 using a mortar and pestle. Frozen powder was transferred to a second mortar containing ice-cold 200 mM HEPES buffer (pH 7.5) with 1 mM DTT, 5mM MgC12, 1 mM EGTA and 10%(w/w) PVPP. One ml of extraction buffer was used for every 100 mg of tissue fresh weight. Buffered extract was centrifuged at 15,000 x g for 10 min to sediment particulate matter. Pellets were saved for salt-solubilized particulate invertase extraction. Supernatant was dialyzed (50,000 MW cutoff) at 4 C for 24 hr against extraction buffer diluted 1:40 (MW cutoff for dialysis was selected to allow escape of proteinaceous invertase inhibitors [Jaynes and Nelson, 1971b]). Buffer was changed twice. Soluble dialyzed extract was centrifuged again at 15,000 x g for 10 min. Supernatant was used for soluble invertase assays as described below.

Insoluble invertase was extracted as described by Doehlert and Felker (1987). Pellets remaining from the above step were washed three times by resuspending in




Full Text
107
also present in the maize clones examined here. Further, these maize invertases
shared considerably greater identity to soluble invertases than to their cell-wall-
bound counterparts. The maize genes examined here thus probably encode soluble
enzymes. This conclusion was also supported by a strong correlation between the
transcript levels of both Ivrl and Ivr2 subfamilies relative to the total soluble
invertase activities in an array of maize tissues and/or developmental stages.
Ivrl and Ivr2 subfamilies of soluble invertase were mapped to two and four
different loci, respectively, each on a different chromosome (in collaboration with
Scott Wright, Genetic Linkage). One of the Ivrl genes mapped to a region near the
miniature mutation in maize (kernels known to lack insoluble invertase). However,
further analysis showed both Ivrl and Ivr2 message to be present in this mutant line,
as well as wild-type levels of soluble invertase activity. Again, this evidence
supports a soluble invertase identity for the invertase family of genes isolated here.
The maize genomic DNA for Ivrl was found to be organized into seven
exons and six introns. The second exon is only 9 nucleotides long, but encodes the
highly conserved domain found in all the invertases (NDPNG, the P-fructosidase
motif). This 9 bp exon is probably the smallest exon thus for identified in the plant
genome (M. Schuler, personal communication).
The expression of two classes of maize invertase (Ivrl and Ivr2) was further
characterized to test the hypothesis that specific genes might be associated with
different developmental stages and/or enlargement of key sets of cells. The two
invertase subgroups were differentially expressed in roots, sink leaves, young


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11
Sugar Levels and Invertase
A number of recent reports demonstrate that various genes involved in
metabolic pathways are either induced or repressed by sugars (Carlson, 1987;
Schuster, 1989; Maas et al., 1990; Sheen, 1990; Koch et al., 1992; Sadka et al.,
1994). Studies of carbohydrate assimilation in potato tubers have revealed that
genes encoding patatin (Rocha-Sosa et al., 1989), sucrose synthase (Salanoubat and
Belliard, 1989) and ADP-glucose pyrophosphorylase (Muller-Rober et al., 1990) can
all be induced by elevated levels of sucrose. Similarly, the tuberous root storage
protein genes of sweet potato (Hattori et al., 1990) and the vegetative storage protein
genes (vegetative storage proteins, Sadka et al., 1994) are upregulated by sugars. In
Arabidopsis, sucrose mimics the light induction of nitrate reductase gene
transcription (Cheng et al., 1992). In maize, elevated carbohydrate levels regulate
the sucrose synthase genes differentially such that Susl is stimulated whereas Shi is
repressed (Maas et al., 1990; Koch et al., 1992).
Repression of gene expression by sugars has also been shown for other plant
genes. In maize mesophyll protoplasts, seven photosynthetic genes are
downregulated by photosynthetic end products sucrose and glucose and by the
exogenous carbon source acetate (Sheen, 1990). In tobacco, the glutamate
dehydrogenase gene is suppressed by feeding glucose (Maestri et al., 1992), and in
cucumber, genes encoding enzymes of the glyoxylate cycle (malate synthase and
isocitrate lyase) are repressed by carbon catabolites (Graham et al., 1994). Together,


AND FACILITATES THEIR DIFFERENTIAL RESPONSIVENESS
TO ABSCISIC ACID 85
Introduction 85
Materials and Methods 87
Results 91
Discussion 99
6 SUMMARY AND CONCLUSIONS 105
REFERENCE LIST 109
BIOGRAPHICAL SKETCH 126
IV


121
Mundy, J., Yamaguchi, S.K. and Chua, N.H. (1990). Nuclear proteins bind
conserved elements in the abscisic acid-responsive promoter of a rice rab
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in the transpiration stream of water plants. Plant Physiol. 88: 703-708.
Nakamura, N., Sado, M. and Arai, Y. (1980). Sucrose metabolism during the
growth of Camellia japnica pollen. Phytochemistry 19: 205-209.
Neales, T.F., Masia, A., Zhang, J., and Davies, W.J. (1989). The effects of partially
drying part of the root system of Helianthus annuus on the abscisic acid
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Nelson,N. (1944). A photometric adaptation of the Somogyi method for the
determination of glucose. J.Biol.Chem. 153: 375-380.
Ng, P.P., Cole, A.L.J., Jameson, P.E., and Mcwha, J.A. (1982). Cytokinin production
by ectomycorrhizal fungi. New Physiol. 91: 57-62.
Nguyen-Quoc, B., Krivitzky, M., Huber, S.C., and Lecharny, A. (1990). Sucrose
synthase in developing maize leaves. Regulation of activity by protein level
during the import to export transition. Plant Physiol. 94, 516-523.
Palme, K Hesse, T., Campos, N., Garbers, C. and Schell, J. (1992). Molecular
analysis of an auxin binding protein gene located on chromosome 4 of
Arabidopsis. Plant Cell 4: 193-201.
Portnoi, L., and Horovitz, A. (1977). Sugars in natural and artificial pollen
germination substrates. Ann. Bot. 41, 21-27.
Pressey, R. (1966). Separation and properties of potato invertase and invertase
inhibitor. Arch. Biochem. Biophys. 113: 667-674.
Pressey, R. (1967). Invertase inhibitor from potatoes: Purification, characterization,
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Pressey, R. (1968). Invertase inhibitors from red beet, sugar beet, and sweet potato
roots. Plant Physiol. 43: 1430-1434.
Pryke, J.A., and Bernier, G. (1978). Acid invertase activity in the apex of Sinapis
alba during transition to flowering. Ann. Bot. 42: 747-749.


32
A. Hydropathy
phob
3.0 -
1.5 -
-1.5-
-3.0
phi I
134
268
402
536
670
B. Structure


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
^aren E. Koch, Chair
Professor of Plant Molecular
and Cellular Biology Program
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Donald Rf Mc(
Associate Professor of
Horticultural Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
L. Curtis Hannah
Professor of Horticultural
Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
OJU.C \\
Alice C. Harmon
Assistant Professor of Botany
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Kenneth J. Boote
Professor of Agronomy


18
containing a 1.2 kb cDNA was obtained. This maize fragment was subcloned and
used to probe for further cDNAs from the same library. Twelve positive clones
ranging from 0.5 to 2.2 kb were identified.
Genomic Library Screening
A maize genomic fragment containing 8 kb DNA was identified by screening
a genomic library (EMBL 3, Clontech, Palo Alto, CA) with the 1.2 kb maize
invertase cDNA clone.
Hybridization with DNA probe
Procedures for library plating and production of filter replicas were conducted
as recommended by Clontech (Palo Alto, CA). Plaques or colonies were blotted to
nylon membranes, and DNA was denatured in situ with NaOH (0.5 M), neutralized
with Tris buffer (1.0 M, pH 7.5), and fixed by baking (80 C, 0.5-2 hr) (Sambrook
et al 1989). Filters were hybridized at either 50 C (low stringency) or at 65 C
(high stringency) in a solution with the selected cDNA, 7% SDS, 250 mM Na2HP04
(pH 7.2) and 1% BSA (Church and Gilbert, 1984). Tomato and/or maize invertase
cDNA fragments were radiolabed by random primer (BRL, Gaithersberg, MD).
Blots were washed as described by Church and Gilbert (1984), and exposed to X-ray
film with intensifying screens at -80 C.


114
Dickinson, C.D., Altabella, T. and Chrispeels, M.J. (1991). Slow-growth phenotype
of transgenic tomato expressing apoplastic invertase. Plant Physiol. 95: 420-
425.
Dickinson, C.D., Evans, R.P. and Neilsen, N.C. (1988). RY repeats are conserved
in the 5-flanking regions of legume seed-protein genes. Nucleic Acids Res.
16: 371-372.
Doehlert, D.C. (1986). Properties of cell wall invertase in developing maize kernels.
In J.C. Shannon, D.P. Knievel, and C.D. Boyer, ed, Regulation of carbon and
nitrogen reduction and utilization in maize. Maryland, American Society of
Plant Physiologists, pp 311-313.
Doehlert, D. (1990). Distribution of enzyme activities within the developing maize
(Zea mays) kernel in relation to starch, oil, and protein accumulation. Physiol.
Plant. 78: 560-576.
Doehlert, D.C., and Felker, F.C. (1987). Characterization and distribution of
invertase activity in developing maize (Zea mays) kernels. Physiol. Plant. 70,
51-57.
Doehlert, D.C., and Kuo, T.M., and Felker, F.C. (1988). Enzymes of sucrose and
hexose metabolism in developing kernels of two inbreeds of maize. Plant
Physiol. 86, 1013-1019.
Duke, E.R., McCarty, D.R., and Koch, K.E. (1991). Organ-specific invertase
deficiency in the primary root of an inbred maize line. Plant Physiol. 97, 523-
527.
Elliott, K,J., Butler, W.O., Dickinson, C.D., Konno, Y., Vedvick, T.S., Fitzmaurice,
L., and Mirkov, T.E. (1993). Isolation and characterization of fruit vacuolar
invertase genes from two tomato species and temporal differences in mRNA
levels during fruit ripening. Plant Mol. Biol. 21: 515-524.
Farrar, J.F. (1985). Fluxes of carbon in root of barley plants. New Physiol. 99, 57-
69.
Gayler, K.R., and Glasziou, K.T. (1969). Plant enzyme synthesis: hormonal
regulation of invertase and peroxidase synthesis in sugar cane. Planta 84:
185-194.
Gersani, M., and Kende, H. (1982). Studies on cytokinin-stimulated translocation in
isolated bean leaves. J. Plant Growth Regul. 1: 161-171.


40
Targeting signals for vacuolar proteins are frequently present in this region as C-
terminal propeptides (Bednarek et al., 1990; Chrispeels, 1991; Bednarek and
Raikhel, 1992). Third, message abundance of lvrl and lvr2 correlates well with
total soluble invertase activities in an array of maize tissues and/or developmental
stages (see Chapter 4).
Invertases of maize and other vascular plants are presumably encoded by
different genes, although in yeast, variable splicing allows a single gene to encode
both cell-wall-bound and soluble invertases (Carlson and Botstein, 1982). There are
at least two 7vr/-like genes in the maize genome, and the lvrl and lvr2 subfamilies
have been tentatively mapped to two and four different loci respectively (data not
shown, collaboration with Scott Wright, Genetic Linkages, Salt Lake, Utah).
The genomic clone of maize invertase has typical CAAT and TATA boxes
located in the upstream untranslated region (Figure 3-2). The second exon is
unusually small (9 bp) in maize lvrl invertase (Figure 3-2) and tomato soluble
invertase genes (Elliott et al., 1993). The amino acids encoded by this 9 bp exon are
located in a highly conserved domain found in all invertase clones (NDPNG, the P-
fructosidase motif, Sturm and Chrispeels, 1990). This represents one of the smallest
exons currently known to function in the plant genome (M. Schuler, personal
communication).
In the lvrl maize invertase genomic gene, several introns (number 1, 3, 4 and
5) are also found to contain one or more copies of an RY sequence motif
(CATGCATG, data not shown), which thus far has been implicated in seed-specific


Figure 4-4. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in
kernels (ovules) sampled daily from 2 days before to 2 days after pollination.
A, RNA gel blots with equal amounts (10 pg) of total RNA from above
tissues were probed with ,2P-labeled Ivrl or Ivr2 representing the two
subfamilies of maize soluble acid invertase. Blots were exposed to X-ray
film for 24 or 12 hr, respectively. B, Total soluble acid invertase activity
from the above tissues. C, Insoluble acid invertase activity from the above
tissues. Values for RNA/protein recovery were ca 0.03 (+ 0.01) with
variability independent of development.


78
the driving force for assimilate movement into normally developing kernels was the
sucrose-gradient between the leaves and the pedicel apoplasm combined with the
monosaccharide-gradient between the pedicel apoplasm and the starchy endosperm
cells (Shannon, 1968; Shannon, 1972; Shannon and Dougherty, 1972). Both
gradients are presumably maintained by the activity of an apoplastic pedicel
invertase (Shannon et al. 1993).
Early phases of kernel growth may differ from previous hypothesis, despite
the apparent importance of insoluble invertase and sucrose synthase activity in later
development (ca 22 DAP) (Tsai et al., 1970; Chourey and Nelson, 1976; Chourey,
1981). Sucrose synthase activity is not detectable prior to 12 DAP (Tsai et al.,1970;
Chourey and Nelson, 1976; Chourey, 1981). Instead, activity of soluble invertase as
well as mRNA for both classes of soluble invertase peak at 8 to 12 DAP (Tsai et al.,
1970; Figure 4-2). Soluble acid invertase may also be important to initial
establishment and maintenance of sink strength.
The potential role of soluble invertase genes in early kernel development may
be related to the difference between tissues in which message classes were most
strongly expressed. Ivrl mRNA levels were greater in endosperm and the upper
kernel whereas Ivr2 message was most abundant in the pedicel region (Figure 4-3A).
This distinction could be important for two reasons. The first of these is the
suggestion that the miniature phenotype results initially from reduced endosperm
invertase and its subsequent effect on pedicel invertase (both presumably insoluble,
Miller and Chourey, 1992). The second is the hypothesis advanced by Hanft and


21
ca 8 kb DNA. Digestion with BamHI and Kpnl generated three fragments, each of
which was subcloned and sequenced.
The invertase coding region was deduced according to the information from
cDNAs, recognition sites for intron splicing (Goodall and Filipowicz, 1989; 1991)
and/or comparision with other invertases from vascular plants (Figure 3-5, 3-6). The
gene for maize invertase 1 (IvrlG) was organized into seven exons and six introns,
as diagrammed in Figure 3-2A. The second exon was only 9 nucleotides long
(Figure 3-4B), and has also been reported in tomato fruit vacuolar invertase (Elliott
et al., 1993). The amino acids encoded by this 9 bp exon are located in a highly
conserved domain found in all invertases cloned thus far (NDPNG, the P-
fructosidase motif, Sturm and Chrispeels, 1990, Figure 3-2B).
The genomic DNA (IvrlG) is almost identical to the Ivrl cDNA clone at the
level of amino acid sequence, except for a few amino acid replacements. Genomic
and cDNA clones are from different maize lines, IvrlG being isolated from a B73
genomic library, and the Ivrl cDNA from a Merit root tip libary.
The deduced amino acid sequence from IvrlG consisted of 670 residues
(Figure 3-3) which predicted a molecular weight of 71,942 and an isoelectric point
of 7.5. This protein also included five potential glycosylation sites (N-X-S/T): N165,
N275, N518, N595 and N639 (Figure 3-3). The amino-terminal sequence of the IvrlG
protein indicated a hydrophobic region between basic N and polar C terminals
(Figure 3-3, 3-4) and other characteristics typical of a signal peptide ([-3,-1] rule,von
Heijne, 1986; K. Cline, personal communication). The predicted excision site for


87
diversity of systems examined included sugarcane stem segments (Sacher et al.,
1963; Glasziou et al., 1966; Gayler and Glasziou, 1969), Avena stem segments
(Kaufman et al., 1973), radish cotyledons (Howard and Witham, 1983), Phaseolus
vulgaris seeds (Morris and Arthur, 1985), soybean seeds (Ackerson, 1985), Citrus
leaves (Schaffer et al., 1987), tobacco crown gall cells (Weil and Rausch, 1990),
etiolated Pisum sativum seedlings (Miyamoto et al., 1993), and shoots of dwarf pea
{Pisum sativum) (Wu et al., 1993).
The present research was motivated by studies which showed that high
invertase activity is usually associated with rapid growth. Action of this enzyme can
provide tissues with not only substrates for their respiratory and synthetic demands
(Morris and Arthur, 1984b; Schaffer et al., 1987), but also elevate turgor for cell
expansion (Avigad, 1982; Schmalstig and Cosgrove, 1988; 1990).
Both cytokinins and ABA are reported to stimulate assimilate translocation
from source to sink (Gersani and Render, 1982; Howard and Witham, 1983; Hein et
al., 1984; Schussler et al., 1984; Ackerson, 1985; Jones et al., 1986; Brokovec and
Prochazka, 1992; Jones et al., 1992). ABA has been called a stress hormone, since
it accumulates during an array of stresses (Chen et al., 1983; Chen and Gusta, 1983;
LaRosa et al., 1985; LaRosa et al., 1987; Davies and Zhang, 1991; Thomas et al.,
1992). Both auxin and gibberellins stimulate cell enlargement, cell elongation and
possibly phototropism and gravitropism (Davies, 1987; Kaufman and Song, 1987;
Kim et al., 1993; Wu et al., 1993a; 1993b).


Figure 4-6. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in silk
sampled daily from 2 days before to 2 days after pollination. A, RNA gel
blots with equal amounts (10 pg) of total RNA from above tissues were
probed with 32P-labeled Ivrl or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively. B, Total soluble acid invertase activity from the above
tissues. C, Insoluble acid invertase activity from the above tissues. Values
for RNA/protein recovery were ca 0.06 (+ 0.02) before pollination and
dropped to 0.02 (+ 0.01) after pollination. Transcription may be markedly
reduced by pollination and/or message longevity may largely determine the
extent of change in types of mRNA predominating.


119
Libbenga, K.R. and Mennes, A.M. (1987). Hormone binding and its role in
hormone action. In P.J. Davies, ed. Plant hormones and their role in plant
growth and development. Dordrecht, Martinus Nijhoff Publishers, pp 194-
221.
Lin, A.Y., Lee, A.S. (1984). Induction of two genes by glucose starvation in hamster
fibroblasts. Proc. Natl. Acad. Sci. USA 81, 988-992.
Lin, W. (1980). Com root protoplasts. Isolation and general characterization of ion
transport. Plant Physiol. 66: 550-554.
Lin, W., Schmitt, M.R., Hitz, W.D. and Giaquinta, R.T. (1984). Sugar transport in
isolated com root protoplasts. Plant Physiol. 76: 894-897.
Long, D.E., Fung, A.K., McGee, E.E.M., Cooke, R.C. and Lewis D.H.
(1975). The activity of invertase and its relevance to the accumulation
of storage polysaccharide in leaves infected by biotrophic fungi. New
Phytologist 74: 173-182.
Loughman, B.C., Ratcliff, R.G., and Southon, T.E. (1989). Observations on the
cytoplasmic and vacuolar orthophosphate pools in leaf tissues using in vivo
3IP spectroscopy. FEBS Lett. 242: 279-284.
Maas, C., Schaal, S., and Werr, W. (1990). A feedback control element near the
transcription start site of the maize Shrunken gene determines promoter
activity. EMBO J. 9, 3447-3452.
Maestri, E., Restivo, F.M., Gulli, M., and Tassi, F. (1991). Glutamate dehydrogenase
regulation in callus cultures of Nicotiana plumbaginifolia: effect of glucose
feeding and carbon source starvation on the isoenzymatic pattern. Plant, Cell
and Env. 14, 613-618.
Marcott, W. Russell, S.H. and Quatrano, R.S. (1989). Abscisic acid-responsive
sequences from the Em gene of wheat. Plant Cell 1: 969-978.
Mason, H., Dewald, D.B., Creelman, R.A., Mullet, J.E. (1992). Coregulation of
soybean vegetative storage protein gene expression by methyl jasmonate and
soluble sugars. Plant Physiol. 98, 859-867.
Matsushita, K. and Urttani, I. (1974). Change in invertase activity of sweet potato in
response to wounding and purification and properties of its invertases. Plant
Physiol. 54: 60-66.


3
Plant growth regulators often have pleiotropic effects on plant growth and
development. When an organism becomes more complex during its development,
communication between its different part requires an appropriate signaling system.
In vascular plants, a complex hormonal system is largely responsible for signaling
such a communication system (Libbenga and Mermes, 1987). Invertase activity can
be upregulated by abscisic acid, auxin, cytokinins and/or gibberellic acid in an array
of vascular plants (Sacher et al., 1963; Glasziou et al., 1966; Kaufman et al., 1973;
Howard and Withan, 1983; Ackerson, 1985; Weil and Rausch, 1990; Miyamota et
al., 1993; Wu et al., 1993).
The purpose of the present research was to test the hypothesis that regulation
of two subfamilies of soluble invertase isozymes in maize could be consistent with a
proposed role for these isozymes in specific aspects of cell expansion during
development and/or environmental adjustment. Specific objectives were as follows.
1. Clone and characterize maize invertase genes.
2. Test the effects of altered carbohydrate availability on soluble invertase activity
and abundance of mRNA from the two invertase subfamilies.
3. Determine the extent to which changes in mRNA abundance and enzyme
activity correspond to those proposed for given physiological roles of these isozymes
in maize development.
4. Characterize the responsiveness of genes for invertase isozymes to
developmental signals at both message and enzyme activity levels.


2
concentration gradient between the phloem and apoplast which facilitated
translocation of sucrose into sugar-utilizing tissues. Although more recent studies
have largely been consistent with this scenario, they also indicate that the entire
story may not be as simple (Shannon et al., 1993).
Rapidly expanding tissues require either invertase or sucrose synthase to
convert sucrose to substrates necessary for respiratory and synthetic processes
(Giaquinta, 1979; Avigad, 1982; Hawker, 1985). Invertase can be especially
important to cell expansion through generation of hexoses and their associated
osmotic potential (Kaufman et al., 1973; Schmalstig and Cosgrove, 1988; 1990).
Genes involved in metabolic pathways are often regulated by levels of
metabolites (Carlson, 1987; Schuster, 1989; Maas et al., 1990; Koch et al., 1992;
Sadka et al., 1994). In vascular plants, sugar-responsive genes have been primarily
characterized in storage organs (Rocha-Sosa et al., 1989; Salanoubat and Belliard,
1989; Muller-Rober et al., 1990; DeWald et al., 1994; Sadka et al., 1994). However,
carbohydrate-induced changes in gene expression have also been identified for
enzymes involved in photosynthesis (Sheen, 1990) and other metabolic pathways
(Maas et al., 1990; Koch et al., 1992; Graham et al., 1994). Data at the enzyme
level have suggested that a similar sugar-regulated gene expression may underlay
responses of invertase to carbohydrate availability (Sacher et al., 1963; Glasziou et
al, 1966; Ricardo et al., 1972; Kaufman et al., 1973). Together, sugar-sensitivity of
these sucrose-metabolizing genes may comprise a system for sensing and
transducing signals of whole plant carbohydrate status.


Invertase activity Probe
(limol glucose mg'1 protein hr'1)
75
Type of sugar


CHAPTER 2
LITERATURE REVIEW
Invertase and Its Functions
Invertase catalyzes one of the only two enzyme reactions known to
breakdown sucrose into its constituent monosaccharides, glucose and fructose, in
vascular plants. Lack of this enzyme will result in abnormal growth and
development of plants, which is evident in root tips of the Oh43 maize mutant
(Robbins, 1958; Duke et al., 1991) and kernels of the miniature-1 (Miller and
Chourey, 1992). Overexpression of invertase in leaves strongly influences growth
and phenotype of transgenic plants (von Schaewen et al., 1990; Dickinson et al.,
1991). Expression of an invertase gene also controls sugar composition in tomato
fruit (Klann et al., 1993).
Invertase may be ubiquitous among multicellular plant species. Acid and
neutral forms can be distinguished based on pH optima, and/or invertases can be
classified as soluble vs cell-wall-bound forms (Avigad, 1982). An "alkaline"
(neutral) invertase has recently been purified from sprouting soybean (Chen and
Black, 1992). However, acidic invertases are generally more common and have
been studied from an array of plants (Hanft and Jones, 1986a; Sturm and Chrispeels,
1990; Arai et al., 1991; Klann et al., 1992; Elliott et al., 1993; Ramloch-Lorenz et
al., 1993; Schwebel-Dugue et al., 1994; Unger et al., 1994).
4


104
A further implication here lies in the response of drought-stressed roots. The
ABA effect on osmotic adjustment would be nominal to non-existent unless
cytokinins were present. Because cytokinins are normally carried away in the xylem
stream, a rapidly transpiring plant may have less cytokinins build up in the root tips
than one with stomata closed during drought stress. Further extension of this
scenario could potentially include root to shoot signalling via cytokinin flow and
subsequent effect on shoot soluble invertase (Davies and Zhang, 1991).
Action of soluble invertase has been implicated in gravitropism (Kaufman
and Song, 1987; Kim et al., 1993; Wu et al., 1993a; 1993b) and may also be
involved in some aspects of phototropism (Davies, 1987; Kuafman and Song, 1987).
Elevation of osmotic potential for cell elongation could readily result from invertase
hydrolysis of sucrose within the vacuole, and the asymmetry of this process across a
stem is consistent with the involvement of similarly distributed plant growth
regulators in action of invertase. This suggestion is supported by the observation
that glucose injection into dwarf pea shoots mimics the effect of GA on cell
elongation (Broughton and McComb, 1971).
For this reason, altered expression of Ivrl and/or Ivr2 was expected in
response to treatment with auxin and/or GA. However, neither the invertase
message levels nor assayed enzyme activity were markedly affected by addition of
exogenous auxin and gibberellic acid in the root tip system used here (Figure 5-3).
One explanation might be as follows. Hormones often have pleiotropic effects, i.e.,
different types of target cells all respond to the same set of signals, but in a different


Figure 3-5. Conserved regions within derived amino acid sequences of higher plant invertases, shown here for the
IvrlG for maize soluble acid invertase gene 1, a mung bean soluble invertase (Arai et al., 1992), a tomato
soluble invertase (Klann et al., 1992), two carrot soluble invertases (Unger et al., 1994), and two carrot insoluble
invertases (Sturm and Chrispeels, 1990; Sturm, unpublished data). Boxes represent highly conserved regions.


100
Discussion
The significance of findings presented here is that they indicate different
environmental and/or developmental signals can regulate the same gene expression
through common and/or different pathways. Thus, the same enzyme reaction could
play multiple roles under various conditions. Sugar-modulated genes are also
responsive to plant growth regulators. These responses provide a potential
mechanism by which import organs may adjust their sucrose-metabolizing capacity
to altered environment and/or developmental stages.
Results shown here also indicate that cytokinin has a positive effect on the
invertase gene system and that both mRNA abundance and soluble acid enzyme
activity are up-regulated by exogenous cytokinin (Figure 5-1). Cytokinin is reported
to stimulate translocation of photosynthate from source leaves to cytokinin-treated
areas thus increasing the sink capacity of importing bean leaves (Gersani and
Render, 1982), radish cotyledons (Howard and Witham, 1983), winter wheat grains
(Borkovec and Procharka, 1992) and developing maize kernels (Jones et al., 1992).
During leaf development of snap bean (Phaseolus vulgaris L.) and Citrus,
soluble acid invertase activity is correlated well with leaf expansion. In contrast,
both insoluble invertase and sucrose synthase activities are low and show little
change during leaf development (Morris and Arthur, 1984; Schaffer et al., 1987).
The authors suggest that soluble acid invertase activity is the primary enzyme
responsible for sucrose catabolism in the expanding bean and citrus leaves. Its


82
Because the Ivr2 subfamily of genes are extremely sensitively to carbohydrate
deprivation (Figures 4-8, 4-9), the invertases encoded by these messages would be
expected to contribute less to physiological process of importing cells under these
circumstances.
However, specific, high-priority developmental processes, such as pollination
and/or reproductive growth, once initiated, should ideally be less sensitive to
changes in carbohydrate availability than is vegetative growth. If the associated
enzymes for sucrose metabolism are less sensitive to de novo down-regulation
during sugar deprivation, then this could provide a mechanism for giving
reproductive and other essential tissues "import priority" during stresses. The Ivrl
and lvr2 subgroup of genes could play contrasting roles in these instances much like
those of Shi and Susl. Ivrl related gene expression is strongly associated with
reproductive structures (Figure 4-1) and is less markedly affected by carbohydrate
deprivation (Figure 4-9). In contrast, the Ivr2 subfamily of messages are widely
distributed and clearly downregulated by in the absence of sugar supply. The altered
pattern of gene expression for Ivrl and Ivr2 classes in response to carbohydrate
deprivation may be an important adaptive strategy during different stresses, in which
plant survival and/or reproduction could depend on the preservation of vital organs
and/or tissues at the expense of others.
This differential regulation of the two invertase subfamilies in response to
sugar suggests that these genes and their respective enzymes may also have an
important function in carbohydrate partitioning between sink and source tissues.


MOLECULAR CHARACTERIZATION AND DIFFERENTIAL EXPRESSION OF
A INVERTASE GENE FAMILY IN MAIZE
By
JIAN XU
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1994

ACKNOWLEDGEMENTS
My sincerest appreciation is extended to the members of my committee, Dr.
Karen Koch, Dr. Alice Harmon, Dr. Ken Boote and Dr. Don McCarty, for their
support and guidance during the completion of this degree. I am also truly grateful
to the other faculty, staff and graduate students for their help and encouragement
during my time here, especially Kurt Nolte, Ed Duke, Wayne Avigne, Don Merhaut,
Gwendolyn Pemberton, Betsy Bihn, Summer Osterman and Aiyu Li.
Finally, I extend my deepest thanks to my wife Naidong Shao, who was
always there whenever I needed her and also to my parents and my brother in
China. They have all given me support and encouragement throughout my
education here, and I can never thank them enough.
11

TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF FIGURES v
ABSTRACT viii
CHAPTERS
1 INTRODUCTION 1
2 LITERATURE ERVIEW 4
Invertase and its Functions 4
Regulation of Invertase Gene Expression 7
3 ISOLATION AND CHARACTERIZATION OF MAIZE
INVERTASE GENES 16
Introduction 16
Materials and Methods 17
Results 20
Discussion 38
4 DEVELOPMENTAL EXPRESSION AND CARBOHYDRATE
RESPONSIVENESS OF TWO MAIZE INVERTASE GENE
SUBFAMILIES 41
Introduction 41
Materials and Methods 44
Results 48
Discussion 75
5 CYTOKININ MIMICS AND SUPERSEDES THE SUGAR-
INDUCIBILITY OF MAIZE INVERTASE FAMILY MEMBERS
iii

AND FACILITATES THEIR DIFFERENTIAL RESPONSIVENESS
TO ABSCISIC ACID 85
Introduction 85
Materials and Methods 87
Results 91
Discussion 99
6 SUMMARY AND CONCLUSIONS 105
REFERENCE LIST 109
BIOGRAPHICAL SKETCH 126
IV

LIST OF FIGURES
Figure 3-1 Restriction maps of Ivr clones for maize soluble acid invertases ... 26
Figure 3-2 Schematic diagram of the genomic organization of the IvrlG .... 28
Figure 3-3 The deduced amino acid sequence for maize invertase 1 gene 30
Figure 3-4 The hydropathy and fold values of the deduced polypeptide for
maize invertase gene 1 32
Figure 3-5 Conserved regions within derived amino acid sequences of higher
plant invertases 34
Figure 3-6 Conserved regions within derived amino acid sequences of the
IvrlG for maize soluble acid invertase and either other soluble
invertases or insoluble invertases from higher plant 36
Figure 3-7 DNA gel blot analysis of cross-reactivity between Ivrl, Ivr2,
Ivr2C-l and Ivr2C-2 38
Figure 4-1 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize
invertases in root tips, a sink leaf, a source leaf, a prop root, anthers,
silk and kernels 55
Figure 4-2 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize
invertases during kernel development 57
Figure 4-3 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize
invertases in pedicel, middle and top portions of kernels at 8, 10, 12
DAP 59
Figure 4-4 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize
v

invertases in kernels sampled daily from 2 days before to 2 days after
pollination 61
Figure 4-5 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize
invertases during the final 3 days of anther development and in mature
pollen 63
Figure 4-6 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble and insoluble maize invertases
in silk sampled daily from 2 days before to 2 days after pollination ... 65
Figure 4-7 Abundance of mRNA from the Ivrl and lvr2 subfamilies of soluble
acid invertase and activity of total soluble and insoluble maize invertases
in tip, mid and low portions of silk sampled at pollination, 3hr later, 6hr
later, or 24hr later 67
Figure 4-8 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase in maize root tips incubated for 24hr in whites basal salts
medium supplemented with glucose or sucrose 69
Figure 4-9 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase during starvation of maize root tips 71
Figure 4-10 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase during post- starvation recovery of maize root
tips 73
Figure 4-11 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble acid invertases in
maize root tips incubated for 24hr in Whites basal salts medium
supplemented with either 2.0% glucose, fructose, sucrose, L-glucose or
mannitol 75
Figure 5-1 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble acid invertases in maize root
tips incubated for 24hr in Whites basal salts medium supplemented
either with (+G) or without (-G) 0.5% glucose and either with (+K) or
without (-K) 5 pM Kinetin 95
Figure 5-2 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble acid invertases in maize root
tips incubated for 24hr in Whites basal salts medium supplemented with
vi

0.5% glucose, either with (+K) or without (-K) 5 pM Kinetin, either with
(+ABA) or without (-ABA) abscisic acid (50 pM) 97
Figure 5-3 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble acid invertases in maize root
tips incubated for 24hr in Whites basal salts medium supplemented with
0.5% glucose, alone (+0) or with either GA or IAA 99
vii

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MOLECULAR CHARACTERIZATION AND DIFFERENTIAL EXPRESSION OF
A INVERTASE GENE FAMILY IN MAIZE
By
Jian Xu
December, 1994
Chairperson: Dr. Karen E. Koch
Major Department: Plant Molecular and Cellular Biology Program
A family of soluble invertase genes in maize (Zea mays L.) were cloned and
characterized to test several hypotheses regarding their potential significance in
specific instances of developmental and/or environmental adjustment. The responses
of two invertase gene subfamilies were examined at the level of both gene
expression and overall enzyme activity.
Five maize cDNA clones (AW, Ivr2, Ivr2C-l, IvrC-2 and IvrC-3) and one
genomic clone (IvrlG) were isolated and found to encode probable isozymes of
soluble invertase. The deduced amino acid sequences show significant identities,
especially to previously characterized soluble acid invertases of higher plants, and
are particularly strong in key regions conserved among these enzymes. One of the
most strongly conserved regions among all invertase sequences (NDPNG) was found
Vlll

to be carried on an unusually small 9 nucleotide exon identified in the maize
genomic DNA.
Two subfamilies of maize soluble invertases (each cross-reactive with either
Ivrl or Ivr2 [Iw2C-l + Ivr2C-2]) were differentially expressed in an array of tissues.
A comparison between message and enzyme activity was consistent with both
subgroups encoding soluble acid invertases. The spatial and temporal patterns of
expression for the two invertase classes, as well as the contrast between them
implicate their potential involvement in several stages of development. Data support
the hypothesis that invertase could be especially important during stages requiring
expansion of specific cells, such as during pollination and early kernel development.
Maize root tips were used to further test the extent to which expression of the
two subfamilies for soluble invertase isozymes may have been regulated by sugar
levels or specific developmental signals.
The mRNA levels from both subgroups were elevated in the presence of
exogenous sugar supplies as long as these were readily metabolizable, however, the
extent of this response differed. The lvr2 group of genes showed a greater
sensitivity to carbohydrate deprivation. The differential responsiveness of invertase
gene subfamilies to carbohydrate availability provides a potential mechanism for
different isozyme genes to predominate in various tissues developmental stages,
and/or altered environmental conditions.
Data also indicated that specific developmental cues could affect expression
of both invertase subgroups as well as soluble activity of acid enzymes. Cytokinin
IX

signals (typically produced by dividing cell, endosperm, root tips and symbionts)
could alone apparently replace and supersede the carbohydrate upregulation of
invertase transcript levels by sugars. Both Ivrl and Ivr2 type mRNA abundance was
upregulated by exogenous ABA (elevated in developing seeds and in response to
some stresses). However, simultaneous presence of cytokinin appeared to be
required before the ABA-induced changes at the message-level could be transduced
at the level of enzyme activity. The differential response of invertase isozyme genes
to sugar levels and specific plant hormones suggests that integration of these types
of signals may mediate developmental responses, symbiosis, and/or adaptation to
stresses.
x

CHAPTER 1
INTRODUCTION
Sucrose is the most abundant long-distance transport carbohydrate in the plant
kingdom. As such, it plays a central and vital role in plant growth and development.
In vascular plants there are two known enzymatic reactions that can breakdown this
sucrose. These are catalyzed by invertase and sucrose synthase.
Invertase is often considered an essential enzyme for carbohydrate
metabolism and partitioning because of the nearly ubiquitous role of sucrose in
photoassimilate translocation (Avigad, 1982; Hawker, 1985; Turgeon, 1989). This is
supported by the observation that invertase deficient kernels of the miniature-1
maize mutant develop abnormally in addition to their reduced size (Miller and
Chourey, 1992). Further, primary root tips of another invertase deficient maize
mutant, OH43, can not grow normally on sucrose agar (Robins, 1958; Duke et al.,
1991).
Invertase activity is widely distributed within and among vascular plants.
Several isoforms of invertase often can be present simultaneously in a given plant
and/or organ. However, the roles of the individual isoforms are not well understood.
Early work with maize kernels (Shannon, 1972; Shannon and Dougherty,
1972; Doehlert and Felker, 1987; Doehlert et al., 1988) indicated that imported
sucrose moved from phloem into the extracellular space where it was hydrolyzed by
a cell-wall-bound, acid invertase. This was presumed to contribute to a sucrose
1

2
concentration gradient between the phloem and apoplast which facilitated
translocation of sucrose into sugar-utilizing tissues. Although more recent studies
have largely been consistent with this scenario, they also indicate that the entire
story may not be as simple (Shannon et al., 1993).
Rapidly expanding tissues require either invertase or sucrose synthase to
convert sucrose to substrates necessary for respiratory and synthetic processes
(Giaquinta, 1979; Avigad, 1982; Hawker, 1985). Invertase can be especially
important to cell expansion through generation of hexoses and their associated
osmotic potential (Kaufman et al., 1973; Schmalstig and Cosgrove, 1988; 1990).
Genes involved in metabolic pathways are often regulated by levels of
metabolites (Carlson, 1987; Schuster, 1989; Maas et al., 1990; Koch et al., 1992;
Sadka et al., 1994). In vascular plants, sugar-responsive genes have been primarily
characterized in storage organs (Rocha-Sosa et al., 1989; Salanoubat and Belliard,
1989; Muller-Rober et al., 1990; DeWald et al., 1994; Sadka et al., 1994). However,
carbohydrate-induced changes in gene expression have also been identified for
enzymes involved in photosynthesis (Sheen, 1990) and other metabolic pathways
(Maas et al., 1990; Koch et al., 1992; Graham et al., 1994). Data at the enzyme
level have suggested that a similar sugar-regulated gene expression may underlay
responses of invertase to carbohydrate availability (Sacher et al., 1963; Glasziou et
al, 1966; Ricardo et al., 1972; Kaufman et al., 1973). Together, sugar-sensitivity of
these sucrose-metabolizing genes may comprise a system for sensing and
transducing signals of whole plant carbohydrate status.

3
Plant growth regulators often have pleiotropic effects on plant growth and
development. When an organism becomes more complex during its development,
communication between its different part requires an appropriate signaling system.
In vascular plants, a complex hormonal system is largely responsible for signaling
such a communication system (Libbenga and Mermes, 1987). Invertase activity can
be upregulated by abscisic acid, auxin, cytokinins and/or gibberellic acid in an array
of vascular plants (Sacher et al., 1963; Glasziou et al., 1966; Kaufman et al., 1973;
Howard and Withan, 1983; Ackerson, 1985; Weil and Rausch, 1990; Miyamota et
al., 1993; Wu et al., 1993).
The purpose of the present research was to test the hypothesis that regulation
of two subfamilies of soluble invertase isozymes in maize could be consistent with a
proposed role for these isozymes in specific aspects of cell expansion during
development and/or environmental adjustment. Specific objectives were as follows.
1. Clone and characterize maize invertase genes.
2. Test the effects of altered carbohydrate availability on soluble invertase activity
and abundance of mRNA from the two invertase subfamilies.
3. Determine the extent to which changes in mRNA abundance and enzyme
activity correspond to those proposed for given physiological roles of these isozymes
in maize development.
4. Characterize the responsiveness of genes for invertase isozymes to
developmental signals at both message and enzyme activity levels.

CHAPTER 2
LITERATURE REVIEW
Invertase and Its Functions
Invertase catalyzes one of the only two enzyme reactions known to
breakdown sucrose into its constituent monosaccharides, glucose and fructose, in
vascular plants. Lack of this enzyme will result in abnormal growth and
development of plants, which is evident in root tips of the Oh43 maize mutant
(Robbins, 1958; Duke et al., 1991) and kernels of the miniature-1 (Miller and
Chourey, 1992). Overexpression of invertase in leaves strongly influences growth
and phenotype of transgenic plants (von Schaewen et al., 1990; Dickinson et al.,
1991). Expression of an invertase gene also controls sugar composition in tomato
fruit (Klann et al., 1993).
Invertase may be ubiquitous among multicellular plant species. Acid and
neutral forms can be distinguished based on pH optima, and/or invertases can be
classified as soluble vs cell-wall-bound forms (Avigad, 1982). An "alkaline"
(neutral) invertase has recently been purified from sprouting soybean (Chen and
Black, 1992). However, acidic invertases are generally more common and have
been studied from an array of plants (Hanft and Jones, 1986a; Sturm and Chrispeels,
1990; Arai et al., 1991; Klann et al., 1992; Elliott et al., 1993; Ramloch-Lorenz et
al., 1993; Schwebel-Dugue et al., 1994; Unger et al., 1994).
4

5
The physiological significance of invertase action has been debated over a
considerable period of time. Work with sugarcane stems (Hawker and Hatch, 1965;
Glasziou and Gayler, 1972) and com kernels (Shannon, 1968; Shannon, 1972;
Shannon and Dougherty, 1972) indicated that imported sucrose moved from phloem
into the extracellular space where it was hydrolyzed by a cell-wall-bound, acid
invertase. This was presumed to contribute to a sucrose concentration gradient
between the phloem and apoplast, enhancing the rate of sucrose transfer into sucrose
utilizing tissues. More recent evidence is also consistent with the initial hypothesis
(Lin et al., 1984; Doehlert, 1986; Doehlert and Felker, 1987; Doehlert et al., 1988;
Doehlert, 1990 ). Nonetheless, work done by other groups indicates that sucrose can
be transported into Zea mays L. endosperm without invertase hydrolysis (Hitz et al.,
1985; Cobb and Hannah, 1986; Schmalstig and Hitz, 1987; Cobb and Hannah,
1988). It is possible that sucrose can move into sucrose-utilizing tissues of maize
kernels by both mechanisms.
Soluble acid invertase activity is closely correlated with leaf expansion in
bean {Phaseolus vulgaris L.) and Citrus whereas sucrose synthase activity is minimal
and fairly constant (Morris and Arthur, 1984; Schaffer et al., 1987). The authors
suggest that soluble acid invertase activity is the primary enzyme responsible for
sucrose catabolism in the expanding bean and citrus leaves. Its activity is
considered to be the primary determinant of sink potential in these systems.
Invertase also predominates over sucrose synthase (which is barely
detectable) in the earliest stages of development for maize kernels (Tsai et al., 1970;

6
Chourey and Nelson, 1976; Chourey, 1981) and snap bean pods (Sung et al., 1994).
In addition, maize kernels induced to abort by high temperature have a much
reduced activity of pedicel soluble invertase than do nonaborting kernels (Hanft and
Jones, 1986a). The rapid expansion characteristic of this early development requires
both osmotic constituents and substrates for respiratory and synthetic processes.
Soluble invertase can be especially important to cell enlargement through generation
of hexoses and their associated osmotic potential (Kaufman, 1973; Avigad, 1982;
Schmalstig and Cosgrove, 1988; 1990). Either invertase or sucrose synthase can
provide an avenue for carbohydrate entry into respiratory and biosynthetic processes
(Giaquinta, 1979; Avigad, 1982; Morris and Arthur, 1984b; Hawker, 1985; Schaffer
et al., 1987).
Soluble invertase activity is also closely associated with other phases of
reproductive development (Tsai et al., 1970; Jaynes and Nelson, 1971a; Shannon and
Dougherty, 1972; Singh and Knox, 1984; Hubbard et al., 1989; Miron and Schaffer,
1991; Klann et al., 1992). Pryke and Berneir (1978) have found that increased
content of sugar and activity of soluble acid invertase in the apices consistently
appear to accompany the transition to flowering in Sinapis alba. Invertase also
appears to be involved in pollen function. The sucrose content from pollen grains of
Camellia japnica decreases rapidly during growth of the tube. Soluble invertase
activity also increases during germination of cultured pollen and a high constant
activity is found during the later stages of pollen tube growth (Nakamura et al.,
1980). Further, invertase is demonstrated to contribute to an in vitro chemotropism

of pearl millet pollen tubes toward stigmatic tissue through its production of glucose
(Reger et al., 1992a; 1992b; 1993).
A number of different genes may be involved in these processes in vascular
plants (Sturm and Chrispeels, 1990; Arai et ah, 1992; Klann et ah, 1992; Elliott et
ah, 1993; Ramloch-Lorenz et ah, 1993; Schwebel-Dugue et ah, 1994; Unger et ah,
1994). In yeast, one gene encodes both cell-wall-bound and soluble invertases
through differential splicing (Carlson and Botstein, 1982). However, in carrot at
least seven different invertase genes have been distinguished (A. Sturm, personal
communication).
Regulation of Invertase Expression
Invertase and Its Endogenous Inhibitors
Proteinaceous invertase inhibitors are found in an array of vascular plants
(Pressey, 1966; 1967; 1968; Jaynes and Nelson, 1971b; Matsushita and Uritani,
1976; Bracho and Whitaker, 1990a; 1990b; Isla et ah, 1992; Weil et ah, 1994). In
Solanum tuberosum they are located in the vacuole (Bracho and Whitaker, 1990a;
1990b; Isla et ah, 1992), whereas in Nicotiana tabacum they are in the extracellular
space (Weil et ah, 1994). These inhibitors bind tightly and specifically to acid
invertase and have molecular weights ranging from 17 to 23 KDa (Bracho and
Whitaker, 1990b; Weil et ah, 1994). However, it remains unclear by what

8
mechanism the endogenous inhibitors may regulate invertase activities either
spatially or temporally.
Plant Growth Regulators and Invertase
Invertase activity appears to be upregulated by abscisic acid, auxin,
cytokinins and/or gibberellic acid depending on the system and tissues involved
(Sacher et al., 1963; Glasziou et al., 1966; Gayler and Glasziou, 1969; Kaufman et
al., 1973; Howard and Witham, 1983; Morris and Arthur, 1984; Ackerson, 1985;
Schaffer et al., 1987; Weil and Rausch, 1990; Miyamoto et al., 1993; Wu et al.,
1993).
Both auxin and gibberellic acid stimulate cell enlargement, cell elongation
and possibly phototropism and gravitropism (Davies, 1987; Kaufman and Song,
1987; Kim et al., 1993; Wu et al., 1993a; 1993b). The concentration of GA
(gibbellic acid), which promotes growth, closely parallels that which increases
invertase activity in Avena stem segments (Kaufman et al., 1973). The increased
rate of hydrolysis of sucrose to hexose following the stimulation of acid invertase
activity by GA is considered one means of generating an elevated level of osmotic
constituents in the growing region of the stem (Morris and Arhtur, 1985). The
stimulation of both invertase activity and stem growth by auxin is consistent with the
finding that invertases are especially active in tissues undergoing rapid cell
enlargement, such as regions near shoot and root apices (Avigad, 1982). The

9
mechanism by which IAA leads to an increase in acid invertase activity, however,
remains obscure. It is not yet clear whether the observed increase in activity is a
cause or a consequence of auxin-induced growth (Morris and Arthur, 1984a).
Both abscisic acid and cytokinins are reported to stimulate assimilate
translocation from source to sink (Gersani and Kender, 1982; Howard and Witham,
1983; Hein et al, 1984; Schussler et al., 1984; Ackerson, 1985; Jones et al., 1986;
Brokovec and Prochazka, 1992; Jones et ah, 1992). Such enhancement may involve
the capacity of invertase to hydrolyze sucrose to hexoses and thus increase sink
potential. This in turn could stimulate the translocation of sugar to seeds (Shannon,
1968; Shannon, 1972; Shannon and Dougherty, 1972; Lin et ah, 1984; Doehlert,
1986; Doehlert and Felker, 1987).
The mechanisms originally proposed to explain the effects of plant hormones on
invertase have been questioned (Sacher et ah, 1963; Glasziou et ah, 1966; Chrispeels
and Varner, 1967; Gayler and Glasziou, 1969; Hagen et ah, 1984). Gayler et ah
(1969) suggested that auxin and gibberellic acid may have aided stabilization of the
mRNA for invertase. They further suggested that the mechanism of abscisic acid
(ABA) action in this instance involved processes subsequent to formation of
invertase-mRNA and prior to invertase destruction. In contrast, Chripeels et ah
(1967) suggested that the gibberellic acid effect required synthesis of enzyme-
specific RNA molecules. They also proposed that abscisin exerted its action either
by inhibiting the synthesis of these enzyme-specific RNA molecules or by
preventing their incorporation into an active enzyme-synthesizing unit.

10
Most recent work at the molecular level, however, indicates that there are
ABA-responsive elements and GA-responsive sequences located on promoter regions
in a number of structural genes (Jacobsen and Beach, 1985; Zwar and Hooley, 1986;
Libbenga and Mermes, 1987; Marcott et al., 1989; Mundy et al., 1990; Salmenkallio
et al., 1990; Jacobsen and Close, 1991; Skriver et al., 1991; Lanahan et al., 1992).
An auxin-responsive promoter appears to be differentially induced by auxin
gradients during tropisms (Li et al., 1991). It is more likely that the effects of
abscisic acid, auxin, cytokinin and/or gibberellic acid on invertase are mediated by
their respective influence on transcription, but these may well occur by different
mechanisms.
Wounding and Invertase
Wounding typically stimulates expression of invertase genes (Matsushita and
Urttani, 1974; Sturm and Chrispeels, 1990). A general increase in the respiratory
activity in response to wounding in various plant storage tissues is well documented
(Matsushita and Urttani, 1974). In root tissue of sweet potato, respiratory activity
doubles within 20 hours after wounding. The increased respiratory activity is
paralleled by increases in RNA content and the de novo synthesis of enzymes
(Shirras and Northcote, 1984). Invertase may well be one of these and would be
advantageous in its enhancement of capacity to initiate sucrose breakdown.

11
Sugar Levels and Invertase
A number of recent reports demonstrate that various genes involved in
metabolic pathways are either induced or repressed by sugars (Carlson, 1987;
Schuster, 1989; Maas et al., 1990; Sheen, 1990; Koch et al., 1992; Sadka et al.,
1994). Studies of carbohydrate assimilation in potato tubers have revealed that
genes encoding patatin (Rocha-Sosa et al., 1989), sucrose synthase (Salanoubat and
Belliard, 1989) and ADP-glucose pyrophosphorylase (Muller-Rober et al., 1990) can
all be induced by elevated levels of sucrose. Similarly, the tuberous root storage
protein genes of sweet potato (Hattori et al., 1990) and the vegetative storage protein
genes (vegetative storage proteins, Sadka et al., 1994) are upregulated by sugars. In
Arabidopsis, sucrose mimics the light induction of nitrate reductase gene
transcription (Cheng et al., 1992). In maize, elevated carbohydrate levels regulate
the sucrose synthase genes differentially such that Susl is stimulated whereas Shi is
repressed (Maas et al., 1990; Koch et al., 1992).
Repression of gene expression by sugars has also been shown for other plant
genes. In maize mesophyll protoplasts, seven photosynthetic genes are
downregulated by photosynthetic end products sucrose and glucose and by the
exogenous carbon source acetate (Sheen, 1990). In tobacco, the glutamate
dehydrogenase gene is suppressed by feeding glucose (Maestri et al., 1992), and in
cucumber, genes encoding enzymes of the glyoxylate cycle (malate synthase and
isocitrate lyase) are repressed by carbon catabolites (Graham et al., 1994). Together,

12
these mechanisms may comprise a means for sensing and transducing signals of
whole plant carbohydrate status, and subsequently altering plant metabolism and/or
development.
Particularly important in such a possibility are the genes encoding those
enzymes which can break down sucrose, sucrose synthase and invertase. Sugar
responsiveness of the former has been characterized (Salanoubat and Belliard, 1989;
Maas et al., 1990; Koch et al., 1992). Although invertase clones have not been
previously characterized, levels of these enzymes appear to show a long-term,
carbohydrate responsiveness (Sacher et al., 1963; Glasziou et al., 1966; Ricardo et
al., 1972; Kaufman et al., 1973; Sarokin and Carlson, 1984).
Kaufman et al. (1973) demonstrated that increased levels of invertase were
correlated with the sustained growth of Avena stem segments in the presence of
sucrose. Their data further indicated that the presence of sucrose greatly enhanced
the GA effect on elevation of invertase activity. They suggested that substrate may
stabilize the enzyme and/or aid its induction. Their studies also support the view
that gibberellic acid, as well as substrate (sucrose) and end products (glucose and
fructose), play a significant role in regulating invertase levels in Avena stem tissue.
Moreover, such regulation could provide a mechanism for increasing the level of
soluble saccharides needed for gibberellic acid-promoted growth.
However, Morris and Arthur (1984a) documented a drop in concentration of
hexose sugars in internodal segments of Phaseolus vulgaris during incubation in the
presence of auxin. The greatest decline in hexose concentrations occurred when

13
segments were treated with compounds which stimulated the most growth. They
also suggested that by reducing sucrose concentrations in the apoplast and/or
symplast of sink tissues, the acid invertases located in these respective compartments
may contribute significantly to maintenance of source-to-sink gradients in sucrose
concentration and hydrostatic pressure which drives phloem transport.
Fungi, Bacteria, and Invertase
Increased invertase activities have been reported in tissues of several plants
infected by biotrophic fungi and/or bacteria (Callow and Ling, 1973; Long et al.,
1975; Billett et al., 1977; Billett and Burnett, 1978; Callow et al., 1980; Krishnan
and Pueppke, 1988; Sturm and Chrispeels, 1990;). In addition, a common feature of
biotrophic fungal infections of vascular plants is an increased translocation of
photosynthetic assimilates into infected plant parts, which is typically accompanied
by accumulation of one or more host carbohydrates (Callow and Ling, 1973; Long et
al., 1975; Billett et al., 1977; Billett and Burnett, 1978; Callow et al., 1980).
Billett et al. (1977) have shown that infection of maize by the corn smut.
Ustilago mayis, stimulates assimilate movement into, and accumulation of soluble
sugars, and starch, in tissues. Smut in maize kernels results in rapid growth, cell
division, and elevated rates of respiration. Enhancement of maize invertase activity
in these regions could facilitate competition with other sinks for the sugars needed to
support these processes. Sucrose import and unloading from phloem could be

14
accelerated by a greater capacity for invertase to remove this sugar from the terminal
end of the transport path.
Little research, however, has been directed toward understanding the
mechanisms by which invertase activity is elevated in response to pathogens (Callow
and Ling, 1973; Long et al., 1975; Billett et al., 1977; Billett and Burnett, 1978;
Callow et al., 1980; Heidecker and Messing, 1986; Collinge and Slusarenko, 1987;
Sheridan, 1988; Sturm and Chrispeels, 1990). The origin of the induced invertase
protein (fungal vs host) has remained controversial (Billett et al., 1977; Callow et al.,
1980).
Agrobacterium tumefaciens and Pseudomonas syringae pv Savastanoi,
however, contain genes that specify the biosynthesis of cytokinin and indoleacetic
acid (Morris, 1986; Morris, 1987; Ishikawa et al., 1988; Weil and Rausch, 1990).
Cytokinins and/or cytokinin-like substances are also reported to be synthesized in
mycorrhizal fungi (Miller, 1967; Crafts and Miller, 1974; Ng et al., 1982) and
Bradyrhizeobium japonicum (Sturtevant and Taller, 1989). Allen et al. (1980) found
that cytokinin levels increase in the host plant following infection by vesicular-
arbuscular mycorrhizae. Elevated levels of IAA and/or cytokinin have also been
implicated in maize tissues infected by Ustilago mayis (Turian and Hamilton, 1960;
Billett et al., 1977; Billett and Burnett, 1978).
Upregulation of invertase expression by fungal infection could facilitate
enhancement and/or establishment of a symbiosis by providing hexoses for those
fungi unable to metabolize sucrose. A resulting question is whether or not plant

15
hormones, such as IAA and/or GA, act as signals to target the elevation of invertase
in plant parts infected by certain biotrophic fungi and/or bacteria (Heidecker and
Messing, 1986; Morris, 1986; Collings and Slusarenko, 1987; Davies, 1987;
Libbenga and Mermes, 1987; Morris, 1987; Ishikawa et al., 1988; Sheriden. 1988;
Weil and Rausch, 1990)?
Sturm and Chrispeels (1990) imply that the homology between extracellular
carrot P-fructosidase and the levan hydrolyzing enzyme, levanase, may allow carrot
p-fructosidase (invertase) to hydrolyze the bacterial slime coat. In this way,
invertase action could inhibit bacterial growth directly or make the pathogen
susceptible to further defense reactions. In this scenario, invertase would function in
a positive, protective role as a new and unrecognized pathogenesis-related protein.

CHAPTER 3
ISOLATION AND CHARACTERIZATION OF MAIZE INVERTASE GENES
Introduction
Only two avenues known for enzymatic breakdown of sucrose exist in
vascular plants. One is catalyzed by sucrose synthase, the other by invertase. Two
distinct types of invertase activities are found in plants (Avigad, 1982). One class
has an optimum pH of 4.5 to 5.0, and includes acid invertases. The second class
hydrolyzes sucrose at a maximal rate at pH 7.5 to 8.0, and is designed as the
alkaline invertases. The existence of these two types of P-fructosidase is evident in
many plants and/or organs (Avigad, 1982). Acid invertases are located either inside
the vacuole (soluble form) or in the extracellular space (varying degrees of soluble
and cell-wall-bound forms). In contrast, alkaline invertases are compartmentalized
in cytoplasm (Hawker, 1985).
Invertase genes encoding cell-wall and vacuolar (soluble) acid invertases have
been characterized from carrot (Sturm and Chrispeels., 1990; Ramloch-Lorenz et al.,
1993; Unger et al., 1994), tomato (Klann et al., 1992; Elliott et al., 1993), mung
bean (Arai et al., 1992), and Arabidopisis (Schwebel-Dugue et al., 1994).
16

17
In the present study, a tomato invertase clone (Klann et al., 1992) was used
to isolate an invertase cDNA from maize, and this, in turn, was used to obtain
additional maize clones. These findings provide the tools for further investigation
along two lines. The first of these will be aimed at combining an analysis of sugar-
responsiveness of these genes with that of the sucrose synthases, to define
carbohydrate regulation of two different avenues for sucrose breakdown. The
second will be to further clarify the potential functional significance for soluble
invertase isozymes in development and/or environmental adjustment in maize.
Materials and Methods
Probe for cDNA Library Screening
A 0.45 Kb fragment from the 5-end of a cDNA encoding a soluble acid
invertase in tomato (Klann et al., 1992) was isolated from tomato clone and
subcloned into a pUC19 vector. The recombinant plasmid was amplified in E. coli
cells, purified, and used to screen a maize cDNA library (Sambrook et al., 1989).
cDNA Library Screening
A maize root tip cDNA library (A.gt 10, Clontech, Palo Alto, CA) was
screened with the 0.h5 kb tomato invertase cDNA fragment. One positive clone

18
containing a 1.2 kb cDNA was obtained. This maize fragment was subcloned and
used to probe for further cDNAs from the same library. Twelve positive clones
ranging from 0.5 to 2.2 kb were identified.
Genomic Library Screening
A maize genomic fragment containing 8 kb DNA was identified by screening
a genomic library (EMBL 3, Clontech, Palo Alto, CA) with the 1.2 kb maize
invertase cDNA clone.
Hybridization with DNA probe
Procedures for library plating and production of filter replicas were conducted
as recommended by Clontech (Palo Alto, CA). Plaques or colonies were blotted to
nylon membranes, and DNA was denatured in situ with NaOH (0.5 M), neutralized
with Tris buffer (1.0 M, pH 7.5), and fixed by baking (80 C, 0.5-2 hr) (Sambrook
et al 1989). Filters were hybridized at either 50 C (low stringency) or at 65 C
(high stringency) in a solution with the selected cDNA, 7% SDS, 250 mM Na2HP04
(pH 7.2) and 1% BSA (Church and Gilbert, 1984). Tomato and/or maize invertase
cDNA fragments were radiolabed by random primer (BRL, Gaithersberg, MD).
Blots were washed as described by Church and Gilbert (1984), and exposed to X-ray
film with intensifying screens at -80 C.

19
DNA Sequencing
Selected cDNA and genomic DNA fragments were subcloned into a pUC 19
vector. The recombinant plasmids were amplified in E. coli cells and purified
through CsCl2 ultracentrifugation and/or with the use of QIAGEN-tip (QIAGEN
Inc., Chatsworth, CA). Both strands of each cDNA and genomic DNA were
sequenced by the Sequence Core Lab of ICBR (Interdiciplinary Center for
Biotechnology Research) located at the University of Florida.
Analysis of DNA and Protein Sequences
Computer-assisted analyses of DNA and protein sequences were carried out
with Geneworks (Release 2.2, IntelliGenetics, Inc., Mountain View, CA).

20
Results
One positive clone containing a 1.2 kb maize cDNA (Ivrl) was obtained
(Figure 3-1) when a cDNA fragment encoding a soluble acid invertase in tomato
(Klann et al., 1992) was used to screen a maize root tip cDNA library (A.gt 10,
Clontech, Palo Alto, CA).
This 1.2 kb maize fragment was used to rescreen the same library. Twelve
positive clones ranging from 0.5 to 2.2 kb were identified. Sequences obtained from
the longest of these indicated that none of them included a full-length cDNA clone.
For this reason, a Hindlll-EcoRI fragment from the longest clone (2.2 kb, Figure 3-
1) was used to rescreen the library a second time. Seven positive clones were
identified.
From the total of twenty clones, five were selected for full length sequencing
based on their sizes, hybridization characteristics, and location of restriction sites
(Figure 3-1). Sequence was provided by the Sequence Core Lab of the ICBR at the
University of Florida. They were designated Ivrl through Ivr2C-3 (Figure 3-1).
Ivr2C-2 was identical to Ivr2, and Ivr2C-3 contained the same but shorter sequence
as Ivr2C-l.
Further information was sought in the corresponding genomic sequence. A
maize seedling genomic DNA library (EMBL 3, Clontech, Palo Alto, CA) was
screened with a 1 kb Kpnl-EcoRl fragment from the Ivrl cDNA Figure 3-1, 3-7).
One positive genomic clone was isolated and characterized. This clone consisted of

21
ca 8 kb DNA. Digestion with BamHI and Kpnl generated three fragments, each of
which was subcloned and sequenced.
The invertase coding region was deduced according to the information from
cDNAs, recognition sites for intron splicing (Goodall and Filipowicz, 1989; 1991)
and/or comparision with other invertases from vascular plants (Figure 3-5, 3-6). The
gene for maize invertase 1 (IvrlG) was organized into seven exons and six introns,
as diagrammed in Figure 3-2A. The second exon was only 9 nucleotides long
(Figure 3-4B), and has also been reported in tomato fruit vacuolar invertase (Elliott
et al., 1993). The amino acids encoded by this 9 bp exon are located in a highly
conserved domain found in all invertases cloned thus far (NDPNG, the P-
fructosidase motif, Sturm and Chrispeels, 1990, Figure 3-2B).
The genomic DNA (IvrlG) is almost identical to the Ivrl cDNA clone at the
level of amino acid sequence, except for a few amino acid replacements. Genomic
and cDNA clones are from different maize lines, IvrlG being isolated from a B73
genomic library, and the Ivrl cDNA from a Merit root tip libary.
The deduced amino acid sequence from IvrlG consisted of 670 residues
(Figure 3-3) which predicted a molecular weight of 71,942 and an isoelectric point
of 7.5. This protein also included five potential glycosylation sites (N-X-S/T): N165,
N275, N518, N595 and N639 (Figure 3-3). The amino-terminal sequence of the IvrlG
protein indicated a hydrophobic region between basic N and polar C terminals
(Figure 3-3, 3-4) and other characteristics typical of a signal peptide ([-3,-1] rule,von
Heijne, 1986; K. Cline, personal communication). The predicted excision site for

22
the signal sequence according to the (-3, -l)-method of von Heijne (1986) was
between A7' and G74.
A comparison between the invertase genes isolated from maize in the present
work and other invertases from vascular plants (Table 3-1; Figure 3-6), IvrlG shared
an approximate 60% amino acid identity with soluble invertases (Arai et al., 1992;
Klann et al., 1992; Elliott et al., 1993; Unger et al., 1994) and 40% with insoluble
forms (Sturm and Chrispeels, 1990; Ramloch-Lorenz et al., 1993); moreover the
conserved key domains (NDPND [P-fructosidase motif, Sturm and Chrispeels,
1990], as well as FRDPTTA, TGMWEC and YASKTF) (Figure 3-5). In addition,
the maize invertase gene has a significantly greater amino acid identity to the soluble
isoforms of invertase than to the cell-wall-bound ones found in other vascular plants,
especially at the C-terminus of this protein (Figure 3-6).
Restriction maps of invertase cDNA and genomic clones from maize are
shown in Figure 3-1. Ivr2 was found to share a 53% sequence similarity at the
amino acid level to IvrlG (Table 3-1), especially have extensive sequence similarity
located at conserved domains (data not shown). The Ivrl probe (1 kb Kpnl-EcoRI
fragment) did not cross-hybridize with the Ivr2 or lvr2C-l cDNAs at high stringency
(Figure 3-7B). The Ivr2 probe (200 bp Pstl-PstI fragment) cross-reacted with the
Ivr2C-l cDNA but not that of Ivrl (Figure 3-7C).
Ivrl was missing its 3end and contained one 5 unspliced (putative) intron
(according to the sizes of 5'RACE products, E. Bihn, unpublished data). Ivr2

23
contained an unusual 5end, missing the NDPNG (sequence indicates possible
incomplete intron splicing). Ivr2C-l was lacking the 3end of its coding sequence.

24
Table 3-1. Percentage comparative sequence similarity shared at the amino acid
level between genomic and cDNA clones for maize soluble invertases (IvrlG and
Ivr2) and those of other invertases from vascular plants
Amino Acid Identity (%)
IvrlG
Ivr2
Tomato3 (S)b
61
48
Mung Bean0 (S)
59
56
Carrotd (SI)
59
49
Carrotd (SII)
59
49
Carrot6 (CWI)f
42
32
Carrote(CWII)f
45
32
IvrlG
100
53
Ivr2
53
100
Tomato soluble invertase (Klann et al., 1992).
bS represents soluble isoform for invertase.
cMung bean soluble invertase (Arai et al., 1992).
dCarrot soluble invertases (Unger et al., 1994)
eCarrot insoluble invertases (Sturm and Chrispeels, 1990; Sturm, unpublished data).
fCW represents cell-wall-bound (insoluble) invertase.

Figure 3-1. Restriction maps of Ivr clones for maize soluble acid invertases.
Restriction maps of maize soluble invertases (IvrlG, Ivrl, Ivr2 and Ivr2C-l).
Sites on the restriction maps are as follows: B, BamH I; H, Hind III; K, Kpn
I; P, Pst I; S, Sma I.

26
IvrlG
H K S B P H
J 1 1 U L
Ivrl
K
J
Ivr2
S S H P P H
1 1 1 1 1 1
Ivr2C-l
K
J
1 Kb

Figure 3-2. Schematic diagram of the genomic organization of the IvrlG gene for soluble invertase from Zea mays L..
A, The entire IvrlG gene for soluble invertase and bordering regions is depicted with exons as solid boxes. The
locations of a putative CAAT box, TATA box, translation start (ATG), translation stop (TGA) are designated
with arrows. B, Enlarged area from A, which encodes the most strongly conserved region among all invertase
sequences (NDPNG, the P-fructosidase motif) (Sturm and Chrispeels, 1990).

below
B

Figure 3-3. The deduced amino acid sequence for maize invertase 1 gene. The
arrow indicates the cleavage site for potential signal peptide. The box
represents P-fructosidase motif (NDPNG, Sturm and Chrispeels, 1990).
Underlined sequences are for putitive glycosylation sites (N-X-S/T).

30
1
ATC
ATC
CCT
occ
OTT
OCT
OAT
CCG
ACG
ACG
CTO
CAC
GGC
OOC
OCC
GCG
CGC
AGG
CCG
H
X
P
A
V
A
O
P
T
T
L
D
O
O
G
A
R
R
P
SI
TTC
CTC
CCG
GAG
ACG
GAC
CCT
COG
GOG
CCT
GCT
GCC
GCC
GGC
OCC
GAG
CAG
AAG
CGG
L
L
P
E
T
D
P
R
G
R
A
A
A
G
A
E
Q
X
R
US
CCG
CCG
GCT
ACG
CCG
ACC
GTT
CTC
ACC
GCC
GTC
OTC
TCC
OCC
GTG
CTC
CTG
CTC
GTC
P
P
A
T
P
T
V
L
T
A
V
V
S
A
V
L
L
L
V
172
CTC
GTC
GCG
GTC
ACA
GTC
CTC
GCG
TCG
CAG
CAC
GTC
GAC
GGG
CAG
GCT
GCG
GGC
GTT
L
V
A
V
T
V
L
A
S
Q
H
V
D
G
0
A
t 0
G
V
229
ccc
GCG
GGC
GAA
CAT
GCC
GTC
GTC
GTC
GAG
GTG
OCC
GCC
TCC
CCT
GGC
GTG
GCT
GAG
p
A
G
E
O
A
V
V
V
E
V
A
A
S
R
G
V
A
E
286
GGC
GTC
TCO
GAG
AAG
TCC
ACG
GCC
CCG
CTC
CTC
GGC
TCC
GGC
GCC
CTC
CAG
GAC
TTC
G
V
S
E
X
S
T
A
P
L
L
G
S
G
A
L
Q
D
F
343
TCC
TGG
ACC
AAC
GCG
ATG
CTO
GCG
TGC
CAG
CGC
ACG
GCG
TTC
CAC
TTC
CAG
CCC
CCC
S
H
T
N
A
M
L
A
W
Q
R
T
A
F
H
F
Q
P
p
400
AAG
AAC
TGG
ATG
AAC
CAT
CCG
AAC
GOT
CCG
CTO
TAT
CAC
AAG
GGC
TGG
TAC
CAC
CTC
X
N
W
M
lN
D
P
N
0
1 f
L
Y
H
X
G
W
Y
H
L
4S7
TTC
TAC
CAC
TOO
AAC
CCG
GAC
TCC
GCG
GTA
TOG
OOC
AAC
ATC
ACC
TOG
OGC
CAC
GCC
F
Y
Q
W
N
P
D
S
A
V
W
G
N
X
T
W
G
H
A
S14
GTC
TCO
CGC
GAC
CTC
CTC
CAC
TOO
CTO
CAC
CTA
CCG
CTG
OCC
ATC
GTG
CCC
GAT
CAC
V
S
R
D
L
L
H
W
L
K
L
P
L
A
M
V
P
D
H
571
CCG
TAC
GAC
GCC
AAC
GGC
GTC
TGG
TCC
GOG
TCG
GCG
ACG
CGC
CTG
CCC
GAC
GGC
CGG
P
Y
D
A
N
O
V
W
S
G
S
A
T
R
L
P
D
G
R
628
ATC
GTC
ATO
CTC
TAC
ACG
GGC
TCC
ACG
GCG
GAG
TCG
TCG
GCG
CAG
CTG
CAG
AAC
CTC
X
V
H
L
Y
T
G
s
T
A
E
S
S
A
Q
V
Q
N
L
685
GCG
GAG
CCG
GCC
GAC
GCG
TCC
GAC
CCG
CTO
CTO
CGC
GAG
TCG
CTC
AAG
TCG
GAC
GCC
A
E
P
A
D
A
S
D
P
L
L
R
E
W
V
X
5
D
A
742
AAC
CCG
GTG
CTO
GTG
CCG
CCG
CCG
GGC
ATC
GGC
CCG
ACG
GAC
TTC
CGC
GAC
CCG
ACG
N
P
V
L
V
P
P
P
G
X
G
P
T
D
F
R
D
P
T
799
ACG
GCG
TGT COO
ACG
CCG GCC
GGC
AAC
GAC
ACG
GCG
TGG
CGG
GTC
GCC
ATC
GGG
TCC
T
A
C
R
T
P
A
G
N
D
T
A
W
R
V
A
X
G
S
856
AAC
GAC
COG
GAC
CAC
GCG
GGG
CTC
GCG
CTC
GTG
TAC
COG
ACG
GAC
GAC
TTC
GTC
CGG
X
D
R
D
H
A
G
L
A
L
V
Y
R
T
B
D
F
V
R
913
TAC
GAC
CCG
GCC
CCG
GCG
CTO
ATG
CAC
OCC
GTG
CCG
GGC
ACC
OGC
ATG
TOO
GAG
TGC
Y
D
P
A
P
A
L
M
H
A
V
P
G
T
G
M
W
E
C
970
GTG
GAC
TTC
TAC
CCG
GTG
GCC
GCG
GGA
TCA
GGC
GCC
GCG
GCG
GGC
AGC
GGG
GAC
GGG
V
D
F
Y
P
V
A
A
G
S
G
A
A
A
G
S
G
D
G
1027
CTG
GAC
ACG
TCC
GCC
GCG
CCG
GGA
CCC
GGG
GTG
AAG
CAC
GTC
CTC
AAG
GCT
AGC
CTC
L
E
T
S
A
A
P
G
P
G
V
X
H
V
L
X
A
S
L
1084
GAC
GAC
GAC
AAC
CAC
GAC
TAC
TAC
GCG
ATC
GGC
ACC
TAC
GAC
CCG
GCG
ACG
GAC
ACC
D
D
0
X
H
0
Y
Y
A
I
G
T
Y
D
P
A
T
D
T
1141
TGG
ACC
CCC
GAC
AGC
GCG
GAG
GAC
GAC
GTC
GGG
ATC
GGC
CTC
CGG
TAC
GAC
TAT
GGC
W
T
P
D
S
A
E
D
D
V
G
I
G
L
R
Y
D
Y
G
1198
AAG
TAC
TAC
GCG
TCO
AAG
ACC
TTC
TAC
GAC
CCC
GTC
CTT
CGC
COG
CGG
GTG
CTC
TGG
X
Y
Y
A
s
X
T
F
Y
D
P
V
L
R
R
R
V
L
W
1255
GGG
TGG
GTC
ooc
GAG
ACC
GAC
AGC
GAG
CGC
GCG
GAC
ATC
CTC
AAG
GGC
TGG GCA TCC
G
W
V
0
E
T
D
S
E
R
A
D
I
L
X
G
W
A
s
1312
GTG
CAC
TCA
ATC
CCC
AGG
ACG
GTC
CTC
CTC
GAC
ACG
AAG
ACG
GGC
AGC
AAC CTC CTC
V
Q
S
X
p
R
T
V
L
L
D
T
X
T
G
5
N
L
L
1369
CAC
TGG
CCG
CTO
GTG
GAG
GTG
GAC
AAC
CTC
CCG
ATG
AGC
OCC
AAG
AGC
TTC GAC GGC
Q
W
P
V
V
E
V
E
N
L
R
M
S
G
X
S
F
D
G
1426
GTC
GCG
CTC
GAC
CGC
GGA
TCC
GTC
GTG
CCC
CTC
CAC
GTC
GGC
AAG
GCG .
ACG CAG TTC
V
A
L
O
R
G
S
V
V
p
L
D
V
G
X
A
T
Q
L
1483
GAC
ATC
GAC
GCT
GTG
TTC
GAG
GTG
GAC
GCG
TCG
GAC
GCG
GCG
GGC
GTC .
ACG GAG GCC
D
X
B
A
V
F
E
V
D
A
S
D
A
A
O
V
T
E
A
1540
GAC
CTG
ACG
TTC
AAC
TGC
AGC
ACC
AGC
GCA
GGC
GCG
CCG
GGC
CGG
GGC i
CTG CTC CGC
0
V
T
F
N
C
S
T
S
A
G
A
A
G
R
G
L
L
G
1597
CCG
TTC
GGC
CTT
CTC
GTC
CTG
GCG
GAC
CAC
GAC
TTC
TCC
GAC
CAG
ACC GCC GTG TAC
P
F
G
L
L
V
L
A
D
D
D
L
S
B
Q
T
A
V
Y
1654
TTC
TAC
CTG
CTC
AAG
GGC
ACG GAC
GGC
AGC
CTC
CAA
ACT
TTC
TTC
TGC CAA GAC GAG
P
Y
L
L
X
G
T
D
G
S
L
Q
T
F
F
C
Q
D
E
1711
CTC
AGG
GCA
TCC
AAG
GCG
AAC
GAT
CTC
GTT
AAG
AGA '
GTA
TAC '
GGG
AGC TTC GTC CCT
L
R
A
S
X
A
N
D
L
V
X
R
V
Y
G
S
L
V
P
1768
GTG
CTA
GAT
GGC
GAG
AAT
CTC
TCO
GTC
ACA
ATA
CTG i
GTT '
GAC
CAC
TCC ATC GTC GAC
V
L
D
O
S
N
L
S
V
R
I
L
V
D
H
S
X
V
E
1825
AGC
TTT
GCT
CAA
GGC
GGG
AGG
ACG
TGC
ATC
ACG
TCC '
CCA
GTC 1
TAC '
CCC ACA CCA GCC
S
F
A
Q
G
G
R
T
C
X
T
S
R
V
Y
P
T
R
A
1882
ATC
TAC
GAC
TCC
GCC
CGC
GTC
TTC
CTC
TTC
AAC .
AAC GCC ACA CAT GCT CAC GTC AAA
I
Y
D
s
A
R
V
F
L
F
N
N
A
T
H
A
H
V
X
1939
GCA
AAA
TCC
GTC
AAG
ATC
TGG
CAC
CTC
AAC
TCC GCC TAC ATC COG CCA TAT CCG GCA
A
K
S
V
X
X
W
Q
L
N
S
A
Y
I
R
P
Y
P
A
1996
ACG
ACG
ACT
TCT
CTA
TGA
T
T
T
S
L

Figure 3-4. The hydropathy and folding values of the deduced polypeptide for
maize invertase gene 1. A, hydropathy. B, folding structure. The dashed
lines indicate the putative signal peptide cleavage site.

32
A. Hydropathy
phob
3.0 -
1.5 -
-1.5-
-3.0
phi I
134
268
402
536
670
B. Structure

Figure 3-5. Conserved regions within derived amino acid sequences of higher plant invertases, shown here for the
IvrlG for maize soluble acid invertase gene 1, a mung bean soluble invertase (Arai et al., 1992), a tomato
soluble invertase (Klann et al., 1992), two carrot soluble invertases (Unger et al., 1994), and two carrot insoluble
invertases (Sturm and Chrispeels, 1990; Sturm, unpublished data). Boxes represent highly conserved regions.

A
5'
end
Ca rot ICMIII
Tumis (ai
Canal (fin
Mmy lata (I)
Cat lot I IvrIO
(allot |CW|)
Conaanaua
)
Canal (aill
Many Ba
canal (81)
IviIO
Canal 10*1)
Cwai
Cano* (0*11)
T'aato (8)
Canal (Bill
Many laan (B|
tanot (ill
IvilO
Calrot (0*11
Conaanaua
Cat cot ICVIII
Tato |S|
Carrot (Stl)
Ming Basil (a)
Catiyl (BII
IvrIO
Carrot (0*11
......... -Aim-- ilv rasca --blpu-sip
ITQC----Y LPKNSABRYT utn- '-yfo somrks uuisaip
{CTHPLPBII DLBMASSYTP RPOSPSTOMB PUPISURTNR RPIRISSSVL
i n pllptssnaa -piss-trad llpvlc ollplss-l
impitismv tplpuhmsp ii/rrnrruo ssrrrsut-- -pvllpsbil
-PAVAOPT TtXMQQAJUir LLPCTDPROR AAAOAIQR-- -RPPATPTVL
-OVTIR-- NUN YDMOS I.PPLOSIX
Carrot (Otlll
Toaato (B|
Canal (Sill
B" Bonn IS)
Canal (8I|
IvrIO
Cauot (0*11
Carcot (0*111
Toasto (81
Carrot (Bill
Ming laan (B|
Canal (SI)
I IvrIO
Corrot I tv I)
Conaanaua
Carrot (0*11)
Tto IS)
Carrot (Sll)
Hong Baa it (S|
Carrot (Sll
a ivrlo
Cauot |0*1|
Conaanaua
Cariot (0*11)
(I
srip LNiN '--o
u vru.uv Arrr-iUM giru^icsi SPA----- -ppsi ; /bqo
LSTLILSPVI PLLVNPMVOO WARBSSKM MU*l)R**KABR BP Ml *PSR
VA--VOUVRA SOVPHAJILSS PTSNHOOOHQ SPTSLPSSRW VPVSf '!
aaci.vmjimv l-ppmso-ns avsrstvvps ktvsva I ; mao
TAVVSAVLLL VLVAVTVLAB OWVOOOAKXiV PAOSUAVWt VAASI i ifJOJ
A ILLY TTTTLMIM '-A
STNRVPPBl.- --OSISAVD* ---VRLV--H
VSCNTTSDV- --AOASNVSY AMSNAMLSWQ
OlCOOlini PRQATAKPSY PWTNCMLSWQ
VBVLM0NI.lv aobooascap pwonshxswq
VSHRSPRSPA L-NASPPANP PWNAHV1JW0
VSKXSTAPU. OMAtQOT SWTNAKLAWU
PMSIMYNL QSVOASN- --VKQV M
VS.X.i
W.N.I
IPPSKPf I
W I > *QPOQ*e
I A fVKUM I
\a VTDMVi I
immm
II 1PPSKP
, tJtvru.tnfc
YljVO*
It' ¡JVQ
' *VQ*
' JVO
* JVU
t M UVQt
\Lvot
ap a n so
BV JVQ A P A >1 SO
M|r *f" JO>
*01
.vfk-RKMR
fu t-1 OAT
If < VNAT
1 it (-LNKT
I if (-IAWP
1 uflDRMIA
-RMRR
falWKRSPHP 1NTKAI
rtna.ujv lmavk
TV SLUM. LMAVPi
(TYBLABXL LRAVPl
(MPTlXIUV LMAVM
fRYUPAPAL MMAVPi
[RVTKARMP 1NS0AI
...L. ... LHAV|M
LI
X Tl
41 TJ
II Tl
IL 1
IV k
SNMNQN
mum
/AA GSOAAAAfMU
S....N
122
111
I
144
1*7
1*1
12C
200
172
21 *
244
2)1
2)4
214
2(4
202
277
200
201
224
>00
240
>10
1)4
240
m
B.
end
Carrot (0*11)
Tuaaalo (S)
Carrot (Sll)
Ming Baan ()
Carrot (SI)
S IvrIO
Cariot (Olll
n < IN
PI \ IN
OIRI
ovni
OVRI
l BVM
t ovw *
OVNI
vri
OVAI
iSTRYr
(MOOt
DORMI
S>CRNI
5L ImoR.tnMM
fmVROPY IpT
XQRNKM 1 PI
iJ'PlNWW 1 PI
XMKVI.K 1 PI
yttrvaoRW \pi
>PArtm* i pi
.TIMOR V I Pi
Carrot (0*11)
T.ar (S|
Canot (Sll)
Mins (S)
Canot (SI)
) IvrIO
Cariot (0*11
Carrot (0*11)
Tcaaato (SI
Carrot (BII)
Mins laan ()
Carrot (SI)
IvrIO
Carrot (0*1)
Conaanaua
S>pf*JS LMHRNORLOM
TV-ROVOLQP
4ajy bi-oovklrp
J vtn EP-ASt.AAAP
IJfMIT Vp.fMVCIMT
I SP-DOVALOR
ijistv RPSRXOOCSK
minaiT
: I ELLAvox
tLVpUlISS
IWSI.OI CT
tVLPl.KIOS
¡wpluvok
ILVCVRCIT
Carrot (0*11)
Toaato (S)
Carrot (Sll)
Ming laan (S)
Carrot (SI)
I IvrIO
Carrot (CM!)
Conaanaua
Carrot (C||)
Twato (S)
Canot (ill)
Hung Baan (S)
Carrot (SI)
> IvrIO
Carrot |CWt|
Conaanaua
l ipi
I TP
l JPI
J IP
>1 P
lLPt
AtkU.VAPSfR
APOVOAOVYO
Asnviatevva
ANOVPAQIFO
AvovoRRivii
ANOLVABVYO
RBai.VRPSPA
A.DV.A..YO
4SS
)
S29
914
917
919
90S
91
914
40B
Carrot (0*11)
Toanato (B)
Carrot (Sll)
Mans Baan (S)
Carrot (SI)
IvrIO
Carrot (0*1)
Conaanaua
Carrot (0*11)
Tcaaato (S)
Carrot (SI!)
Ming Moan (S)
Canot (SI)
i IvrIO
Carrot (041)
Conaanouo
(OR -Arsi
ICS -SL77
OS -MLS'
(TO AA I SI
os -R.a
IMU
Nuwfr
Oil
- af.il
I AO RRMLtftf *
saKoa oRTvn t
(vss) too ORTvn r
SB) tQO ORTCV IP f
I ¡00 ORTCI1 SP r
VSS too CRTCI* St 1
f/BS IAK OATCI.' SA t
ilKoO ORTCITSPlir
.-TIP ITVO-I
tTOA SVTA-BVA
tTOV BVTA-SVPi
tTCA TVTA-aiJtt
tTKA RIIA-BLM
tlMA HVKARBVRi
ISKT ITV8-NU>4*
tTKA .V.A-BLR
Ml-SPS Ml
>I.PSAMIO*r PM) M.
MASATIJIPP PPP.--OI.
MSAPIRPf pniPUOAS
**tta oAyr sPA-n.vi
NJisAYiarv pat-ttsl
Ml----APL R*a*
VN> I
vs*
YO*
YN
99
()
412
19
944
490
4*
490
4
9*2

Figure 3-6. Conserved regions within derived amino acid sequences of the IvrlG for maize soluble acid invertase gene
1 and either A, other soluble invertases or B, insoluble invertases from other vascular plants. A, Derived amino
acid sequence similarities shared between the IvrlG cDNA clone for maize soluble invertase, mung bean soluble
invertase (Arai et al., 1992), tomato soluble invertase (Klann et al., 1992), and carrot soluble invertases (Unger et
al., 1994). B, Derived amino acid sequence similarities shared between the IvrlG and the insoluble invertases of
carrot (Sturm and Chrispeels, 1990; Sturm, unpublished data). Boxes in A and B represent the most highly
conserved regions and underlined sequences are those shared among soluble but not insoluble invertases.

A. Soluble 5/ end
B. Insoluble 5' end
TUmo IS)
Carrot (Sill
Huny Baan (S)
Catcut (SI)
IvrlO
^TUC- 1 0PXNSA8XVT tLMQPO fOHRJCS LKUSOIP
nTHPLPSR DLZHAS8YTP BPOSPtTRHK PDPDASRWR RPIKIBS8VL
Ml-I PI.LPTSSH AA -PTSI-TRKD LLFVLC- -CLLPLSS-L
-HPITISHY TPLPOCBHSP SLTTTNTAEQ SSRRRSLT-- -FVLLPSSIL
l-PAVAOPT TUXWOARRP LLPSTDPRCR AAAOAKQK-- -RPPATPTVL
)7
SO
17
47
44
Conaanaua
L....8..P .L....TR 8.. 88.L SO
TttMto (8)
Carrot (SSI)
Hung baan (8)
Carrot (81)
IvrlO
Conaanaua
US-VfU.LSV APPP--1LNN QSPDtQIMR SPA -PP8I
LSTLILSPVI PLLVHPNVQQ WRKXBSKMS NGkDRHJCASK SPSMJ
VAYOOYRA SQVPilAHLSS PTSHHQQDHQ SPTSLPSSKW YPV8I
AACLVHCTHV L-PPHS0-N8 AVZXSTWPI ETVEVA- -P--1
TAW8AVLLL VLVAVTVLAS QHVDCQAOOV PAOEOAVWI VAAS^ABQ
.A.
.0 P.SffJ/. .0
I : tSQQ
4>PSR
'ssa
'AEG
7*
100
5
I
94
Toacto (8)
Carrot (SIS)
Hung Baan (8)
Caciot (81)
IvrlO
Conaanaua
TMMto (8)
Carrot (8II)
Hung Baan (8)
Carrot (SI)
IvrlO
Conaanaua
Toaato (8)
Carrot (811)
Hung Baan (8)
Carrot (81)
IvrlO
Conaanaua
VSDRTFROV- A0A8XVSY
GBSQGbSEKS PRQATAb'PfiY
VSKXSSNLLF AOBGOASEAP
VSHKSPHHPA l-NAYPPANP
VSKXSTAPLL --GSOALQDP
V8.KS -...A
ToaaCo (8)
Carrot (SIS)
Hung Ban (8)
Carrot (SI)
IvrlO
Conaanaua
Tcaaato (8)
Carrot (SII)
Hung Baan (8)
Carrot (81)
IvrlO
Conaanaua
T
rw
m
fH
*PV
2PV
IPV
AP PPOI
it p ppai
ap ppoi
AP PPOI
idAipvIa p ppoi
;s'
JAl
If) I!
If* OPTTJ
PR DPTTJ
ifR OPTTJ
VR DPTTJ
* DPTTA
> rran
A IK
A ILT1
( -
pg Ho-
ICRO CX
.T8I OK
'AjlRTPI OK
atpa cmmf
I.HQEJLCm^.TP. CK
I gsk
I OSK
I OSK
I OSK
I OSK
-ion: /
VNX1 I
LNX1 :
LHK1 ;
)ADH): *
r SPKUXOVLN
[ TYBLLDNLL8
( TYKLKECLLR
C NFTU.DOVLH
i RYUPAPALHJI
AV 3TGHWEC VPf h
KM
TtGHrtC VD*
il| 3TQMHEC VO
TOHWEC VO
3TCHWEC VD <
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-mum si
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241
294
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212
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)12
))
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324
34)
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Carrot (CWI)
Carrot (CWII)
Conaanaua
giPAVAorrr umcmaarpl
IVTI-RNRN YDHOSL--PP
iRTKILV PSSOS9LP
.T.- D.CS.Pr
f>rroPRORA AAOAXQXRPP atptvltaw
BE EE EE
SB
19
11
SO
IvrlO
Carrot (CHI)
Carrot (CWII)
8AVLLLVLVA VTV l,
-SU.ATU.VT TTI
-SIPSPIP -
tSQHVD ^AOOVPAOI DAVWlffaAS
--HIH
N1H
BCVASOVSIX
HC1KY
- 43ST HRVFP
10#
4S
39
Conaanaua
-S.L...LV. .T1
A. H.V.
100
IvrlO
Carrot (CM!)
Carrot (CWII)
Conaanaua
STAPLLOSOA pfcfSWTMAM LAWjVlfcr Qf*JC>nfi
-N JQ VOAIHVK QV-1 17 n IP Ql CQK
-I l-tpiSAVDVK i-V-l IV \f Qt }KI
14p.8A.NVX LV-lfe3j3v4lLfi mlfDP NO I
IP HOI
iflDP HOI
IDP NO I
IS#
#s
7
ISO
IvrlO
Carrot (CWf)
Carrot (CWII)
Conaanaua
20#
1)5
12
200
IvrlO
Carrot (CWJ)
Carrot (CWII)
Conaanaua
I -YTC r
LYTC I
IT Uffi
vbj n
VSP DPI
> JVQf. K PA D 30P .
LTEJivs. .p.N>cvpty
PA n SDF '
paIi.bdp
34#
1 #5
17#
2S0
IvrlO
Carrot (CWI)
Carrot (CWII)
Conaanaua
tyLVPPPOIO PTT
WANT JO EN ATJ
-VOVUTKN- PSJ
'TfKFDPTTA
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-W...O.. PTAtfiCmA.
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WPO-OGH
W.D.OOM
2##
2)1
22)
)00
IvrlO
Carrot (CWI)
Carrot (CWII)
Conaanaua
TOPA PJ
^ARTICA KHP
XWXRS PHP;
continued
continued
B. Insoluble 3 end
IvrlO
Carrot (CWI)
Carrot (cwit)
Conaanaua
IvrlO
Carrot (CWI)
Carrot (CW(I|
IvrlO
Carrot (CWI)
Carrot (CWII)
IvrlO
Carrot (CWI)
Carrot (CWII)
IvrlO
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Carrot (CWII)
Will
SVRt
mi
nf/DT
Will
H.TRY
ISTRYI
r¡v
t V
vr.
>PAT
TDK
IRVR
(VTjrt urn
TSV
t\(pi rrsv
rrsv.
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II ASKTI III :i
II KA3KTI t 1)1 II
amy bu-i
TiViJpipn
1KT U IW t
rot iiJiynv
rwryf >1 rydv
n.Rvn*
ifprvrp
ifprtu.i:
IKK lllf l
Ufjr
w
P-
P-1
JW I A
JW I IIF
]W I II
aonar-oovA T
t.R SKVXFfiftKQO I
) OSEUIMRHOK p
I 8...P.R.Q. I
U> Vui
^v* or
*rr or
1
AvftevOASDA AOVTXAD-VT f- NC8T8A
VI 1 IfXSLAX RIPmPRWLf YDACXICSLX I
At IfKiUm AX8P0PWIH LDAQOVCOSW |
AitFPXSUW AB.PDP.W.. .DA...C.B. li>T.(J
ft
IP PC
ruplrnwsL orrric
IVI^ lOWTH- xvu .
nl< ITOQKU KVU
.lli^.O..L KVIlfc
a (vela
Carrot (CWI)
Carrot (CWII)
-DOSM
ATDRK
-snxx
^rsr pwywimiij'
wr iakoxtt 1
JtSf IAQRXN1 I
liA.oRTtiiib bvypi
IvrlO THANVXAK6V XI
Carrot (CWI) *FT-ITVEM. Ol
r.urtot (CWII) TtP-ITVWU. Of
conaanaua
T8.-ITV.M. DfUlMS-
JtRHYI xr
mxx
a#is
VTTT8L
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¡EMN--
)9#
114
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a
174
)44
497
43)
415
543
471
4CS
591
531
SIS
44#
S71
544
47#
593
SIS
Soluble 3' end
TumIo (S)
Carrot (all)
Hung Baan (8)
Carrot (81)
> IvrlO
Conaanaua
Tcaaato (SI
Carrot (SII)
Hung Baan (8)
Carrot (SI)
l IvrlO
Coin
vl /
K J
< J
fasi.ro
SLOD
su
SLOO
SLOO
sum.
rt Y(
7T vr
rt Yi
JT YI
xntNXwdp'tyi
IHOXW1 P
yuvur p 1
VBOKM r I
ATOTWIP
.KWJi
iPBi r
IPBl
7VXI
IPE1
1 All f
IPllL
1 1A
sxTpyr Kii
SKTTYI )DKB
tk 1KTTYI )HKD
tk turra itocR
ik aUTfYI^VUl
impJ -x- kJIui
TOMto (1)
Carrot (111)
Hung Baan (fi)
Carrot (SI)
B IvrlO
Conaanaua
TomIo (8)
Carrot |#l!)
Hung Baan (8)
Carrot (SI)
IvrlO
Conaanaua
Toaaato 1*1
Carrot ISIII
Hung Baan (8)
Carrot |S!|
IvrlO
/VI c
I /Rl
IU
JU
HI
TCij HI
NI [-QWPV
L0WPV
L0WPV
L0HI
t ti i>
/Lvfiftttlr MlCowr^A IfOlAJOPTV KOVDtQI
1RBYRI DOVXUCI
BLIP KSLJUKfJ:
NXTVP XHVXII
IP DOVAL
EeIj inLOUn]
/ HI LI
83
bP 74a
....r ..v.t.i
IK
L VI 1
i^V
iV
IV
* Vt I
IVMAA*
(IBBAA0
31XTATQ
IIOSOSQ
7V0XATQ
JiV ViU-X.SA.Q
QT
D> -sr
ht
M
I A> I
Lf I
:clxL 4 u 1
rt Py 1 Af
rt nr
rt py if*
rt PY 4
Uta ntisPU-
:< :\
xc rt
rcMi/r mi >ova rr it
It ngqwi
)f Rl
II Bi
Rffc
bHn- M3
IDVl K
IUVT <
J.DV.
VYb¡
VPVII
sj ^PVl
VPV1
vrvt
PPVI
Toarto (S)
Carrot (811)
Hung Baan (8)
Carrot (S||
s IvrlO
: (psi -
;tin p
;t ils4'
lli(L8U
ymumii
. VDHflVBSf^ poonf/fTtR
VDHSIVK8I
vnisivcsi 2
VDHSIVC8I
yxiRi
ussm
l. vdhsivssfV bocynV^riT
:: rsit
EUL
¡luSUiii
V.A
ffHOA
VBA MtV{Fl|PHHAT
TOA
YHN
YDS
F304A1
i\H r#WAi
UF PWA7
Toadlo (8)
Carrot (SII)
Hung Baan (S)
Carrot ISl)
IvrlO
Conaanaua
CASV1 *
GVSV1 )
XA7V1
CARD *
IIAHV *
.A. Vi
l &LJC.'
; jHAf
JUI
* I IQ SPPIQDL
jruc ppppo-0L
riR rrprwpDOKs
OR OTHfA-DtVI
rift PYPAT-tTSL
V
jyjHHiW.IB PPPP-
341
3 #7
372
37S
393
441
4#7
472
475
493
#41
SBS
57#
571
SOI
411
43S
42#
423
441
437
441
44#
45#
470

Figure 3-7. DNA gel blot analysis of A, Approximate size of the Ivrl, Ivr2, Ivr2C-l and Ivr2C-2 cDNA clones and B,
Extent of cross-reactivity between them. A, EtBr stained DNA gel blot analysis of approximate length for Ivrl,
lvr2, Ivr2C-l and Ivr2C-2. B and C, DNA gel blots with equal amounts (1 pg) of recombinant DNA from each
cDNA (Released from pUC 19 vector by digestion with EcoRI) and probed with 32P-labeled fragments from
either A, Ivrl (Kpn I-EcoR I, 1 kb fragment) or B, Ivr2 (Pst I-Pst I, 200 bp fragment). Both blots were exposed
to X-ray film for 1 hr. Sites on the restriction maps are K, Kpn I; P, Pst I; R, EcoR I.

A
ro

IS} -t* IS}
>
CD
s}
Ivrl
Ivr2
Ivr2C-l
Ivr2C-2
A A
is} i
A
ro
is}

39
Discussion
Soluble invertase genes were cloned and characterized for two reasons. The
first of these was to characterize the extent of their carbohydrate-responsiveness
relative to that of genes for sucrose synthase, ultimately to provide a more complete
picture of how sugars influence the capacity for their own metabolism at the
transcriptional level. The second planned use for the invertase clones was to clarify
the potential significance of these gene family members during development and/or
environmental adjustment by maize tissue and organs.
Three lines of evidence support the designation of these genes not only as
maize invertases but also as soluble ones. First, the full length sequence of the
putative maize invertase clone (IvrlG) has extensive sequence similarity to other
invertases found in vascular plants (Table 3-1), and shares the conserved key
domains identified in other invertases (NDPNG [(3-fructosidase motif, Sturm and
Chrispeels, 1990], plus FRDPTTA, TGMWEC and YASKTF) (Figure 3-5). Second,
the maize invertase gene examined here has a considerably greater amino acid
identity to the soluble isoforms of invertase than to the cell-wall-bound ones found
in other vascular plants (Table 3-1; Figure 3-6). The underlined areas in figure 3-6
are those highly conserved regions which are shared among soluble invertases but
not insoluble ones. In particular, the amino acid sequence at the C-terminus of the
IvrlG protein is significantly more similar to that of soluble vs. insoluble forms.

40
Targeting signals for vacuolar proteins are frequently present in this region as C-
terminal propeptides (Bednarek et al., 1990; Chrispeels, 1991; Bednarek and
Raikhel, 1992). Third, message abundance of lvrl and lvr2 correlates well with
total soluble invertase activities in an array of maize tissues and/or developmental
stages (see Chapter 4).
Invertases of maize and other vascular plants are presumably encoded by
different genes, although in yeast, variable splicing allows a single gene to encode
both cell-wall-bound and soluble invertases (Carlson and Botstein, 1982). There are
at least two 7vr/-like genes in the maize genome, and the lvrl and lvr2 subfamilies
have been tentatively mapped to two and four different loci respectively (data not
shown, collaboration with Scott Wright, Genetic Linkages, Salt Lake, Utah).
The genomic clone of maize invertase has typical CAAT and TATA boxes
located in the upstream untranslated region (Figure 3-2). The second exon is
unusually small (9 bp) in maize lvrl invertase (Figure 3-2) and tomato soluble
invertase genes (Elliott et al., 1993). The amino acids encoded by this 9 bp exon are
located in a highly conserved domain found in all invertase clones (NDPNG, the P-
fructosidase motif, Sturm and Chrispeels, 1990). This represents one of the smallest
exons currently known to function in the plant genome (M. Schuler, personal
communication).
In the lvrl maize invertase genomic gene, several introns (number 1, 3, 4 and
5) are also found to contain one or more copies of an RY sequence motif
(CATGCATG, data not shown), which thus far has been implicated in seed-specific

41
gene expression (Dickinson et al., 1988; Baumlein et al., 1992; Lelievre et al.,
1992). This suggestion is also supported by the preferential expression of the Ivrl
subfamily genes in reproductive tissues (see Chapter 4).
The polypeptide encoded by the IvrlG invertase gene has 670 residues with a
molecular weight of 71,942 (Figure 3-3). The calculated isoelectric point is 7.5,
which is intermediate between that of carrot soluble invertases (SI: 3.8; SII: 5.7,
Unger et al, 1994) and insoluble invertases (carrot CW: 9.9, Sturm and Chrispeels,
1990; Arabidopsis CW: 9.1, Schwebel-Dugue et al., 1994). This protein also
contains five putative glycosylation sites (N-X-S/T) and a potential peptide signal
from M1 to A73 (Figure 3-3).

CHAPTER 4
DEVELOPMENTAL EXPRESSION AND CARBOHYDRATE
RESPONSIVENESS OF TWO MAIZE INVERTASE GENE SUBFAMILIES
Introduction
Invertases ((3-fructosidase, EC 3.2.1.26) play a key role in sugar metabolism.
In vascular plants, different isoforms are located in different cellular compartments
(Avigad, 1982; Hawker, 1985). Isoforms with an acidic pH optimum are found in
the vacuole and/or apoplasm whereas isoforms with a neutral pH optimum are
located in the cytoplasm. Work with sugar cane stems (Hawker and Hatch, 1965;
Glasziou and Gayler, 1972) and com kernels (Shannon, 1968; Shannon, 1972;
Shannon and Doughty, 1972; Shannon et al, 1993) has indicated that imported
sucrose moves from phloem into the extracellular space where it is hydrolyzed by a
cell-wall-bound, acid invertase. This is presumed to contribute to a sucrose
concentration gradient between the phloem and apoplast, facilitating transfer of
sucrose into importing tissues (Lin et al., 1984; Doehlert, 1986; Doehlert and Felker,
1987; Doehlert et al., 1988; Turgon, 1989). Soluble invertase has been found in the
vacuoles of sucrose-storing cells (Avigad, 1982). Thus, soluble invertases with
acidic pH optima are often thought to be localized in the cell vacuoles of other
tissues as well, where they can mobilize sucrose temporarily stored in this compartment.
42

43
Rapidly expanding tissues require either invertase or sucrose synthase to
convert sucrose into substrates necessary for respiratory and synthetic processes
(Giaquinta, 1979; Avigad, 1982; Morris and Arthur, 1984b; Hawker, 1985; Schaffer
et al., 1987). Invertase can be especially important to cell expansion through
generation of hexoses and their associated osmotic potential (Kaufman, 1973;
Avigad, 1982; Schmalstig and Cosgrove, 1988; 1990). Invertase activity is also
associated with reproductive organs (Jaynes and Nelson, 1971a; Shannon and
Dougherty, 1972; Singh and Knox, 1984; Hubbard et al., 1989; Miron and Schaffer,
1991; Klann et al., 1992; Reger et al., 1992). Invertase can aid competition for sink
capacity for reproductive growth. Soluble invertase is the predominant enzyme for
sucrose breakdown during the early developmental stage of maize kernel (Tsai et al.,
1970) and snap bean seed (Sung et al., 1994).
Genes regulated by carbohydrate were first studied in microorganisms
(Carlson, 1987; Schuster, 1989). Those genes are usually involved in metabolic
pathways. In vascular plants, sugar-responsive genes have been primarily
characterized in storage organs (Rocha-Sosa et al., 1989; Salanoubat and Belliard,
1989; Muller-Rober et al., 1990; DeWald et al., 1994; Sadka et al., 1994). Those
genes generally encode storage proteins such as patatin from potato (Rocha-Sosa et
al., 1989), tuberous root storage protein genes from sweet potato (Hattori et al.,
1990), vegetative storage proteins from soybean (Sadka et al., 1994). In addition,
carbohydrate-induced changes in gene expression have also focused on metabolic
pathways, especially those involved in sugar metabolism such as sucrose synthase

44
(Maas et al., 1990; Koch et al., 1992), malate synthase, isocitrate lyase (Graham et
al., 1994) and/or photosynthetic pathway (Sheen, 1990) and are considered critical
mechanisms for sensing environmental and developmental signals.
Invertase is one of the only two enzymes known for sucrose breakdown in
vascular plants and has shown a relatively long-term responsiveness to carbohydrate
availability at the enzyme level (Sacher et al., 1963; Glasziou et al., 1966; Ricardo et
al., 1972; Kaufman et al., 1973).
Previous research indicated that invertase was vital at both the specific organ
level and at the whole plant level. Robbins (1958) found that OH43 primary roots
could not grow on sucrose arga, and Duke et al. (1991) showed these roots to be
invertase deficient. Miller and Chourey (1992) also found that the abnormal
development of miniature kernels was associated with an invertase deficiency. The
present study utilizes two acid invertase gene-probes to determine the effects of
developmental processes and altered carbohydrate availability on expression of the
Ivrl and Ivr2 classes for soluble acid invertase genes. The report presented here
also demonstrates the extent of developmental differences and carbohydrate
responsiveness in two subfamilies of maize genes for acid invertase (probably
soluble). These findings indicate that there may be specific roles for soluble
invertases during development and that these could differentially contribute to
adjustment of sucrose import, cellular volume, and possibly metabolism in vascular
plants.

45
Materials and Methods
Plant Material
The Zea mays hybrid NK 508 was used for all experiments. For analyses of
developmental changes, plants were grown under greenhouse or field conditions.
Samples harvested included leaves, anthers, silk, cobs, pollen, prop roots, and
kernels at different developmental stages.
For experiments with root tips, seeds were first emersed in 20% Clorox for
30 min, followed by 30 min of continuous rinsing with water. Germination took
place in the dark at 18 C on two layers of moist 3 MM paper (Whatman, Inc.,
Clifton, NJ) in 17 x 26 cm glass pans. Air flowed continuously at 1 liter min'1
through each pan for the 6-day period, with 40% 02 supplied during the final 24 hr
before root tip excision. The moisture level was adjusted daily by applying mist and
draining excess water. The terminal 1 cm was cut from root tips (at ca. 3 to 6 cm
total length) under a sterile transfer hood.
Experimental Conditions
Experimental treatments were as described by Koch et al. (1992).
Approximately 100 root tips (~ 500 mg) were used for each experimental treatment.
Excised root tips were incubated in the dark at 18 C for 6 to 48 hr in Whites

46
medium, either with or without an array of supplemental sugars. Each group of root
tips was agitated at 120 cycles per minute in a 125-ml side-arm Erlenmeyer flask
with 50 ml of sterile media. Airflow (40% 02, make sure to supply enough 02)
through air stones in each flask was maintained at 250 ml min'1 throughout the
incubations.
RNA Isolation and Gel Blot Analysis
Root tip samples were rinsed twice in sterile water, blotted dry, weighed, and
frozen in liquid N2. Other samples (as mentioned in the previous text) were
harvested from greenhouse and/or field-grown plants, weighed, and frozen
immediately in liquid N2. Samples were ground into fine power in liquid N2 and
total RNA was extracted (McCarty, 1986). RNA was quantified
spectrophotometrically (Sambrook et al., 1989). Total RNA (10 pg) was separated
by electrophoresis in 1 % agarose gels containing formaldehyde (Thomas, 1980),
blotted to nylon membranes, and fixed by baking and/or UV treatment (Sambrook et
al., 1989). Filters were hybridized at 65 C in a solution with 7 % SDS, 250 mM
Na2HP04 (pH 7.2) and 1 % BSA (Church and Gilbert, 1984). Maize hr 1 and Ivr 2
invertase cDNA clones were radiolabeled by random primer. No cross-reactivity
was observed between the hr 1 and hr 2 gene probes when hybridizations were
conducted at high stringency (data not shown). Blots were washed as described by

47
Church and Gilbert (1984), and exposed against X-ray film with intensifying screens
at -80 C.
Enzvme Extraction
Soluble invertase was extracted as per Duke et al. (1991). Frozen tissue
samples were ground to a fine powder in liquid N2 using a mortar and pestle.
Frozen powder was transferred to a second mortar containing ice-cold 200 mM
HEPES buffer (pH 7.5) with 1 mM DTT, 5mM MgCl2, 1 mM EGTA and 10%(w/w)
PVPP. One ml of extraction buffer was used for each 100 mg of tissue fresh
weight. Buffered extract was centrifuged at 15,000 x g for 10 min to sediment
particulate matter. Pellets were saved for salt-solubilized particulate invertase
extraction. Supernatant was dialyzed (50,000 MW cutoff) at 4 C for 24 hr against
extraction buffer diluted 1:40. Buffer was changed twice. Soluble dialyzed extract
was centrifuged again at 15,000 x g for 10 min and supernatant assayed for soluble
invertase activity as described below.
Insoluble invertase was extracted as described by Doehlert and Felker (1987).
Pellets remaining from extractions of soluble invertase were washed three times by
sequentially resuspending each in 5 to 10 ml extraction buffer and centrifuging at
15,000 x g for 10 min. Salt-solubilized particulate invertase was extracted by
resuspending the pellet in extraction buffer containing 1 M NaCl. Solubilized
particulate invertase was recovered in supernatant following centrifugation at 15,000

x g for 10 min. Pooled supernatant fractions were assayed for insoluble invertase
assay as described below.
48
Enzyme Assay
Both soluble and salt-solubilized invertase activities were assayed for 15 to
30 min at 37 C in an assay medium with 100 mM Na-acetate (pH 4.5) and 100
mM sucrose in a final volume of 500 pi. Activity was determined by measuring
reducing sugars as described by Nelson (1944) and Somogyi (1951).

49
Results
Developmental and organ-level differences were evident in expression of the
two classes of invertase genes (Figure 4-1 A). Message levels for the Ivrl group
were markedly higher in reproductive structures than vegetative tissues, whereas
those of the Ivr2 type transcripts were abundant in essentially all of the sucrose
importing structures examined (loading same amount of total RNA). Message from
both classes of invertase were present in sink leaves, dropping below detectable
levels during sink-to-source transition. Transcript levels of both types were also
evident in those tissues undergoing rapid growth and/or cell division, such as root
tips, anthers, pollen and silk (styles). As observed for relative mRNA abundance,
activity of this enzyme fraction also predominated in the most rapidly elongating
tissues (such as root tips and silk) regardless of whether data were expressed per unit
protein or fresh weight (data not shown). Activity was also generally elevated in
instances of enhanced sucrose import. The greater ratio of RNA/protein recovered
from root tip extracts vs those from other tissues suggests that if changes in total
RNA encoding invertase messages are viewed relative to protein levels, then
invertase mRNA levels in root tips are greater relative to enzyme activity than is
evident in Figure 4-1. The greater values for RNA/protein recovery from root tip
extracts may possibly be due to the extensive meristematic activity in these organs.
Shifts in region of localization were evident during kernel development for
the two subfamilies of maize invertase (Figure 4-2 A and Figure 4-3 A). Message

50
(Days After Pollination), dropping below detection within 16 DAP. However, the
Ivr2 type mRNA was abundant in the pedicel region and barely detectable in the
middle and top portions of the kernels (Figure 4-3A). In contrast, levels of Ivrl-
related messages in the pedicel region were similar or less than those in the middle
and top sections of kernels at the same developmental stage. In addition,
developmental differences in timing were evident, with a narrow peak in Ivrl
transcript abundance at 8 DAP in the upper kernel, vs a broader elevation in Ivr2
message in the pedicel between 8 and 12 days after pollination. Transcript levels of
the Ivrl subgroup were approximately similar in pericarp and endosperm at 10 DAP,
whereas the Ivr2 mRNAs were considerably more abundant in the pericarp (data not
shown).
Figure 4-3 B showed that total soluble acid invertase activity, like that of Ivrl
and Ivr2 mRNA was highest in the pedicel region and lowest in the top area of the
same kernels when expressed per unit total soluble protein (similar results were
observed when data were calculated per unit fresh weight [data not shown] expcept
that peak activity was elevated for two days longer). Total activity of soluble acid
invertase was maximal in extracts of kernels sampled 12 days after pollination,
dropping gradually to below detection in those from between 20 and 24 DAP
(Figure 4-2C). In contrast, salt-solubilized particulate invertase activity (insoluble)
increased gradually in developing kernels, but did so most rapidly between 2 and 6
DAP. Peak activity was observed at ca 16 DAP, and decreased slowly thereafter.
Activity remained detectable at 32 DAP (well past maturity under local growing

51
Activity remained detectable at 32 DAP (well past maturity under local growing
conditions). This salt-solubilized activity was also maximal in the pedicel area and
lowest in the top portion of the same kernels when expressed per unit total salt-
solubilized protein. If decreases in mRNA levels encoding Ivrl and Ivr2 are viewed
relative to protein levels, then the drop in message abundance is more pronounced
than pictured due to the onset of enhanced protein storage in kernels between 10 and
12 DAP.
During the earliest stages of kernel development, message levels for both Ivrl
and lvr2 subfamilies and total soluble invertase activity increased markedly (Figure
4-4). Soluble invertase activity from kernels two days after pollination was twice as
high as that of unpollinated ones (Figure 4-4B) and insoluble activity from the same
kernels (Figure 4-4C).
During anther development, transcript levels of both Ivrl and lvr2 classes of
invertase increased gradually through anthesis (Figure 4-5A). Both message types
were also abundant in RNA extracted from pollen. This was probably not the basis
for localization in young anthers, because shedding anthers had greater apparent
levels of both classes of mRNA than did pollen itself.
Both the Ivrl and lvr2 types of mRNA were abundant in silk if tissue was
sampled before or immediately after pollination (Figure 4-6A). A gradient in
relative message levels for these gene classes was also evident along the length of
the silk, with lowest levels in the top (distal) 1/3 and greatest abundance in the 1/3
closest to the ovary (proximal) (Figure 4-7A). A rapid response to pollination was

52
also observed in a progressive decline of message levels for both classes of invertase
transcripts. The longitudinal gradient of invertase mRNA levels from tip to base of
silks was reduced during this decrease by the rapid decline in message abundance
observed in the basal region of the style. At the enzyme level, temporal and spatial
changes in total soluble activity were consistent with those of the Ivrl and Ivr2
message levels (Figure 4-6B, 4-7B). Salt-solubilized invertase was relatively
constant before and/or after pollination, and no activity gradation was evident along
the length of silk (Figure 4-6C, 4-7C). The drop in mRNA abundance of invertase
is still more pronounced than pictured if considered relative to protein levels.
Differential responses of the Ivrl and Ivr2 class genes to sugar supplies
became apparent when excised root tips were supplemented with a range of glucose
and/or sucrose concentrations and incubated for 24 hr (Figure 4-8). Ivrl class
message levels were maximal with ca 0.5% exogenous glucose (Figure 4-8A) and ca
0.2% sucrose (Figure 4-8B), whereas those of Ivr2 remained relatively constant
when media glucose and/or sucrose levels were between 0.2 and 4.0%. In addition,
levels of the Ivrl subfamily of transcripts appeared to drop less markedly during a
24 hr period without exogenous carbohydrate than did those of Ivr2 (Figure 4-8). In
excised maize root tips, soluble sugars reportedly drop to minimal levels within 10
hr if no supplemental sugars are provided (Saglio and Pradet, 1980). Differential
responses of the Ivrl and Ivr2 classes of invertase to carbohydrate deprivation were
further explored by an analysis of their progression over time in excised root tips
(Figure 4-9). Levels of the Ivrl type mRNAs decreased less rapidly than did those

53
of the Ivr2 subgroup and persisted for considerably longer. Relatively little change
was evident during 24 hr of starvation, and message remained readily apparent for at
least 48 hr. In contrast, levels of the Ivr2 class of mRNA dropped below detection
after between 12 and 18 hr of carbohydrate deprivation (Figure 4-9).
Although Ivrl message abundance appeared to be relatively insensitive to an 18-hr
starvation period or subsequent additions of sugar to media, levels of mRNA for the
Ivr2 subfamily were sensitive to both (Figure 4-10). Glucose replacement after 18
hr of C-depravation appeared to counter initial decreases in levels of message for the
Ivr2 subfamily. These returned to pre-starvation levels after 18 hr incubation in 0.5
% glucose (Figure 4-10).
The responses of the Ivrl and Ivr2 class genes to different types of sugars
(Figure 4-11 A) also showed that expression of both appeared to require a supply of
metabolizable sugars. Transcripts remained abundant in the presence of 2% D-
glucose, fructose, or sucrose in the exogenous media, but dropped when these were
replaced by either L-glucose or mannitol.

Figure 4-1. Abundance of mRNA from the Ivrl and hr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in root
tips, a sink leaf, a source leaf, a prop root, anthers, silk and kernels (2 DAP).
A, RNA gel blots with equal amounts (10 pg) of total RNA from above
tissues were probed with 32P-labeled Ivrl or Ivr2 representing the two
subfamilies of maize soluble acid invertase. Blots were exposed to X-ray
film for 24 or 12 hr, respectively. B, Total soluble acid invertase activity
from the above tissues. C, Insoluble acid invertase activity from the above
tissues. Values for RNA/protein recovery were ca 0.04 (+ 0.02) for tissues
other than root tips and did not otherwise differ significantly between tissue
types. Root tip values were greater (0.15 + 0.04) possibly due to more
extensive meristematic activity.

root tips
sink leaf
H
source leaf
prop root
anthers
silk
H
kernel (2Dap>
}
Invertase activity
(pmol glucose mg'1 protein hr'1)
. Soluble activity
Probe
root tips
sink leaf
source leaf
prop root
anthers
>
3
33
Z
>
silk
kernel (2dap>
Ln
Ln

Figure 4-2. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases during
kernel development. A and B, RNA gel blots with equal amounts (10 pg) of
total RNA from kernels between 6 and 32 DAP (full maturity at ca 30 DAP
under local conditions) were probed with 32P-labeled Ivrl or Ivr2 representing
the two subfamilies of maize soluble acid invertase. Blots were exposed to
X-ray film for two days. Relative abundance of mRNA was quantified by
phosphor image quantifications. C, Total soluble acid invertase activity from
the above tissues. D, Insoluble acid invertase activity from the above tissues.
Values for RNA/protein recovery from this set of kernels were ca 0.04
(+0.02), except at 10 and 12 DAP (0.08 + 0.04) (consistent with changes in
cell division and protein levels during early kernel development).

Invertase activity
(pmol glucose mg1 protein hr1)
H
l
I
H
H
3
O
c
Q
05
O
*<
3

4
-I
O
H
H
Soluble activity
% of maximal response
Ivr1 mRNA

Figure 4-3. Abundance of mRNA from the Ivrl and lvr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in
pedicel, middle and top portions of kernels at 8, 10 and 12 DAP. A, RNA
gel blots with equal amounts (10 fig) of total RNA from above tissues were
probed with 32P-labeled Ivrl or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for two days.
B, Total soluble acid invertase activity from the above tissues. C, Insoluble
acid invertase activity from the above tissues. Values for RNA/protein
recovery were ca 0.03 (+ 0.01) with variability independent of tissue gradient
from kernel top to pedicel. Values for RNA/protein recovery dropped from
ca 0.04 (+ 0.02) to 0.02 (+ 0.01) past 10 DAP, and is consistent with
elevated protein storage in kernels at this stage.

Ivrl
Ivr2
30
20
10
0
300
200
100
0
59
A. mRNA
8 DAP
10 DAP
12 DAP
top mid ped top mid ped top mid ped
m
8 DAP
10 DAP
12 DAP

Figure 4-4. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in
kernels (ovules) sampled daily from 2 days before to 2 days after pollination.
A, RNA gel blots with equal amounts (10 pg) of total RNA from above
tissues were probed with ,2P-labeled Ivrl or Ivr2 representing the two
subfamilies of maize soluble acid invertase. Blots were exposed to X-ray
film for 24 or 12 hr, respectively. B, Total soluble acid invertase activity
from the above tissues. C, Insoluble acid invertase activity from the above
tissues. Values for RNA/protein recovery were ca 0.03 (+ 0.01) with
variability independent of development.

Invertase activity Probe
(|.imol glucose mg'1 protein hr'1)
61
Ivrl
Ivr2
4 -
A. mRNA
Days -/+ pollination (fert)
-2 -1 0 +1 +2
(-3) (-2) (-1) (0) (+1)
B. Soluble activity
X
Ir1
I
C. Insoluble activity
x
i
-2 -1 0 +1 +2
(-3) (-2) (-1) (0) (+1)
Days -/+ pollination (fert)

Figure 4-5. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases during
the final 3 days of anther development and in mature pollen. A, RNA gel
blots with equal amounts (10 pg) of total RNA from above tissues were
probed with 32P-labeled Ivrl or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively. B, Total soluble acid invertase activity from the above
tissues (*, not assayed). C, Insoluble acid invertase activity from the above
tissues (*, not assayed). Values for RNA/protein recovery were ca 0.03
(0.01) for mature and shedding anthers. Values from extracts of young
anthers were greater (0.07 + 0.03), possibly due to more extensive
meristematic activity.

Invertase activity Probe
(nmol glucose mg' protein hr'1)
63
Ivrl
Ivr2
A. mRNA
Days to anthesis
anther pollen
3 10 0
III
20
10
20
10-
tft
B. Soluble activity
X
C. Insoluble activity
X
X
3 10 0
anther pollen
Days to anthesis

Figure 4-6. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in silk
sampled daily from 2 days before to 2 days after pollination. A, RNA gel
blots with equal amounts (10 pg) of total RNA from above tissues were
probed with 32P-labeled Ivrl or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively. B, Total soluble acid invertase activity from the above
tissues. C, Insoluble acid invertase activity from the above tissues. Values
for RNA/protein recovery were ca 0.06 (+ 0.02) before pollination and
dropped to 0.02 (+ 0.01) after pollination. Transcription may be markedly
reduced by pollination and/or message longevity may largely determine the
extent of change in types of mRNA predominating.

Invertase activity Probe
(jimol glucose mg'1 protein hr'1)
Ivrl
Ivr2
60
40
20
20
A. mRNA
Days -/+ pollination (fert)
-2 -1 0 +1 +2
(-3) (-2) (-1) (0) (+1)
!

B. Soluble activity
- T
X
X
InX
C. Insoluble activity
X
X
-2 -1 0 +1 +2
(-3) (-2) (-1) (0) (+1)
Days -/+ pollination (fert)

Figure 4-7. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in tip,
mid, and low portions of silk (portions of silk [ca 4 cm total length] relative
to ovary) sampled at pollination, 3 hr later, 6 hr later, or after 24 hr. A,
RNA gel blots with equal amounts (10 pg) of total RNA from above tissues
were probed with 32P-labeled Ivrl or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively. B, Total soluble acid invertase activity from the above
tissues. C, Insoluble acid invertase activity from the above tissues. Values
for RNA/protein recovery were ca 0.06 (+ 0.02) before pollination and
dropped to 0.04 (+ 0.02) within 3hr after pollination, then to 0.02 (+ 0.01) at
6hr and 24hr after pollination. Transcription may be markedly reduced by
pollination and/or message longevity may largely determine the extent of
change in types of mRNA predominating.

Invertase activity Probe
(l^mol glucose mg'1 protein hr'1)
Ivrl
Ivr2
A. mRNA
+0 hr AP
+3 hr AP
+6 hr AP
+24 hr AP
(ip mid low tip mid low tip mid low tip mid low
Mill | m

100
B. Soluble activity
i
50
X
3.1
X
X
X
X
X
I
C. Insoluble activity
20 -
T
1
X
JL
i
x
pH.
x_
J-
+0 hr AP
+3 hr AP
+6 hr AP
+24 hr AP

Figure 4-8. Abundance of mRNA from the hr l and Ivr2 subfamilies of soluble acid
invertase in maize root tips incubated for 24 hr in Whites basal salts medium
supplemented with either 0, 0.2, 0.5, 2.0, 4.0% glucose or sucrose. RNA gel
blots with equal amounts (10 pg) of total RNA from above tissues were
probed with 32P-labeled Ivrl or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively.

69
glucose (%)
0 0.2 0.5 2.0 4.0

Ivr2
sucrose (%)
0 0.2 0.5 2.0 4.0
Ivrl
7vr2

Figure 4-9. Abundance of mRNA from the Ivrl and lvr2 subfamilies of soluble acid
invertase in maize root tips depleted of carbohydrates for either 6, 12, 18, 24,
36, or 48 hr, respectively, in Whites basal salts medium without an
exogenous sugar supply. RNA gel blots with equal amounts (10 pg) of total
RNA from above tissues were probed with 32P-labeled Ivrl or Ivr2
representing the two subfamilies of maize soluble acid invertase. Blots were
exposed to X-ray film for 24 or 12 hr, respectively.

71
starvation (hr)
6 12 18 24 36 48
Iwl f || .
Ivr2

Figure 4-10. Abundance of mRNA from the hr l and Ivr2 subfamilies of soluble
acid invertase during post-starvation recovery of maize root tips. Sugar
depletion in Whites basal salts without sugars (18 hr) was followed by
incubation for various periods of time (6-18 hr) in media with 0.5% glucose
supplements. RNA gel blots with equal amounts (10 pg) of total RNA from
above tissues were probed with 32P-labeled Ivrl or Ivr2 representing the two
subfamilies of maize soluble acid invertase. Blots were exposed to X-ray
film for 24 or 12 hr, respectively.

73
post-starvation
recovery (hr)
0 6 12 18
Ivrl

Ivr2

Figure 4-11. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble acid invertase in maize root tips
incubated for 24 hr in Whites basal salts medium supplemented with either
2.0% glucose, fructose, sucrose, L-glucose or mannitol respectively. A, RNA
gel blots with equal amounts (10 pg) of total RNA from above tissues were
probed with 32P-labeled Ivr 1 or lvr 2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively. B, Total soluble acid invertase activity from the above
samples. Insoluble invertase activity (not shown) was consistently ca 10-fold
less than that in the soluble fraction of maize root tips. Values for
RNA/protein recovery were ca 0.14 (+ 0.05) with variability independent of
presence or absence of metabolizable C-source.

Invertase activity Probe
(limol glucose mg'1 protein hr'1)
75
Type of sugar

76
Discussion
The significance of findings presented here extends from implications of
special roles for soluble invertases during development (especially pollination and
early kernel development) to broader possible contributions to adjustment of sucrose
import, cell volume, and metabolism in a multi-celled higher plant. The spatial and
temporal patterns of expression for the two invertase subfamilies, as well as the
contrast between them suggest involvement in specific developmental processes.
The availability of these clones has also allowed the hypothesis to be tested that
regulation of transcript level by photosynthate availability could contribute to
adjustment of both avenues for sucrose breakdown in a cell (invertase as well as
sucrose synthase). Moreover, a surprising similarity in differential carbohydrate
responsiveness was evident between the two invertase subfamilies and the two
sucrose synthase genes. In both instances, the more broadly distributed of the two
(Ivr2 or Susl) was found to be readily induced by enhanced carbohydrate
availability, whereas the form which was upregulated during more specific
developmental and environmental signals (Ivrl or Shi) was less sensitive to sugar
supplies (Koch et al., 1992).
The present work indicates that each subfamily of the invertase genes is
expressed differentially depending on developmental stage and the tissue/organ
involved. Although invertase activity was detected in extracts of almost every
sucrose-importing tissue examined, the Ivrl type message was preferentially

77
associated with reproductive organs (Figure 4-1 A). Data shown here for general
association between soluble invertase activity and rapid growth/cell division were
consistent with previous suggestions for the role of this enzyme relative to sucrose
import. Rapidly expanding tissues require either invertase or sucrose synthase to
convert sucrose to substrates necessary for respiratory and synthetic processes
(Glasziou and Gayler, 1972; Giaquinta, 1979; Avigad, 1982; Morris and Arthur,
1984b; Hawker, 1985; Schaffer, 1986; Schaffer et al, 1987). Invertase in particular
can be important to cell expansion through generation of hexoses and their
associated osmotic potential. Changes in both message and activity in the present
study were also consistent with the gradual sink-to-source transition in leaves (Ho,
1988; Turgeon, 1989; Nguyen-Quoc et al., 1990).
Data presented here indicate that soluble invertases may be especially
important during the early stages of maize kernel development. This is consistent
with a hypothesis advanced on the basis of previous work (Hanft and Jones, 1986a;
1986b; Reed and Singletary', 1989), which suggests that the soluble forms of these
enzymes in the pedicel may be critical to initiation of normal kernel development.
The expression pattern of both the Ivrl and Ivr2 classes of invertase, as well as total
soluble activity (Figure 4-2 and Figure 4-4), are also in agreement with this
possibility.
Past research on invertase and kernel development has tended to focus on the
insoluble "cell-wall-bound" form of this enzyme primarily because of its apparent
importance during later stages of kernel fill. Shannon and coworkers proposed that

78
the driving force for assimilate movement into normally developing kernels was the
sucrose-gradient between the leaves and the pedicel apoplasm combined with the
monosaccharide-gradient between the pedicel apoplasm and the starchy endosperm
cells (Shannon, 1968; Shannon, 1972; Shannon and Dougherty, 1972). Both
gradients are presumably maintained by the activity of an apoplastic pedicel
invertase (Shannon et al. 1993).
Early phases of kernel growth may differ from previous hypothesis, despite
the apparent importance of insoluble invertase and sucrose synthase activity in later
development (ca 22 DAP) (Tsai et al., 1970; Chourey and Nelson, 1976; Chourey,
1981). Sucrose synthase activity is not detectable prior to 12 DAP (Tsai et al.,1970;
Chourey and Nelson, 1976; Chourey, 1981). Instead, activity of soluble invertase as
well as mRNA for both classes of soluble invertase peak at 8 to 12 DAP (Tsai et al.,
1970; Figure 4-2). Soluble acid invertase may also be important to initial
establishment and maintenance of sink strength.
The potential role of soluble invertase genes in early kernel development may
be related to the difference between tissues in which message classes were most
strongly expressed. Ivrl mRNA levels were greater in endosperm and the upper
kernel whereas Ivr2 message was most abundant in the pedicel region (Figure 4-3A).
This distinction could be important for two reasons. The first of these is the
suggestion that the miniature phenotype results initially from reduced endosperm
invertase and its subsequent effect on pedicel invertase (both presumably insoluble,
Miller and Chourey, 1992). The second is the hypothesis advanced by Hanft and

79
Jones (1986a; 1986b) which tentatively attributes kernel abortion under water and
heat stresses to reduced activity of soluble invertase in the pedicel. The following
scenario represents one possible explanation for the combination of data on the
greater sensitivity of the Ivr2 genes to carbohydrate deprivation and the abundance
of their transcripts in the pedicel. Any early limitation of assimilate flux into the
endosperm would be expected to reduce soluble sugar concentration in the pedicel
within a relatively short time (Hanft and Jones, 1986a). The depletion of pedicel
sugars could in turn result in decreased levels of the carbohydrate-responsive Ivr2
gene products and a subsequent decrease in soluble invertase activity in this region.
This is consistent with the observation that it is the soluble rather than insoluble acid
invertase activity which is most markedly affected in pedicel of kernels that have
been induced to abort vs. nonaborting kernels (Hanft and Jones, 1986a).
The role of soluble invertases during anther and pollen development is
probably twofold. First, there are no plasmodesmatal connections between
developing pollen grains and the surrounding tapetum layer (Kesselback, 1949). The
tapetum thus lies at the terminal end of the maternal transport path. Any invertase
or sucrose synthase present in these cells could theoretically enhance sugar transport
to pollen grains by creating a sucrose gradient between phloem and the secretory
surface, much as hypothesized for developing kernels (Shannon, 1972; Shannon and
Dougherty, 1972; Lin et al., 1984). Presumably, enhanced hydrolysis could also
benefit the probable elevation in respiratory and biosynthetic demands. Our results

80
indicated that significant amounts of both Ivrl and Ivr2 type message were present
in the anther tissues collectively (Figure 4-5).
Second, invertase message in pollen may be important for subsequent
germination, to facilitate use of endogenous as well as exogenous sucrose. Sucrose
represents the major soluble sugar present in the majority of angiosperm pollen
grains, including maize. Mature pollen grains from diverse plants contain sucrose
but not starch as reserve carbohydrate (Portnoi and Horovitz, 1977; Nakamura et al.,
1980). Germinating pollen grains show an extremely high rate of growth and thus
have a high demand for carbon skeletons required for pollen tube wall synthesis as
well as substrates for respiration (Hoekstra, 1983; Singh and Knox, 1984). The
sucrose content from pollen grains of Camellia japnica decreases rapidly during
pollen growth and the activity of soluble invertase increases during culturing and a
high constant activity is found at the later stages of pollen tube growth (Nakamura et
al., 1980). Our data are consistent with the possibility that invertase has multiple
functions during anther development and pollen grain maturation (Figure 4-5).
Further localization of invertase at the message and/or protein level in situ could
help clarify the functions of invertase in reproductive processes.
The association between high levels of both invertase activity and message
levels with rapidly elongating styles (silk) in maize may have a twofold biological
implication. First, invertase, as one of the important constituents of sink strength
(Morris and Arthur, 1984; Schaffer et al., 1987), can provide an important avenue
for sucrose cleavage. Resulting hexoses can either be subsequently metabolized in

81
support of high respiratory rates and/or compartmentalized in vacuoles to maintain
turgor for cell expansion. Second, invertase is demonstrated to contribute to an in
vitro chemotropism of pearl millet pollen tubes toward stigmatic tissue through its
production of glucose (Reger et al., 1992a; 1992b; 1993). Although other factors,
such as calcium and an ovarian protein, are also important for the chemotropic
response of pearl millet pollen tubes, our results were consistent with an invertase
role in forming gradients of hexoses for pollen growth in maize. The message
gradient of Ivrl and for2 abundance along the length of the silk (Figure 4-6) further
supported the concept that glucose produced by invertase may be at least one key
factor underlying the chemotropic response of pollen tubes, and their pathway
towards the ovule.
Genes encoding carbohydrate metabolizing enzymes are regulated at the
transcriptional level by sugar availability in yeast and vascular plants (Carlson, 1987;
Schuster, 1989; Maas et al., 1990; Koch et al., 1992). Invertase, one of the only two
enzymes known with a capacity to breakdown sucrose in vascular plants (Avigad,
1982), is also shown here to be regulated at the message level by sugar availability.
Further, invertase gene subfamilies are found to be differentially expressed even
within the same organ. The potential exists for these isozyme gene subfamilies to
confer particular biological advantage through their presence in specific tissue at
various stages of development and/or altered environmental conditions.
Photosynthesis and carbohydrate availability are often greatly reduced under
environmental stresses, such as drought, flooding, severe cold and/or insect attack.

82
Because the Ivr2 subfamily of genes are extremely sensitively to carbohydrate
deprivation (Figures 4-8, 4-9), the invertases encoded by these messages would be
expected to contribute less to physiological process of importing cells under these
circumstances.
However, specific, high-priority developmental processes, such as pollination
and/or reproductive growth, once initiated, should ideally be less sensitive to
changes in carbohydrate availability than is vegetative growth. If the associated
enzymes for sucrose metabolism are less sensitive to de novo down-regulation
during sugar deprivation, then this could provide a mechanism for giving
reproductive and other essential tissues "import priority" during stresses. The Ivrl
and lvr2 subgroup of genes could play contrasting roles in these instances much like
those of Shi and Susl. Ivrl related gene expression is strongly associated with
reproductive structures (Figure 4-1) and is less markedly affected by carbohydrate
deprivation (Figure 4-9). In contrast, the Ivr2 subfamily of messages are widely
distributed and clearly downregulated by in the absence of sugar supply. The altered
pattern of gene expression for Ivrl and Ivr2 classes in response to carbohydrate
deprivation may be an important adaptive strategy during different stresses, in which
plant survival and/or reproduction could depend on the preservation of vital organs
and/or tissues at the expense of others.
This differential regulation of the two invertase subfamilies in response to
sugar suggests that these genes and their respective enzymes may also have an
important function in carbohydrate partitioning between sink and source tissues.

83
Under source-limited conditions, invertase involved in certain physiological
processes could act to increase sink activity and stimulate assimilate translocation to
these sinks to compete with others. Under normal growth conditions, assimilate
levels are plentiful. Thus, the Ivr2 class of genes tend to be widely expressed in
sink tissues and their gene products are abundant. This is especially evident in
rapidly growing tissues, which is consistent with the concept that high activity of
"soluble" invertase is usually associated with rapid tissue expansion (Glasziou and
Gayler, 1972; Giaquinta, 1979; Avigad, 1982; Hawker, 1985; Schaffer et al., 1987).
Invertase is considered to facilitate assimilate transportation from the site of phloem
unloading to sink tissues by steepening the gradient of sucrose between source and
sink (Shannon, 1968; Shannon et al, 1972; Shannon and Dougherty, 1972; Shannon
et al., 1993). However, soluble invertase can also promote cell elongation and/or
rapid growth by hydrolyzing sucrose to hexoses, thereby providing osmotically
active solutes and the osmotic pressure necessary to support growth (Kaufman et al.,
1973; Schmalstig and Cosgrove, 1988; 1990).
Gene responses to sugars in vascular plants have been known for some time
(Rocha-Sosa et al., 1989; Salanoubat and Belliard, 1989; Muller-Rober et al., 1990;
Maas et al., 1990; Koch et al., 1992). However, the mechanism, by which the sugar
signal is sensed by plant genes, is not clear. Our results (Figure 4-12) indicated that
naturally occurring, metabolizable sugars, such as sucrose, D-glucose and fructose,
meet the requirement for invertase responsiveness, although data shown here can not
rule out other possibilities for certain non-metabolizable sugars.

84
Sadka et al. (1994) propose that sugar modulates transcription of the soybean
vegetative storage proteins and other sugar-inducible genes by using phosphate as a
signal. In their model, phosphate acts as a negative factor to those sugar-responsive
genes. Carbohydrate activates those genes by accumulation of sugar-phosphates and
concomitant reduction of cellular phosphate levels. High phosphate levels relative to
those of sugars are also found in starved sycamore cells (Rebeille et al., 1985).
Graham et al. (1994), on the other hand, propose that not metabolism per s,
but the phosphorylation by hexokinase per s maybe signaling intracellular sugar-
responsiveness of gene expression. In their experiments, they demonstrate that 2-
deoxyglucose and mannose, like glucose and fructose (which are phosphorylated by
hexokinase but not further metabolized) specifically repress cucumber malate
synthase and isocitrate lyase gene expression. However, 3-methylglucose, an analog
of glucose that is not phosphorylated, does not result in repression of either malate
synthase or isocitrate lyase.
Many of the genes involved in metabolic pathways are subject to regulation
by the fluctuation of internal and external metabolites in multicellular vascular plants
(Maas et al., 1990; Sheen, 1990; Koch et al., 1992; Graham et al., 1994; Sadka et
al., 1994;). The metabolic regulation of gene expression should play a role of
fundamental importance in maintaining an economical balance of the supply and
demand of biomolecules in different organs of vascular plants. Metabolic control of
specific gene expression now appears to be a widespread phenomenon, although the
mechanism of signal transduction and response for different genes will not

85
necessarily be the same. Environmental and developmental signals may also have
contrasting influences, and depending on the role of the gene product, the sensitivity
and degree of the response may also vary.

CHAPTER 5
CYTOKININ MIMICS AND SUPERSEDES THE SUGAR-INDUCIBILITY OF
MAIZE INVERTASE FAMILY MEMBERS AND FACILITATES THEIR
DIFFERENTIAL RESPONSIVENESS TO ABSCISIC ACID
Introduction
Plant growth regulators often affect many different aspects of plant growth
and development. As an organism becomes more complex, communication between
its different parts requires a signaling system with a progressively greater capacity to
integrate distant messages. Hormonal responses belong to such a communication
system (Libbenga and Mermes, 1987). Much of the signalling in vascular plants is
dependent upon a relatively complex array of hormonal signals.
Sucrose, as the major form in which photoassimilates are transported, and as
such plays a central and vital role in plant life (Avigad, 1982; Hawker, 1985).
Invertase is one of the two known enzymes which can initiate breakdown of this
sucrose for further metabolism in vascular plants. It is thus considered a key
enzyme for carbohydrate partitioning and utilization (Robbins, 1958; Glasziou and
Gayler, 1972; Avigad, 1982; Turgeon, 1989; Sturm and Chrispeels, 1990; Duke et
al, 1991; Miller and Chourey, 1992). Early studies of its activity showed that
upregulation of capacity could be observed in response to abscisic acid, auxin,
cytokinins, and/or gibberellic acid depending on species and conditions. The
86

87
diversity of systems examined included sugarcane stem segments (Sacher et al.,
1963; Glasziou et al., 1966; Gayler and Glasziou, 1969), Avena stem segments
(Kaufman et al., 1973), radish cotyledons (Howard and Witham, 1983), Phaseolus
vulgaris seeds (Morris and Arthur, 1985), soybean seeds (Ackerson, 1985), Citrus
leaves (Schaffer et al., 1987), tobacco crown gall cells (Weil and Rausch, 1990),
etiolated Pisum sativum seedlings (Miyamoto et al., 1993), and shoots of dwarf pea
{Pisum sativum) (Wu et al., 1993).
The present research was motivated by studies which showed that high
invertase activity is usually associated with rapid growth. Action of this enzyme can
provide tissues with not only substrates for their respiratory and synthetic demands
(Morris and Arthur, 1984b; Schaffer et al., 1987), but also elevate turgor for cell
expansion (Avigad, 1982; Schmalstig and Cosgrove, 1988; 1990).
Both cytokinins and ABA are reported to stimulate assimilate translocation
from source to sink (Gersani and Render, 1982; Howard and Witham, 1983; Hein et
al., 1984; Schussler et al., 1984; Ackerson, 1985; Jones et al., 1986; Brokovec and
Prochazka, 1992; Jones et al., 1992). ABA has been called a stress hormone, since
it accumulates during an array of stresses (Chen et al., 1983; Chen and Gusta, 1983;
LaRosa et al., 1985; LaRosa et al., 1987; Davies and Zhang, 1991; Thomas et al.,
1992). Both auxin and gibberellins stimulate cell enlargement, cell elongation and
possibly phototropism and gravitropism (Davies, 1987; Kaufman and Song, 1987;
Kim et al., 1993; Wu et al., 1993a; 1993b).

88
The hypotheses tested here are as follows. Invertase gene expression could
be responsive to ABA (aiding osmoregulation), gibberellins and auxin (aiding
gravitropism and phototropism), and/or cytokinins (aiding sink potential and/or
symbiosis).
In this report, we demonstrate that in maize root tips, both Ivrl and Ivr2
expression for soluble acid invertase genes includes an unexpected, differential
responsiveness to specific hormonal signals. These findings indicate that different
invertase isozymes may have specialized functions in a diverse set of developmental
and/or environmental processes.
Materials and Methods
Plant Material
Zea mays hybrid NK 508 was used for all experiments. Seeds were first
emersed in 20 % Clorox for 30 min. followed by 30 min. of continuous rinsing with
water. Germination took place in the dark at 18 C on two layers of moist 3 MM
paper (Whatman, Inc., Clifton, NJ) in 17 x 26 cm glass pans. Air flowed
continuously at 1 liter min'1 through each pan for the 6-day period, with 40% 02
supplied during the final 24 hr before root tip excision. The moisture level was
adjusted daily by applying mist and draining excess water. Root tips (ca 1 cm each)
were excised under a sterile transfer hood.

89
Experimental Conditions
Experimental treatments were as described by Koch et al. (1992).
Approximately 100 root tips (~ 500 mg) were used for each experimental treatment.
Excised root tips were incubated in the dark at 18 C for 6 to 48 hr in Whites
medium, plus 0.5% glucose, either with or without specific supplemental plant
growth regulators (ABA, A1049; GA, G7645; Kinetin, K0753; IAA, 12886; all from
Sigma). Each group of root tips was agitated at 120 cycles per minute in a 125-ml
side-arm Erlenmeyer flask with 50 ml of sterile media. Airflow (40% 02) through
air stones in each flask was maintained at 250 ml min 1 throughout the incubations.
RNA Isolation and Blot Analysis
Root tip samples were rinsed twice in sterile water, blotted dry, weighed, and
frozen in liquid N2. Samples were ground into fine power in liquid N2 and total
RNA was extracted as per McCarty (1986). RNA was quantified
spectrophotometrically (Sambrook et al., 1989).
Total RNA was separated by electrophoresis in 1 % agarose gels containing
formaldehyde (Thomas, 1980), blotted to a nylon membrane, and fixed by baking
and/or UV treatment (Sambrook et al., 1989). Filters were hybridized at 65 C in a
solution containing 7 % SDS, 250 mM Na2HP04, pH 7.2, 1 % BSA (Church and

90
Gilbert, 1984). Maize Ivrl and lvr2 invertase cDNA clones were radiolabeled by
random primer. No cross-reactivity between Ivrl and Ivr2 gene probes was
observed when hybridizations were conducted at high stringency (data not shown).
Blots were washed as described by Church and Gilbert (1984), exposed to X-ray
film with intensifying screens at -80 C.
Enzyme Extraction
Soluble invertase was extracted as per Duke et al. (1991). Frozen tissue
samples were ground to a fine powder in liquid N2 using a mortar and pestle.
Frozen powder was transferred to a second mortar containing ice-cold 200 mM
HEPES buffer (pH 7.5) with 1 mM DTT, 5mM MgCl2, 1 mM EGTA and 10%(w/w)
PVPP. One ml of extraction buffer was used for every 100 mg of tissue fresh
weight. Buffered extract was centrifuged at 15,000 x g for 10 min to sediment
particulate matter. Pellets were saved for salt-solubilized particulate invertase
extraction. Supernatant was dialyzed (50,000 MW cutoff) at 4 C for 24 hr against
extraction buffer diluted 1:40 (MW cutoff for dialysis was selected to allow escape
of proteinaceous invertase inhibitors [Jaynes and Nelson, 1971b]). Buffer was
changed twice. Soluble dialyzed extract was centrifuged again at 15,000 x g for 10
min. Supernatant was used for soluble invertase assays as described below.
Insoluble invertase was extracted as described by Doehlert and Felker (1987).
Pellets remaining from the above step were washed three times by resuspending in

91
extraction buffer and centrifuging at 15,000 x g for 10 min. Salt-solubilized
particulate invertase was extracted by resuspending the pellet in extraction buffer
containing 1 M NaCl. Solubilized particulate invertase was recovered in supernatant
following centrifugation at 15,000 x g for 10 min. Supernatant was used for
insoluble invertase assays as described below.
Enzyme Assay
Both soluble and salt-solubilized invertase activities were assayed for 15 to
30 min at 37 C in a mixture containing 100 mM Na-acetate (pH 4.5) and 100 mM
sucrose in a final volume of 500 pi. Activity was determined by measuring
reducing sugars with the method of Nelson (1944) and Somogyi (1951).

92
Results
Cytokinin (5 pM kinetin) exposure evoked a positive response at the level of
message abundance for both the Ivrl and Ivr2 gene subfamilies as well as at the
level of total soluble acid invertase enzyme activity (Figure 5-1). The responses of
both gene subfamilies to kinetin were similar (elevated 2.5-fold), and maximal at 5
pM (tested range from 1 to 200 pM, data not shown). The same treatment resulted
in ca 1.5-fold elevation of total soluble acid invertase activity within these 24 hr
experiments (Figure 5-1 B).
The positive responses of the Ivrl and lvr2 gene subfamilies to exogenous
cytokinin were also evident when the excised root tips were depleted of
carbohydrates (Figure 5-1). Kinetin mimicked and superseded the sugar-enhanced
expression of both the Ivrl and Ivr2 classes of invertase. The fact that kinetin could
replace and override carbohydrate supply in this respect was also evident at the level
of total soluble acid invertase activity (Figure 5-1 B).
When root tips were incubated with exogenous ABA (50 pM [which gives
the maximal response range from 1 to 200 pM, data not shown]) for 24 hr, levels of
message encoding both the Ivrl and lvr2 invertase subfamilies were elevated 1.5-
fold and 3-fold respectively (Figure 5-2 A). The Ivrl subgroup responded less
markedly than did its Ivr2 counterpart. Although maximal responses to exogenous
ABA were observed at 50 pM for transcripts from both the Ivrl and Ivr2 invertase

93
classes, only minimal affects were evident at the level of total soluble acid invertase
activity (Figure 5-2 B).
The fact that the elevated levels of mRNA encoding Ivrl and Ivr2
subfamilies did not result in altered enzyme activity raised the question of whether
or not ABA upregulated enzyme activity. The possibility remained that the
responses of soluble invertases to ABA at the translational level might be enhanced
by cytokinins as had been observed for sugar-modulated expression of these genes.
To address this question, ABA incubations were supplemented with low
levels (5 pM) of cytokinin. In figure 5-2, total soluble acid invertase activity was
higher when both ABA and kinetin were added to root tips than it was with kinetin
alone (Figure 5-2 B). The responses of Ivrl type genes to ABA were overridden by
kinetin (Figure 5-2 A). Ivrl related message levels were the same when excised
root tips were treated with either kinetin alone or the combination of kinetin and
ABA. Levels of mRNA from the Ivr2 subgroup, on the other hand, were
significantly higher when both ABA and kinetin were applied to the incubation
solution than when ABA or kinetin were present alone. Elevation of the Ivr2 type
message levels was additive for each single treatment.
The responses of both Ivrl and Ivr2 classes of invertase genes were much
less markedly affected by exogenous GA and/or IAA than they were by ABA and/or
kinetin (Figure 5-3 A). No changes were evident in activity of total soluble acid
invertase in response to root tip incubations with either GA or IAA (Figure 5-3 B).

Figure 5-1. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble acid invertase in maize root tips
incubated for 24 hr in Whites basal salts medium supplemented either with
(+G) or without (-G) 0.5% glucose and either with (+K) or without (-K) 5
pM kinetin. A, RNA gel blots with equal amounts (10 pg) of total RNA
from above tissues were probed with 32P-labeled Ivrl or Ivr2 representing the
two subfamilies of maize soluble acid invertase. Blots were exposed to X-
ray film for 24 or 12 hr, respectively. B, Total soluble acid invertase activity
from the above samples. Insoluble invertase activity (not shown) was
consistently ca 10-fold less than that in the soluble fraction of maize root tips.
Values for RNA/protein recovery were ca 0.15 (+0.06) with variability
independent of +K or -K treatments.

+K '
Invertase activity
(jimol glucose mg1 protein hr1)
o
+
o
o
+
o
o
~T
to
o
CO
o
U3
H
H
H
H
. Soluble activity
7vr2
Probe
# I

m t
v£>
Ln

Figure 5-2. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble acid invertase in maize root tips
incubated for 24 hr in Whites basal salts medium supplemented with 0.5%
glucose (all +G), either with (+K) or without (-K) 5 pM kinetin, either with
(+ABA) or without (-ABA) abscisic acid (50 pM). A, RNA gel blots with
equal amounts (10 pg) of total RNA from above tissues were probed with
32P-labeled Ivrl or Ivr2 representing the two subfamilies of maize soluble
acid invertase. Blots were exposed to X-ray film for 24 or 12 hr,
respectively. B, Total soluble acid invertase activity from the above samples.
Insoluble invertase activity (not shown) was consistently ca 10-fold less than
that in the soluble fraction of maize root tips. Values for RNA/protein
recovery were ca 0.20 (0.07) with variability independent of +K or +ABA
treatments.

-ABA 4-ABA -ABA +ABA
Invertase activity
(nmol glucose mg1 protein hr'1)
ro 4* CT>
o o o o
1 1 r~
Probe

Figure 5-3. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble acid invertase in maize root tips
incubated for 24 hr in Whites basal salts medium supplemented with 0.5%
glucose (all +G), alone (+0) or supplemented with either gibberellic acid
(+GA) or auxin (+IAA). A, RNA gel blots with equal amounts (10 pg) of
total RNA from above tissues were probed with 32P-labeled Ivrl or Ivr2
representing the two subfamilies of maize soluble acid invertase. Blots were
exposed to X-ray film for 24 or 12 hr, respectively. B, Total soluble acid
invertase activity from the above samples. Insoluble invertase activity (not
shown) was consistently ca 10-fold less than that in the soluble fraction of
maize root tips. Values for RNA/protein recovery were ca 0.23 (+0.12) with
variability independent of +GA or +IAA treatments.

+ GA +IAA
Invertase activity
(jimol glucose mg'1 protein hr*1)
ro to
o o o o
. Soluble activity
Probe
mRNA

100
Discussion
The significance of findings presented here is that they indicate different
environmental and/or developmental signals can regulate the same gene expression
through common and/or different pathways. Thus, the same enzyme reaction could
play multiple roles under various conditions. Sugar-modulated genes are also
responsive to plant growth regulators. These responses provide a potential
mechanism by which import organs may adjust their sucrose-metabolizing capacity
to altered environment and/or developmental stages.
Results shown here also indicate that cytokinin has a positive effect on the
invertase gene system and that both mRNA abundance and soluble acid enzyme
activity are up-regulated by exogenous cytokinin (Figure 5-1). Cytokinin is reported
to stimulate translocation of photosynthate from source leaves to cytokinin-treated
areas thus increasing the sink capacity of importing bean leaves (Gersani and
Render, 1982), radish cotyledons (Howard and Witham, 1983), winter wheat grains
(Borkovec and Procharka, 1992) and developing maize kernels (Jones et al., 1992).
During leaf development of snap bean (Phaseolus vulgaris L.) and Citrus,
soluble acid invertase activity is correlated well with leaf expansion. In contrast,
both insoluble invertase and sucrose synthase activities are low and show little
change during leaf development (Morris and Arthur, 1984; Schaffer et al., 1987).
The authors suggest that soluble acid invertase activity is the primary enzyme
responsible for sucrose catabolism in the expanding bean and citrus leaves. Its

101
activity is considered to be the primary determinant of sink strength in these
systems. In addition, early development of maize kernels (Tsai et al., 1970) and
snap bean pods (Sung et ah, 1994) takes place with soluble invertase predominating,
whereas sucrose synthase activity in both cases is below level of detection.
Cytokinins stimulate soluble acid invertase gene expression which subsequently
increases sink potential.
Mycorrhizal fungi and/or rhizobia can only metabolize hexoses. Cytokinins
and/or cytokinin-like substances are reported to be synthesized in mycorrhizal fungi
(Miller, 1967; Crafts and Miller, 1974; Ng et ah, 1982) and Bradyrhizobium
japonicum (Sturtevant and Taller, 1989). Allen et ah (1980) report that cytokinin
level increases in the host plant after infection by vesicular-arbuscular mycorrhizae.
Cytokinins originating from mycorrhizal fungi and/or rhizobia act to increase the
sink capacity through elevating invertase expression from the host plant. The
present work therefore indicates that cytokinin-like substances from fungal and/or
rhizobial symbionts could also act through stimulation of invertase.
Figure 5-1 suggested, first, that cytokinin and sugar enhanced the
accumulation of invertase message level independently. Both Ivrl and Ivr2 mRNA
levels were elevated ca 2.5-fold by cytokinin regardless of exogenous carbohydrate
supply. Sugar alone was unable to stimulate invertase expression to the same degree
as was cytokinin. Second, cytokinin alone was sufficient to promote invertase
enzyme activity to the highest level after 24 hr incubation in this system (Figure 5-
1B).

102
The biological significance of the findings presented here is twofold. First,
under stress conditions severe enough to limit photosynthesis, cytokinins still could
maintain sink potential of specific regions by preserving invertase activity for later
recovery of growth. Second, cytokinins could possibly initiate and/or enhance the
capacity for sucrose import and utilization in a given structure simply by stimulating
invertase expression. Jones et al. (1992) found that cytokinin levels increase
dramatically (as much as 400-fold) during the early stages of maize kernel
development and decline subsequently. They suggest that the ultimate capacity for
sucrose import into kernels is established during this first portion of development,
and further, that de novo biosynthesis of cytokinins within kernels plays a role in
this process through regulation of endosperm and/or nucellar development.
The responsiveness of invertase gene expression to exogenous ABA may also
have a distinct biological relevance (Figure 5-2). ABA is considered a stress
hormone due to its accumulation and action during many such conditions (Chen et
al., 1983; Chen and Gusta, 1983; LaRosa et al., 1985; LaRosa et al., 1987; Davies
and Zhang, 1991; Thomas et al., 1992). Thus, ABA could enhance the capacity of a
plant to acclimate to different stresses, such as freezing, drought and high salt, by
inducing invertase expression. This in turn has the capacity to aid adjustment of
osmotic potential.
ABA is also reported to stimulate transport of photosynthate towards
developing seeds in a number of species (Hein et al., 1984; Schussler et al., 1984;
Ackerson, 1985; Jones et al., 1986; Borkovec and Prochazka, 1992). ABA could

103
facilitate this by elevating invertase activity and enhancing early expansion.
Ackerson (1985) found that soluble acid invertase activity in developing soybean
reproductive structures is correlated well with endogenous ABA levels. Sung et al.
(1994) also observed that invertase predominates during early development of pods.
Data here indicated that both Ivrl and Ivr2 mRNA levels were elevated after
exogenous applications of ABA, which supported the above hypotheses (Figure 5-2
A).
Total soluble acid invertase activity, however, remained unchanged (Figure 5-
2 B). At this point, the mechanism by which exogenous ABA treatment could
elevate invertase message levels without affecting enzyme activity remains unclear.
Simultaneous presence of cytokinin appeared to be necessary before message-level
responses could be transduced at the level of enzyme activity (Figure 5-2).
In general, cytokinins are most abundant in young organs (seeds, fruits and
leaves) and in root tips (Salisbury and Ross, 1992). Endogenous cytokinin levels are
similar in the presence or absence of salt stress in Mesembryanthemum crystallinum
(Thomas et al., 1992). During early development of soybean pods, winter wheat and
maize kernels, both endogenous cytokinin and ABA levels are high (Ackerson,
1985; Borkovec and Prochazka, 1992; Jones et al., 1992). Thus, it is possible that
there are sufficient levels of endogenous cytokinins to allow the ABA-
responsiveness of one or more soluble invertase genes to be extended to the level of
enzyme activity. This could be particularly significant to the physiology of young
reproductive structures under stresses and/or during their earliest development.

104
A further implication here lies in the response of drought-stressed roots. The
ABA effect on osmotic adjustment would be nominal to non-existent unless
cytokinins were present. Because cytokinins are normally carried away in the xylem
stream, a rapidly transpiring plant may have less cytokinins build up in the root tips
than one with stomata closed during drought stress. Further extension of this
scenario could potentially include root to shoot signalling via cytokinin flow and
subsequent effect on shoot soluble invertase (Davies and Zhang, 1991).
Action of soluble invertase has been implicated in gravitropism (Kaufman
and Song, 1987; Kim et al., 1993; Wu et al., 1993a; 1993b) and may also be
involved in some aspects of phototropism (Davies, 1987; Kuafman and Song, 1987).
Elevation of osmotic potential for cell elongation could readily result from invertase
hydrolysis of sucrose within the vacuole, and the asymmetry of this process across a
stem is consistent with the involvement of similarly distributed plant growth
regulators in action of invertase. This suggestion is supported by the observation
that glucose injection into dwarf pea shoots mimics the effect of GA on cell
elongation (Broughton and McComb, 1971).
For this reason, altered expression of Ivrl and/or Ivr2 was expected in
response to treatment with auxin and/or GA. However, neither the invertase
message levels nor assayed enzyme activity were markedly affected by addition of
exogenous auxin and gibberellic acid in the root tip system used here (Figure 5-3).
One explanation might be as follows. Hormones often have pleiotropic effects, i.e.,
different types of target cells all respond to the same set of signals, but in a different

105
way. In many cases, these types of target cells have similar perception-and-
transduction mechanisms, but the molecular programs which are elicited by these
mechanisms are different (Libbenga and Mermes, 1987). Gravitropism in shoots is
brought about by auxin-induced elongation on the long side. In roots, however,
auxin does not induce, but rather inhibits elongation. In figure 5-3, the abundance
of both the Ivrl and Ivr2 type messages was downregulated in response to IAA
treatment, however, the total soluble acid invertase activity remained relatively
constant. One explanation may be the apparent longevity of some invertase proteins.
A need for greater activity of invertase has been implicated in the
establishment and enhancement of the capacity for sucrose import (Morris and
Arthur, 1984; Hanft and Jones, 1986a; Schaffer, 1987). Phytohormone modulation
of invertase genes could effectively regulate the processes associated with phloem
unloading and expansion sink potential (Hein et al., 1984; Schussler et al., 1984;
Ackerson, 1985; Jones et al., 1986; Borkovec and Prochazka, 1992; Jones et al.,
1992).
Genes specifying plant growth regulator biosynthesis have been identified in
phytopathogens (Miller, 1967; Crafts and Miller, 1974; Allen et al., 1980; Ng et al.,
1982; Morris, 1986; Weil and Rausch, 1990). Plant hormone regulation of invertase
expression could facilitate its enhancement, in instances such as establishment of
symbiosis, by providing hexoses for those fungi unable to metabolize sucrose.

CHAPTER 6
SUMMARY AND CONCLUSIONS
Sucrose, as the principle form for assimilate transport, plays a central, vital
role in plant growth and development. Invertase catalyzes one of the only two
enzymatic reactions known capable of breaking down sucrose for further metabolism
in vascular plants, and is thus considered an essential enzyme in the control of
carbohydrate partitioning and utilization. High invertase activity is usually
associated with rapid growth. Soluble forms of this enzyme can be especially
important to cell expansion through generation of hexoses and their associated
osmotic potential. The purpose of this study was to test hypotheses relating
expression of soluble invertases to specific developmental processes, carbohydrate
responses, and/or changes in plant growth regulators.
The first step toward this end was accomplished through isolation of five
cDNA and one genomic clones encoding soluble invertase isozymes in maize.
These were obtained by screening maize root tip and a seedling genomic libraries
with a heterologous tomato soluble invertase (Klann et al., 1992). The deduced
amino acid sequences showed significant identities to previously characterized
invertases. Each of the highly conserved domains existing in other invertases were
106

107
also present in the maize clones examined here. Further, these maize invertases
shared considerably greater identity to soluble invertases than to their cell-wall-
bound counterparts. The maize genes examined here thus probably encode soluble
enzymes. This conclusion was also supported by a strong correlation between the
transcript levels of both Ivrl and Ivr2 subfamilies relative to the total soluble
invertase activities in an array of maize tissues and/or developmental stages.
Ivrl and Ivr2 subfamilies of soluble invertase were mapped to two and four
different loci, respectively, each on a different chromosome (in collaboration with
Scott Wright, Genetic Linkage). One of the Ivrl genes mapped to a region near the
miniature mutation in maize (kernels known to lack insoluble invertase). However,
further analysis showed both Ivrl and Ivr2 message to be present in this mutant line,
as well as wild-type levels of soluble invertase activity. Again, this evidence
supports a soluble invertase identity for the invertase family of genes isolated here.
The maize genomic DNA for Ivrl was found to be organized into seven
exons and six introns. The second exon is only 9 nucleotides long, but encodes the
highly conserved domain found in all the invertases (NDPNG, the P-fructosidase
motif). This 9 bp exon is probably the smallest exon thus for identified in the plant
genome (M. Schuler, personal communication).
The expression of two classes of maize invertase (Ivrl and Ivr2) was further
characterized to test the hypothesis that specific genes might be associated with
different developmental stages and/or enlargement of key sets of cells. The two
invertase subgroups were differentially expressed in roots, sink leaves, young

108
kernels, immature and mature anthers, pollen and silk. A comparison between
transcript and enzyme activity was again consistent with both clones encoding
soluble acid invertases. The spatial and temporal patterns of expression for the two
invertase subfamilies, as well as the contrast between them, demonstrated a close
association between soluble invertases and changes in silk during pollination and in
kernels immediately after fertilization.
Maize root tips in 24 hr culture were used to quantify the extent to which
soluble invertase (at the message and enzyme levels) responded to sugar and specific
developmental/environmental signals. In this system, the abundance of mRNA
levels from both classes was upregulated whenever a source of exogenous, readily-
metabolizable, sugars was made available. The extent of response differed from one
invertase subgroup to another, however. The Ivr2 type genes showed greater
sensitivity to carbohydrate deprivation. The differential responsiveness of invertase
gene subfamilies to carbohydrate availability provided a potential mechanism for
different isozyme gene subgroups to predominate in various tissues, developmental
stages, and/or altered environmental conditions.
Several lines of investigation have suggested invertase gene expression may
be responsive to cytokinins (thus enhancing potential for sucrose metabolism, import
and/or symbiosis), water deficit and/or ABA (aiding osmotic alteration), and
gibberellin or auxin (aiding gravitropism and/or phototropism). Kinetin treatments
(5 pM) increased both message levels (Ivrl and hr 2) and elevated total soluble acid
invertase activity. Kinetin alone replaced and superseded the carbohydrate

109
enhancement for invertase expression. Although mRNA abundance was enhanced
by exogenous ABA, no significant change was evident in total soluble acid invertase
activity, when ABA alone was added. In contrast, treatments combining cytokinin
with ABA, resulted in maximal responses at both transcript levels and enzyme
activity levels. Simultaneous presence of ABA and cytokinin appeared to be
necessary before message-level responses could be transduced at the level of enzyme
activity in this case. Neither the invertase message levels nor assayed enzyme
activity were markedly affected by addition of gibberellic acid or auxin. These
findings provide a potential mechanism by which importing organs may adjust their
sucrose-metabolizing capacity to altered environment and/or developmental stages.

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Schwebel-Pugue, N., Mtili, N.E., Kriritzky, M., Jean-Jacques, I., Williams, J.H.H.,
Thomas, M., Kreis, M. and Lechamy, A. (1994). Arabidopsis gene and
cDNA encoding cell-wall invertase. Plant Physiol. 104: 809-810.
Shannon, J.C. (1968). Carbon-14 distribution in carbohydrates of immature Zea mays
kernel following 14C02 treatment of intact plants. Plant Physiol. 43: 1215-
1220.
Shannon, J.C. (1972). Movement of l4C-labelled assimilates into kernels of Zea mays
L. I. Pattern and rate of sugar movement. Plant Physiol. 49: 198-202.
Shannon, J.C., and Dougherty, T.C. (1972). Movement of l4C-labelled assimilates
into kernels of Zea mays L. II. Invertase activity in the pedicel and placento-
chalazal tissues. Plant Physiol. 49: 203-206.
Shannon, J.C., Knievel, D.P., Chourey, P.S. Liu, S. and Liu, K. (1993).
Carbohydrate metabolism in the pedicel and endosperm of miniature maize
kernels. Plant Physiol. 102:42.
Sheen, J. (1990). Metabolic repression of transcription in higher plants. Plant Cell 2:
1027-1038.
Sheridan, W.F. (1988). Maize developmental genetics: Genes of morphogenesis.
Ann. Rev. Gent. 22: 353-385.
Shirras, A.D. and Northcote, D.H. (1984). Molecular cloning and characterization of
cDNA complementary to mRNAs from wounded potato (Solarium tuberosum)
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Sheu-Hwa, C.S., Lewis, D.H. and Walker, D.A. (1975). Stimulation of
photosynthetic starch formation by sequestration of cytoplasmic
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Shuster, J. R. (1989). Regulated transcriptional systems for the production of
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buttersworth. P.J. Barr, A.J. Brike, and P. Valenzuela, eds, London,
Butterworths, pp.83-108.
Singh, M.B., and Knox, R.B. (1984). Invertase of Lilium pollen. Characterization
and activity during in vitro germination. Plant Physiol. 74, 510-515.

125
Skriver, K., Olsen, F.L., Rogers, J.C. and Mundy, J. (1991). cis-Action DNA
elements responsive to gibberellin and its antagonist abscisic acid. Proc. Natl.
Acad. Sci. USA 88: 7266-7270.
Somogyi, M. (1951). Notes on sugar determination. J. Bio. Chem. 181: 19-23.
Sturm, A., and Chrispeels, M.J. (1990). cDNA cloning of carrot extracellular P-
fructosidase and its expression in response to wounding and bacterial
infection. Plant Cell 2, 1107-1119.
Sturtevant, D.B., and Taller, B.J. (1989). Cytokinin production by Bradyrhizobium
japonicum. Plant Physiol. 89: 1247-1251.
Sung, S.S., Sheih, W.J., Geiger, D.R. and Black, C.C. (1994). Growth, sucrose
synthase, and invertase activities of developing Phaseolus vulgaris L. fruits.
Plant Cell and Environ. 17
Thomas, J.C., McElwain, E.F. and Bohnert, H.J. (1992). Convergent induction of
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Plant Physiol. 100: 416-423.
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306.
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Plant Mol. Biol. 40: 119-138.
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carrot (Daucus catota) soluble acid 13-fructofuranosidases and comparing with
the cell wall isoenzyme. Plant Physiol. 104: 1351-1357.
von Heijne, G. (1986). A new method for predicting signal sequence cleavage sites.
Nucleic Acids Res. 14: 4631-4690.
von Schaewen, A., Stitt, M., Schmidt, R., Sonnewald, U. and Willmitzer, L. (1990).
Expression of yeast-derived invertase in the cell wall of tobacco and
Arabidopsis plants leads to accumulation of carbohydrate and inhibition of

126
photosynthesis and strongly influences growth and phenotype of transgenic
tobacco plants. EMBO J. 9: 3033-3044.
Walker, J.C. and Key, J.L. (1982). Isolation of cloned cDNAs to auxin-responsive
poly(A)+RNAs of elongating soybean hypocotyl. EMBO J. 9: 3033-3044.
Walton, D.C., Harrison, M.A., and Cote, P. (1976). The effects of water stress on
abscisic-acid levels and metabolism in roots of Phaseolus vulgaris L. and
other plants. Planta 131: 141-144.
Webster, P.L. (1980). "Stress" protein synthesis in pea root meristem cells? Plant
Sci. Letters 20, 141-145.
Webster, P.L., and Henry, M. (1987). Sucrose regulation of protein synthesis in pea
root meristerm cells. Env. Exp. Bot. 27, 253-262.
Weil, M., Kraisgrill, S., Schuster, A. and Rausch, T. (1994). A 17-kDa Nicotiana
tabacum cell-wall peptide acts as an in-vitro inhibitor of the cell-wall isoform
of acid invertase. Planta 193: 438-445.
Weil, M., and Rausch,. T. (1990). Cell wall invertase in tobacco crown gall cells.
Enzyme properties and regulation by auxin. Plant Physiol. 94, 1575-1581.
Wenzler, H.C., Mignery, G.A., Fisher, L.M., and Park, W.D. (1989). Analysis of a
chimeric class-I patatin-GUS gene in transgenic potato plants: High-level
expression in tubers and sucrose-inducible expression in cultured leaf and
stem explants. Plant Mol. Biol. 12, 41-50.
Wu, L.L., Mitchell, J.P., Cohn,N.S. and Kaufman, P.B. (1993). Gibberellin (GA3)
enhances cell wall invertase activity and mRNA levels in elongating dwarf
pea (Pisum sativum) shoots. Int. Plant Sci. 154(2): 280-289.
Wu, L.L., Song,I. Karuppiah, N. and Kaufman, P.B. (1993). Kinetic induction of
oat shoot pulvinus invertase mRNA by gravistimulation and partial cDNA
cloning by the polymerase chain reaction. Plant Mol. Biol. 21(6): 1175-1179.
Zoeger, D., Scholle, C., Schroder-Lorenz, A., Techel, D., and Rensing, L. (1992).
Some starvation-induced proteins in Neurospora crassa are related to
glucose-regulated proteins. Exp. Mycology 16, 138-145.
Zwar, J.A. and Hooley, R. (1986). Hormonal regulation of a-amylase gene
transcription in wild oat (Avena fatua L) aleurone proplasts. Plant Physiol.
80: 459-463.

BIOGRAPHICAL SKETCH
Jian Xu was bom in Changzhou City, Jiangsu Province, China on May 26,
1963. He attended Nanjing University from 1982 to 1986, where he received his
Bachelor of Science in plant biochemistry and plant physiology.
Jian Xu entered the Shanghai Institute of Plant Physiology, Academia Snica
in September 1986 and received his Master of Science degree in the Plant
Biochemistry Division in August, 1989. In January 1991, Jian Xu began his
program of study for the Doctor of Philosophy degree in the Plant Molecular and
Cellular Biology Program at the University of Florida, working under the direction
of Dr. Karen Koch. He plans to continue in plant science as a postdoctoral research
associate in the laboratory of Dr. Elaine Tobin at the University of California, Los
Angeles. He is the member of American Society of Plant Physiologists and the
Maize Genetics Cooperative.
127

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
^aren E. Koch, Chair
Professor of Plant Molecular
and Cellular Biology Program
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Donald Rf Mc(
Associate Professor of
Horticultural Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
L. Curtis Hannah
Professor of Horticultural
Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
OJU.C \\
Alice C. Harmon
Assistant Professor of Botany
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Kenneth J. Boote
Professor of Agronomy

This dissertation was submitted to the Graduate Faculty of the College of
Agriculture and to the Graduate School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.
December, 1994
j.Jyy
Dean, College of
Agriculture
Dean, Graduate School



12
these mechanisms may comprise a means for sensing and transducing signals of
whole plant carbohydrate status, and subsequently altering plant metabolism and/or
development.
Particularly important in such a possibility are the genes encoding those
enzymes which can break down sucrose, sucrose synthase and invertase. Sugar
responsiveness of the former has been characterized (Salanoubat and Belliard, 1989;
Maas et al., 1990; Koch et al., 1992). Although invertase clones have not been
previously characterized, levels of these enzymes appear to show a long-term,
carbohydrate responsiveness (Sacher et al., 1963; Glasziou et al., 1966; Ricardo et
al., 1972; Kaufman et al., 1973; Sarokin and Carlson, 1984).
Kaufman et al. (1973) demonstrated that increased levels of invertase were
correlated with the sustained growth of Avena stem segments in the presence of
sucrose. Their data further indicated that the presence of sucrose greatly enhanced
the GA effect on elevation of invertase activity. They suggested that substrate may
stabilize the enzyme and/or aid its induction. Their studies also support the view
that gibberellic acid, as well as substrate (sucrose) and end products (glucose and
fructose), play a significant role in regulating invertase levels in Avena stem tissue.
Moreover, such regulation could provide a mechanism for increasing the level of
soluble saccharides needed for gibberellic acid-promoted growth.
However, Morris and Arthur (1984a) documented a drop in concentration of
hexose sugars in internodal segments of Phaseolus vulgaris during incubation in the
presence of auxin. The greatest decline in hexose concentrations occurred when


5
The physiological significance of invertase action has been debated over a
considerable period of time. Work with sugarcane stems (Hawker and Hatch, 1965;
Glasziou and Gayler, 1972) and com kernels (Shannon, 1968; Shannon, 1972;
Shannon and Dougherty, 1972) indicated that imported sucrose moved from phloem
into the extracellular space where it was hydrolyzed by a cell-wall-bound, acid
invertase. This was presumed to contribute to a sucrose concentration gradient
between the phloem and apoplast, enhancing the rate of sucrose transfer into sucrose
utilizing tissues. More recent evidence is also consistent with the initial hypothesis
(Lin et al., 1984; Doehlert, 1986; Doehlert and Felker, 1987; Doehlert et al., 1988;
Doehlert, 1990 ). Nonetheless, work done by other groups indicates that sucrose can
be transported into Zea mays L. endosperm without invertase hydrolysis (Hitz et al.,
1985; Cobb and Hannah, 1986; Schmalstig and Hitz, 1987; Cobb and Hannah,
1988). It is possible that sucrose can move into sucrose-utilizing tissues of maize
kernels by both mechanisms.
Soluble acid invertase activity is closely correlated with leaf expansion in
bean {Phaseolus vulgaris L.) and Citrus whereas sucrose synthase activity is minimal
and fairly constant (Morris and Arthur, 1984; Schaffer et al., 1987). The authors
suggest that soluble acid invertase activity is the primary enzyme responsible for
sucrose catabolism in the expanding bean and citrus leaves. Its activity is
considered to be the primary determinant of sink potential in these systems.
Invertase also predominates over sucrose synthase (which is barely
detectable) in the earliest stages of development for maize kernels (Tsai et al., 1970;


41
gene expression (Dickinson et al., 1988; Baumlein et al., 1992; Lelievre et al.,
1992). This suggestion is also supported by the preferential expression of the Ivrl
subfamily genes in reproductive tissues (see Chapter 4).
The polypeptide encoded by the IvrlG invertase gene has 670 residues with a
molecular weight of 71,942 (Figure 3-3). The calculated isoelectric point is 7.5,
which is intermediate between that of carrot soluble invertases (SI: 3.8; SII: 5.7,
Unger et al, 1994) and insoluble invertases (carrot CW: 9.9, Sturm and Chrispeels,
1990; Arabidopsis CW: 9.1, Schwebel-Dugue et al., 1994). This protein also
contains five putative glycosylation sites (N-X-S/T) and a potential peptide signal
from M1 to A73 (Figure 3-3).


126
photosynthesis and strongly influences growth and phenotype of transgenic
tobacco plants. EMBO J. 9: 3033-3044.
Walker, J.C. and Key, J.L. (1982). Isolation of cloned cDNAs to auxin-responsive
poly(A)+RNAs of elongating soybean hypocotyl. EMBO J. 9: 3033-3044.
Walton, D.C., Harrison, M.A., and Cote, P. (1976). The effects of water stress on
abscisic-acid levels and metabolism in roots of Phaseolus vulgaris L. and
other plants. Planta 131: 141-144.
Webster, P.L. (1980). "Stress" protein synthesis in pea root meristem cells? Plant
Sci. Letters 20, 141-145.
Webster, P.L., and Henry, M. (1987). Sucrose regulation of protein synthesis in pea
root meristerm cells. Env. Exp. Bot. 27, 253-262.
Weil, M., Kraisgrill, S., Schuster, A. and Rausch, T. (1994). A 17-kDa Nicotiana
tabacum cell-wall peptide acts as an in-vitro inhibitor of the cell-wall isoform
of acid invertase. Planta 193: 438-445.
Weil, M., and Rausch,. T. (1990). Cell wall invertase in tobacco crown gall cells.
Enzyme properties and regulation by auxin. Plant Physiol. 94, 1575-1581.
Wenzler, H.C., Mignery, G.A., Fisher, L.M., and Park, W.D. (1989). Analysis of a
chimeric class-I patatin-GUS gene in transgenic potato plants: High-level
expression in tubers and sucrose-inducible expression in cultured leaf and
stem explants. Plant Mol. Biol. 12, 41-50.
Wu, L.L., Mitchell, J.P., Cohn,N.S. and Kaufman, P.B. (1993). Gibberellin (GA3)
enhances cell wall invertase activity and mRNA levels in elongating dwarf
pea (Pisum sativum) shoots. Int. Plant Sci. 154(2): 280-289.
Wu, L.L., Song,I. Karuppiah, N. and Kaufman, P.B. (1993). Kinetic induction of
oat shoot pulvinus invertase mRNA by gravistimulation and partial cDNA
cloning by the polymerase chain reaction. Plant Mol. Biol. 21(6): 1175-1179.
Zoeger, D., Scholle, C., Schroder-Lorenz, A., Techel, D., and Rensing, L. (1992).
Some starvation-induced proteins in Neurospora crassa are related to
glucose-regulated proteins. Exp. Mycology 16, 138-145.
Zwar, J.A. and Hooley, R. (1986). Hormonal regulation of a-amylase gene
transcription in wild oat (Avena fatua L) aleurone proplasts. Plant Physiol.
80: 459-463.


CHAPTER 6
SUMMARY AND CONCLUSIONS
Sucrose, as the principle form for assimilate transport, plays a central, vital
role in plant growth and development. Invertase catalyzes one of the only two
enzymatic reactions known capable of breaking down sucrose for further metabolism
in vascular plants, and is thus considered an essential enzyme in the control of
carbohydrate partitioning and utilization. High invertase activity is usually
associated with rapid growth. Soluble forms of this enzyme can be especially
important to cell expansion through generation of hexoses and their associated
osmotic potential. The purpose of this study was to test hypotheses relating
expression of soluble invertases to specific developmental processes, carbohydrate
responses, and/or changes in plant growth regulators.
The first step toward this end was accomplished through isolation of five
cDNA and one genomic clones encoding soluble invertase isozymes in maize.
These were obtained by screening maize root tip and a seedling genomic libraries
with a heterologous tomato soluble invertase (Klann et al., 1992). The deduced
amino acid sequences showed significant identities to previously characterized
invertases. Each of the highly conserved domains existing in other invertases were
106


Figure 3-2. Schematic diagram of the genomic organization of the IvrlG gene for soluble invertase from Zea mays L..
A, The entire IvrlG gene for soluble invertase and bordering regions is depicted with exons as solid boxes. The
locations of a putative CAAT box, TATA box, translation start (ATG), translation stop (TGA) are designated
with arrows. B, Enlarged area from A, which encodes the most strongly conserved region among all invertase
sequences (NDPNG, the P-fructosidase motif) (Sturm and Chrispeels, 1990).


109
enhancement for invertase expression. Although mRNA abundance was enhanced
by exogenous ABA, no significant change was evident in total soluble acid invertase
activity, when ABA alone was added. In contrast, treatments combining cytokinin
with ABA, resulted in maximal responses at both transcript levels and enzyme
activity levels. Simultaneous presence of ABA and cytokinin appeared to be
necessary before message-level responses could be transduced at the level of enzyme
activity in this case. Neither the invertase message levels nor assayed enzyme
activity were markedly affected by addition of gibberellic acid or auxin. These
findings provide a potential mechanism by which importing organs may adjust their
sucrose-metabolizing capacity to altered environment and/or developmental stages.


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103
facilitate this by elevating invertase activity and enhancing early expansion.
Ackerson (1985) found that soluble acid invertase activity in developing soybean
reproductive structures is correlated well with endogenous ABA levels. Sung et al.
(1994) also observed that invertase predominates during early development of pods.
Data here indicated that both Ivrl and Ivr2 mRNA levels were elevated after
exogenous applications of ABA, which supported the above hypotheses (Figure 5-2
A).
Total soluble acid invertase activity, however, remained unchanged (Figure 5-
2 B). At this point, the mechanism by which exogenous ABA treatment could
elevate invertase message levels without affecting enzyme activity remains unclear.
Simultaneous presence of cytokinin appeared to be necessary before message-level
responses could be transduced at the level of enzyme activity (Figure 5-2).
In general, cytokinins are most abundant in young organs (seeds, fruits and
leaves) and in root tips (Salisbury and Ross, 1992). Endogenous cytokinin levels are
similar in the presence or absence of salt stress in Mesembryanthemum crystallinum
(Thomas et al., 1992). During early development of soybean pods, winter wheat and
maize kernels, both endogenous cytokinin and ABA levels are high (Ackerson,
1985; Borkovec and Prochazka, 1992; Jones et al., 1992). Thus, it is possible that
there are sufficient levels of endogenous cytokinins to allow the ABA-
responsiveness of one or more soluble invertase genes to be extended to the level of
enzyme activity. This could be particularly significant to the physiology of young
reproductive structures under stresses and/or during their earliest development.


This dissertation was submitted to the Graduate Faculty of the College of
Agriculture and to the Graduate School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.
December, 1994
j.Jyy
Dean, College of
Agriculture
Dean, Graduate School


Figure 4-8. Abundance of mRNA from the hr l and Ivr2 subfamilies of soluble acid
invertase in maize root tips incubated for 24 hr in Whites basal salts medium
supplemented with either 0, 0.2, 0.5, 2.0, 4.0% glucose or sucrose. RNA gel
blots with equal amounts (10 pg) of total RNA from above tissues were
probed with 32P-labeled Ivrl or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively.


CHAPTER 1
INTRODUCTION
Sucrose is the most abundant long-distance transport carbohydrate in the plant
kingdom. As such, it plays a central and vital role in plant growth and development.
In vascular plants there are two known enzymatic reactions that can breakdown this
sucrose. These are catalyzed by invertase and sucrose synthase.
Invertase is often considered an essential enzyme for carbohydrate
metabolism and partitioning because of the nearly ubiquitous role of sucrose in
photoassimilate translocation (Avigad, 1982; Hawker, 1985; Turgeon, 1989). This is
supported by the observation that invertase deficient kernels of the miniature-1
maize mutant develop abnormally in addition to their reduced size (Miller and
Chourey, 1992). Further, primary root tips of another invertase deficient maize
mutant, OH43, can not grow normally on sucrose agar (Robins, 1958; Duke et al.,
1991).
Invertase activity is widely distributed within and among vascular plants.
Several isoforms of invertase often can be present simultaneously in a given plant
and/or organ. However, the roles of the individual isoforms are not well understood.
Early work with maize kernels (Shannon, 1972; Shannon and Dougherty,
1972; Doehlert and Felker, 1987; Doehlert et al., 1988) indicated that imported
sucrose moved from phloem into the extracellular space where it was hydrolyzed by
a cell-wall-bound, acid invertase. This was presumed to contribute to a sucrose
1


81
support of high respiratory rates and/or compartmentalized in vacuoles to maintain
turgor for cell expansion. Second, invertase is demonstrated to contribute to an in
vitro chemotropism of pearl millet pollen tubes toward stigmatic tissue through its
production of glucose (Reger et al., 1992a; 1992b; 1993). Although other factors,
such as calcium and an ovarian protein, are also important for the chemotropic
response of pearl millet pollen tubes, our results were consistent with an invertase
role in forming gradients of hexoses for pollen growth in maize. The message
gradient of Ivrl and for2 abundance along the length of the silk (Figure 4-6) further
supported the concept that glucose produced by invertase may be at least one key
factor underlying the chemotropic response of pollen tubes, and their pathway
towards the ovule.
Genes encoding carbohydrate metabolizing enzymes are regulated at the
transcriptional level by sugar availability in yeast and vascular plants (Carlson, 1987;
Schuster, 1989; Maas et al., 1990; Koch et al., 1992). Invertase, one of the only two
enzymes known with a capacity to breakdown sucrose in vascular plants (Avigad,
1982), is also shown here to be regulated at the message level by sugar availability.
Further, invertase gene subfamilies are found to be differentially expressed even
within the same organ. The potential exists for these isozyme gene subfamilies to
confer particular biological advantage through their presence in specific tissue at
various stages of development and/or altered environmental conditions.
Photosynthesis and carbohydrate availability are often greatly reduced under
environmental stresses, such as drought, flooding, severe cold and/or insect attack.


24
Table 3-1. Percentage comparative sequence similarity shared at the amino acid
level between genomic and cDNA clones for maize soluble invertases (IvrlG and
Ivr2) and those of other invertases from vascular plants
Amino Acid Identity (%)
IvrlG
Ivr2
Tomato3 (S)b
61
48
Mung Bean0 (S)
59
56
Carrotd (SI)
59
49
Carrotd (SII)
59
49
Carrot6 (CWI)f
42
32
Carrote(CWII)f
45
32
IvrlG
100
53
Ivr2
53
100
Tomato soluble invertase (Klann et al., 1992).
bS represents soluble isoform for invertase.
cMung bean soluble invertase (Arai et al., 1992).
dCarrot soluble invertases (Unger et al., 1994)
eCarrot insoluble invertases (Sturm and Chrispeels, 1990; Sturm, unpublished data).
fCW represents cell-wall-bound (insoluble) invertase.


Invertase activity Probe
(jimol glucose mg'1 protein hr'1)
Ivrl
Ivr2
60
40
20
20
A. mRNA
Days -/+ pollination (fert)
-2 -1 0 +1 +2
(-3) (-2) (-1) (0) (+1)
!

B. Soluble activity
- T
X
X
InX
C. Insoluble activity
X
X
-2 -1 0 +1 +2
(-3) (-2) (-1) (0) (+1)
Days -/+ pollination (fert)


Figure 3-4. The hydropathy and folding values of the deduced polypeptide for
maize invertase gene 1. A, hydropathy. B, folding structure. The dashed
lines indicate the putative signal peptide cleavage site.


122
Quatrano, R.S. (1987). The role of hormones during seed development. In P.J.
Davies, ed. Plant hormones and their role in plant growth and development.
Dordrecht, Martinus Nijhoff Publisher, pp 494-514.
Ramloch-Lorenz, K., Knudsen, S., and Sturm, A. (1993). Molecular characterization
of gene for carrot cell wall p-fructosidase. Plant J. 4(3): 545-554.
Rebeille, F., Bligny, R., Martin, J.B., and Douce, R. (1985). Effect of sucrose
starvation on sycamore (Acer pseudoplatanus) cell carbohydrate and P, status.
Biochem. J. 226: 679-684.
Reed, A.J., and Singletary, G.W. (1989). Roles of carbohydrate supply and
phytohormones in maize kernel abortion. Plant Physiol. 91, 986-992.
Reger, B.J., Chaubal, R. and Pressey, R. (1992a). Chemotropic responses by pearl
millet pollen tubes. Sex. Plant Report 5: 47-56.
Reger, B.J., Chaubal, R. and Pressey, R. (1993). Chemotropism by pearl millet
pollen tubes. Plant Physiol. 102: 120.
Reger, B.J., Pressey, R. and Chaubal, R. (1992b). In vitro chemotropism of pearl
millet pollen tubes to stigma tissue: a response to glucose produced in the
medium by tissue-bound invertase. Sex. Plant Report 5: 201-205.
Ricardo, C.P.P., ap Rees, T., and Fuller, W.A. (1972). Effects of sugars on invertase
activity of carrot cells. Phytochemistry 11: 2435-2436.
Robbins, W.J. (1958). Sucrose and growth of excised roots of an inbred Zea mays.
Proc. N. A. S. 44, 1210-1212.
Rocha-Saso, M., Sonnewald, U., Frommer, W., Stratmann, M., Schell, J., and
Willmitzer, L. (1989). Both developmental and metabolic signals activate the
promoter of a class I patatin gene. EMBO J. 8, 23-29.
Sacher, J.A., Hatch, M.D., and Glasziou, K.T. (1963). Regulation of invertase
synthesis in sugar cane by an auxin- and sugar-mediated control system.
Physiol. Plantarum 16: 836-842.
Sadka, A., DeWald, D.B., May, G.D., Park, W.D. and Mullet, J.E. (1994).
Phosphate modulates transcription of soybean VspB and other sugar-inducible
genes. Plant Cell. 6: 737-749.
Saglio, P.H., and Pradet, A. (1980). Soluble sugars, respiration, and energy charge
during aging of excised maize root tips. Plant Physiol. 66: 516-519.


Figure 5-1. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble acid invertase in maize root tips
incubated for 24 hr in Whites basal salts medium supplemented either with
(+G) or without (-G) 0.5% glucose and either with (+K) or without (-K) 5
pM kinetin. A, RNA gel blots with equal amounts (10 pg) of total RNA
from above tissues were probed with 32P-labeled Ivrl or Ivr2 representing the
two subfamilies of maize soluble acid invertase. Blots were exposed to X-
ray film for 24 or 12 hr, respectively. B, Total soluble acid invertase activity
from the above samples. Insoluble invertase activity (not shown) was
consistently ca 10-fold less than that in the soluble fraction of maize root tips.
Values for RNA/protein recovery were ca 0.15 (+0.06) with variability
independent of +K or -K treatments.


+ GA +IAA
Invertase activity
(jimol glucose mg'1 protein hr*1)
ro to
o o o o
. Soluble activity
Probe
mRNA


root tips
sink leaf
H
source leaf
prop root
anthers
silk
H
kernel (2Dap>
}
Invertase activity
(pmol glucose mg'1 protein hr'1)
. Soluble activity
Probe
root tips
sink leaf
source leaf
prop root
anthers
>
3
33
Z
>
silk
kernel (2dap>
Ln
Ln


118
Klann, E., Chetelat, R.T. and Bennett, A.B. (1993). Expression of acid invertase
gene controls sugar composition in tomato (Lycopersicon) fruit. Plant Physiol.
103: 863-870.
Klann, E., Yelle, S., and Bennett, A.B. (1992). Tomato fruit acid invertase
complementary DNA. Nucleotide and deduced amino acid sequences. Plant
Physiol. 99, 351-353.
Knox, J.P., and Galfre, G. (1986). Use of monoclonal antibodies to separate the
enantiomers of abscisic acid. Analytical Biochem. 155: 92-94.
Koch, K.E., Nolle, K.D., Duke, E.D., McCarty, D.R., and Avigne, W.T. (1992).
Sugar levels modulate differential expression of maize sucrose synthase
genes. Plant Cell 4, 59-69.
Krishnan, H.B., Blanchette, J.T., and Okita, T.W. (1985). Wheat invertases.
Characterization of cell wall-bound and soluble forms. Plant Physiol. 78, 241 -
245.
Krishnan, H.B., and Pueppke, S.G. (1988). Invertases from rust-infected wheat
leaves. J. Plant Physiol. 133, 336-339.
Lachno, D.R., and Baker, D.A. (1986). Stress induction of abscisic acid in maize
roots. Physiol. Plantarum 68: 215-221.
Lanahan, M.B., Ho, T.H.D., Rogers, S.W. and Rogers, J.C. (1992). A gibberellin
response complex in cereal a-amylase gene promoters. Plant Cell 4: 203-211.
LaRosa, P.C., Handa, A.K., Hasegawa, P.M. and Bressan, R.A. (1985). Abscisic
acid accelerates adaption of cultured tobacco cells to salt. Plant Physiol. 79:
138-142.
LaRosa, P.C., Hasegawa, P.M., Rhodes, D., Clithero, J.M., Watad, A.E.A. and
Bressan, R.A. (1987). Abscisic acid stimulated osmotic adjustment and its
involvement in adaptation of tobacco cells to NaCl. Plant Physiol. 85: 174-
181.
Leliever, J.M., Oliveira, L.O. and Nielson, N.C. (1992). 5-CATGCAT-3 elements
modulate the expression of glycinin genes. Plant Physiol. 98: 387-391.
Li, Y., Hagen, G. and Guilfoyle, T.J. (1991). An auxin-responsive promoter is
differentially induced by auxin gradients during tropisms. Plant Cell 3: 1167-
1175.


71
starvation (hr)
6 12 18 24 36 48
Iwl f || .
Ivr2


of pearl millet pollen tubes toward stigmatic tissue through its production of glucose
(Reger et al., 1992a; 1992b; 1993).
A number of different genes may be involved in these processes in vascular
plants (Sturm and Chrispeels, 1990; Arai et ah, 1992; Klann et ah, 1992; Elliott et
ah, 1993; Ramloch-Lorenz et ah, 1993; Schwebel-Dugue et ah, 1994; Unger et ah,
1994). In yeast, one gene encodes both cell-wall-bound and soluble invertases
through differential splicing (Carlson and Botstein, 1982). However, in carrot at
least seven different invertase genes have been distinguished (A. Sturm, personal
communication).
Regulation of Invertase Expression
Invertase and Its Endogenous Inhibitors
Proteinaceous invertase inhibitors are found in an array of vascular plants
(Pressey, 1966; 1967; 1968; Jaynes and Nelson, 1971b; Matsushita and Uritani,
1976; Bracho and Whitaker, 1990a; 1990b; Isla et ah, 1992; Weil et ah, 1994). In
Solanum tuberosum they are located in the vacuole (Bracho and Whitaker, 1990a;
1990b; Isla et ah, 1992), whereas in Nicotiana tabacum they are in the extracellular
space (Weil et ah, 1994). These inhibitors bind tightly and specifically to acid
invertase and have molecular weights ranging from 17 to 23 KDa (Bracho and
Whitaker, 1990b; Weil et ah, 1994). However, it remains unclear by what


93
classes, only minimal affects were evident at the level of total soluble acid invertase
activity (Figure 5-2 B).
The fact that the elevated levels of mRNA encoding Ivrl and Ivr2
subfamilies did not result in altered enzyme activity raised the question of whether
or not ABA upregulated enzyme activity. The possibility remained that the
responses of soluble invertases to ABA at the translational level might be enhanced
by cytokinins as had been observed for sugar-modulated expression of these genes.
To address this question, ABA incubations were supplemented with low
levels (5 pM) of cytokinin. In figure 5-2, total soluble acid invertase activity was
higher when both ABA and kinetin were added to root tips than it was with kinetin
alone (Figure 5-2 B). The responses of Ivrl type genes to ABA were overridden by
kinetin (Figure 5-2 A). Ivrl related message levels were the same when excised
root tips were treated with either kinetin alone or the combination of kinetin and
ABA. Levels of mRNA from the Ivr2 subgroup, on the other hand, were
significantly higher when both ABA and kinetin were applied to the incubation
solution than when ABA or kinetin were present alone. Elevation of the Ivr2 type
message levels was additive for each single treatment.
The responses of both Ivrl and Ivr2 classes of invertase genes were much
less markedly affected by exogenous GA and/or IAA than they were by ABA and/or
kinetin (Figure 5-3 A). No changes were evident in activity of total soluble acid
invertase in response to root tip incubations with either GA or IAA (Figure 5-3 B).


Benhamou, N., Grenier, J., and Chrispeels, M.J. (1991). Accumulation of P-
fructosidase in the cell walls of tomato roots following infection by a fungal
wilt pathogen. Plant Physiol. 97, 739-750.
Bernier, G., Havelange, A., Houssa, C., Petitjean, and Lejeune, P. (1992).
Physiological signals that induce flowering. Plant Cell 5: 1147-1155.
Billett, E.E., Billett, M.A. and Burnett, J.H. (1977). Stimulation of maize invertase
activity following infection by Ustilago mayis. Phytochemistry 16: 1163-
1166.
Billett, E.E. and Burnett, J.H. (1978). The host-parasite physiology of the maize
smut fungus, Ustilago mayis II. Translocation of ,4C-labelled assimilates in
smutted plant. Physiol. Plant Patho. 12: 103-112.
Borkovec, V. and Prochazka, S. (1992). Interaction of cytokinins with ABA in the
transport of 14C-sucrose in the developing kernels of winter wheat. In M.
Kaminek, D.W.S. Mok and E. Zazimalova, ed. Physiology and biochemistry
of cytokinins in plants. The Hague, SPB Academic Publishing, pp 229-234.
Boyer, C.D., and Shannon, J.C. (1986). Carbohydrate utilization in maize kernels. In
Regulation of carbon and nitrogen reduction and utilization in maize. J.C.
Shannon, D.P. Knievel and C.D. Boyer, eds, Maryland, American Society of
Plant Physiologists, pp. 149-158.
Bracho, G.E., and Whitaker, J.R. (1990a). Charateristics of the inhibition of potato
(Solarium tuberosum) invertase by an endogenous proteinaceous inhibitor in
potatoes. Plant Physiol. 92, 381-385.
Bracho, G.E., and Whitaker, J.R. (1990b). Purification and partial characterization of
potato (Solarium tuberosum) invertase and its endogenous proteinaceous
inhibitor. Plant Physiol. 92, 386-394.
Bray, E.A. (1988). Drought- and ABA-induced changes in polypeptide and mRNA
accumulation in tomato leaves. Plant Physiol. 88: 1210-1214.
Brenner, M.L. (1987). The role of hormones in photosynthate partitioning and seed
filling. In P.J. Davies, ed, Plant hormones and their role in plant growth and
development. Dordrecht, Martinus Nijhoff Publishers, pp 474-493.
Broughton, W.J. and McComb, A.J. (1974). Changes in the pattern of enzyme
development in gibberellin-treated pea intemodes. Ann. Bot. 35:213-228.
Ill


80
indicated that significant amounts of both Ivrl and Ivr2 type message were present
in the anther tissues collectively (Figure 4-5).
Second, invertase message in pollen may be important for subsequent
germination, to facilitate use of endogenous as well as exogenous sucrose. Sucrose
represents the major soluble sugar present in the majority of angiosperm pollen
grains, including maize. Mature pollen grains from diverse plants contain sucrose
but not starch as reserve carbohydrate (Portnoi and Horovitz, 1977; Nakamura et al.,
1980). Germinating pollen grains show an extremely high rate of growth and thus
have a high demand for carbon skeletons required for pollen tube wall synthesis as
well as substrates for respiration (Hoekstra, 1983; Singh and Knox, 1984). The
sucrose content from pollen grains of Camellia japnica decreases rapidly during
pollen growth and the activity of soluble invertase increases during culturing and a
high constant activity is found at the later stages of pollen tube growth (Nakamura et
al., 1980). Our data are consistent with the possibility that invertase has multiple
functions during anther development and pollen grain maturation (Figure 4-5).
Further localization of invertase at the message and/or protein level in situ could
help clarify the functions of invertase in reproductive processes.
The association between high levels of both invertase activity and message
levels with rapidly elongating styles (silk) in maize may have a twofold biological
implication. First, invertase, as one of the important constituents of sink strength
(Morris and Arthur, 1984; Schaffer et al., 1987), can provide an important avenue
for sucrose cleavage. Resulting hexoses can either be subsequently metabolized in


CHAPTER 5
CYTOKININ MIMICS AND SUPERSEDES THE SUGAR-INDUCIBILITY OF
MAIZE INVERTASE FAMILY MEMBERS AND FACILITATES THEIR
DIFFERENTIAL RESPONSIVENESS TO ABSCISIC ACID
Introduction
Plant growth regulators often affect many different aspects of plant growth
and development. As an organism becomes more complex, communication between
its different parts requires a signaling system with a progressively greater capacity to
integrate distant messages. Hormonal responses belong to such a communication
system (Libbenga and Mermes, 1987). Much of the signalling in vascular plants is
dependent upon a relatively complex array of hormonal signals.
Sucrose, as the major form in which photoassimilates are transported, and as
such plays a central and vital role in plant life (Avigad, 1982; Hawker, 1985).
Invertase is one of the two known enzymes which can initiate breakdown of this
sucrose for further metabolism in vascular plants. It is thus considered a key
enzyme for carbohydrate partitioning and utilization (Robbins, 1958; Glasziou and
Gayler, 1972; Avigad, 1982; Turgeon, 1989; Sturm and Chrispeels, 1990; Duke et
al, 1991; Miller and Chourey, 1992). Early studies of its activity showed that
upregulation of capacity could be observed in response to abscisic acid, auxin,
cytokinins, and/or gibberellic acid depending on species and conditions. The
86


Invertase activity
(pmol glucose mg1 protein hr1)
H
l
I
H
H
3
O
c
Q
05
O
*<
3

4
-I
O
H
H
Soluble activity
% of maximal response
Ivr1 mRNA


Figure 4-10. Abundance of mRNA from the hr l and Ivr2 subfamilies of soluble
acid invertase during post-starvation recovery of maize root tips. Sugar
depletion in Whites basal salts without sugars (18 hr) was followed by
incubation for various periods of time (6-18 hr) in media with 0.5% glucose
supplements. RNA gel blots with equal amounts (10 pg) of total RNA from
above tissues were probed with 32P-labeled Ivrl or Ivr2 representing the two
subfamilies of maize soluble acid invertase. Blots were exposed to X-ray
film for 24 or 12 hr, respectively.


47
Church and Gilbert (1984), and exposed against X-ray film with intensifying screens
at -80 C.
Enzvme Extraction
Soluble invertase was extracted as per Duke et al. (1991). Frozen tissue
samples were ground to a fine powder in liquid N2 using a mortar and pestle.
Frozen powder was transferred to a second mortar containing ice-cold 200 mM
HEPES buffer (pH 7.5) with 1 mM DTT, 5mM MgCl2, 1 mM EGTA and 10%(w/w)
PVPP. One ml of extraction buffer was used for each 100 mg of tissue fresh
weight. Buffered extract was centrifuged at 15,000 x g for 10 min to sediment
particulate matter. Pellets were saved for salt-solubilized particulate invertase
extraction. Supernatant was dialyzed (50,000 MW cutoff) at 4 C for 24 hr against
extraction buffer diluted 1:40. Buffer was changed twice. Soluble dialyzed extract
was centrifuged again at 15,000 x g for 10 min and supernatant assayed for soluble
invertase activity as described below.
Insoluble invertase was extracted as described by Doehlert and Felker (1987).
Pellets remaining from extractions of soluble invertase were washed three times by
sequentially resuspending each in 5 to 10 ml extraction buffer and centrifuging at
15,000 x g for 10 min. Salt-solubilized particulate invertase was extracted by
resuspending the pellet in extraction buffer containing 1 M NaCl. Solubilized
particulate invertase was recovered in supernatant following centrifugation at 15,000


116
Hein, M.B., Brenner, M.L. and Brun, W.A. (1984). Concentrations of indole-3-
acetic acid and abscisic acid in soybean seeds during development. Plant
Physiol. 76: 951-954.
Heineke, D., Sonnewald, U., Bssis, D., Gnter, G., Leidreiter, K., Wilke, I.,
Raschke, K., Willmitzer, L., and Heldt, H.W. (1992). Apoplastic expression
of yeast-derived invertase in potato. Effects on photosynthesis, leaf solute
composition, water relations, and tuber composition. Plant Physiol. 100, 301 -
308.
Hitz, W.P., Schmitt, M.R., Card, P.J., and Giaquinta, R.T. (1985). Transport and
metabolism of l-fluorosucrose, a sucrose analog not subject to invertase
hydrolysis. Plant Physiol. 77: 292-295.
Ho, L.C. (1988). Metabolism and compartmentation of imported sugars in sink
organs in relation to sink strength. Ann. Rev. Plant Physiol. Plant Mol. Biol.
39: 355-378.
Howard, H.F., and Witham, F.H. (1983) Invertase activity and the kinetin-stimulated
enlargement of detached radish cotyledons. Plant Physiol. 73, 304-308.
Hubbard, N.L., Huber, S.C., and Pharr, D.M. (1989). Sucrose phosphate synthase
and acid invertase as determinants of sucrose concentration in developing
muskmelon (Cucumis mel L.) fruits. Plant Physiol. 91, 1527-1534.
Huttly, A.K. and Baulcombe, D.C. (1989). A wheat a-Amy 2 promoter is regulated
by gibberellin in transformed oat aleurone protoplasts. EMBO J. 8: 1907-
1913.
Ishikawa, K., Kamada, H., Yamaguchi, I., Takahashi, N. and Harada, H. (1988).
Morphology and hormone levels of tobacco and carrot tissues transformed by
Agrobacterium tumefaciens. I. Auxin and cytokinin contents of cultured
tissues transformed with wild-type and mutant Ti plasmids. Plant Cell
Physiol. 29: 461-466.
Isla, M.I., Leal, D.P., Vattuone, M.A. and Sampietro, A.R. (1992). Cellular
localization of invertase, protienaceous inhibitor and lectin from potato tubers.
Phytochemistry 31: 1115-1118.
Jacobsen, J.V., and Beach, L.R. (1985). Evidence for control of transcription of a-
amylase and ribosomal RNA genes in barley aleurone proplasts by gibberellic
acid and abscisic acid. Nature 316: 275-277.


Figure 5-2. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble acid invertase in maize root tips
incubated for 24 hr in Whites basal salts medium supplemented with 0.5%
glucose (all +G), either with (+K) or without (-K) 5 pM kinetin, either with
(+ABA) or without (-ABA) abscisic acid (50 pM). A, RNA gel blots with
equal amounts (10 pg) of total RNA from above tissues were probed with
32P-labeled Ivrl or Ivr2 representing the two subfamilies of maize soluble
acid invertase. Blots were exposed to X-ray film for 24 or 12 hr,
respectively. B, Total soluble acid invertase activity from the above samples.
Insoluble invertase activity (not shown) was consistently ca 10-fold less than
that in the soluble fraction of maize root tips. Values for RNA/protein
recovery were ca 0.20 (0.07) with variability independent of +K or +ABA
treatments.


6
Chourey and Nelson, 1976; Chourey, 1981) and snap bean pods (Sung et al., 1994).
In addition, maize kernels induced to abort by high temperature have a much
reduced activity of pedicel soluble invertase than do nonaborting kernels (Hanft and
Jones, 1986a). The rapid expansion characteristic of this early development requires
both osmotic constituents and substrates for respiratory and synthetic processes.
Soluble invertase can be especially important to cell enlargement through generation
of hexoses and their associated osmotic potential (Kaufman, 1973; Avigad, 1982;
Schmalstig and Cosgrove, 1988; 1990). Either invertase or sucrose synthase can
provide an avenue for carbohydrate entry into respiratory and biosynthetic processes
(Giaquinta, 1979; Avigad, 1982; Morris and Arthur, 1984b; Hawker, 1985; Schaffer
et al., 1987).
Soluble invertase activity is also closely associated with other phases of
reproductive development (Tsai et al., 1970; Jaynes and Nelson, 1971a; Shannon and
Dougherty, 1972; Singh and Knox, 1984; Hubbard et al., 1989; Miron and Schaffer,
1991; Klann et al., 1992). Pryke and Berneir (1978) have found that increased
content of sugar and activity of soluble acid invertase in the apices consistently
appear to accompany the transition to flowering in Sinapis alba. Invertase also
appears to be involved in pollen function. The sucrose content from pollen grains of
Camellia japnica decreases rapidly during growth of the tube. Soluble invertase
activity also increases during germination of cultured pollen and a high constant
activity is found during the later stages of pollen tube growth (Nakamura et al.,
1980). Further, invertase is demonstrated to contribute to an in vitro chemotropism


invertases in kernels sampled daily from 2 days before to 2 days after
pollination 61
Figure 4-5 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize
invertases during the final 3 days of anther development and in mature
pollen 63
Figure 4-6 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble and insoluble maize invertases
in silk sampled daily from 2 days before to 2 days after pollination ... 65
Figure 4-7 Abundance of mRNA from the Ivrl and lvr2 subfamilies of soluble
acid invertase and activity of total soluble and insoluble maize invertases
in tip, mid and low portions of silk sampled at pollination, 3hr later, 6hr
later, or 24hr later 67
Figure 4-8 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase in maize root tips incubated for 24hr in whites basal salts
medium supplemented with glucose or sucrose 69
Figure 4-9 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase during starvation of maize root tips 71
Figure 4-10 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase during post- starvation recovery of maize root
tips 73
Figure 4-11 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble acid invertases in
maize root tips incubated for 24hr in Whites basal salts medium
supplemented with either 2.0% glucose, fructose, sucrose, L-glucose or
mannitol 75
Figure 5-1 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble acid invertases in maize root
tips incubated for 24hr in Whites basal salts medium supplemented
either with (+G) or without (-G) 0.5% glucose and either with (+K) or
without (-K) 5 pM Kinetin 95
Figure 5-2 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble acid invertases in maize root
tips incubated for 24hr in Whites basal salts medium supplemented with
vi


113
Church, G.M., and Gilbert, W. (1984). Genomic sequencing. Proc. Natl. Acad. Sci.
USA 81, 1991-1995.
Clifford, P.E., Offler, C.E., and Patrick, J.W. (1986). Growth regulators have rapid
effects on photosynthate unloading from seed coats of Phaseolus vulgaris L.
Plant Physiol. 80: 635-637.
Collinge, D.B., and Slusarenko, A.J. (1987). Plant gene expression in response to
pathogens. Plant Mol. Biol. 9: 389-410.
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shrunken-2 maize kernels. Plant Physiol. 80: 609-611.
Cobb, B.G., and Hannah, L.C. (1988). Shrunken-1 encoded sucrose synthase is not
required for sucrose synthesis in the maize endosperm. Plant Physiol. 88,
1219-1221.
Corbin, D.R., Sauer, N., and Lamb, C.J. (1987). Differential regulation of a
hydroxyproline-rich glycoprotein gene family in wounded and infected plants.
Mol. Cell Biol. 7: 4337-4344.
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tissues. Plant Physiol. 72, 326-331.
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root growth in soybean seedlings. Plant Physiol. 92: 205-214.
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PJ Davies, ed, Plant hormones and their role in plant growth and
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DeWald, D.B., Sadka, A. and Mullet, J.E. (1994). Sucrose modulation of soybean
Vsp gene expression is inhibited by auxin. Plant Physiol. 104: 439-444.


Figure 4-11. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble acid invertase in maize root tips
incubated for 24 hr in Whites basal salts medium supplemented with either
2.0% glucose, fructose, sucrose, L-glucose or mannitol respectively. A, RNA
gel blots with equal amounts (10 pg) of total RNA from above tissues were
probed with 32P-labeled Ivr 1 or lvr 2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively. B, Total soluble acid invertase activity from the above
samples. Insoluble invertase activity (not shown) was consistently ca 10-fold
less than that in the soluble fraction of maize root tips. Values for
RNA/protein recovery were ca 0.14 (+ 0.05) with variability independent of
presence or absence of metabolizable C-source.


88
The hypotheses tested here are as follows. Invertase gene expression could
be responsive to ABA (aiding osmoregulation), gibberellins and auxin (aiding
gravitropism and phototropism), and/or cytokinins (aiding sink potential and/or
symbiosis).
In this report, we demonstrate that in maize root tips, both Ivrl and Ivr2
expression for soluble acid invertase genes includes an unexpected, differential
responsiveness to specific hormonal signals. These findings indicate that different
invertase isozymes may have specialized functions in a diverse set of developmental
and/or environmental processes.
Materials and Methods
Plant Material
Zea mays hybrid NK 508 was used for all experiments. Seeds were first
emersed in 20 % Clorox for 30 min. followed by 30 min. of continuous rinsing with
water. Germination took place in the dark at 18 C on two layers of moist 3 MM
paper (Whatman, Inc., Clifton, NJ) in 17 x 26 cm glass pans. Air flowed
continuously at 1 liter min'1 through each pan for the 6-day period, with 40% 02
supplied during the final 24 hr before root tip excision. The moisture level was
adjusted daily by applying mist and draining excess water. Root tips (ca 1 cm each)
were excised under a sterile transfer hood.


45
Materials and Methods
Plant Material
The Zea mays hybrid NK 508 was used for all experiments. For analyses of
developmental changes, plants were grown under greenhouse or field conditions.
Samples harvested included leaves, anthers, silk, cobs, pollen, prop roots, and
kernels at different developmental stages.
For experiments with root tips, seeds were first emersed in 20% Clorox for
30 min, followed by 30 min of continuous rinsing with water. Germination took
place in the dark at 18 C on two layers of moist 3 MM paper (Whatman, Inc.,
Clifton, NJ) in 17 x 26 cm glass pans. Air flowed continuously at 1 liter min'1
through each pan for the 6-day period, with 40% 02 supplied during the final 24 hr
before root tip excision. The moisture level was adjusted daily by applying mist and
draining excess water. The terminal 1 cm was cut from root tips (at ca. 3 to 6 cm
total length) under a sterile transfer hood.
Experimental Conditions
Experimental treatments were as described by Koch et al. (1992).
Approximately 100 root tips (~ 500 mg) were used for each experimental treatment.
Excised root tips were incubated in the dark at 18 C for 6 to 48 hr in Whites


125
Skriver, K., Olsen, F.L., Rogers, J.C. and Mundy, J. (1991). cis-Action DNA
elements responsive to gibberellin and its antagonist abscisic acid. Proc. Natl.
Acad. Sci. USA 88: 7266-7270.
Somogyi, M. (1951). Notes on sugar determination. J. Bio. Chem. 181: 19-23.
Sturm, A., and Chrispeels, M.J. (1990). cDNA cloning of carrot extracellular P-
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Sturtevant, D.B., and Taller, B.J. (1989). Cytokinin production by Bradyrhizobium
japonicum. Plant Physiol. 89: 1247-1251.
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synthase, and invertase activities of developing Phaseolus vulgaris L. fruits.
Plant Cell and Environ. 17
Thomas, J.C., McElwain, E.F. and Bohnert, H.J. (1992). Convergent induction of
osmotic stress-responsive: abscisic acid, cytokinin, and the effect of NaCl.
Plant Physiol. 100: 416-423.
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306.
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Plant Mol. Biol. 40: 119-138.
Unger, C., Hardegger, M., Lienhard, S., and Sturm, A. (1994). cDNA cloning of
carrot (Daucus catota) soluble acid 13-fructofuranosidases and comparing with
the cell wall isoenzyme. Plant Physiol. 104: 1351-1357.
von Heijne, G. (1986). A new method for predicting signal sequence cleavage sites.
Nucleic Acids Res. 14: 4631-4690.
von Schaewen, A., Stitt, M., Schmidt, R., Sonnewald, U. and Willmitzer, L. (1990).
Expression of yeast-derived invertase in the cell wall of tobacco and
Arabidopsis plants leads to accumulation of carbohydrate and inhibition of


120
McCarty, D.R. (1986). A rapid and simple method for extracting RNA from maize
tissues. Maize Gen. Coop. News Lett. 60, 61.
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157: 1055-1057.
Miller, M.E., and Chourey P.S. (1992). The maize invertase-deficient miniature-1
seed mutation is associated with aberrant pedicel and endosperm
development. Plant Cell 4, 279-305.
Miron, D., and Schaffer, A.A. (1991). Sucrose phosphate synthase, sucrose synthase,
and invertase activities in developing fruit of Lycopersicon esculentum mill,
and the sucrose accumulating Lycopersion hirsutum humb. and bonpl. Plant
Physiol. 95, 623-627.
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accumulation in growing subhooks of etiolated Pisum sativum seedlings.
Effects of accumulation and growth. Physiol. Plant. 88: 301-306.
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intemodal segments of Phaseolus vulgaris. Phytochemistry 23, 2163-2167.
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activity and cell growth during leaf expansion in Phaseolus vulgaris L. J.
Exp. Bot. 35: 1369-1379.
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carbohydrate distribution and acid invertase activity in Phaseolus vulgaris.
Physiol. Plant. 65, 257-262.
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phytopathogens. Ann. Rev. Plant Physiol. 37: 509-538.
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136-146.


77
associated with reproductive organs (Figure 4-1 A). Data shown here for general
association between soluble invertase activity and rapid growth/cell division were
consistent with previous suggestions for the role of this enzyme relative to sucrose
import. Rapidly expanding tissues require either invertase or sucrose synthase to
convert sucrose to substrates necessary for respiratory and synthetic processes
(Glasziou and Gayler, 1972; Giaquinta, 1979; Avigad, 1982; Morris and Arthur,
1984b; Hawker, 1985; Schaffer, 1986; Schaffer et al, 1987). Invertase in particular
can be important to cell expansion through generation of hexoses and their
associated osmotic potential. Changes in both message and activity in the present
study were also consistent with the gradual sink-to-source transition in leaves (Ho,
1988; Turgeon, 1989; Nguyen-Quoc et al., 1990).
Data presented here indicate that soluble invertases may be especially
important during the early stages of maize kernel development. This is consistent
with a hypothesis advanced on the basis of previous work (Hanft and Jones, 1986a;
1986b; Reed and Singletary', 1989), which suggests that the soluble forms of these
enzymes in the pedicel may be critical to initiation of normal kernel development.
The expression pattern of both the Ivrl and Ivr2 classes of invertase, as well as total
soluble activity (Figure 4-2 and Figure 4-4), are also in agreement with this
possibility.
Past research on invertase and kernel development has tended to focus on the
insoluble "cell-wall-bound" form of this enzyme primarily because of its apparent
importance during later stages of kernel fill. Shannon and coworkers proposed that


20
Results
One positive clone containing a 1.2 kb maize cDNA (Ivrl) was obtained
(Figure 3-1) when a cDNA fragment encoding a soluble acid invertase in tomato
(Klann et al., 1992) was used to screen a maize root tip cDNA library (A.gt 10,
Clontech, Palo Alto, CA).
This 1.2 kb maize fragment was used to rescreen the same library. Twelve
positive clones ranging from 0.5 to 2.2 kb were identified. Sequences obtained from
the longest of these indicated that none of them included a full-length cDNA clone.
For this reason, a Hindlll-EcoRI fragment from the longest clone (2.2 kb, Figure 3-
1) was used to rescreen the library a second time. Seven positive clones were
identified.
From the total of twenty clones, five were selected for full length sequencing
based on their sizes, hybridization characteristics, and location of restriction sites
(Figure 3-1). Sequence was provided by the Sequence Core Lab of the ICBR at the
University of Florida. They were designated Ivrl through Ivr2C-3 (Figure 3-1).
Ivr2C-2 was identical to Ivr2, and Ivr2C-3 contained the same but shorter sequence
as Ivr2C-l.
Further information was sought in the corresponding genomic sequence. A
maize seedling genomic DNA library (EMBL 3, Clontech, Palo Alto, CA) was
screened with a 1 kb Kpnl-EcoRl fragment from the Ivrl cDNA Figure 3-1, 3-7).
One positive genomic clone was isolated and characterized. This clone consisted of


52
also observed in a progressive decline of message levels for both classes of invertase
transcripts. The longitudinal gradient of invertase mRNA levels from tip to base of
silks was reduced during this decrease by the rapid decline in message abundance
observed in the basal region of the style. At the enzyme level, temporal and spatial
changes in total soluble activity were consistent with those of the Ivrl and Ivr2
message levels (Figure 4-6B, 4-7B). Salt-solubilized invertase was relatively
constant before and/or after pollination, and no activity gradation was evident along
the length of silk (Figure 4-6C, 4-7C). The drop in mRNA abundance of invertase
is still more pronounced than pictured if considered relative to protein levels.
Differential responses of the Ivrl and Ivr2 class genes to sugar supplies
became apparent when excised root tips were supplemented with a range of glucose
and/or sucrose concentrations and incubated for 24 hr (Figure 4-8). Ivrl class
message levels were maximal with ca 0.5% exogenous glucose (Figure 4-8A) and ca
0.2% sucrose (Figure 4-8B), whereas those of Ivr2 remained relatively constant
when media glucose and/or sucrose levels were between 0.2 and 4.0%. In addition,
levels of the Ivrl subfamily of transcripts appeared to drop less markedly during a
24 hr period without exogenous carbohydrate than did those of Ivr2 (Figure 4-8). In
excised maize root tips, soluble sugars reportedly drop to minimal levels within 10
hr if no supplemental sugars are provided (Saglio and Pradet, 1980). Differential
responses of the Ivrl and Ivr2 classes of invertase to carbohydrate deprivation were
further explored by an analysis of their progression over time in excised root tips
(Figure 4-9). Levels of the Ivrl type mRNAs decreased less rapidly than did those


Figure 4-3. Abundance of mRNA from the Ivrl and lvr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in
pedicel, middle and top portions of kernels at 8, 10 and 12 DAP. A, RNA
gel blots with equal amounts (10 fig) of total RNA from above tissues were
probed with 32P-labeled Ivrl or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for two days.
B, Total soluble acid invertase activity from the above tissues. C, Insoluble
acid invertase activity from the above tissues. Values for RNA/protein
recovery were ca 0.03 (+ 0.01) with variability independent of tissue gradient
from kernel top to pedicel. Values for RNA/protein recovery dropped from
ca 0.04 (+ 0.02) to 0.02 (+ 0.01) past 10 DAP, and is consistent with
elevated protein storage in kernels at this stage.


90
Gilbert, 1984). Maize Ivrl and lvr2 invertase cDNA clones were radiolabeled by
random primer. No cross-reactivity between Ivrl and Ivr2 gene probes was
observed when hybridizations were conducted at high stringency (data not shown).
Blots were washed as described by Church and Gilbert (1984), exposed to X-ray
film with intensifying screens at -80 C.
Enzyme Extraction
Soluble invertase was extracted as per Duke et al. (1991). Frozen tissue
samples were ground to a fine powder in liquid N2 using a mortar and pestle.
Frozen powder was transferred to a second mortar containing ice-cold 200 mM
HEPES buffer (pH 7.5) with 1 mM DTT, 5mM MgCl2, 1 mM EGTA and 10%(w/w)
PVPP. One ml of extraction buffer was used for every 100 mg of tissue fresh
weight. Buffered extract was centrifuged at 15,000 x g for 10 min to sediment
particulate matter. Pellets were saved for salt-solubilized particulate invertase
extraction. Supernatant was dialyzed (50,000 MW cutoff) at 4 C for 24 hr against
extraction buffer diluted 1:40 (MW cutoff for dialysis was selected to allow escape
of proteinaceous invertase inhibitors [Jaynes and Nelson, 1971b]). Buffer was
changed twice. Soluble dialyzed extract was centrifuged again at 15,000 x g for 10
min. Supernatant was used for soluble invertase assays as described below.
Insoluble invertase was extracted as described by Doehlert and Felker (1987).
Pellets remaining from the above step were washed three times by resuspending in


signals (typically produced by dividing cell, endosperm, root tips and symbionts)
could alone apparently replace and supersede the carbohydrate upregulation of
invertase transcript levels by sugars. Both Ivrl and Ivr2 type mRNA abundance was
upregulated by exogenous ABA (elevated in developing seeds and in response to
some stresses). However, simultaneous presence of cytokinin appeared to be
required before the ABA-induced changes at the message-level could be transduced
at the level of enzyme activity. The differential response of invertase isozyme genes
to sugar levels and specific plant hormones suggests that integration of these types
of signals may mediate developmental responses, symbiosis, and/or adaptation to
stresses.
x


10
Most recent work at the molecular level, however, indicates that there are
ABA-responsive elements and GA-responsive sequences located on promoter regions
in a number of structural genes (Jacobsen and Beach, 1985; Zwar and Hooley, 1986;
Libbenga and Mermes, 1987; Marcott et al., 1989; Mundy et al., 1990; Salmenkallio
et al., 1990; Jacobsen and Close, 1991; Skriver et al., 1991; Lanahan et al., 1992).
An auxin-responsive promoter appears to be differentially induced by auxin
gradients during tropisms (Li et al., 1991). It is more likely that the effects of
abscisic acid, auxin, cytokinin and/or gibberellic acid on invertase are mediated by
their respective influence on transcription, but these may well occur by different
mechanisms.
Wounding and Invertase
Wounding typically stimulates expression of invertase genes (Matsushita and
Urttani, 1974; Sturm and Chrispeels, 1990). A general increase in the respiratory
activity in response to wounding in various plant storage tissues is well documented
(Matsushita and Urttani, 1974). In root tissue of sweet potato, respiratory activity
doubles within 20 hours after wounding. The increased respiratory activity is
paralleled by increases in RNA content and the de novo synthesis of enzymes
(Shirras and Northcote, 1984). Invertase may well be one of these and would be
advantageous in its enhancement of capacity to initiate sucrose breakdown.


Figure 5-3. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble acid invertase in maize root tips
incubated for 24 hr in Whites basal salts medium supplemented with 0.5%
glucose (all +G), alone (+0) or supplemented with either gibberellic acid
(+GA) or auxin (+IAA). A, RNA gel blots with equal amounts (10 pg) of
total RNA from above tissues were probed with 32P-labeled Ivrl or Ivr2
representing the two subfamilies of maize soluble acid invertase. Blots were
exposed to X-ray film for 24 or 12 hr, respectively. B, Total soluble acid
invertase activity from the above samples. Insoluble invertase activity (not
shown) was consistently ca 10-fold less than that in the soluble fraction of
maize root tips. Values for RNA/protein recovery were ca 0.23 (+0.12) with
variability independent of +GA or +IAA treatments.


39
Discussion
Soluble invertase genes were cloned and characterized for two reasons. The
first of these was to characterize the extent of their carbohydrate-responsiveness
relative to that of genes for sucrose synthase, ultimately to provide a more complete
picture of how sugars influence the capacity for their own metabolism at the
transcriptional level. The second planned use for the invertase clones was to clarify
the potential significance of these gene family members during development and/or
environmental adjustment by maize tissue and organs.
Three lines of evidence support the designation of these genes not only as
maize invertases but also as soluble ones. First, the full length sequence of the
putative maize invertase clone (IvrlG) has extensive sequence similarity to other
invertases found in vascular plants (Table 3-1), and shares the conserved key
domains identified in other invertases (NDPNG [(3-fructosidase motif, Sturm and
Chrispeels, 1990], plus FRDPTTA, TGMWEC and YASKTF) (Figure 3-5). Second,
the maize invertase gene examined here has a considerably greater amino acid
identity to the soluble isoforms of invertase than to the cell-wall-bound ones found
in other vascular plants (Table 3-1; Figure 3-6). The underlined areas in figure 3-6
are those highly conserved regions which are shared among soluble invertases but
not insoluble ones. In particular, the amino acid sequence at the C-terminus of the
IvrlG protein is significantly more similar to that of soluble vs. insoluble forms.


Brouqisse, R., James, F., Raymond, P., and Pradet, A. (1991). Study of glucose
starvation in excised maize root tips. Plant Physiol. 96, 619-626.
112
Callow, J.A. and Ling, I T. (1973). Histology of neoplasms and chlorotic lesions in
maize seedlings following injection of sporidia of Ustilago mayis (DC),
Corda. Physiol. Plant Patho. 3: 489-494.
Callow, J.A., Long, D.E., and Lithgow, E.D. (1980). Multiple molecular forms of
invertase in maize smut infections. Physiol. Plant Patho. 16, 93-107.
Carlson, M. (1987). Regulation of sugar utilization in Saccharomyces species. J.
Bacteriol. 169: 4873-4877.
Carlson, M., and Botstein, D. (1982). Two differentially regulated mRNAs with
different 5 ends encode secreted and intracellular forms of yeast invertase.
Cell 28, 145-154.
Chen, H.H., Li, P.H. and Brenner, M.L. (1983). Involvement of abscisic acid in
potato cold acclimation. Plant Physiol. 71: 362-365.
Chen, J.Q. and Black, C.C. (1992). Biochemical and immunological properties of
alkaline invertase isolated from sprouting soybean hypocotyls. 295: 61-69.
Chen, T.H.H. and Gusta, L.V. (1983). Abscisic acid-induced freezing resistance in
cultured plant cells. Plant Physiol. 73: 71-75.
Cheng, C.L., Acedo, G.N., Cristinsin, M., and Conkling, M.A. (1992). Sucrose
mimics the light induction of Arabidopsis nitrate reductase gene transcription.
Proc. natl. Acad. Sci. USA 89: 1861-1864.
Chourey, P.S. (1981). Genetic control of sucrose synthase in maize endosperm.
Mol. Gen. Genet. 184: 372-376.
Chourey, P.S., and Nelson, O.E. (1976). The enzymatic deficiency conditioned by
the Shrunken-1 mutations in maize. Biochem. Genet. 14: 1041-1055.
Chrispeels, M.J. (1991). Sorting of proteins in the secretory system. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 42: 21-53.
Chrispeels, M.J., and Varner, J.E. (1967). Hormonal control of enzyme synthesis:
on the mode of action of gibberellic acid and abscisin in aleurone layers of
barley. Plant Physiol. 42: 1008-1016.


9
mechanism by which IAA leads to an increase in acid invertase activity, however,
remains obscure. It is not yet clear whether the observed increase in activity is a
cause or a consequence of auxin-induced growth (Morris and Arthur, 1984a).
Both abscisic acid and cytokinins are reported to stimulate assimilate
translocation from source to sink (Gersani and Kender, 1982; Howard and Witham,
1983; Hein et al, 1984; Schussler et al., 1984; Ackerson, 1985; Jones et al., 1986;
Brokovec and Prochazka, 1992; Jones et ah, 1992). Such enhancement may involve
the capacity of invertase to hydrolyze sucrose to hexoses and thus increase sink
potential. This in turn could stimulate the translocation of sugar to seeds (Shannon,
1968; Shannon, 1972; Shannon and Dougherty, 1972; Lin et ah, 1984; Doehlert,
1986; Doehlert and Felker, 1987).
The mechanisms originally proposed to explain the effects of plant hormones on
invertase have been questioned (Sacher et ah, 1963; Glasziou et ah, 1966; Chrispeels
and Varner, 1967; Gayler and Glasziou, 1969; Hagen et ah, 1984). Gayler et ah
(1969) suggested that auxin and gibberellic acid may have aided stabilization of the
mRNA for invertase. They further suggested that the mechanism of abscisic acid
(ABA) action in this instance involved processes subsequent to formation of
invertase-mRNA and prior to invertase destruction. In contrast, Chripeels et ah
(1967) suggested that the gibberellic acid effect required synthesis of enzyme-
specific RNA molecules. They also proposed that abscisin exerted its action either
by inhibiting the synthesis of these enzyme-specific RNA molecules or by
preventing their incorporation into an active enzyme-synthesizing unit.


Invertase activity Probe
(l^mol glucose mg'1 protein hr'1)
Ivrl
Ivr2
A. mRNA
+0 hr AP
+3 hr AP
+6 hr AP
+24 hr AP
(ip mid low tip mid low tip mid low tip mid low
Mill | m

100
B. Soluble activity
i
50
X
3.1
X
X
X
X
X
I
C. Insoluble activity
20 -
T
1
X
JL
i
x
pH.
x_
J-
+0 hr AP
+3 hr AP
+6 hr AP
+24 hr AP


Invertase activity Probe
(|.imol glucose mg'1 protein hr'1)
61
Ivrl
Ivr2
4 -
A. mRNA
Days -/+ pollination (fert)
-2 -1 0 +1 +2
(-3) (-2) (-1) (0) (+1)
B. Soluble activity
X
Ir1
I
C. Insoluble activity
x
i
-2 -1 0 +1 +2
(-3) (-2) (-1) (0) (+1)
Days -/+ pollination (fert)


22
the signal sequence according to the (-3, -l)-method of von Heijne (1986) was
between A7' and G74.
A comparison between the invertase genes isolated from maize in the present
work and other invertases from vascular plants (Table 3-1; Figure 3-6), IvrlG shared
an approximate 60% amino acid identity with soluble invertases (Arai et al., 1992;
Klann et al., 1992; Elliott et al., 1993; Unger et al., 1994) and 40% with insoluble
forms (Sturm and Chrispeels, 1990; Ramloch-Lorenz et al., 1993); moreover the
conserved key domains (NDPND [P-fructosidase motif, Sturm and Chrispeels,
1990], as well as FRDPTTA, TGMWEC and YASKTF) (Figure 3-5). In addition,
the maize invertase gene has a significantly greater amino acid identity to the soluble
isoforms of invertase than to the cell-wall-bound ones found in other vascular plants,
especially at the C-terminus of this protein (Figure 3-6).
Restriction maps of invertase cDNA and genomic clones from maize are
shown in Figure 3-1. Ivr2 was found to share a 53% sequence similarity at the
amino acid level to IvrlG (Table 3-1), especially have extensive sequence similarity
located at conserved domains (data not shown). The Ivrl probe (1 kb Kpnl-EcoRI
fragment) did not cross-hybridize with the Ivr2 or lvr2C-l cDNAs at high stringency
(Figure 3-7B). The Ivr2 probe (200 bp Pstl-PstI fragment) cross-reacted with the
Ivr2C-l cDNA but not that of Ivrl (Figure 3-7C).
Ivrl was missing its 3end and contained one 5 unspliced (putative) intron
(according to the sizes of 5'RACE products, E. Bihn, unpublished data). Ivr2


ACKNOWLEDGEMENTS
My sincerest appreciation is extended to the members of my committee, Dr.
Karen Koch, Dr. Alice Harmon, Dr. Ken Boote and Dr. Don McCarty, for their
support and guidance during the completion of this degree. I am also truly grateful
to the other faculty, staff and graduate students for their help and encouragement
during my time here, especially Kurt Nolte, Ed Duke, Wayne Avigne, Don Merhaut,
Gwendolyn Pemberton, Betsy Bihn, Summer Osterman and Aiyu Li.
Finally, I extend my deepest thanks to my wife Naidong Shao, who was
always there whenever I needed her and also to my parents and my brother in
China. They have all given me support and encouragement throughout my
education here, and I can never thank them enough.
11


79
Jones (1986a; 1986b) which tentatively attributes kernel abortion under water and
heat stresses to reduced activity of soluble invertase in the pedicel. The following
scenario represents one possible explanation for the combination of data on the
greater sensitivity of the Ivr2 genes to carbohydrate deprivation and the abundance
of their transcripts in the pedicel. Any early limitation of assimilate flux into the
endosperm would be expected to reduce soluble sugar concentration in the pedicel
within a relatively short time (Hanft and Jones, 1986a). The depletion of pedicel
sugars could in turn result in decreased levels of the carbohydrate-responsive Ivr2
gene products and a subsequent decrease in soluble invertase activity in this region.
This is consistent with the observation that it is the soluble rather than insoluble acid
invertase activity which is most markedly affected in pedicel of kernels that have
been induced to abort vs. nonaborting kernels (Hanft and Jones, 1986a).
The role of soluble invertases during anther and pollen development is
probably twofold. First, there are no plasmodesmatal connections between
developing pollen grains and the surrounding tapetum layer (Kesselback, 1949). The
tapetum thus lies at the terminal end of the maternal transport path. Any invertase
or sucrose synthase present in these cells could theoretically enhance sugar transport
to pollen grains by creating a sucrose gradient between phloem and the secretory
surface, much as hypothesized for developing kernels (Shannon, 1972; Shannon and
Dougherty, 1972; Lin et al., 1984). Presumably, enhanced hydrolysis could also
benefit the probable elevation in respiratory and biosynthetic demands. Our results


108
kernels, immature and mature anthers, pollen and silk. A comparison between
transcript and enzyme activity was again consistent with both clones encoding
soluble acid invertases. The spatial and temporal patterns of expression for the two
invertase subfamilies, as well as the contrast between them, demonstrated a close
association between soluble invertases and changes in silk during pollination and in
kernels immediately after fertilization.
Maize root tips in 24 hr culture were used to quantify the extent to which
soluble invertase (at the message and enzyme levels) responded to sugar and specific
developmental/environmental signals. In this system, the abundance of mRNA
levels from both classes was upregulated whenever a source of exogenous, readily-
metabolizable, sugars was made available. The extent of response differed from one
invertase subgroup to another, however. The Ivr2 type genes showed greater
sensitivity to carbohydrate deprivation. The differential responsiveness of invertase
gene subfamilies to carbohydrate availability provided a potential mechanism for
different isozyme gene subgroups to predominate in various tissues, developmental
stages, and/or altered environmental conditions.
Several lines of investigation have suggested invertase gene expression may
be responsive to cytokinins (thus enhancing potential for sucrose metabolism, import
and/or symbiosis), water deficit and/or ABA (aiding osmotic alteration), and
gibberellin or auxin (aiding gravitropism and/or phototropism). Kinetin treatments
(5 pM) increased both message levels (Ivrl and hr 2) and elevated total soluble acid
invertase activity. Kinetin alone replaced and superseded the carbohydrate


69
glucose (%)
0 0.2 0.5 2.0 4.0

Ivr2
sucrose (%)
0 0.2 0.5 2.0 4.0
Ivrl
7vr2


Figure 4-5. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases during
the final 3 days of anther development and in mature pollen. A, RNA gel
blots with equal amounts (10 pg) of total RNA from above tissues were
probed with 32P-labeled Ivrl or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively. B, Total soluble acid invertase activity from the above
tissues (*, not assayed). C, Insoluble acid invertase activity from the above
tissues (*, not assayed). Values for RNA/protein recovery were ca 0.03
(0.01) for mature and shedding anthers. Values from extracts of young
anthers were greater (0.07 + 0.03), possibly due to more extensive
meristematic activity.


14
accelerated by a greater capacity for invertase to remove this sugar from the terminal
end of the transport path.
Little research, however, has been directed toward understanding the
mechanisms by which invertase activity is elevated in response to pathogens (Callow
and Ling, 1973; Long et al., 1975; Billett et al., 1977; Billett and Burnett, 1978;
Callow et al., 1980; Heidecker and Messing, 1986; Collinge and Slusarenko, 1987;
Sheridan, 1988; Sturm and Chrispeels, 1990). The origin of the induced invertase
protein (fungal vs host) has remained controversial (Billett et al., 1977; Callow et al.,
1980).
Agrobacterium tumefaciens and Pseudomonas syringae pv Savastanoi,
however, contain genes that specify the biosynthesis of cytokinin and indoleacetic
acid (Morris, 1986; Morris, 1987; Ishikawa et al., 1988; Weil and Rausch, 1990).
Cytokinins and/or cytokinin-like substances are also reported to be synthesized in
mycorrhizal fungi (Miller, 1967; Crafts and Miller, 1974; Ng et al., 1982) and
Bradyrhizeobium japonicum (Sturtevant and Taller, 1989). Allen et al. (1980) found
that cytokinin levels increase in the host plant following infection by vesicular-
arbuscular mycorrhizae. Elevated levels of IAA and/or cytokinin have also been
implicated in maize tissues infected by Ustilago mayis (Turian and Hamilton, 1960;
Billett et al., 1977; Billett and Burnett, 1978).
Upregulation of invertase expression by fungal infection could facilitate
enhancement and/or establishment of a symbiosis by providing hexoses for those
fungi unable to metabolize sucrose. A resulting question is whether or not plant


51
Activity remained detectable at 32 DAP (well past maturity under local growing
conditions). This salt-solubilized activity was also maximal in the pedicel area and
lowest in the top portion of the same kernels when expressed per unit total salt-
solubilized protein. If decreases in mRNA levels encoding Ivrl and Ivr2 are viewed
relative to protein levels, then the drop in message abundance is more pronounced
than pictured due to the onset of enhanced protein storage in kernels between 10 and
12 DAP.
During the earliest stages of kernel development, message levels for both Ivrl
and lvr2 subfamilies and total soluble invertase activity increased markedly (Figure
4-4). Soluble invertase activity from kernels two days after pollination was twice as
high as that of unpollinated ones (Figure 4-4B) and insoluble activity from the same
kernels (Figure 4-4C).
During anther development, transcript levels of both Ivrl and lvr2 classes of
invertase increased gradually through anthesis (Figure 4-5A). Both message types
were also abundant in RNA extracted from pollen. This was probably not the basis
for localization in young anthers, because shedding anthers had greater apparent
levels of both classes of mRNA than did pollen itself.
Both the Ivrl and lvr2 types of mRNA were abundant in silk if tissue was
sampled before or immediately after pollination (Figure 4-6A). A gradient in
relative message levels for these gene classes was also evident along the length of
the silk, with lowest levels in the top (distal) 1/3 and greatest abundance in the 1/3
closest to the ovary (proximal) (Figure 4-7A). A rapid response to pollination was


to be carried on an unusually small 9 nucleotide exon identified in the maize
genomic DNA.
Two subfamilies of maize soluble invertases (each cross-reactive with either
Ivrl or Ivr2 [Iw2C-l + Ivr2C-2]) were differentially expressed in an array of tissues.
A comparison between message and enzyme activity was consistent with both
subgroups encoding soluble acid invertases. The spatial and temporal patterns of
expression for the two invertase classes, as well as the contrast between them
implicate their potential involvement in several stages of development. Data support
the hypothesis that invertase could be especially important during stages requiring
expansion of specific cells, such as during pollination and early kernel development.
Maize root tips were used to further test the extent to which expression of the
two subfamilies for soluble invertase isozymes may have been regulated by sugar
levels or specific developmental signals.
The mRNA levels from both subgroups were elevated in the presence of
exogenous sugar supplies as long as these were readily metabolizable, however, the
extent of this response differed. The lvr2 group of genes showed a greater
sensitivity to carbohydrate deprivation. The differential responsiveness of invertase
gene subfamilies to carbohydrate availability provides a potential mechanism for
different isozyme genes to predominate in various tissues developmental stages,
and/or altered environmental conditions.
Data also indicated that specific developmental cues could affect expression
of both invertase subgroups as well as soluble activity of acid enzymes. Cytokinin
IX


53
of the Ivr2 subgroup and persisted for considerably longer. Relatively little change
was evident during 24 hr of starvation, and message remained readily apparent for at
least 48 hr. In contrast, levels of the Ivr2 class of mRNA dropped below detection
after between 12 and 18 hr of carbohydrate deprivation (Figure 4-9).
Although Ivrl message abundance appeared to be relatively insensitive to an 18-hr
starvation period or subsequent additions of sugar to media, levels of mRNA for the
Ivr2 subfamily were sensitive to both (Figure 4-10). Glucose replacement after 18
hr of C-depravation appeared to counter initial decreases in levels of message for the
Ivr2 subfamily. These returned to pre-starvation levels after 18 hr incubation in 0.5
% glucose (Figure 4-10).
The responses of the Ivrl and Ivr2 class genes to different types of sugars
(Figure 4-11 A) also showed that expression of both appeared to require a supply of
metabolizable sugars. Transcripts remained abundant in the presence of 2% D-
glucose, fructose, or sucrose in the exogenous media, but dropped when these were
replaced by either L-glucose or mannitol.


124
Schussler, J.R., Brenner, M.L., and Brun, W.A. (1984). Abscisic acid and its
relationship to seed filling in soybeans. Plant Physiol. 76: 301-306.
Schwebel-Pugue, N., Mtili, N.E., Kriritzky, M., Jean-Jacques, I., Williams, J.H.H.,
Thomas, M., Kreis, M. and Lechamy, A. (1994). Arabidopsis gene and
cDNA encoding cell-wall invertase. Plant Physiol. 104: 809-810.
Shannon, J.C. (1968). Carbon-14 distribution in carbohydrates of immature Zea mays
kernel following 14C02 treatment of intact plants. Plant Physiol. 43: 1215-
1220.
Shannon, J.C. (1972). Movement of l4C-labelled assimilates into kernels of Zea mays
L. I. Pattern and rate of sugar movement. Plant Physiol. 49: 198-202.
Shannon, J.C., and Dougherty, T.C. (1972). Movement of l4C-labelled assimilates
into kernels of Zea mays L. II. Invertase activity in the pedicel and placento-
chalazal tissues. Plant Physiol. 49: 203-206.
Shannon, J.C., Knievel, D.P., Chourey, P.S. Liu, S. and Liu, K. (1993).
Carbohydrate metabolism in the pedicel and endosperm of miniature maize
kernels. Plant Physiol. 102:42.
Sheen, J. (1990). Metabolic repression of transcription in higher plants. Plant Cell 2:
1027-1038.
Sheridan, W.F. (1988). Maize developmental genetics: Genes of morphogenesis.
Ann. Rev. Gent. 22: 353-385.
Shirras, A.D. and Northcote, D.H. (1984). Molecular cloning and characterization of
cDNA complementary to mRNAs from wounded potato (Solarium tuberosum)
tuber tissue. Planta. 162: 353-360.
Sheu-Hwa, C.S., Lewis, D.H. and Walker, D.A. (1975). Stimulation of
photosynthetic starch formation by sequestration of cytoplasmic
orthophosphate. New Phytol. 74: 385-392.
Shuster, J. R. (1989). Regulated transcriptional systems for the production of
proteins in yeast: Regulation by carbon source. In Yeast genetic engineering
buttersworth. P.J. Barr, A.J. Brike, and P. Valenzuela, eds, London,
Butterworths, pp.83-108.
Singh, M.B., and Knox, R.B. (1984). Invertase of Lilium pollen. Characterization
and activity during in vitro germination. Plant Physiol. 74, 510-515.


23
contained an unusual 5end, missing the NDPNG (sequence indicates possible
incomplete intron splicing). Ivr2C-l was lacking the 3end of its coding sequence.


49
Results
Developmental and organ-level differences were evident in expression of the
two classes of invertase genes (Figure 4-1 A). Message levels for the Ivrl group
were markedly higher in reproductive structures than vegetative tissues, whereas
those of the Ivr2 type transcripts were abundant in essentially all of the sucrose
importing structures examined (loading same amount of total RNA). Message from
both classes of invertase were present in sink leaves, dropping below detectable
levels during sink-to-source transition. Transcript levels of both types were also
evident in those tissues undergoing rapid growth and/or cell division, such as root
tips, anthers, pollen and silk (styles). As observed for relative mRNA abundance,
activity of this enzyme fraction also predominated in the most rapidly elongating
tissues (such as root tips and silk) regardless of whether data were expressed per unit
protein or fresh weight (data not shown). Activity was also generally elevated in
instances of enhanced sucrose import. The greater ratio of RNA/protein recovered
from root tip extracts vs those from other tissues suggests that if changes in total
RNA encoding invertase messages are viewed relative to protein levels, then
invertase mRNA levels in root tips are greater relative to enzyme activity than is
evident in Figure 4-1. The greater values for RNA/protein recovery from root tip
extracts may possibly be due to the extensive meristematic activity in these organs.
Shifts in region of localization were evident during kernel development for
the two subfamilies of maize invertase (Figure 4-2 A and Figure 4-3 A). Message


Figure 4-9. Abundance of mRNA from the Ivrl and lvr2 subfamilies of soluble acid
invertase in maize root tips depleted of carbohydrates for either 6, 12, 18, 24,
36, or 48 hr, respectively, in Whites basal salts medium without an
exogenous sugar supply. RNA gel blots with equal amounts (10 pg) of total
RNA from above tissues were probed with 32P-labeled Ivrl or Ivr2
representing the two subfamilies of maize soluble acid invertase. Blots were
exposed to X-ray film for 24 or 12 hr, respectively.


13
segments were treated with compounds which stimulated the most growth. They
also suggested that by reducing sucrose concentrations in the apoplast and/or
symplast of sink tissues, the acid invertases located in these respective compartments
may contribute significantly to maintenance of source-to-sink gradients in sucrose
concentration and hydrostatic pressure which drives phloem transport.
Fungi, Bacteria, and Invertase
Increased invertase activities have been reported in tissues of several plants
infected by biotrophic fungi and/or bacteria (Callow and Ling, 1973; Long et al.,
1975; Billett et al., 1977; Billett and Burnett, 1978; Callow et al., 1980; Krishnan
and Pueppke, 1988; Sturm and Chrispeels, 1990;). In addition, a common feature of
biotrophic fungal infections of vascular plants is an increased translocation of
photosynthetic assimilates into infected plant parts, which is typically accompanied
by accumulation of one or more host carbohydrates (Callow and Ling, 1973; Long et
al., 1975; Billett et al., 1977; Billett and Burnett, 1978; Callow et al., 1980).
Billett et al. (1977) have shown that infection of maize by the corn smut.
Ustilago mayis, stimulates assimilate movement into, and accumulation of soluble
sugars, and starch, in tissues. Smut in maize kernels results in rapid growth, cell
division, and elevated rates of respiration. Enhancement of maize invertase activity
in these regions could facilitate competition with other sinks for the sugars needed to
support these processes. Sucrose import and unloading from phloem could be


85
necessarily be the same. Environmental and developmental signals may also have
contrasting influences, and depending on the role of the gene product, the sensitivity
and degree of the response may also vary.


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MOLECULAR CHARACTERIZATION AND DIFFERENTIAL EXPRESSION OF
A INVERTASE GENE FAMILY IN MAIZE
By
Jian Xu
December, 1994
Chairperson: Dr. Karen E. Koch
Major Department: Plant Molecular and Cellular Biology Program
A family of soluble invertase genes in maize (Zea mays L.) were cloned and
characterized to test several hypotheses regarding their potential significance in
specific instances of developmental and/or environmental adjustment. The responses
of two invertase gene subfamilies were examined at the level of both gene
expression and overall enzyme activity.
Five maize cDNA clones (AW, Ivr2, Ivr2C-l, IvrC-2 and IvrC-3) and one
genomic clone (IvrlG) were isolated and found to encode probable isozymes of
soluble invertase. The deduced amino acid sequences show significant identities,
especially to previously characterized soluble acid invertases of higher plants, and
are particularly strong in key regions conserved among these enzymes. One of the
most strongly conserved regions among all invertase sequences (NDPNG) was found
Vlll


91
extraction buffer and centrifuging at 15,000 x g for 10 min. Salt-solubilized
particulate invertase was extracted by resuspending the pellet in extraction buffer
containing 1 M NaCl. Solubilized particulate invertase was recovered in supernatant
following centrifugation at 15,000 x g for 10 min. Supernatant was used for
insoluble invertase assays as described below.
Enzyme Assay
Both soluble and salt-solubilized invertase activities were assayed for 15 to
30 min at 37 C in a mixture containing 100 mM Na-acetate (pH 4.5) and 100 mM
sucrose in a final volume of 500 pi. Activity was determined by measuring
reducing sugars with the method of Nelson (1944) and Somogyi (1951).


105
way. In many cases, these types of target cells have similar perception-and-
transduction mechanisms, but the molecular programs which are elicited by these
mechanisms are different (Libbenga and Mermes, 1987). Gravitropism in shoots is
brought about by auxin-induced elongation on the long side. In roots, however,
auxin does not induce, but rather inhibits elongation. In figure 5-3, the abundance
of both the Ivrl and Ivr2 type messages was downregulated in response to IAA
treatment, however, the total soluble acid invertase activity remained relatively
constant. One explanation may be the apparent longevity of some invertase proteins.
A need for greater activity of invertase has been implicated in the
establishment and enhancement of the capacity for sucrose import (Morris and
Arthur, 1984; Hanft and Jones, 1986a; Schaffer, 1987). Phytohormone modulation
of invertase genes could effectively regulate the processes associated with phloem
unloading and expansion sink potential (Hein et al., 1984; Schussler et al., 1984;
Ackerson, 1985; Jones et al., 1986; Borkovec and Prochazka, 1992; Jones et al.,
1992).
Genes specifying plant growth regulator biosynthesis have been identified in
phytopathogens (Miller, 1967; Crafts and Miller, 1974; Allen et al., 1980; Ng et al.,
1982; Morris, 1986; Weil and Rausch, 1990). Plant hormone regulation of invertase
expression could facilitate its enhancement, in instances such as establishment of
symbiosis, by providing hexoses for those fungi unable to metabolize sucrose.


84
Sadka et al. (1994) propose that sugar modulates transcription of the soybean
vegetative storage proteins and other sugar-inducible genes by using phosphate as a
signal. In their model, phosphate acts as a negative factor to those sugar-responsive
genes. Carbohydrate activates those genes by accumulation of sugar-phosphates and
concomitant reduction of cellular phosphate levels. High phosphate levels relative to
those of sugars are also found in starved sycamore cells (Rebeille et al., 1985).
Graham et al. (1994), on the other hand, propose that not metabolism per s,
but the phosphorylation by hexokinase per s maybe signaling intracellular sugar-
responsiveness of gene expression. In their experiments, they demonstrate that 2-
deoxyglucose and mannose, like glucose and fructose (which are phosphorylated by
hexokinase but not further metabolized) specifically repress cucumber malate
synthase and isocitrate lyase gene expression. However, 3-methylglucose, an analog
of glucose that is not phosphorylated, does not result in repression of either malate
synthase or isocitrate lyase.
Many of the genes involved in metabolic pathways are subject to regulation
by the fluctuation of internal and external metabolites in multicellular vascular plants
(Maas et al., 1990; Sheen, 1990; Koch et al., 1992; Graham et al., 1994; Sadka et
al., 1994;). The metabolic regulation of gene expression should play a role of
fundamental importance in maintaining an economical balance of the supply and
demand of biomolecules in different organs of vascular plants. Metabolic control of
specific gene expression now appears to be a widespread phenomenon, although the
mechanism of signal transduction and response for different genes will not


-ABA 4-ABA -ABA +ABA
Invertase activity
(nmol glucose mg1 protein hr'1)
ro 4* CT>
o o o o
1 1 r~
Probe


117
Jacobsen, J.V. and Close, T.J. (1991). Control of transient expression of chimeric
genes by gibberellic acid and abscisic acid in protoplasts prepared from
mature barley aleurone layers. Plant Mol. Biol. 16:713-724.
Jameson, P.E., Letham, D.S., Zhang, R., Parker, C.W., and Badenoch-Jones, J.
(1987). Cytokinin translocation and metabolism in lupin species. I. zeatin
riboside introduced into the xylem at the base of Lupinus angustifolius stems.
Aust. J. Plant Physiol. 14: 695-718.
Jaynes, T.A., and Nelson, O.E. (1971a). Invertase activity in normal and mutant
maize endosperms during development. Plant Physiol. 47, 623-628.
Jaynes, T.A., and Nelson, O.E. (1971b). An invertase inactivator in maize
endosperm and factors affecting inactivation. Plant Physiol. 47, 629-634.
Jones, R.J., Griffith, S.M. and Brenner, M.L. (1986). Sink regulation of source
activity:regulation by hormonal control. In J. Shannon, ed. Regulation of
carbon and nitrogen reduction and utilization. Maryland, American Society of
Plant Physiologists, pp 233-246.
Jones, R.J., Schreiber, B.M., McNeil, K., Brenner, M.L., and Foxon, G. (1992).
Cytokinin levels and oxidase activity during maize kernel development. In M.
Kaminek, D.W.S. Mok and E. Zazimalova, ed. Physiology and biochemistry
of cytokinins in plants. The Hague, SPB academic publishing, pp 235-239.
Karuppiah, N., Vadlamudi, B., and Kaufman, P.B. (1989). Purification and
characterization of soluble (cytosolic) and bound (cell wall) isoforms of
invertases in barley (Hoedeum vulgare) elongating stem tissue. Plant Physiol.
91, 993-998.
Kaufman, P.B., Ghosheh, N.S., Lacroix, J.D., Soni, S.L., and Ikuma, H. (1973).
Regulation of invertase levels in Avena stem segments by gibberellic acid,
sucrose, glucose, and fructose. Plant Physiol. 52, 221-228.
Kaufman, P.B. and Song.I. (1987). Hormones and the orientation of growth. In P.J.
Davies, ed. Plant hormones and their role in plant growth and development.
Dordrecht, Martinus Nijhoff Publishers, pp 375-392.
Kiesselbach, T.A. (1949). The structure and reproduction of com. Lincoln,
University of Nebraska Press.
Kim, D., Wu, L., Ghosheh, N. and Kaufman, P.B. (1993). Molecular studies on
tissue sensitivuy to auxin during the gravitropic response in oat shoot pulvini.
Plant Physiol. 102:118.


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83
Under source-limited conditions, invertase involved in certain physiological
processes could act to increase sink activity and stimulate assimilate translocation to
these sinks to compete with others. Under normal growth conditions, assimilate
levels are plentiful. Thus, the Ivr2 class of genes tend to be widely expressed in
sink tissues and their gene products are abundant. This is especially evident in
rapidly growing tissues, which is consistent with the concept that high activity of
"soluble" invertase is usually associated with rapid tissue expansion (Glasziou and
Gayler, 1972; Giaquinta, 1979; Avigad, 1982; Hawker, 1985; Schaffer et al., 1987).
Invertase is considered to facilitate assimilate transportation from the site of phloem
unloading to sink tissues by steepening the gradient of sucrose between source and
sink (Shannon, 1968; Shannon et al, 1972; Shannon and Dougherty, 1972; Shannon
et al., 1993). However, soluble invertase can also promote cell elongation and/or
rapid growth by hydrolyzing sucrose to hexoses, thereby providing osmotically
active solutes and the osmotic pressure necessary to support growth (Kaufman et al.,
1973; Schmalstig and Cosgrove, 1988; 1990).
Gene responses to sugars in vascular plants have been known for some time
(Rocha-Sosa et al., 1989; Salanoubat and Belliard, 1989; Muller-Rober et al., 1990;
Maas et al., 1990; Koch et al., 1992). However, the mechanism, by which the sugar
signal is sensed by plant genes, is not clear. Our results (Figure 4-12) indicated that
naturally occurring, metabolizable sugars, such as sucrose, D-glucose and fructose,
meet the requirement for invertase responsiveness, although data shown here can not
rule out other possibilities for certain non-metabolizable sugars.


76
Discussion
The significance of findings presented here extends from implications of
special roles for soluble invertases during development (especially pollination and
early kernel development) to broader possible contributions to adjustment of sucrose
import, cell volume, and metabolism in a multi-celled higher plant. The spatial and
temporal patterns of expression for the two invertase subfamilies, as well as the
contrast between them suggest involvement in specific developmental processes.
The availability of these clones has also allowed the hypothesis to be tested that
regulation of transcript level by photosynthate availability could contribute to
adjustment of both avenues for sucrose breakdown in a cell (invertase as well as
sucrose synthase). Moreover, a surprising similarity in differential carbohydrate
responsiveness was evident between the two invertase subfamilies and the two
sucrose synthase genes. In both instances, the more broadly distributed of the two
(Ivr2 or Susl) was found to be readily induced by enhanced carbohydrate
availability, whereas the form which was upregulated during more specific
developmental and environmental signals (Ivrl or Shi) was less sensitive to sugar
supplies (Koch et al., 1992).
The present work indicates that each subfamily of the invertase genes is
expressed differentially depending on developmental stage and the tissue/organ
involved. Although invertase activity was detected in extracts of almost every
sucrose-importing tissue examined, the Ivrl type message was preferentially


Figure 4-7. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in tip,
mid, and low portions of silk (portions of silk [ca 4 cm total length] relative
to ovary) sampled at pollination, 3 hr later, 6 hr later, or after 24 hr. A,
RNA gel blots with equal amounts (10 pg) of total RNA from above tissues
were probed with 32P-labeled Ivrl or Ivr2 representing the two subfamilies of
maize soluble acid invertase. Blots were exposed to X-ray film for 24 or 12
hr, respectively. B, Total soluble acid invertase activity from the above
tissues. C, Insoluble acid invertase activity from the above tissues. Values
for RNA/protein recovery were ca 0.06 (+ 0.02) before pollination and
dropped to 0.04 (+ 0.02) within 3hr after pollination, then to 0.02 (+ 0.01) at
6hr and 24hr after pollination. Transcription may be markedly reduced by
pollination and/or message longevity may largely determine the extent of
change in types of mRNA predominating.


19
DNA Sequencing
Selected cDNA and genomic DNA fragments were subcloned into a pUC 19
vector. The recombinant plasmids were amplified in E. coli cells and purified
through CsCl2 ultracentrifugation and/or with the use of QIAGEN-tip (QIAGEN
Inc., Chatsworth, CA). Both strands of each cDNA and genomic DNA were
sequenced by the Sequence Core Lab of ICBR (Interdiciplinary Center for
Biotechnology Research) located at the University of Florida.
Analysis of DNA and Protein Sequences
Computer-assisted analyses of DNA and protein sequences were carried out
with Geneworks (Release 2.2, IntelliGenetics, Inc., Mountain View, CA).


Figure 3-3. The deduced amino acid sequence for maize invertase 1 gene. The
arrow indicates the cleavage site for potential signal peptide. The box
represents P-fructosidase motif (NDPNG, Sturm and Chrispeels, 1990).
Underlined sequences are for putitive glycosylation sites (N-X-S/T).


102
The biological significance of the findings presented here is twofold. First,
under stress conditions severe enough to limit photosynthesis, cytokinins still could
maintain sink potential of specific regions by preserving invertase activity for later
recovery of growth. Second, cytokinins could possibly initiate and/or enhance the
capacity for sucrose import and utilization in a given structure simply by stimulating
invertase expression. Jones et al. (1992) found that cytokinin levels increase
dramatically (as much as 400-fold) during the early stages of maize kernel
development and decline subsequently. They suggest that the ultimate capacity for
sucrose import into kernels is established during this first portion of development,
and further, that de novo biosynthesis of cytokinins within kernels plays a role in
this process through regulation of endosperm and/or nucellar development.
The responsiveness of invertase gene expression to exogenous ABA may also
have a distinct biological relevance (Figure 5-2). ABA is considered a stress
hormone due to its accumulation and action during many such conditions (Chen et
al., 1983; Chen and Gusta, 1983; LaRosa et al., 1985; LaRosa et al., 1987; Davies
and Zhang, 1991; Thomas et al., 1992). Thus, ABA could enhance the capacity of a
plant to acclimate to different stresses, such as freezing, drought and high salt, by
inducing invertase expression. This in turn has the capacity to aid adjustment of
osmotic potential.
ABA is also reported to stimulate transport of photosynthate towards
developing seeds in a number of species (Hein et al., 1984; Schussler et al., 1984;
Ackerson, 1985; Jones et al., 1986; Borkovec and Prochazka, 1992). ABA could


46
medium, either with or without an array of supplemental sugars. Each group of root
tips was agitated at 120 cycles per minute in a 125-ml side-arm Erlenmeyer flask
with 50 ml of sterile media. Airflow (40% 02, make sure to supply enough 02)
through air stones in each flask was maintained at 250 ml min'1 throughout the
incubations.
RNA Isolation and Gel Blot Analysis
Root tip samples were rinsed twice in sterile water, blotted dry, weighed, and
frozen in liquid N2. Other samples (as mentioned in the previous text) were
harvested from greenhouse and/or field-grown plants, weighed, and frozen
immediately in liquid N2. Samples were ground into fine power in liquid N2 and
total RNA was extracted (McCarty, 1986). RNA was quantified
spectrophotometrically (Sambrook et al., 1989). Total RNA (10 pg) was separated
by electrophoresis in 1 % agarose gels containing formaldehyde (Thomas, 1980),
blotted to nylon membranes, and fixed by baking and/or UV treatment (Sambrook et
al., 1989). Filters were hybridized at 65 C in a solution with 7 % SDS, 250 mM
Na2HP04 (pH 7.2) and 1 % BSA (Church and Gilbert, 1984). Maize hr 1 and Ivr 2
invertase cDNA clones were radiolabeled by random primer. No cross-reactivity
was observed between the hr 1 and hr 2 gene probes when hybridizations were
conducted at high stringency (data not shown). Blots were washed as described by


x g for 10 min. Pooled supernatant fractions were assayed for insoluble invertase
assay as described below.
48
Enzyme Assay
Both soluble and salt-solubilized invertase activities were assayed for 15 to
30 min at 37 C in an assay medium with 100 mM Na-acetate (pH 4.5) and 100
mM sucrose in a final volume of 500 pi. Activity was determined by measuring
reducing sugars as described by Nelson (1944) and Somogyi (1951).


Figure 4-1. Abundance of mRNA from the Ivrl and hr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases in root
tips, a sink leaf, a source leaf, a prop root, anthers, silk and kernels (2 DAP).
A, RNA gel blots with equal amounts (10 pg) of total RNA from above
tissues were probed with 32P-labeled Ivrl or Ivr2 representing the two
subfamilies of maize soluble acid invertase. Blots were exposed to X-ray
film for 24 or 12 hr, respectively. B, Total soluble acid invertase activity
from the above tissues. C, Insoluble acid invertase activity from the above
tissues. Values for RNA/protein recovery were ca 0.04 (+ 0.02) for tissues
other than root tips and did not otherwise differ significantly between tissue
types. Root tip values were greater (0.15 + 0.04) possibly due to more
extensive meristematic activity.


MOLECULAR CHARACTERIZATION AND DIFFERENTIAL EXPRESSION OF
A INVERTASE GENE FAMILY IN MAIZE
By
JIAN XU
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1994


Invertase activity Probe
(nmol glucose mg' protein hr'1)
63
Ivrl
Ivr2
A. mRNA
Days to anthesis
anther pollen
3 10 0
III
20
10
20
10-
tft
B. Soluble activity
X
C. Insoluble activity
X
X
3 10 0
anther pollen
Days to anthesis


Figure 3-7. DNA gel blot analysis of A, Approximate size of the Ivrl, Ivr2, Ivr2C-l and Ivr2C-2 cDNA clones and B,
Extent of cross-reactivity between them. A, EtBr stained DNA gel blot analysis of approximate length for Ivrl,
lvr2, Ivr2C-l and Ivr2C-2. B and C, DNA gel blots with equal amounts (1 pg) of recombinant DNA from each
cDNA (Released from pUC 19 vector by digestion with EcoRI) and probed with 32P-labeled fragments from
either A, Ivrl (Kpn I-EcoR I, 1 kb fragment) or B, Ivr2 (Pst I-Pst I, 200 bp fragment). Both blots were exposed
to X-ray film for 1 hr. Sites on the restriction maps are K, Kpn I; P, Pst I; R, EcoR I.


73
post-starvation
recovery (hr)
0 6 12 18
Ivrl

Ivr2


LITERATURE CITED
Ackerson, R.C. (1985). Invertase activity and abscisic acid in relation to
carbohydrate status in developing soybean reproductive structures. Crop Sci.
25: 615-618.
Allen, M.F., Moore, J.T.S., and Christensen, M. (1980). Phytohormone changes in
Bouteloua gracilis infected by vesicular-arbuscular mycorrhizae: I. cytokinin
increases in the host plant. Can. J. Bot. 58: 371-374.
Arai, M., Mori, H., and Imaseki, H. (1992). Cloning and sequence of cDNA for an
intracellular acid invertase from etiolated hypocotyls of mung bean and
expression of the gene during growth of seedlings. Plant Cell Physiol. 33 (3):
245-252.
Avigad, G. (1982). Sucrose and other disaccharides. In F.A. Loewus, W. Tanner, ed,
Encyclopedia of plant physiology, new series. Berlin and New York,
Springer-Verlag, pp 216-347.
Baumlein, H., Nagy, I., Villarroel, R. Inze, D. and Wobus, U. (1992). Cis-analysis
of a seed protein gene promoter: the conservative RY repeat CATGCATG
within the legumin box is essential for tissue-specific expression of legumin
gene. Plant J. 2: 233-239.
Baysdorfer, C., and Vanderwoude, W.J. (1988). Carbohydrate responsive proteins in
the roots of Pennisetum americanum. Plant Physiol. 87, 566-570.
Baysdorfer, C., Warmbrodt, R.D., and Vanderwoude, W.J. (1988). Mechanisms of
starvation tolerance in pearl millet. Plant Physiol. 88, 1381-1387.
Bednarek, S.Y. and Raikhel, N.V. (1992). Intracellular trafficking of secretor
proteins. Plant Mol. Biol. 20: 133-150.
Bednarek, S.Y., Wilkins, T.A., Dombowski, J.E., and Raikhel, N.V. (1990). A
carboxyl-terminal propeptide is necessary for proper sorting of barley lectin
to vacuoles of tobacco. Plant Cell 2: 1145-1155.
110


50
(Days After Pollination), dropping below detection within 16 DAP. However, the
Ivr2 type mRNA was abundant in the pedicel region and barely detectable in the
middle and top portions of the kernels (Figure 4-3A). In contrast, levels of Ivrl-
related messages in the pedicel region were similar or less than those in the middle
and top sections of kernels at the same developmental stage. In addition,
developmental differences in timing were evident, with a narrow peak in Ivrl
transcript abundance at 8 DAP in the upper kernel, vs a broader elevation in Ivr2
message in the pedicel between 8 and 12 days after pollination. Transcript levels of
the Ivrl subgroup were approximately similar in pericarp and endosperm at 10 DAP,
whereas the Ivr2 mRNAs were considerably more abundant in the pericarp (data not
shown).
Figure 4-3 B showed that total soluble acid invertase activity, like that of Ivrl
and Ivr2 mRNA was highest in the pedicel region and lowest in the top area of the
same kernels when expressed per unit total soluble protein (similar results were
observed when data were calculated per unit fresh weight [data not shown] expcept
that peak activity was elevated for two days longer). Total activity of soluble acid
invertase was maximal in extracts of kernels sampled 12 days after pollination,
dropping gradually to below detection in those from between 20 and 24 DAP
(Figure 4-2C). In contrast, salt-solubilized particulate invertase activity (insoluble)
increased gradually in developing kernels, but did so most rapidly between 2 and 6
DAP. Peak activity was observed at ca 16 DAP, and decreased slowly thereafter.
Activity remained detectable at 32 DAP (well past maturity under local growing


CHAPTER 3
ISOLATION AND CHARACTERIZATION OF MAIZE INVERTASE GENES
Introduction
Only two avenues known for enzymatic breakdown of sucrose exist in
vascular plants. One is catalyzed by sucrose synthase, the other by invertase. Two
distinct types of invertase activities are found in plants (Avigad, 1982). One class
has an optimum pH of 4.5 to 5.0, and includes acid invertases. The second class
hydrolyzes sucrose at a maximal rate at pH 7.5 to 8.0, and is designed as the
alkaline invertases. The existence of these two types of P-fructosidase is evident in
many plants and/or organs (Avigad, 1982). Acid invertases are located either inside
the vacuole (soluble form) or in the extracellular space (varying degrees of soluble
and cell-wall-bound forms). In contrast, alkaline invertases are compartmentalized
in cytoplasm (Hawker, 1985).
Invertase genes encoding cell-wall and vacuolar (soluble) acid invertases have
been characterized from carrot (Sturm and Chrispeels., 1990; Ramloch-Lorenz et al.,
1993; Unger et al., 1994), tomato (Klann et al., 1992; Elliott et al., 1993), mung
bean (Arai et al., 1992), and Arabidopisis (Schwebel-Dugue et al., 1994).
16


below
B


CHAPTER 4
DEVELOPMENTAL EXPRESSION AND CARBOHYDRATE
RESPONSIVENESS OF TWO MAIZE INVERTASE GENE SUBFAMILIES
Introduction
Invertases ((3-fructosidase, EC 3.2.1.26) play a key role in sugar metabolism.
In vascular plants, different isoforms are located in different cellular compartments
(Avigad, 1982; Hawker, 1985). Isoforms with an acidic pH optimum are found in
the vacuole and/or apoplasm whereas isoforms with a neutral pH optimum are
located in the cytoplasm. Work with sugar cane stems (Hawker and Hatch, 1965;
Glasziou and Gayler, 1972) and com kernels (Shannon, 1968; Shannon, 1972;
Shannon and Doughty, 1972; Shannon et al, 1993) has indicated that imported
sucrose moves from phloem into the extracellular space where it is hydrolyzed by a
cell-wall-bound, acid invertase. This is presumed to contribute to a sucrose
concentration gradient between the phloem and apoplast, facilitating transfer of
sucrose into importing tissues (Lin et al., 1984; Doehlert, 1986; Doehlert and Felker,
1987; Doehlert et al., 1988; Turgon, 1989). Soluble invertase has been found in the
vacuoles of sucrose-storing cells (Avigad, 1982). Thus, soluble invertases with
acidic pH optima are often thought to be localized in the cell vacuoles of other
tissues as well, where they can mobilize sucrose temporarily stored in this compartment.
42


Ivrl
Ivr2
30
20
10
0
300
200
100
0
59
A. mRNA
8 DAP
10 DAP
12 DAP
top mid ped top mid ped top mid ped
m
8 DAP
10 DAP
12 DAP


Figure 3-1. Restriction maps of Ivr clones for maize soluble acid invertases.
Restriction maps of maize soluble invertases (IvrlG, Ivrl, Ivr2 and Ivr2C-l).
Sites on the restriction maps are as follows: B, BamH I; H, Hind III; K, Kpn
I; P, Pst I; S, Sma I.


101
activity is considered to be the primary determinant of sink strength in these
systems. In addition, early development of maize kernels (Tsai et al., 1970) and
snap bean pods (Sung et ah, 1994) takes place with soluble invertase predominating,
whereas sucrose synthase activity in both cases is below level of detection.
Cytokinins stimulate soluble acid invertase gene expression which subsequently
increases sink potential.
Mycorrhizal fungi and/or rhizobia can only metabolize hexoses. Cytokinins
and/or cytokinin-like substances are reported to be synthesized in mycorrhizal fungi
(Miller, 1967; Crafts and Miller, 1974; Ng et ah, 1982) and Bradyrhizobium
japonicum (Sturtevant and Taller, 1989). Allen et ah (1980) report that cytokinin
level increases in the host plant after infection by vesicular-arbuscular mycorrhizae.
Cytokinins originating from mycorrhizal fungi and/or rhizobia act to increase the
sink capacity through elevating invertase expression from the host plant. The
present work therefore indicates that cytokinin-like substances from fungal and/or
rhizobial symbionts could also act through stimulation of invertase.
Figure 5-1 suggested, first, that cytokinin and sugar enhanced the
accumulation of invertase message level independently. Both Ivrl and Ivr2 mRNA
levels were elevated ca 2.5-fold by cytokinin regardless of exogenous carbohydrate
supply. Sugar alone was unable to stimulate invertase expression to the same degree
as was cytokinin. Second, cytokinin alone was sufficient to promote invertase
enzyme activity to the highest level after 24 hr incubation in this system (Figure 5-
1B).


+K '
Invertase activity
(jimol glucose mg1 protein hr1)
o
+
o
o
+
o
o
~T
to
o
CO
o
U3
H
H
H
H
. Soluble activity
7vr2
Probe
# I

m t
v£>
Ln


43
Rapidly expanding tissues require either invertase or sucrose synthase to
convert sucrose into substrates necessary for respiratory and synthetic processes
(Giaquinta, 1979; Avigad, 1982; Morris and Arthur, 1984b; Hawker, 1985; Schaffer
et al., 1987). Invertase can be especially important to cell expansion through
generation of hexoses and their associated osmotic potential (Kaufman, 1973;
Avigad, 1982; Schmalstig and Cosgrove, 1988; 1990). Invertase activity is also
associated with reproductive organs (Jaynes and Nelson, 1971a; Shannon and
Dougherty, 1972; Singh and Knox, 1984; Hubbard et al., 1989; Miron and Schaffer,
1991; Klann et al., 1992; Reger et al., 1992). Invertase can aid competition for sink
capacity for reproductive growth. Soluble invertase is the predominant enzyme for
sucrose breakdown during the early developmental stage of maize kernel (Tsai et al.,
1970) and snap bean seed (Sung et al., 1994).
Genes regulated by carbohydrate were first studied in microorganisms
(Carlson, 1987; Schuster, 1989). Those genes are usually involved in metabolic
pathways. In vascular plants, sugar-responsive genes have been primarily
characterized in storage organs (Rocha-Sosa et al., 1989; Salanoubat and Belliard,
1989; Muller-Rober et al., 1990; DeWald et al., 1994; Sadka et al., 1994). Those
genes generally encode storage proteins such as patatin from potato (Rocha-Sosa et
al., 1989), tuberous root storage protein genes from sweet potato (Hattori et al.,
1990), vegetative storage proteins from soybean (Sadka et al., 1994). In addition,
carbohydrate-induced changes in gene expression have also focused on metabolic
pathways, especially those involved in sugar metabolism such as sucrose synthase


BIOGRAPHICAL SKETCH
Jian Xu was bom in Changzhou City, Jiangsu Province, China on May 26,
1963. He attended Nanjing University from 1982 to 1986, where he received his
Bachelor of Science in plant biochemistry and plant physiology.
Jian Xu entered the Shanghai Institute of Plant Physiology, Academia Snica
in September 1986 and received his Master of Science degree in the Plant
Biochemistry Division in August, 1989. In January 1991, Jian Xu began his
program of study for the Doctor of Philosophy degree in the Plant Molecular and
Cellular Biology Program at the University of Florida, working under the direction
of Dr. Karen Koch. He plans to continue in plant science as a postdoctoral research
associate in the laboratory of Dr. Elaine Tobin at the University of California, Los
Angeles. He is the member of American Society of Plant Physiologists and the
Maize Genetics Cooperative.
127


17
In the present study, a tomato invertase clone (Klann et al., 1992) was used
to isolate an invertase cDNA from maize, and this, in turn, was used to obtain
additional maize clones. These findings provide the tools for further investigation
along two lines. The first of these will be aimed at combining an analysis of sugar-
responsiveness of these genes with that of the sucrose synthases, to define
carbohydrate regulation of two different avenues for sucrose breakdown. The
second will be to further clarify the potential functional significance for soluble
invertase isozymes in development and/or environmental adjustment in maize.
Materials and Methods
Probe for cDNA Library Screening
A 0.45 Kb fragment from the 5-end of a cDNA encoding a soluble acid
invertase in tomato (Klann et al., 1992) was isolated from tomato clone and
subcloned into a pUC19 vector. The recombinant plasmid was amplified in E. coli
cells, purified, and used to screen a maize cDNA library (Sambrook et al., 1989).
cDNA Library Screening
A maize root tip cDNA library (A.gt 10, Clontech, Palo Alto, CA) was
screened with the 0.h5 kb tomato invertase cDNA fragment. One positive clone


A. Soluble 5/ end
B. Insoluble 5' end
TUmo IS)
Carrot (Sill
Huny Baan (S)
Catcut (SI)
IvrlO
^TUC- 1 0PXNSA8XVT tLMQPO fOHRJCS LKUSOIP
nTHPLPSR DLZHAS8YTP BPOSPtTRHK PDPDASRWR RPIKIBS8VL
Ml-I PI.LPTSSH AA -PTSI-TRKD LLFVLC- -CLLPLSS-L
-HPITISHY TPLPOCBHSP SLTTTNTAEQ SSRRRSLT-- -FVLLPSSIL
l-PAVAOPT TUXWOARRP LLPSTDPRCR AAAOAKQK-- -RPPATPTVL
)7
SO
17
47
44
Conaanaua
L....8..P .L....TR 8.. 88.L SO
TttMto (8)
Carrot (SSI)
Hung baan (8)
Carrot (81)
IvrlO
Conaanaua
US-VfU.LSV APPP--1LNN QSPDtQIMR SPA -PP8I
LSTLILSPVI PLLVHPNVQQ WRKXBSKMS NGkDRHJCASK SPSMJ
VAYOOYRA SQVPilAHLSS PTSHHQQDHQ SPTSLPSSKW YPV8I
AACLVHCTHV L-PPHS0-N8 AVZXSTWPI ETVEVA- -P--1
TAW8AVLLL VLVAVTVLAS QHVDCQAOOV PAOEOAVWI VAAS^ABQ
.A.
.0 P.SffJ/. .0
I : tSQQ
4>PSR
'ssa
'AEG
7*
100
5
I
94
Toacto (8)
Carrot (SIS)
Hung Baan (8)
Caciot (81)
IvrlO
Conaanaua
TMMto (8)
Carrot (8II)
Hung Baan (8)
Carrot (SI)
IvrlO
Conaanaua
Toaato (8)
Carrot (811)
Hung Baan (8)
Carrot (81)
IvrlO
Conaanaua
VSDRTFROV- A0A8XVSY
GBSQGbSEKS PRQATAb'PfiY
VSKXSSNLLF AOBGOASEAP
VSHKSPHHPA l-NAYPPANP
VSKXSTAPLL --GSOALQDP
V8.KS -...A
ToaaCo (8)
Carrot (SIS)
Hung Ban (8)
Carrot (SI)
IvrlO
Conaanaua
Tcaaato (8)
Carrot (SII)
Hung Baan (8)
Carrot (81)
IvrlO
Conaanaua
T
rw
m
fH
*PV
2PV
IPV
AP PPOI
it p ppai
ap ppoi
AP PPOI
idAipvIa p ppoi
;s'
JAl
If) I!
If* OPTTJ
PR DPTTJ
ifR OPTTJ
VR DPTTJ
* DPTTA
> rran
A IK
A ILT1
( -
pg Ho-
ICRO CX
.T8I OK
'AjlRTPI OK
atpa cmmf
I.HQEJLCm^.TP. CK
I gsk
I OSK
I OSK
I OSK
I OSK
-ion: /
VNX1 I
LNX1 :
LHK1 ;
)ADH): *
r SPKUXOVLN
[ TYBLLDNLL8
( TYKLKECLLR
C NFTU.DOVLH
i RYUPAPALHJI
AV 3TGHWEC VPf h
KM
TtGHrtC VD*
il| 3TQMHEC VO
TOHWEC VO
3TCHWEC VD <
.Y.LLDOLU1 UviETCHWEC VDftll /
-mum si
l- 8VT053I
SUCHO) 31
8TPOB 11
/AAOS CAAAOSOC 31,
Uli
241
294
279
212
29)
)12
))
33)
324
34)
IvrlO
Carrot (CWI)
Carrot (CWII)
Conaanaua
giPAVAorrr umcmaarpl
IVTI-RNRN YDHOSL--PP
iRTKILV PSSOS9LP
.T.- D.CS.Pr
f>rroPRORA AAOAXQXRPP atptvltaw
BE EE EE
SB
19
11
SO
IvrlO
Carrot (CHI)
Carrot (CWII)
8AVLLLVLVA VTV l,
-SU.ATU.VT TTI
-SIPSPIP -
tSQHVD ^AOOVPAOI DAVWlffaAS
--HIH
N1H
BCVASOVSIX
HC1KY
- 43ST HRVFP
10#
4S
39
Conaanaua
-S.L...LV. .T1
A. H.V.
100
IvrlO
Carrot (CM!)
Carrot (CWII)
Conaanaua
STAPLLOSOA pfcfSWTMAM LAWjVlfcr Qf*JC>nfi
-N JQ VOAIHVK QV-1 17 n IP Ql CQK
-I l-tpiSAVDVK i-V-l IV \f Qt }KI
14p.8A.NVX LV-lfe3j3v4lLfi mlfDP NO I
IP HOI
iflDP HOI
IDP NO I
IS#
#s
7
ISO
IvrlO
Carrot (CWf)
Carrot (CWII)
Conaanaua
20#
1)5
12
200
IvrlO
Carrot (CWJ)
Carrot (CWII)
Conaanaua
I -YTC r
LYTC I
IT Uffi
vbj n
VSP DPI
> JVQf. K PA D 30P .
LTEJivs. .p.N>cvpty
PA n SDF '
paIi.bdp
34#
1 #5
17#
2S0
IvrlO
Carrot (CWI)
Carrot (CWII)
Conaanaua
tyLVPPPOIO PTT
WANT JO EN ATJ
-VOVUTKN- PSJ
'TfKFDPTTA
RDPTTA
-W...O.. PTAtfiCmA.
RTPAONDTA
WLOKSOH
WPO-OGH
W.D.OOM
2##
2)1
22)
)00
IvrlO
Carrot (CWI)
Carrot (CWII)
Conaanaua
TOPA PJ
^ARTICA KHP
XWXRS PHP;
continued
continued
B. Insoluble 3 end
IvrlO
Carrot (CWI)
Carrot (cwit)
Conaanaua
IvrlO
Carrot (CWI)
Carrot (CW(I|
IvrlO
Carrot (CWI)
Carrot (CWII)
IvrlO
Carrot (CWI)
Carrot (CWII)
IvrlO
Carrot (CWI)
Carrot (CWII)
Will
SVRt
mi
nf/DT
Will
H.TRY
ISTRYI
r¡v
t V
vr.
>PAT
TDK
IRVR
(VTjrt urn
TSV
t\(pi rrsv
rrsv.
(VfrASKTfV piVi
II ASKTI III :i
II KA3KTI t 1)1 II
amy bu-i
TiViJpipn
1KT U IW t
rot iiJiynv
rwryf >1 rydv
n.Rvn*
ifprvrp
ifprtu.i:
IKK lllf l
Ufjr
w
P-
P-1
JW I A
JW I IIF
]W I II
aonar-oovA T
t.R SKVXFfiftKQO I
) OSEUIMRHOK p
I 8...P.R.Q. I
U> Vui
^v* or
*rr or
1
AvftevOASDA AOVTXAD-VT f- NC8T8A
VI 1 IfXSLAX RIPmPRWLf YDACXICSLX I
At IfKiUm AX8P0PWIH LDAQOVCOSW |
AitFPXSUW AB.PDP.W.. .DA...C.B. li>T.(J
ft
IP PC
ruplrnwsL orrric
IVI^ lOWTH- xvu .
nl< ITOQKU KVU
.lli^.O..L KVIlfc
a (vela
Carrot (CWI)
Carrot (CWII)
-DOSM
ATDRK
-snxx
^rsr pwywimiij'
wr iakoxtt 1
JtSf IAQRXN1 I
liA.oRTtiiib bvypi
IvrlO THANVXAK6V XI
Carrot (CWI) *FT-ITVEM. Ol
r.urtot (CWII) TtP-ITVWU. Of
conaanaua
T8.-ITV.M. DfUlMS-
JtRHYI xr
mxx
a#is
VTTT8L
Mi--
¡EMN--
)9#
114
Ilf
a
174
)44
497
43)
415
543
471
4CS
591
531
SIS
44#
S71
544
47#
593
SIS
Soluble 3' end
TumIo (S)
Carrot (all)
Hung Baan (8)
Carrot (81)
> IvrlO
Conaanaua
Tcaaato (SI
Carrot (SII)
Hung Baan (8)
Carrot (SI)
l IvrlO
Coin
vl /
K J
< J
fasi.ro
SLOD
su
SLOO
SLOO
sum.
rt Y(
7T vr
rt Yi
JT YI
xntNXwdp'tyi
IHOXW1 P
yuvur p 1
VBOKM r I
ATOTWIP
.KWJi
iPBi r
IPBl
7VXI
IPE1
1 All f
IPllL
1 1A
sxTpyr Kii
SKTTYI )DKB
tk 1KTTYI )HKD
tk turra itocR
ik aUTfYI^VUl
impJ -x- kJIui
TOMto (1)
Carrot (111)
Hung Baan (fi)
Carrot (SI)
B IvrlO
Conaanaua
TomIo (8)
Carrot |#l!)
Hung Baan (8)
Carrot (SI)
IvrlO
Conaanaua
Toaaato 1*1
Carrot ISIII
Hung Baan (8)
Carrot |S!|
IvrlO
/VI c
I /Rl
IU
JU
HI
TCij HI
NI [-QWPV
L0WPV
L0WPV
L0HI
t ti i>
/Lvfiftttlr MlCowr^A IfOlAJOPTV KOVDtQI
1RBYRI DOVXUCI
BLIP KSLJUKfJ:
NXTVP XHVXII
IP DOVAL
EeIj inLOUn]
/ HI LI
83
bP 74a
....r ..v.t.i
IK
L VI 1
i^V
iV
IV
* Vt I
IVMAA*
(IBBAA0
31XTATQ
IIOSOSQ
7V0XATQ
JiV ViU-X.SA.Q
QT
D> -sr
ht
M
I A> I
Lf I
:clxL 4 u 1
rt Py 1 Af
rt nr
rt py if*
rt PY 4
Uta ntisPU-
:< :\
xc rt
rcMi/r mi >ova rr it
It ngqwi
)f Rl
II Bi
Rffc
bHn- M3
IDVl K
IUVT <
J.DV.
VYb¡
VPVII
sj ^PVl
VPV1
vrvt
PPVI
Toarto (S)
Carrot (811)
Hung Baan (8)
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470


TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF FIGURES v
ABSTRACT viii
CHAPTERS
1 INTRODUCTION 1
2 LITERATURE ERVIEW 4
Invertase and its Functions 4
Regulation of Invertase Gene Expression 7
3 ISOLATION AND CHARACTERIZATION OF MAIZE
INVERTASE GENES 16
Introduction 16
Materials and Methods 17
Results 20
Discussion 38
4 DEVELOPMENTAL EXPRESSION AND CARBOHYDRATE
RESPONSIVENESS OF TWO MAIZE INVERTASE GENE
SUBFAMILIES 41
Introduction 41
Materials and Methods 44
Results 48
Discussion 75
5 CYTOKININ MIMICS AND SUPERSEDES THE SUGAR-
INDUCIBILITY OF MAIZE INVERTASE FAMILY MEMBERS
iii


Figure 3-6. Conserved regions within derived amino acid sequences of the IvrlG for maize soluble acid invertase gene
1 and either A, other soluble invertases or B, insoluble invertases from other vascular plants. A, Derived amino
acid sequence similarities shared between the IvrlG cDNA clone for maize soluble invertase, mung bean soluble
invertase (Arai et al., 1992), tomato soluble invertase (Klann et al., 1992), and carrot soluble invertases (Unger et
al., 1994). B, Derived amino acid sequence similarities shared between the IvrlG and the insoluble invertases of
carrot (Sturm and Chrispeels, 1990; Sturm, unpublished data). Boxes in A and B represent the most highly
conserved regions and underlined sequences are those shared among soluble but not insoluble invertases.


30
1
ATC
ATC
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occ
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1312
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V
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p
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L
L
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5
N
L
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1369
CAC
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GAG
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CTC
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Q
W
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V
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V
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N
L
R
M
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1426
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CTC
GAC
CGC
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TCC
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GTG
CCC
CTC
CAC
GTC
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ACG CAG TTC
V
A
L
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V
V
p
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1483
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GAC
GCG
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ACG GAG GCC
D
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B
A
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F
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V
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0
V
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1597
CCG
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CTC
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L
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1654
TTC
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CTC
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CTC
CAA
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P
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L
L
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L
Q
T
F
F
C
Q
D
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1711
CTC
AGG
GCA
TCC
AAG
GCG
AAC
GAT
CTC
GTT
AAG
AGA '
GTA
TAC '
GGG
AGC TTC GTC CCT
L
R
A
S
X
A
N
D
L
V
X
R
V
Y
G
S
L
V
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1768
GTG
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GAT
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GAG
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CTC
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CTG i
GTT '
GAC
CAC
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V
L
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1825
AGC
TTT
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ATC
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CCA
GTC 1
TAC '
CCC ACA CCA GCC
S
F
A
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R
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C
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T
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R
V
Y
P
T
R
A
1882
ATC
TAC
GAC
TCC
GCC
CGC
GTC
TTC
CTC
TTC
AAC .
AAC GCC ACA CAT GCT CAC GTC AAA
I
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D
s
A
R
V
F
L
F
N
N
A
T
H
A
H
V
X
1939
GCA
AAA
TCC
GTC
AAG
ATC
TGG
CAC
CTC
AAC
TCC GCC TAC ATC COG CCA TAT CCG GCA
A
K
S
V
X
X
W
Q
L
N
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1996
ACG
ACG
ACT
TCT
CTA
TGA
T
T
T
S
L


123
Salanoubat, M, and Belliard, G. (1989) The steady-state level of potato sucrose
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115
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89
Experimental Conditions
Experimental treatments were as described by Koch et al. (1992).
Approximately 100 root tips (~ 500 mg) were used for each experimental treatment.
Excised root tips were incubated in the dark at 18 C for 6 to 48 hr in Whites
medium, plus 0.5% glucose, either with or without specific supplemental plant
growth regulators (ABA, A1049; GA, G7645; Kinetin, K0753; IAA, 12886; all from
Sigma). Each group of root tips was agitated at 120 cycles per minute in a 125-ml
side-arm Erlenmeyer flask with 50 ml of sterile media. Airflow (40% 02) through
air stones in each flask was maintained at 250 ml min 1 throughout the incubations.
RNA Isolation and Blot Analysis
Root tip samples were rinsed twice in sterile water, blotted dry, weighed, and
frozen in liquid N2. Samples were ground into fine power in liquid N2 and total
RNA was extracted as per McCarty (1986). RNA was quantified
spectrophotometrically (Sambrook et al., 1989).
Total RNA was separated by electrophoresis in 1 % agarose gels containing
formaldehyde (Thomas, 1980), blotted to a nylon membrane, and fixed by baking
and/or UV treatment (Sambrook et al., 1989). Filters were hybridized at 65 C in a
solution containing 7 % SDS, 250 mM Na2HP04, pH 7.2, 1 % BSA (Church and


92
Results
Cytokinin (5 pM kinetin) exposure evoked a positive response at the level of
message abundance for both the Ivrl and Ivr2 gene subfamilies as well as at the
level of total soluble acid invertase enzyme activity (Figure 5-1). The responses of
both gene subfamilies to kinetin were similar (elevated 2.5-fold), and maximal at 5
pM (tested range from 1 to 200 pM, data not shown). The same treatment resulted
in ca 1.5-fold elevation of total soluble acid invertase activity within these 24 hr
experiments (Figure 5-1 B).
The positive responses of the Ivrl and lvr2 gene subfamilies to exogenous
cytokinin were also evident when the excised root tips were depleted of
carbohydrates (Figure 5-1). Kinetin mimicked and superseded the sugar-enhanced
expression of both the Ivrl and Ivr2 classes of invertase. The fact that kinetin could
replace and override carbohydrate supply in this respect was also evident at the level
of total soluble acid invertase activity (Figure 5-1 B).
When root tips were incubated with exogenous ABA (50 pM [which gives
the maximal response range from 1 to 200 pM, data not shown]) for 24 hr, levels of
message encoding both the Ivrl and lvr2 invertase subfamilies were elevated 1.5-
fold and 3-fold respectively (Figure 5-2 A). The Ivrl subgroup responded less
markedly than did its Ivr2 counterpart. Although maximal responses to exogenous
ABA were observed at 50 pM for transcripts from both the Ivrl and Ivr2 invertase


44
(Maas et al., 1990; Koch et al., 1992), malate synthase, isocitrate lyase (Graham et
al., 1994) and/or photosynthetic pathway (Sheen, 1990) and are considered critical
mechanisms for sensing environmental and developmental signals.
Invertase is one of the only two enzymes known for sucrose breakdown in
vascular plants and has shown a relatively long-term responsiveness to carbohydrate
availability at the enzyme level (Sacher et al., 1963; Glasziou et al., 1966; Ricardo et
al., 1972; Kaufman et al., 1973).
Previous research indicated that invertase was vital at both the specific organ
level and at the whole plant level. Robbins (1958) found that OH43 primary roots
could not grow on sucrose arga, and Duke et al. (1991) showed these roots to be
invertase deficient. Miller and Chourey (1992) also found that the abnormal
development of miniature kernels was associated with an invertase deficiency. The
present study utilizes two acid invertase gene-probes to determine the effects of
developmental processes and altered carbohydrate availability on expression of the
Ivrl and Ivr2 classes for soluble acid invertase genes. The report presented here
also demonstrates the extent of developmental differences and carbohydrate
responsiveness in two subfamilies of maize genes for acid invertase (probably
soluble). These findings indicate that there may be specific roles for soluble
invertases during development and that these could differentially contribute to
adjustment of sucrose import, cellular volume, and possibly metabolism in vascular
plants.


Figure 4-2. Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble acid
invertase and activity of total soluble and insoluble maize invertases during
kernel development. A and B, RNA gel blots with equal amounts (10 pg) of
total RNA from kernels between 6 and 32 DAP (full maturity at ca 30 DAP
under local conditions) were probed with 32P-labeled Ivrl or Ivr2 representing
the two subfamilies of maize soluble acid invertase. Blots were exposed to
X-ray film for two days. Relative abundance of mRNA was quantified by
phosphor image quantifications. C, Total soluble acid invertase activity from
the above tissues. D, Insoluble acid invertase activity from the above tissues.
Values for RNA/protein recovery from this set of kernels were ca 0.04
(+0.02), except at 10 and 12 DAP (0.08 + 0.04) (consistent with changes in
cell division and protein levels during early kernel development).


15
hormones, such as IAA and/or GA, act as signals to target the elevation of invertase
in plant parts infected by certain biotrophic fungi and/or bacteria (Heidecker and
Messing, 1986; Morris, 1986; Collings and Slusarenko, 1987; Davies, 1987;
Libbenga and Mermes, 1987; Morris, 1987; Ishikawa et al., 1988; Sheriden. 1988;
Weil and Rausch, 1990)?
Sturm and Chrispeels (1990) imply that the homology between extracellular
carrot P-fructosidase and the levan hydrolyzing enzyme, levanase, may allow carrot
p-fructosidase (invertase) to hydrolyze the bacterial slime coat. In this way,
invertase action could inhibit bacterial growth directly or make the pathogen
susceptible to further defense reactions. In this scenario, invertase would function in
a positive, protective role as a new and unrecognized pathogenesis-related protein.


0.5% glucose, either with (+K) or without (-K) 5 pM Kinetin, either with
(+ABA) or without (-ABA) abscisic acid (50 pM) 97
Figure 5-3 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of soluble
acid invertase and activity of total soluble acid invertases in maize root
tips incubated for 24hr in Whites basal salts medium supplemented with
0.5% glucose, alone (+0) or with either GA or IAA 99
vii


LIST OF FIGURES
Figure 3-1 Restriction maps of Ivr clones for maize soluble acid invertases ... 26
Figure 3-2 Schematic diagram of the genomic organization of the IvrlG .... 28
Figure 3-3 The deduced amino acid sequence for maize invertase 1 gene 30
Figure 3-4 The hydropathy and fold values of the deduced polypeptide for
maize invertase gene 1 32
Figure 3-5 Conserved regions within derived amino acid sequences of higher
plant invertases 34
Figure 3-6 Conserved regions within derived amino acid sequences of the
IvrlG for maize soluble acid invertase and either other soluble
invertases or insoluble invertases from higher plant 36
Figure 3-7 DNA gel blot analysis of cross-reactivity between Ivrl, Ivr2,
Ivr2C-l and Ivr2C-2 38
Figure 4-1 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize
invertases in root tips, a sink leaf, a source leaf, a prop root, anthers,
silk and kernels 55
Figure 4-2 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize
invertases during kernel development 57
Figure 4-3 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize
invertases in pedicel, middle and top portions of kernels at 8, 10, 12
DAP 59
Figure 4-4 Abundance of mRNA from the Ivrl and Ivr2 subfamilies of
soluble acid invertase and activity of total soluble and insoluble maize
v


8
mechanism the endogenous inhibitors may regulate invertase activities either
spatially or temporally.
Plant Growth Regulators and Invertase
Invertase activity appears to be upregulated by abscisic acid, auxin,
cytokinins and/or gibberellic acid depending on the system and tissues involved
(Sacher et al., 1963; Glasziou et al., 1966; Gayler and Glasziou, 1969; Kaufman et
al., 1973; Howard and Witham, 1983; Morris and Arthur, 1984; Ackerson, 1985;
Schaffer et al., 1987; Weil and Rausch, 1990; Miyamoto et al., 1993; Wu et al.,
1993).
Both auxin and gibberellic acid stimulate cell enlargement, cell elongation
and possibly phototropism and gravitropism (Davies, 1987; Kaufman and Song,
1987; Kim et al., 1993; Wu et al., 1993a; 1993b). The concentration of GA
(gibbellic acid), which promotes growth, closely parallels that which increases
invertase activity in Avena stem segments (Kaufman et al., 1973). The increased
rate of hydrolysis of sucrose to hexose following the stimulation of acid invertase
activity by GA is considered one means of generating an elevated level of osmotic
constituents in the growing region of the stem (Morris and Arhtur, 1985). The
stimulation of both invertase activity and stem growth by auxin is consistent with the
finding that invertases are especially active in tissues undergoing rapid cell
enlargement, such as regions near shoot and root apices (Avigad, 1982). The