Isolation, purification and characterization of Zn-binding factors associated with the citrus blight syndrome

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Title:
Isolation, purification and characterization of Zn-binding factors associated with the citrus blight syndrome
Uncontrolled:
Citrus blight syndrome
Physical Description:
xiii, 85 leaves : ill. ; 28 cm.
Language:
English
Creator:
Taylor, Kathryn Campbell, 1958-
Publication Date:

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Subjects / Keywords:
Citrus -- Diseases and pests   ( lcsh )
Zinc enzymes   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1987.
Bibliography:
Includes bibliographical references (leaves 77-84).
Statement of Responsibility:
by Kathryn Campbell Taylor.
General Note:
Typescript.
General Note:
Vita.

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Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000952681
notis - AER5029
oclc - 16995971
System ID:
AA00003799:00001

Full Text











ISOLATION, PURIFICATION AND CHARACTERIZATION OF ZN-BINDING
FACTORS ASSOCIATED WITH THE CITRUS BLIGHT SYNDROME


By

KATHRYN CAMPBELL TAYLOR


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


1987

































To Bill, for his love
and unwavering loyalty.















ACKNOWLEDGeMENTS


Appreciation is extended to Dr. L. Gene Albrigo for his support,

advice and guidance as chairman and Drs. R.H. Biggs, C.D. Chase, K.E.

Koch, I. Stewart and J.J. Street for their assistance as members of the

supervisory committee. For the use of laboratory facilities I would

like to thank Drs. R.H. Biggs, C.D. Chase, K.C. Cline, D.H. Hubbell,

K.E. Koch and J.J. Street.

Gratitude is expressed to the students, faculty and staff of the

Department of Horticultural Science and the members of Dr J.J. Street's

lab for their enlightening discussions and assistance. For direction

and assistance in the execution of many laboratory procedures and for

her thoughtful discussion, I extend special thanks to Patricia Tolson

Tomlinson. I would like to express my appreciation to Chris Thomas for

many long hours of typing and manuscript changes.

To my son Charles, I am thankful for helping me maintain some

balance. To my husband, Bill, I am most grateful. He gave many hours

of babysitting, lab assistance and always his support in this, as in all

endeavors.


iii















TABLE OF CONTENTS


PAGE

ACKNOWLEDGEMENTS.....................................................iii

TABLE OF CONTENTS.....................................................iv

LIST OF TABLES........................................................vi

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

KEY TO ABBREVIATIONS...................................................x

ABSTRACT.............................................................xii

CHAPTER

i LITERATURE REVIEW.................................................1

Tree Physiology of Citrus and Citrus Blight.......................
Zinc Metabolism.................................................4
Metal Transport and Localization .................................11
The Metal Binding Polypeptides,
Metallothionein and Phytochelatin.............................15
Objectives and Justification for Study............................17

2 MATERIALS AND METHODS ...........................................21

Phloem Tissue Sampling and Extraction........................21
Isolation and Purification of Zn-binding Factor ..................22
Eletrophoretic Evaluation of Isolated Zn-binding Factor..........22
Characterization of Zn-binding Factor..........................23
Methods of Assay............................................23
RCC Comparisons...............................................26
Temperature and pH Stability .................................26
Plant Tissue Localization of Zn-Binding Factor in
Predecline Trees ...........................................26

3 RESULTS AND DISCUSSION ..........................................28

Preliminary Analyses ............................................28
Evidence for a Zn-binding Factor with the
Occurrence of Citrus Blight...................................31
DEAE-Ion Exchange Chromatography (0.25-2.0 M
NaC1 Gradient) ..............................................31









DEAE-Ion Exchange Chromatography (0.50-3.0 M
NaCl Gradient)..................................... ... 42
Correlation of A-254 nm, Total Zn and RCC........................61
Electrophoretic Evaluation of Zn-binding Factor..................63
Plant Tissue Localization of Zn-binding Factor
in Predecline Trees..........................................68
4 SUMMARY AND DISCUSSION ..........................................73

LITERATURE CITED.................................................... .77

BIOGRAPHICAL SKETCH............... ..................................85















LIST OF TABLES


TABLE ,, PAGE

1. Zinc metallo-enzymes involved in plant metabolism...............6

2. Comparative Pb-, Cd-, and Zn-RCC by phloem extracts from
healthy and blight-affected citrus trees, as assayed by DPP.
Samples represent activity of 100 iM of phloem tissue
extract placed in 10 ml of pH 7.0, 0.1 M KN03. (All data
are given on the basis of extractions made on an equivalent
fresh weight basis of 10 g phloem tissue/30 mls 50 mM Tris-
HC1 buffer, pH 7.8.)........................................30

3. NaCl concentrations required to elute the corresponding Zn-
binding peaks within the DEAE-IEC (0.25-2.0 M NaCl) elution
profile. (All data are given on the basis of extractions
made on equivalent fresh weight basis of 10 g phloem tissue/
30 mls 50 mM Tris-HCl buffer, pH 7.8.).......................36

4. Comparison of the maximum levels of A-254 am, ppm total Zn and
ppm RCC in the composite gel filtered material (from 0.5-3.0
M NaCI DRAE-IEC) in 'Valencia' sweet orange and 'Marsh'
grapefruit. (All data are given on the basis of extractions
made on an equivalent fresh weight basis of 10 g phloem
tissue/30 mls 50 mM Tris-HCl buffer, pH 7.8.)................54


5. R values for A-254 nm and TCC correlation. (All data are
given on the basis of extractions made on an equivalent
fresh weight basis of 10 g phloem tissue/30 mls 50 mM
Tris-HCl buffer, pH 7.8.)....................................

6. Comparative Pb-, Cd-, and Zn-RCC by purified Zn-binding factor
from predecline tissue extract, as assayed by DPP. Samples
represent activity of 100 iL of purified material placed in
10 ml of pH 7.0, 0.1M KNO3. DPP was performed with a 25 ppm
Pb, Cd, and Zn solution. (All data are given on the basis of
extractions made on an equivalent fresh weight basis of 10 g
phloem tissue/30 ls 50 mM Tris-HCl buffer, pH 7.8.).........67
















LIST OF FIGURES


FIGURES PAGE

1. Calibration curve for differential pulse polarography assay....25

2. Effect of temperature and pH on the capacity of crude phloem
tissue extract from blight-affected 'Valencia' sweet orange
to complex Zn. (All data are given on the basis of
extractions made on an equivalent fresh weight basis of 10 g
phloem tissue/30 mls 50 mM Tris-HC1 buffer, pH 7.8.).........29

3. Absorbance at 254 nm of phloem tissue extract from healthy,
predecline and decline stage, blight-affected 'Valencia'
sweet orange after IEC on DEAE Sephadex-A-50 with an elution
gradient of 0.25-2.0 M NaC1 (a, b, c, and d represent
fractions with maximum activities). (All data are given on
the basis of extractions made on an equivalent fresh weight
basis of 10 g phloem tissue/30 mls 50 mM Tris-HCl buffer,
pH 7.8.).....................................................33

4. Concentration (ppm) of total Zn in phloem tissue extract from
healthy, predecline and decline stage, blight-affected
'Valencia' sweet orange after IEC on DEAE Sephadex-A-50 with
an elution gradient of 0.25-2.0 M NaC1 (a, b, c, and d
represent fractions with maximum activities). (All data are
given on the basis of extractions made on an equivalent
fresh weight basis of 10 g phloem tissue/30 mls 50 mM Tris-
HC1 buffer, pH 7.8.)....................... ................34

5. RCC (ppm) of phloem tissue extract from healthy, predecline and
decline stage of blight-affected 'Valencia' sweet orange
after IEC on DEAE Sephadex-A-50 with an elution gradient of
0.25-2.0 M NaC1 (a, b, c, and d represent fractions with
maximum activities). (All data are given on the basis of
extractions made on an equivalent fresh weight basis of 10 g
phloem tissue/30 mis 50 mM Tris-HC1 buffer, pH 7.8.).........38

6. Comparison of A-254 nm, ppm total Zn and ppm RCC (showing
coincidence in all 3 assays) in phloem tissue extract from
blight-affected 'Marsh' grapefruit after IEC on DEAE
Sephadex-A-50 with an elution gradient of 0.25-2.0 M NaC1 (a,
b, c, and d represent fractions with maximum activities).
(All data are given on the basis of extractions made on an
equivalent fresh weight basis of 10 g phloem tissue/30 mis
50 mM Tris-HC1 buffer, pH 7.8.)..............................41









7. Comparison of A-254 nm, ppm total Zn and ppm RCC after gel
filtration on Sephadex G-50 of pooled fractions #28-38 from
DEAE Sephadex-A-50 elutionn gradient 0.25-2.0 M NaCl.
Coincidence in all 3 assays was shown in the phloem tissue
extracts from blight-affected 'Valencia' sweet orange.
Zn-binding factor has an apparent molecular weight of 4kd.
(All data are given on the basis of extractions made on an
equivalent fresh weight basis of 10 g phloem tissue/30 mls
50 mM Tris-HCl buffer, pH 7.8.) .............................44

8. Comparison of A-254 nm, ppm total Zn and ppm RCC (showing
coincidence in all 3 assays) in phloem tissue extract from
blight-affected 'Marsh' grapefruit after IEC on DEAE
Sephadex-A-50 with an elution gradient of 0.25-2.0 M NaCI,
with subsequent gel filtration on Sephadex G-50. Zn-binding
factor has an apparent molecular weight of 4kd. (All data
are given on the basis of extractions made on an equivalent
fresh weight basis of 10 g phloem tissue/30 mis 50 mM
Tris-HC1 buffer, pH 7.8.).................................46

9. Comparison of A-254 nm, ppm total Zn and ppm RCC (showing
coincidence in all 3 assays) in phloem tissue extract from
blight-affected 'Valencia' sweet orange after IEC on DEAE
Sephadex-A-50 with an elution gradient of 0.50-3.0 M NaC1.
(All data are given on the basis of extractions made on an
equivalent fresh weight basis of 10 g phloem tissue/30 mis
50 mM Tris-HC1 buffer, pH 7.8.).............................49

10. Comparison of A-254 nm, ppm total Zn and ppm RCC (showing
coincidence in all 3 assays) in phloem tissue extract from
healthy 'Valencia' sweet orange after IEC on DEAE Sephadex-
A-50 with an elution gradient of 0.50-3.0 M NaCI. (All data
are given on the basis of extractions made on an equivalent
fresh weight basis of 10 g phloem tissue/30 mis 50 mM Tris-
HCI buffer, pH 7.8.)..................................50

11. Comparison of A-254 nm, ppm total Zn and ppm RCC (showing
coincidence in all 3 assays) in phloem tissue extract from
blight-affected 'Marsh' grapefruit after IEC on DEAE
Sephadex-A-50 with an elution gradient of 0.50-3.0 M NaC1.
(All data are given on the basis of extractions made on an
equivalent fresh weight basis of 10 g phloem tissue/30 mis
50 mM Tris-HC1 buffer, pH 7.8.).............................52

12. Comparison of A-254 nm, ppm total Zn and ppm RCC (showing
coincidence in all 3 assays) in phloem tissue extract from
healthy 'Marsh' grapefruit after IEC on DEAE Sephadex-A-50
with an elution gradient of 0.50-3.0 M NaC1. (All data are
given on the basis of extractions made on an equivalent
fresh weight basis of 10 g phloem tissue/30 mis 50 mM Tris-
HC1 buffer, pH 7.8.) ....................................53


viii









13. Comparison of A-254 nm, ppm total Zn and ppm RCC after gel
filtration on Sephadex G-50 of pooled fractions 2-13 from
DEAE Sephadex-A-50 elutionn gradient = 0.50-3.0 M NaC1).
Coincidence in all 3 assays was shown in the phloem tissue
extracts from blight-affected 'Valencia' sweet orange.
Zn-binding factor has an apparent molecular weight of 4kd.
(All data are given on the basis of extractions made on an
equivalent fresh weight basis of 10 g phloem tissue/30 mls
50 mM Tris-HCl buffer, pH 7.8.)..............................56

14. Comparison of A-254 nm, ppm total Zn and ppm RCC after gel
filtration on Sephadex G-50 of pooled fractions 2-13 from
DEAE Sephadex-A-50 elutionn gradient = 0.50-3.0 M NaC1).
Coincidence in all 3 assays was shown in the phloem tissue
extracts from blight-affected 'Marsh" grapefruit. Zn-binding
factor has an apparent molecular weight of 4kd. (All data
are given on the basis of extractions made on an equivalent
fresh weight basis of 10 g phloem tissue/30 mis 50 mM Tris-
HCl buffer, pH 7.8.)........................................58

15. Absorbance at 254 nm and 280 nm of gel filtration pooled
fractions #9-20 after polyacrylamide gel electrophoresis.
The predecline stage, blight-affected 'Valencia' sweet
orange phloem tissue extract was partially purified on DEAE-
IEC elutionn gradient 0.50-3.0M NaCI) with subsequent gel
filtration on Sephadex G-50 (1-4 represent species peaks
isolated from PAGE). (All data are given on the basis of
extractions made on an equivalent fresh weight basis of 10 g
phloem tissue/30 mls 50 mM Tris-HCl buffer, pH 7.8.).........64

16. Concentration (ppm) of total Zn in gel filtration pooled
in fractions #9-20 after polyacrylamide gel electrophoresis.
The predecline stage, blight-affected 'Valencia' sweet
orange phloem tissue extract was partially purified on DEAE-
IEC elutionn gradient = 0.50-3.0 M NaCI), with subsequent gel
filtration on Sephadex G-50 (1-4 represent species peaks
isolated from PAGE). (All data are given on the basis of
extractions made on an equivalent fresh weight basis of 10 g
phloem tissue/30 mls 50 mM Tris-HCl buffer, pH 7.8.).........65

17. Comparisons of A-254 nm, ppm total Zn and ppm RCC in healthy
phloem tissue and predecline stage, blight-affected leaf,
stem, phloem, wood, root phloem and feeder root tissue
extract from 'Valencia' sweet orange after IEC on DEAE
Sephadex-A-50 with an elution gradient of 0.50-3.0 M NaC1,
with subsequent gel filtration on Sephadex G-50 of DEAE-IEC
pooled fractions #9-20. These samples were concentrated
approximately 5-fold after gel filtration, prior to assay.
(All data are given on the basis of extractions made on an
equivalent fresh weight basis of 10 g phloem tissue/30 mls
50 mM Tris-HCl buffer, pH 7.8.).............................. 70
















KEY TO ABBREVIATIONS


A-254 nm Absorbance at 254 nm

A-280 na Absorbance at 280 nm

ACTH Adrenocorticotropic hormone

ATP Adenosine 5'-triphosphate

Cys Cysteine

CTP Cytidine 5'-triphosphate

DHAP Dihydroxyacetone phosphate

DNA Deoxyribonucleic acid

DNP 2,4-dinitrophenol

DEAE Diethylaminoethyl

DPP Differential pulse polarography

F-1,6-P Fructose-1,6-phosphate

F-6-P Fructose-6-phosphate

G-1-P Glucose-l-phosphate

G-6-P Glucose-6-phosphate

Glu Glutamic acid

Gly Glycine

GTP Guanosine 5'-triphosphate

IAA Indole 3-acetic acid

IEC Ion exchange chromatography

NAD Nicotinamide adenine dinucleotide

NADH Nicotinamide adenine dihydroxynucleotide

NDP Nucleotide 5'-diphosphate

x









NMP Nucleotide 5'-monophosphate

NTP Nucleotide 5'-triphosphate

OAA Oxaloacetic acid

PAGE Polyacrylamide electrophoresis

PEP Phosphoenolpyruvate

3PGAld 3-phosphoglyceraldehyde

PVPP Polyvinylpolypyrrolidone

RCC Residual complexation capacity

RuBP Ribulose bisphosphate

SOD Superoxide dismutase

TEMED N,N,N',N'-tetramethylethylenediamine

t-RNA transfer ribonucleic acid

TCA Trichloroacetic acid

TCC Total complexation capacity

Tris Tris[hydroxymethyl]-aminomethane

UTP Uridine 5'-triphosphate















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


ISOLATION, PURIFICATION AND CHARACTERIZATION OF ZN-BINDING
FACTORS ASSOCIATED WITH THE CITRUS BLIGHT SYNDROME


By

KATHRYN CAMPBELL TAYLOR


May, 1987


Chairman: L. Gene Albrigo
Major Department: Horticultural Science


Redistribution of Zn within citrus trees has been found associated

with the predecline and decline stages of the disease, citrus blight.

An accumulation of Zn occurs in trunk phloem above the bud union in

blight-affected trees at the predecline and decline stages. This study

determined that Zn-binding factors also were associated with the

occurrence of citrus blight in predecline and decline stages, and that

the Zn-binding factor accumulated in trunk phloem above the bud union.

The Zn binding factors were found to be highly anionic and composed of

at least 4 anionic species by DEAE Sephadex-A-50 ion exchange

chromatography and polyacrylamide gel electrophoresis. Two anionic

species were associated with the healthy tree, while all 4 were

associated with the occurrence of citrus blight. The metal- binding

factors displayed absorbance at 254 nm, indicating their metal-thiol

chromophore character and they lacked absorbance at 280 nm. These are

xii









common traits of certain metal-binding molecules, phytochelatins. They

were capable of removing Zn from a Zn-containing solution, as assayed by

differential pulse polarography (DPP). The fractions from

chromatography which displayed absorbance at 254 nm and which were

capable of removing Zn from a Zn-containing solution also contained

elevated levels of Zn. These characteristics combined are

circumstantial evidence that this metal binding factor is a Zn-binding

factor and a phytochelatin. All the Zn-binding factors were isolated

with an apparent molecular weight of 4kd, using Sephadex G-50, fine, gel

filtration.

Phytochelatins are reported to be involved in heavy metal

homeostasis in plants. In the case of citrus blight, the Zn-binding

factors may be produced in excess, or new forms of these Zn-binding

factors may be produced and remove Zn from availability for Zn-requiring

enzymes. In this way Zn-binding factors may alter Zn metabolism of the

tree somehow contributing to the occurrence of citrus blight, which is

manifested as a decreased water conductivity and general decline of the

tree.


xiii















CHAPTER 1
LITERATURE REVIEW


Tree Physiology of Citrus and Citrus Blight


Citrus blight is a major cause of tree loss and reduced fruit yield

in citrus production areas in Florida (Smith, 1974) and around the

world, with the exception of citrus production areas in Mediterranean

climates (Wutscher et al., 1977; Graca and van Vuuren, 1979; Childs,

1979b; Lima, 1982). It is second only to freezes as a cause of mature

tree loss in Florida. Several comprehensive reviews of blight

literature are available (Childs, 1979a, 1979b; Cohen, 1968; Rhoads,

1936; Smith, 1974; Smith and Reitz, 1977).

The blight disorder has received attention by citrus growers and

scientists in Florida for nearly 100 years. The earliest descriptions

of citrus blight were reported in 1891 by Underwood and 1896 by Swingle

and Webber (Rhoads, 1936). Two possible causes of blight were cited:

1) local soil conditions or 2) a contagion (Rhoads, 1936). The latter

was considered less likely because no organism had been found associated

with blight and it had not been transmitted in budwood to trees

propagated from buds from wilted portions of typical blighted trees.

The cause of blight still remains unknown. The disease is regarded

as an extremely complex decline problem due to the fact that on occasion

it appears to be influenced by cultural, climatic and edaphic factors

(Young et al., 1980; Nemec et al., 1982; Wutscher and Hardesty, 1979;

Wutscher, 1981b). Additionally, there is evidence of transmission

1









by continuous root grafts (Tucker et al., 1984; Tucker et al., personal

communication, 1986); however, no transmitted organism has been

isolated. With a scattered array of influences and effects of blight, a

single cause seems contraindicated. Yet the fact that it is

transmissible suggests a single cause. Because symptom development is

the best documented information available regarding citrus blight, a

system of working backwards through symptom development ultimately to

the cause has seemed the most suitable approach. There are three recent

findings which appear to hold promise in understanding the citrus blight

syndrome.

1) Water conductivity decreases with progression of the blight

syndrome (Young and Garnsey, 1977). This decrease is due to

increased plugging of the inner xylem vessels associated with

wilt and the decline stage (Brlansky et al., 1984). Amorphous

plugging is responsible for the reduced H20 conductivity which

leads to wilt.

2) Zinc accumulates first in the phloem and then in the outer wood

in the very early stages of decline, prior to xylem plugging

(Albrigo et al., 1986).

3) Blight is transmitted from blight-affected trees to healthy

trees through root grafts in 2-3 years. In contrast, in

side-by-side plantings of non-grafted healthy and blight-

affected trees, the healthy trees do not develop blight in that

period (Tucker et al., 1984). Transmission is indicated when

trees show Zn accumulation followed by reduced H20

conductivity.









Of these three recent findings, most significant (because it is the

first known change) is the finding that Zn accumulates first in the

phloem and then in the outer (youngest) wood of the trunk prior to the

development of xylem plugging in the inner (older) trunk wood (Albrigo

et al., 1986; Young et al., 1980). This finding suggests the Zn

redistribution may result in Zn deficiencies observed in other portions

of the tree during the development of the blight syndrome. Zinc

deficiency patterns in leaves can accompany Zn accumulation in the

phloem and wood if trees are not provided foliar applications of Zn

(Rhoads, 1936).

There are other alterations in metabolite levels which occur either

concurrently or following wilt of the tree. Wood pH (Wutscher et al.,

1983) and wood phenolics (Wutscher et al., 1977; Wutscher, 1981a;

Wutscher, 1983) increase as do scion bark and leaf proline (a typical

water stress response) and trunk nitrogen (an indication of metabolite

imbalance) (Hanks and Feldman, 1974; Syvertsen and Albrigo, 1984). ATP,

carbonic anhydrase (Bausher, 1979) and IAA oxidase (Bausher, 1982)

activities decrease. Carbonic anhydrase and IAA oxidase are

Zn-requiring enzymes (Randall and Bouma, 1973; Lindskog, 1983; Vallee,

1983). Decreased activities of these enzymes may be a secondary

response to Zn redistribution within the tree. The other metabolite

changes also may be secondary responses due to stresses brought about by

the Zn redistribution.

The reduction of feeder roots beneath the tree is an additional

change occurring in blight-affected trees (Burnett et al., 1982; Nemec

et al., 1982). This occurs during the later stages of the blight

syndrome (Albrigo et al., 1986; Cohen, 1968). Such root loss may be due









to a secondary infection of Fusarium solani Mart. It has been suggested

that reduced carbohydrate supply to the roots and the consequent

reduction in root vigor would be permissive to such secondary infection

(Graham et al., 1983).

Several mineral element concentrations are altered as well

(Wutscher and Hardesty, 1978; Williams and Albrigo, 1984). All

elemental changes with the exception of Zn occur either concurrently or

after visible wilt (Albrigo et al., 1986). Zinc accumulates first in

the active phloem and then in the outer wood. The Zn redistribution

occurs one to three years prior to xylem plugging which develops in the

inner wood and is responsible for H20 deficits and wilt symptoms

(Wutscher et al., 1982; Albrigo et al., 1986). Zinc accumulation occurs

in a different location from the xylem plugging. Thus Zn accumulation

and xylem plugging are not directly linked in symptom development, in

terms of location and time. Zinc accumulation may also be a secondary

physiological response to a blight inducing agent which may be

transmissible through root grafts. However, it is the earliest known

response associated with the development of blight since it occurs prior

to reduced water conductivity (Tucker et al., 1984: Albrigo et al.,

1986). Therefore the mechanism which mediates this redistribution needs

to be studied.


Zinc Metabolism


Many enzymes in higher plants have been determined to require the

presence of Zn as a cofactor for their optimum activity. Zinc is a IIB

transition metal with 2s electrons which combine in the 2+ oxidation

state. Zinc is neither oxidized nor reduced in biological reactions.









Zinc is considered to be poised for its intended catalytic function in

enzymes due to its chemical characteristics and because it is capable of

octahedral, tetrahedral and pentagonal coordination (Vallee, 1983).

The first nutritional study of Zn was performed by Raulin in 1869 when

he demonstrated the necessity of Zn for the growth of Aspergillus niger

Mich. Keilin and Mann (1940) isolated the first Zn-requiring enzyme,

carbonic anhydrase, from mammalian erythrocytes. They demonstrated that

the protein contained 0.33% Zn. Since that time almost 200 Zn-enzymes

have been isolated from plant and animal systems (Vallee, 1983). A list

of some of the Zn-requiring enzymes and their roles in plant metabolism

is given in Table 1.

Some of the key areas where Zn imbalance within the plant may have

significant effects on plant metabolism are discussed below. The

information given in the following paragraphs was taken from Goodwin and

Mercer (1983), except where noted.

Superoxide dismutase (SOD) is important for quenching the free

radicals that are naturally generated in the high energy light reactions

of photosynthesis. If the superoxide anionic free radical is not

reduced via the SOD mediated reaction (02-10-h202+ 02), the cell's

membranes (especially chloroplast membranes) would be irreversibly

damaged and photo-oxidation would occur. This refers to the destruction

of the "photosynthetic machinery." The energy for photosynthetic carbon

reduction, which fuels carbon biosynthesis, would not be available

without the completion of the photosynthetic light reactions.

Carbonic anhydrase catalyzes the attainment of equilibrium between

CO2 (substrate for RuBP carboxylase) and HCO3 (substrate for PEP

carboxylase) (Randall and Bouma, 1973; Goodwin and Mercer, 1983). In


























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the chloroplast, stromal pH increases from 7 to 8 favoring the formation

of HCO3 in the light. Carbonic anhydrase maintains the equilibrium so

that RuBP carboxylase activity and hence CO2 fixation is favored.

Through this equilibrium, substrate is provided for carbon storage as

starch or for immediate use in ATP generation via the tricarboxylic acid

cycle. If HCO3 formation were favored, little CO2 would be stored as

starch. This would leave the plant unprotected against periods of

carbon deficit. Carbonic anhydrase activity and photosynthesis are

reduced and respiration is increased in Zn-deficient soybeans (Glycine

max L. Merrill) (Ohki, 1978). Carbonic anhydrase also was substantially

reduced in Zn deficient Citrullus vulgaris Schrad. (Sharma et al.,

1981).

One of the enolases is responsible for the formation of PEP which

is a pivotal high energy intermediate in carbohydrate biosynthesis.

From PEP, carbohydrate biosynthesis may proceed in either a

gluconeogenic or glycolytic direction.

Nucleotide pyrophosphatase is responsible for the synthesis of

nucleotide diphosphates which are the precursors of GTP, CTP, UTP and

ATP. Adenosine 5'-triphosphate (ATP) is the primary energy currency in

organisms. The nucleotide 5'-triphosphate, ATP, would be decreased due

to lack of substrate if the level of nucleotide 5'-diphosphate, ATP,is

decreased. Adenosine 5'-triphosphate synthesis is found decreased in Zn

deficient Alyssum bertolonii Desv. (Grossi, 1985). Sharma et al. (1981)

report increased inorganic phosphate and decreased organic phosphate in

Zn-deficient Citrullus vulgaris Schrad.

Phosphoglucomutase is a pivotal enzyme for gluconeogenesis versus

oligo/polysaccharide synthesis. A changed rate of reaction for this

enzyme could alter the normal ratio of glucose-l-phosphate to









glucose-6-phosphate. Glucose-6-phosphate is the immediate precursor for

the glucose moiety which is used in sucrose biosynthesis.

Glucose-1-phosphate is a precursor for the formation of the

oligo/polysaccharides. A reduction in phosphoglucomutase activity would

decrease the level of sucrose (a non-reducing sugar). Sharma et al.

(1981) noted a marked accumulation of reducing sugars and decrease in

non-reducing sugars. The above may be a possible explanation for this

observation.

Nason et al. (1951) reported that the activity of tryptophan

synthase was decreased in Zn-deficient Neurospora sp. It has been

observed that Zn-deficient plants behave as if they are auxin deficient

(Skoog, 1940). Tryptophan synthase has been shown to be involved in IAA

biosynthesis (Goodwin and Mercer, 1983), as tryptorhan is the key

precursor for IAA via the indole 3-pyruvic acid pathway.

In the studies of Zn-stressed Citrullus vulgaris Schrad., other

noted metabolic changes were marked accumulation of non-protein nitrogen

[similar to the non-protein nitrogen increase reported in blight-

affected citrus (Syvertsen and Albrigo, 1984)] and a decrease in protein

nitrogen and nucleic acids; and stimulated acid phosphatase and

ribonuclease activities (Sharma et al., 1981). The reason for these

changes may be that the activities of RNA and DNA polymerases as well as

t-RNA synthetase would be reduced with Zn-deficiency. This would result

in an accumulation of untranslated RNA's which would require the

increased activity of ribonuclease. Nucleic acid levels and substrate

for protein synthesis would be reduced, decreasing the level of protein

nitrogen. There would likely be a consequent accumulation of

non-protein nitrogen.









Zinc also appears necessary for the maintenance of ribosome

stability and in the binding of activators and/or repressors which

affect gene expression. Specifically, the species of histones present

in Euglena gracilis cells grown in (-Zn) culture are quite different

than when grown in (+Zn) culture. Thus Zn could be potentially growth

limiting, as uncommon histones may be formed that could have a negative

effect on growth potential via altered gene expression. Modification of

histones by Zn appears to be through the metal-dependent methylation or

phosphorylation of DNA affecting transcription or through interaction

with arginine-rich peptides which inhibit activity of RNA polymerase II

(Vallee, 1983). Prask and Polcke (1971) reported that cytoplasmic

ribosomes of E. gracilis normally contain substantial amounts of Zn and

that they become extremely unstable with Zn deficiency. Instability of

ribosomes with Zn deficiency may cause an increase in RNA simply because

there is no protein synthesis occurring. This too would account for a

required increase in ribonuclease activity, such as that noted by Sharma

et al. (1981). The reduction of RNA and ribosome cell contents is

suggested as the earliest possible causal event in symptom development

in the course of Zn deficiency (Price et al., 1972).

Likewise, Zn redistribution in citrus trees may cause a metabolic

upset of proportions significant enough to cause the blight syndrome.

An altered Zn distribution, causing the accumulation of Zn in one tissue

which affects Zn-deprivation in another tissue, would reduce the

availability of the cofactor for Zn-requiring enzymes. As the above

discussion emphasizes, reduction of activity of these enzymes may have

significant detrimental effects on the overall metabolism of a plant.









Metal Transport and Localization


Linehan (1984) provided a model for Zn adsorption by roots and

subsequent plant uptake. The model suggests that metals adsorbed on

root surfaces are remobilized via organic ligands which leak from root

cells. This ligand leakage is induced by the presence of the metal ion.

Lack of an external liquid diffusion path away from the root, due to the

transpirational stream, prevents the accumulation of metal-ligand

complexes in the free space and superficial water film of the root.

Therefore the organo-metal complex is taken into the root cells and

translocated along the plant axis. Thus the level of metal that is

xylem transported is related to the level of metal adsorbed by their

roots.

Linehan's model would accommodate the commonly used citrate model

for metal binding and displacement reactions. Citrate is often used in

this model due to its affinity for a diverse number of metals (Tiffin,

1972). In addition, citrate has been found to be a primary if not the

primary organic ligand for Zn as well as several other metals (Chino and

Baba, 1981; Godbold et al., 1984; Linehan, 1984; McGrath and Robson,

1984a; Tiffin, 1967; Tiffin, 1972; Van Goor and Wiersma, 1976). McGrath

and Robson (1984a) were able to show that more Zn passed into excised

stems treated with the citrate-ligand, than those treated with Zn:alone.

In studies of the adaptive mechanisms of plants to toxic levels of

metals, Godbold et al. (1984) found that the Zn accumulation in tolerant

ecotypes of Dechampsia caespitosa (L.) Beauv. as compared to non-

tolerant ecotypes was correlated with the accumulation of citrate in the

root sap.









Linehan suggests a concentration dependent component of metal in

the soil solution that directs the level of metal moving through the

plant axis. McGrath and Robson (1984b) found a similar correlation with

less Zn moving up the axis of Pinus radiata D. Don under Zn deficiency

conditions. In addition, they found xylary movement of Zn to be

extremely slow. There was movement of Zn along the tree's axis 2 weeks

after tracer application, even with sufficient levels of Zn.

The citrate model incorporates a displacement component. One

report suggests a Cd-Zn antagonism (Wong et al., 1984). At low Cd

levels Zn uptake is stimulated, and at high Cd levels Zn uptake is

reduced. Cutler and Rains (1974) found a competitive interaction

between Cd and Zn in excised barley roots.

Linehan's model and the citrate model appear to suggest passive

metal uptake. In contrast, Chino and Baba (1981) suggested a

requirement of metabolic energy for uptake, since they found that low

temperatures and shading decreased the rate of Zn and Cd uptake as

organo-metal complexes. It could be argued that these factors merely

reduced the rate of transpiration thereby decreasing the rate of uptake.

However, Cataldo et al. (1983) used DNP to show that the metabolicallyy

absorbed" fraction represented 75-80% of the absorbed fraction of Cd.

Zinc, Mn, Cu and Fe competed well with Cd for absorption in the study

reported by Cataldo et al. (1983). This suggested the existence of a

common transport site or process.

While study of the mechanism of root absorption and xylem transport

is well documented, the mechanism of ion-phloem transport is not. The

information available is mainly qualitative. An exception to the above,

is the wealth of information concerning K -phloem transport. However,









this review is mainly concerned with the transport of Zn and any cations

that may be related to Zn in terms of charge and size.

The phloem integrity comprising leaf, petiole, and stem traces

indicates that leaf nutrients can move to other organs without extensive

diversion to the xylem (Tiffin, 1972). A 1C-acetate study has

demonstrated assymetrical seed head development of sunflower when source

leaves from one side of the stalk were removed, limiting lateral

transfer of assimilates to deprived seeds (Prokofyev et al., 1957).

Although much is known about macronutrient and sugar transport in the

phloem, few studies deal quantitatively with micronutrient movement out

of source-leaves or their form of transit.

In his review, Tiffin (1972) reports evidence of foliar applied Cu

moving into new leaves and fruit; Fe into roots, or from seed to

seedling; and physiological and seasonal changes in micronutrient levels

of foliage (suggesting remobilization to other tissues, e.g., to

developing fruit, seeds or from senescing tissues into storage tissues).

When Bukovac and Riga (1962) followed Zn, P and Ca distribution from

bean cotyledons to other plant parts, they found that 51% of Zn moved

out of the cotyledons within 6 days of cotyledon emergence (Zn was most

readily translocated). These are all suggestive of phloem involvement

in metal transport.

In tracer experiments using steam girdled and non-girdled

nonsenescent geranium petioles, Neumann and Chamel (1986) report of the

phloem mobility of Ni, another divalent transition metal of a molecular

weight similar to Zn. They demonstrated that 63Ni was one-fourth as

mobile as 86Rb (a very mobile cation used as a K-analogue) and 25 times

as mobile as Ca (a very non-mobile cation). Mengel and Kirkby (1981)









report that Ni is very phloem mobile. Zinc, Ni and Co have been

detected in phloem exudates from bark incisions in stems of Ricinis

communis L. (Wiersma and Van Goor, 1979). In addition, it has been

demonstrated that Ni and other minerals are mobilized and transported to

seed sinks via the phloem (Hocking and Pate, 1977; Misha and Kar, 1974;

Neumann and Noodin, 1984). At senescence, >70% of the Ni present in

soybean shoots was remobilized to seeds (Cataldo et al., 1978), >90% of

which was associated with the soluble tissue fraction. With

ultrafiltration of the soluble fraction >77% was shown to be in the

range of 0.5-10.0 kd. Several organic complexes were resolved from the

soluble fraction. Most of the remobilized Ni was associated with the

cotyledons, 80% was soluble, 70% of the soluble fraction was composed of

Ni-complexes with molecular weights <10kd. A soluble organic-Zn complex

has been isolated in Ricinis communis L. (Van Goor and Wiersma, 1976).

The molecular weight of this complex is estimated at approximately

0.5kd.

In summary, the literature suggests that Zn moves into the root via

citrate through an exchange site which may be common to Cd, Mn, Cu and

Fe. This uptake may be dependent on a metabolic component, although

soil-Zn concentration appears important to the magnitude of Zn moving up

the plant axis. This movement via the xylem appears to be very slow.

Little information is available concerning phloem transport and

even less on Zn-phloem mobility. It is apparent that Zn is phloem

mobile and possibly more mobile in the phloem than xylem, comparing the

results of the Bukovac and Riga (1962) study with those of the ticGrath

and Robson (1984a;b) studies. Zinc moves through Ricinis communis L.

phloem as a soluble small molecular weight complex of 0.5kd (Van Goor









and Wiersma, 1976). Similarly, Ni appears to move through the phloem as

an organo-metal complex of 0.5-10.0 kd. The Ni is in a highly soluble

fraction that is very mobile. Are the organo-metal complexes formed in

the phloem in association with Zn and Ni related? In addition, are

there similar complexes in the phloem that may be responsible for the

transport of other heavy metal divalent cations?


The Metal Binding Polypeptides, Metallothionein and Phytochelatin


Recent concern over heavy metal contamination of the environment

(Varma and Katz, 1978) has stimulated research on plant response to

toxic levels of heavy metals (Bartolf et al., 1980; Casterline and

Barnett, 1982; Girling and Peterson, 1981; Jarvis et al., 1976; Kaneta

et al., 1983; Page et al., 1972; Petit and Van De Geijn, 1978; Rauser

and Curvetto, 1980). Because metallothionein is an extremely effective

sequestering agent of heavy metals in mammalian systems (Kagi et al.,

1980), several groups have investigated a similar role in plants

(Bartolf et al., 1980; Casterline and Barnett, 1982; Hardiman and

Jacoby, 1984; Hogan and Rauser, 1981; Jackson et al., 1985; Wagner and

Trotter, 1982).

Metallothioneins are low molecular weight cysteine-rich proteins

which appear responsible for heavy metal homeostasis in animals (Kagi

and Vallee, 1961; Nordberg and Kojima, 1979). The role of

metallothioneins in heavy metal homeostasis involves the sequestering of

toxic metals (Hg and Cd) and the regulation of essential elements such

as Zn and Cu. Other functions have been proposed, including regulation

of cellular metabolism and growth, detoxification of free radicals and

protection against ionizing radiation (Karin, 1985).








In their review, Kagi et al. (1980) reported that these highly

anionic proteins are characterized by a single polypeptide chain 61

amino acid residues in length. The chain contained 20 cysteine

residues. Because they lack histidine and aromatic residues, these

proteins do not show absorbance at 280 nm. Serine, lysine and alanine

are also abundant in these proteins. Metallothioneins sequester the IB

and IIB transition metals at distorted tetrahedral coordination sites of

-cys-x-cys- in a ratio of 1:3, metal ion: cysteine residues. Metal-

thiol chromophores exhibit absorbance at 254 nm. Integrity of these

chromophores is reversibly lost below pH 2.0. A decrease in absorption

with acidification occurs due to the breakage of the metal-mercaptide

bonds (Macara, 1978). The metal-thiol complex is heat stable.

Metallothionein synthesis is induced by the presence of IB and IIB

transition metals (Durnam and Palmiter, 1981). This increase is

accomplished by the increased transcription of a multigene family (Karin

and Richards, 1982) and in some cases by the amplification of a single

gene member (Beach and Palmiter, 1981).

Recent isolation and characterization of metal binding polypeptides

(phytochelatins) from higher plant cell suspension cultures has revealed

that their structure and performance may differ somewhat from their

functional analogues in animal systems (Grill et al., 1985; Jackson et

al., 1985; Robinson et al., 1985; Wagner and Reese, personal

communication, 1986; Robinson and Jackson, 1986). Although they also

are cysteine-rich, these polypeptides do not contain serine, lysine and

alanine, but they are rich in glutamic acid (Lolkema et al., 1984; Kondo

et al., 1985; Rauser, 1984; Rauser and Glover, 1984; Webb and Cain,

1982). The peptide chain contains 7-15 amino acids of which 3-8 are


cysteine.








The latest evidence indicated that phytochelatins have a polymeric

structure of (y-glutamyl-cysteine) -glycine (n= 3-8) (Grill et al.,

1985; 1986; Jackson et al., 1985). The Cd-binding peptides cadystin B

and cadystin A, from Schizosacchoromyces pombe, are structurally similar

to the metal-binding polypeptides in plants. However, the (y-glu-cys)

unit is repeated only 2 times (n- 2). Thus the repeating unit of the

metal-binding polypeptide may have a value of n- 2-8 (Kondo et al.,

1985). Phytochelatins from Agrostis gigantea Roth. have a greater

binding efficiency of 1:2, Cu: cysteine residue (Rauser and Curvetto,

1980) when compared to metallothionein proteins.

Presence of the y-glu-cys linkage rather than a peptide linkage

suggests that this polypeptide is not a primary gene product but rather

a biosynthetic product. Enzymes involved in glutathione ( glutamyl-

cysteinyl-glycine) synthesis may be involved in the synthesis of

completing peptides based on their structural similarities.

This class of polypeptide (phytochelatin) may bind not only IB and

IIB but also transition metals from groups VIII, IVA and VA (Grill et

al., 1985; 1986). Phytochelatins, like metallothioneins, also exhibit

absorbance at 254 un but lack absorbance at 280 nm. Their synthesis is

metal inducible. Phytochelatins are heat stable, labile at or below pH

2 and highly anionic, like the animal polypeptides, metallothioneins.



Objectives and Justification for Study


Two questions were key to the understanding of the etiology of

citrus blight, because Zn redistribution is the earliest change in

symptom development of blight, and the activities of at least 2








Zn-requiring enzymes are known to be reduced at the decline stage of

blight (after Zn redistribution in the predecline stage). The first is

the one of primary concern in this study: is the Zn redistribution

directly associated with blight and what is the mechanism of the

redistribution? Second, if so, does this redistribution alter Zn

metabolism and cause blight symptoms?

In this study the mechanism and the path of the Zn redistribution

were sought. The literature suggests that Zn is more phloem mobile than

xylem mobile (Bukovac and Riga, 1962; McGrath and Robson, 1984a;b). In

addition, the phloem above the bud union is the site of Zn accumulation

(Albrigo et al., 1986). Therefore, the phloem above the bud union was

the primary tissue sampled in the study. There is evidence of a

soluble, low molecular weight organo-metal complex in the phloem

(Cataldo et al., 1978, Van Goor and Weisma, 1976). Because

phytochelatins are documented as low molecular weight, heavy metal

completing agents, methods for their study were implemented, as the

nature of the mechanism of Zn redistribution was sought in this study.

Early, at the predecline stage in the developmental progression of

the blight syndrome, Zn accumulated in the phloem (outer trunk).

However, 1-2 years later, when xylem plugging occurred at the decline

stage, there was no accumulation of Zn in the plugged xylem (inner wood)

(Albrigo et al., 1986; Young et al., 1980). Separation of these events

in time and space suggests that Zn accumulation and xylem plugging are

not directly related. Yet, the fact that the latter never takes place

without first the occurrence of the former would imply a possible

relationship. Therefore, study of the occurrence of a Zn completing

agent during development of the blight syndrome and with regard to








location within the tree was performed to better understand the implied

relationship, if it exists. For the study of a completing agent during

blight development, phloem tissue was sampled from healthy, predecline

and decline stage trees. In addition, to determine the presence of the

completing agent with regard to location, several sites were sampled

within predecline stage trees.

Since Zn accumulated in one plant tissue, the trunk phloem above

the bud union (Albrigo and Young, 1981), while at times it became

deficient in another, the young leaves (Rhoads, 1936), the accumlation

of a completing agent in the trunk phloem was suggested. To generate

excessive levels of the completing agent, its synthesis must be induced.

The nature of this induction was of interest, with regard to synthesis

of a new Zn-complexing metabolite versus amplified synthesis of an

existing metabolite. This was of particular interest in view of

evidence of blight transmission (Tucker et al., 1984). The data of

Tucker et al. (1984) data suggest the involvement of a transmitted

agent. This agent could have two possible roles. One, it may be the

Zn-complexing metabolite. Two, it could induce the synthesis of the

Zn-complexing metabolite.

Ultimately, information about the mechanism of the blight-induction

is sought. Does the transmitted agent simply amplify transcription of a

normally expressed gene or does it induce expression of a normally non-

expressed portion of the genome?

Phytochelatins are regarded as normal metabolites for maintaining

heavy metal homeostasis. If a Zn-complexing agent associated with

blight is a phytochelatin, then induction of amplified synthesis by the

transmitted agent is implied. In this event, even the phytochelatin








could be the transmitted agent. However, somewhere synthesis of the

phytochelatin would have been amplified, either in a plant source or

possibly in a microbe capable of phytochelatin synthesis.

The primary objective of this study was to determine if a

Zn-binding molecule exists and to isolate, purify and characterize the

molecule if its presence correlated with the occurrence of citrus

blight. A secondary objective was to determine the primary location of

the accumulated completing agent. To determine if the Zn-complexing

agent was a phytochelatin, methods for their isolation and purification

and assays for their characterization were used in the study.

Isolation and purification procedures for plant and animal

metal-binding polypeptides are very similar. Most laboratories working

with these systems use various combinations of gel filtration, ion

exchange chromatography, electrophoresis, ammonium sulfate or heat

precipitation, ultrafiltration, dialysis and lyophilization (Bartolf et

al., 1980; Casterline and Barnett, 1982; Grill et al., 1985; Jackson et

al., 1984; Lerch, 1980; Lolkema et al., 1984; Rauser and Glover, 1984;

Wagner and Trotter, 1982). Precipitation procedures are more

appropriate for the larger molecular weight proteins from animal

systems. Casterline and Barnett (1982) explained that such treatment

resulted in some loss of Cd-containing fractions of low molecular weight

in soybeans. Concentrating procedures are most useful when detectable

levels of the metal-mercaptide complex are limited. The major

purification procedures used are based on molecular sieving and ionic

characteristics, i.e. gel filtration and ion exchange, respectively.















CHAPTER 2
MATERIALS AND METHODS


Phloem Tissue Sampling and Extraction


'Valencia' sweet orange and 'Marsh' grapefruit cultivars on rough

lemon or carrizo rootstocks from 3 groves were sampled for phloem tissue

from healthy, predecline and decline stages of citrus blight. Healthy,

predecline and decline trees were screened and separated on the basis of

Zn levels and water conductivity. Decline trees had higher Zn

concentrations with reduced water conductivity. Predecline trees had an

elevated Zn concentration and normal water conductivity, while healthy

trees had lower Zn levels and normal water conductivity (Albrigo et al.,

1986). One 10 x 10 cm bark patch was removed from each sampled tree

approximately 25 cm above the bud union. The bark patch was divided

into ten, 1 cm strips and transported in a plastic bag on ice from the

grove to the lab. Bark strips were rinsed in deionized water and the

phloem was separated from the periderm with a stainless steel blade.

Ten grams (f.w.) of phloem tissue were ground for 20 minutes in a 50 ml

sorvall grinder cup cooled in an ice bath. For grinding, the ten gram

tissue sample was placed in approximately 25 mls of cold buffer

containing 50 aM Tris-HC1, pH 7.8 with 50 mM 2-B-mercaptoethanol and 5%

PVPP. The resulting homogenate was filtered through Whatman No. 1

filter paper with buffer washes up to a final volume of 30 mls/1O g

phloem tissue (f.w.). The filtrate was centrifuged at 4%C for 20

21








minutes at 15,000 x gmax The supernatant was filtered through a

0.45 Un filter and transferred to a 0.2 Ua millipore filter apparatus

for final filtration, in order to eliminate bacterial contamination.


Isolation and Purification of Zn-binding Factor


A 10 ml aliquot of the resulting phloem tissue extract was loaded

onto a 5 x 4.1 cm column of DEAE-Sephadex A-50 for ion exchange

chromatography (IEC). The column was equilibrated with 50 mM Tris-HC1,

pH 7.8. A 280 ml linear gradient of either 0.25-2.0 M NaC1 or 0.5-3.0 M

NaC1 in 50 mM Tris-HC1, pH 7.8 was applied. The flow rate was

approximately 40 ml/h with either 5.0 0.2 ml or 9.5 0.2 ml fractions

collected at 4C. Fractions with absorbance at 254 nm and coincident Zn

peaks were pooled and a 5 ml aliquot applied to a 20 x 4.1 cm column of

Sephadex G-50 (fine) in the same buffer also at 4C. The column was

developed at a rate of 30 ml/h. Fractions of either 5.0 0.2 ml or

9.5 0.2 ml were collected. Appropriate fractions were pooled and

lyophilized.


Electrophoretic Evaluation of Isolated Zn-binding Factor


Purity of isolated material was assessed on a polyacrylamide gel

electrophoresis (PAGE) run in 0.15 M Tricene, pH 6.95. This gel system

was specifically designed for detection of anionic, low molecular

weight, metal-binding proteins from suspension cultured cabbage and

tobacco cells (Reese and Wagner, personal communication, 1986). The gel

was a 20% aqueous acrylamide solution (0.8% bisacrylamide) and contained

0.04% (w/v) TEMED, and 0.04% (w/v) ammonium persulfate in 1.0 M Tris-

HC1, pH 7.6. Electrophoresis was performed at room temperature at a








constant current of 30 mA using 0.002% bromophenol blue as the tracking

dye. Prior to electrophoresis, 45 1i of sample was mixed with 5 1i of

glycerol containing 2.0% bromophenol blue in 0.15M Tricene as a sample

buffer. Duplicate gels were run. One gel was sliced into 1 x 1.5 cm

sections, eluted in distilled/deionized H20 and analyzed for total Zn,

A-254 nm and A-280 nm. The other gel was fixed for 5 min. in 12.5% TCA

and stained for 30 min. in 0.2% coomassie blue and 12.5% TCA. This

stained gel was developed in 10% TCA (Chrambach and Rodbard, 1981).

Coomassie blue is a protein stain. It is unable to stain the small

Y-polypeptide, phytochelatin. The absence of staining and A-280 nm

activity was used as confirmation of the purity of the isolated material

from classical proteins.


Characterization of Zn-binding Factor


Methods of Assay

Presence of coincident metal-thiol chromophore and Zn concentration

in chromatography fractions was determined as evidence of the presence

of a Zn-binding factor. Metal-thiol chromophore was assayed at A-254 nm

with a Perkin-Elmer Lambda 3A UV/Vis Spectrophotometer. Total Zn was

determined by atomic absorption at 213.9 nm with a Perkin-Elmer 460

Atomic Absorption Spectrophotometer.

Differential pulse polarography (DPP) is an electroanalytical

technique which differentiates between free-Zn in solution (Zn ) and

completed Zn (Zn ) (Street and Petersen, 1978). This is in contrast to

atomic absorption spectrophotometry which measures both free and

completed Zn in solution (total Zn). DPP was used to determine the

capacity of each sample to reduce the level of free Zn in a 5 ppm








Zn-solution. An EG and G Princeton Applied Research Model 384B

Polarographic Analyzer and the Model 303A Static Mercury Electrode were

used in the assay. DPP was performed over the range of -0.800 to -1.200

V with a drop/step time of 0.6 mV/sec. Each aliquot for assay was added

to a 0.1M KNO3 matrix of 5.0 ppm Zn, pH 7.0. The 100 ul aliquot plus

the 5.0 ppm Zn in KN03 equaled a total volume of 10 ml. The standard

curve is given in Figure 1. Loss or increase in free Zn concentration

in solutions containing each sample aliquot was determined and reported.

(Calculations of free Zn concentration were made on the basis of the

total 10 ml assay mixture.) A reduction of the concentration of free Zn

was assumed to be an indication of the level of Zn completed rather than

precipitated by the sample. This assumption was made on the basis that

no cloudiness (signaling precipitation) was noted during the DPP

analysis; Zn is found in the phloem as an organo-metal complex (Van Goor

and Wiersma, 1976); and the complex is apparently soluble since it can

be extracted using an aqueous buffer and several species of a

metal-binding factor are recovered after chromatographic separations.

Given this assumption, complexation occurred when <5.0 ppm Zn remained

in solution after the assay. The sample was considered lacking in such

capacity when there was >5.0 ppm Zn in solution. That level of Zn

greater than the 5.0 ppm was considered the level of intrinsic free Zn

ions in the original aliquot prior to assay. Because each aliquot may

have its own intrinsic concentration of completed Zn prior to assay, any

complexation reduction of free Zn that occurred would be residual.

Therefore this capacity was termed residual complexation capacity, RCC.

Each level of purification from crude extract through gel filtration

eluate was assayed as described above, for healthy, predecline and

decline stages of citrus blight.






25












1500


S1413 nA











1000














500










n A
5654 nA














56.5 3nA


5 10 15 20 25

ppm Zn Std n 0.1 M KNO3

Figure 1. Calibration curve for differential pulse polarography
assay.









RCC Comparisons

RCC of healthy,, predecline and decline crude phloem tissue extracts

were compared for their ability to complex a known amount of Pb, Cd, and

Zn. The experiment was run on all extracts at varied metal

concentrations of 2.5, 5.0, 7.5 and 25.0 ppm in 0.1M KNO3. A 100 ul

aliquot of each extract was contained in the 10 ml volume of extract +

metal + 0.1 M KNO3 (1Z phloem extract). This experiment was performed

only on 2 replicates and data are therefore considered preliminary.

Upon partial purification of the crude extract via DEAE-IEC, followed by

gel filtration on Sephadex G-50, 3 aliquots of predecline completing

agent were electrophoresed and four completing species obtained. These

were each assayed for RCC of Pb, Cd and Zn. Sample fractions showing

A-254 nm activity and containing elevated Zn were compared and assayed.

Temperature and pH Stability

Ability to complex Zn was assayed when crude extracts of phloem

tissue from decline stage trees were tested for temperature and pH

stability. Prior to assay for temperature stability, sampled extracts

were either incubated at room temperature (22C) or 38C for 2 hours, or

boiled for 5 minutes. To determine pH stability, samples were adjusted

to pH 1.5, 2.0, 2.8, 4.0, 6.0, 7.8, 9.0 and 10.0 and assayed for RCC.

After IEC and gel filtration of a predecline crude extract, temperature

was again tested. Temperatures of:20, 40, 60, 80 and 100%C were used in

that portion of the experiment.

Plant Tissue Localization of Zn-Binding Factor in Predecline Trees

Each of eight tissues from 2 predecline trees was sampled for Zn

binding factors: leaf and stem tissues, phloem above and below the bud









union, wood above and below the bud union, root phloem and feeder root

tissues. These tissues were extracted and purified in the usual manner,

except that the gel filtered material was concentrated 5-fold prior to

assay. The eight tissues from the 2 predecline trees were compared for

level of metal-thiol chromophore (A-254 nm), but total Zn and residual

complexation capacity were determined on the eight tissues of a single

predecline tree. Therefore the results from this experiment may only be

considered preliminary. Values of A-254 nm, total Zn and RCC were

compared to those values for the extract from healthy phloem tissue

above the bud union (taken from the isolation and purification portion

of the study).














CHAPTER 3
RESULTS AND DISCUSSION


Preliminary Analyses


Crude extracts from 2 decline trees were analyzed for temperature

and pH effect on RCC (Figure 2). If the Zn-binding factor was not

affected by temperature and pH, RCC would not be expected to change

substantially from 220C and pH 7.8. At pH's ranging from 2.8-10 there

was little change in Zn completed (range- 22.3- 24.9 ppm Zn). But with

decreased pH of 2.8 to 1.5 there was a marked decrease in Zn

complexation (22.3 to 1.25 ppm Zn). This reduced RCC was reversible

with an increase in pH from 1.5 to 2.8 (data not shown). Temperatures

ranging from 22-1000C had no effect on Zn completed. When partially

purified predecline extracts were assayed in the same manner as above

for temperature effect on RCC of Zn, there was no significant change

(data not shown). Over the temperature range of 100-20*C, Zn-RCC

changed from 22.7 to 25.0 ppm. Classical proteins would be expected to

denature and precipitate with very high temperatures, losing activity.

However, this completing agent did not. The above data suggest that

this Zn-complexing agent is not a classical protein.

Crude extracts from healthy, predecline and decline tissues were

placed in solutions containing 2.5, 5.0, 7.5 or 25 ppm Pb, Cd and Zn.

Predecline and decline extracts apparently completed more metal than

healthy extracts. Predecline and decline extracts completed Zn more

than Cd which was completed more than Pb (Table 2). The predecline and

28



























































5 15 25


0
C




-60 "




,40
1>


ppm Zn Chelated


Effect of temperature and pH on the capacity
of crude phloem tissue extract from
blight-affected 'Valencia' sweet orange to
complex Zn. (All data are given on the
basis of extractions made on an equivalent
fresh weight basis of 10 g phloem tissue/
30 mis 50 mM Tris-HCl buffer, pH 7.8.)


Figure 2.


...
..
..
....'
.Y



















Table 2.
Comparative Pb-, Cd-, and Zn-RCC by phloem extracts from healthy and
blight-affected citrus trees, as assayed by DPP. Samples represent
activity of 100 pu of phloem tissue extract placed in 10 ml of pH 7.0,
0.1M KNO3. (All data are given on the basis of extractions made on an
equivalent fresh weight basis of 10 g phloem tissue/30 mis 50 M
Tris-HC1 buffer, pH 7.8.)


Phloem ppm metal in ppm RCC
Tissue KNO3 solution ________
Extract Pb Cd Zn


Healthy 2.5 0.388 0.155 1.180
5.0 2.029 0.505 2.206
7.5 3.829 0.509 3.333
25.0 5.132 0.578 4.974

Predecline 2.5 2.495 2.500 2.500
5.0 3.312 5.998 5.000
7.5 4.830 7.495 7.500
25.0 18.622 20.933 25.000

Decline 2.5 1.304 2.495 2.500
5.0 2.944 3.798 5.000
7.5 4.515 5.178 7.500
25.0 14.207 14.950 25.000


al0 gm (f.w.) phloem/30 ml 50 mM Tris-HC1 buffer, pH 7.8.









decline phloem tissue extracts appear to possess a metal-binding factor

with an apparent higher affinity for Zn. This completing factor does

not appear to exist in substantial quantities in healthy phloem tissue

extracts. At this stage there appeared to be a difference in the

behavior of crude extracts from healthy and blight-affected trees which

was significant enough to warrant further investigation of the existence

of a Zn completing agent associated with the occurrence of citrus

blight.




Evidence for a Zn-binding Factor with the
Occurrence of Citrus Blight


Absorbance of UV at 254 nm (A-254nm), concentration of total Zn and

RCC were assayed as indicators of the presence of a Zn-binding factor.

Elevated A-254 nm (which demonstrates the presence of metal-thiol

chromophores) coincided with elevated total Zn concentration. This

provides strong evidence that the metal-thiol chromophore is a Zn-thiol

chromophore. If in addition, RCC (measure of the capacity of a sample

to complex metal) coincides with A-254 nm and total Zn, further capacity

of the Zn-thiol chromophore to complex Zn is demonstrated. The A-254 nm

and total metal concentration assays are standard for demonstrating the

presence of the metal-complexing, cysteine-rich phytochelatins. The RCC

assay was added to this study to examine the surplus Zn completing

capacity of any Zn-binding factor associated with citrus blight.

DEAE-Ion Exchange Chromatography (0.25 2.0 M NaCl Gradient)

A-254 nm. Trunk phloem tissue extracts from healthy, predecline

and decline 'Valencia' sweet orange were assayed for A-254 nm after ion








exchange chromatography (IEC) on a DEAE-Sephadex-A-50 column elutionn

gradient 0.25-2.0 M NaCl). There were 4 peaks of elevated A-254 nm

activity within the elution profiles (Figure 3). The healthy phloem

profile contained two peaks and the predecline and decline phloem

profiles contained all 4 peaks. The first two peaks, a and b (maximum

values at fractions #17 and #22-24, respectively), were common to all 3

phloem extracts and there was no difference in the level of each. It

may be argued that peak b is a shoulder within peak a. For reasons that

will become more apparent, it is being referred to as a peak in this

discussion. The next peak (c) of A-254 nm activity (maximum values at

fractions #31-33) was more evident in decline stage than the predecline

phloem extract. A fourth, less evident, peak of A-254 nm activity

occurred at fraction 36 (point d) of the elution profiles of the

predecline and decline phloem extracts. There was no difference in the

quantity of this activity between these tissues.

These data demonstrate that there are 2 metal-thiol chromophores

present in healthy tissue that remain in nearly equal quantity with

progression to the predecline and decline stages of blight. However,

two other metal-thiol chromophore species appear at the predecline and

decline stages of citrus blight and are absent when citrus blight

appears absent (i.e. no Zn accumulation and/or reduction in H20

conductivity).

Total Zn. After the A-254 nm assay the above phloem extracts were

assayed for total Zn (Figure 4). There were 4 coincident peaks (with

regard to A-254 nm data) of elevated total Zn concentration. Again, the

healthy phloem profile contained a peak around fraction #17 (peak a).

This peak of elevated Zn also was contained in the predecline and




























E o@



x

















FRACTION NUMBER
Figure 3. Absorbance at 254 nm of phloem tissue extract from
healthy, predecline and decline stage, blight-
affected 'Valencia' sweet orange after IEC on
DEAE Sephadex-A-50 with an elution gradient of
0.25-2.0 M NaCl (a, b, c, and d represent
fractions with maximum activities). (All data
are given on the basis of extractions made on an
equivalent fresh weight basis of 10 g phloem
tissue/30 ms 50 m Tris-HC1 buffer, p 7.8.)
tissue/30 mis 50 mM Tris-HCl buffer, ph 7.8.)






















.---- Decllne

---- Predeclne


..... 0 Healthy


10 20 30 4(
FRACTION NUMBER



Concentration (ppm) of total Zn in phloem tissue
extract from healthy, predecline and decline
stage, blight-affected 'Valencia' sweet orange
after IEC on DEAE Sephadex-A-50 with an elution
gradient of 0.25-2.0 M NaC1 (a, b, and d
represent fractions with maximum activities).
(All data are given on the basis of extractions
made on an equivalent fresh weight basis of 10 g
phloem tissue/30 mis 50 mM Tris-HC1 buffer, pH
7.8.)


1.2








c
N
o.8-

I
E







0.4-


Figure 4.









decline profiles with little or no difference evident among healthy,

predecline and decline phloem extracts. Shouldering (peak b) was

evident in the decline and almost absent in the healthy and predecline

profiles. At the predecline and healthy stages, the metal-thiol

chromophores indicated by peaks at b (Figure 3) may be complexes of

metals other than Zn.

Peaks c and d (corresponding to fractions #30-32 and #35,

respectively) were combined in the predecline and decline elution

profiles and absent in the healthy elution profile. The concomitance of

total Zn with A-254 nm at peak c and d in the predecline and decline

elution profiles provides strong evidence of 2 Zn-thiol chromophores

that were not apparent in healthy phloem extracts. This also provides

evidence, by comparison (Grill et al., 1985; Jackson et al., 1985;

Rauser and Glover, 1984), that the Zn-binding factors associated with

citrus blight are phytochelatins. This is especially evident because

those peaks of activity were strongly anionic since they require

relatively concentrated NaC1 for elution. Others working with

phytochelatins have reported similar results (Bartolf et al, 1980;

Casterline and Barnett, 1982; Rauser and Curvetto, 1980) (Table 3).

The Zn-binding factors (peaks a and b) common to all phloem extracts

were strongly anionic in addition to having Zn-chromophore activity.

Therefore, a phytochelatin which remains even during development of the

citrus blight syndrome is suggested in healthy phloem tissue.

RCC. Coincidence was only somewhat evident when the same IEC

fractions were assayed for RCC (Figure 5). There was a broad area of

residual Zn-complexing capacity in elution profiles of all 3 phloem

tissue extracts. This area of complexation occurred at several single








Table 3.

NaCI concentrations required
to elute the corresponding
Zn-binding peaks within the
DEAE-IEC (0.25-2.0 M NaCI)
elution profile. (All data
are given on the basis of
extractions made on an
equivalent fresh weight I
basis of 10 g phloem tissue/
30 mls 50mM Tris-HCl buffer,
pH 7.8)

Concentration
Peak of NaC1 (M)

a 0.60 0.80

b 0.97 1.05

c 1.45 1.50

d 1.55 1.60




fraction peaks in fractions #14-25. The corresponding areas of elevated

A-254 nm activity and total Zn, encompassing points a and b were

contained in fractions #10-27 and #8-24, respectively (Figures 3 and 4).

Generally, there is evidence of Zn-RCC in the common region of

Zn-chromophore presence in healthy, predecline and decline elution

profiles. There are single fraction peaks of RCC at fractions #33 and

#35 of the predecline and decline elution profiles which may correspond

to peaks c and d from the A-254 nm and total Zn assays. It should be

noted that fraction #35 actually shows no RCC as defined earlier.

However, there is a decline in Zn concentration relative to adjacent

fractions. Thus, RCC at peak d is debatable. Very significant RCC

occurs at fraction #40 in decline and predecline elution profiles. This














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significant activity might have been due to a completing agent which did

not contain a significant level of thiolates and/or intrinsic level of

Zn. There was no elevated A-254 rn activity or total Zn in the

corresponding fractions for those assays. To determine if the apparent

peak at fraction #40 returns to the RCC baseline, the column should be

run with a somewhat extended elution gradient (possibly 0.25-2.5 M

NaCI). This may effect complete elution of the peak at fraction #40.

To prevent loss of resolution of the 4 other peaks and possibly increase

that resolution, a greater volume of the 2 buffers composing the elution

gradient should be used. This would serve to extend the number of

fractions over which elution will occur, so that peaks would be clearly

separate.

There appeared to be much variability within the RCC values.

However, the level of variability was <5%. This appearance of

variability in RCC may be characteristic of DEAE-purified material,

since this variability was not apparent in material after gel

filtration. A reverse order of column application (Sephadex G-50 prior

to DEAE-Sephadex A-50) might be useful as a test of this hypothesis.

Absorbance at A-254 nm, Zn concentration and RCC means for 2

decline stage 'Marsh' grapefruit are given in Figure 6. The

chromatography profile, though slightly different, corresponds well with

that for 'Valencia' sweet orange. Peak a was centered at fraction #16,

shoulder bwas around fraction #22, peak c at fraction #33 and peak d at

fraction #37. Again there was a Zn completing peak of RCC at fraction

#40, for which there is no clear explanation.

The concomitance of peaks a and b for orange and grapefruit

(Figures 3-6) is an indication that 2 species of Zn-binding factors are

































Comparison of A-254 nm, ppm total Zn and ppm RCC (showing
coincidence in all 3 assays) in phloem tissue extract
from blight-affected 'Marsh' grapefruit after IEC on DEAE
Sephadex-A-50 with an elution gradient of 0.25-2.0 M NaCl
(a, b, c, and d represent fractions with maximum
activities). (All data are given on the basis of
extractions made on an equivalent fresh weight basis of
10 g phloem tissue/30 mis 50 mM Tris-HCI buffer, pH
7.8.)


Figure 6.


























.or
It


0



0


0

*0


I
/


I

p'


I.I
I.r
/5.

~: '

0r


........ A-254 nm


---.. Total Zn (ppm)
*.

b _- RCC, (ppm)


*









\ q













rIt
1 10

9

.* -
,, ..


FRACTION NUMBER


E
0.4


N




E 0.2.
a.



E
.1
4,



0.1








normal metabolites in the healthy citrus tree (possibly responsible for

normal heavy metal homeostasis). Various assay measurements were

concomitant for peak a and shoulder b in initial separations of all

stages of healthy and blight-affected trees. Therefore, they were not

considered in the subsequent gel filtration column series (Figures 7-8).

Only those fractions which composed peaks c and d from decline and

predecline tissues were pooled and gel filtered.

Gel Filtration. Values from predecline and decline samples were

pooled to form the means presented in Figure 7 ('Valencia' sweet orange)

and Figure 8 ('Marsh' grapefruit). Peak maximums of A-254 na, Zn

concentration and RCC were all coincident at fraction #20. An apparent

molecular weight of 4 kd was determined based on a gel filtration

profile which was calibrated with cytochrome c (12.3 kd), ACTH (4.5 kd)

and bacitracin (1.5 kd).

DEAE-Ion Exchange Chromatoghraphy (0.5-3.0 M NaC1 Gradient)

The above ion exchange series for sweet orange and grapefruit was

performed using a 0.25-2.0 M NaC1 gradient. To examine the molecular

weight characteristics of all anionic species [peaks a, b, c and d

(Figures 3-5)], another ion exchange series was performed using a

steeper gradient of 0.5-3.0 M NaCl. This served to merge all anionic

species (peaks a, b, c and d) into an apparent single anionic species

which was placed as a single aliquot on Sephadex G-50 for gel

filtration. With this approach, it could be determined whether the 4

anionic species represented one molecular weight species.

With ion exchange chromatography of healthy and blight-affected

tissues of 'Valencia' sweet orange and 'Marsh' grapefruit using the

0.5-3.0 M NaCl gradient, all elevated A-254 nm activity, total Zn and







































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RCC of Zn were merged into single peaks around fractions #5-6

(Figures 9-12). The DEAE-IEC elution profiles of sweet orange and

grapefruit were very similar. However, the apparent level of Zn-binding

factor detected was higher in grapefruit than sweet orange samples

(Table 4). Inability to load known levels of protein polypeptidee of

interest) onto the columns made it difficult to determine what the

actual difference was. But sweet orange and grapefruit levels of

Zn-binding factor were different when isolated, purified and assayed on

the basis of equal fresh weights (Table 4).

On this basis, the 2 scions were quite different, and on a fresh

weight basis, it was quite evident that the level of Zn-binding factor

present in predecline trees was significantly higher than in healthy

trees for both scion cultivars of citrus (Table 4). This difference is

very likely due to the presence of the anionic species eluting at peaks

c and d of the 0.25-2.0 M NaCl elution profile.

Gel Filtration. Fractions #2-18 from the above DEAE-IEC were

pooled and an aliquot placed on the gel filtration column (Figure 13 and

14). Upon gel filtration of these IEC peaks (sweet orange and

grapefruit) which contained 4 different anionic species of Zn completing

agent, an apparent single peak of Zn complexation eluted with a single

apparent molecular weight of 4 kd (Figures 13-14). This however does

not resolve the question of the number of molecular weight species.

Within the resolving power of column chromatography (Sephadex G-50, in

particular), one molecular weight species would appear likely since

small differences in molecular weight would be undetected. However,

Robinson et al. (1985) used an extremely long Sephadex G-50 column under

dissociating conditions followed by gel filtration on low-molecular-



































Comparison of A-254 nm, ppm total Zn and ppm RCC (showing
coincidence in all 3 assays) in phloem tissue extract
from blight-affected 'Valencia' sweet orange after IEC on
DEAE Sephadex-A-50 with an elution gradient of 0.50-3.0 M
NaCl. (All data are given on the basis of extractions
made on an equivalent fresh weight basis of 10 g phloem
tissue/30 mls 50 mM Tris-HC1 buffer, pH 7.8.)


Figure 9.






49









2.0 ..
**

........ A- 254 nm



---- Total Zn (ppm)


--- RCC (ppm)




E I
0
a *1.0 *
0*
I \ *.
a 1
N 1 4


i \ "
- 1


0 1 \
E 4




I 0
4 0 ****. ...

02 *. -


0.2







10 20 30

FRACTION NUMBER







































E
CL
CL

E E
c L
N CL

C-4
4, I- E








I


50
























wi U~ 0
zt.Eoo






ui 0 0 0
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c T UIU-I

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-' U~J >

S upd e -4-









00


-4
Dk
Lip) XI)
-4 ~m~-


S14 cm

Doll wdd
uZ tu1o.Lwdd wuvtsa-v



































Figure 11. Comparison of A-254 nm, ppm total Zn and ppm RCC (showing
coincidence in all 3 assays) in phloem tissue extract
from blight-affected 'Marsh' grapefruit after IEC on DEAE
Sephadex-A-50 with an elution gradient of 0.50-3.0 M
NaCl. (All data are given on the basis of extractions
made on an equivalent fresh weight basis of 10 g phloem
tissue/30 mis 50 mM Tris-HCl buffer, pH 7.8.)


























*... A-254nm


STotal Zn


2.0 RCC (pp




'
*5





I I



S1

I 10 i .

: I
CI

































FRACTION NUMBER
a.I



9; &


o .1 i o.
o I %
.9 "0.
o0 i
















0 o 20


FRACTION NUMBER


(ppm)


R)


30
















































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Figure 13. Comparison of A-254 nm, ppm total Zn and ppm RCC after gel
filtration on Sephadex G-50 of pooled fractions 2-13 from
DEAE Sephadex-A-50 elutionn gradient 0.50-3.0 M NaCI).
Coincidence in all 3 assays was shown in the phloem
tissue extracts from blight-affected 'Valencia' sweet
orange. Zn-binding factor has an apparent molecular
weight of 4kd. (All data are given on the basis of
extractions made on an equivalent fresh weight basis of
10 g phloem tissue/30 mis 50 mM Tris-HCl buffer, pH
7.8.)



















1.4 r
II
Mi
II
........ A-254

I.
1.2 4 --. Total Zn (ppr)w


S--- RCC (ppm)

I i

SI




0.8 I
,8

g
af,
a. i



i





I I



+ **2 ;:*|[ '*- e-
.
















04 i .1 "


FRACTION NUMBER

































Figure 14.


Comparison of A-254 nm, ppm total Zn and ppm RCC after gel
filtration on Sephadex G-50 of pooled fractions 2-13 from
DEAE Sephadex-A-50 elutionn gradient = 0.50-3.0 M NaCI).
Coincidence in all 3 assays was shown in the phloem
tissue extracts from blight-affected 'Marsh' grapefruit.
Zn-binding factor has an apparent molecular weight of
4kd. (All data are given on the basis of extractions
made on an equivalent fresh weight basis of 10 g phloem
tissue/30 mis 50 mM Tris-HCl buffer, pH 7.8.)























A-254 nm


total Zn ppm


- RCC ppm


*' S
















:S.
*ai

'I `
*: I

.4 I

I )

*
.5c I S,



',
h ~ `O


0.*

,~ g..
I'
r\ i


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0.2






0.4


10 20
FRACTION NUMBER









weight selectivity columns, resolving 2 peptides with approximate

molecular weights of 600-800 from Datura inoxia, Mill. This gel

filtration was performed in the presence of the denaturant guanidine HC1

and yielded molecular weight data in agreement with amino acid sequence

data that had suggested the presence of Cd-binding multimers. Based on

reports of those working with phytochelatins, the molecular weight of

the denatured form of Zn-complexing agent associated with blight would

be lower than 4kd (Grill et al., 1985; Jackson, personal communication,

1985). The denaturant removes metal ions and associated H20 molecules,

leaving only the amino acids. The higher level of resolution obtainable

with HPLC may allow a distinction between molecular weight species.

Thus HPLC may be an appropriate separation method for this application.

The finding of several metal binding species is not unprecedented.

Jackson et al. (personal communication, 1986) have found that different

species of (y-glu-cys)n-gly polymer from Datura inoxia Mill. have

preference for different heavy metals. Using 1C-NMR, they have

demonstrated that the n- 2,3 species of the phytochelatin preferentially

binds Cu, while the n- 5,7 species binds Cd. Jackson et al. (1985) have

suggested that the n- 9 species may bind Zn. Grill et al. (1986) have

reported similar findings for these and other metals. All forms appear

capable of binding other metals, though they prefer one metal ion over

the others. Of the 4 apparent species isolated in predecline trees, one

or more may bind Zn, with certain species having more or less preference

for Zn. If the Zn-complexing agents are phytochelatins, the synthesis

of all 4 could be induced by the same agent. The n-3 and n=5 forms of

phytochelatin reported by Robinson et al. (1986) were induced by Cu and

Cd, respectively. They suggested the multiple forms were biosynthetic









products of a single enzyme of glutathione synthesis, I-gldtamyl

transpeptidase. This enzyme may be responsible for the assembly of

(1-glutamyl-cysteinyl) -glycine. It has been hypothesized that it uses

glutathione and an activated form of y-glu-cys as precursors.


(Y-glytamyl transpeptidase).
Y-glu-cys-gly + --glu-cys ( -glu-cys)2-gly


Binding of Cd to (y-glu-cys) -gly elicits product removal. Increased

synthesis of the peptides in response to Cd is accompanied by increased

production of certain mRNA species (Robinson et al., 1986). This may

indicate amplification of normally expressed portions) of the genome.

To address the question of whether the Zn-binding factor is a

normal or new metabolite, it is first assumed that this completing agent

is a phytochelatin. This assumption is based on the coincidence of the

3 assays which indicate the presence of the Zn-binding factors. This

coincidence has been demonstrated by others as evidence of

phytochelatins (Rauser and Curvetto, 1980; Grill et al., 1985; Jackson

et al., 1985). The low molecular weight of the Zn-binding factors,

their highly anionic character and their heat stability also add

credence to this assumption. It then follows from the above that

synthesis of more of a normal metabolite is being induced and that

either extended or truncated forms of the (y-glu-cys) -gly may be

synthesized as well to give the additional 2 species found in the blight

tree. These forms may vary in anionic character simply because there

are more or less of the cysteine residues which provide the (-) charges

for the binding of Zn Their addition would cause small differences

in molecular weight of approximately 268, exclusive of any additional Zn

bound to the new species. It may be significant that the new species









noted in blight-affected trees are more highly anionic, and that a

possible fifth species, at fraction #40 (DEAE-IEC 0.25-2.0 M NaC1

gradient) is extremely anionic. As new species appear with citrus

blight progression, there may be more extended forms of those existing

in healthy trees (species-peaks a and b). This is reasonable since the

"fifth species" at fraction #40 showed no A-254 nm activity or elevated

total Zn but showed a very high Zn-RCC. In this case the

(Y-glu-cys) -gly may be present, but may not yet be associated with a

metal. In the RCC assay, an aliquot of Zn was added to the test

solution (to form 10 ml of test solution at 5 ppm Zn). At that point a

metal was available to form the metal-thiol chromophore (in the RCC

assay, there was a reduction of Zn in solution from 5 ppm). This data

would have been more conclusive if a follow-up A-254 nm assay had been

performed. As a further investigation of this phenomenon it would be

helpful to treat the individual species with saturation levels of Zn,

dialyze them to remove the excess Zn and then assay to see if their

anionic character is altered (are the same species present?) and assay

for their behavior regarding A-254 nm, total Zn and RCC. It is

certainly possible that the various species only differ in the amount of

Zn they have sequestered. They may appear anionically different on this

basis. The Zn-saturation experiment outlined above may allow a

distinction between possible forms.

Correlation of A-254 nm, Total Zn and RCC

Because A-254 nm, total Zn and RCC assays had a high degree of

coincidence at points a, b, c and d, a correlation analysis was

performed (Table 5). This analysis considered gel filtration data

(pooled fractions #27-38) from the 0.25-2.0 M NaC1 column series or









(pooled fractions #2-13) from the 0.50-3.0 M NaC1 column series. In

order to correlate the 3 assays used to show the presence of the

Zn-binding factor, A-254 nm was compared to the sum of total Zn and RCC,

yielding total complexation capacity (TCC). TCC represents the total Zn

in a given fraction + the additional Zn it is capable of binding as

determine using DPP. The comparisons were made in tissue extracts after

gel filtration (Table 5). All were found to be significant at p>5 or

p<10. The lack of correlation at p<5 may be due to the high variability

found from sample to sample. This may be reduced if a constant quantity

of the Zn completing agent can be loaded onto the chromatography

columns. As yet, a reliable assay for quantification of phytochelatin

is unknown. The standard protein assays will not work because of the

small size of the completing agent, the Y-carboxyl linkage or the lack

of histidine and aromatic residues. Ninhydrin which quantifies the

terminal amino groups in a sample (Jackson, personal communication,

1985) or Ellman's reagent which quantifies the level of thiol groups


Table 5.
R values for A-254 nm and TCC
correlation. (All data are
given on the basis of extractions
made on an equivalent fresh weight
basis of 10 g phloem tissue/30 mls
50mM Tris-HCl buffer, pH 7.8)

Column R Values
Series


0.25-2.0 M NaC1 0.6017 (9)a

0.50-3.0 M NaCI 0.5873 (10)


Values in parenthesis represent
the number of replicates
considered in the correlation.








(Ellman, 1959) may offer possible quantification procedures. In this

study, samples were applied to the columns on the basis of fresh weight

of active phloem.

On the other hand the level of correlation may be as much as one

could expect. With reference to Table 4, study of the relationship of

A-254 nm with TCC at the peak maximum (given as a ratio of A-254 nm/TCC)

would suggest that no relationship should exist. Apparently, the

integration achieved by the pairwise comparison of A-254 nm and TCC

values of each fraction along the elution profile was adequate to give

the levels of correlation given in Table 5. The analyses were performed

on 9 pairs of data from the DEAE-IEC (0.25-2.0 M NaC1) and 10 pairs from

the DEAE-IEC (0.50-3.0 M NaCl).


Electrophoretic Evaluation of Zn-Binding Factor

The Zn-binding factors isolated by ion exchange and gel filtration

chromatography of 4 predecline phloem tissue extracts were found

essentially free of classical protein contaminants when stained with

coomassie-blue after PAGE. Newly purified sample immediately

electrophoresed was found to be free of contaminants. A small amount of

contamination was found in 2 lanes in samples stored for several weeks

at 4"C. This may have been due to microbial growth. Under the

coomassie-blue stain, the Zn completing agent is free of any stainable

activity. Verification of this may be achieved with the more sensitive

silver stain (Chrambach and Rodbard, 1981), or with ninhydrin staining

of denatured material (Jackson, personal communication, 1985).

Two gels were run in duplicate. Non-stained gels (representing 4

purified samples) were sectioned, eluted and assayed for A-254 nm, A-280

nm (Figure 15) and total Zn (Figure 16). The A-280 nm data provided




















4


Ca .05


Figure 15.


6 12 18
SECTION NUMBER
Absorbance at 254 nm and 280 nm of gel filtration pooled
fractions #9-20 after polyacrylamide gel electrophoresis.
The predecline stage, blight-affected 'Valencia' sweet
orange phloem tissue extract was partially purified on
DEAE-IEC elutionn gradient 0.50-3.0M NaC1) with
subsequent gel filtration on Sephadex G-50 (1-4
represent species peaks isolated from PAGE). (All data
are given on the basis of extractions made on an
equivalent fresh weight basis of 10 g phloem tissue/30
mls 50 mM Tris-HC1 buffer, pH 7.8.)































0.10


0.05


8 12 18
SECTION NUMBER


Figure 16.


Concentration (ppm) of total Zn in gel filtration pooled
in fractions #9-20 after polyacrylamide gel
electrophoresis. The predecline stage, blight-affected
'Valencia' sweet orange phloem tissue extract was
partially purified on DEAE-IEC elutionn gradient =
0.50-3.0 M NaCl), with subsequent gel filtration on
Sephadex G-50 (1-4 represent species peaks isolated from
PAGE). (All data are given on the basis of extractions
made on an equivalent fresh weight basis of 10 g phloem
tissue/30 ils 50 mM Tris-HCl buffer, pH 7.8.)









further evidence that the isolated completing agents were free of

protein contamination. Findings, based on the use of a native gel

system which operated on the basis of charge, indicated the presence of

4 anionic species. Coinciding peaks of A-254 nm and total Zn were

apparent at sections 1, 9, 14 and 16 of the sectioned gels (Figures 15

and 16). These data contribute further evidence to that from the

DEAE-IEC run with 0.25-2.0 M NaC1 gradient which demonstrated the

presence of 4 species. Since the sample preparation prior to

electrophoresis involved the use of the 0.5-3.0 M NaCI gradient, it is

not certain that the peak apparent at fraction #40 of the RCC data (from

the DEAE-IEC run with 0.25-2.0 M NaC1 gradient) was included as part of

the sample placed on the gel. This gel system was not useful for

determination of molecular weight. As yet, no one working with the

small molecular weight (non-aggregated) phytochelatins has been able to

make a molecular weight determination based on electrophoretic data.

An experiment was performed comparing the relative abilities of the

4 electrophoretically separated anionic species to complex Pb, Cd and

Zn. The 4 anionic species were assessed separately (Table 6). The

test-solution contained 25 ppm each of Pb, Cd and Zn. The data

indicated that the different species have varied abilities to complex

the three cations. The first two anionic species, represented by the

data for section-peaks 1 and 2, completed more Zn and Cd than Pb. Of Zn

and Cd, Zn was completed in the greatest quantity. This is reasonable,

since these species, being the least anionic of the 4, probably

correspond to the species found in healthy phloem tissue from DEAE-IEC

data. It is also interesting that species-peak 2 which may correspond

to peak b from DEAE-IEC (0.50-3.0 M NaCl) contained the greatest overall



















Table 6.
Comparative Pb-, Cd-, and Zn-RCC by purified
Zn-binding factor from predecline tissue extract,
as assayed by DPP. Samples represent activity of
100 pO of purified material placed in 10 ml of pH
7.0, 0.1M KNO DPP was performed with a 25 ppm
Pb, Cd, and Zn solution. (All data are given on
the basis of extractions made on an equivalent
fresh weight basis of 10 g phloem tissue/30 mls 50
mM Tris-HC1 buffer, pH 7.8.)


Species- ppm RCC
Peaks
Pb Cd Zn


1 0.00 0.64 0.89
2 1.57 2.21 2.73
3 1.34 0.89 0.36
4 1.07 1.24 0.33


apooled fractions #2-13 from DEAE-IEC (0.5-3.0 M
NaC1 gradient) were placed on Sephadex G-50 for
gel filtration. Subsequent pooled fractions
#9-20 were separated by PAGE to yield species
1-4 which were assayed for Pb-, Cd- and Zn-RCC.









RCC. This peak had very little total Zn (Figure 4). Possibly this

species had very high potential metal-thiol chromophore content,

possessing a large quantity of non-complexed metal-binding factor. The

species represented by peak 3 sequestered Pb>Cd>Zn and peak 4 Cd>Pb>Zn.

The latter peaks appear to have an affinity for different ions, as

compared to the 2 former peaks. This might be expected as they are

probably somewhat different forms. These are probably extended forms of

those existing in healthy tissue.

The varied abilities of the 4 species to bind these 3 metals may

have had some impact on the low correlation of A-254 nm with TCC. If

TCC had been measured on the basis of these 3 (or more) metals, the R

values may have been increased.

This experiment would have been better performed on the basis of

the relative activities of the three metals in relation to the 4

ligands. This requires more knowledge about the ligand-molecules.

Amperometric titration of the ligand with various metals would yield

formation constants for each metal, from which activities for each metal

could be determined (Hoyle and Thorpe, 1978). To do this experiment,

ligand standards are required. The decision as to what should be used

as the standards requires knowledge as to the exact nature of the

completing molecules. Therefore the amperometric titrations may have to

be performed after amino acid sequencing.



Plant Tissue Localization of Zn-Binding
Factor in Predecline Trees


Two replicates were assayed for A-254 nm and 1 for total Zn

concentration and RCC to develop preliminary data on the morphological































Figure 17. Comparisons of A-254 nm, ppm total Zn and ppm RCC in
healthy phloem tissue and predecline stage,
blight-affected leaf, stem, phloem, wood, root phloem and
feeder root tissue extract from 'Valencia' sweet orange
after IEC on DEAE Sephadex-A-50 with an elution gradient
of 0.50-3.0 M NaCI, with subsequent gel filtration on
Sephadex G-50 of DEAE-IEC pooled fractions #9-20. These
samples were concentrated approximately 5-fold after gel
filtration, prior to assay. (All data are given on the
basis of extractions made on an equivalent fresh weight
basis of 10 g phloem tissue/30 mls 50 mM Tris-HCl buffer,
pH 7.8.)
















Phloem
- Above
S Bud Union


Leaf


Stem


Bu



Bu


Bu


Iviii .
0-6.00
~bam U


Phloem -. .
Above ..g 0 i" V-
d Union m u -- -------- -

Phloem
Below*
d Union

Wood9
Above
d UnionI


Wood
Below
Bud Union

Root
Phloem


Feeder
Roots


ZbA


3I Total Zn
(ppm)


BI A- 254nm


/ RCC
(ppm)
-1
1 x10









location of the Zn-binding factor accumulating in citrus blight-affected

trees at the predecline stage (Figure 17). Eight tissues have been

compared graphically to the mean from 2 healthy phloem extracts of

tissue removed above the bud union. In general, based on A-254 nm and

total Zn, extracts of predecline trunk phloem taken from above the bud

union contained at least 3 times the level (on a fresh weight basis) of

Zn-binding factor found in all other tissues including healthy trunk

phloem from above the bud union. RCC was comparable in all tissues

except the wood below the bud union, which had very low RCC. A-254 nm

and total Zn assays also indicated that the wood tissues were deficient

in Zn-binding factor (as compared to those levels found in healthy

tissue). Thus, there is little evidence of the Zn-binding factor in the

wood at the predecline stage of blight. This agrees with the lack of Zn

in the wood at early predecline (Albrigo et al., 1986).

In addition, the levels of Zn-binding factor (data from all 3

assays) in all tissues excluding phloem above the bud union (> healthy

tissue) and wood below the bud union (< healthy tissue) are comparable

to that found in healthy tissue. Therefore, Zn-binding factor was

accumulating only in the tissue (phloem above the bud union) in which Zn

was accumulating at the predecline stage of blight. In relation to this

finding, an X-ray microanalytical study of the distribution of Cd in

root cross-sections of Zea mays L. has shown Cd accumulation in walls of

sieve elements and middle lamella separating the endodermis from the

pericycle (Khan et al., 1984). This was in comparison with other

tissues in the root cross-section. In the Khan et al. (1984) study, Zn

competed well for Cd-sequestering agents. Possibly a normal metal

binding factor resides primarily in plant phloem tissues. But with









blight, the predecline tree may manufacture an abnormally high level of

the metal-binding molecule.

In addition to the data presented here, 2 samples of soil solution

taken proximal to the roots of decline and healthy trees were

assayed for RCC. Neither contained significant levels of RCC. This

gives some indication that the agent responsible for the overproduction

of the Zn-binding factor does not reside in the soil. However, data

establishing the absence of elevated A-254 nm associated with Zn are

necessary to confirm this. A blight-inducing agent can be transmitted

plant to plant (Tucker et al., 1984). It is likely that the transmitted

agent is responsible for inducing the synthesis of the Zn-binding

factor. Two possible inducing agents are the metal itself, Zn (Grill et

al., 1985; 1986; Jackson et al., 1985; Rauser and Glover, 1984), or an

organism. Certainly, transmission supports the possibility that the

transmitted agent is an organism. However, no such organism has been

found consistently at the predecline stage in blight-affected trees.

On the other hand, transmission of high Zn levels may be possible, and

certainly there is abundant evidence that elevated levels of Zn are

present in association with blight. This is not to suggest that one is

more likely to be the blight transmitting agent than the other, but is

only to suggest that they are both possible transmitting agents.
















CHAPTER 4
SUMMARY AND CONCLUSIONS


In this research, 4 anionic species of Zn-binding factors were

separated. Two of the anionic species were present in healthy,

predecline and decline phloem tissue extracts, while the other two were

present only in predecline and decline phloem tissue extracts. This

occurred in sweet orange on 2 rootstocks on different sites and

grapefruit at a third site. The verity of these findings was shown by

the coincidence of A-254 nm, total Zn and RCC data. However, the

quantitative values for these assays were not shown to be highly

correlated. Assuming that the 4 completing species are

(y-glu-cys) -gly, better correlation of the assays may be possible with

a y-polypeptide quantification method (possibly ninhydrin or Ellman's

assays). Additionally, the correlation may be improved if the species

are treated with saturating levels of Zn and examined separately using

the 3 standard assays. Such correlation of the assays is important

because A-254 nm and total Zn are the standard assays used in

phytochelatin research.

The characterization of the Zn-binding factors revealed several

points of circumstantial data which suggest that the isolated species

are phytochelatins. First, the 4 completing species bind Zn, a IIB

transition metal which is reportedly sequestered by phytochelatins. In

addition the 4 species are capable of completing other transition metals

which are commonly completed by phytochelatins. Second, they are

73









temperature stable and ph labile at pH 2 and below. Third, the

fractions containing Zn-binding factor absorbed UV at 254 nm but did not

at 280 nm. These same fractions had increased levels of total Zn.

Fourth, the Zn-binding factor had a low molecular weight of 4kd. Amino

acid sequencing is necessary to demonstrate unequivocally that this

Zn-binding factor is a phytochelatin.

The pooled fractions containing the Zn-binding factors were shown

to be reasonably pure (free of classical proteins) on coomassie-blue

stained polyacrylamide gels. Assay of the lanes for A-254 or total Zn

as indicators of a Zn-binding factor gave further evidence of the

presence of at least 4 anionic species. This agrees with data from the

DEAE-IEC (0.25-2.0 M NaC1 gradient), where 4 anionic species were

separated in predecline samples.

Preliminary data indicated that the Zn-binding factor accumulated

in the active phloem above the bud union of a predecline tree up to 3

times the level of the healthy tissue or other tissues in the predecline

tree (data was based on equivalent fresh weights of extracted tissues).

Wood above and below the bud union of the predecline trees sampled

provided no more indication of Zn-binding activity than healthy phloem

tissue and in the case of the wood below the bud union there was less.

This study should be replicated and performed using corresponding tissue

from healthy and decline stage extracts for further verification.

Two Zn-binding factors appear to be normally synthesized

metabolites. Other studies suggest that amplified synthesis may be

responsible for the production in blight-affected citrus trees of

extended forms of the Zn-binding factors which are found in the healthy

citrus tree. This is based on the finding that healthy and blight









tissue extracts contained a Zn-binding factor with similar charge in the

same fractions (a and b) and on the finding that all the anionic species

are very near the same molecular weight. In addition, the new

Zn-binding forms in phloem tissue from blight-affected citrus were more

highly anionic (c, d and fraction #40) than the existing forms in

healthy phloem tissue. The greater anionic character of the c and d

forms may be due to addition of cysteine units through the y-glu-cys

unit of the phytochelatin molecule.

Lack of RCC in soil solution taken from the rhizosphere of healthy

and decline trees along with transmission data (Tucker et al., 1984)

indicates that the agent which induces increased or new production of

the Zn-binding factors is probably not of edaphic origin. However, this

agent is likely to be Zn or an organism. Either way, the transmitted

agent must induce synthesis of an existing or new metabolite in order to

form the 4 species separated in this study. It is intriguing that the

induced synthesis may be of extended forms of this metal-binding

molecule, in view of evidence that the 4 species, obtained from PAGE

show varied affinities for Pb, Cd and Zn. This adds further credence to

the hypothesis that the 4 species may be several forms of phytochelatin.

With regard to future research, it will be useful to separate the

species and collect them in preparative quantity. It is important to

use a silver stain which has greater sensitivity, to assay the purity of

each isolated completing agent. Possibly, affinity chromatography will

be necessary to achieve optimum purification. With isoelectric

focusing, the Zn-binding factors could be further characterized in terms

of the isoelectric points of the 4 species. Once optimum purity is









achieved, amino acid composition may be determined. This would provide

final verification that the Zn-binding factors associated with citrus

blight are phytochelatins. Once the metal-binding molecules have been

identified as to content and structure, amperometric titrations may be

performed which will enable the comparison of the 4 species as to their

relative abilities to bind several metals based on equal activities of

those metals.

The effect of the Zn-binding factors on Zn-metabolism and the

mechanism by which an altered Zn metabolism affects the manifestation of

blight are of interest. This question could be partially addressed by

determining if the Zn-binding factors affect the activity of known

Zn-requiring enzymes. In addition, determination of the activities of

these enzymes in healthy versus predecline and decline trees might

contribute evidence as to the directness of the involvement of Zn

metabolism in development of the citrus blight syndrome. The integrated

consequence of their reduced activity on the tree's metabolism would be

much more difficult to ascertain.

An extension of the information about the biosynthesis of the

Zn-binding factor is pertinent to blight research. Therefore,

determination of whether there is induction of synthesis of new mRNA

species or amplification of normal mRNA species is of interest. The

data from this study suggest the latter. Further, localization of this

increased aRNA within predecline trees should indicate the source-site

of the Zn-binding factor.















LITERATURE CITED


Albrigo, L. G. (in press). Xylem plugging and mineral status of blight-
affected citrus trees...a review. Proc. Int. Soc. Citriculture. 1984

Albrigo, L. G. Syvertsen, J. P., and R. H. Young. 1986. Stress
symptoms of citrus trees in successive stages of decline due to blight.
J. Amer. Soc. Short. Sci. 111(3):465-470.

Albrigo, L. G. and R. H. Young. 1981. Phloem zinc accumulation in
citrus trees affected with blight. HortScience 16:158-160.

Bartolf, M., T. Brennan, and C. A. Price. 1980. Partial character-
ization of a cadmium binding protein from the roots of cadmium-treated
tomato. Plant Physiol. 66:438-441.

Bausher, M. G. 1979. Changes in ATP levels and carbonic anhydrase in
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BIOGRAPHICAL SKETCH


Kathryn Campbell Taylor was born August 8, 1958,'in Greenville,

South Carolina. She received an Associate of Arts and Science degree

from Southside Virginia Community College in June, 1977. Her Bachelor

of Science degree in biology was received from the University of South

Carolina in December 1979.

After graduation, Kathryn was employed as a laboratory technician

by the South Carolina Department of Agriculture, Columbia, and by

Clemson University's Sandhill Experiment Station, Elgin, South Carolina.

She managed a greenhouse for initiating vegetable seedlings before

transplant to the field, a hydroponic nutrition study and a nutrient

analysis laboratory.

She enrolled as a graduate student in the Department of

Horticulture at Clemson University in August, 1981. The degree of

Master of Science was conferred in December, 1983. Kathryn was accepted

for graduate study in the Fruit Crops Department of the University of

Florida for January, 1984, enrollment. She will receive the degree of

Doctor of Philosophy in May, 1987.

Kathryn was married to William Thomas Taylor, Jr., August, 1979.

They have a son, Charles William, born December, 1984, and expect their

second child in March, 1987. She plans to continue her research

interests in the context of a university environment. She begins a

post-doctoral fellowship in the Vegetable Crops Department of the

University of Florida, January, 1987.

85
















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. Gene Albrigo, Chairmanq,
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.

-- v: 1.1 -z.
R. Hilton Biggs //
Professor of Biochemistry
and Molecular Biology


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.


Christine D. Chase
Assistant 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.


Karen E. Koch
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.


Ivan Stewart
Professor of Biochemistry
and Molecular Biology


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.


Jif y J. !treet
'Asociate Professor of
Soil Science


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 Docto of Philosophy.

May, 1987 ;
Dean, College of Agriculture


Dean, Graduate School