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Genetic Analyses of the Pretty Few Seeds2 Locus on Ovule Development

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Genetic Analyses of the Pretty Few Seeds2 Locus on Ovule Development
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Hwang, Soon
Hauser, Bernard ( Mentor )
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Gainesville, Fla.
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Genetic Analyses of the Pretty Few Seeds2 Locus on Ovule Development

Soon Hwang


ABSTRACT


Ovules are the developmental precursors of seed. The maternal tissues surrounding the growing embryo are

critical for its growth and development. A number of mutations affecting the development of these maternal

organs result in female sterility. This investigation describes a new genetic locus that is necessary to the

development of these maternal tissues and fertility. The pretty few seeds2 (pfs2) mutant exhibits specific defects

in gametophyte formation and integument morphogenesis. This locus is inherited as a maternal trait, which

indicates that the defects in gametophyte development are secondary to development of the maternal

organs. Examination of genetic interactions of the pfs2 locus with other well-characterized ovule loci has clarified

the role of this locus on ovule development. Interestingly, the double-mutant phenotypes of the PFS2 locus with

the inner no outer and strubbelig loci all had integuments that appear similar to telomes. Based on the

prehistoric fossil record, telomes are believed to be the evolutionary precursors of ovules. Based on these data,

we hypothesize that the PFS2 gene might have played an important role in integument evolution.



INTRODUCTION


A small angiosperm, Arabidopsis thaliana, has been utilized as a model organism to study plant reproduction.

The growth of A. thaliana ovules serves as a general model for angiosperm ovule development. Numerous

studies have characterized the development of wild-type A. thaliana ovules (Modrusan et al., 1994; Robinson-

Beers et al., 1992; Schneitz et al., 1995). In addition, molecular and genetic studies have made steady progress

to identify and predict additional loci that regulate steps of Arabidopsis ovule development (for reviews see, Gasser

et al., 1998; Grossniklaus and Schneitz, 1998). For these reasons, it is practical to use this member of the

mustard family to define genetic steps of ovule development.

Herein, we describe a new locus that regulates ovule development in Arabidopsis thaliana. Through the

genetic analysis of the interactions between the PFS2 locus and other ovule loci, the function of gene in the

ovule developmental pathways will be examined. Identification of the function of the PFS2 gene on

ovule development could provide a tool to explore important aspects of plant reproduction.



METHODS


Plant growth and genetics








Plants were grown on sterile soil (Fafard Mix #2, Conrad Fafard Inc., Agawam, MA) at 220C with a fluence rate

of ~100 pE/m2/s. The pfs2 mutant was crossed with the following ovule mutants: aintegumenta (ant), bell

(bell), fiddle faddle (ffd), inner no outer (/no), nozzle (nzz), and strubbelling (sub). These crosses were made

by fertilizing pfs2 pistils with pollen from other mutants. Segregating F2 plants were examined as described

below. The probabilities of observed segregation ratios in double-mutant populations were determined by chi

squared analyses.


Ovule morphology and anatomy



An MZFL3 stereomicroscope was used to examine the morphology (Leica Microsystems,

Heidelberg, Germany) and determine phenotype of ovules in segregating double mutant populations.

The internal anatomy of cleared ovules was visualized using Normarsky optics, where differences in

the refractive index of the samples generated contrast. Ovules were cleared in BB4.5 (Herr, 1971).


Scanning electron microscopy (SEM)



Single and double mutants were examined using an SEM. Each pistil was dissected along the length of

a carpel, which exposed the ovules, and fixed in FAA (10% formalin, 5% acetic acid, 50%

ethanol). Specimens were dehydrated in a graded ethanol series and dried with a critical point

dryer. Dried specimens were mounted on stubs, dissected, sputter-coated with platinum, and

examined using a Hitachi S-4000 field emission scanning electron microscope (Tokyo,

Japan). Microscopic images were processed using Adobe Photoshop 5.5 (Adobe Systems, Inc., San

Jose, California).



RESULTS


Ovule development in wild-type plants


The development of wild-type Arabidopsis ovules has been characterized (Robinson-Beer et al.,

1992). An ovule initiates from the growth of cells in the placenta. Ovule primordia differentiate into

three distinct zones: funiculus, chalaza, and nucellus (Fig. 1A). Within the nucellus, a megaspore

mother cell undergoes meiosis. A single meiotic product divides and differentiates into a seven-

celled gametophyte (Fig. 1C). Integument primoridia originate from the chalazal tissue and

differentiate into the inner and outer integuments (Fig. 1B). These integuments protect and nourish

the nucellus and developing embryo sac. Asymmetric growth of the outer integument leads to

the curvature of the distal end of the ovule, until it is adjacent to the funiculus (Fig. 1C).






A B C
es

S0oi 0i

placenta




Figure 1. A) Ovule primordia emerge from the placenta. These primordia differentiate into three

distinct zones: the nucellus (n), chalaza (c), and funiculus (f). B) Integument primordia emerge from

the chalaza region and develop into the inner integument (ii) and outer integument (oi). A cell within

the nucellus undergoes meiosis, forming three polar bodies and a single functional megaspore. C)

This megaspore divides three times to produce the embryo sac (es), which contains the

antipodals, synergids, egg, and central cell. The outer integument undergoes asymmetric growth,

causing the distal tip of the ovule to curve until it is adjacent to the funiculus.



Morphology of pfs2 ovules


The pfs2 mutants displayed defects in megaspore and gametophyte differentiation. The embryo sac

in pfs2 mutants was disorganized, often containing fewer cells than normal (Figure 2). In addition,

the growth of integuments was retarded and often displayed aberrant morphology (Figure 2). Only

a small number of ovules made anatomically normal embryo sacs; most had fewer than the

normal complement of seven cells.




stage 1-11 stage 2-111 stage 3-VI


Figure 2. The ovule anatomy of pfs2 and of wild-type were compared. Whole-mount preparations

were depicted with optical sections. A) At stage 1-11, ovule primordia began to differentiate into





the funiculus, chalaza, and nucellus regions. B) At stage of 2-III, the inner integument primordia

(iip) and outer integument primordia (oip) emerged from the chalaza region in the wild type. C) In

the pfs2 mutant, the size of the megaspore mother cell (mmc) was highly reduced compared to the

mmc of the wild type. Additional cells, marked with arrows, occupied the region where normally

there would be the megaspore mother cell. D) The embryo sac of a wild-type ovule contained polar

nuclei (pn), an egg cell (e), and synergids (s). (E) In the pfs2 mutant, a reduced number of cells

was detected in its embryo sac.



Effects of pfs2 locus on fecundity


The number of viable ovules in randomly selected pistils was counted in wild-type and pfs2

mutant plants. Carpels from the pfs2 mutants formed an average of 2.0 �1.2 viable seeds, while

wild-type carpels averaged 47.2 �4.5 ovules. Thus, the pfs2 mutation clearly caused a

significant reduction in fertility (P = 3.7 X 10-22).




Table 1

The frequencies of ovule phenotypes in the F2 populations that were segregating for multiple mutations were tabulated.

F2 phenotypes
Parent genotype Parent phenotype WT:pfs2:SM:DM Confidence level (X2) p-value

pfs2/+bel/+ Wild type 15:1:6:2 3.56 0.31

pfs2/+ffd/+ Wild type 19:5:3:1 1.97 0.58

pfs2/-Hno/+ Wild type 30:8:4:2 3.52 0.32

pfs2/+nzz/+ Wild type 21:6:9:2 0.74 0.86

pfs2/+sub/+ Wild type 23:12:4:3 4.14 0.25


The segregations of mutations closely correlated with the expected Mendelian ratio of 9:3:3:1. [WT: pfs2 : single mutant (SM): double mutant (DM)]. A chi-

squared test, which compared the observed frequencies with the expected frequencies, was used to determine the probability of these distributions (p-value).




Double-mutant analysis




The phenotypes of ovules from segregating F2 populations were determined using a

stereomicroscope (Table 1). The populations segregated for mutations closely correlate with

the expected Mendelian ratio of 9:3:3:1 (wild-type: single mutant A: single mutant

B: double mutant AB). We found the following results in the double-mutant analysis: (1) ant is

epistatic to pfs2 (Fig. 3); (2) bell and ffd exhibited additive genetic interaction with pfs2 (Fig. 4-5);

(3) synergistic genetic interactions existed between pfs2 and ino, nzz, and sub (Fig. 6-8).































Figure 3. A&C) Scanning electron micrographs of ovules. B&D) Optical section of ovules. A) In ant

mutants, integuments failed to emerge from the chalaza. B) The pfs2/-ant/- double mutant was

not distinguishable from the ant single mutant. C) The megaspore mother cell was absent from

the nucellus (n) of ant mutants. D) In the internal anatomy of the pfs2/-ant/- double mutant, the

phenotypic characteristics of the ant single mutant were observed. This evidence indicated that

the ANT was epistatic to the PFS2.


1i


Figure 4. A&C) Scanning electron micrographs of ovules. B&D) Optical section of ovules. A) The

bell single mutant failed to form either an inner or outer integument. Instead, the mutant

developed into an abnormal integument-like structure (ils). B) The embryo sac was absent in the

bell single mutant. C) The pfs2/-bell/- double mutant exhibited a bell-shaped integument

structure, which was a typical characteristic of the bell single mutant. In addition, the growth of

the integument-like structure was reduced. D) The pfs2/-bell/- double mutant failed to develop

an embryo sac.































Figure 5. A&C) Scanning electron micrographs of ovules. B&D) Optical section of ovules. A) In the

ffd single mutant, the orientation and shape of the integument cells were irregular, which led an

abnormal ovule morphology. In the ffd single mutant, the cells of the adjacent ovules sometimes

fused. B) The ffd mutant's outer integument (oi) did not fully develop, so did not encapsulate the

inner integument (ii). C) In addition to having the ffd single-mutant characteristics, the pfs2/-

ffd/- double mutant had pfs2 characteristics, such as aberrant integument expansion and

shortened integuments. D) No embryo sac developed in pfs2/-ffd/- double mutants.




DISCUSSION


PFS2 regulates ovule zonation


Examining genetic interactions is a valuable tool for determining the function of loci in

developmental pathways. There are three common outcomes of double mutant analysis:

additive, epistatic, and synergistic interactions.

An epistatic interaction results when one mutation masks the phenotypic expression of another

mutation. Data revealed that the ant pfs2 double mutant was indistinguishable from the ant

single mutant (Fig. 3); ant was epistatic to pfs2. Reduction in cell division in the integument primordia

of ant mutants resulted in premature termination of ovule development (Baker et al., 1997). For

epistatic interactions, it is generally concluded that one mutation precedes another on a

developmental pathway. These data indicated that ant acts before pfs2 in ovule development.

When two mutations regulate independent and/or parallel pathways, double mutants are termed

additive phenotypes. This type of interaction was observed in pfs2 mutants in the bel 1 and ffd

mutant backgrounds. The bel 1 mutant is involved in determining ovule identity (Modrusan et al.,

1994; Robinson-Beers et al., 1992). Regulating a pathway independent from the bel 1 mutant, the






ffd mutant is involved in epidermal differentiation of ovules. The observed additive interactions

indicated that BEL 1 and FFD act independently of PFS.

Synergistic interactions were observed between pfs2 and other mutations that affect ovule

patterning (Figs. 6-8). When two loci perform similar functions, the double mutant, when compared

to the single mutants, exhibits a synergistic phenotype. Differentiation of the chalaza, integuments,

and nucellus is regulated by patterning genes. The phenotype of pfs2 indicated that PFS2

regulates differentiation of the integuments and nucellus (Fig. 2). The nzz locus encodes a novel

protein that is reported to be essential for nucellus zonation and sporocyte formation (Schiefthaler et

al., 1999; Yang et al., 1999). The SUB locus encodes a leucine-rich repeat kinase. The Sub-

phenotype and gene indicate this locus is involved in communication during the morphogenesis of

outer integuments. The INO locus is required for zonation and differentiation of the outer

integuments (Villanueva et al., 1999). The SUB, INO, and NZZ loci all regulate ovule patterning

and morphogenesis. The synergistic interaction of pfs2 with these three mutants indicates

PFS2 regulates similar processes. Thus, our analyses suggest that pfs2 orchestrates ovule patterning

of the integuments and nucellus in conjunction with SUB, INO, and NZZ.




























Figure 6. A&C) Scanning electron micrographs of ovules. B&D) Optical section of ovules. A) In the

ino mutant, the outer integument (oi) primordia failed to develop. B) An embryo sac (es) formed

in the ino mutant. C) The pfs2/-ino/- double mutant had reduced growth of the outer integument,

thus resembling the development of the ino single mutant. In addition, the inner integument (ii)

was bifurcated. D) The double mutant lacked an embryo sac. In addition, the inner integument

bifurcated into finger-like projections. These projections appeared similar to telomes, which were

the evolutionary progenitors of the inner integument.




























Figure 7. A&C) Scanning electron micrographs of ovules. B&D) Optical section of ovules. A) In the

nzz mutants, the integument morphology was similar to that of wild-type, although, the ovules

were reduced in size. B) In the nzz mutants, the embryo sac failed to form. C&D) In the pfs2/-

nzz/- double mutant, the integument primordia failed to develop. The reduction in size of the

inner and outer integument (ii, oi, respectively) led to the nucellus being exposed.


Figure 8. A&C) Scanning electron micrographs of ovules. B&D) Optical section of ovules. A) The

integument morphogenesis was disrupted in the sub mutant. In the sub single mutant, the

orientation of the integument cells was abnormal. B) In the sub single mutant, the megaspore

mother cell failed to differentiate into an embryo sac. C) The pfs2/-sub/- double mutant had

finger-like structures, which appear analogous to telomes. D) The embryo sac was absent in the

pfs2/-sub/- double mutant.


Ovule evolution






Integuments protect and nourish developing gametes and seeds. Paleobotanists have proposed that

the inner integument originated from the fusion of the sterilized sporangia (for a review see, Kenrick

and Crane, 1997). In a number of extinct taxa, the fossils had sterilized sporangia (termed telomes)

that partially or completely surrounded a fertile sporangium (Kenrick and Crane, 1997). As

female reproductive structures evolved, telomes fused together along their entire length, producing

an integument. In our phenotypic analyses of pfs2 double mutants, involving the ino, and sub loci,

ovules had telome-like structures. This ovule morphology is believed to be the evolutionary precursors

of ovules. The mimicking of telomes indicates that the radially symmetrical sporangia would have

to organize their growth around the centrally located fertile sporangia. Each of these genes was

involved in ovule patterning and acted as a communication system during the growth of

sterilized sporangia around fertile sporangium. Thus, it is probable that PFS2, SUB, and INO loci took

on roles in ovule patterning as telomes evolved into an integument.



CONCLUSION


Genetic models of ovule development demonstrate a close interplay between the development of

the gametophyte and integuments (Gasser et al., 1998; Grossniklaus and Schneitz, 1998). Our

present investigation of the pfs2 mutant phenotype revealed that this locus is necessary for

appropriate integument growth and megaspore mother cell differentiation. Our morphological

and histological analyses of the double mutant of pfs2 with other ovule mutants provided evidence

that PFS2 gene plays a vital role in the morphogensis of the ovule. Further work will be done

to determine if PFS2 gene specifies identity along the proximal-distal axis during ovule development

or by regulates cell polarity in a developmental field.






REFERENCES


Baker, S. C., Robinson-Beers, K., Villanueva, J. M., Gaiser, J. C. and Gasser, C. S. (1997).

Interactions among genes regulating ovule development in Arabidopsis thaliana. Genetics 145,

1109-1124.


Gasser, C. S., Broadhvest, J. and Hauser, B. A. (1998). Genetic analysis of ovule development. Ann.

Rev. Plant Physiol. Plant Mol. Biol. 49, 1-24.


Grossniklaus, U. and Schneitz, K. (1998). The molecular and genetic basis of ovule and

megagametophyte development. Sem. Cell & Devel. Biol. 9, 227-238.


Herr, J. M. (1971). A new clearing technique for the study of ovule development in angiosperms. Am.

J. Bot. 58, 785-790.




Kenrick, P. and Crane, P. R. (1997). The origin and early diversification of land plants: a cladistic

study. Washington, DC: Smithsonian Institution Press.

Modrusan, Z., Reiser, L., Feldmann, K. A., Fischer, R. L. and Haughn, G. W. (1994).

Homeotic transformation of ovules into carpel-like structures in Arabidopsis. Plant Cell 6, 333-349.

Robinson-Beers, K., Pruitt, R. E. and Gasser, C. S. (1992). Ovule development in wild-type

Arabidopsis and two female-sterile mutants. Plant Cell 4, 1237-1249.

Schiefthaler, U., Balasubramanian, S., Sieber, P., Chevalier, D., Wisman, E. and Schneitz, K.

(1999). Molecular analysis of NOZZLE, a gene involved in pattern formation and early

sporogenesis during sex organ development in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 96,

11664-11669.

Schneitz, K., Hulskamp, M. and Pruitt, R. E. (1995). Wild-type ovule development in Arabidopsis

thaliana: a light microscope study of cleared whole-mount tissue. Plant J. 7, 731-749.

Villanueva, J. M., Broadhvest, J., Hauser, B. A., Meister, R. J., Schneitz, K. and Gasser, C. S. (1999).

INNER NO OUTER regulates abaxial-adaxial patterning in Arabidopsis ovules. Genes & Development

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Yang, C.-D., Ye, D., Xu, J. and Sundaresan, V. (1999). The SPOROCYTELESS gene of Arabidopsis

is required for initiation of sporogenesis and encodes a novel nuclear protein. 13, 2108-2117.


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