Male Sterility in Pea VI. Gene action duplicity

_??_ 1994 The Japan Mendel Society Cytologia 59: 195-201, 1994

Male Sterility in Pea VI. Gene action duplicity

C. Nirmala and M. L. H. Kaul

Department of Botany, Kurukshetra University, Kurukshetra-132119 (Haryana), India

Accepted March 18, 1994

In a developing flower bud, the meristematic cells choose their developmental fate and differentiate either into reproductive (stamens and carpels) or into non-reproductive (sepals and petals or perianth) parts. Whereas, only mitosis occurs in the latter, meiosis is characteristic of the former organs. Meiosis is controlled by a special set of genes termed as the "meiotic

genes". Mutations of one or more of these genes derail meiotic tract and result in spore sterility. Though majority of the meiotic genes are common in male and female sex organs, a few are either male- or female-sex specific. Mutations of these result in male or female sterility, respectively (Kaul 1988, Skorupska et al. 1993). The male sterile mutant genes either inhibit or distract meiosis or gametogenesis (Kaul 1988, Myers et al. 1992, Denis et al. 1993). Majority of these genes exhibit uniform gene action and are sex-, stage-, site- and time-specific (Gottschalk and Kaul 1974, Graybosch and Palmer 1988, Kaul 1988, Theis and Robbelen 1990, Kaul and Singh 1991, Nirmala and Kaul 1991). But a male sterile mutant gene msg2 in Pisum sativum, exhibits dual and diffuse gene action that does not follow time-, stage-, and site-specifities. In this mutant, whereas in some PMCs, the gene inhibits male meiosis completely, in others, the gene permits meiosis to continue but makes it abnormal by its variable and diffuse gene action. Description of this dual gene action comprises text of this paper.

Materials and methods

Mutant induction and isolationSeeds of the pea variety Bonneville presoaked in distilled water for 12 hr were subjected to

5 kR gamma ray treatment. From the treated seed, M1 progeny was raised. The M2, M3 and M4 generation segregation and back crosses results revealed the male sterility to be controlled by a single recessive gene.

Sample sizeFrom each of the 20 randomly selected male sterile plants, 20 flower buds per plant/geno

type were used for meiotic investigation. Plants of 3 generations were utilized for the study. Thus in all, 1200 floral buds from 60 male sterile plants were cytologically examined.

Meiotic analysis

For meiotic studies, young flower buds from the male steriles were fixed between 10-11 am

during November-December (temp. range 14-20•Ž day, 5-9•Ž night) in freshly prepared

acetic alcohol mixture (1:3). Fertile flower buds were also fixed for comparison. The material

was preserved in 70% ethanol and squashed in 1% acetocarmine.

Meiotic event quantificationThe major meiotic events were quantified (Tables 1, 2). To determine the impact of a

cytological anomaly, its mean value was divided by the mean value of the prime cytological event. This yielded the absolute value.

196 C. Nirmala and M. L. H. Kaul Cytologia 59

Table 1. Major meiotic events in msg2 mutant

SD=Standard deviation. N=1200.

Table 2. Mean with its statistical parameters and significance of difference

* Mean values not followed by the same alphabet (a, b, c, •c) differ significantly from each other at 5P level as

revealed by Duncan's multiple range test and paired t-test. SE=Standard error; CV=Coefficient of variability.

N=1200.

Statistical analysisFor computation of various statistical constants, method outlined by Steel and Torrie

(1980) was followed. The significance of mean difference between various cytological events was determined by using Duncan's multiple range test (Gomez and Gomez 1984). Wherever overlaps occurred, t-test was utilized to decide the significance of mean difference.

Observations

In this mutant, about 5% floral buds termed as early formed flowers, appear 8-14 days

prior to the main flowering flush and drop off subsequently. In all the PMCs of these buds,

1994 Male Sterility in Pea VI 197

cytomixis occurs during premeiosis and the PMCs degenerate without undergoing any meiosis. The remaining 95% flowers are formed during normal flowering period. In these flowers,

whereas in 66% PMCs meiosis continues abnormally, in 34% PMCs meiosis is inhibited and cytomixis is rampant. Thus the normally formed flowers of this mutant exhibit a two tract male meiosis. This is detailed below.

a) Cytomictic PMCs (34%)Prior to the meiotic initiation, all the PMCs of these florets are fused with each other by

cytoplasmic connections through which transmigration of chromatin and/or nucleolus occurs. Whereas in 40% PMCs, both chromatin and nucleolus transmigrate (Fig. 1), in 60% PMCs, only chromatin migrates (Fig. 2). This chromatin migration is either directional or random. In about 9% PMCs, the chromatin undergoes condensation, low to extensive fragmentation and

Figs. 1-4. Cytomictic PMCs. 1, Chromatin migration along with nucleolus. 2, Chromatin

migration without nucleolus. 3, Extensive chromatin fragmentation and transmigration. 4,

Chromatin fragmentation and migration. •~1500.


Figs. 5-12. Non-cytomictic PMCs. 5, 5II and 4I at diplotene. 6, Complete univalency at MI.

7, Irregular AI disjunction. 8, Chromosome bridges and fragments at AI. 9, Chromosome

bridges and fragments at AII. 10, Coenocyte with multiple and fragmented nucleoli. 11,

Nucleolar persistance and chromatin degeneration in microspores. 12, End-to-end attachment of

different sized degenerating microspores. •~1500.

transmigration (Fig. 3). In some such PMCs, the fragments move out of the cell and degeneration ensues thereafter (Fig. 4).

b) Non-cytomictic PMCs (66%)In these, the male meiotic progression is highly abnormal. Homologous chromosomal

pairing in 88% PMCs is partial. The bivalent associations in the PMCs ranges from 2 to 6, the modal value being 5 (Table 1, Fig. 5). In the 66% PMCs, dys-synapsis is followed by chromosomal stickiness. The MI plate is incipient and precocious disjunction of bivalents occurs in about 18% PMCs. This creates univalency at MI (Fig. 6). The subsequent male meiotic events are abnormal (Table 1) the main anomalies being: irregular AI and AII disjunctions (Fig. 7) chromosome breakages and reunions, bridges and fragment formations


(Figs. 8, 9). Consequently unequal sized dyads, tetrads and polyads are formed. In about 18% PMCs, coenocytes with multiple nuclei are formed (Fig . 10). Due to these meiotic abberations, the microspores are of variable sizes and shapes (Fig. 11). Whereas the larger ones have dense cytoplasm, the smaller ones have scanty chromatin . But nucleolus in all of them is prominent and persistent throughout the chromatin degeneration. Some microspores exhibit wall to wall fusions (Fig. 12). All the microspores degenerate and no viable pollen is produced by this mutant. Hence the mutant is completely male sterile but is fully female fertile as evidenced by the normal seed set after hand pollination.

Discussion

Gene action specificityBoth sporogenesis and gametogenesis are developmentally complex processes that are

controlled and co-ordinated by a spatially sequenced genetic programme. Whereas majority of

genes for micro- and mega-sporogenesis and gametogenesis are common, a few are unique to each sex. Accordingly, male sex specific and female sex specific genes differentiate megasporo

genesis from microsporogenesis. These two sets of genes also exhibit different mutation frequency and spectrum. Both these are much higher for male sex specific genes than for female specific genes (Kaul 1988, Kaul and Nirmala 1989, Mascarenhas 1990). This is evidenced by the presence of a large number of spontaneously arisen and mutagen induced male sterile genes in higher plants (Kaul 1988, Kaul and Nirmala 1993, 1994). One of the characteristic features of male sterile genes is their specificity for time, site, stage and sex (Kaul 1988, Kaul and Singh 1991, Mariani et al. 1991). This is also true for the male sterile genes of P. sativum (Gottschalk and Kaul 1974, Kaul 1988, Myers et al. 1992).

Exceptions to the above mentioned type of ms gene action do exist in pea. For instance, one male sterile mutant msg3 defies this generality of gene specificity as its action is diffuse and staggered. The ms gene acts during either meiotic initiation or its progression or at the end

stage (Nirmala and Kaul 1993). Though like msg3, the msg2 mutant does not show the typical ms gene specificity in action, but unlike msg3, it does not exhibit diffuse or staggered gene action. Instead, this mutant gene exhibits duplicity of gene action. Whereas in 34% PMCs, the

gene causes cytomixis and meiotic arrest, in the remaining 66% PMCs, the male meiosis continues abnormally exhibiting constellation of meiotic anomalies and complete pollen sterility

(Table 1). Though the mean values of most of the meiotic anomalies differ significantly from one another, both the variance and coefficient of variability values are high only for the meiotic anomalies i and iii of the Table 2. This indicates a strong correlation between these two events, the first being the cause and the second, the result of this anomaly. Insignificant mean differences between the anomalies iv, v and vi indicate that almost all the PMCs that undergo male meiosis exhibit these three anomalies during meiotic progression. Mean increase in the

number of variable sized microspores points towards cumulation of other minor anomalies towards the contribution to microspore degeneration of this mutant.

CytomixisIn addition to the abovementioned meiotic anomalies occurring in the 66% PMCs of msg2

anthers, 34% PMCs of this mutant exhibit cytomixis involving the migration of chromatin, nucleus, nucleolus, nucleolar bits, chromosomes and other cell contents from one cell to

another through intercellular connections designated as cytomictic channels. Whereas in many other plants cytomixis occurs in meristematic, tapetal integumental, nucellar and ovary cells, epidermal cell of scales and leaves, in msg2 mutant, it is confined only to one third of PMCs of the flowers developed during the usual flowering time and impedes meiotic progression


Table 3. Plants exhibiting high cytomixis frequency

completely. Since both the occurrence and absence of cytomixis are male sex specific in msg2 mutant and are constantly inherited, the anomaly appears to be either caused by the msg2 gene or the gene responsible for it is closely linked to the ms gene. No segregation between male sterility and cytomixis occurs. Despite this non-segregation, why and how only a certain

proportion of PMCs exhibit cytomixis and meiotic arrest in normally formed flowers is not known. If cytomixis is gene controlled, it is intringuing why the gene action is total in all the PMCs of early formed flowers and only one-third PMCs of the normally formed flowers. Also how such gene action segregation occurs in the PMCs of normally formed flowers or how 66%

PMCs evade or endure the ms gene action and continue to undergo meiosis, is not known.Sporadic occurrence of nonrecurrent or recurrent cytomixis is known in many plants. But

in only a few, the frequency is high (Table 3). This frequency is altered by environmental factors, especially photoperiod and/or temperature. Whether any of these two factors influence msg2 gene action is not known. But why only 34% PMCs exhibit cytomixis is difficult to comprehend. Since the cytomixis is male sex specific, isolation of the ms gene expressed during male meiosis could reveal the processes involved in the gene regulation and the nature of the

products this uniquely expressed ms gene encodes.

Summary

Two track male meiosis is exhibited by a ƒÁ-ray induced monogenic recessive male sterile

mutant of pea (Pisum sativum L). Whereas the ms gene causes cytomixis and meiotic arrest in

one third of the PMCs, in the remaining PMCs, the meiosis is highly abnormal; the main

meiotic anomalies being dys-synapsis, incipient MI plate formation, irregular AI and AII

disjunctions and unequal sized dyad, tetrad and polyad production. All the microspores formed

degenerate and the mutant is completely male sterile. Since only 34% PMCs exhibit cytomixis

and meiotic arrest, it is intriguing how and why do the remaining 66% PMCs escape cytomixis

and continue meiosis abnormally.

Key words: Sex-specific genes, dual gene action, chromatin transmigration.

Acknowledgements

We are grateful to University Grants Commission and Council of Scientific & Industrial

Research, New Delhi for financial assistance. For laboratory facilities and helpful discussions , M. L. H. Kaul is thankful to Prof. T. Kinoshita, Prof. K. Mori and Dr. I. Takamure of Hokkaido University, Sapporo, Japan, where part of this work was done.


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Male Sterility in Pea VI. Gene action duplicity