Ramamurthy Baskar

LACHESIS Restricts Gametic Cell Fate in the Female Gametophyte of Arabidopsis

In flowering plants, the egg and sperm cells form within haploid gametophytes. The female gametophyte of Arabidopsis consists of two gametic cells, the egg cell and the central cell, which are flanked by five accessory cells. Both gametic and accessory cells are vital for fertilization; however, the mechanisms that underlie the formation of accessory versus gametic cell fate are unknown. In a screen for regulators of egg cell fate, we isolated the lachesis (lis) mutant which forms supernumerary egg cells. In lis mutants, accessory cells differentiate gametic cell fate, indicating that LIS is involved in a mechanism that prevents accessory cells from adopting gametic cell fate. The temporal and spatial pattern of LIS expression suggests that this mechanism is generated in gametic cells. LIS is homologous to the yeast splicing factor PRP4, indicating that components of the splice apparatus participate in cell fate decisions.

Dosage-sensitive function of retinoblastoma related and convergent epigenetic control are required during the Arabidopsis life cycle.

The plant life cycle alternates between two distinct multi-cellular generations, the reduced gametophytes and the dominant sporophyte. Little is known about how generation-specific cell fate, differentiation, and development are controlled by the core regulators of the cell cycle. In Arabidopsis, RETINOBLASTOMA RELATED (RBR), an evolutionarily ancient cell cycle regulator, controls cell proliferation, differentiation, and regulation of a subset of Polycomb Repressive Complex 2 (PRC2) genes and METHYLTRANSFERASE 1 (MET1) in the male and female gametophytes, as well as cell fate establishment in the male gametophyte. Here we demonstrate that RBR is also essential for cell fate determination in the female gametophyte, as revealed by loss of cell-specific marker expression in all the gametophytic cells that lack RBR. Maintenance of genome integrity also requires RBR, because diploid plants heterozygous for rbr (rbr/RBR) produce an abnormal portion of triploid offspring, likely due to gametic genome duplication. While the sporophyte of the diploid mutant plants phenocopied wild type due to the haplosufficiency of RBR, genetic analysis of tetraploid plants triplex for rbr (rbr/rbr/rbr/RBR) revealed that RBR has a dosage-dependent pleiotropic effect on sporophytic development, trichome differentiation, and regulation of PRC2 subunit genes CURLY LEAF (CLF) and VERNALIZATION 2 (VRN2), and MET1 in leaves. There were, however, no obvious cell cycle and cell proliferation defects in these plant tissues, suggesting that a single functional RBR copy in tetraploids is capable of maintaining normal cell division but is not sufficient for distinct differentiation and developmental processes. Conversely, in leaves of mutants in sporophytic PRC2 subunits, trichome differentiation was also affected and expression of RBR and MET1 was reduced, providing evidence for a RBR-PRC2-MET1 regulatory feedback loop involved in sporophyte development. Together, dosage-sensitive RBR function and its genetic interaction with PRC2 genes and MET1 must have been recruited during plant evolution to control distinct generation-specific cell fate, differentiation, and development.

Arabidopsis GLAUCE promotes fertilization-independent endosperm development and expression of paternally inherited alleles

Early seed development of sexually reproducing plants requires both maternal and paternal genomes but is prominently maternally influenced. A novel gametophytic maternal-effect mutant defective in early embryo and endosperm development, glauce (glc), has been isolated from a population of Arabidopsis Ds transposon insertion lines. The glc mutation results from a deletion at the Ds insertion site, and the molecular identity of GLC is not known. glc embryos can develop up to the globular stage in the absence of endosperm and glc central cells appear to be unfertilized. glc suppresses autonomous endosperm development observed in the fertilization-independent seed (fis) class mutants. glc is also epistatic to mea, one of the fis class mutants, in fertilized seeds, and is essential for the biparental embryonic expression of PHE1, a repressed downstream target of MEA. In addition, maternal GLC function is required for the paternal embryonic expression of the ribosome protein gene RPS5a and the AMP deaminase gene FAC1, both of which are essential for early embryo and endosperm development. These results indicate that factors derived from the female gametophyte activate a subset of the paternal genome of fertilized seeds.

Female gametophytic cell specification and seed development require the function of the putative Arabidopsis INCENP ortholog WYRD

In plants, gametes, along with accessory cells, are formed by the haploid gametophytes through a series of mitotic divisions, cell specification and differentiation events. How the cells in the female gametophyte of flowering plants differentiate into gametes (the egg and central cell) and accessory cells remains largely unknown. In a screen for mutations that affect egg cell differentiation in Arabidopsis, we identified the wyrd (wyr) mutant, which produces additional egg cells at the expense of the accessory synergids. WYR not only restricts gametic fate in the egg apparatus, but is also necessary for central cell differentiation. In addition, wyr mutants impair mitotic divisions in the male gametophyte and endosperm, and have a parental effect on embryo cytokinesis, consistent with a function of WYR in cell cycle regulation. WYR is upregulated in gametic cells and encodes a putative plant ortholog of the inner centromere protein (INCENP), which is implicated in the control of chromosome segregation and cytokinesis in yeast and animals. Our data reveal a novel developmental function of the conserved cell cycle-associated INCENP protein in plant reproduction, in particular in the regulation of egg and central cell fate and differentiation.

Regulation of aggregate size and pattern by adenosine and caffeine in cellular slime molds

BackgroundMulticellularity in cellular slime molds is achieved by aggregation of several hundreds to thousands of cells. In the model slime mold Dictyostelium discoideum, adenosine is known to increase the aggregate size and its antagonist caffeine reduces the aggregate size. However, it is not clear if the actions of adenosine and caffeine are evolutionarily conserved among other slime molds known to use structurally unrelated chemoattractants. We have examined how the known factors affecting aggregate size are modulated by adenosine and caffeine.ResultAdenosine and caffeine induced the formation of large and small aggregates respectively, in evolutionarily distinct slime molds known to use diverse chemoattractants for their aggregation. Due to its genetic tractability, we chose D. discoideum to further investigate the factors affecting aggregate size. The changes in aggregate size are caused by the effect of the compounds on several parameters such as cell number and size, cell-cell adhesion, cAMP signal relay and cell counting mechanisms. While some of the effects of these two compounds are opposite to each other, interestingly, both compounds increase the intracellular glucose level and strengthen cell-cell adhesion. These compounds also inhibit the synthesis of cAMP phosphodiesterase (PdsA), weakening the relay of extracellular cAMP signal. Adenosine as well as caffeine rescue mutants impaired in stream formation (pde4- and pdiA- ) and colony size (smlA- and ctnA- ) and restore their parental aggregate size.ConclusionAdenosine increased the cell division timings thereby making large number of cells available for aggregation and also it marginally increased the cell size contributing to large aggregate size. Reduced cell division rates and decreased cell size in the presence of caffeine makes the aggregates smaller than controls. Both the compounds altered the speed of the chemotactic amoebae causing a variation in aggregate size. Our data strongly suggests that cytosolic glucose and extracellular cAMP levels are the other major determinants regulating aggregate size and pattern. Importantly, the aggregation process is conserved among different lineages of cellular slime molds despite using unrelated signalling molecules for aggregation.

Hybridization alters spontaneous mutation rates in a parent-of-origin-dependent fashion in Arabidopsis.

Hybridization alters mutation rates in Arabidopsis. Over 70 years ago, increased spontaneous mutation rates were observed in Drosophila spp. hybrids, but the genetic basis of this phenomenon is not well understood. The model plant Arabidopsis (Arabidopsis thaliana) offers unique opportunities to study the types of mutations induced upon hybridization and the frequency of their occurrence. Understanding the mutational effects of hybridization is important, as many crop plants are grown as hybrids. Besides, hybridization is important for speciation and its effects on genome integrity could be critical, as chromosomal rearrangements can lead to reproductive isolation. We examined the rates of hybridization-induced point and frameshift mutations as well as homologous recombination events in intraspecific Arabidopsis hybrids using a set of transgenic mutation detector lines that carry mutated or truncated versions of a reporter gene. We found that hybridization alters the frequency of different kinds of mutations. In general, Columbia (Col) × Cape Verde Islands and Col × C24 hybrid progeny had decreased T→G and T→A transversion rates but an increased C→T transition rate. Significant changes in frameshift mutation rates were also observed in some hybrids. In Col × C24 hybrids, there is a trend for increased homologous recombination rates, except for the hybrids from one line, while in Col × Cape Verde Islands hybrids, this rate is decreased. The overall genetic distance of the parents had no influence on mutation rates in the progeny, as closely related accessions on occasion displayed higher mutation rates than accessions that are separated farther apart. However, reciprocal hybrids had significantly different mutation rates, suggesting parent-of-origin-dependent effects on the mutation frequency.

Engineering of Apomixis in Crop Plants: What Can We Learn from Sexual Model Systems?

The development of apomixis technology in crop plants is a desirable goal. Apomixis is the asexual reproduction through seeds, which occurs in over 400 flowering plants (Nogler, 1984). The introduction of clonal reproduction to crop plants will allow the indefinite propagation of any desirable genotype (including that of heterozygous F1 hybrids) and will completely transform current breeding and seed production strategies. Developmental aspects of apomixis (Koltunow, 1993; Grossniklaus, 2001; Spillane et al., 2001), its genetic control (Savidan, 2000; Grossniklaus et al., 2001a; Grimanelli et al., 2001), and its potential use in agriculture (Koltunow et al., 1995; Hanna et al., 1998; Jefferson and Bicknell, 1996; Thoenissen, 2001) have been extensively reviewed. Here, we provide a short summary of developmental and genetic aspects and report on our program using sexual model systems to identify genes and promoters relevant to the engineering of apomixis.

Parental Age Affects Somatic Mutation Rates in the Progeny of Flowering Plants

The reproductive age of the parents has a significant influence on the kind and rate of somatic mutations in their progeny. In humans, it is well known that the parental reproductive age has a strong influence on mutations transmitted to their progeny. Meiotic nondisjunction is known to increase in older mothers, and base substitutions tend to go up with paternal reproductive age. Hence, it is clear that the germinal mutation rates are a function of both maternal and paternal ages in humans. In contrast, it is unknown whether the parental reproductive age has an effect on somatic mutation rates in the progeny, because these are rare and difficult to detect. To address this question, we took advantage of the plant model system Arabidopsis (Arabidopsis thaliana), where mutation detector lines allow for an easy quantitation of somatic mutations, to test the effect of parental age on somatic mutation rates in the progeny. Although we found no significant effect of parental age on base substitutions, we found that frameshift mutations and transposition events increased in the progeny of older parents, an effect that is stronger through the maternal line. In contrast, intrachromosomal recombination events in the progeny decrease with the age of the parents in a parent-of-origin-dependent manner. Our results clearly show that parental reproductive age affects somatic mutation rates in the progeny and, thus, that some form of age-dependent information, which affects the frequency of double-strand breaks and possibly other processes involved in maintaining genome integrity, is transmitted through the gametes.

Regulation of multiple tip formation by caffeine in cellular slime molds

BackgroundThe multicellular slug in Dictyostelium has a single tip that acts as an organising centre patterning the rest of the slug. High adenosine levels at the tip are believed to be responsible for this tip dominance and the adenosine antagonist, caffeine overrides this dominance promoting multiple tip formation.ResultsCaffeine induced multiple tip effect is conserved in all the Dictyostelids tested. Two key components of cAMP relay namely, cAMP phosphodiesterase (Pde4) and adenyl cyclase-A (AcaA) levels get reduced during secondary tip formation in Dictyostelium discoideum. Pharmacological inhibition of cAMP phosphodiesterase also resulted in multiple tips. Caffeine reduces cAMP levels by 16.4, 2.34, 4.71 and 6.30 folds, respectively in D. discoideum, D. aureostipes, D. minutum and Polysphondylium pallidum. We propose that altered cAMP levels, perturbed cAMP gradient and impaired signalling may be the critical factors for the origin of multiple tips in other Dictyostelids as well. In the presence of caffeine, slug cell movement gets impaired and restricted. The cell type specific markers, ecmA (prestalk) and pspA (prespore) cells are not equally contributing during additional tip formation. During additional tip emergence, prespore cells transdifferentiate to compensate the loss of prestalk cells.ConclusionCaffeine decreases adenyl cyclase–A (AcaA) levels and as a consequence low cAMP is synthesised altering the gradient. Further if cAMP phosphodiesterase (Pde4) levels go down in the presence of caffeine, the cAMP gradient breaks down. When there is no cAMP gradient, directional movement is inhibited and might favour re-differentiation of prespore to prestalk cells.

Functional interaction of PkcA and PldB regulate aggregation and development in Dictyostelium discoideum

Multicellular development in Dictyostelium discoideum involves tightly regulated signaling events controlling the entry into development, initiation of aggregation and chemotaxis, and cellular differentiation. Here we show that PkcA, a Dictyostelium discoideum Protein Kinase C-orthologue, is involved in quorum sensing and the initiation of development, as well as cAMP sensing during chemotaxis. Additionally, by epistasis analysis we provide evidence that PkcA and PldB (a Phospholipase D-orthologue) functionally interact to regulate aggregation, differentiation, and cell-cell adhesion during development. Finally, we show that PkcA acts as a positive regulator of intracellular PLD-activity during development. Taken together, our results suggest that PkcA act through PldB, by regulating PLD-activity, in order to control events during development.