Functional recapitulation of transitions in sexual systems by homeosis during the evolution of dioecy in Thalictrum.
ABSTRACT: Sexual systems are highly variable in flowering plants and an important contributor to floral diversity. The ranunculid genus Thalictrum is especially well-suited to study evolutionary transitions in sexual systems. Homeotic transformation of sexual organs (stamens and carpels) is a plausible mechanism for the transition from hermaphroditic to unisexual flowers in this lineage because flowers of dioecious species develop unisexually from inception. The single-copy gene PISTILLATA (PI) constitutes a likely candidate for rapid switches between stamen and carpel identity. Here, we first characterized the expression pattern of all B class genes in the dioecious species T. dioicum. As expected, all B class orthologs are expressed in stamens from the earliest stages. Certain AP3 lineages were also expressed late in sepal development. We then tested whether orthologs of PI could potentially control sexual system transitions in Thalictrum, by knocking-down their expression in T. dioicum and the hermaphroditic species T. thalictroides. In T. dioicum, we found that ThdPI-1/2 silencing caused stamen primordia to develop into carpels, resulting in male to female flower conversions. In T. thalictroides, we found that ThtPI silencing caused stamen primordia to develop into supernumerary carpels, resulting in hermaphroditic to female flower conversions. These phenotypes illustrate the ability for homeotic mutations to bring about sudden and potentially adaptive changes by altering the function of a single gene. We propose a two-step evolutionary model where transitions from hermaphroditic to unisexual plants in Thalictrum result from two independent mutations at a B class gene locus. Our PI knockdown experiments in T. thalictroides recapitulate the second step in this model: the evolution of female plants as a result of a loss-of-function mutation in a B class gene.
Project description:The Arabidopsis SUPERMAN (SUP) gene encodes a C2H2 type zinc finger protein that is required for maintaining the boundaries between stamens and carpels, and for regulating development of ovule outer integument. Orthologs of SUP have been characterized in bisexual flowers as well as dioecious species, but it remains elusive in monoecious plants with unisexual flowers on the same individual. Here we isolate the SUP ortholog in Cucumis sativus L (CsSUP), a monoecious vegetable. CsSUP is predominantly expressed in female specific organs: the female flower buds and ovules. Ectopic expression of CsSUP in Arabidopsis can partially complement the fruit development in sup-5 mutant, and its over-expression in wide-type leads to reduced silique length, suppressed stamen development and distorted petal patterning. Our data suggest that CsSUP plays conserved as well as distinct roles during flower and fruit development, and it may function in the boundaries and ovules to balance petal patterning, stamen and ovule development in Arabidopsis.
Project description:Females of Thalictrum pubescens produce stamens that contain sterile pollen, whereas males are both functionally and morphologically unisexual. This study examines the investment in stamen production by females of T. pubescens by comparing the female structures with those of their fully functional male counterparts. Stamens from females had the same biomass and contained the same amount of nitrogen and phosphorus as stamens from males. Anther size was the same in males and females, but filaments were longer in stamens from males. Females produced more pollen per anther than males, and pollen size was the same in both sexes. Within flowers, there was a positive correlation between the amount of pollen per anther and the length of anthers in males, but not in females. This would be expected if males growing in better environmental conditions or with greater vigour invested more resources in pollen production, thereby increasing fitness. Females, who receive no fitness benefits from increased pollen production, did not show this pattern. There was also evidence of a trade-off within female flowers between the number of stamens and the number of pistils. This trade-off was noted in conditions when variance among plants was reduced, namely in the field during a year when flower size was particularly small and in a previous glasshouse study. Therefore, it appears that when environmental variance is low, stamens are produced at the expense of producing more pistils, and hence seeds. In conclusion, stamen production does not appear to be inconsequential to females of Thalictrum pubescens.
Project description:<h4>Premise</h4>Multiple transitions from insect to wind pollination are associated with polyploidy and unisexual flowers in <i>Thalictrum</i> (Ranunculaceae), yet the underlying genetics remains unknown. We generated a draft genome of <i>Thalictrum thalictroides</i>, a representative of a clade with ancestral floral traits (diploid, hermaphrodite, and insect pollinated) and a model for functional studies. Floral transcriptomes of <i>T. thalictroides</i> and of wind-pollinated, andromonoecious <i>T. hernandezii</i> are presented as a resource to facilitate candidate gene discovery in flowers with different sexual and pollination systems.<h4>Methods</h4>A draft genome of <i>T. thalictroides</i> and two floral transcriptomes of <i>T. thalictroides</i> and <i>T. hernandezii</i> were obtained from HiSeq 2000 Illumina sequencing and de novo assembly.<h4>Results</h4>The <i>T. thalictroides</i> de novo draft genome assembly consisted of 44,860 contigs (N50 = 12,761 bp, 243 Mbp total length) and contained 84.5% conserved embryophyte single-copy genes. Floral transcriptomes contained representatives of most eukaryotic core genes, and most of their genes formed orthogroups.<h4>Discussion</h4>To validate the utility of these resources, potential candidate genes were identified for the different floral morphologies using stepwise data set comparisons. Single-copy gene analysis and simple sequence repeat markers were also generated as a resource for population-level and phylogenetic studies.
Project description:Dasiphora fruticosa comprises male, female and hermaphrodite plants, which are distributed sympatrically in some populations on the Qinghai-Tibet Plateau. To explore what governs the coexistence of these three sexual phenotypes, we investigated the DNA contents, pollen and ovule production, pollen deposition, and performed hand-pollination in both hermaphroditic and dioecious individuals of D. fruticosa. Flow cytometry confirmed that the DNA content of males and females were almost twice as much as that of the hermaphrodites. Male and female flowers produced more pollen grains and ovules than hermaphroditic flowers. Hand-pollinated treatments showed that unisexual flowers were sterile in one sexual function and bisexual flowers were fertile for both functions, but no sterile seeds were produced between unisexual and bisexual flowers. Our findings imply that polyploidy is related to gender dimorphism, and both are likely to play a strong role in the coexistence of two cryptic biological species of D. fruticosa (low ploidy hermaphroditic species and high ploidy dioecious species) in the Qinghai-Tibet Plateau.
Project description:BACKGROUND: Evolution of unisexual flowers entails one of the most extreme changes in plant development. Cultivated spinach, Spinacia oleracea L., is uniquely suited for the study of unisexual flower development as it is dioecious and it achieves unisexually by the absence of organ development, rather than by organ abortion or suppression. Male staminate flowers lack fourth whorl primordia and female pistillate flowers lack third whorl primordia. Based on theoretical considerations, early inflorescence or floral organ identity genes would likely be directly involved in sex-determination in those species in which organ initiation rather than organ maturation is regulated. In this study, we tested the hypothesis that sexual dimorphism occurs through the regulation of B class floral organ gene expression by experimentally knocking down gene expression by viral induced gene silencing. RESULTS: Suppression of B class genes in spinach resulted in the expected homeotic transformation of stamens into carpels but also affected the number of perianth parts and the presence of fourth whorl. Phenotypically normal female flowers developed on SpPI-silenced male plants. Suppression of the spinach C class floral organ identity gene, SpAG, resulted in loss of reproductive organ identity, and indeterminate flowers, but did not result in additional sex-specific characteristics or structures. Analysis of the genomic sequences of both SpAP3 and SpPI did not reveal any allelic differences between males and females. CONCLUSION: Sexual dimorphism in spinach is not the result of homeotic transformation of established organs, but rather is the result of differential initiation and development of the third and fourth whorl primordia. SpAG is inferred to have organ identity and meristem termination functions similar to other angiosperm C class genes. In contrast, while SpPI and SpAP3 resemble other angiosperms in their essential functions in establishing stamen identity, they also appear to have an additional function in regulating organ number and identity outside of the third whorl. We present a model for the evolution of dioecy in spinach based on the regulation of B class expression.
Project description:In hermaphroditic Arabidopsis, the phytohormone gibberellin (GA) stimulates stamen development by opposing the DELLA repression of B and C classes of floral homeotic genes. GA can promote male flower formation in cucumber (Cucumis sativus L.), a typical monoecious vegetable with unisexual flowers, and the molecular mechanism remains unknown. Here we characterized a DELLA homolog CsGAIP in cucumber, and we found that CsGAIP is highly expressed in stem and male flower buds. In situ hybridization showed that CsGAIP is greatly enriched in the stamen primordia, especially during the hermaphrodite stage of flower development. Further, CsGAIP protein is located in nucleus. CsGAIP can partially rescue the plant height, stamen development and fertility phenotypes of Arabidopsis rga-24/gai-t6 mutant, and ectopic expression of CsGAIP in wide-type Arabidopsis results in reduced number of stamens and decreased transcription of B class floral homeotic genes APETALA3 (AP3) and PISTILLATA (PI). Our data suggest that monoecious CsGAIP may inhibit staminate development through transcriptional repression of B class floral homeotic genes in Arabidopsis.
Project description:In the model plant Arabidopsis thaliana, a core eudicot, the floral homeotic C-class gene AGAMOUS (AG) has a dual role specifying reproductive organ identity and floral meristem determinacy. We conduct a functional analysis of the putative AG ortholog ThtAG1 from the ranunculid Thalictrum thalictroides, a representative of the sister lineage to all other eudicots. Down-regulation of ThtAG1 by virus-induced gene silencing resulted in homeotic conversion of stamens and carpels into sepaloid organs and loss of flower determinacy. Moreover, flowers exhibiting strong silencing of ThtAG1 phenocopied the double-flower ornamental cultivar T. thalictroides 'Double White.' Molecular analysis of 'Double White' ThtAG1 alleles revealed the insertion of a retrotransposon causing either nonsense-mediated decay of transcripts or alternative splicing that results in mutant proteins with K-domain deletions. Biochemical analysis demonstrated that the mutation abolishes protein-protein interactions with the putative E-class protein ThtSEP3. C- and E-class protein heterodimerization is predicted by the floral quartet model, but evidence for the functional importance of this interaction is scarce outside the core eudicots. Our findings therefore corroborate the importance and conservation of the interactions between C- and E-class proteins. This study provides a functional description of a full C-class mutant in a noncore ("basal") eudicot, an ornamental double flower, affecting both organ identity and meristem determinacy. Using complementary forward and reverse genetic approaches, this study demonstrates deep conservation of the dual C-class gene function and of the interactions between C- and E-class proteins predicted by the floral quartet model.
Project description:Perennial woodland herbs in the genus Thalictrum exhibit high diversity of floral morphology, including four breeding and two pollination systems. Their phylogenetic position, in the early-diverging eudicots, makes them especially suitable for exploring the evolution of floral traits and the fate of gene paralogs that may have shaped the radiation of the eudicots. A current limitation in evolution of plant development studies is the lack of genetic tools for conducting functional assays in key taxa spanning the angiosperm phylogeny. We first show that virus-induced gene silencing (VIGS) of a PHYTOENE DESATURASE ortholog (TdPDS) can be achieved in Thalictrum dioicum with an efficiency of 42% and a survival rate of 97%, using tobacco rattle virus (TRV) vectors. The photobleached leaf phenotype of silenced plants significantly correlates with the down-regulation of endogenous TdPDS (P<0.05), as compared to controls. Floral silencing of PDS was achieved in the faster flowering spring ephemeral T. thalictroides. In its close relative, T. clavatum, silencing of the floral MADS box gene AGAMOUS (AG) resulted in strong homeotic conversions of floral organs. In conclusion, we set forth our optimized protocol for VIGS by vacuum-infiltration of Thalictrum seedlings or dormant tubers as a reference for the research community. The three species reported here span the range of floral morphologies and pollination syndromes present in Thalictrum. The evidence presented on floral silencing of orthologs of the marker gene PDS and the floral homeotic gene AG will enable a comparative approach to the study of the evolution of flower development in this group.
Project description:BACKGROUND: Coccinia grandis is a dioecious species of Cucurbitaceae having heteromorphic sex chromosomes. The chromosome constitution of male and female plants is 22?+?XY and 22?+?XX respectively. Y chromosome of male sex is conspicuously large and plays a decisive role in determining maleness. Sex modification has been studied in hypogynous Silene latifolia (Caryophyllaceae) but there is no such report in epigynous Coccinia grandis. Moreover, the role of organ identity genes during sex expression in Coccinia has not been evaluated earlier. Investigations on sexual phenotypes of C. grandis including a rare gynomonoecious (GyM) form and AgNO3 mediated sex modification have added a new dimension to the understanding of sex expression in dioecious flowering plants. RESULTS: Morphometric analysis showed the presence of staminodes in pistillate flowers and histological study revealed the absence of carpel initials in male flowers. Though GyM plant had XX sex chromosomes, the development of stamens occurred in hermaphrodite flowers but the pollens were not fertile. Silver nitrate (AgNO3) application enhanced stamen growth in wild type female flowers like that of GyM plant but here also the pollens were sterile. Differential expression of CgPI could be involved in the development of different floral phenotypes. CONCLUSIONS: The three principle factors, Gynoecium Suppression (SuF), Stamen Promoting Factor (SPF) and Male Fertility (mF) that control sex expression in dioecious C. grandis assumed to be located on Y chromosome, play a decisive role in determining maleness. However, the characteristic development of stamens in hermaphrodite flowers of GyM plant having XX sex chromosomes indicates that Y-linked SPF regulatory pathway is somehow bypassed. Our experimental findings together with all other previous chromosomal and molecular cytogenetical data strongly support the view that C. grandis could be used as a potential model system to study sex expression in dioecious flowering plant.
Project description:Hermaphroditic flowers have evolved primarily under the selection on male function. Evolutionary modification often leads to stamen differentiation within flowers, or "heteranthery", a phenomenon intrigued scientists since the 18(th) century until recently. However, the genetic basis and molecular regulation mechanism has barely been touched. Here we conducted comparative transcriptome profiling in Cassia biscapsularis L., a heterantherous species with representative patterns of stamen differentiation. Numerous differentially expressed genes (DEGs) were detected between the staminodes (the degenerated stamens) and fertile stamens, while much fewer genes differentially expressed among the three sets of fertile stamens. GO term enrichment and KEGG pathway analysis characterized functional properties of DEGs in different stamen types. Transcripts showing close correlation between expression pattern and stamen types were identified. Transcription factors from the bHLH family were suggested to have taken crucial part in the formation of staminodes. This first global transcriptomic analysis focusing on stamen differentiation opens the door toward a more comprehensive understanding on the molecular regulation of floral organ evolution. Especially, the generated unigene resource would be valuable for developing male sterile lines in agronomy.