MADS8 is indispensable for female reproductive development at high ambient temperatures in cereal crops.

Temperature is a major factor that regulates plant growth and phenotypic diversity. To ensure reproductive success at a range of temperatures, plants must maintain developmental stability of their sexual organs when exposed to temperature fluctuations. However, the mechanisms integrating plant floral organ development and temperature responses are largely unknown. Here, we generated barley and rice loss-of-function mutants in the SEPALLATA-like MADS-box gene MADS8. The mutants in both species form multiple carpels that lack ovules at high ambient temperatures. Tissue-specific markers revealed that HvMADS8 is required to maintain floral meristem determinacy and ovule initiation at high temperatures, and transcriptome analyses confirmed that temperature-dependent differentially expressed genes in Hvmads8 mutants predominantly associate with floral organ and meristem regulation. HvMADS8 temperature-responsive activity relies on increased binding to promoters of downstream targets, as revealed by a cleavage under targets and tagmentation (CUT&Tag) analysis. We also demonstrate that HvMADS8 directly binds to 2 orthologs of D-class floral homeotic genes to activate their expression. Overall, our findings revealed a new, conserved role for MADS8 in maintaining pistil number and ovule initiation in cereal crops, extending the known function of plant MADS-box proteins in floral organ regulation.

Rice SEPALLATA genes OsMADS5 and OsMADS34 cooperate to limit inflorescence branching by repressing the TERMINAL FLOWER1-like gene RCN4.

The spatiotemporal control of meristem identity is critical for determining inflorescence architecture, and thus yield, of cereal plants. However, the precise mechanisms underlying inflorescence and spikelet meristem determinacy in cereals are still largely unclear. We have generated loss-of-function and overexpression mutants of the paralogous OsMADS5 and OsMADS34 genes in rice (Oryza sativa), and analysed their panicle phenotypes. Using chromatin immunoprecipitation, electrophoretic mobility-shift and dual-luciferase assays, we have also identified RICE CENTRORADIALIS 4 (RCN4), a TFL1-like gene, as a direct downstream target of both OsMADS proteins, and have analysed RCN4 mutants. The osmads5 osmads34 mutant lines had significantly enhanced panicle branching with increased secondary, and even tertiary and quaternary, branches, compared to wild-type (WT) and osmads34 plants. The osmads34 mutant phenotype could largely be rescued by also knocking out RCN4. Moreover, transgenic panicles overexpressing RCN4 had significantly increased branching, and initiated development of c. 7× more spikelets than WT. Our results reveal a role for OsMADS5 in panicle development, and show that OsMADS5 and OsMADS34 play similar functions in limiting branching and promoting the transition to spikelet meristem identity, in part by repressing RCN4 expression. These findings provide new insights to better understand the molecular regulation of rice inflorescence architecture.

SMALL REPRODUCTIVE ORGANS, a SUPERMAN-like transcription factor, regulates stamen and pistil growth in rice.

Organ size is determined mainly by cell division and cell expansion. Several genetic factors regulating development of plant lateral organs have been characterized, but those involved in determining reproductive organ size and separation in rice (Oryza sativa) remain unknown. We have isolated the rice gene SMALL REPRODUCTIVE ORGANS (SRO) encoding a nucleus-localized Cys2His2 (C2 H2 ) zinc finger protein orthologous to Arabidopsis transcription factor (TF) SUPERMAN (SUP). Combined developmental, genetic, histological and transcriptomic analyses were used to determine the function of SRO in regulating reproductive organ size. SRO affects genes involved in cell division, cell expansion and phytohormone signalling in the rice flower. SRO is specifically expressed in the first stages of stamen filament development to regulate their correct formation and separation. In addition, SRO noncell-autonomously regulates the size and functionality of male and female reproductive organs. The B-class MADS-box gene OsMADS16/SPW1 is epistatic to SRO, whereas SRO regulates reproductive organ specification and floral meristem determinacy synergistically with C-class genes OsMADS3 and OsMADS58. These findings provide insights into how an evolutionarily conserved TF has a pivotal role in reproductive organ development in core eudicots and monocots, through partially conserved expression, function and regulatory network.

A Tea Plant (Camellia sinensis) FLOWERING LOCUS C-like Gene, CsFLC1, Is Correlated to Bud Dormancy and Triggers Early Flowering in Arabidopsis.

Flowering and bud dormancy are crucial stages in the life cycle of perennial angiosperms in temperate climates. MADS-box family genes are involved in many plant growth and development processes. Here, we identified three MADS-box genes in tea plant belonging to the FLOWERING LOCUS C (CsFLC) family. We monitored CsFLC1 transcription throughout the year and found that CsFLC1 was expressed at a higher level during the winter bud dormancy and flowering phases. To clarify the function of CsFLC1, we developed transgenic Arabidopsis thaliana plants heterologously expressing 35S::CsFLC1. These lines bolted and bloomed earlier than the WT (Col-0), and the seed germination rate was inversely proportional to the increased CsFLC1 expression level. The RNA-seq of 35S::CsFLC1 transgenic Arabidopsis showed that many genes responding to ageing, flower development and leaf senescence were affected, and phytohormone-related pathways were especially enriched. According to the results of hormone content detection and RNA transcript level analysis, CsFLC1 controls flowering time possibly by regulating SOC1, AGL42, SEP3 and AP3 and hormone signaling, accumulation and metabolism. This is the first time a study has identified FLC-like genes and characterized CsFLC1 in tea plant. Our results suggest that CsFLC1 might play dual roles in flowering and winter bud dormancy and provide new insight into the molecular mechanisms of FLC in tea plants as well as other plant species.

Transcriptome analysis reveals rice MADS13 as an important repressor of the carpel development pathway in ovules.

In angiosperms, floral homeotic genes encoding MADS-domain transcription factors regulate the development of floral organs. Specifically, members of the SEPALLATA (SEP) and AGAMOUS (AG) subfamilies form higher-order protein complexes to control floral meristem determinacy and to specify the identity of female reproductive organs. In rice, the AG subfamily gene OsMADS13 is intimately involved in the determination of ovule identity, since knock-out mutant plants develop carpel-like structures in place of ovules, resulting in female sterility. Little is known about the regulatory pathways at the base of rice gynoecium development. To investigate molecular mechanisms acting downstream of OsMADS13, we obtained transcriptomes of immature inflorescences from wild-type and Osmads13 mutant plants. Among a total of 476 differentially expressed genes (DEGs), a substantial overlap with DEGs from the SEP-family Osmads1 mutant was found, suggesting that OsMADS1 and OsMADS13 may act on a common set of target genes. Expression studies and preliminary analyses of two up-regulated genes encoding Zinc-finger transcription factors indicated that our dataset represents a valuable resource for the identification of both OsMADS13 target genes and novel players in rice ovule development. Taken together, our study suggests that OsMADS13 is an important repressor of the carpel pathway during ovule development.

Loss of LOFSEP Transcription Factor Function Converts Spikelet to Leaf-Like Structures in Rice.

SEPALLATA (SEP)-like genes, which encode a subfamily of MADS-box transcription factors, are essential for specifying floral organ and meristem identity in angiosperms. Rice (Oryza sativa) has five SEP-like genes with partial redundancy and overlapping expression domains, yet their functions and evolutionary conservation are only partially known. Here, we describe the biological role of one of the SEP genes of rice, OsMADS5, in redundantly controlling spikelet morphogenesis. OsMADS5 belongs to the conserved LOFSEP subgroup along with OsMADS1 and OsMADS34OsMADS5 was expressed strongly across a broad range of reproductive stages and tissues. No obvious phenotype was observed in the osmads5 single mutants when compared with the wild type, which was largely due to the functional redundancy among the three LOFSEP genes. Genetic and molecular analyses demonstrated that OsMADS1, OsMADS5, and OsMADS34 together regulate floral meristem determinacy and specify the identities of spikelet organs by positively regulating the other MADS-box floral homeotic genes. Experiments conducted in yeast also suggested that OsMADS1, OsMADS5, and OsMADS34 form protein-protein interactions with other MADS-box floral homeotic members, which seems to be a typical, conserved feature of plant SEP proteins.

Interactions between FLORAL ORGAN NUMBER4 and floral homeotic genes in regulating rice flower development.

The floral meristem (FM) is self-maintaining at the early stages of flower development, but it is terminated when a fixed number of floral organs are produced. The FLORAL ORGAN NUMBER4 (FON4; also known as FON2) gene, an ortholog of Arabidopsis CLAVATA3 (CLV3), is required for regulating FM size and determinacy in rice. However, its interactions with floral homeotic genes remain unknown. Here, we report the genetic interactions between FON4 and floral homeotic genes OsMADS15 (an A-class gene), OsMADS16 (also called SUPERWOMAN1, SPW1, a B-class gene), OsMADS3 and OsMADS58 (C-class genes), OsMADS13 (a D-class gene), and OsMADS1 (an E-class gene) during flower development. We observed an additive phenotype in the fon4 double mutant with the OsMADS15 mutant allele dep (degenerative palea). The effect on the organ number of whorl 2 was enhanced in fon4 spw1. Double mutant combinations of fon4 with osmads3, osmads58, osmads13, and osmads1 displayed enhanced defects in FM determinacy and identity, respectively, indicating that FON4 and these genes synergistically control FM activity. In addition, the expression patterns of all the genes besides OsMADS13 had no obvious change in the fon4 mutant. This work reveals how the meristem maintenance gene FON4 genetically interacts with C, D, and E floral homeotic genes in specifying FM activity in monocot rice.

Regulatory network and genetic interactions established by OsMADS34 in rice inflorescence and spikelet morphogenesis.

Grasses display highly diversified inflorescence architectures that differ in the arrangement of spikelets and flowers and determine cereal yields. However, the molecular basis underlying grass inflorescence morphogenesis remains largely unknown. Here we investigate the role of a functionally diversified SEPALLATA MADS-box transcription factor, OsMADS34, in regulating rice (Oryza sativa L.) inflorescence and spikelet development. Microarray analysis showed that, at the very early stages of inflorescence formation, dysfunction of OsMADS34 caused altered expression of 379 genes that are associated with protein modification and degradation, transcriptional regulation, signaling and metabolism activity. Genetic analysis revealed that OsMADS34 controls different aspects of inflorescence structure, branching and meristem activity synergistically with LAX PANICLE1 (LAX1) and FLORAL ORGAN NUMBER4 (FON4), as evidenced by the enhanced phenotypes of osmads34 lax1 and osmads34 fon4 compared with the single mutants. Additionally, double mutant between osmads34 and the sterile lemma defective mutant elongated empty glume (ele) displayed an enhanced phenotype, that is, longer and wider sterile lemmas that were converted into lemma/palea-like organs, suggesting that ELE and OsMADS34 synergistically control the sterile lemma development. OsMADS34 may act together with OsMADS15 in controlling sterile lemma development. Collectively, these findings provide insights into the regulatory function of OsMADS34 in rice inflorescence and spikelet development.

Genome-Wide Transcriptome Analysis During Anthesis Reveals New Insights into the Molecular Basis of Heat Stress Responses in Tolerant and Sensitive Rice Varieties.

Rice is one of the main food crops in the world. In the near future, yield is expected to be under pressure due to unfavorable climatic conditions, such as increasing temperatures. Therefore, improving rice germplasm in order to guarantee rice production under harsh environmental conditions is of top priority. Although many physiological studies have contributed to understanding heat responses during anthesis, the most heat-sensitive stage, molecular data are still largely lacking. In this study, an RNA-sequencing approach of heat- and control-treated reproductive tissues during anthesis was carried out using N22, one of the most heat-tolerant rice cultivars known to date. This analysis revealed that expression of genes encoding a number of transcription factor families, together with signal transduction and metabolic pathway genes, is repressed. On the other hand, expression of genes encoding heat shock factors and heat shock proteins was highly activated. Many of these genes are predominantly expressed at late stages of anther development. Further physiological experiments using heat-tolerant N22 and two sensitive cultivars suggest that reduced yield in heat-sensitive plants may be associated with poor pollen development or production in anthers prior to anthesis. In parallel, induction levels of a set of heat-responsive genes in these tissues correlated well with heat tolerance. Altogether, these findings suggest that proper expression of protective chaperones in anthers is needed before anthesis to overcome stress damage and to ensure fertilization. Genes putatively controlling this process were identified and are valuable candidates to consider for molecular breeding of highly productive heat-tolerant cultivars.

Flower development: the evolutionary history and functions of the AGL6 subfamily MADS-box genes.

AGL6 is an ancient subfamily of MADS-box genes found in both gymnosperms and angiosperms. Its functions remained elusive despite the fact that the MADS-box genes and the ABC model have been studied for >20 years. Nevertheless, recent discoveries in petunia, rice, and maize support its involvement in the 'E' function of floral development, very similar to the closely related AGL2 (SEPALLATA) subfamily which has been well characterized. The known functions of AGL6 span from ancient conserved roles to new functions acquired in specific plant families. The AGL6 genes are involved in floral meristem regulation, in floral organs, and ovule (integument) and seed development, and have possible roles in both male and female germline and gametophyte development. In grasses, they are also important for the development of the first whorl of the flower, whereas in Arabidopsis they may play additional roles before floral meristem formation. This review covers these recent insights and some other aspects that are not yet fully elucidated, which deserve more studies in the future.

OsJAR1 is required for JA-regulated floret opening and anther dehiscence in rice.

Jasmonates are important phytohormones regulating reproductive development. We used two recessive rice Tos17 alleles of OsJAR1, osjar1-2 and osjar1-3, to study the biological function of jasmonates in rice anthesis. The florets of both osjar1 alleles stayed open during anthesis because the lodicules, which control flower opening in rice, were not withering on time. Furthermore, dehiscence of the anthers filled with viable pollen, was impaired, resulting in lower fertility. In situ hybridization and promoter GUS transgenic analysis confirmed OsJAR1 expression in these floral tissues. Flower opening induced by exogenous applied methyl jasmonate was impaired in osjar1 plants and was restored in a complementation experiment with transgenics expressing a wild type copy of OsJAR1 controlled by a rice actin promoter. Biochemical analysis showed that OsJAR1 encoded an enzyme conjugating jasmonic acid (JA) to at least Ile, Leu, Met, Phe, Trp and Val and both osjar1 alleles had substantial reduction in content of JA-Ile, JA-Leu and JA-Val in florets. We conclude that OsJAR1 is a JA-amino acid synthetase that is required for optimal flower opening and closing and anther dehiscence in rice.

MADS reloaded: evolution of the AGAMOUS subfamily genes.

AGAMOUS subfamily proteins are encoded by MADS-box family genes. They have been shown to play key roles in the determination of reproductive floral organs such as stamens, carpels and ovules. However, they also play key roles in ensuring a fixed number of floral organs by controlling floral meristem determinacy. Recently, an enormous amount of sequence data for nonmodel species have become available together with functional data on AGAMOUS subfamily members in many species. Here, we give a detailed overview of the most important information about this interesting gene subfamily and provide new insights into its evolution.

Functional analysis of all AGAMOUS subfamily members in rice reveals their roles in reproductive organ identity determination and meristem determinacy.

Reproductive organ development is one of the most important steps in the life cycle of plants. Studies using core eudicot species like thale cress (Arabidopsis thaliana) and snapdragon (Antirrhinum majus) have shown that MADS domain transcription factors belonging to the AGAMOUS (AG) subfamily regulate the identity of stamens, carpels, and ovules and that they are important for floral meristem determinacy. Here, we investigate the genetic interactions between the four rice (Oryza sativa) AG subfamily members, MADS3, MADS13, MADS21, and MADS58. Our data show that, in contrast with previous reports, MADS3 and MADS58 determine stamen and carpel identity and, together with MADS13, are important for floral meristem determinacy. In the mads3 mads58 double mutant, we observed a complete loss of reproductive organ identity and massive accumulation of lodicules in the third and fourth floral whorls. MADS21 is an AGL11 lineage gene whose expression is not restricted to ovules. Instead, its expression profile is similar to those of class C genes. However, our genetic analysis shows that MADS21 has no function in stamen, carpel, or ovule identity determination.

Rice MADS6 interacts with the floral homeotic genes SUPERWOMAN1, MADS3, MADS58, MADS13, and DROOPING LEAF in specifying floral organ identities and meristem fate.

AGAMOUS-LIKE6 (AGL6) genes play essential roles in flower development, but whether and how they work with floral organ identity genes remain less understood. Here, we describe interactions of the rice (Oryza sativa) AGL6 gene MADS6 with other rice floral homeotic genes in flower development. Genetic analyses revealed that MADS6 specifies the identity of the three inner whorls and floral meristem determinacy redundantly with SUPERWOMAN1/MADS16 (B-gene) or MADS3 (C-gene). MADS6 was shown to define carpel/ovule development and floral determinacy by interacting with MADS13 (D-gene) and control the palea and floral meristem identities together with the YABBY gene DROOPING LEAF. Expression analyses revealed that the transcript levels of six B-, C-, and E-class genes were reduced in mads6-1 at the early flower developmental stage, suggesting that MADS6 is a key regulator of early flower development. Moreover, MADS6 can directly bind to a putative regulatory motif on MADS58 (C-gene), and mads6-1 mads58 displayed phenotypes similar to that of mads6-1. These results suggest that MADS6 is a key player in specifying flower development via interacting with other floral homeotic genes in rice, thus providing new insights into the mechanism by which flower development is controlled.

The ABC of Flower Development in Monocots: The Model of Rice Spikelet.

The initial seminal studies of flower developmental genetics were made from observations in several eudicot model species, particularly Arabidopsis and Antirrhinum. However, an increasing amount of research in monocot model and crop species is finally giving the credit that monocots deserve for their position in the evolutionary history of Angiosperms, their astonishing diversification and adaptation, their diversified floral structures, their pivotal function in most ecosystems on Earth and, finally, their importance in agriculture and farming, economy, landscaping and feeding mankind. Rice is a staple crop and the major monocot model to study the reproductive phase and flower evolution. Inspired by this, this chapter reviews a story of highly conserved functions related to the ABC model of flower development. Nevertheless, this model is complicated in rice by cases of gene neofunctionalization, like the recruitment of MADS-box genes for the development of the unique organs known as lemma and palea, subfunctionalization, and rewiring of conserved molecular pathways.

Tracing the Evolution of the SEPALLATA Subfamily across Angiosperms Associated with Neo- and Sub-Functionalization for Reproductive and Agronomically Relevant Traits.

SEPALLATA transcription factors (SEP TFs) have been extensively studied in angiosperms as pivotal components of virtually all the MADS-box tetrameric complex master regulators of floral organ identities. However, there are published reports that suggest that some SEP members also regulate earlier reproductive events, such as inflorescence meristem determinacy and inflorescence architecture, with potential for application in breeding programs in crops. The SEP subfamily underwent a quite complex pattern of duplications during the radiation of the angiosperms. Taking advantage of the many whole genomic sequences now available, we present a revised and expanded SEP phylogeny and link it to the known functions of previously characterized genes. This snapshot supports the evidence that the major SEP3 clade is highly specialized for the specification of the three innermost floral whorls, while its sister LOFSEP clade is functionally more versatile and has been recruited for diverse roles, such as the regulation of extra-floral bract formation and inflorescence determinacy and shape. This larger pool of angiosperm SEP genes confirms previous evidence that their evolution was driven by whole-genome duplications rather than small-scale duplication events. Our work may help to identify those SEP lineages that are the best candidates for the improvement of inflorescence traits, even in far distantly related crops.

Reversible male sterility in eggplant (Solanum melongena L.) by artificial microRNA-mediated silencing of general transcription factor genes.

Since decades, plant male sterility is considered a powerful tool for biological containment to minimize unwanted self-pollination for hybrid seed production. Furthermore, prevention of pollen dispersal also answers to concerns regarding transgene flow via pollen from Genetically Modified (GM) crops to traditional crop fields or wild relatives. We induced male sterility by suppressing endogenous general transcription factor genes, TAFs, using anther-specific promoters combined with artificial microRNA (amiRNA) technology (Schwab et al., 2006). The system was made reversible by the ethanol inducible expression of an amiRNA-insensitive form of the target gene. We provide proof of concept in eggplant, a cultivated crop belonging to the Solanaceae family that includes many important food crops. The transgenic eggplants that we generated are completely male sterile and fertility can be fully restored by short treatments with ethanol, confirming the efficiency but also the reliability of the system in view of open field cultivation. By combining this system with induced parthenocarpy (Rotino et al., 1997), we provide a novel example of complete transgene containment in eggplant, which enables biological mitigation measures for the benefit of coexistence or biosafety purposes for GM crop cultivation.

Molecular Control of Carpel Development in the Grass Family.

Carpel is the ovule-bearing female reproductive organ of flowering plants and is required to ensure its protection, an efficient fertilization, and the development of diversified types of fruits, thereby it is a vital element of most food crops. The origin and morphological changes of the carpel are key to the evolution and adaption of angiosperms. Progresses have been made in elucidating the developmental mechanisms of carpel establishment in the model eudicot plant Arabidopsis thaliana, while little and fragmentary information is known in grasses, a family that includes many important crops such as rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare), and wheat (Triticum aestivum). Here, we highlight recent advances in understanding the mechanisms underlying potential pathways of carpel development in grasses, including carpel identity determination, morphogenesis, and floral meristem determinacy. The known role of transcription factors, hormones, and miRNAs during grass carpel formation is summarized and compared with the extensively studied eudicot model plant Arabidopsis. The genetic and molecular aspects of carpel development that are conserved or diverged between grasses and eudicots are therefore discussed.

Functionally Divergent Splicing Variants of the Rice AGAMOUS Ortholog OsMADS3 Are Evolutionary Conserved in Grasses.

Within the MADS-box gene family, the AGAMOUS-subfamily genes are particularly important for plant reproduction, because they control stamen and carpel identity. A number of studies in the last three decades have demonstrated that the AGAMOUS (AG) function has been conserved during land plant evolution. However, gene duplication events have led to subfunctionalization and neofunctionalization of AG-like genes in many species. Here we show that alternative splicing in Oryza sativa produces two variants of the AG ortholog OsMADS3 which differ in just one serine residue, S109. Interestingly, this alternative splicing variant is conserved and specific to the grass family. Since in eudicots the S109 residue is absent in AG proteins, stamen and carpel identity determination activity of the two rice isoforms was tested in Arabidopsis thaliana. These experiments revealed that only the eudicot-like OsMADS3 isoform, lacking the serine residue, had ability to specify stamens and carpels in ag mutant flowers, suggesting an important functional role for the serine residue at position 109 in AG proteins of grasses.