MtSUPERMAN plays a key role in compound inflorescence and flower development in Medicago truncatula.

Legumes have unique features, such as compound inflorescences and a complex floral ontogeny. Thus, the study of regulatory genes in these species during inflorescence and floral development is essential to understand their role in the evolutionary origin of developmental novelties. The SUPERMAN (SUP) gene encodes a C2H2 zinc-finger transcriptional repressor that regulates the floral organ number in the third and fourth floral whorls of Arabidopsis thaliana. In this work, we present the functional characterization of the Medicago truncatula SUPERMAN (MtSUP) gene based on gene expression analysis, complementation and overexpression assays, and reverse genetic approaches. Our findings provide evidence that MtSUP is the orthologous gene of SUP in M. truncatula. We have unveiled novel functions for a SUP-like gene in eudicots. MtSUP controls not only the number of floral organs in the inner two whorls, but also in the second whorl of the flower. Furthermore, MtSUP regulates the activity of the secondary inflorescence meristem, thus controlling the number of flowers produced. Our work provides insight into the regulatory network behind the compound inflorescence and flower development in this angiosperm family.

Maize YABBY genes drooping leaf1 and drooping leaf2 regulate floret development and floral meristem determinacy.

Floral morphology is shaped by factors that modulate floral meristem activity and size, and the identity, number and arrangement of the lateral organs they form. We report here that the maize CRABS CLAW co-orthologs drooping leaf1 (drl1) and drl2 are required for development of ear and tassel florets. Pistillate florets of drl1 ears are sterile with unfused carpels that fail to enclose an expanded nucellus-like structure. Staminate florets of drl1 tassels have extra stamens and fertile anthers. Natural variation and transposon alleles of drl2 enhance drl1 mutant phenotypes by reducing floral meristem (FM) determinacy. The drl paralogs are co-expressed in lateral floral primordia, but not within the FM. drl expression together with the more indeterminate mutant FMs suggest that the drl genes regulate FM activity and impose meristem determinacy non-cell-autonomously from differentiating cells in lateral floral organs. We used gene regulatory network inference, genetic interaction and expression analyses to suggest that DRL1 and ZAG1 target each other and a common set of downstream genes that function during floret development, thus defining a regulatory module that fine-tunes floret patterning and FM determinacy.

Tomato CRABS CLAW paralogues interact with chromatin remodelling factors to mediate carpel development and floral determinacy.

CRABS CLAW (CRC) orthologues play a crucial role in floral meristem (FM) determinacy and gynoecium formation across angiosperms, the key developmental processes for ensuring successful plant reproduction and crop production. However, the mechanisms behind CRC mediated FM termination are far from fully understood. Here, we addressed the functional characterization of tomato (Solanum lycopersicum) paralogous CRC genes. Using mapping-by-sequencing, RNA interference and CRISPR/Cas9 techniques, expression analyses, protein-protein interaction assays and Arabidopsis complementation experiments, we examined their potential roles in FM determinacy and carpel formation. We revealed that the incomplete penetrance and variable expressivity of the indeterminate carpel-inside-carpel phenotype observed in fruit iterative growth (fig) mutant plants are due to the lack of function of the S. lycopersicum CRC homologue SlCRCa. Furthermore, a detailed functional analysis of tomato CRC paralogues, SlCRCa and SlCRCb, allowed us to propose that they operate as positive regulators of FM determinacy by acting in a compensatory and partially redundant manner to safeguard the proper formation of flowers and fruits. Our results uncover for the first time the physical interaction of putative CRC orthologues with members of the chromatin remodelling complex that epigenetically represses WUSCHEL expression through histone deacetylation to ensure the proper termination of floral stem cell activity.

Characterization of a new rice OsMADS1 null mutant generated by homologous recombination-mediated gene targeting.

MAIN CONCLUSION: A new, stable, null mutant of OsMADS1 generated by homologous recombination-based gene targeting in an indica rice confirms its regulatory role for floral meristem identity, its determinate development and floral organ differentiation. OsMADS1, an E-class MADS-box gene, is an important regulator of rice flower development. Studies of several partial loss-of-function and knockdown mutants show varied floret organ defects and degrees of meristem indeterminacy. The developmental consequences of a true null mutant on floret meristem identity, its determinate development and differentiation of grass-specific organs such as the lemma and palea remain unclear. In this study, we generated an OsMADS1 null mutant by homologous recombination-mediated gene targeting by inserting a selectable marker gene (hpt) in OsMADS1 and replacing parts of its cis-regulatory and coding sequences. A binary vector was constructed with diphtheria toxin A chain gene (DT-A) as a negative marker to eliminate random integrations and the hpt marker for positive selection of homologous recombination. Precise disruption of the endogenous OsMADS1 locus in the rice genome was confirmed by Southern hybridization. The homozygous osmads1ko null mutant displayed severe defects in all floral organs including the lemma and palea. We also noticed striking instances of floral reversion to inflorescence and vegetative states which has not been reported for other mutant alleles of OsMADS1 and further reinforces the role of OsMADS1 in controlling floral meristem determinacy. Our data suggest, OsMADS1 commits and maintains determinate floret development by regulating floral meristem termination, carpel and ovule differentiation genes (OsMADS58, OsMADS13) while its modulation of genes such as OsMADS15, OsIG1 and OsMADS32 could be relevant in the differentiation and development of palea. Further, our study provides an important perspective on developmental stage-dependent modulation of some OsMADS1 target genes.

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.

Grass inflorescence architecture and meristem determinacy.

The grass inflorescence is striking not only for its beauty and diversity, but also for its developmental complexity. While models of inflorescence architecture have been proposed in both eudicots and grasses, these are inadequate to fully explain the complex branching events that occur during the development of the grass inflorescence. Key to understanding grass inflorescence architecture is the meristem determinacy/indeterminacy decision, which regulates the number of branching events that occur. Here we review what has been learned about meristem determinacy from grass mutants with defects in inflorescence development. A picture is emerging of a complex network of signaling molecules and meristem identity factors that interact to regulate inflorescence meristem activity, many of which have been modified during crop domestication directly affecting yield traits.

Crop reproductive meristems in the genomic era: a brief overview.

Modulation of traits beneficial for cultivation and yield is one of the main goals of crop improvement. One of the targets for enhancing productivity is changing the architecture of inflorescences since in many species it determines fruit and seed yield. Inflorescence shape and organization is genetically established during the early stages of reproductive development and depends on the number, arrangement, activities, and duration of meristems during the reproductive phase of the plant life cycle. Despite the variety of inflorescence architectures observable in nature, many key aspects of inflorescence development are conserved among different species. For instance, the genetic network in charge of specifying the identity of the different reproductive meristems, which can be indeterminate or determinate, seems to be similar among distantly related species. The availability of a large number of published transcriptomic datasets for plants with different inflorescence architectures, allowed us to identify transcription factor gene families that are differentially expressed in determinate and indeterminate reproductive meristems. The data that we review here for Arabidopsis, rice, barley, wheat, and maize, particularly deepens our knowledge of their involvement in meristem identity specification.

Rice Flower Development Revisited: Regulation of Carpel Specification and Flower Meristem Determinacy.

The ABC model in flower development represents a milestone of plant developmental studies and is essentially conserved across a wide range of angiosperm species. Despite this overall conservation, individual genes in the ABC model are not necessarily conserved and sometimes play a species-specific role, depending on the plant. We previously reported that carpels are specified by the YABBY gene DROOPING LEAF (DL) in rice (Oryza sativa), which bears flowers that are distinct from those of eudicots. In contrast, another group reported that carpels are specified by two class C genes, OsMADS3 and OsMADS58. Here, we have addressed this controversial issue by phenotypic characterization of floral homeotic gene mutants. Analysis of a complete loss-of-function mutant of OsMADS3 and OsMADS58 revealed that carpel-like organs expressing DL were formed in the absence of the two class C genes. Furthermore, no known flower organs including carpels were specified in a double mutant of DL and SUPERWOMAN1 (a class B gene), which expresses only class C genes in whorls 3 and 4. These results suggest that, in contrast to Arabidopsis, class C genes are not a key regulator for carpel specification in rice. Instead, they seem to be involved in the elaboration of carpel morphology rather than its specification. Our phenotypic analysis also revealed that, similar to its Arabidopsis ortholog CRABS CLAW, DL plays an important function in regulating flower meristem determinacy in addition to carpel specification.

Regulation of meristem maintenance and organ identity during rice reproductive development.

Grasses have evolved complex inflorescences, where the primary unit is the specialized short branch called a spikelet. Detailed studies of the cumulative action of the genetic regulators that direct the progressive change in axillary meristem identity and their terminal differentiation are crucial to understanding the complexities of the inflorescence and the development of a determinate floret. Grass florets also pose interesting questions concerning the morphologies and functions of organs as compared to other monocots and eudicots. In this review, we summarize our current knowledge of the regulation of the transitions that occur in grass inflorescence meristems, and of the specification of floret meristems and their determinate development. We primarily use rice as a model, with appropriate comparisons to other crop models and to the extensively studied eudicot Arabidopsis. The role of MADS-domain transcription factors in floral organ patterning is well documented in many eudicots and in grasses. However, there is evidence to suggest that some of these rice floral regulators have evolved distinctive functions and that other grass species-specific factors and regulatory pathways occur - for example the LOFSEP 'E' class genes OsMADS1 and OsMAD34, and ramosa genes. A better understanding of these systems and the epigenetic regulators and hormone signaling pathways that interact with them will provide new insights into the rice inflorescence meristem and the differentiation of its floret organs, and should indicate genetic tools that can be used to control yield-related traits in both rice and other cereal crops.

FAR-RED ELONGATED HYPOCOTYL3 activates SEPALLATA2 but inhibits CLAVATA3 to regulate meristem determinacy and maintenance in Arabidopsis.

Plant meristems are responsible for the generation of all plant tissues and organs. Here we show that the transcription factor (TF) FAR-RED ELONGATED HYPOCOTYL3 (FHY3) plays an important role in both floral meristem (FM) determinacy and shoot apical meristem maintenance in Arabidopsis, in addition to its well-known multifaceted roles in plant growth and development during the vegetative stage. Through genetic analyses, we show that WUSCHEL (WUS) and CLAVATA3 (CLV3), two central players in the establishment and maintenance of meristems, are epistatic to FHY3 Using genome-wide ChIP-seq and RNA-seq data, we identify hundreds of FHY3 target genes in flowers and find that FHY3 mainly acts as a transcriptional repressor in flower development, in contrast to its transcriptional activator role in seedlings. Binding motif-enrichment analyses indicate that FHY3 may coregulate flower development with three flower-specific MADS-domain TFs and four basic helix-loop-helix TFs that are involved in photomorphogenesis. We further demonstrate that CLV3, SEPALLATA1 (SEP1), and SEP2 are FHY3 target genes. In shoot apical meristem, FHY3 directly represses CLV3, which consequently regulates WUS to maintain the stem cell pool. Intriguingly, CLV3 expression did not change significantly in fhy3 and phytochrome B mutants before and after light treatment, indicating that FHY3 and phytochrome B are involved in light-regulated meristem activity. In FM, FHY3 directly represses CLV3, but activates SEP2, to ultimately promote FM determinacy. Taken together, our results reveal insights into the mechanisms of meristem maintenance and determinacy, and illustrate how the roles of a single TF may vary in different organs and developmental stages.

Domestication-driven Gossypium profilin 1 (GhPRF1) gene transduces early flowering phenotype in tobacco by spatial alteration of apical/floral-meristem related gene expression.

BACKGROUND: Plant profilin genes encode core cell-wall structural proteins and are evidenced for their up-regulation under cotton domestication. Notwithstanding striking discoveries in the genetics of cell-wall organization in plants, little is explicit about the manner in which profilin-mediated molecular interplay and corresponding networks are altered, especially during cellular signalling of apical meristem determinacy and flower development. RESULTS: Here we show that the ectopic expression of GhPRF1 gene in tobacco resulted in the hyperactivation of apical meristem and early flowering phenotype with increased flower number in comparison to the control plants. Spatial expression alteration in CLV1, a key meristem-determinacy gene, is induced by the GhPRF1 overexpression in a WUS-dependent manner and mediates cell signalling to promote flowering. But no such expression alterations are recorded in the GhPRF1-RNAi lines. The GhPRF1 transduces key positive flowering regulator AP1 gene via coordinated expression of FT4, SOC1, FLC1 and FT1 genes involved in the apical-to-floral meristem signalling cascade which is consistent with our in silico profilin interaction data. Remarkably, these positive and negative flowering regulators are spatially controlled by the Actin-Related Protein (ARP) genes, specifically ARP4 and ARP6 in proximate association with profilins. This study provides a novel and systematic link between GhPRF1 gene expression and the flower primordium initiation via up-regulation of the ARP genes, and an insight into the functional characterization of GhPRF1 gene acting upstream to the flowering mechanism. Also, the transgenic plants expressing GhPRF1 gene show an increase in the plant height, internode length, leaf size and plant vigor. CONCLUSIONS: Overexpression of GhPRF1 gene induced early and increased flowering in tobacco with enhanced plant vigor. During apical meristem determinacy and flower development, the GhPRF1 gene directly influences key flowering regulators through ARP-genes, indicating for its role upstream in the apical-to-floral meristem signalling cascade.

Analysis of the Arabidopsis superman allelic series and the interactions with other genes demonstrate developmental robustness and joint specification of male-female boundary, flower meristem termination and carpel compartmentalization.

BACKGROUND AND AIMS: SUPERMAN is a cadastral gene controlling the sexual boundary in the flower. The gene's functions and role in flower development and evolution have remained elusive. The analysis of a contrasting SUP allelic series (for which the names superman, superwoman and supersex have been coined) makes it possible to distinguish early vs. late regulatory processes at the flower meristem centre to which SUP is an important contributor. Their understanding is essential in further addressing evolutionary questions linking bisexuality and flower meristem homeostasis. METHODS: Inter-allelic comparisons were carried out and SUP interactions with other boundary factors and flower meristem patterning and homeostasis regulators (such as CLV, WUS, PAN, CUC, KNU, AG, AP3/PI, CRC and SPT) have been evaluated at genetic, molecular, morphological and histological levels. KEY RESULTS: Early SUP functions include mechanisms of male-female (sexual) boundary specification, flower mersitem termination and control of stamen number. A SUP-dependent flower meristem termination pathway is identified and analysed. Late SUP functions play a role in organ morphogenesis by controlling intra-whorl organ separation and carpel medial region formation. By integrating early and late SUP functions, and by analyzing in one single experiment a series of SUP genetic interactions, the concept of meristematic 'transference' (cascade) - a regulatory bridging process redundantly and sequentially co-ordinating the triggering and completion of flower meristem termination, and carpel margin meristem and placenta patterning - is proposed. CONCLUSIONS: Taken together, the results strongly support the view that SUP(-type) function(s) have been instrumental in resolving male/female gradients into sharp male and female identities (whorls, organs) and in enforcing flower homeostasis during evolution. This has probably been achieved by incorporating the meristem patterning system of the floral axis into the female/carpel programme.

The diversification of the shoot branching system: A quantitative and comparative perspective in meristem determinacy.

Reiterative shoot branching largely defines important yield components of crops and is essentially controlled by programs that direct the initiation, dormancy release, and differentiation of meristems in the axils of leaves. Here, we focus on meristem determinacy, defining the number of reiterations that shape the shoot architectures and exhibit enormous diversity in a wide range of species. The meristem determinacy per se is hierarchically complex and context-dependent for the successively emerged meristems, representing a crucial mechanism in shaping the complexity of the shoot branching. In addition, we have highlighted that two key components of axillary meristem developmental programs may have been co-opted in controlling flower/ear number of an axillary inflorescence in legumes/maize, hinting at the diversification of axillary-meristem-patterning programs in different lineages. This begs the question how axillary meristem patterning programs may have diversified during plant evolution and hence helped shape the rich variation in shoot branching systems.

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.

SEPALLATA--like genes of Isatis indigotica can affect the architecture of the inflorescences and the development of the floral organs.

BACKGROUND: The architecture of inflorescence and the development of floral organs can influence the yield of seeds and have a significant impact on plant propagation. E-class floral homeotic MADS-box genes exhibit important roles in regulation of floral transition and differentiation of floral organs. Woad (Isatis indigotica) possesses unique inflorescence, floral organs and fruit. However, very little research has been carried out to determine the function of MADS-box genes in this medicinal cruciferous plant species. RESULTS: SEPALLATA orthologs in I. indigotica were cloned by degenerate PCR. The sequence possessing the highest identity with SEP2 and SEP4 of Arabidopsis were named as IiSEP2 and IiSEP4, respectively. Constitutive expression of IiSEP2 in Columbia (Col-0) ecotype of Arabidopsis led to early flowering, and the number of the flowers and the lateral branches was reduced, indicating an alteration in architecture of the inflorescences. Moreover, the number of the floral organs was declined, the sepals were turned into carpelloid tissues bearing stigmatic papillae and ovules, and secondary flower could be produced in apetalous terminal flowers. In 35S::IiSEP4-GFP transgenic Arabidopsis plants in Landsberg erecta (Ler) genetic background, the number of the floral organs was decreased, sepals were converted into curly carpelloid structures, accompanied by generation of ovules. Simultaneously, the size of petals, stamens and siliques was diminished. In 35S::IiSEP4-GFP transgenic plants of apetalous ap1 cal double mutant in Ler genetic background, the cauliflower phenotype was attenuated significantly, and the petal formation could be rescued. Occasionally, chimeric organs composed of petaloid and sepaloid tissues, or petaloid and stamineous tissues, were produced in IiSEP4 transgenic plants of apl cal double mutant. It suggested that overexpression of IiSEP4 could restore the capacity in petal differentiation. Silencing of IiSEP4 by Virus-Induced Gene Silencing (VIGS) can delay the flowering time, and reduce the number and size of the floral organs in woad flowers. CONCLUSION: All the results showed that SEPALLATA-like genes could influence the architecture of the inflorescence and the determinacy of the floral meristems, and was also related to development of the floral organs.

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.

AUXIN RESPONSE FACTOR3 plays distinct role during early flower development.

AUXIN RESPONSE FACTOR3 (ARF3), one of the auxin response factors family of transcription factors, is well characterized by its functions in polarity identification and organ patterning. We recently demonstrated that ARF3 plays important roles in floral meristem (FM) maintenance and termination by regulating cytokinin biosynthesis and signaling. However, the relationship of its multiple roles in differently developmental stage is still unclear. Here, we present data that ARF3 plays distinct roles during early flower development that are different from its roles in organ polarity determination and pattering. Thus, our findings shed light on the functional diversity of one specific transcription factor in plant development.

FAR-RED ELONGATED HYPOCOTYL3 promotes floral meristem determinacy in Arabidopsis.

The transposase-derived transcription factor genes FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and FAR-RED IMPAIRED RESPONSE1 (FAR1) have redundant and multifaceted roles in plant growth and development during the vegetative stage, including phytochrome A-mediated far-red light (FR) signaling and circadian clock entrainment. Little is known about their functions in the reproductive stage. We recently demonstrated that FHY3 plays important roles in shoot apical meristem (SAM) maintenance and floral meristem (FM) determinacy through its target genes CLAVATA3 (CLV3), SEPALLATA1 (SEP1) and SEP2. Here we present data that FHY3 but not its homolog, FAR1, has a distinct role in FM determinacy in a manner independent of its light signaling and circadian pathway functions. Moreover, genome-wide gene expression profiling showed that the homeostasis of the FM is critical for the regulation of FM activity.

Overexpression of the JAZ factors with mutated jas domains causes pleiotropic defects in rice spikelet development.

In a determinate meristem, such as a floral meristem, a genetically determined number of organs are produced before the meristem is terminated. In rice, iterative formation of organs during flower development with defects in meristem determinacy, classically called 'proliferation', is caused by several mutations and observed in dependence on environmental conditions. Here we report that overexpression of several JAZ proteins, key factors in jasmonate signaling, with mutations in the Jas domains causes an increase in the numbers of organs in florets, aberrant patterns of organ formation and repetitious organ production in spikelets. Our results imply that JAZ factors modulate mechanisms that regulate meristem functions during spikelet development.