The ALOG domain defines a family of plant-specific transcription factors acting during Arabidopsis flower development.

The ALOG (Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 (LSH1) and Oryza G1) proteins are conserved plant-specific Transcription Factors (TFs). They play critical roles in the development of various plant organs (meristems, inflorescences, floral organs, and nodules) from bryophytes to higher flowering plants. Despite the fact that the first members of this family were originally discovered in Arabidopsis, their role in this model plant has remained poorly characterized. Moreover, how these transcriptional regulators work at the molecular level is unknown. Here, we study the redundant function of the ALOG proteins LSH1,3,4 from Arabidopsis. We uncover their role in the repression of bract development and position them within a gene regulatory network controlling this process and involving the floral regulators LEAFY, BLADE-ON-PETIOLE, and PUCHI. Next, using in vitro genome-wide studies, we identified the conserved DNA motif bound by ALOG proteins from evolutionarily distant species (the liverwort Marchantia polymorpha and the flowering plants Arabidopsis, tomato, and rice). Resolution of the crystallographic structure of the ALOG DNA-binding domain in complex with DNA revealed the domain is a four-helix bundle with a disordered NLS and a zinc ribbon insertion between helices 2 and 3. The majority of DNA interactions are mediated by specific contacts made by the third alpha helix and the NLS. Taken together, this work provides the biochemical and structural basis for DNA-binding specificity of an evolutionarily conserved TF family and reveals its role as a key player in Arabidopsis flower development.

Assessing the role of REM13, REM34 and REM46 during the transition to the reproductive phase in Arabidopsis thaliana.

REM (reproductive meristem) transcription factors have been proposed as regulators of plant reproductive development mainly based on their specific expression patterns in reproductive structures, but their roles are still largely unknown probably because of their redundancy. We selected three REM genes (REM13, REM34 and REM46) for functional analysis, based on their genome position and/or co-expression data.Our results suggest that these genes have a role in flowering time regulation and may modulate cell cycle progression. In addition, protein interaction experiments revealed that REM34 and REM46 interact with each other, suggesting that they might work cooperatively to regulate cell division during inflorescence meristem commitment.Previous attempts of using co-expression data as a guide for functional analysis of REMs were limited by the transcriptomic data available at the time. Our results uncover previously unknown functions of three members of the REM family of Arabidopsis thaliana and open the door to more comprehensive studies of the REM family, where the combination of co-expression analysis followed by functional studies might contribute to uncovering the biological roles of these proteins and the relationship among them.

The ALOG family members OsG1L1 and OsG1L2 regulate inflorescence branching in rice.

The architecture of the rice inflorescence is an important determinant of crop yield. The length of the inflorescence and the number of branches are among the key factors determining the number of spikelets, and thus grains, that a plant will develop. In particular, the timing of the identity transition from indeterminate branch meristem to determinate spikelet meristem governs the complexity of the inflorescence. In this context, the ALOG gene TAWAWA1 (TAW1) has been shown to delay the transition to determinate spikelet development in Oryza sativa (rice). Recently, by combining precise laser microdissection of inflorescence meristems with RNA-seq, we observed that two ALOG genes, OsG1-like 1 (OsG1L1) and OsG1L2, have expression profiles similar to that of TAW1. Here, we report that osg1l1 and osg1l2 loss-of-function CRISPR mutants have similar phenotypes to the phenotype of the previously published taw1 mutant, suggesting that these genes might act on related pathways during inflorescence development. Transcriptome analysis of the osg1l2 mutant suggested interactions of OsG1L2 with other known inflorescence architecture regulators and the data sets were used for the construction of a gene regulatory network (GRN), proposing interactions among genes potentially involved in controlling inflorescence development in rice. In this GRN, we selected the homeodomain-leucine zipper transcription factor encoding the gene OsHOX14 for further characterization. The spatiotemporal expression profiling and phenotypical analysis of CRISPR loss-of-function mutants of OsHOX14 suggests that the proposed GRN indeed serves as a valuable resource for the identification of new proteins involved in rice inflorescence development.

Cell wall modifications by α-XYLOSIDASE1 are required for control of seed and fruit size in Arabidopsis.

Cell wall modifications are of pivotal importance during plant development. Among cell wall components, xyloglucans are the major hemicellulose polysaccharide in primary cell walls of dicots and non-graminaceous monocots. They can connect the cellulose microfibril surface to affect cell wall mechanical properties. Changes in xyloglucan structure are known to play an important role in regulating cell growth. Therefore, the degradation of xyloglucan is an important modification that alters the cell wall. The α-XYLOSIDASE1 (XYL1) gene encodes the only α-xylosidase acting on xyloglucans in Arabidopsis thaliana. Here, we showed that mutation of XYL1 strongly influences seed size, seed germination, and fruit elongation. We found that the expression of XYL1 is directly regulated in developing seeds and fruit by the MADS-box transcription factor SEEDSTICK. We demonstrated that XYL1 complements the stk smaller seed phenotype. Finally, by atomic force microscopy, we investigated the role of XYL1 activity in maintaining cell stiffness and growth, confirming the importance of cell wall modulation in shaping organs.

Cauliflower fractal forms arise from perturbations of floral gene networks.

Throughout development, plant meristems regularly produce organs in defined spiral, opposite, or whorl patterns. Cauliflowers present an unusual organ arrangement with a multitude of spirals nested over a wide range of scales. How such a fractal, self-similar organization emerges from developmental mechanisms has remained elusive. Combining experimental analyses in an Arabidopsis thaliana cauliflower-like mutant with modeling, we found that curd self-similarity arises because the meristems fail to form flowers but keep the "memory" of their transient passage in a floral state. Additional mutations affecting meristem growth can induce the production of conical structures reminiscent of the conspicuous fractal Romanesco shape. This study reveals how fractal-like forms may emerge from the combination of key, defined perturbations of floral developmental programs and growth dynamics.

The RNA-dependent DNA methylation pathway is required to restrict SPOROCYTELESS/NOZZLE expression to specify a single female germ cell precursor in Arabidopsis.

In higher plants, the female germline is formed from the megaspore mother cell (MMC), a single cell in the premeiotic ovule. Previously, it was reported that mutants in the RNA-dependent DNA methylation (RdDM) pathway might be involved in restricting the female germline to a single nucellus cell. We show that the DRM methyltransferase double mutant drm1drm2 also presents ectopic enlarged cells, consistent with supernumerary MMC-like cells. In wild-type ovules, MMC differentiation requires SPOROCYTELESS/NOZZLE (SPL/NZZ), as demonstrated by the spl/nzz mutant failing to develop an MMC. We address the poorly understood upstream regulation of SPL/NZZ in ovules, showing that the RdDM pathway is important to restrict SPL/NZZ expression. In ago9, rdr6 and drm1drm2 mutants, SPL/NZZ is expressed ectopically, suggesting that the multiple MMC-like cells observed might be attributable to the ectopic expression of SPL/NZZ. We show that the ovule identity gene, SEEDSTICK, directly regulates AGO9 and RDR6 expression in the ovule and therefore indirectly regulates SPL/NZZ expression. A model is presented describing the network required to restrict SPL/NZZ expression to specify a single MMC.

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.

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.

MADS-Box and bHLH Transcription Factors Coordinate Transmitting Tract Development in Arabidopsis thaliana.

The MADS-domain transcription factor SEEDSTICK (STK) controls several aspects of plant reproduction. STK is co-expressed with CESTA (CES), a basic Helix-Loop-Helix (bHLH) transcription factor-encoding gene. CES was reported to control redundantly with the brassinosteroid positive signaling factors BRASSINOSTEROID ENHANCED EXPRESSION1 (BEE1) and BEE3 the development of the transmitting tract. Combining the stk ces-4 mutants led to a reduction in ovule fertilization due to a defect in carpel fusion which, caused the formation of holes at the center of the septum where the transmitting tract differentiates. Combining the stk mutant with the bee1 bee3 ces-4 triple mutant showed an increased number of unfertilized ovules and septum defects. The transcriptome profile of this quadruple mutant revealed a small subset of differentially expressed genes which are mainly involved in cell death, extracellular matrix and cell wall development. Our data evidence a regulatory gene network controlling transmitting tract development regulated directly or indirectly by a STK-CES containing complex and reveal new insights in the regulation of transmitting tract development by bHLH and MADS-domain transcription factors.

Spatiotemporal Restriction of FUSCA3 Expression by Class I BPCs Promotes Ovule Development and Coordinates Embryo and Endosperm Growth.

Spatiotemporal regulation of gene expression is critical for proper developmental timing in plants and animals. The transcription factor FUSCA3 (FUS3) regulates developmental phase transitions by acting as a link between hormonal pathways in Arabidopsis (Arabidopsis thaliana). However, the mechanisms governing its spatiotemporal expression pattern are poorly understood. Here, we show that FUS3 is repressed in the ovule integuments and seed endosperm. FUS3 repression requires class I BASIC PENTACYSTEINE (BPC) proteins, which directly bind GA/CT cis-elements in FUS3 and restrict its expression pattern. During vegetative and reproductive development, FUS3 derepression in bpc1-1 bpc2 (bpc1/2) double mutant or misexpression in ProML1:FUS3 lines causes dwarf plants carrying defective flowers and aborted ovules. After fertilization, ectopic FUS3 expression in bpc1/2 endosperm or ProML1:FUS3 endosperm and endothelium increases endosperm nuclei proliferation and seed size, causing delayed or arrested embryo development. These phenotypes are rescued in bpc1/2 fus3-3 Finally, class I BPCs interact with FIS-PRC2 (FERTILIZATION-INDEPENDENT SEED-Polycomb Repressive Complex2), which represses FUS3 in the endosperm during early seed development. We propose that BPC1 and 2 promote the transition from reproductive to seed development by repressing FUS3 in ovule integuments. After fertilization, BPC1 and 2 and FIS-PRC2 repress FUS3 in the endosperm to coordinate early endosperm and embryo growth.

CRISPR-mediated accelerated domestication of African rice landraces.

African Oryza glaberrima and Oryza sativa landraces are considered valuable resources for breeding traits due to their adaptation to local environmental and soil conditions. They often possess superior resistance to endemic pests and tolerance to drought and nutrient deficiencies when compared to the "imported" high production Asian rice varieties. In contrast, "domestication traits" such as seed shattering, lodging, and seed yield are not well established in these African landraces. Therefore, the use of these African varieties for high production agriculture is limited by unpredictable yield and grain quality. We are addressing this shortcoming by developing protocols for genetically transforming African landraces to allow the use of CRISPR-Cas mediated breeding approaches. Here we use as proof of concept the cultivated African landrace Kabre to target selected known "domestication loci" and improve the agronomic potential of Kabre rice. Stable genetic transformation with CRISPR-Cas9-based vectors generated single and simultaneous multiple gene knockouts. Plants with reduced stature to diminish lodging were generated by disrupting the HTD1 gene. Furthermore, three loci shown to control seed size and/or yield (GS3, GW2 and GN1A) were targeted using a multiplex CRISPR-Cas9 construct. This resulted in mutants with significantly improved seed yield. Our study provides an example of how new breeding technologies can accelerate the development of highly productive African landrace rice varieties, an important advancement considering that Africa is a hotspot for worldwide population growth and therefore prone to food shortage.

BPC transcription factors and a Polycomb Group protein confine the expression of the ovule identity gene SEEDSTICK in Arabidopsis.

The BASIC PENTACYSTEINE (BPC) GAGA (C-box) binding proteins belong to a small plant transcription factor family. We previously reported that class I BPCs bind directly to C-boxes in the SEEDSTICK (STK) promoter and the mutagenesis of these cis-elements affects STK expression in the flower. The MADS-domain factor SHORT VEGETATIVE PHASE (SVP) is another key regulator of STK. Direct binding of SVP to CArG-boxes in the STK promoter are required to repress its expression during the first stages of flower development. Here we show that class II BPCs directly interact with SVP and that MADS-domain binding sites in the STK promoter region are important for the correct spatial and temporal expression of this homeotic gene. Furthermore, we show that class I and class II BPCs act redundantly to repress STK expression in the flower, most likely by recruiting TERMINAL FLOWER 2/LIKE HETEROCHROMATIN PROTEIN 1 (TFL2/LHP1) and mediating the establishment and the maintenance of H3K27me3 repressive marks on DNA. We investigate the role of LHP1 in the regulation of STK expression. In addition to providing a better understanding of the role of BPC transcription factors in the regulation of STK expression, our results suggest the existence of a more general regulatory complex composed of BPCs, MADS-domain factors and Polycomb Repressive Complexes that co-operate to regulate gene expression in reproductive tissues. We believe that our data along with the molecular model described here could provide significant insights for a more comprehensive understanding of gene regulation in plants.

REM34 and REM35 Control Female and Male Gametophyte Development in Arabidopsis thaliana.

The REproductive Meristem (REM) gene family encodes for transcription factors belonging to the B3 DNA binding domain superfamily. In Arabidopsis thaliana, the REM gene family is composed of 45 members, preferentially expressed during flower, ovule, and seed developments. Only a few members of this family have been functionally characterized: VERNALIZATION1 (VRN1) and, most recently, TARGET OF FLC AND SVP1 (TFS1) regulate flowering time and VERDANDI (VDD), together with VALKYRIE (VAL) that control the death of the receptive synergid cell in the female gametophyte. We investigated the role of REM34, REM35, and REM36, three closely related and linked genes similarly expressed in both female and male gametophytes. Simultaneous silencing by RNA interference (RNAi) caused about 50% of the ovules to remain unfertilized. Careful evaluation of both ovule and pollen developments showed that this partial sterility of the transgenic RNAi lines was due to a postmeiotic block in both female and male gametophytes. Furthermore, protein interaction assays revealed that REM34 and REM35 interact, which suggests that they work together during the first stages of gametogenesis.

Gene expression profiling of reproductive meristem types in early rice inflorescences by laser microdissection.

In rice, inflorescence architecture is established at early stages of reproductive development and contributes directly to grain yield potential. After induction of flowering, the complexity of branching, and therefore the number of seeds on the panicle, is determined by the activity of different meristem types and the timing of transitions between them. Although some of the genes involved in these transitions have been identified, an understanding of the network of transcriptional regulators controlling this process is lacking. To address this we used a precise laser microdissection and RNA-sequencing approach in Oryza sativa ssp. japonica cv. Nipponbare to produce quantitative data that describe the landscape of gene expression in four different meristem types: the rachis meristem, the primary branch meristem, the elongating primary branch meristem (including axillary meristems), and the spikelet meristem. A switch in expression profile between apical and axillary meristem types followed by more gradual changes during transitions in axillary meristem identity was observed, and several genes potentially involved in branching were identified. This resource will be vital for a mechanistic understanding of the link between inflorescence development and grain yield.

SHORT VEGETATIVE PHASE reduces gibberellin biosynthesis at the Arabidopsis shoot apex to regulate the floral transition.

In Arabidopsis thaliana environmental and endogenous cues promote flowering by activating expression of a small number of integrator genes. The MADS box transcription factor SHORT VEGETATIVE PHASE (SVP) is a critical inhibitor of flowering that directly represses transcription of these genes. However, we show by genetic analysis that the effect of SVP cannot be fully explained by repressing known floral integrator genes. To identify additional SVP functions, we analyzed genome-wide transcriptome data and show that GIBBERELLIN 20 OXIDASE 2, which encodes an enzyme required for biosynthesis of the growth regulator gibberellin (GA), is upregulated in svp mutants. GA is known to promote flowering, and we find that svp mutants contain elevated levels of GA that correlate with GA-related phenotypes such as early flowering and organ elongation. The ga20ox2 mutation suppresses the elevated GA levels and partially suppresses the growth and early flowering phenotypes of svp mutants. In wild-type plants, SVP expression in the shoot apical meristem falls when plants are exposed to photoperiods that induce flowering, and this correlates with increased expression of GA20ox2. Mutations that impair the photoperiodic flowering pathway prevent this downregulation of SVP and the strong increase in expression of GA20ox2. We conclude that SVP delays flowering by repressing GA biosynthesis as well as integrator gene expression and that, in response to inductive photoperiods, repression of SVP contributes to the rise in GA at the shoot apex, promoting rapid induction of flowering.

Analysis of the arabidopsis REM gene family predicts functions during flower development.

BACKGROUND AND AIMS: The REM (Reproductive Meristem) gene family of Arabidopsis thaliana is part of the B3 DNA-binding domain superfamily. Despite the fact that several groups have worked on the REM genes for many years, little is known about the function of this transcription factor family. This study aims to identify a set of REM genes involved in flower development and to characterize their function. METHODS: In order to provide an overview of the REM gene family, a detailed expression analysis for all REM genes of A. thaliana was performed and combined with a meta-analysis of ChIP-sequencing and microarray experiments. KEY RESULTS: Two sets of phylogenetically closely related REM genes, namely REM23, REM24 and REM25, and REM34, REM35 and REM36, were identified as possibly being involved in the early stages of flower development. Single- and double-mutant combinations were analysed for these genes, and no phenotypic effects were detected during flower development. CONCLUSIONS: The data suggest that the REM34, REM35 and REM36 group is the most interesting one, as REM34 is co-expressed with the floral meristem identity (FMI) genes, they are bound by AP1, SVP, AP3 and PI, and they are expressed in the floral meristem and during the earliest stages of flower development. However, it appears that high levels of functional redundancy may conceal the exact function of these transcription factor genes.

Gene coexpression patterns during early development of the native Arabidopsis reproductive meristem: novel candidate developmental regulators and patterns of functional redundancy.

During very early stages of flower development in Arabidopsis thaliana, a series of key decisions are taken. Indeed, the position and the basic patterning of new flowers are determined in less than 4 days. Given that the scientific literature provides hard evidence for the function of only 10% of A. thaliana genes, we hypothesized that although many essential genes have already been identified, many poorly characterized genes are likely to be involved in floral patterning. In the current study, we use high-throughput sequencing to describe the transcriptome of the native inflorescence meristem, the floral meristem and the new flower immediately after the start of organ differentiation. We provide evidence that our experimental system is reliable and less affected by experimental artefacts than a widely used floral induction system. Furthermore, we show how these data can be used to identify candidate genes for functional studies, and to generate hypotheses of functional redundancies and regulatory interactions.

Flower development: open questions and future directions.

Almost three decades of genetic and molecular analyses have resulted in detailed insights into many of the processes that take place during flower development and in the identification of a large number of key regulatory genes that control these processes. Despite this impressive progress, many questions about how flower development is controlled in different angiosperm species remain unanswered. In this chapter, we discuss some of these open questions and the experimental strategies with which they could be addressed. Specifically, we focus on the areas of floral meristem development and patterning, floral organ specification and differentiation, as well as on the molecular mechanisms underlying the evolutionary changes that have led to the astounding variations in flower size and architecture among extant and extinct angiosperms.

Identification of pathways directly regulated by SHORT VEGETATIVE PHASE during vegetative and reproductive development in Arabidopsis.

BACKGROUND: MADS-domain transcription factors play important roles during plant development. The Arabidopsis MADS-box gene SHORT VEGETATIVE PHASE (SVP) is a key regulator of two developmental phases. It functions as a repressor of the floral transition during the vegetative phase and later it contributes to the specification of floral meristems. How these distinct activities are conferred by a single transcription factor is unclear, but interactions with other MADS domain proteins which specify binding to different genomic regions is likely one mechanism. RESULTS: To compare the genome-wide DNA binding profile of SVP during vegetative and reproductive development we performed ChIP-seq analyses. These ChIP-seq data were combined with tiling array expression analysis, induction experiments and qRT-PCR to identify biologically relevant binding sites. In addition, we compared genome-wide target genes of SVP with those published for the MADS domain transcription factors FLC and AP1, which interact with SVP during the vegetative and reproductive phases, respectively. CONCLUSIONS: Our analyses resulted in the identification of pathways that are regulated by SVP including those controlling meristem development during vegetative growth and flower development whereas floral transition pathways and hormonal signaling were regulated predominantly during the vegetative phase. Thus, SVP regulates many developmental pathways, some of which are common to both of its developmental roles whereas others are specific to only one of them.

Basic pentacysteine proteins mediate MADS domain complex binding to the DNA for tissue-specific expression of target genes in Arabidopsis.

Basic pentacysteine (BPC) transcription factors have been identified in a large variety of plant species. In Arabidopsis thaliana there are seven BPC genes, which, except for BPC5, are expressed ubiquitously. BPC genes are functionally redundant in a wide range of developmental processes. Recently, we reported that BPC1 binds to guanine and adenine (GA)-rich consensus sequences in the seedstick (STK) promoter in vitro and induces conformational changes. Here we show by chromatin immunoprecipitation experiments that in vivo BPCs also bind to the consensus boxes, and when these were mutated, expression from the STK promoter was derepressed, resulting in ectopic expression in the inflorescence. We also reveal that short vegetative phase (SVP) is a direct regulator of STK. SVP is a floral meristem identity gene belonging to the MADS box gene family. The SVP-APETALA1 (AP1) dimer recruits the SEUSS (SEU)-LEUNIG (LUG) transcriptional cosuppressor to repress floral homeotic gene expression in the floral meristem. Interestingly, we found that GA consensus sequences in the STK promoter to which BPCs bind are essential for recruitment of the corepressor complex to this promoter. Our data suggest that we have identified a new regulatory mechanism controlling plant gene expression that is probably generally used, when considering BPCs' wide expression profile and the frequent presence of consensus binding sites in plant promoters.