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.

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.

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.

Genes of the RAV Family Control Heading Date and Carpel Development in Rice.

In plants, correct formation of reproductive organs is critical for successful seedset and perpetuation of the species. Plants have evolved different molecular mechanisms to coordinate flower and seed development at the proper time of the year. Among the plant-specific RELATED TO ABI3 AND VP1 (RAV) family of transcription factors, only TEMPRANILLO1 (TEM1) and TEM2 have been shown to affect reproductive development in Arabidopsis (Arabidopsis thaliana). They negatively regulate the floral transition through direct repression of FLOWERING LOCUS T and GIBBERELLIN 3-OXIDASE1/2, encoding major components of the florigen. Here we identify RAV genes from rice (Oryza sativa), and unravel their regulatory roles in key steps of reproductive development. Our data strongly suggest that, like TEMs, OsRAV9/OsTEM1 has a conserved function as a repressor of photoperiodic flowering upstream of the floral activators OsMADS14 and Hd3a, through a mechanism reminiscent of that one underlying floral transition in temperate cereals. Furthermore, OsRAV11 and OsRAV12 may have acquired a new function in the differentiation of the carpel and the control of seed size, acting downstream of floral homeotic factors. Alternatively, this function may have been lost in Arabidopsis. Our data reveal conservation of RAV gene function in the regulation of flowering time in monocotyledonous and dicotyledonous plants, but also unveil roles in the development of rice gynoecium.

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.

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.

Gynoecium size and ovule number are interconnected traits that impact seed yield.

Angiosperms form the largest group of land plants and display an astonishing diversity of floral structures. The development of flowers greatly contributed to the evolutionary success of the angiosperms as they guarantee efficient reproduction with the help of either biotic or abiotic vectors. The female reproductive part of the flower is the gynoecium (also called pistil). Ovules arise from meristematic tissue within the gynoecium. Upon fertilization, these ovules develop into seeds while the gynoecium turns into a fruit. Gene regulatory networks involving transcription factors and hormonal communication regulate ovule primordium initiation, spacing on the placenta, and development. Ovule number and gynoecium size are usually correlated and several genetic factors that impact these traits have been identified. Understanding and fine-tuning the gene regulatory networks influencing ovule number and pistil length open up strategies for crop yield improvement, which is pivotal in light of a rapidly growing world population. In this review, we present an overview of the current knowledge of the genes and hormones involved in determining ovule number and gynoecium size. We propose a model for the gene regulatory network that guides the developmental processes that determine seed yield.

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.

SEEDSTICK is a master regulator of development and metabolism in the Arabidopsis seed coat.

The role of secondary metabolites in the determination of cell identity has been an area of particular interest over recent years, and studies strongly indicate a connection between cell fate and the regulation of enzymes involved in secondary metabolism. In Arabidopsis thaliana, the maternally derived seed coat plays pivotal roles in both the protection of the developing embryo and the first steps of germination. In this regard, a characteristic feature of seed coat development is the accumulation of proanthocyanidins (PAs - a class of phenylpropanoid metabolites) in the innermost layer of the seed coat. Our genome-wide transcriptomic analysis suggests that the ovule identity factor SEEDSTICK (STK) is involved in the regulation of several metabolic processes, providing a strong basis for a connection between cell fate determination, development and metabolism. Using phenotypic, genetic, biochemical and transcriptomic approaches, we have focused specifically on the role of STK in PA biosynthesis. Our results indicate that STK exerts its effect by direct regulation of the gene encoding BANYULS/ANTHOCYANIDIN REDUCTASE (BAN/ANR), which converts anthocyanidins into their corresponding 2,3-cis-flavan-3-ols. Our study also demonstrates that the levels of H3K9ac chromatin modification directly correlate with the active state of BAN in an STK-dependent way. This is consistent with the idea that MADS-domain proteins control the expression of their target genes through the modification of chromatin states. STK might thus recruit or regulate histone modifying factors to control their activity. In addition, we show that STK is able to regulate other BAN regulators. Our study demonstrates for the first time how a floral homeotic gene controls tissue identity through the regulation of a wide range of processes including the accumulation of secondary metabolites.

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.

MADS domain transcription factors mediate short-range DNA looping that is essential for target gene expression in Arabidopsis.

MADS domain transcription factors are key regulators of eukaryotic development. In plants, the homeotic MIKC MADS factors that regulate floral organ identity have been studied in great detail. Based on genetic and protein-protein interaction studies, a floral quartet model was proposed that describes how these MADS domain proteins assemble into higher order complexes to regulate their target genes. However, despite the attractiveness of this model and its general acceptance in the literature, solid in vivo proof has never been provided. To gain deeper insight into the mechanisms of transcriptional regulation by MADS domain factors, we studied how SEEDSTICK (STK) and SEPALLATA3 (SEP3) directly regulate the expression of the reproductive meristem gene family transcription factor-encoding gene VERDANDI (VDD). Our data show that STK-SEP3 dimers can induce loop formation in the VDD promoter by binding to two nearby CC(A/T)6GG (CArG) boxes and that this is essential for promoter activity. Our in vivo data show that the size and position of this loop, determined by the choice of CArG element usage, is essential for correct expression. Our studies provide solid in vivo evidence for the floral quartet model.

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.

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.

Peptide aptamers: The versatile role of specific protein function inhibitors in plant biotechnology.

In recent years, peptide aptamers have emerged as novel molecular tools that have attracted the attention of researchers in various fields of basic and applied science, ranging from medicine to analytical chemistry. These artificial short peptides are able to specifically bind, track, and inhibit a given target molecule with high affinity, even molecules with poor immunogenicity or high toxicity, and represent a remarkable alternative to antibodies in many different applications. Their use is on the rise, driven mainly by the medical and pharmaceutical sector. Here we discuss the enormous potential of peptide aptamers in both basic and applied aspects of plant biotechnology and food safety. The different peptide aptamer selection methods available both in vivo and in vitro are introduced, and the most important possible applications in plant biotechnology are illustrated. In particular, we discuss the generation of broad-based virus resistance in crops, "reverse genetics" and aptasensors in bioassays for detecting contaminations in food and feed. Furthermore, we suggest an alternative to the transfer of peptide aptamers into plant cells via genetic transformation, based on the use of cell-penetrating peptides that overcome the limits imposed by both crop transformation and Genetically Modified Organism commercialization.

TAF13 interacts with PRC2 members and is essential for Arabidopsis seed development.

TBP-Associated Factors (TAFs) are components of complexes like TFIID, TFTC, SAGA/STAGA and SMAT that are important for the activation of transcription, either by establishing the basic transcription machinery or by facilitating histone acetylation. However, in Drosophila embryos several TAFs were shown to be associated with the Polycomb Repressive Complex 1 (PRC1), even though the role of this interaction remains unclear. Here we show that in Arabidopsis TAF13 interacts with MEDEA and SWINGER, both members of a plant variant of Polycomb Repressive Complex 2 (PRC2). PRC2 variants play important roles during the plant life cycle, including seed development. The taf13 mutation causes seed defects, showing embryo arrest at the 8-16 cell stage and over-proliferation of the endosperm in the chalazal region, which is typical for Arabidopsis PRC2 mutants. Our data suggest that TAF13 functions together with PRC2 in transcriptional regulation during seed development.

Arabidopsis plants lacking PsbQ and PsbR subunits of the oxygen-evolving complex show altered PSII super-complex organization and short-term adaptive mechanisms.

The oxygen-evolving complex of eukaryotic photosystem II (PSII) consists of four extrinsic subunits, PsbO (33 kDa), PsbP (23 kDa), PsbQ (17 kDa) and PsbR (10 kDa), encoded by seven nuclear genes, PsbO1 (At5g66570), PsbO2 (At3g50820), PsbP1 (At1g06680), PsbP2 (At2g30790), PsbQ1 (At4g21280), PsbQ2 (At4g05180) and PsbR (At1g79040). Using Arabidopsis insertion mutant lines, we show that PsbP1, but not PsbP2, is essential for photoautotrophic growth, whereas plants lacking both forms of PsbQ and/or PsbR show normal growth rates. Complete elimination of PsbQ has a minor effect on PSII function, but plants lacking PsbR or both PsbR and PsbQ are characterized by more pronounced defects in PSII activity. Gene expression and immunoblot analyses indicate that accumulation of each of these proteins is highly dependent on the presence of the others, and is controlled at the post-transcriptional level, whereas PsbO stability appears to be less sensitive to depletion of other subunits of the oxygen-evolving complex. In addition, comparison of levels of the PSII super-complex in wild-type and mutant leaves reveals the importance of the individual subunits of the oxygen-evolving complex for the supramolecular organization of PSII and their influence on the rate of state transitions.

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.