Rice florigens control a common set of genes at the shoot apical meristem including the F-BOX BROADER TILLER ANGLE 1 that regulates tiller angle and spikelet development.

Rice flowering is triggered by transcriptional reprogramming at the shoot apical meristem (SAM) mediated by florigenic proteins produced in leaves in response to changes in photoperiod. Florigens are more rapidly expressed under short days (SDs) compared to long days (LDs) and include the HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1) phosphatidylethanolamine binding proteins. Hd3a and RFT1 are largely redundant at converting the SAM into an inflorescence, but whether they activate the same target genes and convey all photoperiodic information that modifies gene expression at the SAM is currently unclear. We uncoupled the contribution of Hd3a and RFT1 to transcriptome reprogramming at the SAM by RNA sequencing of dexamethasone-inducible over-expressors of single florigens and wild-type plants exposed to photoperiodic induction. Fifteen highly differentially expressed genes common to Hd3a, RFT1, and SDs were retrieved, 10 of which still uncharacterized. Detailed functional studies on some candidates revealed a role for LOC_Os04g13150 in determining tiller angle and spikelet development and the gene was renamed BROADER TILLER ANGLE 1 (BRT1). We identified a core set of genes controlled by florigen-mediated photoperiodic induction and defined the function of a novel florigen target controlling tiller angle and spikelet development.

Environmental control of rice flowering time.

Correct measurement of environmental parameters is fundamental for plant fitness and survival, as well as for timing developmental transitions, including the switch from vegetative to reproductive growth. Important parameters that affect flowering time include day length (photoperiod) and temperature. Their response pathways have been best described in Arabidopsis, which currently offers a detailed conceptual framework and serves as a comparison for other species. Rice, the focus of this review, also possesses a photoperiodic flowering pathway, but 150 million years of divergent evolution in very different environments have diversified its molecular architecture. The ambient temperature perception pathway is strongly intertwined with the photoperiod pathway and essentially converges on the same genes to modify flowering time. When observing network topologies, it is evident that the rice flowering network is centered on EARLY HEADING DATE 1, a rice-specific transcriptional regulator. Here, we summarize the most important features of the rice photoperiodic flowering network, with an emphasis on its uniqueness, and discuss its connections with hormonal, temperature perception, and stress pathways.

Two florigens and a florigen-like protein form a triple regulatory module at the shoot apical meristem to promote reproductive transitions in rice.

Many plant species monitor and respond to changes in day length (photoperiod) for aligning reproduction with a favourable season. Day length is measured in leaves and, when appropriate, leads to the production of floral stimuli called florigens that are transmitted to the shoot apical meristem to initiate inflorescence development1. Rice possesses two florigens encoded by HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1)2. Here we show that the arrival of Hd3a and RFT1 at the shoot apical meristem activates FLOWERING LOCUS T-LIKE 1 (FT-L1), encoding a florigen-like protein that shows features partially differentiating it from typical florigens. FT-L1 potentiates the effects of Hd3a and RFT1 during the conversion of the vegetative meristem into an inflorescence meristem and organizes panicle branching by imposing increasing determinacy to distal meristems. A module comprising Hd3a, RFT1 and FT-L1 thus enables the initiation and balanced progression of panicle development towards determinacy.

OsFD4 promotes the rice floral transition via florigen activation complex formation in the shoot apical meristem.

In rice, the florigens Heading Date 3a (Hd3a) and Rice Flowering Locus T 1 (RFT1), OsFD-like basic leucine zipper (bZIP) transcription factors, and Gf14 proteins assemble into florigen activation/repressor complexes (FACs/FRCs), which regulate transition to flowering in leaves and apical meristem. Only OsFD1 has been described as part of complexes promoting flowering at the meristem, and little is known about the role of other bZIP transcription factors, the combinatorial complexity of FAC formation, and their DNA-binding properties. Here, we used mutant analysis, protein-protein interaction assays and DNA affinity purification (DAP) sequencing coupled to in silico prediction of binding syntaxes to study several bZIP proteins that assemble into FACs or FRCs. We identified OsFD4 as a component of a FAC promoting flowering at the shoot apical meristem, downstream of OsFD1. The osfd4 mutants are late flowering and delay expression of genes promoting inflorescence development. Protein-protein interactions indicate an extensive network of contacts between several bZIPs and Gf14 proteins. Finally, we identified genomic regions bound by bZIPs with promotive and repressive effects on flowering. We conclude that distinct bZIPs orchestrate floral induction at the meristem and that FAC formation is largely combinatorial. While binding to the same consensus motif, their DNA-binding syntax is different, suggesting discriminatory functions.

Control of flowering in rice through synthetic microProteins.

Photoperiod-dependent flowering in rice is regulated by HEADING DATE 1 (Hd1), which acts as both an activator and repressor of flowering in a daylength-dependent manner. To investigate the use of microProteins as a tool to modify rice sensitivity to the photoperiod, we designed a synthetic Hd1 microProtein (Hd1miP) capable of interacting with Hd1 protein, and overexpressed it in rice. Transgenic OX-Hd1miP plants flowered significantly earlier than wild type plants when grown in non-inductive long day conditions. Our results show the potential of microProteins to serve as powerful tools for modulating crop traits and unraveling protein function.

A transcription factor coordinating internode elongation and photoperiodic signals in rice.

In several plant species, inflorescence formation is accompanied by stem elongation. Both processes are accelerated in rice upon perception of shortening days. Here, we show that PREMATURE INTERNODE ELONGATION 1 (PINE1), encoding a rice zinc-finger transcription factor, reduces the sensitivity of the stem to gibberellin (GA). The florigens reduce PINE1 expression to increase stem responsiveness to GA and promote flowering. These data indicate the existence of a regulatory network coordinating flowering and GA-dependent growth.

The Enhancement of Plant Disease Resistance Using CRISPR/Cas9 Technology.

Genome editing technologies have progressed rapidly and become one of the most important genetic tools in the implementation of pathogen resistance in plants. Recent years have witnessed the emergence of site directed modification methods using meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). Recently, CRISPR/Cas9 has largely overtaken the other genome editing technologies due to the fact that it is easier to design and implement, has a higher success rate, and is more versatile and less expensive. This review focuses on the recent advances in plant protection using CRISPR/Cas9 technology in model plants and crops in response to viral, fungal and bacterial diseases. As regards the achievement of viral disease resistance, the main strategies employed in model species such as Arabidopsis and Nicotiana benthamiana, which include the integration of CRISPR-encoding sequences that target and interfere with the viral genome and the induction of a CRISPR-mediated targeted mutation in the host plant genome, will be discussed. Furthermore, as regards fungal and bacterial disease resistance, the strategies based on CRISPR/Cas9 targeted modification of susceptibility genes in crop species such as rice, tomato, wheat, and citrus will be reviewed. After spending years deciphering and reading genomes, researchers are now editing and rewriting them to develop crop plants resistant to specific pests and pathogens.

Antagonistic Transcription Factor Complexes Modulate the Floral Transition in Rice.

Plants measure day or night lengths to coordinate specific developmental changes with a favorable season. In rice (Oryza sativa), the reproductive phase is initiated by exposure to short days when expression of HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1) is induced in leaves. The cognate proteins are components of the florigenic signal and move systemically through the phloem to reach the shoot apical meristem (SAM). In the SAM, they form a transcriptional activation complex with the bZIP transcription factor OsFD1 to start panicle development. Here, we show that Hd3a and RFT1 can form transcriptional activation or repression complexes also in leaves and feed back to regulate their own transcription. Activation complexes depend on OsFD1 to promote flowering. However, additional bZIPs, including Hd3a BINDING REPRESSOR FACTOR1 (HBF1) and HBF2, form repressor complexes that reduce Hd3a and RFT1 expression to delay flowering. We propose that Hd3a and RFT1 are also active locally in leaves to fine-tune photoperiodic flowering responses.

The Importance of Being on Time: Regulatory Networks Controlling Photoperiodic Flowering in Cereals.

Flowering is the result of the coordination between genetic information and environmental cues. Gene regulatory networks have evolved in plants in order to measure diurnal and seasonal variation of day length (or photoperiod), thus aligning the reproductive phase with the most favorable season of the year. The capacity of plants to discriminate distinct photoperiods classifies them into long and short day species, depending on the conditions that induce flowering. Plants of tropical origin and adapted to short day lengths include rice, maize, and sorghum, whereas wheat and barley were originally domesticated in the Fertile Crescent and are considered long day species. In these and other crops, day length measurement mechanisms have been artificially modified during domestication and breeding to adapt plants to novel areas, to the extent that a wide diversity of responses exists within any given species. Notwithstanding the ample natural and artificial variation of day length responses, some of the basic molecular elements governing photoperiodic flowering are widely conserved. However, as our understanding of the underlying mechanisms improves, it becomes evident that specific regulators exist in many lineages that are not shared by others, while apparently conserved components can be recruited to novel functions during evolution.

Transcriptional and Post-transcriptional Mechanisms Limit Heading Date 1 (Hd1) Function to Adapt Rice to High Latitudes.

Rice flowering is controlled by changes in the photoperiod that promote the transition to the reproductive phase as days become shorter. Natural genetic variation for flowering time has been largely documented and has been instrumental to define the genetics of the photoperiodic pathway, as well as providing valuable material for artificial selection of varieties better adapted to local environments. We mined genetic variation in a collection of rice varieties highly adapted to European regions and isolated distinct variants of the long day repressor HEADING DATE 1 (Hd1) that perturb its expression or protein function. Specific variants allowed us to define novel features of the photoperiodic flowering pathway. We demonstrate that a histone fold domain scaffold formed by GRAIN YIELD, PLANT HEIGHT AND HEADING DATE 8 (Ghd8) and several NF-YC subunits can accommodate distinct proteins, including Hd1 and PSEUDO RESPONSE REGULATOR 37 (PRR37), and that the resulting OsNF-Y complex containing Hd1 can bind a specific sequence in the promoter of HEADING DATE 3A (Hd3a). Artificial selection has locally favored an Hd1 variant unable to assemble in such heterotrimeric complex. The causal polymorphism was defined as a single conserved lysine in the CCT domain of the Hd1 protein. Our results indicate how genetic variation can be stratified and explored at multiple levels, and how its description can contribute to the molecular understanding of basic developmental processes.

Y flowering? Regulation and activity of CONSTANS and CCT-domain proteins in Arabidopsis and crop species.

Changes in day length regulate the proper timing of flowering in several plant species. The genetic architecture of this process is based on CCT-domain proteins, many of which interact with NF-Y subunits to regulate transcription of target genes. In the model plant Arabidopsis thaliana, the CONSTANS CCT-domain protein is a central photoperiodic sensor. We will discuss how the diurnal rhythms of its transcription and protein accumulation are generated, and how the protein engages into multiple complexes to control production of a systemic flowering signal. Regulatory parallels will be drawn between Arabidopsis and major crops that indicate conservation of some CCT/NF-Y modules during plant evolution. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.

Alternative splicing enhances transcriptome complexity in desiccating seeds.

Before being dispersed in the environment, mature seeds need to be dehydrated. The survival of seeds after dispersal depends on their low hydration in combination with high desiccation tolerance. These characteristics are established during seed maturation. Some key seed maturation genes have been reported to be regulated by alternative splicing (AS). However, so far AS was described only for single genes and a comprehensive analysis of AS during seed maturation has been lacking. We investigated gene expression and AS during Arabidopsis thaliana seed development at a global level, before and after desiccation. Bioinformatics tools were developed to identify differentially spliced regions within genes. Our data suggest the importance and shows the peculiar features of AS during seed desiccation. We identified AS in 34% of genes that are expressed at both timepoints before and after desiccation. Most of these AS transcript variants had not been found before in other tissues. Among the AS genes some seed master regulators could be found. Interestingly, 6% of all expressed transcripts were not transcriptionally regulated during desiccation, but only modified by AS. We propose that AS should be more routinely taken into account in the analysis of transcriptomic data to prevent overlooking potentially important regulators.

Loss of floral repressor function adapts rice to higher latitudes in Europe.

The capacity to discriminate variations in day length allows plants to align flowering with the most favourable season of the year. This capacity has been altered by artificial selection when cultivated varieties became adapted to environments different from those of initial domestication. Rice flowering is promoted by short days when HEADING DATE 1 (Hd1) and EARLY HEADING DATE 1 (Ehd1) induce the expression of florigenic proteins encoded by HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1). Repressors of flowering antagonize such induction under long days, maintaining vegetative growth and delaying flowering. To what extent artificial selection of long day repressor loci has contributed to expand rice cultivation to Europe is currently unclear. This study demonstrates that European varieties activate both Hd3a and RFT1 expression regardless of day length and their induction is caused by loss-of-function mutations at major long day floral repressors. However, their contribution to flowering time control varies between locations. Pyramiding of mutations is frequently observed in European germplasm, but single mutations are sufficient to adapt rice to flower at higher latitudes. Expression of Ehd1 is increased in varieties showing reduced or null Hd1 expression under natural long days, as well as in single hd1 mutants in isogenic backgrounds. These data indicate that loss of repressor genes has been a key strategy to expand rice cultivation to Europe, and that Ehd1 is a central node integrating floral repressive signals.

Molecular control of seasonal flowering in rice, arabidopsis and temperate cereals.

BACKGROUND: Rice (Oryza sativa) and Arabidopsis thaliana have been widely used as model systems to understand how plants control flowering time in response to photoperiod and cold exposure. Extensive research has resulted in the isolation of several regulatory genes involved in flowering and for them to be organized into a molecular network responsive to environmental cues. When plants are exposed to favourable conditions, the network activates expression of florigenic proteins that are transported to the shoot apical meristem where they drive developmental reprogramming of a population of meristematic cells. Several regulatory factors are evolutionarily conserved between rice and arabidopsis. However, other pathways have evolved independently and confer specific characteristics to flowering responses. SCOPE: This review summarizes recent knowledge on the molecular mechanisms regulating daylength perception and flowering time control in arabidopsis and rice. Similarities and differences are discussed between the regulatory networks of the two species and they are compared with the regulatory networks of temperate cereals, which are evolutionarily more similar to rice but have evolved in regions where exposure to low temperatures is crucial to confer competence to flower. Finally, the role of flowering time genes in expansion of rice cultivation to Northern latitudes is discussed. CONCLUSIONS: Understanding the mechanisms involved in photoperiodic flowering and comparing the regulatory networks of dicots and monocots has revealed how plants respond to environmental cues and adapt to seasonal changes. The molecular architecture of such regulation shows striking similarities across diverse species. However, integration of specific pathways on a basal scheme is essential for adaptation to different environments. Artificial manipulation of flowering time by means of natural genetic resources is essential for expanding the cultivation of cereals across different environments.

Molecular control of flowering in response to day length in rice.

Flowering at the most appropriate times of the year requires careful monitoring of environmental conditions and correct integration of such information with an endogenous molecular network. Rice (Oryza sativa) is a facultative short day plant, and flowers quickly under short day lengths, as opposed to Arabidopsis thaliana whose flowering is accelerated by longer days. Despite these physiological differences, several genes controlling flowering in response to day length (or photoperiod) are conserved between rice and Arabidopsis, and the molecular mechanisms involved are similar. Inductive day lengths trigger expression of florigenic proteins in leaves that can move to the shoot apical meristem to induce reproductive development. As compared to Arabidopsis, rice also possesses unique factors that regulate expression of florigenic genes. Here, we discuss recent advances in understanding the molecular mechanisms involved in day length perception, production of florigenic signals, and molecular responses of the shoot apical meristem to florigenic proteins.

Seed maturation in Arabidopsis thaliana is characterized by nuclear size reduction and increased chromatin condensation.

Most plant species rely on seeds for their dispersal and survival under unfavorable environmental conditions. Seeds are characterized by their low moisture content and significantly reduced metabolic activities. During the maturation phase, seeds accumulate storage reserves and become desiccation-tolerant and dormant. Growth is resumed after release of dormancy and the occurrence of favorable environmental conditions. Here we show that embryonic cotyledon nuclei of Arabidopsis thaliana seeds have a significantly reduced nuclear size, which is established at the beginning of seed maturation. In addition, the chromatin of embryonic cotyledon nuclei from mature seeds is highly condensed. Nuclei regain their size and chromatin condensation level during germination. The reduction in nuclear size is controlled by the seed maturation regulator ABSCISIC ACID-INSENSITIVE 3, and the increase during germination requires two predicted nuclear matrix proteins, LITTLE NUCLEI 1 and LITTLE NUCLEI 2. Our results suggest that the specific properties of nuclei in ripe seeds are an adaptation to desiccation, independent of dormancy. We conclude that the changes in nuclear size and chromatin condensation in seeds are independent, developmentally controlled processes.

Chromatin dynamics during seed dormancy.

The chromatin structure determines gene expression and thereby regulates developmental processes in the plant. The molecular mechanisms regulating the induction and release of seed dormancy are still largely unknown and the underlying changes in chromatin organization have hardly been analyzed. Most chromatin studies in plants have been performed on vegetative tissues and have focused on seedlings. The composition of seeds hampers molecular analyses and requires adaptation of the methods that are used for other tissues. Here, we give an overview of the current methods that are used to study different aspects of chromatin organization in seeds. Cytogenetic methods, like fluorescence in situ hybridization and immunolocalization, are used to study chromatin at the microscopic level. Changes in DNA methylation and histone modifications can be studied with molecular methods, like bisulfite sequencing, immunoblotting, and chromatin immunoprecipitation.

The conserved splicing factor SUA controls alternative splicing of the developmental regulator ABI3 in Arabidopsis.

ABSCISIC ACID INSENSITIVE3 (ABI3) is a major regulator of seed maturation in Arabidopsis thaliana. We detected two ABI3 transcripts, ABI3-alpha and ABI3-beta, which encode full-length and truncated proteins, respectively. Alternative splicing of ABI3 is developmentally regulated, and the ABI3-beta transcript accumulates at the end of seed maturation. The two ABI3 transcripts differ by the presence of a cryptic intron in ABI3-alpha, which is spliced out in ABI3-beta. The suppressor of abi3-5 (sua) mutant consistently restores wild-type seed features in the frameshift mutant abi3-5 but does not suppress other abi3 mutant alleles. SUA is a conserved splicing factor, homologous to the human protein RBM5, and reduces splicing of the cryptic ABI3 intron, leading to a decrease in ABI3-beta transcript. In the abi3-5 mutant, ABI3-beta codes for a functional ABI3 protein due to frameshift restoration.

Genetic interaction between AINTEGUMENTA (ANT) and the ovule identity genes SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2).

AINTEGUMENTA (ANT) promotes initiation and growth of ovule integuments which cell fate is specified by ovule identity factors, such as SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2). To study the genetic interaction between ANT and the ovule identity genes, we have obtained a stk shp1 shp2 ant quadruple mutant. The molecular and morphological characterization of the quadruple mutant and its comparison with the stk shp1 shp2 triple mutant, the shp1 shp2 ant triple mutant and the stk ant double mutant are here presented.

A new role for the SHATTERPROOF genes during Arabidopsis gynoecium development.

Gynoecium development is a complex process which is regulated by key factors that control the spatial formation of the apical, medial and basal parts. SHATTERPROOF1 (SHP1) and SHP2, two closely related MADS-box genes, redundantly control the differentiation of the dehiscence zone and promote the lignification of adjacent cells. Furthermore, SHP1 and SHP2 have shown to play an important role in ovule identity determination. The present work identifies a new function for these two genes in promoting stigma, style and medial tissue development. This new role was discovered by combining the shp1 shp2 double mutant with the aintegumenta (ant) and crabs claw (crc) mutants. In quadruple mutant flowers, the inner whorl is composed of unfused carpels which lack almost completely apical and medial tissues, a phenotype similar to the previously reported fil ant and lug ant double mutants.