Comparative Genomics of Flowering Time Pathways Using Brachypodium distachyon as a Model for the Temperate Grasses

Brachypodium distachyon (Brachypodium) is a model for the temperate grasses which include important cereals such as barley, wheat and oats. Comparison of the Brachypodium genome (accession Bd21) with those of the model dicot Arabidopsis thaliana and the tropical cereal rice (Oryza sativa) provides an opportunity to compare and contrast genetic pathways controlling important traits. We analysed the homologies of genes controlling the induction of flowering using pathways curated in Arabidopsis Reactome as a starting point. Pathways include those detecting and responding to the environmental cues of day length (photoperiod) and extended periods of low temperature (vernalization). Variation in these responses has been selected during cereal domestication, providing an interesting comparison with the wild genome of Brachypodium. Brachypodium Bd21 has well conserved homologues of circadian clock, photoperiod pathway and autonomous pathway genes defined in Arabidopsis and homologues of vernalization pathway genes defined in cereals with the exception of VRN2 which was absent. Bd21 also lacked a member of the CO family (CO3). In both cases flanking genes were conserved showing that these genes are deleted in at least this accession. Segmental duplication explains the presence of two CO-like genes in temperate cereals, of which one (Hd1) is retained in rice, and explains many differences in gene family structure between grasses and Arabidopsis. The conserved fine structure of duplications shows that they largely evolved to their present structure before the divergence of the rice and Brachypodium. Of four flowering-time genes found in rice but absent in Arabidopsis, two were found in Bd21 (Id1, OsMADS51) and two were absent (Ghd7, Ehd1). Overall, results suggest that an ancient core photoperiod pathway promoting flowering via the induction of FT has been modified by the recruitment of additional lineage specific pathways that promote or repress FT expression.     

Brahma is required for proper expression of the floral repressor FLC in Arabidopsis

Background

BRAHMA (BRM) is a member of a family of ATPases of the SWI/SNF chromatin remodeling complexes from Arabidopsis. BRM has been previously shown to be crucial for vegetative and reproductive development.

Methodology/Principal Findings

Here we carry out a detailed analysis of the flowering phenotype of brm mutant plants which reveals that, in addition to repressing the flowering promoting genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), BRM also represses expression of the general flowering repressor FLOWERING LOCUS C (FLC). Thus, in brm mutant plants FLC expression is elevated, and FLC chromatin exhibits increased levels of histone H3 lysine 4 tri-methylation and decreased levels of H3 lysine 27 tri-methylation, indicating that BRM imposes a repressive chromatin configuration at the FLC locus. However, brm mutants display a normal vernalization response, indicating that BRM is not involved in vernalization-mediated FLC repression. Analysis of double mutants suggests that BRM is partially redundant with the autonomous pathway. Analysis of genetic interactions between BRM and the histone H2A.Z deposition machinery demonstrates that brm mutations overcome a requirement of H2A.Z for FLC activation suggesting that in the absence of BRM, a constitutively open chromatin conformation renders H2A.Z dispensable.

Conclusions/Significance

BRM is critical for phase transition in Arabidopsis. Thus, BRM represses expression of the flowering promoting genes CO, FT and SOC1 and of the flowering repressor FLC. Our results indicate that BRM controls expression of FLC by creating a repressive chromatin configuration of the locus.     

The molecular mechanism of vernalization in Arabidopsis and cereals: role of Flowering Locus C and its homologs

Winter varieties of plants can flower only after exposure to prolonged cold. This phenomenon is known as vernalization and has been widely studied in the model plant Arabidopsis thaliana as well as in monocots. Through the repression of floral activator genes, vernalization prevents flowering in winter. In Arabidopsis, FLOWERING LOCUS C or FLC is the key repressor during vernalization, while in monocots vernalization is regulated through VRN1, VRN2 and VRN3 (or FLOWERING LOCUS T). Interestingly, VRN genes are not homologous to FLC but FLC homologs are found to have a significant role in vernalization response in cereals. The presence of FLC homologs in monocots opens new dimensions to understand, compare and retrace the evolution of vernalization pathways between monocots and dicots. In this review, we discuss the molecular mechanism of vernalization-induced flowering along with epigenetic regulations in Arabidopsis and temperate cereals. A better understanding of cold-induced flowering will be helpful in crop breeding strategies to modify the vernalization requirement of economically important temperate cereals.     

Diversified mechanisms for regulating flowering time in a short-day plant rice

Flowering in rice is influenced by not only endogenous factors that comprise an autonomous pathway, but also environmental effects, such as photoperiod, water availability, and temperature just before floral initiation. Recent molecular genetics studies have elucidated the functional roles of genes involved in the photoperiod pathway, e.g., photoreceptors, circadian clock components, and short-day (SD) promotion factors. Although these molecular players are well conserved between rice andArabidopsis, their actual genetic functions are distinct. This is exemplified byHd1 (aCO counterpart) and phytochromes, in particular, ricePHYA. Hd1 has a dual role in regulating flowering time and the expression ofHd3a (anFT counterpart) repression under long-day (LD) conditions while promotion under SDs. Models have been proposed to explain these photoperiod-dependent antagonistic activities. Some regulatory factors are present in only one of the model systems, e.g.,FLC inArabidopsis orEhd1 in rice. Furthermore, epistatic relationships vary among such flowering regulators asHd3a (FT), OsMADS50 (SOCT), andOsMADS14 (AP1). Further experiments to probe these differences will be essential to enlarging our understanding of the diversified flowering regulation mechanisms in rice.     

Heterologous Expression of Wheat VERNALIZATION 2 (TaVRN2) Gene in Arabidopsis Delays Flowering and Enhances Freezing Tolerance

The vernalization gene 2 (VRN2), is a major flowering repressor in temperate cereals that is regulated by low temperature and photoperiod. Here we show that the gene from Triticum aestivum (TaVRN2) is also regulated by salt, heat shock, dehydration, wounding and abscissic acid. Promoter analysis indicates that TaVRN2 regulatory region possesses all the specific responsive elements to these stresses. This suggests pleiotropic effects of TaVRN2 in wheat development and adaptability to the environment. To test if TaVRN2 can act as a flowering repressor in species different from the temperate cereals, the gene was ectopically expressed in the model plant Arabidopsis. Transgenic plants showed no alteration in morphology, but their flowering time was significantly delayed compared to controls plants, indicating that TaVRN2, although having no ortholog in Brassicaceae, can act as a flowering repressor in these species. To identify the possible mechanism by which TaVRN2 gene delays flowering in Arabidopsis, the expression level of several genes involved in flowering time regulation was determined. The analysis indicates that the late flowering of the 35S::TaVRN2 plants was associated with a complex pattern of expression of the major flowering control genes, FCA, FLC, FT, FVE and SOC1. This suggests that heterologous expression of TaVRN2 in Arabidopsis can delay flowering by modulating several floral inductive pathways. Furthermore, transgenic plants showed higher freezing tolerance, likely due to the accumulation of CBF2, CBF3 and the COR genes. Overall, our data suggests that TaVRN2 gene could modulate a common regulator of the two interacting pathways that regulate flowering time and the induction of cold tolerance. The results also demonstrate that TaVRN2 could be used to manipulate flowering time and improve cold tolerance in other species.     

Role of VIN3-LIKE 2 in facultative photoperiodic flowering response in Arabidopsis

In Arabidopsis, expression of FLC and FLC-related genes (collectively called FLC clade) contributes to flowering time in response to environmental changes, such as day length and temperature, by acting as floral repressors. VIN3 is required for vernalization-mediated FLC repression and a VIN3 related protein, VIN3-LIKE 1/VERNALIZATION 5 (VIL1/VRN5), acts to regulate FLC and FLM in response to vernalization.^1–3 VIN3 also exists as a small family of PHD finger proteins in Arabidopsis, including VIL1/VRN5, VIL2/VEL1, VIL3/VEL2 and VIL4/VEL3. We showed that the PHD finger protein, VIL2, is required for proper repression of MAF5, an FLC clade member, to accelerate flowering under non-inductive photoperiods. VIL2 acts together with POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) to repress MAF5 in a photoperiod dependent manner.Key words: photoperiod, chromatin, floweringThe decision to flower is critical to the survival of flowering plants. Thus, plants sense environmental cues to initiate floral transition at a time that both ensures and optimizes their own reproductive fitness. Using a model plant, Arabidopsis thaliana, genetic studies have shown that the regulation of floral transition mainly consists of four genetic pathways: the inductive photoperiod pathway, the autonomous pathway, the vernalization pathway and the gibberellin pathway.⁴ In Arabidopsis, these four flowering pathways eventually merge into a group of genes called floral integrators, including FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and LEAFY (LFY). Based on the response to specific photoperiod conditions, the flowering behaviors of plants can be classified into three groups: long day (LD), short day (SD) and day neutral response.^5,6 Depending on the requirement of day length, plants show either obligate or facultative responses. For example, henbane, carnation and ryegrass are obligate long day (LD) flowering plants which flower under increasing inductive photoperiod but do not flower at all under non-inductive photoperiod.⁵ On the other hand, plants including Arabidopsis, wheat, lettuce and barley, are considered to be facultative flowering plants. Thus, these plants exhibit early flowering under LD and late-flowering under non-inductive short days (SD). Studies on photoperiodic flowering time mainly focus on the inductive LD-photoperiod pathway in Arabidopsis.     

The Role of VIN3-LIKE Genes in Environmentally Induced Epigenetic Regulation of Flowering

Given their sessile nature, it is critical for the survival of plants to adapt to their environment. Accordingly, plants have evolved the ability to sense seasonal changes to govern developmental fates such as the floral transition. Temperature and day length are among the seasonal cues that plants sense. We recently reported that VIN3-LIKE 1 (VIL1) is involved in mediating the flowering response to both cold and day length via regulation of two related genes, FLOWERING LOCUS C (FLC) and FLOWERING LOCUS M (FLM), respectively.Key Words: flowering, vernalization, photoperiod, chromatin, histone, gene expressionVernalization renders plants competent to flower after exposure to the prolonged cold of winter.^1,2 Arabidopsis exhibits facultative responses to both vernalization and photoperiod to initiate the floral transition. The facultative nature of the responses makes Arabidopsis a tractable genetic system to study these aspects of flowering time control.In Arabidopsis, vernalization creates competence to flower via silencing of the potent floral repressor, FLC, in a mitotically stable manner.^3,4 Thus, the vernalization response is an environmentally induced epigenetic switch in that exposure to cold permanently affects the plants'' developmental program. This epigenetic switch is associated with increased levels of FLC chromatin methylation on Histone H3 Lys 9 and Lys 27.^5,6 VERNALIZATION INSENSITIVE 3 (VIN3) plays an essential role in this switch since no modifications to FLC chromatin occur in vin3 mutants.⁵ Furthermore, the levels of expression of VIN3 mRNA are tightly correlated with the degree of the vernalization response.⁵ VIN3 encodes Plant HomeoDomain (PHD) finger-containing protein. PHD finger-containing proteins are often associated with protein complexes that are involved in chromatin remodeling.⁷We performed a yeast two-hybrid screen to identify potential protein partners of VIN3. VIN3-LIKE 1 (VIL1) was identified by this screen.⁸ VIL1 encodes a PHD finger-containing protein that is related to VIN3. As expected for proteins that are associated with VIN3, plants containing loss-of-function alleles of VIL1 do not respond to vernalization. Furthermore, no vernalization-mediated histone modifications occur at FLC in vil1 mutants similar to the situation in vin3 mutants. Thus, by yeast two hybrid and genetic analysis, VIL1 is a bona fide VIN3 partner that is required for vernalization-mediated histone modifications at FLC chromatin. Unlike VIN3, the expression of VIL1 does not change over the course of cold exposure. Rather, VIL1 mRNA levels are affected by photoperiod. VIL1 expression is significantly increased in non-inductive photoperiods (short days; SD). Consistent with this expression pattern, vil1 mutants in the Columbia accession exhibit a SD-specific late-flowering phenotype. Furthermore, VIL1 is required for attenuating expression of FLOWERING LOCUS M, a FLC-related gene, in a SD-specific manner. It is possible that the attenuation of FLM by VIL1 has a role in creating the facultative nature of photoperiod response in Arabidopsis since vil1 mutants tend towards an obligate photoperiod response (i.e., vil1 mutants often fail to flower in SD).In Arabidopsis, there are four VIN3-related genes, which we named as VIL1 ∼ VIL4,⁸ and which have also been called VRN5 and VEL1 ∼ VEL3.⁹ The C-terminal domain is highly conserved among these genes and was named the VIN3-Interacting Domain (VID) since it is required for protein-protein interaction between VIN3 and VIL1. The effect of cold on the expression patterns of VIN3-related genes varies. For example, VIL2 and VIL3 are induced specifically by vernalizing cold exposures whereas others such as VIL1 are, for the most part, constitutively expressed. It will be interesting to determine the functions of the remaining VIL genes.FLC is the main target for vernalization in Arabidopsis. Interestingly, FLC orthologs have not been found in vernalization-responsive varieties of cereals. However, in wheat, VRN2 appears to have a role equivalent to that of FLC in Arabidopsis.¹⁰ VRN2 encodes a ZCCT type zinc-finger protein that does not have a homolog in the Arabidopsis genome. In diploid wheat, down regulation of VRN2 is correlated with the vernalization response.¹¹ Interestingly, wheat contains three VIN3-LIKE (VIL) genes, TmVIL1, TmVIL2 and TmVIL3.¹² Furthermore, TmVIL1 is up-regulated by vernalization.¹² However, whether TmVIL1 has a direct role in the vernalization-mediated repression of VRN2 in wheat has not yet been addressed. Similar to VIL1, TmVIL3 shows elevated level of expression in SD. Furthermore, VRN2 is downregulated in SD;^13,14 thus there is a correlation between the induction of TmVIL genes and the downregulation of the floral repressor VRN2 similar to the VIN3/FLC and VIL1/FLM relationships (Fig. 1). Perhaps VIN3-related genes have similar roles both in Arabidopsis and in temperate wheat, but act on different target genes, possibly as a result of convergent evolution. Interestingly, the wheat gene TmVRN3 is homologous to FLOWERING LOCUS T (FT) of Arabidopsis, and TmVRN3 is repressed by TmVRN2 as FT is repressed by FLC,¹⁵ suggesting another similarity in the regulation of flowering time between Arabidopsis and temperate wheat (Fig. 1).Open in a separate windowFigure 1Proposed relationship of VIN3 family genes to the regulatory network controlling flowering time in response to environmental cues in Arabidopsis and diploid wheat (adapted from ref. ¹⁶).Although the PHD finger can be found in various eukaryotes, the VID is unique to plants. It is also noteworthy that VIN3-related genes can be found in various plant species, including rice, which does not have a vernalization response. It will be interesting to address whether the VIN3-related genes from various plant species are more broadly involved in relaying environmental signals to developmental programs.     

Comparison of rice flowering-time genes under paddy conditions

Heading date is one of most important agronomic traits in rice. Flowering regulatory mechanisms have been elucidated in many cultivars through various approaches. Although study about flowering has been extensively examined in rice, but contributions of floral regulators had been poorly understood in a common genetic background for rice grown under paddy conditions. Thus, we compared the expression of 10 flowering-time genes — OsMADS50, OsMADS51, OsVIL2, OsPhyA, OsPhyB, OsPhyC, Ghd7, Hd1, OsGI, and OsTrx1 — in the same genetic background for ‘Dongjin’ rice (Oryza sativa) grown under paddy conditions when days were longer than 13.5 h. Whereas the wild type (WT) rice flowered 105 days after sowing, the latest mutant to do so was ostrx1, flowering 53 d later. This indicated that the gene is the strongest inducer among all of those examined. Mutations in OsMADS50 delayed flowering by 45 d when compared with the WT, suggesting that this MADS gene is another strong positive element. The third positive element was OsVIL2; mutations in the gene caused plants to flower 27 d late. In contrast, the double phytochrome mutant osphyA osphyB flowered 44 d earlier than the WT. The single mutant osphyB and the double mutant osphyB osphyC did the same, although not as early as the osphyA osphyB double mutant. These results demonstrated that phytochromes are major inhibitors under paddy conditions. Mutations in Ghd7 accelerated flowering by 34 d, indicating that the gene is also a major inhibitor. The hd1 mutants flowered 16 d earlier than the WT while a mutation in OsGI hastened flowering by 10 d, suggesting that both are weak flowering repressors. Of the two florigen genes (Hd3a being the other one), RFT1 played a major role under paddy conditions. Its expression was strongly promoted by Ehd1, which was negatively controlled by Ghd7. Here we show that phytochromes strongly inhibit flowering and OsTrx1 and OsMADS50 significantly induce flowering under paddy conditions through Ghd7-Ehd1-RFT1 pathway. Thus, we may be able to control heading date under paddy conditions through manipulating those genes, Ghd7, Ehd1 and RFT1.     

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.     

Developmental transcriptome analysis of floral transition in <Emphasis Type="Italic">Rosa odorata</Emphasis> var. <Emphasis Type="Italic">gigantea</Emphasis>

Key message

Expression analyses revealed that floral transition of Rosa odorata var. gigantea is mainly regulated by VRN1, COLs, DELLA and KSN, with contributions by the effects of phytohormone and starch metabolism.

Abstract

Seasonal plants utilize changing environmental and developmental cues to control the transition from vegetative growth to flowering at the correct time of year. This study investigated global gene expression profiles at different developmental stages of Rosa odorata var. gigantea by RNA-sequencing, combined with phenotypic characterization and physiological changes. Gene ontology enrichment analysis of the differentially expressed genes (DEGs) between four different developmental stages (vegetative meristem, pre-floral meristem, floral meristem and secondary axillary buds) indicated that DNA methylation and the light reaction played a large role in inducing the rose floral transition. The expression of SUF and FLC, which are known to play a role in delaying flowering until vernalization, was down-regulated from the vegetative to the pre-floral meristem stage. In contrast, the expression of VRN1, which promotes flowering by repressing FLC expression, increased. The expression of DELLA proteins, which function as central nodes in hormone signaling pathways, and probably involve interactions between GA, auxin, and ABA to promote the floral transition, was well correlated with the expression of floral integrators, such as AGL24, COL4. We also identified DEGs associated with starch metabolism correlated with SOC1, AGL15, SPL3, AGL24, respectively. Taken together, our results suggest that vernalization and photoperiod are prominent cues to induce the rose floral transition, and that DELLA proteins also act as key regulators. The results summarized in the study on the floral transition of the seasonal rose lay a foundation for further functional demonstration, and have profound economic and ornamental values.

     

<Emphasis Type="Italic">CaVIL1</Emphasis>, a plant homeodomain gene that promotes flowering in pepper

Key message

CaVIL1 is a homolog of VIL1, a regulator of vernalization response in Arabidopsis and acts as a flowering promoter in pepper which does not respond to vernalization and photoperiod.

Abstract

As part of our goal to study the genetic and molecular basis of transition to flowering in pepper, we isolated the late-flowering mutant E-2698. Aside from late flowering, multiple pleiotropic alterations of the shoot structure, such as enlarged and distorted leaves, weak apical dominance, and reduced angle of the lateral branches were observed, indicating a broad role for the mutated gene in pepper development. Genetic mapping and sequence analyses revealed that the disrupted gene in E-2698 is the pepper homolog of VERNALIZATION INSENSITIVE 3-LIKE 1 (VIL1) that acts as a regulator of vernalization in Arabidopsis through chromatin modification. The pepper gene, CaVIL1, contains a plant homeodomain motif associated with chromatin modification and a VERNALIZATION INSENSITIVE 3-interacting domain that is truncated in E-2698 and in two other allelic mutants. Because pepper flowering does not respond to vernalization, we postulate that CaVIL1 regulates flowering time via chromatin modification of unknown targets. Expression analysis indicated that CaVIL1 activates the flowering promoter CaFLOWERING LOCUS T and represses the flowering repressor CaAPETALA2. Furthermore, CaVIL1 represses several genes from the FLOWERING LOCUS C (FLC)-LIKE clade that are clustered together in the pepper genome. This indicates their possible involvement in flowering regulation in this species. Our results show that CaVIL1 is a major regulator of flowering and interacts with other flowering promoters and repressors, as well as with FLC-LIKE genes whose function in flowering regulation is not yet known in pepper.