Modulation of Ambient Temperature-Dependent Flowering in Arabidopsis thaliana by Natural Variation of FLOWERING LOCUS M

Plants integrate seasonal cues such as temperature and day length to optimally adjust their flowering time to the environment. Compared to the control of flowering before and after winter by the vernalization and day length pathways, mechanisms that delay or promote flowering during a transient cool or warm period, especially during spring, are less well understood. Due to global warming, understanding this ambient temperature pathway has gained increasing importance. In Arabidopsis thaliana, FLOWERING LOCUS M (FLM) is a critical flowering regulator of the ambient temperature pathway. FLM is alternatively spliced in a temperature-dependent manner and the two predominant splice variants, FLM-ß and FLM-δ, can repress and activate flowering in the genetic background of the A. thaliana reference accession Columbia-0. The relevance of this regulatory mechanism for the environmental adaptation across the entire range of the species is, however, unknown. Here, we identify insertion polymorphisms in the first intron of FLM as causative for accelerated flowering in many natural A. thaliana accessions, especially in cool (15°C) temperatures. We present evidence for a potential adaptive role of this structural variation and link it specifically to changes in the abundance of FLM-ß. Our results may allow predicting flowering in response to ambient temperatures in the Brassicaceae.     

Genetic interactions between FLM and other flowering-time genes in Arabidopsis thaliana

FLOWERING LOCUS M (FLM) is a MADS-domain gene that acts as an inhibitor of flowering in Arabidopsis. Here we describe the genetic interaction of FLM with genes in the photoperiod and autonomous flowering pathways. Although the sequence of FLM is most similar to that of FLC, FLM and FLC interact with different flowering pathways. It has been previously shown that flc lesions suppress the late-flowering phenotype of FRI-containing lines and autonomous-pathway mutants. However, flm lesions suppress the late-flowering phenotype of photoperiod-pathway mutants but not that of FRI-containing lines or autonomous-pathway mutants. Another MADS-domain flowering repressor with a mutant phenotype similar to FLM is SVP. The late-flowering phenotype of FLM over-expression is suppressed by the svp mutation, and an svp flm double mutant behaves like the single mutants. Thus FLM and SVP are in the same flowering pathway which interacts with the photoperiod pathway. Abbreviations: CO, CONSTANS; FLC, FLOWERING LOCUS C; FLM, FLOWERING LOCUS M; FRI, FRIGIDA; GI, GIGANTEA; LD, LUMINIDEPENDENS; SVP, SHORT VEGETATIVE PHASE; FCA is not an abbreviation     

Identification of a MADS-box gene, FLOWERING LOCUS M, that represses flowering

The timing of flowering is important for the reproductive success of plants. Here we describe the identification and characterization of a new MADS-box gene, FLOWERING LOCUS M (FLM), which is involved in the transition from vegetative to reproductive development. FLM is similar in amino-acid sequence to FLC, another MADS-box gene involved in flowering-time control. flm mutants are early flowering in both inductive and non-inductive photoperiods, and flowering time is sensitive to FLM dosage. FLM overexpression produces late-flowering plants. Thus FLM acts as an inhibitor of flowering. FLM is expressed in areas of cell division such as root and shoot apical regions and leaf primordia.     

The FLX gene of Arabidopsis is required for FRI-dependent activation of FLC expression

The Arabidopsis FLOWERING LOCUS C (FLC) gene encodes a MADS box protein that acts as a dose-dependent repressor of flowering. Mutants and ecotypes with elevated expression of FLC are late flowering and vernalization responsive. In this study we describe an early flowering mutant in the C24 ecotype, flc expressor (flx), that has reduced expression of FLC. FLX encodes a protein of unknown function with putative leucine zipper domains. FLX is required for FRIGIDA (FRI)-mediated activation of FLC but not for activation of FLC in autonomous pathway mutants. FLX is also required for expression of the FLC paralogs MADS AFFECTING FLOWERING 1 (MAF1) and MAF2.     

Florigen trafficking integrates photoperiod and temperature signals in Arabidopsis

The transition to flowering is the most dramatic phase change in flowering plants and is crucial for reproductive success. A complex regulatory network in plants has evolved to perceive and integrate the endogenous and environmental signals. These signals perceived, including day length and temperature, converge to regulate FLOWERING LOCUS T (FT), which encodes a mobile stimulus required for floral induction in Arabidopsis. Despite the discovery of modulation of FT messenger RNA (mRNA) expression by ambient temperature, whether the trafficking of FT protein is controlled in response to changes in growth temperature is so far unknown. Here, we show that FT transport from companion cells to sieve elements is controlled in a temperature‐dependent manner. This process is mediated by multiple C2 domain and transmembrane region proteins (MCTPs) and a soluble N‐ethylmaleimide‐sensitive factor protein attachment protein receptor (SNARE). Our findings suggest that ambient temperatures regulate both FT mRNA expression and FT protein trafficking to prevent precocious flowering at low temperatures and ensure plant reproductive success under favorable environmental conditions.     

Requirement of histone acetyltransferases HAM1 and HAM2 for epigenetic modification of FLC in regulating flowering in Arabidopsis

Histone acetylation is an important posttranslational modification associated with gene activation. In Arabidopsis, two MYST histone acetyltransferases HAM1 and HAM2 work redundantly to acetylate histone H4 lysine 5 (H4K5ace) in vitro. The double mutant ham1/ham2 is lethal, which suggests the critical role of HAM1 and HAM2 in development. Here, we used an artificial microRNA (amiRNA) strategy in Arabidopsis to uncover a novel function of HAM1 and HAM2. The amiRNA-HAM1/2 transgenic plants showed early flowering and reduced fertility. In addition, they responded normally to photoperiod, gibberellic acid treatment, and vernalization. The expression of flowering-repressor FLOWERING LOCUS C (FLC) and its homologues, MADS-box Affecting Flowering genes 3/4 (MAF3/4), were decreased in amiRNA-HAM1/2 lines. HAM1 overexpression caused late flowering and elevated expression of FLC and MAF3/4. Mutation of FLC almost rescued the late flowering with HAM1 overexpression, which suggests that HAM1 regulation of flowering time depended on FLC. Global H4 acetylation was decreased in amiRNA-HAM1/2 lines, but increased in HAM1-OE lines, which further confirmed the acetyltransferase activity of HAM1 in vivo. Chromatin immunoprecipitation revealed that H4 hyperacetylation and H4K5ace at FLC and MAF3/4 were less abundant in amiRNA-HAM1/2 lines than the wild type, but were enriched in HAM1-OE lines. Thus, HAM1 and HAM2 may affect flowering time by epigenetic modification of FLC and MAF3/4 chromatins at H4K5 acetylation.     

Role of SEPALLATA3 (SEP3) as a downstream gene of miR156-SPL3-FT circuitry in ambient temperature-responsive flowering

SEPALLATA3 (SEP3) is important in determining flowering time as well as floral organ identity. Although much is known about the regulation of floral organ identity by SEP3, its role as a downstream gene of FLOWERING LOCUS T (FT) for the regulation of ambient temperature-responsive flowering is poorly understood. Here, we show that SEP3 as a downstream gene of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) and FT modulates the flowering time in response to different ambient temperatures. SEP3 overexpression showed temperature-insensitive flowering at 23°C and 16°C. This suggests that altered SEP3 activity affects ambient temperature-responsive flowering. However, a lesion in SEP3 did not obviously affect ambient temperature-responsive flowering. SEP3 expression was affected by altered SPL3 and FT activities in the leaf and shoot apical regions at different temperatures. These results suggest that the miR156-SPL3-FT circuitry directly or indirectly regulates SEP3 expression for the regulation of ambient temperature-responsive flowering in Arabidopsis.     

A splicing site mutation in <Emphasis Type="Italic">BrpFLC1</Emphasis> and repressed expression of <Emphasis Type="Italic">BrpFLC</Emphasis> genes are associated with the early flowering of purple flowering stalk

FLOWERING LOCUS C (FLC), which encodes a MADS-box domain protein, is a flowering repressor involved in the key position of Arabidopsis (Arabidopsis thaliana) flowering network. In Brassica species, several FLC homologues are involved in flowering time like Arabidopsis FLC. Here, we report the analysis of splicing variation in BrpFLC1 and the expression of BrpFLC homologues associated with early flowering of Purple Flowering Stalk (Brassica campestris L. ssp. chinensis L. var. purpurea Bailey). It was indicated that a splice site mutation happened in intron 6 with G to A at the 5′ splice site. Three alternative splicing patterns of BrpFLC1, including the entire exon 6 excluded and 24 bp or 87 bp of intron 6 retained, were identified in Purple Flowering Stalk. But there was only one normal splicing pattern in Pakchoi (Brassica campestris ssp. chinensis var. communis). Northern blotting and semi-quantitative RT-PCR revealed that the expression levels of the three FLC homologues in Purple Flowering Stalk were lower than that in Pakchoi. However, the expression levels of downstream genes, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FLOWERING LOCUS T (FT), were higher in Purple Flowering Stalk. These results suggest that a natural splicing site mutation in BrpFLC1 gene and repressed expression of all BrpFLC genes contribute significantly to flowering time variation in Purple Flowering Stalk.     

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 MADS-Domain Factors AGAMOUS-LIKE15 and AGAMOUS-LIKE18, along with SHORT VEGETATIVE PHASE and AGAMOUS-LIKE24, Are Necessary to Block Floral Gene Expression during the Vegetative Phase

Multiple factors, including the MADS-domain proteins AGAMOUS-LIKE15 (AGL15) and AGL18, contribute to the regulation of the transition from vegetative to reproductive growth. AGL15 and AGL18 were previously shown to act redundantly as floral repressors and upstream of FLOWERING LOCUS T (FT) in Arabidopsis (Arabidopsis thaliana). A series of genetic and molecular experiments, primarily focused on AGL15, was performed to more clearly define their role. agl15 agl18 mutations fail to suppress ft mutations but show additive interactions with short vegetative phase (svp) mutations in ft and suppressor of constans1 (soc1) backgrounds. Chromatin immunoprecipitation analyses with AGL15-specific antibodies indicate that AGL15 binds directly to the FT locus at sites that partially overlap those bound by SVP and FLOWERING LOCUS C. In addition, expression of AGL15 in the phloem effectively restores wild-type flowering times in agl15 agl18 mutants. When agl15 agl18 mutations are combined with agl24 svp mutations, the plants show upward curling of rosette and cauline leaves, in addition to early flowering. The change in leaf morphology is associated with elevated levels of FT and ectopic expression of SEPALLATA3 (SEP3), leading to ectopic expression of floral genes. Leaf curling is suppressed by sep3 and ft mutations and enhanced by soc1 mutations. Thus, AGL15 and AGL18, along with SVP and AGL24, are necessary to block initiation of floral programs in vegetative organs.Appropriate timing of the shift from vegetative to reproductive growth is an important determinant of plant fitness. The time at which a plant flowers is determined through integration of signals reflecting extrinsic and intrinsic conditions, such as photoperiod, the duration of cold, plant health, and age (for review, see Amasino, 2010). One of the most important pathways regulating the timing of the floral transition is the photoperiod pathway (for review, see Imaizumi and Kay, 2006). Under long-day (LD) inductive conditions in Arabidopsis (Arabidopsis thaliana), photoperiod pathway components act to promote flowering by inducing CONSTANS (CO) and downstream genes. The floral integrator FLOWERING LOCUS T (FT) is a major target of multiple flowering pathways and the photoperiod pathway in particular. It is directly activated by CO (Samach et al., 2000). Under LD conditions, the peak of CO expression is coincident with the presence of light, and CO activates FT expression in the leaf vascular system (Yanovsky and Kay, 2003). FT travels through the phloem to the shoot apex (Corbesier et al., 2007), where, together with FLOWERING LOCUS D (Abe et al., 2005; Wigge et al., 2005), it activates APETALA1 (AP1) and other floral meristem identity genes, starting the flowering process. Other flowering time pathways converge on FT and/or directly impact gene expression in the meristem. The changes in gene expression that accompany the floral transition must be rapid, robust, largely irreversible, and strictly controlled spatially. This is achieved through positive feed-forward and negative feedback loops involving multiple regulatory factors (for recent review, see Kaufmann et al., 2010).Members of the MADS-box family of regulatory factors are central players in the regulatory loops controlling the floral transition (for a recent review, see Smaczniak et al., 2012a). MADS-domain factors typically act in large multimeric complexes and are well suited for regulation that involves combinatorial action. During the floral transition, MADS-domain proteins can act either as repressors or activators. In Arabidopsis, important floral repressors include SHORT VEGETATIVE PHASE (SVP) and members of the FLOWERING LOCUS C (FLC)-like group, including FLC, FLOWERING LOCUS M (FLM)/MADS AFFECTING FLOWERING1 (MAF1), and MAF2 to MAF5. Promoters of flowering include such MADS-domain factors as SUPPRESSOR OF CONSTANS1 (SOC1) and AGAMOUS-LIKE24 (AGL24). Together with non-MADS-box proteins FT and TWIN SISTER OF FT, SOC1 and AGL24 function as floral integrators. These operate downstream of the flowering time pathways but upstream of the meristem identity regulators such as LEAFY (LFY) and the MADS-domain factor AP1.The MADS-domain factors AGL15 and AGL18 also contribute to regulation of the floral transition in Arabidopsis. While single mutants have no phenotype, agl15 agl18 double mutants flower earlier than the wild type (Adamczyk et al., 2007). Therefore, AGL15 and AGL18 appear to act in a redundant fashion in seedlings, and like SVP, FLC, and MAF1 to MAF5, they act as floral repressors. The contributions of AGL15 and AGL18 are most apparent in the absence of strong photoperiodic induction: the agl15 agl18 double mutant combination partially suppresses the delay in flowering observed in co mutants, as well as the flowering delay associated with growth under short-day (SD) noninductive conditions. The earlier flowering in agl15 agl18 mutants under these conditions is associated with up-regulation of FT, and both AGL15 and AGL18 are expressed in the vascular system and shoot apex of young seedlings (Adamczyk et al., 2007), raising the possibility that AGL15 and AGL18 act directly on FT in leaves, as well as other targets in the meristem.AGL15, and to a lesser extent AGL18, have been further implicated in the networks that control flowering through molecular studies. Zheng et al. (2009) performed a chromatin immunoprecipitation (ChIP) analysis using AGL15-specific antibodies, tissue derived from embryo cultures, and a tiling array. Floral repressors (SVP and FLC), floral integrators (FT and SOC1), and a microRNA targeting AP2-like factors (miR172a) were identified as possible AGL15 targets (Zheng et al., 2009), suggesting that AGL15 may contribute to regulation through multiple avenues during the floral transition. AGL15 itself is directly bound and activated by AP2, which is both an A-class floral identity gene and a floral repressor (Yant et al., 2010). AGL15 is down-regulated in ap2 mutants, which are early flowering, while AGL18 is the nearest locus to multiple AP2-bound sites (Yant et al., 2010). Both AGL15 and AGL18 were identified as SOC1 targets through ChIP analyses (Immink et al., 2009; Tao et al., 2012). In yeast (Saccharomyces cerevisiae) two-hybrid assays, AGL15 interacts with a number of other MADS-domain proteins (de Folter et al., 2005), and in a one-hybrid study based on the SOC1 promoter, AGL15-SVP, AGL15-AGL24, and AGL15-SOC1 heterodimers were shown to bind to regions containing CArG boxes (Immink et al., 2012). AGL18 may act redundantly to AGL15 in these contexts. However, AGL18 either does not interact or only interacts weakly with other proteins in yeast two-hybrid assays (de Folter et al., 2005; Hill et al., 2008; Causier et al., 2012). It remains to be determined whether this truly reflects weaker or nonredundant in planta interactions or a technical problem in the artificial yeast system.Guided by the knowledge gained about AGL15 targets and interactions from molecular studies, we asked the following question: what is the functional significance of these molecular relationships in the context of the floral transition? We performed a series of genetic experiments combining agl15 agl18 mutations and mutations in interacting factors such as SVP, AGL24, and SOC1, as well as targets such as FT and SOC1. We also performed further molecular experiments focused on AGL15, for which a variety of tools are available. Among other things, we show that AGL15 and AGL18, along with AGL24 and SVP, play a role in blocking expression of the floral MADS-domain factor SEPALLATA3 (SEP3) during the vegetative phase. In the absence of these four factors, reproductive programs are initiated early, and floral genes are expressed in the youngest rosette leaf and cauline leaves.