Late-flowering Phenotype Research Articles

ORTH/VIM proteins that regulate DNA methylation are functional ubiquitin E3 ligases

Appropriate methylation of genomes is essential for gene regulation. Here, we describe the six-member ORTHRUS (ORTH) gene family of Arabidopsis thaliana that plays a role in DNA methylation in vivo. ORTH1- ORTH5 are predicted to encode proteins that contain one plant homeodomain (PHD), two really interesting new gene (RING) domains, and one set ring associated (SRA) domain, whereas ORTHlike-1 encodes a protein with only one RING and SRA domain. cDNAs for ORTH1, ORTH2, ORTH5 and ORTHlike-1 were isolated, and when expressed as glutathione-S-transferase (GST) fusion proteins, were capable of promoting ubiquitylation in vitro with the E2 AtUBC11. ORTH1 promotes ubiquitylation when paired with additional AtUBC8 family members. ORTH1 proteins with substitutions in metal-ligand binding residues in each ORTH1 RING domain individually, and ORTH1 truncation derivatives lacking one or both RING domains, were tested for their ability to catalyze ubiquitylation in vitro. In these assays, either ORTH1 RING domain is capable of promoting ubiquitylation. The PHD alone is not active as an E3 ligase, nor is it required for ligase activity. GFP-ORTH1 and GFP-ORTH2 are nuclear-localized in transgenic Arabidopsis plants. Overexpression of ORTH1 or ORTH2 in Arabidopsis leads to an altered flowering time. Inspection of DNA methylation at FWA and Cen180 repeats revealed hypomethylation when ORTH proteins were overexpressed. Once initiated, a late-flowering phenotype persisted in the absence of the ORTH transgene, consistent with epigenetic effects at FWA. We conclude that ORTH proteins are E3 ligases mediating DNA methylation status in vivo.

Circadian Clock Proteins LHY and CCA1 Regulate SVP Protein Accumulation to Control Flowering in Arabidopsis

The floral regulators GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) play key roles in the photoperiodic flowering responses of the long-day plant Arabidopsis thaliana. The GI-CO-FT pathway is highly conserved in plants. Here, we demonstrate that the circadian clock proteins LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) not only repressed the floral transition under short-day and long-day conditions but also accelerated flowering when the plants were grown under continuous light (LL). LHY and CCA1 accelerated flowering in LL by promoting FT expression through a genetic pathway that appears to be independent of the canonical photoperiodic pathway involving GI and CO proteins. A genetic screen revealed that the late-flowering phenotype of the lhy;cca1 double mutant under LL was suppressed through mutations in SHORT VEGETATIVE PHASE (SVP), a MADS box transcription factor. Yeast two-hybrid analysis demonstrated an interaction between SVP and FLOWERING LOCUS C, and genetic analysis indicated that these two proteins act as partially redundant repressors of flowering time. SVP protein accumulated in lhy;cca1 plants under LL. We propose a model in which LHY and CCA1 accelerate flowering in part by reducing the abundance of SVP and thereby antagonizing its capacity to repress FT expression under LL.

The small glycine‐rich RNA binding protein AtGRP7 promotes floral transition in Arabidopsis thaliana

The RNA binding protein AtGRP7 is part of a circadian slave oscillator in Arabidopsis thaliana that negatively autoregulates its own mRNA, and affects the levels of other transcripts. Here, we identify a novel role for AtGRP7 as a flowering-time gene. An atgrp7-1 T-DNA mutant flowers later than wild-type plants under both long and short days, and independent RNA interference lines with reduced levels of AtGRP7, and the closely related AtGRP8 protein, are also late flowering, particularly in short photoperiods. Consistent with the retention of a photoperiodic response, the transcript encoding the key photoperiodic regulator CONSTANS oscillates with a similar pattern in atgrp7-1 and wild-type plants. In both the RNAi lines and in the atgrp7-1 mutant transcript levels for the floral repressor FLC are elevated. Conversely, in transgenic plants ectopically overexpressing AtGRP7, the transition to flowering is accelerated mainly in short days, with a concomitant reduction in FLC abundance. The late-flowering phenotype of the RNAi lines is suppressed by introducing the flc-3 loss-of-function mutation, suggesting that AtGRP7 promotes floral transition, at least partly by downregulating FLC. Furthermore, vernalization overrides the late-flowering phenotype. Retention of both the photoperiodic response and vernalization response are features of autonomous pathway mutants, suggesting that AtGRP7 is a novel member of the autonomous pathway.

Cloning and characterization of a flowering time gene from <italic>Thellungiella halophila</italic>

<title>Abstract</title> <italic>Thellungiella halophila (T. halophila)</italic> (salt cress) is a close relative of <italic>Arabidopsis</italic> and a model plant for salt tolerance research. However, the nature of its later flowering causes some difficulties in genetic analysis. The <italic>FRIGIDA (FRT)</italic> gene plays a key role in the <italic>Arabidopsis</italic> vernalization flowering pathway, whose homolog in <italic>T. halophila</italic> may also be a key factor in controlling flowering time. In order to study the molecular mechanism of vernalization responses in <italic>T. halophila</italic>, a full length cDNA named <italic>ThFRI (Thellungiella halophila FRIGIDA)</italic> was isolated from the young seedlings of <italic>T. halophila</italic> by RT-PCR and RACE. The <italic>ThFRI cDNA</italic> was 2017 bp in length and contained an open reading frame encoding a putative protein of 605 ami no acids. The <italic>ThFRI</italic> showed significant homology to <italic>AtFRI</italic> (74.5% at the nucleotide level and 63.9% at the ami no acid level). To study its function, <italic>ThFRI</italic> cDNA was transformed into <italic>Arabidopsis thaliana</italic>, driven by CaMV 35S promoter. Transgenic plants expressing <italic>ThFRI exhibited</italic> late-flowering phenotype, which suggests that <italic>ThFRI</italic> is the funtional FRI homolog in <italic>T. halophila</italic>. The cloning and funtional characterization of the <italic>FRI</italic> homolog of <italic>T. halophila</italic> will faciliate further study of flowering time control in <italic>T. halophila</italic>. <b>FullText for HTML: </b><a target="_blank" class="col_4A90E2" href="https://doi.org/10.1093/abbs/40.8.747">https://doi.org/10.1093/abbs/40.8.747</a>

Cloning and characterization of a flowering time gene fromThellungiella halophila

Thellungiella halophila (T. halophila) (salt cress) is a close relative of Arabidopsis and a model plant for salt tolerance research. However, the nature of its later flowering causes some difficulties in genetic analysis. The FRIGIDA (FRI) gene plays a key role in the Arabidopsis vernalization flowering pathway, whose homolog in T. halophila may also be a key factor in controlling flowering time. In order to study the molecular mechanism of vernalization responses in T. halophila, a full length cDNA named ThFRI (Thellungiella halophila FRIGIDA) was isolated from the young seedlings of T. halophila by RT-PCR and RACE. The ThFRI cDNA was 2017 bp in length and contained an open reading frame encoding a putative protein of 605 amino acids. The ThFRI showed significant homology to AtFRI (74.5% at the nucleotide level and 63.9% at the amino acid level). To study its function, ThFRI cDNA was transformed into Arabidopsis thaliana, driven by CaMV 35S promoter. Transgenic plants expressing ThFRI exhibited late-flowering phenotype, which suggests that ThFRI is the funtional FRI homolog in T. halophila. The cloning and funtional characterization of the FRI homolog of T. halophila will faciliate further study of flowering time control in T. halophila.

Involvement of the MADS-Box GeneZMM4in Floral Induction and Inflorescence Development in Maize

AbstractThe switch from vegetative to reproductive growth is marked by the termination of vegetative development and the adoption of floral identity by the shoot apical meristem (SAM). This process is called the floral transition. To elucidate the molecular determinants involved in this process, we performed genome-wide RNA expression profiling on maize (Zea mays) shoot apices at vegetative and early reproductive stages using massively parallel signature sequencing technology. Profiling revealed significant up-regulation of two maize MADS-box (ZMM) genes, ZMM4 and ZMM15, after the floral transition. ZMM4 and ZMM15 map to duplicated regions on chromosomes 1 and 5 and are linked to neighboring MADS-box genes ZMM24 and ZMM31, respectively. This gene order is syntenic with the vernalization1 locus responsible for floral induction in winter wheat (Triticum monococcum) and similar loci in other cereals. Analyses of temporal and spatial expression patterns indicated that the duplicated pairs ZMM4-ZMM24 and ZMM15-ZMM31 are coordinately activated after the floral transition in early developing inflorescences. More detailed analyses revealed ZMM4 expression initiates in leaf primordia of vegetative shoot apices and later increases within elongating meristems acquiring inflorescence identity. Expression analysis in late flowering mutants positioned all four genes downstream of the floral activators indeterminate1 (id1) and delayed flowering1 (dlf1). Overexpression of ZMM4 leads to early flowering in transgenic maize and suppresses the late flowering phenotype of both the id1 and dlf1 mutations. Our results suggest ZMM4 may play roles in both floral induction and inflorescence development.

Sugar beet contains a large CONSTANS-LIKE gene family including a CO homologue that is independent of the early-bolting (B) gene locus

Floral transition in the obligate long-day (LD) plant sugar beet (Beta vulgaris ssp. vulgaris) is tightly linked to the B gene, a dominant early-bolting quantitative trait locus, the expression of which is positively regulated by LD photoperiod. Thus, photoperiod regulators like CONSTANS (CO) and CONSTANS-LIKE (COL) genes identified in many LD and short-day (SD)-responsive plants have long been considered constituents and/or candidates for the B gene. Until now, the photoperiod response pathway of sugar beet (a Caryophyllid), diverged from the Rosids and Asterids has not been identified. Here, evidence supporting the existence of a COL gene family is provided and the presence of Group I, II, and III COL genes in sugar beet, as characterized by different zinc-finger (B-box) and CCT (CO, CO-like, TOC) domains is demonstrated. BvCOL1 is identified as a close-homologue of Group 1a (AtCO, AtCOL1, AtCOL2) COL genes, hence a good candidate for flowering time control and it is shown that it maps to chromosome II but distant from the B gene locus. The late-flowering phenotype of A. thaliana co-2 mutants was rescued by over-expression of BvCOL1 thereby suggesting functional equivalence with AtCO, and it is shown that BvCOL1 interacts appropriately with the endogenous downstream genes, AtFT and AtSOC1 in the transgenic plants. Curiously, BvCOL1 has a dawn-phased diurnal pattern of transcription, mimicking that of AtCOL1 and AtCOL2 while contrasting with AtCO. Taken together, these data suggest that BvCOL1 plays an important role in the photoperiod response of sugar beet.

Functional Redundancy and New Roles for Genes of the Autonomous Floral-Promotion Pathway

The early-flowering habit of rapid-cycling accessions of Arabidopsis (Arabidopsis thaliana) is, in part, due to the genes of the autonomous floral-promotion pathway (AP). The AP promotes flowering by repressing expression of the floral inhibitor FLOWERING LOCUS C (FLC). AP mutants are therefore late flowering due to elevated levels of FLC, and this late-flowering phenotype is eliminated by loss-of-function mutations in FLC. To further investigate the role of the AP, we created a series of double mutants. In contrast to the phenotypes of single mutants, which are largely limited to delayed flowering, a subset of AP double mutants show a range of defects in growth and development. These phenotypes include reduced size, chlorophyll content, growth rate, and fertility. Unlike the effects of the AP on flowering time, these phenotypes are FLC independent. Recent work has also shown that two AP genes, FCA and FPA, are required for the repression and, in some cases, proper DNA methylation of two transposons. We show that similar effects are seen for all AP genes tested. Microarray analysis of gene expression in AP single and double mutants, however, suggests that the AP is not likely to play a broad role in the repression of gene expression through DNA methylation: very few of the genes that have been reported to be up-regulated in DNA methylation mutants are misexpressed in AP mutants. Together, these data indicate that the genes of the AP play important and sometimes functionally redundant roles in aspects of development in addition to flowering time.

The FLOWERING LOCUS T/TERMINAL FLOWER 1 Family in Lombardy Poplar

Genes in the FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1) family have been shown to be important in the control of the switch between vegetative and reproductive growth in several plant species. We isolated nine members of the FT/TFL1 family from Lombardy poplar (Populus nigra var. italica Koehne). Sequence analysis of the members of the FT/TFL1 family revealed considerable homology within their coding regions both among family members and to the members of the same family in Arabidopsis, tomato and grapevine. Moreover, members of this family in all four species examined display a common exon-intron organization. Phylogenetic analysis revealed that the genes fall into four different clades: two into the TFL1 clade; five into the FT clade; and one each into the MOTHER OF FT AND TFL1 and BROTHER OF FT AND TFL1 clades. One gene in the TFL1 clade, PnTFL1, is expressed in vegetative meristems, and transgenic Arabidopsis that ectopically expressed PnTFL1 had a late-flowering phenotype. The expression patterns of two genes in the FT clade, PnFT1 and PnFT2, suggested a role for them in the promotion of flowering, and transgenic Arabidopsis that ectopically expressed either PnFT1 or PnFT2 had an early-flowering phenotype.

Insight into Missing Genetic Links Between Two Evening-Expressed Pseudo-Response Regulator Genes TOC1 and PRR5 in the Circadian Clock-Controlled Circuitry in Arabidopsis thaliana

In Arabidopsis thaliana, many circadian clock-associated genes have been identified. Among them, the evening-expressed TOC1 (TIMING OF CAB EXPRESSION 1) gene plays a role by forming a transcriptional feedback core loop together with the morning-expressed CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) gene and its homologous LHY (LATE ELONGATED HYPOCOTYL) gene. TOC1 encodes a member of the PSEUDO-RESPONSE REGULATOR (PRR) family, including PRR9, PRR7, PRR5, PRR3,and PRR1/TOC1. The PRR genes other than TOC1 (or PRR1) also appear to be crucial for certain circadian-associated events. To clarify missing genetic linkages amongst these PRR genes, here we constructed a toc1 prr5 double knockdown mutant. In free-running circadian rhythms, the resulting toc1-2 prr5-11 mutant plants showed an extremely short period and reduced amplitude phenotype, which was more severe than that of the toc1-2 single mutant plant, suggesting a non-linear genetic interaction between TOC1 and PRR5. Surprisingly, the hallmark early flowering phenotype of toc1-2 in the short-day conditions had been converted to a markedly late flowering phenotype in the long-day conditions, when combined with the prr5-11 allele, which itself showed a subtle flowering phenotype. This unexpected genetic result (i.e. phenotypic sign conversion) suggested that the TOC1 and PRR5 genes are coordinately implicated in a non-linear and closed genetic circuitry. In the toc1-2 prr5-11 double mutant, the diurnal expression profile of CDF1 (CYCLING DOF FACTOR 1) was markedly de-repressed in the evening in the long-day conditions. These and other results of this study led us to propose the novel view that TOC1 might play bipartite roles in the control of flowering time within a closed circuitry; the one is a GI (GIGANTEA)-dependent negative role through CCA1/LHY, and the other is a CDF1-dependent positive role through cooperating closely with PRR5.

Di- and Tri- but Not Monomethylation on Histone H3 Lysine 36 Marks Active Transcription of Genes Involved in Flowering Time Regulation and Other Processes in Arabidopsis thaliana

Histone lysines can be mono-, di-, or trimethylated, providing an ample magnitude of epigenetic information for transcription regulation. In fungi, SET2 is the sole methyltransferase responsible for mono-, di-, and trimethylation of H3K36. Here we show that in Arabidopsis thaliana, the degree of H3K36 methylation is regulated by distinct methyltransferases. The SET2 homologs SDG8 and SDG26 each can methylate oligonucleosomes in vitro, and both proteins are localized in the nucleus. While the previously reported loss-of-function sdg8 mutants have an early-flowering phenotype, the loss-of-function sdg26 mutants show a late-flowering phenotype. Consistently, several MADS-box flowering repressors are down-regulated by sdg8 but up-regulated by sdg26. The sdg8 but not the sdg26 mutant plants show a dramatically reduced level of both di- and trimethyl-H3K36 and an increased level of monomethyl-H3K36. SDG8 is thus specifically required for di- and trimethylation of H3K36. Our results further establish that H3K36 di- and tri- but not monomethylation correlates with transcription activation. Finally, we show that SDG8 and VIP4, which encodes a component of the PAF1 complex, act independently and synergistically in transcription regulation. Together our results reveal that the deposition of H3K36 methylation is finely regulated, possibly to cope with the complex regulation of growth and development in higher eukaryotes.

Mutations in theArabidopsis SWC6gene, encoding a component of the SWR1 chromatin remodelling complex, accelerate flowering time and alter leaf and flower development

Mutations affecting the Arabidopsis SWC6 gene encoding a putative orthologue of a component of the SWR1 chromatin remodelling complex in plants have been characterized. swc6 mutations cause early flowering, shortened inflorescence internodes, and altered leaf and flower development. These phenotypic defects resemble those of the photoperiod independent early flowering 1 (pie1) and early in short days 1 (esd1) mutants, also affected in homologues of the SWR1 complex subunits. SWC6 is a ubiquitously expressed nuclear HIT-Zn finger-containing protein, with the highest levels found in pollen. Double mutant analyses suggest that swc6 abolishes the FLC-mediated late-flowering phenotype of plants carrying active alleles of FRI and of mutants of the autonomous pathway. It was found that SWC6 is required for the expression of the FLC repressor to levels that inhibit flowering. However, the effect of swc6 in an flc null background and the down-regulation of other FLC-like/MAF genes in swc6 mutants suggest that flowering inhibition mediated by SWC6 occurs through both FLC- and FLC-like gene-dependent pathways. Both genetic and physical interactions between SWC6 and ESD1 have been demonstrated, suggesting that both proteins act in the same complex. Using chromatin immunoprecipitation, it has been determined that SWC6, as previously shown for ESD1, is required for both histone H3 acetylation and H3K4 trimethylation of the FLC chromatin. Altogether, these results suggest that SWC6 and ESD1 are part of an Arabidopsis SWR1 chromatin remodelling complex involved in the regulation of diverse aspects of plant development, including floral repression through the activation of FLC and FLC-like genes.

AnAGAMOUS-Related MADS-Box Gene,XAL1(AGL12), Regulates Root Meristem Cell Proliferation and Flowering Transition in Arabidopsis

MADS-box genes are key components of the networks that control the transition to flowering and flower development, but their role in vegetative development is poorly understood. This article shows that the sister gene of the AGAMOUS (AG) clade, AGL12, has an important role in root development as well as in flowering transition. We isolated three mutant alleles for AGL12, which is renamed here as XAANTAL1 (XAL1): Two alleles, xal1-1 and xal1-2, are in Columbia ecotype and xal1-3 is in Landsberg erecta ecotype. All alleles have a short-root phenotype with a smaller meristem, lower rate of cell production, and abnormal root apical meristem organization. Interestingly, we also encountered a significantly longer cell cycle in the strongest xal1 alleles with respect to wild-type plants. Expression analyses confirmed the presence of XAL1 transcripts in roots, particularly in the phloem. Moreover, XAL1beta-glucuronidase expression was specifically up-regulated by auxins in this tissue. In addition, mRNA in situ hybridization showed that XAL1 transcripts were also found in leaves and floral meristems of wild-type plants. This expression correlates with the late-flowering phenotypes of the xal1 mutants grown under long days. Transcript expression analysis suggests that XAL1 is an upstream regulator of SOC, FLOWERING LOCUS T, and LFY. We propose that XAL1 may have similar roles in both root and aerial meristems that could explain the xal1 late-flowering phenotype.

Functional characterization of AP3, SOC1 and WUS homologues from citrus (Citrus sinensis)

Flowering and flower formation are defining features of angiosperms and the control of these developmental processes involves a common repertoire of genes which are shared among different species of flowering plants. These genes were first identified using various homeotic and flowering time mutants of Arabidopsis and snapdragon, and homologous genes have subsequently been isolated from a wide range of different plant species based on the conservation of protein sequence and function. Using degenerate reverse-transcriptase polymerase chain reaction, we have isolated one APETALA3-like (CitMADS8) and two SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1)-like (CsSL1 and CsSL2) homologues from sweet orange (Citrus sinensis L.). Although the translated amino acid sequence of CitMADS8 shares many similarities with other higher plant APETALA3 proteins, CitMADS8 fails to complement the floral organ identity defects of the Arabidopsis ap3-3 mutant. By contrast, the two citrus SOC1-like genes, particularly CsSL1, are able to shorten the time taken to flower in the Arabidopsis wild-type ecotypes Columbia and C24, and functionally complement the late flowering phenotype of the soc1 mutant, essentially performing the endogenous function of Arabidopsis SOC1. Once flowering has commenced, interactions between specific flowering genes and a gene required for meristem maintenance, WUSCHEL, ensure that the Arabidopsis flower is a determinate structure with four whorls. We have isolated a citrus WUSCHEL homologue (CsWUS) that is capable of restoring most of the meristem function to the shoots and flowers of the Arabidopsis wus-1 mutant, implying that CsWUS is the functional equivalent of Arabidopsis WUSCHEL.

Overexpression of TaVRN1 in Arabidopsis Promotes Early Flowering and Alters Development

TaVRN1, a member of the APETALA1 (AP1) subfamily of MADS-box transcription factors, is a key gene that controls transition from vegetative to reproductive phase in wheat. The accumulation of TaVRN1 transcripts in winter wheat probably requires the down-regulation of TaVRT2, a MADS-box factor that binds and represses the TaVRN1 promoter, and of the flowering repressor TaVRN2. However, the molecular mechanisms by which TaVRN1 functions as an activator of phase transition is unknown. To address this, a combination of gene expression and functional studies was used. RNA in situ hybridization studies showed that TaVRN1 transcripts accumulate in all meristems and primordia associated with flower development. An interaction screen in yeast revealed that TaVRN1 interacts with several proteins involved in different processes of plant development such as transcription factors, kinases and a cyclophilin. Arabidopsis plants overexpressing TaVRN1 flower early and show various levels of modified plant architecture. The ectopic expression causes an overexpression of the AP1 and MAX4 genes, which are associated with flowering and auxin regulation, respectively. The induction of gene expression probably results from the binding of TaVRN1 to CArG motifs present on the AP1 and MAX4 promoters. In contrast, Arabidopsis plants that overexpress TaVRT2, which encodes a putative flowering repressor, show an opposite late flowering phenotype. Together, the data provide molecular evidence that TaVRN1 may have pleiotropic effects in various processes such as control of axillary bud growth, transition to flowering and development of floral organs.

Control of Flowering Time and Cold Response by a NAC-Domain Protein in Arabidopsis

BackgroundPlants must integrate complex signals from environmental and endogenous cues to fine-tune the timing of flowering. Low temperature is one of the most common environmental stresses that affect flowering time; however, molecular mechanisms underlying the cold temperature regulation of flowering time are not fully understood.Methodology/Principal FindingsWe report the identification of a novel regulator, LONG VEGETATIVE PHASE 1 (LOV1), that controls flowering time and cold response. An Arabidopsis mutant, long v egetative phase 1-1D (lov1-1D) showing the late-flowering phenotype, was isolated by activation tagging screening. Subsequent analyses demonstrated that the phenotype of the mutant resulted from the overexpression of a NAC-domain protein gene (At2g02450). Both gain- and loss-of-function alleles of LOV1 affected flowering time predominantly under long-day but not short-day conditions, suggesting that LOV1 may act within the photoperiod pathway. The expression of CONSTANS (CO), a floral promoter, was affected by LOV1 level, suggesting that LOV1 controls flowering time by negatively regulating CO expression. The epistatic relationship between CO and LOV1 was consistent with this proposed regulatory pathway. Physiological analyses to elucidate upstream signalling pathways revealed that LOV1 regulates the cold response in plants. Loss of LOV1 function resulted in hypersensitivity to cold temperature, whereas a gain-of-function allele conferred cold tolerance. The freezing tolerance was accompanied by upregulation of cold response genes, COLD-REGULATED 15A (COR15A) and COLD INDUCED 1 (KIN1) without affecting expression of the C-repeat-binding factor/dehydration responsive element-binding factor 1 (CBF/DREB1) family of genes.ConclusionsOur study shows that LOV1 functions as a floral repressor that negatively regulates CO expression under long-day conditions and acts as a common regulator of two intersecting pathways that regulate flowering time and the cold response, respectively. Our results suggest an overlapping pathway for controlling cold stress response and flowering time in plants.

Regulation of Plant Developmental Processes by a Novel Splicing Factor

Serine/arginine-rich (SR) proteins play important roles in constitutive and alternative splicing and other aspects of mRNA metabolism. We have previously isolated a unique plant SR protein (SR45) with atypical domain organization. However, the biological and molecular functions of this novel SR protein are not known. Here, we report biological and molecular functions of this protein. Using an in vitro splicing complementation assay, we showed that SR45 functions as an essential splicing factor. Furthermore, the alternative splicing pattern of transcripts of several other SR genes was altered in a mutant, sr45-1, suggesting that the observed phenotypic abnormalities in sr45-1 are likely due to altered levels of SR protein isoforms, which in turn modulate splicing of other pre-mRNAs. sr45-1 exhibited developmental abnormalities, including delayed flowering, narrow leaves and altered number of petals and stamens. The late flowering phenotype was observed under both long days and short days and was rescued by vernalization. FLC, a key flowering repressor, is up-regulated in sr45-1 demonstrating that SR45 influences the autonomous flowering pathway. Changes in the alternative splicing of SR genes and the phenotypic defects in the mutant were rescued by SR45 cDNA, further confirming that the observed defects in the mutant are due to the lack of SR45. These results indicate that SR45 is a novel plant-specific splicing factor that plays a crucial role in regulating developmental processes.

Pea LATE BLOOMER1 is a GIGANTEA ortholog with roles in photoperiodic flowering, deetiolation, and transcriptional regulation of circadian clock gene homologs.

Genes controlling the transition to flowering have been studied in several species, including Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), but have not yet received much attention in legumes. Here, we describe a new allelic series of late-flowering, photoperiod-insensitive mutants in the pea (Pisum sativum) LATE BLOOMER1 (LATE1) gene and show that LATE1 is an ortholog of Arabidopsis GIGANTEA. Mutants display defects in phytochrome B-dependent deetiolation under red light and in the diurnal regulation of pea homologs of several Arabidopsis circadian clock genes, including TIMING OF CAB1, EARLY FLOWERING4, and CIRCADIAN CLOCK ASSOCIATED1/LATE ELONGATED HYPOCOTYL. LATE1 itself shows strongly rhythmic expression with a small but distinct acute peak following dark-to-light transfer. Mutations in LATE1 prevent the induction of a FLOWERING LOCUS T (FT) homolog FTL in long days but cause only minor alteration to the rhythmic expression pattern of the only known group Ia CONSTANS homolog COLa. The late-flowering phenotype of late1 mutants can be completely rescued by grafting to the wild type, but this rescue is not associated with a significant increase in FTL transcript level in shoot apices. Genetic interactions of late1 with the photoperiod-insensitive, early-flowering sterile nodes (sn) mutant and impairment of the LATE1 diurnal expression rhythm in sn plants suggest that SN may also affect the circadian clock. These results show that several functions of Arabidopsis GIGANTEA are conserved in its pea ortholog and demonstrate that genetic pathways for photoperiodic flowering are likely to be conserved between these two species. They also suggest that in addition to its role in the floral transition, LATE1 also acts throughout reproductive development.

SKB1-mediated symmetric dimethylation of histone H4R3 controls flowering time in Arabidopsis

Plant flowering is a crucial developmental transition from the vegetative to reproductive phase and is properly timed by a number of intrinsic and environmental cues. Genetic studies have identified that chromatin modification influences the expression of FLOWERING LOCUS C (FLC), a MADS-box transcription factor that controls flowering time. Histone deacetylation and methylation at H3K9 and H3K27 are associated with repression of FLC; in contrast, methylation at H3K4 and H3K36 activates FLC expression. However, little is known about the functions of histone arginine methylation in plants. Here, we report that Arabidopsis Shk1 binding protein 1 (SKB1) catalyzes histone H4R3 symmetric dimethylation (H4R3sme2). SKB1 lesion results in upregulation of FLC and late flowering under both long and short days, but late flowering is reversed by vernalization and gibberellin treatments. An skb1-1flc-3 double mutant blocks late-flowering phenotype, which suggests that SKB1 promotes flowering by suppressing FLC transcription. SKB1 binds to the FLC promoter, and disruption of SKB1 results in reduced H4R3sme2, especially in the promoter of FLC chromatin. Thus, SKB1-mediated H4R3sme2 is a novel histone mark required for repression of FLC expression and flowering time control.

Involvement of the Histone Acetyltransferase AtHAC1 in the Regulation of Flowering Time via Repression of FLOWERING LOCUS C in Arabidopsis

Histone acetylation is an important posttranslational modification correlated with gene activation. In Arabidopsis (Arabidopsis thaliana), the histone acetyltransferase AtHAC1 is homologous to animal p300/CREB (cAMP-responsive element-binding protein)-binding proteins, which are the main histone acetyltransferases participating in many physiological processes, including proliferation, differentiation, and apoptosis. The functions of p300/CREB-binding proteins in animals are well characterized, whereas little is known about the roles of AtHAC1 in developmental control in Arabidopsis. Lesions in AtHAC1 caused pleiotropic developmental defects, including delayed flowering, a shortened primary root, and partially reduced fertility. Analysis of the molecular basis of late flowering in hac1 mutants showed that the hac1 plants respond normally to day length, gibberellic acid treatment, and vernalization. Furthermore, the expression level of the flowering repressor FLOWERING LOCUS C (FLC) is increased in hac1 mutants, indicating that the late-flowering phenotype of hac1 mutants is mediated by FLC. Since histone acetylation is usually associated with the activation of gene expression, histone modifications of FLC chromatin are not affected by mutations in HAC1 and expression levels of all known autonomous pathway genes are unchanged in hac1 plants, we propose that HAC1 affects flowering time by epigenetic modification of factors upstream of FLC.