Regulation Of Flowering Time Research Articles

Changing Responses to Changing Seasons: Natural Variation in the Plasticity of Flowering Time.

The mechanisms by which the environment regulates flowering time have evolved as crops and wild populations have adapted to diverse climates, and the specific variants involved are increasingly known.

An ortholog of LEAFY in Jatropha curcas regulates flowering time and floral organ development.

Jatropha curcas seeds are an excellent biofuel feedstock, but seed yields of Jatropha are limited by its poor flowering and fruiting ability. Thus, identifying genes controlling flowering is critical for genetic improvement of seed yield. We isolated the JcLFY, a Jatropha ortholog of Arabidopsis thaliana LEAFY (LFY), and identified JcLFY function by overexpressing it in Arabidopsis and Jatropha. JcLFY is expressed in Jatropha inflorescence buds, flower buds, and carpels, with highest expression in the early developmental stage of flower buds. JcLFY overexpression induced early flowering, solitary flowers, and terminal flowers in Arabidopsis, and also rescued the delayed flowering phenotype of lfy-15, a LFY loss-of-function Arabidopsis mutant. Microarray and qPCR analysis revealed several flower identity and flower organ development genes were upregulated in JcLFY-overexpressing Arabidopsis. JcLFY overexpression in Jatropha also induced early flowering. Significant changes in inflorescence structure, floral organs, and fruit shape occurred in JcLFY co-suppressed plants in which expression of several flower identity and floral organ development genes were changed. This suggests JcLFY is involved in regulating flower identity, floral organ patterns, and fruit shape, although JcLFY function in Jatropha floral meristem determination is not as strong as that of Arabidopsis.

Leaf-Like Sepals Induced by Ectopic Expression of a SHORT VEGETATIVE PHASE (SVP)-Like MADS-Box Gene from the Basal Eudicot Epimedium sagittatum.

Epimedium L. (Berberidaceae, Ranales), a perennial traditional Chinese medicinal herb, has become a new popular landscape plant for ground cover and pot culture in many countries based on its excellent ornamental characteristics and, distinctive and diverse floral morphology. However, little is known about the molecular genetics of flower development in Epimedium sagittatum. Here, we describe the characterization of EsSVP that encodes a protein sharing 68, 54, and 35% similarity with SVP, AGAMOUS-like 24 (AGL24) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) in Arabidopsis, respectively. Quantitative RT-PCR (qRT-PCR) indicated that EsSVP transcripts were principally found in petiole and leaf tissues, with little expression in roots and flowers and no in fruits. The highest EsSVP expression was observed in leaves. The flowering time of 35S::EsSVP in most Arabidopsis thaliana and in all petunia plants was not affected in both photoperiod conditions, but 35S::EsSVP 5# and 35S::EsSVP 1# Arabidopsis lines induced late and early flowering under long day (LD, 14 h light/10 h dark) and short day (SD, 10 h light/14 h dark) conditions, respectively. The 35S::EsSVP Arabidopsis produced extra secondary inflorescence or floral meristems in the axils of the leaf-like sepals with excrescent trichomes, and leaf-like sepals not able to enclose the inner three whorls completely. Moreover, almost all transgenic Arabidopsis plants showed persistent sepals around the completely matured fruits. Upon ectopic expression of 35S::EsSVP in Petunia W115, sepals were enlarged, sometimes to the size of leaves; corollas were greenish and did not fully open. These results suggest that EsSVP is involved in inflorescence meristem identity and flowering time regulation in some conditions. Although, the SVP homologs might have suffered functional diversification among diverse species between core and basal eudicots, the protein functions are conserved between Arabidopsis/Petunia and Epimedium.

The K Domain Mediates Homologous and Heterologous Interactions Between FLC and SVP Proteins of Brassica juncea

The transcription factors FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE (SVP) can interact to form homologous and heterologous protein complexes that regulate flowering time in Brassica juncea Coss. (Mustard).Previous studies showed that protein interactions were mediated by the K domain, which contains the subdomains K1, K2 and K3. However, it remains unknown how the subdomains mediate the interactions between FLC and SVP. In the present study, we constructed several mutants of subdomains K1–K3 and investigated the mechanisms involved in the heterologous interaction of BjFLC/BjSVP and in the homologous interaction of BjFLC/BjFLC or BjSVP/BjSVP. Yeast two-hybrid and β-Galactosidase activity assays showed that the 19 amino acids of the K1 subdomain in BjSVP and the 17 amino acids of the K1 subdomain in BjFLC were functional subdomains that interact with each other to mediate hetero-dimerization. The heterologous interaction was enhanced by the K2 subdomain of BjSVP protein, but weakened by its interhelical domain L2. The heterologous interaction was also enhanced by the K2 subdomain of BjFLC protein, but weakened by its K3 subdomain. The homologous interaction of BjSVP was mediated by the full K-domain. However, the homologous interaction of BjFLC was regulated only by its K1 and weakened by its K2 and K3 subdomains. The results provided new insights into the interactions between FLC and SVP, which will be valuable for further studies on the molecular regulation mechanisms of the regulation of flowering time in B. juncea and other Brassicaceae.

Down-regulation of GIGANTEA-like genes increases plant growth and salt stress tolerance in poplar.

SummaryThe flowering time regulator GIGANTEA (GI) connects networks involved in developmental stage transitions and environmental stress responses in Arabidopsis. However, little is known about the role of GI in growth, development and responses to environmental challenges in the perennial plant poplar. Here, we identified and functionally characterized three GI‐like genes (PagGIa, PagGIb and PagGIc) from poplar (Populus alba × Populus glandulosa). PagGIs are predominantly nuclear localized and their transcripts are rhythmically expressed, with a peak around zeitgeber time 12 under long‐day conditions. Overexpressing PagGIs in wild‐type (WT) Arabidopsis induced early flowering and salt sensitivity, while overexpressing PagGIs in the gi‐2 mutant completely or partially rescued its delayed flowering and enhanced salt tolerance phenotypes. Furthermore, the PagGIs‐PagSOS2 complexes inhibited PagSOS2‐regulated phosphorylation of PagSOS1 in the absence of stress, whereas these inhibitions were eliminated due to the degradation of PagGIs under salt stress. Down‐regulation of PagGIs by RNA interference led to vigorous growth, higher biomass and enhanced salt stress tolerance in transgenic poplar plants. Taken together, these results indicate that several functions of Arabidopsis GI are conserved in its poplar orthologues, and they lay the foundation for developing new approaches to producing salt‐tolerant trees for sustainable development on marginal lands worldwide.

Natural Variation in Brachypodium Links Vernalization and Flowering Time Loci as Major Flowering Determinants.

The domestication of plants is underscored by the selection of agriculturally favorable developmental traits, including flowering time, which resulted in the creation of varieties with altered growth habits. Research into the pathways underlying these growth habits in cereals has highlighted the role of three main flowering regulators: VERNALIZATION1 (VRN1), VRN2, and FLOWERING LOCUS T (FT). Previous reverse genetic studies suggested that the roles of VRN1 and FT are conserved in Brachypodium distachyon yet identified considerable ambiguity surrounding the role of VRN2 To investigate the natural diversity governing flowering time pathways in a nondomesticated grass, the reference B. distachyon accession Bd21 was crossed with the vernalization-dependent accession ABR6. Resequencing of ABR6 allowed the creation of a single-nucleotide polymorphism-based genetic map at the F4 stage of the mapping population. Flowering time was evaluated in F4:5 families in five environmental conditions, and three major loci were found to govern flowering time. Interestingly, two of these loci colocalize with the B. distachyon homologs of the major flowering pathway genes VRN2 and FT, whereas no linkage was observed at VRN1 Characterization of these candidates identified sequence and expression variation between the two parental genotypes, which may explain the contrasting growth habits. However, the identification of additional quantitative trait loci suggests that greater complexity underlies flowering time in this nondomesticated system. Studying the interaction of these regulators in B. distachyon provides insights into the evolutionary context of flowering time regulation in the Poaceae as well as elucidates the way humans have utilized the natural variation present in grasses to create modern temperate cereals.

Chloroplast retrograde signal regulates flowering

Light is a major environmental factor regulating flowering time, thus ensuring reproductive success of higher plants. In contrast to our detailed understanding of light quality and photoperiod mechanisms involved, the molecular basis underlying high light-promoted flowering remains elusive. Here we show that, in Arabidopsis, a chloroplast-derived signal is critical for high light-regulated flowering mediated by the FLOWERING LOCUS C (FLC). We also demonstrate that PTM, a PHD transcription factor involved in chloroplast retrograde signaling, perceives such a signal and mediates transcriptional repression of FLC through recruitment of FVE, a component of the histone deacetylase complex. Thus, our data suggest that chloroplasts function as essential sensors of high light to regulate flowering and adaptive responses by triggering nuclear transcriptional changes at the chromatin level.

A Novel Role for Banana MaASR in the Regulation of Flowering Time in Transgenic Arabidopsis.

The abscisic acid (ABA)-, stress-, and ripening-induced (ASR) protein is a plant-specific hydrophilic transcriptional factor involved in fruit ripening and the abiotic stress response. To date, there have been no studies on the role of ASR genes in delayed flowering time. Here, we found that the ASR from banana, designated as MaASR, was preferentially expressed in the banana female flowers from the eighth, fourth, and first cluster of the inflorescence. MaASR transgenic lines (L14 and L38) had a clear delayed-flowering phenotype. The number of rosette leaves, sepals, and pedicel trichomes in L14 and L38 was greater than in the wild type (WT) under long day (LD) conditions. The period of buds, mid-flowers, and full bloom of L14 and L38 appeared later than the WT. cDNA microarray and quantitative real-time PCR (qRT-PCR) analyses revealed that overexpression of MaASR delays flowering through reduced expression of several genes, including photoperiod pathway genes, vernalization pathway genes, gibberellic acid pathway genes, and floral integrator genes, under short days (SD) for 28 d (from vegetative to reproductive transition stage); however, the expression of the autonomous pathway genes was not affected. This study provides the first evidence of a role for ASR genes in delayed flowering time in plants.

Genome-wide transcriptomic analysis uncovers the molecular basis underlying early flowering and apetalous characteristic in Brassica napus L.

Floral transition and petal onset, as two main aspects of flower development, are crucial to rapeseed evolutionary success and yield formation. Currently, very little is known regarding the genetic architecture that regulates flowering time and petal morphogenesis in Brassica napus. In the present study, a genome-wide transcriptomic analysis was performed with an absolutely apetalous and early flowering line, APL01, and a normally petalled line, PL01, using high-throughput RNA sequencing. In total, 13,205 differential expressed genes were detected, of which 6111 genes were significantly down-regulated, while 7094 genes were significantly up-regulated in the young inflorescences of APL01 compared with PL01. The expression levels of a vast number of genes involved in protein biosynthesis were altered in response to the early flowering and apetalous character. Based on the putative rapeseed flowering genes, an early flowering network, mainly comprised of vernalization and photoperiod pathways, was built. Additionally, 36 putative upstream genes possibly governing the apetalous character of line APL01 were identified, and six genes potentially regulating petal origination were obtained by combining with three petal-related quantitative trait loci. These findings will facilitate understanding of the molecular mechanisms underlying floral transition and petal initiation in B. napus.

Flowering Time-Regulated Genes in Maize Include the Transcription Factor ZmMADS1.

Flowering time (FTi) control is well examined in the long-day plant Arabidopsis (Arabidopsis thaliana), and increasing knowledge is available for the short-day plant rice (Oryza sativa). In contrast, little is known in the day-neutral and agronomically important crop plant maize (Zea mays). To learn more about FTi and to identify novel regulators in this species, we first compared the time points of floral transition of almost 30 maize inbred lines and show that tropical lines exhibit a delay in flowering transition of more than 3 weeks under long-day conditions compared with European flint lines adapted to temperate climate zones. We further analyzed the leaf transcriptomes of four lines that exhibit strong differences in flowering transition to identify new key players of the flowering control network in maize. We found strong differences among regulated genes between these lines and thus assume that the regulation of FTi is very complex in maize. Especially genes encoding MADS box transcriptional regulators are up-regulated in leaves during the meristem transition. ZmMADS1 was selected for functional studies. We demonstrate that it represents a functional ortholog of the central FTi integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) of Arabidopsis. RNA interference-mediated down-regulation of ZmMADS1 resulted in a delay of FTi in maize, while strong overexpression caused an early-flowering phenotype, indicating its role as a flowering activator. Taken together, we report that ZmMADS1 represents a positive FTi regulator that shares an evolutionarily conserved function with SOC1 and may now serve as an ideal stating point to study the integration and variation of FTi pathways also in maize.

Genome-wide associations with flowering time in switchgrass using exome-capture sequencing data.

Flowering time is a major determinant of biomass yield in switchgrass (Panicum virgatum), a perennial bioenergy crop, because later flowering allows for an extended period of vegetative growth and increased biomass production. A better understanding of the genetic regulation of flowering time in switchgrass will aid the development of switchgrass varieties with increased biomass yields, particularly at northern latitudes, where late-flowering but southern-adapted varieties have high winter mortality. We use genotypes derived from recently published exome-capture sequencing, which mitigates challenges related to the large, highly repetitive and polyploid switchgrass genome, to perform genome-wide association studies (GWAS) using flowering time data from a switchgrass association panel in an effort to characterize the genetic architecture and genes underlying flowering time regulation in switchgrass. We identify associations with flowering time at multiple loci, including in a homolog of FLOWERING LOCUS T and in a locus containing TIMELESS, a homolog of a key circadian regulator in animals. Our results suggest that flowering time variation in switchgrass is due to variation at many positions across the genome. The relationship of flowering time and geographic origin indicates likely roles for genes in the photoperiod and autonomous pathways in generating switchgrass flowering time variation.

GL2-type homeobox gene Roc4 in rice promotes flowering time preferentially under long days by repressing Ghd7

Under long day (LD) lengths, flowering can be delayed in rice by modulating several regulatory genes. We found activation tagging lines that showed an early flowering phenotype preferentially under LD conditions. Expression of Rice outermost cell-specific gene 4 (Roc4), encoding a homeodomain Leu-zipper class IV family protein, was significantly increased. Transcript levels of Grain number, plant height, and heading date7 (Ghd7) were significantly reduced while those of Ghd7 downstream genes were increased. However, other flowering regulators were unaffected. Whereas constitutive overexpression of Roc4 in ‘Dongjin’ japonica rice, which carries active Ghd7, also caused LD-preferential early flowering, its overexpression in ‘Longjing27′ rice, which is defective in functional Ghd7, did not produce the same result. This confirmed that Roc4 regulates flowering time mainly through Ghd7. Phytochromes and O. sativa GIGANTEA (OsGI) function upstream of Roc4. Transgenic plants showed ubiquitous expression of the β-glucuronidase reporter gene under the Roc4 promoter. Furthermore, Roc4 had transcriptional activation activity in the N-terminal region of the StAR-related lipid-transfer domain. All of these findings are evidence that Roc4 is an LD-preferential flowering enhancer that functions downstream of phytochromes and OsGI, but upstream of Ghd7.

Implications of High Temperature and Elevated CO2 on Flowering Time in Plants.

Flowering is a crucial determinant for plant reproductive success and seed-set. Increasing temperature and elevated carbon-dioxide (e[CO2]) are key climate change factors that could affect plant fitness and flowering related events. Addressing the effect of these environmental factors on flowering events such as time of day of anthesis (TOA) and flowering time (duration from germination till flowering) is critical to understand the adaptation of plants/crops to changing climate and is the major aim of this review. Increasing ambient temperature is the major climatic factor that advances flowering time in crops and other plants, with a modest effect of e[CO2].Integrated environmental stimuli such as photoperiod, temperature and e[CO2] regulating flowering time is discussed. The critical role of plant tissue temperature influencing TOA is highlighted and crop models need to substitute ambient air temperature with canopy or floral tissue temperature to improve predictions. A complex signaling network of flowering regulation with change in ambient temperature involving different transcription factors (PIF4, PIF5), flowering suppressors (HvODDSOC2, SVP, FLC) and autonomous pathway (FCA, FVE) genes, mainly from Arabidopsis, provides a promising avenue to improve our understanding of the dynamics of flowering time under changing climate. Elevated CO2 mediated changes in tissue sugar status and a direct [CO2]-driven regulatory pathway involving a key flowering gene, MOTHER OF FT AND TFL1 (MFT), are emerging evidence for the role of e[CO2] in flowering time regulation.

Structural determinants of miR156a precursor processing in temperature-responsive flowering in Arabidopsis.

MicroRNAs originate from primary transcripts (pri-miRNAs) containing hairpin structures. Plant pri-miRNAs have highly variable structures and little is known about the information encoded in their secondary structures. Arabidopsis miR156 is an ambient temperature-responsive miRNA and plays an important role in regulating flowering time. To identify the structural determinants for miR156 processing, we analyzed the effects of mutations introduced in the upper stem of pri-miR156a on its temperature-dependent processing and flowering time. The levels of pri-miR156a and mature miR156 were opposite at different temperatures. Mutations in the upper stem, especially the region closer to the miR156a/miR156a* duplex, reduced miR156 processing at 23 °C and 16 °C and caused a less severe phenotype compared with the un-mutated construct. Mutation in the second stem near the first cleavage site of pri-miR156a affected miR156 processing at 23 °C, but not at 16 °C. This was also seen in pri-miR172a, another ambient temperature-responsive miRNA. Replacement of the upper stem of pri-miR156a with that of pri-miR172a severely affected miR156 processing and flowering time. These results suggested that the upper stem of pri-miR156a is important for miR156 processing at different temperatures. In particular, the second stem adjacent to the first cleavage site plays a role in the regulation of ambient temperature-responsive flowering.

Heterologous expression of an RNA-binding protein affects flowering time as well as other developmental processes in Solanaceae

Flowering time in members of the Solanaceae plant family, such as pepper (Capsicum spp.) and tomato (Solanum lycopersicum), is an important agronomic trait for controlling shoot architecture and improving yield. To investigate the feasibility of flowering time regulation in tomato, an RNA-binding protein (RBP) encoding gene homologous to human Nucleolar protein interacting with the forkhead-associated (FHA) domain of pKI-67 (NIFK), CaRBP, was isolated from hot pepper. The function of CaRBP was determined in transgenic tomato. The deduced amino acid sequence includes an RNA recognition motif (RRM) and showed most similarity to the RRM present in a putative RBP encoded by human NIFK. CaRBP was highly expressed in the vegetative and reproductive tissues, such as leaves and fruits, respectively. Subcellular localization analysis indicated that CaRBP is a nucleolar protein. Heterologous expression of CaRBP under 35S promoter in tomato plants induced severe alteration of flowering with additional defects of vegetative organs. This floral retardation was associated with the alteration of SFT/SP3D and SlSOC1s as floral integrators. Furthermore, CaRBP reduces the expression levels of SlCOLs/TCOLs via changes in the expression of SlCDF3, SlFBHs, and SlFKF1s. This indicates a repressive effect of CaRBP on the regulation of flowering time in tomato. Overall, these results suggest that alteration in CaRBP expression levels may provide an effective means of controlling flowering time in day-neutral Solanaceae.

LATE ELONGATED HYPOCOTYL regulates photoperiodic flowering via the circadian clock in Arabidopsis.

BackgroundPlants constantly monitor changes in photoperiod or day length to trigger the flowering cycle at the most appropriate time of the year. It is well established that photoperiodic flowering is intimately associated with the circadian clock in Arabidopsis. In support of this notion, many clock-defective mutants exhibit altered photoperiodic sensitivity in inducing flowering. LATE ELONGATED HYPOCOTYL (LHY) and its functional paralogue CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) constitute the core of the circadian clock together with TIMING OF CAB EXPRSSION 1 (TOC1). While it is known that TOC1 contributes to the timing of flowering entirely by modulating the clock function, molecular mechanisms by which LHY and CCA1 regulate flowering time have not been explored.ResultsWe investigated how LHY and CCA1 regulate photoperiodic flowering through molecular genetic and biochemical studies. It was found that LHY-defective mutants (lhy-7 and lhy-20) exhibit accelerated flowering under both long days (LDs) and short days (SDs). Consistent with the accelerated flowering phenotypes, gene expression analysis revealed that expression of the floral integrator FLOWERING LOCUS T (FT) is up-regulated in the lhy mutants. In addition, the expression peaks of GIGANTEA (GI) and FLAVIN-BINDING, KELCH REPEAT, F-BOX PROTEIN 1 (FKF1) genes, which constitute the clock output pathway that is linked with photoperiodic flowering, were advanced by approximately 4 h in the mutants. Furthermore, the up-regulation of FT disappeared when the endogenous circadian period is matched to the external light/dark cycles in the lhy-7 mutant. Notably, whereas CCA1 binds strongly to FT gene promoter, LHY does not show such DNA-binding activity.ConclusionsOur data indicate that the advanced expression phases of photoperiodic flowering genes are associated with the clock defects in the lhy mutants and responsible for the reduced photoperiodic sensitivity of the mutant flowering, demonstrating that LHY regulates photoperiodic flowering via the circadian clock, similar to what has been shown with TOC1. It is notable that while LHY regulates photoperiodic flowering in a similar manner as with TOC1, the underlying molecular mechanism would be somewhat distinct from that exerted by CCA1 in Arabidopsis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0810-8) contains supplementary material, which is available to authorized users.

Genome-Wide Analysis of Gene Regulatory Networks of the FVE-HDA6-FLD Complex in Arabidopsis.

FVE/MSI4 is a homolog of the mammalian RbAp48 protein. We found that FVE regulates flowering time by repressing FLC through decreasing histone H3K4 trimethylation and H3 acetylation. Furthermore, FVE interacts with the histone deacetylase HDA6 and the histone demethylase FLD, suggesting that these proteins may form a protein complex to regulate flowering time. To further investigate the function of the FVE-FLD-HDA6 complex, we compared the gene expression profiles of fve, fld, and hda6 mutant plants by using RNA-seq analysis. Among the mis-regulated genes found in fve plants, 51.8 and 36.5% of them were also mis-regulated in fld and hda6 plants, respectively, suggesting that FVE, HDA6, and FLD may regulate the gene expression in the same developmental processes in Arabidopsis. Gene ontology analysis revealed that among 383 genes co-regulated by FVE, HDA6, and FLD, 15.6% of them are involved in transcription, 8.2% in RNA metabolic process, 7.7% in response to abiotic stress, and 6.3% in hormone stimulus. Taken together, these results indicate that HDA6, FVE, and FLD co-regulate the gene expression in multiple development processes and pathways.

Mutation of OsGIGANTEA Leads to Enhanced Tolerance to Polyethylene Glycol-Generated Osmotic Stress in Rice.

Water deficit is one of the most important environmental stresses limiting plant growth and crop yield. While the identification of many key factors involved in the plant water deficit response has greatly increased our knowledge about the regulation system, the mechanisms underlying dehydration tolerance in plants are still not well understood. In our current study, we investigated the roles of the key flowering time regulator, OsGIGANTEA (OsGI), in the osmotic stress tolerance in rice. Results showed that mutation of OsGI conferred tolerance to osmotic stress generated by polyethylene glycol (PEG), increased proline and sucrose contents, and accelerated stomata movement. In addition, qRT-PCR and microarray analysis revealed that the transcript abundance of some osmotic stress response genes, such as OsDREB1E, OsAP37, OsAP59, OsLIP9, OsLEA3, OsRAB16A, and OsSalT, was significantly higher in osgi than in WT plants, suggesting that OsGI might be a negative regulator in the osmotic stress response in rice.

CHD3 chromatin-remodeling factor PICKLE regulates floral transition partially via modulating LEAFY expression at the chromatin level in Arabidopsis.

PICKLE (PKL), a putative CHD3 chromatin remodeling factor, has been suggested to be involved in multiple processes in Arabidopsis. Here, we confirmed the late-flowering phenotype caused by pkl mutation with pkl mutants in two different ecotypes, and investigated the possible mechanisms that account for PKL regulation of flowering time. Quantitative RT-PCR and RNA-seq assays showed that expression of the LEAFY gene (LFY) and a number of LFY-regulated floral homeotic genes were down-regulated in seedlings of the pkl mutants. As predicted, overexpression of LFY restored normal flowering time of pkl mutants. Our results suggest that PKL may be involved in regulating flowering time via LFY expression. To uncover the underlying mechanism, ChIP-PCR using anti-PKL was performed on materials from three developmental stages of seedlings. Our results showed that PKL associated with the genomic sequences of LFY, particularly at 10-day and 25-day after germination. We also showed that loss of PKL affected H3K27me3 level at the promoter of LFY. Taken together, our data suggest that transcriptional regulation of LFY at the chromatin level by PKL may at least partially account for the late-flowering phenotype of pkl mutants.

Genome-Wide Identification of Soybean U-Box E3 Ubiquitin Ligases and Roles of GmPUB8 in Negative Regulation of Drought Stress Response in Arabidopsis.

Plant U-box (PUB) E3 ubiquitin ligases play important roles in hormone signaling pathways and response to abiotic stresses, but little is known about them in soybean, Glycine max. Here, we identified and characterized 125 PUB genes from the soybean genome, which were classified into eight groups according to their protein domains. Soybean PUB genes (GmPUB genes) are broadly expressed in many tissues and are a little more abundant in the roots than in the other tissues. Nine GmPUB genes, GmPUB1-GmPUB9, showed induced expression patterns by drought, and the expression of GmPUB8 was also induced by exogenous ABA and NaCl. GmPUB8 was localized to post-Golgi compartments, interacting with GmE2 protein as demonstrated by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments, and showed E3 ubiquitin ligase activity by in vitro ubiquitination assay. Heterogeneous overexpression of GmPUB8 in Arabidopsis showed decreased drought tolerance, enhanced sensitivity with respect to osmotic and salt stress inhibition of seed germination and seedling growth, and inhibited ABA- and mannitol-mediated stomatal closure. Eight drought stress-related genes were less induced in GmPUB8-overexpressing Arabidopsis after drought treatment compared with the wild type and the pub23 mutant. Taken together, our results suggested that GmPUB8 might negatively regulate plant response to drought stress. In addition, Y2H and BiFC showed that GmPUB8 interacted with soybean COL (CONSTANS LIKE) protein. GmPUB8-overexpressing Arabidopsis flowered earlier under middle- and short-day conditions but later under long-day conditions, indicating that GmPUB8 might regulate flowering time in the photoperiod pathway. This study helps us to understand the functions of PUB E3 ubiquitin ligases in soybean.