A trans-acting long non-coding RNA represses flowering in Arabidopsis

Eukaryotic genomes give rise to thousands of long non-coding RNAs (lncRNAs), yet the purpose of lncRNAs remains largely enigmatic. Functional characterization of lncRNAs is challenging due to multiple orthogonal hypothesis for molecular activities of lncRNA loci. Here, we identified a flowering associated intergenic lncRNA (FLAIL) that represses flowering in Arabidopsis. An allelic series of flail loss-of-function mutants generated by CRISPR/Cas9 and T-DNA mutagenesis showed an early flowering phenotype. Gene expression analyses in flail mutants revealed differentially expressed genes linked to the regulation of flowering. A genomic rescue fragment of FLAIL introduced in flail mutants complemented gene expression defects and early flowering, consistent with trans-acting effects of the FLAIL RNA. Knock-down of FLAIL RNA levels using the artificial microRNA approach revealed an early flowering phenotype shared with genomic mutations, indicating a trans-acting role of FLAIL RNA in the repression of flowering time. Genome-wide detection of FLAIL-DNA interactions by ChIRP-seq suggested that FLAIL may directly bind genomic regions. FLAIL bound to genes involved in regulation of flowering that were differentially expressed in flail, consistent with the interpretation of FLAIL as a trans-acting lncRNA directly shaping gene expression. Our findings highlight FLAIL as a trans-acting lncRNA that affects flowering in Arabidopsis, likely through mediating transcriptional regulation of genes directly bound by FLAIL.

ZjSEP3 modulates flowering time by regulating the LHY promoter

Background SEPALLATA3 (SEP3), which is conserved across various plant species, plays essential and various roles in flower and fruit development. However, the regulatory network of the role of SEP3 in flowering time at the molecular level remained unclear. Results Here, we investigated that SEP3 in Ziziphus jujuba Mill. (ZjSEP3) was expressed in four floral organs and exhibited strong transcriptional activation activity. ZjSEP3 transgenic Arabidopsis showed an early-flowering phenotype and altered the expression of some genes related to flowering. Among them, the expression of LATE ELONGATED HYPOCOTYL (AtLHY), the key gene of circadian rhythms, was significantly suppressed. Yeast one-hybrid (Y1H) and electrophoretic mobility shift assays (EMSAs) further verified that ZjSEP3 inhibited the transcription of AtLHY by binding to the CArG-boxes in its promoter. Moreover, ZjSEP3 also could bind to the ZjLHY promoter and the conserved binding regions of ZjSEP3 were found in the LHY promoter of various plant species. The ectopic regulatory pathway of ZjSEP3-AtLHY was further supported by the ability of 35S::AtLHY to rescue the early-flowering phenotype in ZjSEP3 transgenic plants. In ZjSEP3 transgenic plants, total chlorophyll content and the expression of genes involved in chlorophyll synthesis increased during vegetative stages, which should contribute to its early flowering and relate to the regulatory of AtLHY. Conclusion Overall, ZjSEP3-AtLHY pathway represents a novel regulatory mechanism that is involved in the regulation of flowering time.

Genome sequencing and analysis of two early-flowering cherry (Cerasus × kanzakura) varieties, ‘Kawazu-zakura’ and ‘Atami-zakura’

To gain genetic insights into the early-flowering phenotype of ornamental cherry, also known as sakura, we determined the genome sequences of two early-flowering cherry (Cerasus × kanzakura) varieties, ‘Kawazu-zakura’ and ‘Atami-zakura’. Since the two varieties are interspecific hybrids, likely derived from crosses between Cerasus campanulata (early-flowering species) and Cerasus speciosa, we employed the haplotype-resolved sequence assembly strategy. Genome sequence reads obtained from each variety by single molecule real-time sequencing (SMRT) were split into two subsets, based on the genome sequence information of the two probable ancestors, and assembled to obtain haplotype-phased genome sequences. The resultant genome assembly of ‘Kawazu-zakura’ spanned 519.8 Mb with 1,544 contigs and an N50 value of 1,220.5 kb, while that of ‘Atami-zakura’ totaled 509.6 Mb with 2,180 contigs and an N50 value of 709.1 kb. A total of 72,702 and 72,528 potential protein-coding genes were predicted in the genome assemblies of ‘Kawazu-zakura’ and ‘Atami-zakura’, respectively. Gene clustering analysis identified 2,634 clusters uniquely presented in the C. campanulata haplotype sequences, which might contribute to its early-flowering phenotype. Genome sequences determined in this study provide fundamental information for elucidating the molecular and genetic mechanisms underlying the early-flowering phenotype of ornamental cherry tree varieties and their relatives.

Identification and characterization of regulatory pathways involved in early flowering in the new leaves of alfalfa (Medicago sativa L.) by transcriptome analysis

Background Alfalfa (Medicago sativa L.) is a perennial legume extensively planted throughout the world as a high nutritive value livestock forage. Flowering time is an important agronomic trait that contributes to the production of alfalfa hay and seeds. However, the underlying molecular mechanisms of flowering time regulation in alfalfa are not well understood. Results In this study, an early-flowering alfalfa genotype 80 and a late-flowering alfalfa genotype 195 were characterized for the flowering phenotype. Our analysis revealed that the lower jasmonate (JA) content in new leaves and the downregulation of JA biosynthetic genes (i.e. lipoxygenase, the 12-oxophytodienoate reductase-like protein, and salicylic acid carboxyl methyltransferase) may play essential roles in the early-flowering phenotype of genotype 80. Further research indicated that genes encode pathogenesis-related proteins [e.g. leucine rich repeat (LRR) family proteins, receptor-like proteins, and toll-interleukin-like receptor (TIR)-nucleotide-binding site (NBS)-LRR class proteins] and members of the signaling receptor kinase family [LRR proteins, kinases domain of unknown function 26 (DUF26) and wheat leucine-rich repeat receptor-like kinase10 (LRK10)-like kinases] are related to early flowering in alfalfa. Additionally, those involved in secondary metabolism (2-oxoglutarate/Fe (II)-dependent dioxygenases and UDP-glycosyltransferase) and the proteasome degradation pathway [really interesting new gene (RING)/U-box superfamily proteins and F-box family proteins] are also related to early flowering in alfalfa. Conclusions Integrated phenotypical, physiological, and transcriptomic analyses demonstrate that hormone biosynthesis and signaling pathways, pathogenesis-related genes, signaling receptor kinase family genes, secondary metabolism genes, and proteasome degradation pathway genes are responsible for the early flowering phenotype in alfalfa. This will provide new insights into future studies of flowering time in alfalfa and inform genetic improvement strategies for optimizing this important trait.

UGT85A53 promotes flowering via mediating abscisic acid glucosylation and FLC transcription in Camellia sinensis

Uridine diphosphate (UDP)-dependent glycosyltransferases catalyse the glycosylation of small molecules and play important roles in maintaining cell homeostasis and regulating plant development. Glycosyltransferases are widely distributed, but their detailed roles in regulating plant growth and development are largely unknown. In this study, we identified a UDP-glycosyltransferase, UGT85A53, from Camellia sinensis, the expression of which was strongly induced by various abiotic stress factors and its protein product was distributed in both the cytoplasm and nucleus. Ectopic overexpression of CsUGT85A53 in Arabidopsis resulted in an early-flowering phenotype under both long- and short-day conditions. The transcript accumulation of the flowering repressor genes FLC and ABI5, an activator of FLC in ABA-regulated flowering signaling, were both significantly decreased in transgenic Arabidopsis compared with wild-type plants. The decreased expression level of FLC might be associated with an increased level of DNA methylation that was observed in CsUGT85A53-overexpressing (OE) plants. Biochemical analyses showed that CsUGT85A53 could glucosylate ABA to form inactive ABA-glycoside in vitro and in planta. Overexpression of CsUGT85A53 in Arabidopsis resulted in a decreased concentration of free ABA and increased concentration of ABA-glucoside. The early-flowering phenotype in the CsUGT85A53-OE transgenic lines was restored by ABA application. Furthermore, CsUGT85A53-OE plants displayed an ABA-insensitive phenotype with higher germination rates compared with controls in the presence of low concentrations of exogenous ABA. Our findings are the first to identify a UGT in tea plants that catalyses ABA glucosylation and enhance flowering transition as a positive regulator.

ARGONAUTE5 Represses Age-Dependent Induction of Flowering through Physical and Functional Interaction with miR156 in Arabidopsis

Flowering time is a finely tuned process in plants, in part controlled by the age-regulated microRNA156 (miR156), which functions by suppressing the transcripts of SQUAMOSA-PROMOTER BINDING LIKE (SPL) transcription factors. ARGONAUTE (AGO) proteins are essential effectors of miRNA-mediated gene regulation. However, which AGO(s) mediate(s) the control of flowering time remains unclear. Here, we demonstrate a role of AGO5 in controlling flowering time by modulating the expression of SPL transcription factors. We show that AGO5 interacts physically and functionally with miR156 and that ago5 mutants present an early flowering phenotype in Arabidopsis. Furthermore, in ago5 mutants, the repression of flowering caused by miR156 overexpression is largely reversed, whereas leaf morphology remains unaffected. Our results thus indicate a specific role for AGO5 in mediating miR156 activity in meristematic, but not vegetative, tissue. As such, our data suggest a spatiotemporal regulation of the miR156 aging pathway mediated through different AGO proteins in different tissues.

ARGONAUTE5 Mediates Fine-Tuning of Vegetative-to-Reproductive Phase Transition Through Its Interaction with miR156 in Arabidopsis

ABSTRACTVegetative-to-reproductive phase change is a finely tuned process in plants, largely controlled by the age-regulated microRNA156 (miR156), which functions by suppressing the transcripts of SQUAMOSA-PROMOTER BINDING LIKE (SPL) transcription factors. ARGONAUTE proteins (AGO) are essential effectors of miRNA-mediated gene regulation. However, which AGO(s) mediate(s) the control of flowering time remains unclear. Here, we demonstrate a role for AGO5 in vegetative-to-reproductive phase transition through the modulation of SPL transcription factors. We show that AGO5 interacts physically and functionally with miR156 and that ago5 mutants present an early flowering phenotype in Arabidopsis. Furthermore, in ago5 mutants, the repression of flowering caused by miR156 overexpression is largely reversed, whereas leaf morphology remains unaffected. Our results thus indicate a specific role for AGO5 in mediating miR156 activity in meristematic, but not vegetative, tissue. As such, our data suggest a spatiotemporal regulation of the miR156 aging pathway, mediated through different AGO proteins in different tissues.