Molecular cloning and characterization of a gene regulating flowering time from Alfalfa (Medicago sativa L.)

Genes that regulate flowering time play crucial roles in plant development and biomass formation. Based on the cDNA sequence of Medicago truncatula (accession no. AY690425), the LFY gene of alfalfa was cloned. Sequence similarity analysis revealed high homology with FLO/LFY family genes of other plants. When fused to the green fluorescent protein, MsLFY protein was localized in the nucleus of onion (Allium cepa L.) epidermal cells. The RT-qPCR analysis of MsLFY expression patterns showed that the expression of MsLFY gene was at a low level in roots, stems, leaves and pods, and the expression level in floral buds was the highest. The expression of MsLFY was induced by GA₃ and long photoperiod. Plant expression vector was constructed and transformed into Arabidopsis by the agrobacterium-mediated methods. PCR amplification with the transgenic Arabidopsis genome DNA indicated that MsLFY gene had integrated in Arabidopsis genome. Overexpression of MsLFY specifically caused early flowering under long day conditions compared with non-transgenic plants. These results indicated MsLFY played roles in promoting flowering time.     

Inflorescence abnormalities occur with overexpression of Arabidopsis lyrata FT in the fwa mutant of Arabidopsis thaliana

Arabidopsis thaliana is a quantitative long-day plant with the timing of the floral transition being regulated by both endogenous signals and multiple environmental factors. fwa is a late-flowering mutant, and this phenotype is due to ectopic FWA expression caused by hypomethylation at the FWA locus. The floral transition results in the activation of the floral development process, the key regulators being the floral meristem identity genes, AP1 (APETALA1) and LFY (LEAFY). In this study, we describe inflorescence abnormalities in plants overexpressing the Arabidopsis lyrata FT (AlFT) and A. thaliana FWA (AtFWA) genes simultaneously. The inflorescence abnormality phenotype was present in only a proportion of plants. All plants overexpressing both AlFT and AtFWA flowered earlier than fwa, suggesting that the inflorescence abnormality and earlier flowering time are caused independently. The inflorescence abnormality phenotype was similar to that of the double mutant of ap1 and lfy, and AP1 and LFY genes were down-regulated in the abnormal inflorescences. From these results, we suggest that not only does ectopic AtFWA expression inhibit AtFT/AlFT function to delay flowering but that overexpression of AtFWA and AlFT together inhibits AP1 and LFY function to produce abnormal inflorescences.     

YCZ-18 Is a New Brassinosteroid Biosynthesis Inhibitor

Plant hormone brassinosteroids (BRs) are a group of polyhydroxylated steroids that play critical roles in regulating broad aspects of plant growth and development. The structural diversity of BRs is generated by the action of several groups of P450s. Brassinazole is a specific inhibitor of C-22 hydroxylase (CYP90B1) in BR biosynthesis, and the application use of brassinazole has emerged as an effective way of complementing BR-deficient mutants to elucidate the functions of BRs. In this article, we report a new triazole-type BR biosynthesis inhibitor, YCZ-18. Quantitative analysis the endogenous levels of BRs in Arabidopsis indicated that YCZ-18 significantly decreased the BR contents in plant tissues. Assessment of the binding affinity of YCZ-18to purified recombinant CYP90D1 indicated that YCZ-18 induced a typical type II binding spectrum with a K_d value of approximately 0.79 μM. Analysis of the mechanisms underlying the dwarf phenotype associated with YCZ-18 treatment of Arabidopsis indicated that the chemically induced dwarf phenotype was caused by a failure of cell elongation. Moreover, dissecting the effect of YCZ-18 on the induction or down regulation of genes responsive to BRs indicated that YCZ-18 regulated the expression of genes responsible for BRs deficiency in Arabidopsis. These findings indicate that YCZ-18 is a potent BR biosynthesis inhibitor and has a new target site, C23-hydroxylation in BR biosynthesis. Application of YCZ-18 will be a good starting point for further elucidation of the detailed mechanism of BR biosynthesis and its regulation.     

Disruption of the Homogentisate Solanesyltransferase Gene Results in Albino and Dwarf Phenotypes and Root,Trichome and Stomata Defects in Arabidopsis thaliana

Homogentisate solanesyltransferase (HST) plays an important role in plastoquinone (PQ) biosynthesis and acts as the electron acceptor in the carotenoids and abscisic acid (ABA) biosynthesis pathways. We isolated and identified a T-DNA insertion mutant of the HST gene that displayed the albino and dwarf phenotypes. PCR analyses and functional complementation also confirmed that the mutant phenotypes were caused by disruption of the HST gene. The mutants also had some developmental defects, including trichome development and stomata closure defects. Chloroplast development was also arrested and chlorophyll (Chl) was almost absent. Developmental defects in the chloroplasts were consistent with the SDS-PAGE result and the RNAi transgenic phenotype. Exogenous gibberellin (GA) could partially rescue the dwarf phenotype and the root development defects and exogenous ABA could rescue the stomata closure defects. Further analysis showed that ABA and GA levels were both very low in the pds2-1 mutants, which suggested that biosynthesis inhibition by GAs and ABA contributed to the pds2-1 mutants'' phenotypes. An early flowering phenotype was found in pds2-1 mutants, which showed that disruption of the HST gene promoted flowering by partially regulating plant hormones. RNA-sequencing showed that disruption of the HST gene resulted in expression changes to many of the genes involved in flowering time regulation and in the biosynthesis of PQ, Chl, GAs, ABA and carotenoids. These results suggest that HST is essential for chloroplast development, hormone biosynthesis, pigment accumulation and plant development.     

Overexpression of a Kunitz-type trypsin inhibitor (AtKTI1) causes early flowering in Arabidopsis

An early flowering mutant of Arabidopsis, elf32-D was isolated from activation tagging screening. The mutant flowered earlier than wild type under both long day and short day conditions. The mutant phenotype was caused by overexpression of a Kunitz-type trypsin inhibitor gene (AtKTI1). The expression of AtKTI1 was detected in leaves, flowers, siliques and roots. In the vegetative state, no change of flowering integrator gene expression was observed for AtKTI1 overexpressing plants. In contrast, at the reproductive stage, its overexpression resulted in the down-regulation of FLC, a strong floral repressor which integrates the autonomous and vernalization pathways and also the up-regulation of FT and AP1, which are downstream floral integrator genes. It is probable that the AtKTI1 overexpression inhibits components of the flowering signaling pathway upstream of FLC, eventually regulating expression of FLC, or causing perturbations in plant metabolism and thus indirectly affecting flowering.     

Photoperiodic Control of Carbon Distribution during the Floral Transition in Arabidopsis

Flowering is a crucial process that demands substantial resources. Carbon metabolism must be coordinated with development through a control mechanism that optimizes fitness for any physiological need and growth stage of the plant. However, how sugar allocation is controlled during the floral transition is unknown. Recently, the role of a CONSTANS (CO) ortholog (Cr-CO) in the control of the photoperiod response in the green alga Chlamydomonas reinhardtii and its influence on starch metabolism was demonstrated. In this work, we show that transitory starch accumulation and glycan composition during the floral transition in Arabidopsis thaliana are regulated by photoperiod. Employing a multidisciplinary approach, we demonstrate a role for CO in regulating the level and timing of expression of the GRANULE BOUND STARCH SYNTHASE (GBSS) gene. Furthermore, we provide a detailed characterization of a GBSS mutant involved in transitory starch synthesis and analyze its flowering time phenotype in relation to its altered capacity to synthesize amylose and to modify the plant free sugar content. Photoperiod modification of starch homeostasis by CO may be crucial for increasing the sugar mobilization demanded by the floral transition. This finding contributes to our understanding of the flowering process.     

Knockdown of NtMed8, a Med8-like gene, causes abnormal development of vegetative and floral organs in tobacco (Nicotiana tabacum L.)

Med8, a subunit of mediator complex, has proved to possess crucial functions in many organisms from yeast to human. In plant, the med8 mutant of Arabidopsis thaliana displayed delayed anthesis and increased number of leaves during the vegetative period. However, the roles of Med8 in other flowering plants are still unknown. To investigate the function of Med8 ortholog in tobacco (Nicotiana tabacum L.; named as NtMed8), we created transgenic tobacco plants with repressed NtMed8 expression mediated by RNA interference (RNAi). Compared with the wild type, the NtMed8-RNAi plants exhibited: more leaves with smaller but thicker blades; larger cells and vascular bundles with lower stomata density in leaves; swelled chloroplasts with thicker and lumen-enlarged thylakoids; weaker root system with fewer lateral roots; larger flowers and floral organs; flowering earlier under long day, but later under short day conditions; and male sterile with larger but less germinable pollens. In addition, quantitative RT-PCR indicated that NtMed8 is expressed in both vegetative and floral tissues. Subcellular localization analysis by transient expression of fusion protein in Nicotiana benthamiana leaves showed that NtMed8 was located in both plasma membrane and nucleus. These results suggest that NtMed8 plays important roles in both vegetative and reproductive development, and the function of Med8 appears to be, at least partially, conserved in flowering plants.