Photosynthetic photon flux density and phytochrome B interact to regulate branching in Arabidopsis

Branching is regulated by environmental signals including phytochrome B (phyB)-mediated responses to the ratio of red to far red light. While the mechanisms associated with phytochrome regulation of branching are beginning to be elucidated, there is little information regarding other light signals, including photosynthetic photon flux density (PPFD) and how it influences phytochrome-mediated responses. This study shows that Arabidopsis (Arabidopsis thaliana) branching is modified by both varying PPFD and phyB status and that significant interactions occur between these variables. While phyB deficiency decreased branching when the PPFD was low, the effect was suppressed by high PPFD and some branching aspects were actually promoted. Photosynthesis measurements showed that PPFD may influence branching in phyB-deficient plants at least partially through a specific signalling pathway rather than directly through energy effects on the shoot. The expression of various genes in unelongated buds of phyB-deficient and phyB-sufficient plants grown under high and low PPFD demonstrated potential roles for several hormones, including auxin, cytokinins and ABA, and also showed imperfect correlation between expression of the branching regulators BRC1 and BRC2 and bud fate. These results may implicate additional undiscovered bud autonomous mechanisms and/or components contributing to bud outgrowth regulation by environmental signals.     

Shoot bending promotes flower bud formation by miRNA‐mediated regulation in apple (Malus domestica Borkh.)

Flower induction in apple (Malus domestica Borkh.) trees plays an important life cycle role, but young trees produce fewer and inferior quality flower buds. Therefore, shoot bending has become an important cultural practice, significantly promoting the capacity to develop more flower buds during the growing seasons. Additionally, microRNAs (miRNAs) play essential roles in plant growth, flower induction and stress responses. In this study, we identified miRNAs potentially involved in the regulation of bud growth, and flower induction and development, as well as in the response to shoot bending. Of the 195 miRNAs identified, 137 were novel miRNAs. The miRNA expression profiles revealed that the expression levels of 68 and 27 known miRNAs were down‐regulated and up‐regulated, respectively, in response to shoot bending, and that the 31 differentially expressed novel miRNAs between them formed five major clusters. Additionally, a complex regulatory network associated with auxin, cytokinin, abscisic acid (ABA) and gibberellic acid (GA) plays important roles in cell division, bud growth and flower induction, in which related miRNAs and targets mediated regulation. Among them, miR396, 160, 393, and their targets associated with AUX, miR159, 319, 164, and their targets associated with ABA and GA, and flowering‐related miRNAs and genes, regulate bud growth and flower bud formation in response to shoot bending. Meanwhile, the flowering genes had significantly higher expression levels during shoot bending, suggesting that they are involved in this regulatory process. This study provides a framework for the future analysis of miRNAs associated with multiple hormones and their roles in the regulation of bud growth, and flower induction and formation in response to shoot bending in apple trees.     

Effect of the interaction between MiR‐200b‐3p and DNMT3A on cartilage cells of osteoarthritis patients

The aim of this research is to explore the effect of miR‐200b‐3p targeting DNMT3A on the proliferation and apoptosis of osteoarthritis (OA) cartilage cells. Quantitative RT‐PCR was performed to analyse the expression of miR‐200b‐3p, DNMT3A, MMP1, MMP3, MMP9, MMP13 and COL II in normal and OA cartilage tissues. The dual‐luciferase reporter assay and Western blot assay were conducted to confirm the targeting relationship between miR‐200b‐3p and DNMT3A. We also constructed eukaryotic expression vector to overexpress miR‐200b‐3p and DNMT3A. We detected the expression level of MMPs and COL II in stable transfected cartilage cells using RT‐PCR and Western blot. Cell proliferation and apoptosis were evaluated using the MTS, pellet culture and Hoechst 33342 staining method. Finally, we explored the effect of miR‐200b‐3p targeting DNMT3A on the proliferation and apoptosis of OA cartilage cells. The results of RT‐PCR indicated that both miR‐200b‐3p and COL II were down‐regulated in OA cartilage tissues, while the expression of DNMT3A and MMPs was up‐regulated in OA cartilage tissues. The expressions of DNMT3A, MMPs and COL II detected by Western blot showed the same trend of the results of RT‐PCR. The dual‐luciferase reporter assay and Western blot assay confirmed the targeting relationship between miR‐200b‐3p and DNMT3A. In overexpressed miR‐200b‐3p cartilage cells, DNMT3A and MMPs were significantly down‐regulated, COL II was significantly up‐regulated, cell viability was enhanced and apoptosis rate was decreased (P < 0.05). In overexpressed DNM3T cartilage cells, MMPs were significantly up‐regulated, COL II was significantly down‐regulated, cell viability was weakened and apoptosis rate was increased (P < 0.05). MiR‐200b‐3p inhibited the secretion of MMPs, promoted the synthesis of COL II and enhanced the growth and proliferation of OA cartilage cells through inhibiting the expression of DNMT3A.     

Florigen is involved in axillary bud development at multiple stages in Arabidopsis

The wide variety of plant architectures is largely based on diverse and flexible modes of axillary shoot development. In Arabidopsis, floral transition (flowering) stimulates axillary bud development. The mechanism that links flowering and axillary bud development is, however, largely unknown. We recently showed that FLOWERING LOCUS T (FT) protein, which acts as florigen, promotes the phase transition of axillary meristems, whereas BRANCHED1 (BRC1) antagonizes the florigen action in axillary buds. Here, we present evidences for another possible role of florigen in axillary bud development. Ectopic overexpression of FT or another florigen gene TWIN SISTER OF FT (TSF) with LEAFY (LFY) induces ectopic buds at cotyledonary axils, confirming the previous proposal that these genes are involved in formation of axillary buds. Taken together with our previous report that florigen promotes axillary shoot elongation, we propose that florigen regulates axillary bud development at multiple stages to coordinate it with flowering in Arabidopsis.     

A photo‐responsive F‐box protein FOF2 regulates floral initiation by promoting FLC expression in Arabidopsis

Floral initiation is regulated by various genetic pathways in response to light, temperature, hormones and developmental status; however, the molecular mechanisms underlying the interactions between different genetic pathways are not fully understood. Here, we show that the photoresponsive gene FOF2 (F‐box of flowering 2) negatively regulates flowering. FOF2 encodes a putative F‐box protein that interacts specifically with ASK14, and its overexpression results in later flowering under both long‐day and short‐day photoperiods. Conversely, transgenic plants expressing the F‐box domain deletion mutant of FOF2 (FOF2ΔF), or double loss of function mutant of FOF2 and FOL1 (FOF2‐LIKE 1) present early flowering phenotypes. The late flowering phenotype of the FOF2 overexpression lines is suppressed by the flc‐3 loss‐of‐function mutation. Furthermore, FOF2 mRNA expression is regulated by autonomous pathway gene FCA, and the repressive effect of FOF2 in flowering can be overcome by vernalization. Interestingly, FOF2 expression is regulated by light. The protein level of FOF2 accumulates in response to light, whereas it is degraded under dark conditions via the 26S proteasome pathway. Our findings suggest a possible mechanistic link between light conditions and the autonomous floral promotion pathway in Arabidopsis.     

Down‐regulation of BnDA1, whose gene locus is associated with the seeds weight,improves the seeds weight and organ size in Brassica napus

Brassica napus L. is an important oil crop worldwide and is the main raw material for biofuel. Seed weight and seed size are the main contributors to seed yield. DA1 (DA means big in Chinese) is an ubiquitin receptor and negatively regulates seed size. Down‐regulation of AtDA1 in Arabidopsis leads to larger seeds and organs by increasing cell proliferation in integuments. In this study, BnDA1 was down‐regulated in B. napus by over expressed of AtDA1^R358K, which is a functional deficiency of DA1 with an arginine‐to‐lysine mutation at the 358th amino acid. The results showed that the biomass and size of the seeds, cotyledons, leaves, flowers and siliques of transgenic plants all increased significantly. In particular, the 1000 seed weight increased 21.23% and the seed yield per plant increased 13.22% in field condition. The transgenic plants had no negative traits related to yield. The candidate gene association analysis demonstrated that the BnDA1 locus was contributed to the seeds weight. Therefore, our study showed that regulation of DA1 in B. napus can increase the seed yield and biomass, and DA1 is a promising target for crop improvement.     

The GRAS protein SCL13 is a positive regulator of phytochrome-dependent red light signaling, but can also modulate phytochrome A responses

Phytochrome photoreceptors enable plants to perceive divergent light signals leading to adaptive changes in response to differing environmental conditions. However, the mechanism of light signal transduction is not fully understood. Here we report the identification of a new signaling intermediate from Arabidopsis thaliana, Scarecrow-like (SCL)13, which serves as a positive regulator of continuous red light signals downstream of phytochrome B (phyB). SCL13 antisense lines exhibit reduced sensitivity towards red light, but only a distinct subset of phyB-mediated responses is affected, indicating that SCL13 executes its major role in hypocotyl elongation during de-etiolation. Genetic evidence suggests that SCL13 is also needed to modulate phytochrome A (phyA) signal transduction in a phyB-independent way. The SCL13 protein is localized in the cytoplasm, but can also be detected in the nucleus. Overexpression of both a nuclear and cytoplasmic localized SCL13 protein leads to a hypersensitive phenotype under red light indicating that SCL13 is biologically active in both compartments. SCL13 is a member of the plant-specific GRAS protein family, which is involved in various different developmental and signaling pathways. A previously identified phytochrome A signaling intermediate, PAT1, belongs to the same subbranch of GRAS proteins as SCL13. Although both proteins are involved in phytochrome signaling, each is specific for a different light condition and regulates a different subset of responses.     

From model to crop: functional characterization of SPL8 in M. truncatula led to genetic improvement of biomass yield and abiotic stress tolerance in alfalfa

Biomass yield, salt tolerance and drought tolerance are important targets for alfalfa (Medicago sativa L.) improvement. Medicago truncatula has been developed into a model plant for alfalfa and other legumes. By screening a Tnt1 retrotransposon‐tagged M. truncatula mutant population, we identified three mutants with enhanced branching. Branch development determines shoot architecture which affects important plant functions such as light acquisition, resource use and ultimately impacts biomass production. Molecular analyses revealed that the mutations were caused by Tnt1 insertions in the SQUAMOSA PROMOTER BINDING PROTEIN‐LIKE 8 (SPL8) gene. The M. truncatula spl8 mutants had increased biomass yield, while overexpression of SPL8 in M. truncatula suppressed branching and reduced biomass yield. Scanning electron microscopy (SEM) analysis showed that SPL8 inhibited branching by directly suppressing axillary bud formation. Based on the M. truncatula SPL8 sequence, alfalfa SPL8 (MsSPL8) was cloned and transgenic alfalfa plants were produced. MsSPL8 down‐regulated or up‐regulated alfalfa plants exhibited similar phenotypes to the M. truncatula mutants or overexpression lines, respectively. Specifically, the MsSPL8 down‐regulated alfalfa plants showed up to 43% increase in biomass yield in the first harvest. The impact was even more prominent in the second harvest, with up to 86% increase in biomass production compared to the control. Furthermore, down‐regulation of MsSPL8 led to enhanced salt and drought tolerance in transgenic alfalfa. Results from this research offer a valuable approach to simultaneously improve biomass production and abiotic stress tolerance in legumes.     

Do Strigolactones Regulate Bud Winter Dormancy and Charactrisitc Secondary Metabolism in Tea?

Tea (Camellia sinensis [L.] O. Kuntze.) is an important cash crop, which mainly uses tender shoots and young leaves for manufacturing. Due to the marketing characteristic that earlier made tea has higher price, the time of the breaking of winter dormancy buds in spring is extremely important in tea industry. Strigolactones are a group of carotenoids-derived metabolites which regulates bud outgrowth, shoot branching, tiller angle and environmental stress responses. The role of strigolactones in tea plant was briefly summarized in the current review, with an emphasis of the association of strigolactones on bud ecodormancy and shoot branching. The involvement of strigolactones on the biosynthesis of the tea characteristic metabolites flavonoids, caffeine and theanine were also discussed. Moreover, recent advances on the biosynthesis of strigolactones and its regulation by microRNAs and environmental stresses were also presented. This review provides a basis for future investigations underlying the mechanisms of strigolactones on bud winter dormancy and tea secondary metabolism.     

A root chicory MADS box sequence and the Arabidopsis flowering repressor FLC share common features that suggest conserved function in vernalization and de‐vernalization responses

Root chicory (Cichorium intybus var. sativum) is a biennial crop, but is harvested to obtain root inulin at the end of the first growing season before flowering. However, cold temperatures may vernalize seeds or plantlets, leading to incidental early flowering, and hence understanding the molecular basis of vernalization is important. A MADS box sequence was isolated by RT‐PCR and named FLC‐LIKE1 (CiFL1) because of its phylogenetic positioning within the same clade as the floral repressor Arabidopsis FLOWERING LOCUS C (AtFLC). Moreover, over‐expression of CiFL1 in Arabidopsis caused late flowering and prevented up‐regulation of the AtFLC target FLOWERING LOCUS T by photoperiod, suggesting functional conservation between root chicory and Arabidopsis. Like AtFLC in Arabidopsis, CiFL1 was repressed during vernalization of seeds or plantlets of chicory, but repression of CiFL1 was unstable when the post‐vernalization temperature was favorable to flowering and when it de‐vernalized the plants. This instability of CiFL1 repression may be linked to the bienniality of root chicory compared with the annual lifecycle of Arabidopsis. However, re‐activation of AtFLC was also observed in Arabidopsis when a high temperature treatment was used straight after seed vernalization, eliminating the promotive effect of cold on flowering. Cold‐induced down‐regulation of a MADS box floral repressor and its re‐activation by high temperature thus appear to be conserved features of the vernalization and de‐vernalization responses in distant species.     

MicroRNA156 as a promising tool for alfalfa improvement

A precursor of miR156 (MsmiR156d) was cloned and overexpressed in alfalfa (Medicago sativa L.) as a means to enhance alfalfa biomass yield. Of the five predicted SPL genes encoded by the alfalfa genome, three (SPL6, SPL12 and SPL13) contain miR156 cleavage sites and their expression was down‐regulated in transgenic alfalfa plants overexpressing miR156. These transgenic plants had reduced internode length and stem thickness, enhanced shoot branching, increased trichome density, a delay in flowering time and elevated biomass production. Minor effects on sugar, starch, lignin and cellulose contents were also observed. Moreover, transgenic alfalfa plants had increased root length, while nodulation was maintained. The multitude of traits affected by miR156 may be due to the network of genes regulated by the three target SPLs. Our results show that the miR156/SPL system has strong potential as a tool to substantially improve quality and yield traits in alfalfa.