Shumei Zhou

The role of the F-box gene TaFBA1 from wheat (Triticum aestivum L.) in drought tolerance.

Drought is one of the most important factors limiting plant growth and development. We identified a gene in wheat (Triticum aestivum L.) under drought stress named TaFBA1. TaFBA1 encodes a putative 325-amino-acid F-box protein with a conserved N-terminal F-box domain and a C-terminal AMN1 domain. Real-time RT-PCR analysis revealed that TaFBA1 transcript accumulation was upregulated by high-salinity, water stress, and abscisic acid (ABA) treatment. To evaluate the functions of TaFBA1 in the regulation of drought stress responses, we produced transgenic tobacco lines overexpressing TaFBA1. Under water stress conditions, the transgenic tobacco plants had a higher germination rate, higher relative water content, net photosynthesis rate (Pn), less chlorophyll loss, and less growth inhibition than WT. These results demonstrate the high tolerance of the transgenic plants to drought stress compared to the WT. The enhanced oxidative stress tolerance of these plants, which may be involved in their drought tolerance, was indicated by their lower levels of reactive oxygen species (ROS) accumulation, MDA content, and cell membrane damage under drought stress compared to WT. The antioxidant enzyme activities were higher in the transgenic plants than in WT, which may be related to the upregulated expression of some antioxidant genes via overexpression of TaFBA1.

De Novo Assembly and Annotation of the Chinese Chive ( Allium tuberosum Rottler ex Spr.) Transcriptome Using the Illumina Platform

Chinese chive (A. tuberosum Rottler ex Spr.) is one of the most widely cultivated Allium species in China. However, minimal transcriptomic and genomic data are available to reveal its evolution and genetic diversity. In this study, de novo transcriptome sequencing was performed to produce large transcript sequences using an Illumina HiSeq 2000 instrument. We produced 51,968,882 high-quality clean reads and assembled them into 150,154 contigs. A total of 60,031 unigenes with an average length of 631 bp were identified. Of these, 36,523 unigenes were homologous to existing database sequences, 35,648 unigenes were annotated in the NCBI non-redundant (Nr) sequence database, and 23,509 unigenes were annotated in the Swiss-Prot database. A total of 26,798 unigenes were assigned to 57 Gene Ontology (GO) terms, and 13,378 unigenes were assigned to Cluster of Orthologous Group categories. Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, we mapped 21,361 unigenes onto 128 pathways. Furthermore, 2,125 sequences containing simple sequence repeats (SSRs) were identified. This new dataset provides the most comprehensive resource currently available for gene expression, gene discovery, and future genomic research on Chinese chive. The sequence resources developed in this study can be used to develop molecular markers that will facilitate further genetic research on Chinese chive and related species.

The Involvement of Wheat F-Box Protein Gene TaFBA1 in the Oxidative Stress Tolerance of Plants

As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT). The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD), were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants’ tolerance to multiple stress conditions.

Overexpression of wheat ubiquitin gene, Ta-Ub2, improves abiotic stress tolerance of Brachypodium distachyon.

Ubiquitination plays an important role in regulating plants development and adaptability to abiotic stress. To investigate the possible functions of a wheat monoubiquitin gene Ta-Ub2 in abiotic stress in monocot and compare it with that in dicot, we generated transgenic Brachypodium plants overexpressing Ta-Ub2 under the control of CaMV35s and stress-inducible RD29A promoters. The constitutive expression of Ta-Ub2 displayed slight growth inhibition in the growth of transgenic Brachypodium distachyon under the control conditions. However, this inhibition was minimized by expression of Ta-Ub2 under the control of stress-inducible RD29A promoter. Compared with WT, the transgenic plants preserved more water and showed higher enzymatic antioxidants under drought stress, which might be related to the change in the expression of some antioxidant genes. The expression of C-repeat binding factors transcription factor genes in the transgenic B. distachyon lines were upregulated under water stress. Salt and cold tolerances of transgenic B. distachyon were also improved. Although the phenotypic changes in the transgenic plants were different, overexpression of Ta-Ub2 improved the abiotic stress tolerance in both dicot and monocot plants. The improvement in Ta-Ub2 transgenic plants in abiotic stress tolerance might be, at least partly, through regulating the gene expression and increasing the enzymatic antioxidants.

The improvement of salt tolerance in transgenic tobacco by overexpression of wheat F-box gene TaFBA1

F-box protein is a major subunit of the Skp1-Cullin-F-box (SCF) complex. We previously isolated an F-box gene from wheat, TaFBA1, and here we show that overexpression of TaFBA1 in transgenic plants under salt stress increases germination rate, root elongation, and biomass accumulation compared with WT plants. Improvements in the photosynthetic rate and its corresponding parameters were also found in the transgenic plants. These results suggest that overexpression of TaFBA1 improves salt stress tolerance in transgenic tobacco. Further, the transgenic plants displayed less membrane damage, higher antioxidant enzyme activity, and less accumulation of ROS under salt stress. The transgenic plants also had lower Na+ content and higher K+ content than WT plants in leaves and roots. The activity of H+-ATPase on the plasma membrane in the transgenic plants was higher than in WT plants, and was accompanied by a net Na+ efflux. In the tonoplast, the activity levels of V-ATPase and PPase were also higher in the transgenic plants, thus helping to maximize intracellular Na+ compartmentalization. The expression of some stress-related genes was upregulated by salt stress. This suggests that the enhancement of plant salt stress tolerance may be associated with an improvement in antioxidative competition and Na+/K+ ion regionalization.

Stress-Inducible Expression of an F-box Gene TaFBA1 from Wheat Enhanced the Drought Tolerance in Transgenic Tobacco Plants without Impacting Growth and Development

E3 ligase plays an important role in the response to many environment stresses in plants. In our previous study, constitutive overexpression of an F-box protein gene TaFBA1 driven by 35S promoter improved the drought tolerance in transgenic tobacco plants, but the growth and development in transgenic plants was altered in normal conditions. In this study, we used stress-inducible promoter RD29A instead of 35S promoter, as a results, the stress-inducible transgenic tobacco plants exhibit a similar phenotype with wild type (WT) plants. However, the drought tolerance of the transgenic plants with stress-inducible expressed TaFBA1 was enhanced. The improved drought tolerance of transgenic plants was indicated by their higher seed germination rate and survival rate, greater biomass and photosynthesis than those of WT under water stress, which may be related to their greater water retention capability and osmotic adjustment. Moreover, the transgenic plants accumulated less reactive oxygen species, kept lower MDA content and membrane leakage under water stress, which may be related to their higher levels of antioxidant enzyme activity and upregulated gene expression of some antioxidant enzymes. These results suggest that stress induced expression of TaFBA1 confers drought tolerance via the improved water retention and antioxidative compete ability. Meanwhile, this stress-inducible expression strategy by RD29A promoter can minimize the unexpectable effects by 35S constitutive promoter on phenotypes of the transgenic plants.

NtLTP4, a lipid transfer protein that enhances salt and drought stresses tolerance in Nicotiana tabacum

Lipid transfer proteins (LTPs), a class of small, ubiquitous proteins, play critical roles in various environmental stresses. However, their precise biological functions remain unknown. Here we isolated an extracellular matrix-localised LTP, NtLTP4, from Nicotiana tabacum. The overexpression of NtLTP4 in N. tabacum enhanced resistance to salt and drought stresses. Upon exposure to high salinity, NtLTP4-overexpressing lines (OE lines) accumulated low Na+ levels. Salt-responsive genes, including Na+/H+ exchangers (NHX1) and high-affinity K+ transporter1 (HKT1), were dramatically higher in OE lines than in wild-type lines. NtLTP4 might regulate transcription levels of NHX1 and HKT1 to alleviate the toxicity of Na+. Interestingly, OE lines enhanced the tolerance of N. tabacum to drought stress by reducing the transpiration rate. Moreover, NtLTP4 could increase reactive oxygen species (ROS)-scavenging enzyme activity and expression levels to scavenge excess ROS under drought and high salinity conditions. We used a two-hybrid yeast system and screened seven putative proteins that interact with NtLTP4 in tobacco. An MAPK member, wound-induced protein kinase, was confirmed to interact with NtLTP4 via co-immunoprecipitation and a firefly luciferase complementation imaging assay. Taken together, this is the first functional analysis of NtLTP4, and proves that NtLTP4 positively regulates salt and drought stresses in N. tabacum.

TaPUB1, a Putative E3 Ligase Gene from Wheat, Enhances Salt Stress Tolerance in Transgenic Nicotiana benthamiana

High salinity is one of the most severe environmental stresses and limits the growth and yield of diverse crop plants. We isolated a gene named TaPUB1 from wheat (Triticum aestivum L. cv HF9703) that encodes a novel protein containing a U-box domain, the precursor RNA processing 19p (Prp19) superfamily and WD-40 repeats. Real-time reverse transcription-PCR analysis showed that TaPUB1 transcript accumulation was up-regulated by high salinity, drought and phytohormones, suggesting that it plays a role in the abiotic-related defense response. We overexpressed TaPUB1 in Nicotiana benthamiana to evaluate the function of TaPUB1 in the regulation of the salt stress response. Transgenic N. benthamiana plants (OE) with constitutively overexpressed TaPUB1 under the control of the Cauliflower mosaic virus 35S (CaMV 35S) promoter exhibited a higher germination rate, less growth inhibition, less Chl loss and higher photosynthetic capacity than wild-type (WT) plants under salt stress conditions. These results demonstrated the increased tolerance of OE plants to salt stress compared with the WT. The OE plants had lower osmotic potential (OP), reduced Na+ toxicity and less reactive oxygen species accumulation compared with the WT, which may be related to their higher level of osmolytes, lower Na+/K+ ratio and higher antioxidant enzyme activities under salt stress conditions. Consistent with these results, the up-regulated expression of osmic- and antioxidant-related genes in OE plants indicated a role for TaPUB1 in plant salt tolerance.

Altered gibberellin content affects growth and development in transgenic tobacco lines overexpressing a wheat gene encoding F-box protein

In a previous study, we have identified and characterized gene from wheat (Triticum aestivum L.) encoding F-box protein and named it TaFBA. In this paper, transgenic tobacco (Nicotiana tabacum L.) plants overexpressing TaFBA1 displayed accelerated growth early, but the rate slowed gradually at later stages of growth, and the mature transgenic plants were even shorter in stature and flowered later than did the wild type (WT). Treatment with gibberellin (GA) conferred an accelerated growth rate to the transgenic tobacco plants at later stages, similar to that of WT, whereas growth was inhibited more seriously in WT than in transgenic tobacco when plants were treated with a GA biosynthesis inhibitor. The content of GA in transgenic tobacco plants was higher at early developmental stages, but it was lower at later growth stages than in WT. Some GA biosynthesis genes were down regulated, which was accompanied with elevated expression of a GA catabolism gene. Thus, our results suggest that TaFBA1 is possibly involved in the regulation of plant growth and development, and that it may be related to the production, metabolism, and proper function of GA.

Molecular cloning and functional characterisation of the tomato E3 ubiquitin ligase SlBAH1 gene

Emerging evidence suggests that E3 ligases play critical roles in diverse biological processes, including pathogen resistance in plants. In the present study, an ubiquitin ligase gene (SlBAH1) was cloned from a tomato plant, and the functions of the gene were studied. The SlBAH1 gene contained 1002 nucleotides and encodes a protein with 333 amino acids. The SlBAH1 protein contains a SPX domain and a RING domain. SlBAH1 displayed E3 ubiquitin ligase activity in vitro. SlBAH1 was shown to localise in the nucleus, cytoplasm and plasma membrane by a subcellular localisation assay. The expression of SlBAH1 was induced by various hormones and Botrytis cinerea Pers. treatment. SlBAH1-silencing in plants obtained by virus-induced gene silencing (VIGS) technology enhanced resistance to B. cinerea, and the expression of pathogenesis-related (PR) genes, including PR1, PR2, PR4, PR5, and PR7, was significantly increased. These results indicate that the SlBAH1-dependent activation of defence-related genes played a key role in the enhanced fungal resistance observed in the SlBAH1-silenced plants and may be related to the SA-dependent and JA-dependent signalling pathways.