Functional Analysis Of A Wheat Expansin Gene TaEXPB23

Wheat (Triticum aestivum L) is the second important food crop and its planting area only less than that of rice. The growth of wheat is often inhabited by environmental stresses, thus leads to the decrease of production. Expansins are a class of cell wall proteins that can regulate the extension of cell wall and the plant growth, and they exist in almost all the growing tissues and fruits. They were first isolated from cucumber hypocotyls by Cosgrove in1989because of their ability to restore long-term extension to cell walls. In recent years, the research on expansins deeply and the results gradually enriched. It has been found that expansins involved in many process in plant growth and development, and responsed to various environmental stresses.Base on our previous works of cloning a wheat expansin gene TaEXPB23and its overexpressing tobacco plants, here, we want to study the function of TaEXPB23in plant adaptability to water stress, salt stress, high temperature stress and phosphorus stress. Moreover, we want to analyze the physiological mechanisms of this gene underlying its stress adaptability. First, we cloned the promoter region of TaEXPB23and analyzed its spatial and temporal expression patterns in tobacco. Then, we studied the functions of TaEXPB23in various environmental stresses using TaEXPB23overexpressing tobacco plants. The main results are listed as follows:(1) In subcellular localization assays, TaEXPB23localized to the onion epidermal cell wall. Expression analysis using qRT-PCR demonstrated that the transcription pattern of TaEXPB23corresponded to the growth course of wheat coleop tiles.(2) Using the TAIL-PCR, we cloned the promoter region of TaEXPB23, and got996bp region upstreamof the initiation codon(GenBank ID:HQ391898). PlantCAREand PLACE databases analysis indicated that it had light responsive elements, tissue-specific and development-related elements, phytohormone (SA, MeJA) response elements and some abiotic stress (drought, ABA and salt) responsive elements. In2-week-old transgenic tobacco seedlings carrying the ProTaEXPB23::GUS plasmid, GUS expression was detected not only in the stem but also in the root. (3) To investigate the response of TaEXPB23expression to a variety of phytohormones, six kinds of phyto hormones, including ABA, MeJA, IAA, ET, GA3and NAA, were exogenous applied to the wheat seedlings. TaEXPB23expression levels were examined using qRT-PCR. When the wheat seedlings were exposed to GA3, ET and NAA, the expression levels of TaEXPB23were obviously inhibited compared to controls. While the expression levels of TaEXPB23were enhanced following MeJA treatment. Following IAA treatment, the expression of TaEXPB23was first inhibited and then induced. ABA decreased the expression level of TaEXPB23at the late stage. Besides, the mRNA level of TaEXPB23were significantly upregulated by salt stress, while downregulated by high temperature (HT).(4) Studies on adaptability of TaEXPB23overexpressing tobacco plants indicated that the transgenic tobacco plants showed enhanced tolerance to drought and salt stresses, while decreased tolerance to high temperature compared to wild-type (WT). Overexpression of TaEXPB23in tobacco conferred tolerance to salt stress by enhancing water retention ability (WRA) and decreasing osmotic potential (OP). The improved tolerance was indicated by better phenotype, longer primary root and higher survival rate than that of WT. Under drought stress, the transgenic plants showed improved growth phenotype and higher survival rate than WT plants. However, transgenic plants overexpressing TaEXPB23did not show any improvement in the tolerance to HT stress compared to WT.(5) The wheat coleoptiles showed significant changes in length under different P levels, further study suggested that both excess and low P levels may induce the expansion of cortex cell walls in wheat. Multiple expansins (five α-and nine β-expansin genes) were up-or down-regulated in response to changes of phosphorus levels and showed different expression patterns in wheat. Meanwhile, the expression level of TaEXPB23was up-regulated at excess-P condition, suggesting the involvement of TaEXPB23in phosphorus adaptability.(6) Under excess-P and low-P conditions, overexpression of the TaEXPB23resulted in improved phenotypes, particularly improved root system architecture, as indicated by the increased number of lateral roots in transgenic tobacco plants compared to WT. Thus, these transgenic plants maintained better photosynthetic gas exchange ability than the control (normal P level) under both P-sufficient and P-deficient conditions.(7) The expression level of TaEXPB23was obviously induced at both12and24h compared to control under MV-induced stress. The transgenic tobacco seedlings showed better growth, longer primary roots, higher fresh weights, and higher chlorophyll contents than WT seedlings following exposure to MV. The MDA content was lower in TaEXPB23over express ing plants than in WT. All of these results together suggest that overexpression of the wheat expansin TaEXPB23improved the tolerance to oxidative stress in transgenic tobacco plants.(8) Overexpressing TaEXPB23influenced the activity of antioxidant enzymes; in particular, the activity of the cell wall-bound peroxidase was strongly increased. The role of this enzyme was confirmed by the decreased oxidative stress tolerance of the Arabidopsis expansin mutant atexpb2. Not only the decreased cell wall-bound peroxidase activity, atexpb2also exhibited decreased chlorophyll contents and the germination rate compared to Col-0.