| Wheat is the most important food crops in China only to rice and corn and it has rich nutritional value and the high economic value. There are two types of wheat: winter wheat and spring wheat. Throughout the whole winter, the growing period of winter wheat is from its tillering stage to jointing stage. Because that winter wheat planting is restricted in the cold area of northern China, such as Heilongjiang province, the temperature in winter is very low. These areas become restricted zones of winter wheat planting, but have to plant spring wheat. Moreover, spring wheat also has its deficiencies, for example, poor tillering, low spike rate, and restricted by bad environmental factors spring drought, spring floods, high temperature and wet in harvest time, which results in unstable annual yield and high cost. If winter wheat is planted in those cold areas, bad environmental factors could be avoided of spring wheat planting and the output could be increased in 30%."Dongnongdongmai 1"is the only cultivar of winter wheat which could pass the winter securely in Heilongjiang province, and its greening rate is more than 85%.The previous researches on cold tolerance are mainly about comparing cold tolerance among species at the simulative low temperature indoor conditions, which is different from temperature and light (which related to the cold tolerance directly) of the natural growth conditions, especially can not realize the cooling exercise at the continually low temperature. In addition, the studies of cold tolerance of wheat seedling may use the young leaves as the materials, while in the northern cold area, underground organs are main part of winter wheat to pass the whole winter. Therefore, the studies simulated cold resistance under low-temperature indoor is difficult to represent their real findings at the actual situation and has limitations in guiding cold crop breeding and production.In this study, cold resistant variety Dongnongdongmai 1 (Jimai22, from Shan-dong province, as control) was used as experimental material. The seedling habit, greening rate, physiological and biochemical characteristics, and anatomical structure were investigated to reveal the physiological and structural mechanisms for cold resistance of winter wheat. In addition, the regulatory mechanism of hormone was also explored by using exogenous hormone at different ways and different concentrations. Moreover, cold proteins were obtained through the two-dimensional gel electrophoresis and mass spectrometry to reveal the molecular mechanism of cold resistance of winter wheat. This research provided the theoretical bases for revealing the mechanism of cold tolerance and finding the cold-related proteins of winter wheat, and had important theoretical and practical significance in selecting cold-resistant winter wheat germplasm resources and breeding new varieties in northern cold area of China.The main experimental results were as follows. (1)Seedling habits and greening rateThe seedlings of Donongdongmai 1 grew prostratly in the field overwintering , and theirs greening rate had significant correlation with soil water content and temperature. The soil water content of 25% was suitable environment for growing and greening of Donongdongmai 1. Under low temperature and long time of duration of low temperature, the greening rate decreased about 60%~100%. In certain extent, greening rate had a significantly positive correlation with temperature, and negative correlation with water content.(2)The characteristics of physiological and biochemical before overwinteringThe organs (root, tillering node and leaf) of Dongnongdongmai1 showed the following laws in metabolic adaptation and material preparations for resisting cold: Relative water content, tillering node> root> leaf; soluble sugar content , tillering node> root> leaf; protein content, leaf> tillering node > root; proline content, tillering node > root> leaf in early stage , root> tillering node > leaf in middle stage, and tillering node > leaf> root in late stage; POD activity, tillering node > root> leaf; SOD activity, tillering node > leaves> roots in early stage, leaf > tillering node > root in late stage; relative conductivity, root> tillering node > leaf; ABA content, leaf> tillering node > root in early stage, tillering node > leaf> root in late stage; GA content, leaf> root> tillering node; IAA content, leaf> tillering node > root in early stage, leaf > tillering node > root in late stage; ZR content, tillering node > leaf> root. The general results showed that tillering node was the most important organ of winter wheat for safely overwintering because of high cold resistant metabolic adaptation and strong ability for material preparations. And it directly affected greening rate of winter wheat in next year.The SOD activity, soluble sugar, soluble protein, proline, ABA, ZR and IAA content in the tillering node of Dongnongdongmai 1 showed the correlation at different levels, indicating that these physiological indices had synergistic effect to cold resistance of wheat. The soluble sugar had significant correlation with other six indices and it was the important index to show cold resistance of winter wheat dongnongdongmai 1.(3)Regulation of plant growth regulatorABA had significant effects on cold resistance of dongnongdongmai 1,the low concentration could promote the growth of wheat under low temperature,but the high concentrations inhibit that. For increasing the wheat cold resistance,the optimal concentration of ABA was 10-7M. Under low temperature at seeding stage, the ABA could increase chlorophyll content, soluble sugar content, and soluble protein content, improve the activity of SOD and decrease membrane permeability. The root absorption of ABA was better than leaf-spraying. In addition, the next year greening rate was increased by watering rhizosphere with different concentrations (0, 10-4, 10-5, 10-6, 10-7 M) of the ABA, GA and 6-BA in the field at early tillering seedlings. The general results showed that ABA and 6-BA could increase the greening rate at 10-5 M, but GA had the contrary effect, and decrease the greening rate to 0 at 10-4 M. (4)Cold resistance ccorelated proteinThe expression level of 55 protein spots were significantly different (±more than 2 folds) after low temperature stress -30℃in protein profiles of tillering node Dongnongdongmai 1 from pH4 to pH7. The high expressed protein spots were detected by matrix-assisted laser desorption ionization time-of -flight mass spectrometry (MALDI-TOF MS), and analyzed in retrieval database. According to the peptide mass fingerprints, 14.3% were stress protein(TIR-NBS-LRR-TIR type disease resistance protein, heat shock protein 70 precursor, non-cell-autonomous heat shock cognate protein 70, chilling-inducible protein,heat shock protein, 23.5 kDa heat-shock protein, heat shock protein 90, disease resistance protein-like, resposive to dessication 22, ascorbate peroxidase and class III peroxidase 59 precursor ) ; 23.2% were metabolism correlated protein(glucose-6-phosphate dehydrogenase, subfamily of Adenine nucleotide alpha hydrolases superfamily, phosphoenolpyruvate carboxylase, glyceraldehyde-3-phosphate dehydrogenase subunit B, ribulose-1,5- bisphosphate carboxylase, F0-F1 ATPase alpha subunit, lypoxygenase, alpha-glucosidase, 5,10-methylene-tetrahydrofolate reductase, starch synthase II, cytosolic malate dehydrogenase, self-incompatibility ribonuclease); 17.9% were signaling molecule(incle Protein tyrosine kinase, CBF-like protein, calmodulin,calmodulin binding protein-like, mitigen activated protein kinase, serin/threonine protein kinase, transcription factor< PWWP domain protein> -like protein); 17.9% were un-known function protein, and other protein took up 26.8%. The results indicated that these high or low expressed proteins might play an important role in dongnongdongmai1 cold resistance. Moreover, the shape of ice crystals significant changed as the temperature decreased, which indicated that antifreeze proteins were produced in tillering node.(5)Anatomic structure of tissue and cell for cold resistance of winter wheatStructure of Dongnongdongmai 1 (leaf, root and tillering node) was integrity at about -15℃. The inter-space among the leaf cells was big, and the root ray cells were small, and there are many interlace bulks arranged in a crisscross pattern in the tillering node. After low temperature, the chloroplast was ellipse, and grana lamellae and stroma lamellae of thylakoid arranged regularly and parallel-aligned among long axis of chloroplast, distributed uniformly in chloroplast. The mitochondria were a little intumesces, but the cristae were not absent. These structural features furtherly confirmed the high cold resistance of Dongnongdongmai 1. |
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