The Physiological Mechanisms Underlying Over-expressing BADH Gene Improves Chilling Tolerance Of Wheat

In natural environments, productivity and geographical distribution of plants are limited by adverse conditions, such as cold, salinity and drought. Among their various protective mechanisms for ensuring survival, one is the accumulation of compatible solutes, such as organic metabolites that are of low molecular weight. Glycinebetaine (GB) is a quaternary ammonium compound that regarded as an extremely effective compatible solute. GB level can be increased by GB transgenic engineering or GB application. Chilling stress disturbs the growth and development of wheat, therefore, decreases the production and quality of wheat seeds. In this study, the improvement of GB and its physiological mechanism involved in were researched with one wild-type wheat cultivar (Triticum aestivum L.) Shi 4185 and three BADH transgenic lines T1, T4, T6. The transgenic wheat lines were produced by transforming BADH gene from Atriplex hortensis L. The protection of over accumulating glycinebetaine (GB) to wheat plasma membrane and thylakoid membrane under chilling stress (0-2℃) and the underlying mechanisms involved in were detected. Indexes determined include content of GB, osmotic adjustment ability, plasma membrane H⁺-ATPase activity, superoxide radical (O₂^-) production rate and H₂O₂ content, anti-oxidant enzymes activities, content of compatible solutes, components of plasma membrane lipids, photosynthesis and chlorophyll fluorescence parameters, chlorophyll content, Hill reaction, chloroplast Ca²⁺-ATPase and Mg²⁺-ATPase activities, and the components of thylakoid membrane lipids. The main results are as follows:1. Over accumulated GB protected the structure and function of the plasma membrane in wheat under chilling stress.(1) Introducing of foreign BADH gene visibly enhanced the accumulation of GB in wheat leaves, and its accumulation is induced by chilling stress conditions.(2) Chilling stress destroyed the membrane integrity and stability, and ultimately resulted in evident increase in malondialdehyde (MDA) content and electrolyte leakage. Over accumulating GB effectively protected the PM of transgenic lines from chilling stress presented by lower MDA content and electrolyte leakage than WT.(3) Chilling stress induced the decrease of the activities of PM H⁺-ATPase in leaves of both WT and transgenic plants. This decrease was might due to the deactivation of membrane proteins induced by chilling. The fact that the decline in H⁺-ATPase activities was more in WT plant but less in transgenic plants suggested that the accumulation of GB in leaves of transgenic wheat plants could provide protection to the membrane proteins including H⁺-ATPase. Our results also showed that, upon transferred wheat plants to normal growth condition (25℃) from chilling (0-2℃), about 98 % of PM H⁺-ATPase activity in the transgenic plants was recovered compared with only 80 % of that in the WT plants, this was important for the cells in transformed lines to recover their functions after stress.(4) Chilling stress caused increase in superoxide radical (O₂^- ) production and H₂O₂ content. Some main antioxidant enzymes such as SOD, CAT, POD and APX of transgenic lines with over accumulating GB maintained higher activity under chilling stress, which then inhibited increase of O₂^- and H₂O₂ production. And ultimately, over accumulating GB was effectively decrease the chilling induced oxidative stress.(5) Besides osmolytes, the over accumulated GB could sustain a higher level of osmotic adjustment via accumulating some other compatible solutes such as proline, soluble sugar and soluble protein, these compatible solutes also contributed to the integrity and function of PM at chilling.(6) The destabilization of PM is one of the primary results of chilling stress. Transgenic lines with over accumulating GB can maintain a relatively stable status of PM under chilling stress in components and levels of lipids. It is suggested that over-accumulating GB endows the PM in the transgenic wheat lines with enhanced chilling tolerance.2. Over accumulating GB maintained higher level of photosynthesis through improving thylakoid membrane lipid components and function of protein complexes under chilling stress.(1) Upon exposure to chilling stress of 0² oC, the transgenic lines could maintain higher photosynthetic rate (Pn) and the maximal quantum yield of PSâ…¡photochemistry (Fv/Fm) than wild type (WT) plants, and the smaller decrease in chlorophyll content, Ca²⁺-ATPase and Mg²⁺-ATPase activities of thylakoid membrane, and Hill-reaction activity were also observed in transgenic lines compared to that of WT.(2) Thylakoid membrane lipids in WT and T6 transgenic line were also examined. The change in the lipid composition of the thylakoid membrane under chilling stress include (1) There was a significant decrease in the proportion of monogalactosyldiacylglycerols (MGDG), (2) the proportions of diagalactosyldiacylglycerols (DGDG) and phosphatidylglycerol (PG) were increased, (3) There were no significant changes in the proportions of sulfoquinovosyldiglyceride (SQDG) and phosphatidylcholin (PC). It was also shown that under chilling stress the relative content of MGDG was lower and the relative contents of DGDG and PG were higher in T6 than WT.(3) The fatty acid compositions of thylakoid membrane in WT and T6 were also examined. It was shown that the stabilization of index of unsaturated fatty acid (IUFA) of the thylakoid membrane in T6 was more than that in Shi 4185. Our results suggested that over-accumulation of GB as a consequence of the expression of BADH gene in the transgenic wheat plants increase the stability of thylakoid membrane compositions, including chlorophylls, proteins and lipids as well, which result in the higher photosynthetic activity in transgenic plants under chilling stress and recovery, and resultantly, the growth of the transgenic plants can recover quickly after chilling stress.From the results in this work, it showed that over-accumulating GB endows the plasma membrane and thylakoid membrane in the transgenic wheat lines with enhanced chilling tolerance. We propose that, the enhancement in chilling tolerance in transgenic lines might be relative to these aspects below: Firstly, GB ameliorates water status of cell by enhancing osmotic adjustment, which functioning by GB itself and other osmoyltes induced by GB. These compatible solutes are able to maintain membrane integrity by protecting membrane-bound proteins from chilling-induced inactivation and denaturation. Secondly, through maintaining or enhancing the activity of antioxidant enzymes, the accumulation of ROS and peroxidant level of membrane lipids are decreased, and the integrity and fluidity of membrane are sustained. Thirdly, over accumulating GB can protect the structure and function of protein complexes embed in PM and thylakoid membrane under chilling stress.