Researches On The Effects Of Cold Stress On Ultra Structure,Resistant Physiology And Relative Gene Expressions In Neosinocalamus Affinis

Cold is one of the most important environmental stresses affecting plant growth and crop productivity.Low temperature around 0? would limit the geographical distribution and growing season of many crops and cause significant crop losses.As a woody perennial grass,Bamboo is distinguished by its growth speed,that almost faster than any of other woody plants.The rapid growth,high specific gravity and long fiber length of bamboo make it preferred for the paper and pulp production,and it is also widely used in architecture,furniture,foods and care industry.Wheras,low temperature is one of the key factors to limit the growing and geographical distribution of bamboo.Here,we used Neosinocalamus affinis and Bambusa pervariabilis × Dendrocalamopsis grandis from Ya'an,Sichuan as materials,and simulated the extreme situation the bamboo species may encounter in southern China,in order to explore the mechanisms of responses to cold stress in physiological,subcellular and molecular levels of bamboo.(1)The symptoms occurred in electron micrographs of bamboo under cold stress,including swelling and disorganization of chloroplast,reduced size and number of starch grains and lipid droplet accumulation in chloroplasts,are largely similar to other species.According to the classification offered by Kratsch and Wise,bamboo is one of the most chilling-sensitive species.Hydrolyzation of starch could produce soluble sugars to lower stromal water potential in time when chloroplast photosynthate is reduced,thus to protect cells from loss of water or even plasmolysis at last.However,the figure of ultrastructure showed that the reduction of starch grains was more obvious in Neosinocalamus affinis than hybrid bamboo.As a result,the hydrolyzation of starch grains is one of the mechanisms to improve cold-resistance of Neosinocalamus affinis under cold stress.(2)Under cold stress,Ch1 contents of both bamboo species gradually decreased with decreasing temperature,while the membrane permeability and malondialdehyde(MDA),the soluble sugar content,free proline content and electrolyte leakage significantly increased,and the dismutase(SOD),peroxidase(POD)and catalase(CAT)activities remarkably increased.All above the physiological indexes except for MDA and electrolyte leakage in the leaves of Neosinocalamus affinis were significantly higher than those of hybrid bamboo under the same low temperature stress.Results indicated that under cold stress,Neosinocalamus affinis could actively adjust its physiological function to adapt for the low temperature and to increase the resistance to cold.Neosinocalamus affinis showed highly stronger resistance to the cold than hybrid bamboo.(3)We obtained partial sequences of FAD,Cu/Zn-SOD,Mn-SOD and ACT genes in Neosinocalamus affinis,they are 633bp,503bp,459b and 756bp.Among them,Cu/Zn-SOD contained one complete CDS and the others covered partial CDS.Real-time quantitative PCR analysis revealed that FAD,Cu/Zn-SOD and Mn-SOD were up-regulated to different extents,indicating that they are all cold-inducible genes,(4)Cu/Zn-SOD and Mn-SOD presented similar patterns of dynamic responses in Neosinocalamus affinis under cold stress.The regularity of SOD isoenzymes response to cold stress was not as clear as FAD,because SODs activities are not only transcriptionally regulated,but also influenced by the interactions of Catalase,Peroxidase and Superoxide in antioxidative system.(5)FAD gene was more strongly induced by cold stress than SODs.In addition,the transcripts accumulation of FAD was more aboundant as the temperature went down.We proposed that the up-regulation of FAD was relative to the cold-resistance mechanism of Neosinocalamus affinis.(6)In this experiment,ultrastructural changes and physiological researches showed that Neosinocalamus affinis is more cold-endurable and cold-resistant contrast to hybrid bamboo.By real-time quantitative PCR analysis,we identified that FAD,Cu/Zn-SOD and Mn-SOD are all cold-inducible genes,and the up-regulation of FAD was relative to the cold-resistance mechanism of Neosinocalamus affinis.The identification of bamboo cold-responsive genes could be an important resource in bamboo molecular breeding programs.