Comparative Proteomics Of Manihot Esculenta Leaves And Chloroplasts Under Drought Stress

Environment stress has a significant impact on plant growth, development and agricultural production. Cassava (Manihot esculenta Crantz), as a Euphorbiaceae Manihot of Perennial plants, has some excellent characteristics including high environment stress resistance ability, high starch yield and survive in barren soil environment. The research involving in clarifying the drought resistance mechanism of cassava plants has some guiding significance to generate new varieties of drought-resistant crops.The proteomics can be implied in many fields which cannot be solved by genomics. At present, research on proteomics has reached subcellular level and post-translational modification level, which have lead to better understanding of the mechanism of stress resistance in plant. In plants, about30%of total proteins were phosphorylated. Protein phosphorylation and dephosphorylation played important roles in plant growth, development and stress regulation.In this study, cassava (SC8) was used as experimental materials. To understand the response mechanism of cassava under different levels of drought stress, morphological characteristics and ultramicroscopic structure of mesophyll cell in cassava were studied; also, phosphoproteomics and subcellular proteomics were carried out to find out the key proteins of associated with drought stress. The results resulted in the following conclusions:1. The result of the morphology analysis as to cassava under different degrees of drought stress as follows:In control,At the earlier stage of drought stress, cassava leaves began wilting; From low degree to heavy degree of drought stress, cassava leaves turn browning and dropping, but the uppermost leaves usually remain green.The research indicating cassava give priority to ensure the photosynthesis process and physiological activities to continue normally when suffer drought stress2. The ultrastructural feature of cassava mesophyll cells under different levels of drought stress shows:The shape of chloroplasts in mesophyll cells was changed from shapes of kidney, spindle or arciform to ellipse or irregular; Chloroplasts location in mesophyll cell occurs change; The grana lamella structures of choroplasts become loose; The number of starch grains and osmiophilic particles in chloroplasts was increased, and the size was enlarged.3. In order to further study the drought response mechanism of Manihot esculenta Crantz on protein level, phosphoproteomics research was carried out. With plus or minus1.5fold protein expression as a standard,33up-regulation protein spots and32down-regulation protein spots under drought stress for five days and49up-regulation protein spots and36down-regulation protein spots under drought stress for fifteen days. We selected49significantly altered phosphoproteins spots for In-gel enzymatic digestion and MALDI TOF/TOF MS/MS analysis; According to the NCBI database searching results,28differentially expressed phosphoproteins were identified in all. These proteins can be divided into7categories, including:(1)28%photosynthesis related proteins;(2)18%energy metabolism related proteins;(3)14%carbon metabolism related proteins;(4)11%defense and stress tolerance related proteins;(5)11%protein binding proteins;(6)7%transcription and translation related proteins;(7)11%predicted proteins.4. Study of comparative proteomics on Manihot esculenta Crantz chloroplast under different degrees of drought stress was carried out.57differential expression chloroplast proteins were positively identified by MALDI TOF/TOF MS/MS through the combination of peptide mass fingerprinting (PMF) and peptide fragmentation fingerprinting (PFF) analysis, including29up-regulation protein spots and28down-regulation protein spots. The majority of these proteins involved in photosynthesis and energy metabolism, minority concentrated in carbon metabolism, antioxidant, a transcription factor and a defense reaction.Among them, Rubisco large subunit and ATP synthase subunit protein content were the most abundant ones.In summary, during drought stress, leaves on the top of cassava keep normal growth state by means of resisting the external environment stress resistance; mesophyll cell changes in the structure plays an important role in drought resistance;and effective regulation of drought response protein also plays an important role in cassava drought resistance. Comparative proteomics of Manihot esculenta leaves and chloroplasts under drought stress provide preliminary data for further study cassava drought tolerance mechanism.