The Mechanism Of Small Heat Shock26(sHSP26) Protecting Maize Chloroplast From Heat Stress

small heat shock proteins （sHSPs） play an important role in response to stresstolerance in plants. Chloroplast-localized small heat shock proteins, members of a super-family ofheat shock proteins, function in the protection of PSⅡduring heat stress. Recent studies have shownthat, the expression of sHSP26in maize is induced only under the heat stress and the combinationof drought and heat Stress, but not induced under normal and drought conditions.The subcellularlocalization of sHSP26was only speculated located in the chloroplasts by software analysis and invitro experiments, To our knowledge, no direct data are available for sHSP26subcellularlocalization, and it is not clear whether sHSP26play a role in protecting PS II from heat stress.Therefore, Use "Zhengdan958" as experimental material, it is important to study intracellular sitesand expression characteristics of sHSP26under the heat stress in maize, which is helpful to clarifythe influence of sHSP26on chloroplast proteome and PSII function in maize.The main results areas follows:1. To precisely define the subcellular distribution of the sHSP26protein, electron microscopyimmunogold labeling was performed. Our results showed that immunogold labeling with thesHSP26antiserum was found in the chloroplasts. To investigate the H₂O₂accumulation in theleaves of maize plants exposed to heat stress, a cytochemical technique with CeCl3, which reactswith H₂O₂to produce electron-dense deposits of cerium perhydroxides was used. Our resultsshowed that CeCl3deposits, indicative of the accumulation of H₂O₂, were not observed in themesophyll cells in maize leaves under normal conditions. H₂O₂accumulation was observed in thecell walls facing intercellular spaces and in chloroplasts under heat stress, but was visible only inthe cell walls facing intercellular spaces under drought stress.2. In this study, the PEG-mediated transfection procedure was applied to establish theprotoplast transformation.To determine the optimum concentration of dsRNA for PEG-mediatedtransformation, dsRNA was transformed into maize protoplasts at concentrations of0,10,15,20,25and30μg/106protoplasts/ml.Transformation efficiencies were determined by analyzing sHSP26gene expression, concentrations above20μg （20,25or30μg） significantly suppressed the sHSP26gene expression, but25or30μg almost fully suppressed the expression of sHSP26gene.To further prove the effectiveness of25μg dsRNA transient silencing, we compared sHSP26gene expression among protoplast without any treatment, protoplast with heat treatment, protoplast with heat treatment after PEG transfer water, protoplast with heat treatment after PEG transferwater and protoplast with heat treatment after PEG transfer dsRNA. The results showed thatsHSP26expression in protoplast without any treatment was dimly detected, but sHSP26expressionin protoplast with heat treatment after PEG transfer water and protoplast with heat treatment afterPEG transfer water, strongly detected and was no significantly difference.3. The concentrations25μg dsRNA was used in this study. A proteomics approach was adopted to identifythe expression of proteins of maize leaves after transient RNAi under heat stress. In the case of the2-DE analysisof protein samples, more than350protein spots were reproducibly detected in each CBB-stained gel, among theseproteins,45specific proteins, which were significantly influnced by transient suppresse of sHSP26expression.The45proteins were identified by using MALDI-TOF MS and the tools of Protein analysis.which were classifiedinto twelve functional categories including ATP synthesis （12）, antioxidant defense （3）, CO₂fixation （4）, lightharvesting （2）, electron transporter （7）, chlorophyll synthesis （1）, cytoskeleton （1）, chaperone function （2）, signaltransduction （3）, protein synthesis （1）, chlorophyll fluorescence （1） and uncharacterized protein （8）.The35chloroplast proteins are related to the photosynthetic electron transport chain, carbon assimilation,anti-oxidation protection system protein.4. In order to further proving the proteins associated with sHSP26, co-immunoprecipitationmethod was used, and72proteins belonging to the chloroplast were identified by massspectrometry. among these proteins,11specific proteins were the same to2-DE after RNAi, as thefollows, ATP synthase（subunit alpha, subunit beta, subunit gamma）, Phosphoglycerate kinasechloroplastic, Ribulose bisphosphate carboxylase large chain, Oxygen-evolving enhancer protein1,2-cys peroxiredoxin BAS1, Photosystem I reaction center subunit VI, Chlorophyll a-b bindingprotein, Germin-like protein and Actin-1.5. After transient suppresse of sHSP26expression, the gene expression of psbA which is agene coding D1protein, psbQ that is16kDa protein of Oxygen-evolving complex, RUBP did notchange significantly compared to no RNAi under heat stress. This shows that sHSP26did not affectthe gene expression of RUBP, psbA and psbQ, may only affect their protein expression.In summary, these results confirmed that sHSP26played an important role in protecting thechloroplast protein and PSII function of maize leaves from heat stress. These findings don’t onlyprovide a theoretical basis for understanding the mechanism of sHSP26protecting maize underHeat stress, but also to provid a theoretical foundation for cloning and building sHSP26expressionvector, further cultivating new maize varieties with a comprehensive resistance.