| It has been demonstrated that the chloroplast small heat shock proteins (Cp-sHSP) are correlated with increased thermotolerance in a number of plant species. They appear to improve heat tolerance by protecting Photosystem II during high temperature stress. Our research is focused on determining the functional significance of the qualitative and quantitative variation in Cp-sHSP in plant species and ecotypes with differing levels of heat tolerance. To accomplish this we isolated and characterized Cp-sHSP genes from two heat-sensitive species Agrostis stononifera, Chenopodium album (New York and Mississippi ecotypes), two moderately heat-tolerant species, S partina alterniflora and Amaranthus retroflexus, and two very heat-tolerant species, Agave americana and Ferocactus wislizenii.; Gene-specific primers were designed from the methionine-rich region of the Cp-sHSP and these core sequences were used for PCR-based genome walking and 5' and 3' cDNA RACE to isolate the corresponding genes from all the plants. A total of eight different genes were isolated and characterized from plant species mentioned above.; We also compared the sequences of these genes and proteins at the nucleotide and amino acid level to determine their evolutionary relationships. Real-Time PCR was to quantify mRNA expression levels of the four Cp-sHSP family members isolated from C. album. CaHsp26.26m was constitutively expressed at very low levels in C. album (MS), but not in C. album (NY). The transcript levels of all of the four Cp-sHSP genes increased 10-20 folds under heat stress in both Chenopodium ecotypes. The expression Cp-sHSPs in response to heat stress was examined by immunoblot analysis using antibodies specific to met-rich region.; Analysis of the promoter sequences of these genes are additional number of "GAA" repeats, which are essential for the binding of heat shock factor, in heat shock elements (HSE) of heat tolerant plants. The pattern of 'GAA' is also distinct in each group of plant. The presence of these additional elements may be correlated with the greater thermotolerance.; We use the proteomics approach to study the effect of heat stress on chloroplast of a non-model CAM plant, A. americana. To better understand the function and CAM metabolism in chloroplast, we utilized multidimensional protein identification technology (MudPIT) to analyze proteomes for control and heat stressed plants. |
