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Abstract
Plants, like all organisms, produce heat stress proteins (HSPs) in response to high temperature treatment. Some of the low molecular weight HSPs are related to the mammalian eye lens alpha-crystallin proteins. These proteins are called the small HSPs (sHSPs) and are particularly diverse and abundant in plants. In this dissertation, the maize (Zea mays L. inbred B73) mitochondrial 22-kD sHSP (HSP22) and the mitochondrial homologs of the Escherichia coli molecular chaperone proteins DnaK (HSP70) and GroEL (cpn60) are identified and characterized. A partiai cDNA for maize mitochondrial HSP22 was cloned and the full length sequence was obtained with data from the Pioneer Hi-Bred EST database.
Monoclonal antibodies were developed to maize mitochondrial HSP70, cpn60, and HSP22, and their cross-reactivity to homologs in other subcellular fractions and to different species was analyzed. Heat stress (42$\sp\circ$C for 4 h) induced mitochondrial accumulation of HSP22 but the levels of HSP70 and cpn60 proteins did not change significantly. Expression of mitochondrial HSP22 was evaluated with respect to induction temperature in etiolated seedlings and green maize leaves. The accumulation and degradation of HSP22 protein and mRNA during prolonged heat stress and the subsequent relief of stress were also analyzed.
HSP22 was purified by anion-exchange, hydrophobic interaction and reverse phase liquid chromatography. HSP22 was resolved into two peaks that were analyzed using electrospray ionization mass spectrometry. In each peak two forms of HSP22, which differed in mass by 80 D were identified. The two proteins (19,425 and 19,505 D) in the first HSP22 peak were 57 D larger than the two proteins (19,368 and 19,448 D) in the second peak. The mono-phosphorylation of HSP22, indicated by the presence of a +80 D modification, was confirmed by in organello phosphorylation of HSP22 by (gamma-$\sp{32}$P) ATP.
The effects of prolonged heat stress on maize seedling mitochondrial enzyme activity and membrane integrity were analyzed. Maize mitochondrial HSP22 was found to be expressed under field conditions, indicating that mitochondrial sHSP expression is widespread in nature.
