Rice (Oryza sativa L.) is sensitive to chilling particularly during early seedling development. Given the biochemical complexity of tolerance mechanisms, genetic potential for this trait depends on highly coordinated expression of many genes. We used a simple cDNA subtraction strategy to develop Expressed Sequence Tags (ESTs) that represent an important subset of cold stress-upregulated genes. The 3,084 subtracted cDNA clones represent a total of 1,967 unigenes from 1,354 singletons and 613 contigs. As expected in the developing seedlings, genes involved in basic cellular processes, i.e., metabolism, growth and development, protein synthesis, folding and destination, cellular transport, cell division and DNA replication were widely represented. Genes with stress-related and regulatory functions comprised 23.17% of the total ESTs. These categories included proteins with known function in cellular defenses against abiotic (drought, cold and salinity) and biotic (pathogen) stresses, and proteins involved in developmental and stress response signalling and transcription. Based on the types of genes represented, tolerance mechanisms rely on precise integration of developmental processes with stress-related responses. A large fraction of the ESTs (38.7%) represents unknown proteins. This EST library is a rich source of cold stress-related genes, and supplements for other publicly available libraries for comprehensive analysis of the stress-response transcriptome.

Mechanical wounding of 2-week-old maize (Zea mays L.) leaves, one of the first steps in both pathogen infection and herbivore attack, stimulates metabolism and activates signal transduction pathways dedicated to defense and recovery. The signaling pathways include reversible protein phosphorylation which can modulate protein activities, and transmit signals within cellular pathways and networks. We have used multiplex-staining of high-resolution 2D gels for protein (Sypro Ruby) and phosphorylation (Pro-Q Diamond) as a strategy for quantifying changes in the stoichiometry of phosphorylation after wounding for 270 protein spots. Rigorous statistical analysis of the time-index data allowed us to accept patterns of change in 125 of the spots as non-random, and these patterns were assigned to five clusters. A reliable identity was assigned to 21 selected proteins, most of which have been previously described as phospho-proteins. The results suggest that analysis of protein spots from high-resolution 2D gels by multiplex-staining for protein plus phosphorylation is a strategy that can be broadly useful for study of how the phospho-proteome responds to abiotic stress.

The sodium/lithium tolerance gene previously isolated from Arabidopsis thaliana (AtSLT1) and Nicotiana tabacum (NtSLT1) has been implicated in the regulation of ion homeostasis via the CaN and SPK1/Hal4 signal transduction pathways. A homologous gene from rice (OsSLT1) encoded by an intronless open-reading frame (1563 bp) on chromosome-1 was studied. The 58-kDa OsSLT1 protein contains a highly conserved C-terminal domain characteristic of the small heatshock protein (smHSP) and a-crystallin classes of molecular chaperons. Biochemical analysis of recombinant full-length, N-terminal-truncated (lacking aa-1 to aa-217), and C-terminal-truncated (lacking aa-443 to aa-521) versions of OsSLT1 indicated that removal of the C-terminal extension enhances chaperon activity as shown by the ability of the truncated protein to prevent thermal and non-thermal aggregation of client proteins in an ATP-independent manner. The C-terminal-truncated OsSLT1 also enhanced thermotolerance of recombinant Escherichia coli. Unlike the smHSPs, which are transcriptionally activated during stress, the activity of SLT1 appears to be modulated via complex mechanisms of protein cleavage. The implication of the results in relation to the immediate cellular defenses against protein denaturation during stress and to the previously demonstrated role of SLT1 in the CaN and SPK1/Hal4 signaling pathways is discussed.