Metabolic profiling analyses were performed to determine metabolite temporal dynamics associated with the induction of acquired thermotolerance in response to heat shock and acquired freezing tolerance in response to cold shock. Low-M r polar metabolite analyses were performed using gas chromatography-mass spectrometry. Eighty-one identified metabolites and 416 unidentified mass spectral tags, characterized by retention time indices and specific mass fragments, were monitored. Cold shock influenced metabolism far more profoundly than heat shock. The steady-state pool sizes of 143 and 311 metabolites or mass spectral tags were altered in response to heat and cold shock, respectively. Comparison of heat-and cold-shock response patterns revealed that the majority of heat-shock responses were shared with cold-shock responses, a previously unknown relationship. Coordinate increases in the pool sizes of amino acids derived from pyruvate and oxaloacetate, polyamine precursors, and compatible solutes were observed during both heat and cold shock. In addition, many of the metabolites that showed increases in response to both heat and cold shock in this study were previously unlinked with temperature stress. This investigation provides new insight into the mechanisms of plant adaptation to thermal stress at the metabolite level, reveals relationships between heat-and cold-shock responses, and highlights the roles of known signaling molecules and protectants.Environmental stresses arise from conditions that are unfavorable for the optimal growth and development of organisms (Levitt, 1972;Guy, 1999). Environmental stresses can be classified either as abiotic or biotic. Abiotic stresses are produced by inappropriate levels of physical components of the environment, including temperature extremes. Biotic stresses are caused by pathogens, parasites, predators, and other competing organisms. Even though biotic and abiotic stresses cause injury through unique mechanisms that result in specific responses, all forms of stress seem to elicit a common set of responses (Levitt, 1972). For instance, both biotic and abiotic stresses can result in oxidative stress through the formation of free radicals, which are highly destructive to lipids, nucleic acids, and proteins (Mittler, 2002). Another example is water stress, which is produced as a secondary stress by chilling, freezing, heat, and salt, as a tertiary stress by radiation, and, of course, as a primary stress during drought (Levitt, 1972).The ability of most organisms to survive and recover from unfavorable conditions is a function of basal and acquired tolerance mechanisms. Acquired tolerance involves a set of mechanisms that can transiently extend or improve overall stress tolerance (Levitt, 1972;Hallberg et al., 1985;Guy, 1999;Thomashow, 1999) following exposure to moderate stress conditions. For example, if plants are preexposed to a nonlethal high temperature, they can acquire enhanced tolerance to otherwise lethal high temperatures. Similarly, many plants can tolerate a gre...

The 70-kD heat-shock proteins (HSP70s) are encoded by a multigene family in eukaryotes. In plants, the 70-kD heat-shock cognate (HSC70) proteins are located in organellar and cytosolic compartments of cells in most tissues. Previous work has indicated that HSC7O proteins of spinach (Spinacia oleracea) are adively synthesized during cold-acclimating conditions. We have isolated, sequenced, and characterized cDNA and genomic clones for the endoplasmic reticulum (ER) luminal HSC70 protein (immunoglobulin heavy chain-binding protein; BiP) of spinach. The spinach ERluminal HSC70 is a constitutively expressed gene consisting of eight exons. Spinach BiP mRNA appears to be up-regulated during cold acclimation but is not expressed during water stress or heat shock. In contrast to the differential regulation of mRNA, the ERluminal HSC70 protein levels remain constant in response to various environmental stresses. Two other members of the spinach 70-kD heat-shock (HS70) multigene family also show differential expression in response to a variety of environmental stresses. A constitutively expressed cytosolic HSC70 protein in spinach appears also to be up-regulated in response to both cold-acclimating and heat-shock treatments. Spinach also contains a cold-shockinduced HS70 gene that is not expressed during heat shock or water stress. Since HSP70s are considered to be involved with the chaperoning and folding of proteins, the data further support the concept that they may be important for maintaining cellular homeostasis and proper protein biogenesis during cold acclimation of spinach.HSP70s are a ubiquitous group of evolutionarily conserved 70-kD proteins (Craig, 1989) encoded by a multigene family in eukaryotes and by a single gene, dnaK, in Escherichia coli. In yeast, nine HS70 genes have been identified and subdivided into four subgroups of which members of severa1 subgroups are considered essential for growth under various conditions (Craig, 1989). HS70 genes have been further classified into two groups: (a) cognate (HSC70) genes, which are expressed under normal growth conditions, frequently contain introns and are not strongly induced by heat shock and (b) those that are not expressed under normal conditions

The characterization of a cDNA for an 85 kDa spinach protein, CAP85 (cold acclimation protein) that is responsive to cold acclimation and water stress is described. Both transcript and protein levels are increased during cold acclimation and water stress. A novel characteristic of CAP85 is the presence of an 11 amino acid, lysine-rich repeat, common to Group 2 LEAs (late embryogenesis abundant proteins), which is included within a larger repeating motif present in 11 copies. Two other motifs of 8 and 16 residues are also found in three and four copies, respectively. CAP85 like other dehydrins and cold-regulated polypeptides remains soluble upon boiling. Protein blot analyses indicate that CAP85 protein is expressed in all aerial tissues as well as in roots. RNA blots show the presence of mRNA for the 85 kDa protein in leaf, petiole, and root tissue. Cell fractionation studies suggest that CAP85 is predominantly found in the cytosol.

Spinach (Spinacia oleracea L.) seedlings exposed to low nonfreezing temperatures (0-10° C) that promote cold acclimation, synthesize a variety cold-acclimation proteins and at the same time acquire a greater ability to withstand cellular dehydration imposed by the freezing of tissue water. Two of these proteins (160 and 85 kDa) become more abundant over time at low temperature. In addition, a small decline in tissue water status from a maximally hydrated state also appears to be associated with an initiation of the accumulation of these proteins at a noninductive temperature. Imposing a severe water stress on young seedlings grown at 25° C by withholding water leads to substantial accumulation of the 160- and 85-kDa proteins, and maximal induction of freezing tolerance. This evidence implies that responses to cold acclimation and water stress involve common mechanisms, and further establishes the linkage of these two proteins with stresses having an osmotic component.