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...

We isolated cDNA clones for two nuclear-encoded, organellar members of the Arabidopsis hsp70 gene family, mtHsc70-2 (AF217458) and cpHsc70-2 (AF217459). Together with the completion of the genome sequence, the hsp70 family in Arabidopsis consists of 14 members unequally distributed among the five chromosomes. To establish detailed expression data of this gene family, a comprehensive reverse transcriptase-polymerase chain reaction analysis for 11 hsp70s was conducted including analysis of organ-specific and developmental expression and expression in response to temperature extremes. All hsp70s showed 2-to 20-fold induction by heat shock treatment except cpHsc70-1 and mtHsc70-1, which were unchanged or repressed. The expression profiles in response to low temperature treatment were more diverse than those evoked by heat shock treatment. Both mitochondrial and all cytosolic members of the family except Hsp70b were strongly induced by low temperature, whereas endoplasmic reticulum and chloroplast members were not induced or were slightly repressed. Developmentally regulated expression of the heat-inducible Hsp70 in mature dry seed and roots in the absence of temperature stress suggests prominent roles in seed maturation and root growth for this member of the hsp70 family. This reverse transcriptase-polymerase chain reaction analysis establishes the complex differential expression pattern for the hsp70s in Arabidopsis that portends specialized functions even among members localized to the same subcellular compartment.

SummaryExposure of Arabidopsis to low temperatures results in cold acclimation where freezing tolerance is enhanced. To achieve a wider view of the role of transcriptome to biochemical changes that occur during cold acclimation, analyses of concurrent transcript and metabolite changes during cold acclimation was performed revealing the dynamics of selected gene-metabolite relationships. Exposure to low temperature resulted in broad transcriptional and metabolite responses. Principal component analysis revealed sequentially progressive, global changes in both gene expression and metabolite profiles during cold acclimation. Changes in transcript abundance for many metabolic processes, including protein amino acid biosynthetic pathways and soluble carbohydrates, during cold acclimation were observed. For some metabolic processes, changes in transcript abundance temporally correlated with changes in metabolite levels. For other metabolic processes, changes in transcript levels were not correlated with changes in metabolite levels. The present findings demonstrate that regulatory processes independent of transcript abundance represent a key part of the metabolic adjustments that occur during cold acclimation.

Hsp70s function as molecular chaperones. The protective chaperone activities of hsp70 help to confer tolerance to heat, glucose deprivation, and drought. Overexpression of hsp70s in many organisms correlates with enhanced thermotolerance, altered growth, and development. To better understand the roles of hsp70 proteins in Arabidopsis, the molecular and physiological consequences of altered expression of the major heat shock cognate, Hsc70-1, were analyzed. Extensive efforts to achieve underexpression of Hsc70-1 mRNA using a full-length antisense cDNA resulted in no viable transgenic plants, suggesting that reduced expression is lethal. Constitutive overexpression of Hsc70-1 also appeared to be deleterious to viability, growth, and development because fewer transformants were recovered, and most were dwarfed with altered root systems. Despite being dwarfed, the overexpression plants progressed normally through four selected developmental stages. Heat treatment revealed that Hsc70-1 overexpression plants were more tolerant to heat shock (44°C for 10 min). The elevated basal levels of HSC70-1 in transgenic plants led to delayed heat shock response of several heat shock genes. The data in this study suggest that tight regulation of Hsc70-1 expression is critical for the viability of Arabidopsis and that the functions of HSC70-1 contribute to optimum growth, development, thermotolerance, and regulation of the heat shock response.