To investigate the importance of different processes to heat stress tolerance, 45 Arabidopsis (Arabidopsis thaliana) mutants and one transgenic line were tested for basal and acquired thermotolerance at different stages of growth. Plants tested were defective in signaling pathways (abscisic acid, salicylic acid, ethylene, and oxidative burst signaling) and in reactive oxygen metabolism (ascorbic acid or glutathione production, catalase) or had previously been found to have temperature-related phenotypes (e.g. fatty acid desaturase mutants, uvh6). Mutants were assessed for thermotolerance defects in seed germination, hypocotyl elongation, root growth, and seedling survival. To assess oxidative damage and alterations in the heat shock response, thiobarbituric acid reactive substances, heat shock protein 101, and small heat shock protein levels were determined. Fifteen mutants showed significant phenotypes. Abscisic acid (ABA) signaling mutants (abi1 and abi2) and the UV-sensitive mutant, uvh6, showed the strongest defects in acquired thermotolerance of root growth and seedling survival. Mutations in nicotinamide adenine dinucleotide phosphate oxidase homolog genes (atrbohB and D), ABA biosynthesis mutants (aba1, aba2, and aba3), and NahG transgenic lines (salicylic acid deficient) showed weaker defects. Ethylene signaling mutants (ein2 and etr1) and reactive oxygen metabolism mutants (vtc1, vtc2, npq1, and cad2) were more defective in basal than acquired thermotolerance, especially under high light. All mutants accumulated wild-type levels of heat shock protein 101 and small heat shock proteins. These data indicate that, separate from heat shock protein induction, ABA, active oxygen species, and salicylic acid pathways are involved in acquired thermotolerance and that UVH6 plays a significant role in temperature responses in addition to its role in UV stress.

The small heat shock proteins (sHSPs) recently have been reported to have molecular chaperone activity in vitro; however, the mechanism of this activity is poorly defined. We found that HSP18.1, a dodecameric sHSP from pea, prevented the aggregation of malate dehydrogenase (MDH) and glyceraldehyde‐3‐phosphate dehydrogenase heated to 45°C. Under conditions in which HSP18.1 prevented aggregation of substrates, size‐exclusion chromatography and electron microscopy revealed that denatured substrates coated the HSP18.1 dodecamers to form expanded complexes. SDS–PAGE of isolated complexes demonstrated that each HSP18.1 dodecamer can bind the equivalent of 12 MDH monomers, indicating that HSP18.1 has a large capacity for non‐native substrates compared with other known molecular chaperones. Photoincorporation of the hydrophobic probe 1,1′‐bi(4‐anilino)naphthalene‐5,5′‐disulfonic acid (bis‐ANS) into a conserved C‐terminal region of HSP18.1 increased reversibly with increasing temperature, but was blocked by prior binding of MDH, suggesting that bis‐ANS incorporates proximal to substrate binding regions and that substrate–HSP18.1 interactions are hydrophobic. We also show that heat‐denatured firefly luciferase bound to HSP18.1, in contrast to heat‐aggregated luciferase, can be reactivated in the presence of rabbit reticulocyte or wheat germ extracts in an ATP‐dependent process. These data support a model in which sHSPs prevent protein aggregation and facilitate substrate refolding in conjunction with other molecular chaperones.

Plants are sessile organisms, and their ability to adapt to stress is crucial for survival in natural environments. Many observations suggest a relationship between stress tolerance and heat shock proteins (HSPs) in plants, but the roles of individual HSPs are poorly characterized. We report that transgenic Arabidopsis plants expressing less than usual amounts of HSP101, a result of either antisense inhibition or cosuppression, grew at normal rates but had a severely diminished capacity to acquire heat tolerance after mild conditioning pretreatments. The naturally high tolerance of germinating seeds, which express HSP101 as a result of developmental regulation, was also profoundly decreased. Conversely, plants constitutively expressing HSP101 tolerated sudden shifts to extreme temperatures better than did vector controls. We conclude that HSP101 plays a pivotal role in heat tolerance in Arabidopsis. Given the high evolutionary conservation of this protein and the fact that altering HSP101 expression had no detrimental effects on normal growth or development, one should be able to manipulate the stress tolerance of other plants by altering the expression of this protein.