Heat-shock protein 90 (Hsp90) chaperones the maturation of many regulatory proteins and, in the fruitfly Drosophila melanogaster, buffers genetic variation in morphogenetic pathways. Levels and patterns of genetic variation differ greatly between obligatorily outbreeding species such as fruitflies and self-fertilizing species such as the plant Arabidopsis thaliana. Also, plant development is more plastic, being coupled to environmental cues. Here we report that, in Arabidopsis accessions and recombinant inbred lines, reducing Hsp90 function produces an array of morphological phenotypes, which are dependent on underlying genetic variation. The strength and breadth of Hsp90's effects on the buffering and release of genetic variation suggests it may have an impact on evolutionary processes. We also show that Hsp90 influences morphogenetic responses to environmental cues and buffers normal development from destabilizing effects of stochastic processes. Manipulating Hsp90's buffering capacity offers a tool for harnessing cryptic genetic variation and for elucidating the interplay between genotypes, environments and stochastic events in the determination of phenotype.

Our understanding of gene regulation in plants is constrained by our limited knowledge of plant cis-regulatory DNA and its dynamics. We mapped DNase I hypersensitive sites (DHSs) in A. thaliana seedlings and used genomic footprinting to delineate ∼ 700,000 sites of in vivo transcription factor (TF) occupancy at nucleotide resolution. We show that variation associated with 72 diverse quantitative phenotypes localizes within DHSs. TF footprints encode an extensive cis-regulatory lexicon subject to recent evolutionary pressures, and widespread TF binding within exons may have shaped codon usage patterns. The architecture of A. thaliana TF regulatory networks is strikingly similar to that of animals in spite of diverged regulatory repertoires. We analyzed regulatory landscape dynamics during heat shock and photomorphogenesis, disclosing thousands of environmentally sensitive elements and enabling mapping of key TF regulatory circuits underlying these fundamental responses. Our results provide an extensive resource for the study of A. thaliana gene regulation and functional biology.

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. INTRODUCTIONOrganisms have evolved a wide array of mechanisms for adapting to stressful environments. One of the most closely studied of these is the induction of heat shock proteins (HSPs), which comprise several evolutionarily conserved protein families. All of the major HSPs (that is, those expressed in very high amounts in response to heat and other stresses) have related functions: they ameliorate problems caused by protein misfolding and aggregation. However, each major HSP family has a unique mechanism of action. Some promote the degradation of misfolded proteins (Lon, ubiquitin, and various ubiquitin-conjugating enzymes); others bind to different types of folding intermediates and prevent them from aggregating (Hsp70 and Hsp60); and still another (Hsp100) promotes the reactivation of proteins that have already aggregated Lindquist, 1993, 1994).Although all organisms synthesize HSPs in response to heat, the balance of proteins synthesized and the relative importance of individual HSP families in stress tolerance vary greatly among organisms. For example, in yeast , a member of the Hsp100 (ClpB/C) family, Hsp104, is strongly expressed in the nuclear-cytoplasmic compartment in response to stress and plays a particularly pivotal role in tolerance to extreme conditions (Sanchez et al., 1992;. Yeast cells expressing Hsp104 survive exposure to high temperatures or high concentrations of ethanol 1000-to 10,000-fold better than do cells not expressing Hsp104. Members of the Hsp100 family also play critical roles in the stress tolerance of bacterial cells (Schirmer et al., 1996), including photosynthetic cyanobacteria (Eriksson and Clarke, 1996). In contrast, the fruit fly Drosophila makes no protein of this type in response to stress; instead, the induction of Hsp70 plays the central role in stress tolerance in this organism (Solomon et al., 19...