Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis

Abstract

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.

Figure 1.

Altered HSP101 Expression in Transgenic Plants. Protein gel blot analysis of representative transgenic plants from the No-0 and Col-0 ecotypes. (A) Vector control line No-V1 and antisense lines No-AS1, No-AS2, and No-AS6. (B) Vector control line Col-V1 and cosuppression lines Col-SUP1 and Col-SUP2. (C) Vector control line No-V1 and constitutive expression lines No-C1, Col-C1, and Col-C2. Total cellular proteins from whole plants maintained at 22°C or heat shocked at 38°C for 90 min were electrophoretically separated on SDS–polyacrylamide gels and transferred to filters for reaction with an antiserum specific for HSP101 and a monoclonal antibody that recognized both constitutive and inducible members of the Hsp70 family. Immune complexes were detected with radiolabeled protein A and visualized by using a PhosphorImager. Samples prepared from different individual plants in the same experiment (I and II) illustrate the reproducibility of HSP101 alterations.

Figure 2.

Altered HSP101 Expression Has No Noticeable Effect on Growth and Development. Representative plants from two vector control lines (No-V1 and Col-V1), an antisense line (No-AS1), a cosuppression line (Col-SUP1), and a constitutive expression line (Col-C1) are shown at different stages of development. (Top) After 14 days. (Center) After 3 weeks. (Bottom) After 5 weeks. Ecotype-specific morphological differences exist between Col-0 and No-0 lines, but no growth rate or morphological changes were associated with HSP101 transgenes.

Figure 3.

Reducing HSP101 Expression Impairs the Acquisition of Thermotolerance in a Dosage-Dependent Manner. Fourteen-day-old seedlings grown at 22°C were pretreated at 38°C for 90 min, immediately subjected to a severe heat shock at 45°C for 2 hr, and then returned to 22°C for recovery. (A) Representative Col-0 vector control plants (Col-V1) and cosuppression plants from the lines with the greatest reductions in HSP101 expression (Col-SUP1 or Col-SUP2) were photographed after 6 days of recovery at 22°C. (B) Representative No-0 vector control plants (No-V1) and plants from the two antisense lines with the most severe reductions in HSP101 (No-AS1 or No-AS2) at 5 days after return to 22°C. (C) Representative plants from the antisense line that had an intermediate decrease (No-AS6) after 5 days of recovery at 22°C.

Figure 4.

Quantitative Hypocotyl Elongation Assay. (A) HSP101, HSP17.6, and HSP22 expression in 2.5-day-old vector control (V) and antisense (AS) seedlings. Total proteins from 40 to 46 seedlings that had been maintained at 22°C (C) or heated to 38°C for 90 min (H) were electrophoretically separated on SDS–polyacrylamide gels, transferred to filters, and reacted with antibodies against HSP101, HSP17.6, and HSP22. The HSP101 blot was exposed longer than those in Figure 1 to visualize the low amount of developmentally induced HSP101 remaining in the vector control seedlings at 22°C (lane C). (B) Schematic representation of a hypocotyl elongation assay with adapted and nonadapted seedlings. Seedlings grown at 22°C for 2.5 days (d) were transferred directly to 45°C (Nonadapted). Seedlings pretreated (PreT) at 38°C for 90 min were allowed to recover for 2 hr (2 H R) before the 45°C treatment (Adapted). After the 45°C heat shock, all seedlings were returned to 22°C for 2.5 days of recovery before the hypocotyl elongation was measured. (C) Results of a representative hypocotyl elongation assay. One vector control (V; No-V1) and five antisense lines (AS1 to AS5 and No-AS1 to No-AS5) were subjected to the experimental conditions described in (B). Vector control seedlings showed marked hypocotyl elongation when heat shocked after a conditioning mild pretreatment (adapted seedlings). In contrast, hypocotyl elongation of antisense seedlings was greatly affected despite adaptation. (D) Graphical presentation of combined results of two independent hypocotyl elongation assays for five antisense lines and one vector control. Error bars represent standard deviation and are based on data for at least 22 seedlings of each genotype. HS, heat shock.

Figure 5.

Germinating Seedlings Have High Thermotolerance, Which Is Lost after 2 Days of Growth. Seeds germinated on plates at 22°C for 30 min, 30 hr, 36 hr, 48 hr, or 72 hr were exposed to 47°C for 2 hr (HS). Representative plants were photographed 5 days after heat stress. The arrow marks the time (48 hr of germination) after which all heat-shocked seedlings died.

Figure 6.

Reduced HSP101 Expression Impairs Basal Thermotolerance in Seeds from Antisense Plants. (A) Levels of developmentally regulated HSP101 and HSP17.6 expression in mature seeds of a vector control line (V; No-V1) and three antisense lines (AS1 to AS3 and No-AS1 to No-AS3). Seeds (10 mg) of each genotype were used to prepare protein samples, and the proteins were electrophoretically separated on SDS–polyacrylamide gels. For HSP101 and HSP17.6, 0.5 mg of total protein was loaded per lane; for 5 × HSP101, 2.5 mg of total protein per lane was loaded. (B) Seeds of the three antisense lines and two control lines analyzed in (A) were germinated for 30 hr and then exposed directly to 47°C for 2 hr. Representative plates were photographed 10 days after heat shock.

Figure 7.

Constitutive Expression of HSP101 Provides a Growth Advantage to Unconditioned 14-Day-Old Plants. Fourteen-day-old plants grown at 22°C were shifted directly to 45°C for 30, 45, or 60 min and then returned to 22°C. Representative plates containing vector controls (No-V1 and Col-V1) and constitutive expression plants (No-C1, Col-C1, and Col-C2) were photographed 5 days after return to 22°C (6 days for Col-V1 and Col-C2 after 60 min of heat shock).

Figure 8.

Constitutive Expression of HSP101 Provides a Growth Advantage to 3-Day-Old Seedlings. (A) Analysis of HSP101 expression in 3-day-old seedlings of one vector control line (No-V1) and three constitutive expression lines (No-C1, Col-C1, and Col-C2). Total proteins from pooled seedlings of each genotype grown at 22°C were analyzed as in Figure 1. In constitutive lines, expression of HSP101 at day 3 was not as high relative to Hsp70 as in 14-day-old plants of the same genotype (see Figure 1). Vector controls did not contain HSP101 at this developmental stage. Samples prepared from different individual plants in the same experiment (I and II) illustrate the reproducibility of HSP101 alterations. (B) Seeds of vector controls and constitutive lines were plated together and germinated for 3 days. Seedlings were heat shocked at 47°C for 30 min and then returned to 22°C. Representative seedlings of vector control (No-V1) and constitutive line No-C1 are shown 2 days after exposure to heat shock. All four photographs were taken at the same magnification. (C) A lower magnification photograph of plants from the same experiment shown in (B) at 10 days after heat shock.