Abstract
The genes and proteins of the HSP70 family, are involved in important processes in cells and organelles at normal temperature and after heat stress. ConstitutiveHsc70 and heat-inducibleHsp70 genes are known in all organisms including plants. The goal of our present investigation was to generate anHsp70 mutation inArabidopsis thaliana. In a transgenic approach a heat-inducible antisenseHsp70 gene was constructed, plants were transformed and screened for lack of heat-inducible HSP70 mRNA; two such lines were further investigated. In these plants theHsp70 gene was not induced by heat shock, and the level of HSC70 RNA was also greatly reduced. This negative antisense effect was specific for genes of the HSP70 family and the induction of mRNAs encoding the small HSP18 class of heat shock protein (HSP) was not affected. The level of HSP70/HSC70 proteins was significantly reduced in transgenic plants, but HSP18 was induced to the same level in different transgenic lines and in untransformed plants. The acquisition of thermotolerance was negatively affected in artisense plants, the survival temperature being 2° C below the survival temperature of the wild type and other transgenic lines. Another major effect concerning the regulation of the endogenous heat shock transcription factor HSF was detected by testing the ability to form heterotrimers between authentic HSF and recombinant HSF-GUS (β-glucuronidase) proteins. The shut-off time, required to turn off HSF activity during recovery from heat stress, was significantly prolonged in antisense plants compared with wild-type and other transgenic lines. Our results imply a dual role of HSP70 in plants, a protective role in thermotolerance and a regulatory effect on HSF activity and hence the autoregulation of the heat shock response.
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Abravaya K, Myers MP, Murphy SP, Morimoto RI (1992) The human heat shock protein hsp70 interacts with HSF, the transcription factor that regulates heat-shock gene expression. Genes Dev 6: 1153–1164
Baler R, Welch WJ, Voellmy R (1992) Heat shock gene regulation by nascent polypeptides and denatured proteins: hsp70 as a potential autoregulatory factor. J Cell Biol 117:1151–1159
Boorstein WR, Craig EA (1990) Structure and regulation of the SSA Hsp70 gene ofSaccharomyces cerevisiae. J Biol Chem 256: 18912–18921
Craig EA, Gross CA (1991) Is hsp70 the cellular thermometer? Trends Biochem Sci 16:135–140
Craig EA, Jacobsen K (1984) Mutations in the heat-inducible 70 kilodalton gene confer temperature sensitive growth. Cell 38:841–849
Craig EA, Jacobsen K (1985) Mutations in cognate genes inSaccharomyces cerevisiae hsp70 result in reduced growth rate at low temperature. Mol Cell Biol 5:3517–3524
Engvall E, Perlmann PJ (1972) Enzyme linked immunosorbent assay (ELISA), III. Quantification of specific antibodies by enzyme-labelled anti-immunoglobulin in antigen-coated tubes. J Immunol 109:129–135
Helm KW, Vierling E (1989) AnArabidopsis cDNA clone encoding a low molecular weight heat shock protein. Nucleic Acids Res 17:7995
Hübel A, Schöffl F (1994)Arabidopsis heat shock factor: isolation and characterization of the gene and the recombinant protein. Plant Mol Biol 26:353–362
Hübel A, Lee J-H, Wu C, Schöffl F (1995)Arabidopsis heat shock factor is constitutively active inDrosophila and human cells. Mol Gen Genet 248:136–141
Jorgenson R (1990) Altered gene expression in plants due to trans interactions between homolgous genes. Trends Biotechnol 8: 340–344
Kim D, Ouyang H, Li G (1995) Heat shock protein hsp70 accelerates the recovery of heat-shocked mammalian cells through its modulation of heat shock transcription factor HSF1. Proc Natl Acad Sci USA 92:2126–2130
Kyhse-Andersen J (1984) Electroblotting of multiple gels: a simple apparatus without buffer for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10:203–209
Landry J, Chretien P, Lambert H, Hickey E, Weber LA (1989) Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells. J Cell Biol 109:7–15
Lee J-H, Schöffl F (1995) GUS activity staining in gels — a powerful tool for studying protein interactions in plants. Plant Mol Biol Rep 13:346–354
Lee J-H, Hübel A, Schöffl F (1995) Derepression of the activity of genetically engineered heat shock factor causes constitutve synthesis of heat shock proteins and increased thermotolerance in transgenicArabidopsis. Plant J 8:101–110
Li GC, Li L, Liu Y-K, Mak JY, Chen L, Lee WMF (1991) Thermal response of rat fibroblasts, stably transfected with the human 70-kDa heat shock protein-encoding gene. Proc Natl Acad Sci USA 88:1681–1685
Li GC, Li L, Liu RY, Rehman M, Lee WMF (1992) Heat shock protein hsp70 protects cells from thermal stress even after deletion of its ATP-binding domain. Proc Natl Acad Sci USA 89:2036–2040
Morimoto RI, Tissières A, Georgopoulos C (1990) Stress proteins in biology and medicine. Cold Spring Harbor Laboratory press, Cold Spring Harbor, NY
Mosser DD, Theodorakis NG, Morimoto RI (1988) Coordinate changes in heat shock element binding activity and HSP70 gene transcription rates in human cells. Mol Cell Biol 8:4736–4744
Nagao RT, Czarnecka E, Gurley WB, Schöffl F, Key JL (1985) Genes for low molecular weight heat shock proteins of soybean: sequence analysis of a multigene family. Mol Cell Biol 5: 3417–3428
Pelham HRB (1984) HSP70 accelerates the recovery of nucleolar morphology after heat shock. EMBO J 3:3095–3100
Petko L, Lindquist S (1986) Hsp26 is not required for growth at high temperatures, nor for thermotolerance, spore development, or germination. Cell 45:885–894
Rabindran SK, Wisniewski J, Li L, Li GC, Wu C (1994) Interaction between HSF and hsp70 is insufficient to suppress induction of DNA binding activityin vivo. Mol Cell Biol 14:6552–6560
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Sanchez Y, Lindquist S (1990) Hsp104 required for induced thermotolerance. Science 248:1112–1115
Sanchez Y, Parsell DA, Taulien J, Vogel JL, Craig EA, Lindquist S (1993) Genetic evidence for a functional relationship between Hsp104 and Hsp70. J Bacteriol 175:6484–6491
Sanger F, Nicklen S, Coulsen A (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schöffl F, Rieping M, Baumann G (1987) Constitutive transcription of a soybean heat shock gene by a cauliflower mosaic virus promoter in transgenic tobacco. Dev Genet 8:365–374
Severin K, Schöffl F (1990) Heat-inducible hygromycin resistance in transgenic tobacco. Plant Mol Biol 15:827–833
Severin K, Wagner A, Schöffl F (1995) A heat-inducibleAdh gene as a reporter for a negative selection in transgenicArabidopsis. Transgenic Res 4:163–172
Solomon JM, Rossi JM, Golic K, McGarry T, Lindquist S (1991) Changes in hsp70 alter thermotolerance and heat-shock regulation inDrosophila. New Biologist 3:1106–1120
Sorger PK (1991) Heat shock factor and heat shock response. Cell 65:363–366
Stone DE, Craig EA (1990) Self-regulation of 70-kilodalton heat shock proteins inSaccharomyces cerevisiae. Mol Cell Biol 10:1622–1632
Straus DB, Walter WA, Gross CA (1989) Activity of σ32 is reduced under conditions of excess heat shock protein production inEscherichia coli. Genes Dev 2:1851–1858
Straus DB, Walter W, Gross CA (1990) DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of σ32. Genes Dev 4:2202–2209
Suzuki K, Watanabe M (1994) Modulation of cell growth and mutation induction by introduction of the expression vector of human hsp70 gene. Exp Cell Res 215:75–81
Tilly K, McKittrick N, Zylicz M, Georgopoulos C (1983) The DnaK protein modulates the heat shock response ofEscherichia coli. Cell 34:641–646
Tilly K, Spence J, Georgopoulos C (1989) Modulation of the stability of theEscherichia coli heat shock regulatory factor σ32. J Bacteriol 171:1585–1589
Torres R, Hemleben V (1994) Pattern and degree of methylation in ribosomal RNA genes ofCucurbita pepo. Plant Mol Biol 26:1167–1179
Valvekens D, van Montagu M, Lijsebettens M (1988)Agrobacterium tumefaciens-mediated transformation ofArabidopsis thaliana root explants by using kanamycin selection. Proc Natl Acad Sci USA 85:5536–5548
Vierling E (1991) The role of heat shock proteins in plants. Annu Rev Plant Physiol Plant Mol Biol 42:579–620
Vogel JL, Parsell DA, Lindquist S (1995) Heat-shock proteins Hsp104 and Hsp70 reactive mRNA splicing after heat inactivation. Curr Biol 5:306–317
Welte MA, Tetrault JM, Dellavalle RP, Lindquist S (1993) A new method for manipulating transgenes: engineering heat tolerance in a complex, multicellular organism. Curr Biol 3:842–853
Werner-Washburne M, Stone DE, Craig EA (1987) Complex interactions among members of an essential subfamily of hsp70 genes inSaccharomyces cerevisiae. Mol Cell Biol 7:2568–2577
Westwood JT, Wu C (1993) Activation ofDrosophila heat shock factor: conformational change associated with monomer to trimer transition. Mol Cell Biol 13:3481–3486
Wu CH, Caspar T, Browse J, Lindquist S, Somerville C (1988) Characterization of an Hsp70 cognate gene family inArabidopsis. Plant Physiol 88:731–740
Wu S-H, Wang C, Chen J, Lin B-L (1994) Isolation of a cDNA encoding a 70 kDa heat-shock cognate protein expressed in vegetative tissues ofArabidopsis thaliana. Plant Mol Biol 25:577–583
Zabaleta E, Oropeza A, Assad N, Mandel A, Salerno G, Herrera-Estrella L (1994) Antisense expression of chaperonin 60β in transgenic tobacco plants leads to abnormal phenotypes and altered distribution of photoassimilates. Plant J 6:425–432
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Lee, J.H., Schöffl, F. AnHsp70 antisense gene affects the expression of HSP70/HSC70, the regulation of HSF, and the acquisition of thermotolerance in transgenicArabidopsis thaliana . Molec. Gen. Genet. 252, 11–19 (1996). https://doi.org/10.1007/BF02173200
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DOI: https://doi.org/10.1007/BF02173200