Isolation and Analysis of the Expression of Two Genes for the 81-Kilodalton Heat-Shock Proteins from Arabidopsis

Abstract

We have cloned and characterized two members of the family of genes for the 81-kilodalton heat-shock proteins from Arabidopsis thaliana, HSP81-1 and HSP81-2. Comparison of the entire genomic sequence of the HSP81-1 gene with the corresponding full-length cDNA previously reported as AtHS83 (TW Conner, PR LaFayette, RT Nagao, JL Key [1990] Plant Physiol 94: 1689-1695 reveals the presence of three introns of 315, 83, and 88 base pairs. By contrast, analysis of the HSP81-2 genomic and partial cDNA sequences suggests that the HSP81-2 gene is interrupted by only two introns of 304 and 106 base pairs. The 5'-initiation sites of the two corresponding mRNAs were mapped from results of experiments with SI nuclease. The deduced amino acid sequences of the proteins encoded by these two genes show 88% identity, and they also show striking similarities to the hsp90 family of proteins in yeast and animal cells. From the results of northem blot analysis of transcripts, it appears that the expression of the HSP81-1 gene occurs at only very low levels in the absence of heat shock and is strongly induced by heat (350C).The HSP81-2 gene is constitutively expressed at much higher levels, and its expression is moderately enhanced by elevated temperatures. Severe heat shock appears to block the splicing of the pre-mRNA transcribed from HSP81-1. We also examined the effects of arsenite and cadmium on the expression of the HSP81 genes, as well as on other groups of hsp genes in Arabidopsis. Treatment with cadmium was marginally effective in inducing hsp genes, whereas arsenite stress strongly stimulated the accumulation of each mRNA in a coordinated fashion. transcriptional level. Nucleotide sequence analysis of plant hsp genes has revealed the presence of a positive control element (25) that is homologous to the HSE, which was originally identified upstream of the hsp genes in Drosophila (19). This finding suggests that, in plants, transcriptional regulation of hsp genes is mediated by the binding to HSEs of trans-acting factors, referred to as heat-shock factors, in a similar way to that reported for other eukaryotes, such as fruit flies, yeast cells, and HeLa cells (32).The expression of hsp genes in plants is also induced by adverse environmental stimuli other than heat shock. These stimuli include heavy metal ions, arsenite, phytohormones (6,8,9,28), and insecticides (22). The regulation of hsp genes has also been shown to be dependent on the developmental stage of the plant (27). However, the molecular mechanisms of plant responses to a wide range of stresses and the physiological significance of hsps under stressed conditions remain obscure.As an initial approach to resolving these issues, we previously isolated and characterized two genes that encode small hsps in Arabidopsis thaliana (26). We have now extended our analysis of the hsp genes from Arabidopsis and report here the cloning of two distinct members of the family of genes for 81 -kD hsps, as well as their complete nucleotide sequences. Examination of the ups...

The chlorate‐resistant and photomorphogenesis‐defective mutant cr88 encodes a chloroplast‐targeted HSP90

“…Recently, Queitsch et al (2002) showed that HSP90 may act as a capacitor, as had been proposed previously in Drosophila (Rutherfort and Lindquist, 1998). Genes encoding HSP90, along with their pattern of expression at the mRNA level, have been reported in maize (Marrs et al, 1993), barley (Walther-Larsen et al, 1993), tomato (Koning et al, 1992), periwinkle (Schroder et al, 1993), Brassica (Krishna et al, 1995;Neumann et al, 1993;Reddy et al, 1998), Arabidopsis (Conner et al, 1990;Takahashi et al, 1992), Pharbitis (Felsheim and Das, 1992), and rye (Schmitz et al, 1996), but the functional signi®cance of their expression patterns is not clear. In Arabidopsis, there are at least six HSP90 genes in addition to CR88.…”

“…In Arabidopsis, there are at least six HSP90 genes in addition to CR88. Three cytoplasmic HSP90 genes have been previously reported (Conner et al, 1990;Takahashi et al, 1992). The fourth cytoplasmic homolog (Accession No.…”

Combinatorial Interaction of Cis Elements Specifies the Expression of the Arabidopsis AtHsp90-1Gene

“…The proteins are localized in different cell compartments, including the cytoplasm, the endoplasmic reticulum, and chloroplasts (Koning et al, 1992;Takahashi et al, 1992;Marrs et al, 1993;Schrö der et al, 1993;Krishna et al, 1995;Schmitz et al, 1996;Milioni and Hatzopoulos, 1997). The corresponding genes were shown to be specifically expressed during embryogenesis, pollen development (Marrs et al, 1993), and seed germination (Reddy et al, 1998) in young and rapidly divid-ing tissues such as shoot and root apices (Koning et al, 1992) and in flowers (Takahashi et al, 1992;Krishna et al, 1995). In oilseed rape (Brassica napus) and tomato (Lycopersicon esculentum) seedlings, HSP90 protein levels were found to increase by exogenous 24-epibrassinolide application (Dhaubhadel et al, 1999), whereas a glucosinolate-deficient Arabidopsis mutant was shown to be thermosensitive and defective in the cytosolic HSP90 expression after heat stress (Ludwig-Muller et al, 2000).…”

“…In oilseed rape (Brassica napus) and tomato (Lycopersicon esculentum) seedlings, HSP90 protein levels were found to increase by exogenous 24-epibrassinolide application (Dhaubhadel et al, 1999), whereas a glucosinolate-deficient Arabidopsis mutant was shown to be thermosensitive and defective in the cytosolic HSP90 expression after heat stress (Ludwig-Muller et al, 2000). It has also been reported that the hsp90 genes are stimulated by chemical treatments such cadmium or arsenite (Takahashi et al, 1992;Milioni and Hatzopoulos, 1997) and by exogenous treatment with indoleacetic acid or 0.1 m NaCl (Yabe et al, 1994). Also, in rice (Oryza sativa) seedlings, a putative HSP90 protein was shown to accumulate in response to salinity, low temperature stress, and exogenous abscisic acid application (Pareek et al, 1995).…”

OsHsfA2c and OsHsfB4b are involved in the transcriptional regulation of cytoplasmic OsClpB (Hsp100) gene in rice (Oryza sativa L.)

“…At the genetic level, significant modulation of gene expression is noted at the transcriptional level accompanying exposure of cells to high temperature. Promoters of a range of different plant Hsp genes have been analyzed for characterization of developmental and tissue specific expression patterns, both with and without heat stress (Crone et al 2001;Prandl et al 1995;Takahashi et al 1992;Yabe et al 1994). Analyses of 5′ and 3′ promoter deletions of the GmHsp17.3B in transgenic tobacco showed that the functional unit underlying this regulation process is the heat shock element (HSE) for the HS-dependent transcription of the native gene or with fused reporter genes.…”