Plant HsplO0 family with special reference to rice SNEH LATA SINGLA, ASHWANI PAREEK and ANIL GROVER* Department of Plant Molecular Biology, University of Delhi South Campus, Benito J~arez Road, Dhaula Kuan, New Delhi 110 021, India *Corresponding author (Fax, 91-11-6886427; Emait, [email protected]). Heat shock proteins (Hsps) represent a group of specific proteins which are synthesized primarily in response to heat shock in almost all biological systems. Members of Hspl00 family have been directly implicated in induction of thermotolerance in microbial and animal cells. Yeast cells harbouring defective hspl04 gene do not show thermoto]erance under conditions in which the normal cells do. Several plant species have been shown to synthesize Hsps in the range of 100 kDa. Rice Hspl04 (OsHspl04) is rapidly and predominantly accumulated in heat-shocked cells. Western blotting anaIysis show that anti rice Hspt04 antibodies (generated against purified Hspl04 protein from cultivated rice Oryza sativa L.) cross-react with the same-sized high temperature inducible protein in 15 different wild rices. It was further found that anti rice Hspl04 antibodies also cross-react with a major high temperature regulated protein of Escherichia coll. We have previously shown that a 110 kDa stress regulated protein in rice (OsHspll0) is immunologicalIy related to yeast Hspl04 protein. In this paper, we present a comparative account of characteristics of the OsHspl04 and OsHspI10 proteins. l. Introduction ture response of rice in molecular terms (Khush and Toenniessen 1991). Rice is a major crop for the southeast Asian countries. Different living systems respond differentially to Abiotic stresses such as high and low temperatures, increased temperatures. Basal thermototerance is a salinity, alkalinity, drought and flooding conditions affect measure of the inherent capacity of an organism to rice cultivation to a significant extent (Widawsky and tolerate high temperature (Lee et izt 1995). It is also a O'Toole 1990; Khush and Toenniessen 1991; Christou matter of common observation that living systems sub- 1994). While high temperature stress affects rice pro- jected to sublethal temperature conditions combat lethal duction at almost every stage of its life cycle, more temperature stress much better than when they are directly severe effects of high temperature stress are noted at exposed to lethal temperature stress (Linet al 1984; the seed germination stage and at the time of anthesis. Vierling 1991). The terms 'acquired' or 'induced' thermo- Germination process of rice seeds is drastically affected tolerance are used to denote tolerance developed in this once the ambient temperature exceeds 40-45~ (yoshida manner (Lin et al 1984). Tissie~es et aZ (1974) employing I977; Pareek et af 1997b). In many parts of the world, Drosophila cells provided evidence that specific proteins, including Punjab in India, high temperature causes pollen referred to as heat shock protein (Hsp), are synthesized and spikelet sterility in rice (Satake and Yoshida I978). in response to heat shock (HS). In plants, Barnett et al This effect of high temperature at the time of anthesis (I980) reported that Hsps equivalent to those in animal is ~o fatal that even I~ rise in ambient temperature systems are synthesized in tobacco and soybean cells. for just l h can lead to high levels of spikelet sterility 1,12ey et al (1981) subsequently showed that synthesis of (Yoshida et al 198l). Unfortunately, no major efforts Hsps is a conspicuous shift in metabolism in response have been made in characterization of the high tempera- to HS in soybean seedlings. Following these initial Keyworfls, Heat shock proteins; Hspl00 family; OsHspl04; OsHsplI0; rice J. Biosci., 23, No. 4, October 1998, pp 337-345. Indian Academy of Sciences 337 338 Sneh Lata Singla, Ashwani Pareek and Anil Grover observations, Hsps have been analysed in a range of Accumulation of Hsps has been correlated with plant species growing under diverse ecosystems (Vierting acquisition of therrnotolerance in both animal and plant 1991; Singla et al 1997b). Essentially, Hsps represent systems. For instance, Lin et al (1984) demonstrated proteins which are preferentially synthesized during high that soybean seedlings which have accumulated Hsps temperature stress. Hsps are either high (80-100 kDa), (due to pre-adaptation at 40~ combat lethal stress of intermediate (60-80 kDa) or low molecular weight (8- 45~ in a more effective way than seedlings which were 20 kDa) proteins. Remarkable progress has been made directly taken from 28 ~ to 45~ and thus had little in the characterization and regulation of genes governing opportunity of synthesizing and accumulating Hsps. Direct synthesis of Hsps (hsp genes) (Howarth and Ougham role of Hsps in governing thermotolerance is implicated I993; Morimoto 1993; Parsell and Lindquist 1993; Singla by the observation that ceils which fail to synthesize et al 1997b). Apart from HS, Hsps are noted to be Flsps by either selective mutagenesis of hsp genes or synthesized in response to a large number of factors/ by inactivation of Hsps through antibody binding are conditions including a range of other abiotic stresses incapable of developing thermotolerance (RiabowoI et al such as water stress, salinity stress, chilling and anoxic 1988; Sanchez and Lindquist 1990). Lee et al (1995) conditions (Mocquot et at 1987; Borkird et al 1991; raised transgenic Arabidopsis thaliana plants with con- Neven et al 1992; Cabane et al 1993; Pareek et al stitutive expression of heat shock transcription factors I995). (HSFs) and thus of Hsps and showed that such plants Table 1. Hspl00 in higher plants. Molecular weight Species (kDa) Characteristic features Reference Glycine max 103, 99 Synthesized at 37 ~ and 40~ as se_.enby in vivo labelling Barnett et al (1980) of proteins Gossypium hirsutum 1O0 Accumulated under field conditions with canopy Burke et at (1985) temperature of 40~ for several days as seen in Coomassie blue stained SDS-polyacrylamide gels. Also accumulated in growth chamber grown plants by HS as noted by radioIabelling Nicotiana tabacum ] 10, i00 Accumulated at 35~43~ as seen by autoradiography Barnett et al (1980) N. tabacum 1O0 Accumulated in mesophyll protoplasts by 40~ (3 h) Meyer and Chart[er HS as seen by autoradiography (1983) Opuntia ficus indiea 1 I0, 103 Accumulated in roots at 45~ as seen by Somers et al (1991) autoradiography Prosopis chilensis 108, 103 Accumulated in seedling axis by 40 or 45~ 2 h HS Medina and as seen by fluorography. The 103 kDa Hsp was stable Cardemil (1993) at 50~ (2 h) while the 108kDa disappeared at 50~ Saccharum. officinarum 97 Noted in autoradiograms by temperature upshift to 38~ Moisyadi and Harrington (1989) Secale cereate 103 Noted in roots in response to HS as seen by Necchi et al (1987) au toradiography Triticum aesEvum 118 Induced in seeds imbibed in water by heat hardening Kraus et al (1995) treatments at 40~ (2h)as seen by fluorography T. aestivum, T. durum 103 Accumulated in roots and coleoptiles by 4D~ HS as Necchi et al (1987) noted by autoradiography Vigna radiata 114 Accumulated in excised hypocotyls at 40~ (1 h) as Collins et al (1995) seen by autoradiography. Declines within 4 h of HS Zea mays 108 Accumulated in plumules by 1 h HS at 4I~ as seen Baszczynski and by radiolabelling. Dispappears within 6-8 h of recovery Walden (1982) at 27~ Resolves in two spots of pI 7.7 and 8.2 on 2-dimensional fluorograms Z. mays 108 Accumulation noted in in vitro translated products of Sinibaldi and RNA from 4I~ treated seedlings. Also noted in Turpen (t985) autoradiograms of in vivo labelled proteins at 41 ~ and 45~ Plant HsplO0 family with special reference to rice 339 exhibit improved basal thermotolerance. These results et al 1995; Singla 1996; Singla et at 1997a,b, 1998) indicate that with the availability o[' hsp genes and/or and have established the following characterisitics of other regulatory strategies, thermotolerance of the orga- these proteins. nism can be modulated. In microbial and animal systems, Hsp60, Hsp70, Hsp90 3.1 OsHspl04 is a promb,ent proteb~ in aod Hspl00 family members have been mote directly heat-shocked rice cells implicated in the induction of thermotoierance (Johnston and Kucey 1988; Riabowol et al 1988; Sanchez and In rice, heat shock-induced 104kDa polypeptide was Lindquist 1990; Parself and Lindquist t993; Kimura et detected in de novo synthesized protein profiles as wetl at 1994). Hspl00 family members have been intensively as in profile of steady-state proteins stained with silver analysed in recent years in this regard. Hspl04 is a nitrate or Coomassie brilliant blue dye (Singla and Grover major Hsp in yeast and its role in thermotolerance has 1994). Further work has shown that the rice HspI04 is been unequivocally shown as mutant yeast cells harbour- accumulated to as high as 1.2% and 0.4% of the total ing defective hspl04 gone do not show thermotolerance soluble protein content in shoot tissues of young seedlings under conditions in which the normal wild type cells and mature Ieaves, respectively (Singla and Grover 1994; do (Sanchez and Lindquist 1990). Apart from high Singla t996). OsHspl04 thus represents a class of Hsps temperature, yeast Hspl04 protein plays a critical role which is accumulated to appreciable extent in response in the tolerance of these cells to high concentrations of to temperature stress conditions. ethanol, arsenite and long-term storage in cold (Sanchez et al 1992). It has recently been found that the yeast 3.2 OsHspl04 is a conserved stress protein Hspl04 mediates the resolubilization of heat-inactivated proteins from insoluble aggregates (Parsell et at t994). Accumulation of OsHspI04 in leaf segments in response The dissolution of protein aggregates through a Hsp is to HS was detected at four different stages of development a unique feature for the Hspl04 protein.