Plant HsplO0 family with special reference to rice

SNEH LATA SINGLA, ASHWANI PAREEK and ANIL GROVER* Department of Plant , University of South Campus, Benito J~arez Road, Dhaula Kuan, 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. Information on (i.e., 30, 55, 90 and 110 days after sowing) of rice the plant Hspl00 family with special reference to rice plant, apart from the seedlings (Singla 1996). Thus, plant is presented in this paper. regardless of the growth stage, accumulation of this 2, Plant HsplO0 proteins protein in leaf tissues is an integral component of the response of rice plants to high temperature stress. Com- The current status of information on plant Hspl00 proteins parable levels of OsHspI04 were accumulated in six is summarized in table [. Hsps are considered to be different indica and japonica rice cultivars (SingIa and highly conserved since related proteins from diverse Grover 1994), suggesting that quantitative levels of species show a marked homology in their structure and OsHspa04 protein in various cultivated rice types may function (Singla et al 1997b). Work from our laboratory as well be a conserved response. Immunological homo- showed, for the first time, that immunological homotogue logues of OsHspl04 with molecular weight in almost of yeast Hspl04 is accumulated in heat-shocked rice the same range (100 kDa) were noted, in response to cells (Singla and Grover 1993). Cross-reactivity of anti high temperature stress, in five other crop plants namely yeast Hspt04 antibodies has been further detected in Triticum aestivum, Sorghum bicolor, Pisum sativum, Zea several other plant species (Lee et al 1994; Schirmer et mays and Brassica juncea (Pareek et af 1995). at I994). Functional resemblance of the plant Hspl00 family proteins has been proven in the following two recent studies. It has been shown that A. thaliana AthsplOt 3.3 Time and temperature-kinetics of OsHspl04 gone (homologue of yeast hspI04) can partially substitute accumulation for the function of hsp[04 in yeast, restoring induced thermotoIerance in strains carrying mutation of their own Most J-Isps in cells are reported to be transiently syn- hspl04 gene (Schirmer et af 1994). Similarly, soybean thesized (Nagao et aI 1990; Howarth I991). However, homologue of yeast Hspi04 (GmhsplO1) has been found the synthesis and accumulation of different Hsps are to provide partial complementation of the thermotolerance reported to vary in time- and temperature-dependent defect of the Hspl04 mutant of yeast (Lee et at 1994). patterns (Lin et af 1984; Hsieh et al 1992). OsHspl04 These-studies demonstrate that yeast Hspl04 and related in stained gels showed high level accumulation following proteins from plant systems are structurally and func- 45~ HS for 2 to 4 h in case of intact seedlings (Singla tionally homologous. and Grover 1994), Following the stress, high levels of OsHsp]04 persisted till 8 h of recovery (28~ it appears from an analysis of de novo synthesized proteins that 3, Rice Hspl00 family synthesis of OsHspl04 ceases after 2 h (Singla 1996). We have been analysing the rice Hsp[00 family for the Put together, these observations indicate that Hspl04 is past few years (Singla and Grove[- 1993, 1994; Pareek a relatively stable protein. According to Howarth and 340 Sneh Lata Singla, Ashwcmi Pareek and Anil Grover

Ougham (1993), stable Hsps may have a more crucial 3.6 OsHspl04 is responsive to exogenously applied role to play in governing thermotolerance. It should be abscisic acid interesting to compare the role(s) of such stable Hsps In recent years, plant hormone abscisic acid (ABA) has with those which are transiently accumulated in governing been widely implicated in stress responses. The level of thermotolerance. this hormone has been found to increase in response to various abiotic stress conditions in a variety of plant 3.4 OsHspl04 is a stress-associated protein in the genera (Chandler and Robertson 1994). Furthermore, it has been noted that the expression of many stress res- rice system ponsive genes can be triggered under control conditions On the basis of silver-stained SDS-gels, we found that if plants are treated with exogenous ABA (Singla and OsHspl04 is specifically accumulated in response to Grover 1993; Chandler and Robertson 1994). On the high temperature stress as subjecting intact seedlings to basis of such observations, ABA is considered a common low temperature, salinity or air drying caused no apparent denominator of the stress responses. Accumulation of change in the levels of this protein (Singla and Grover OsHspl04 was triggered in shoots of rice seedlings in 1994). However, with Western blotting, rice Hspt04 response to ABA, indicating that ABA might have a protein was found to accumulate against salinity, air role as a cellular messenger governing stress inducibility drying and low temperature stresses too. Since OsHsp104 of OsHspl04 (Pareek et al 1995). There is as yet no protein accumulates in response to a number of different work on actual quantitation of the endogenous ABA stress conditions, we also refer this protein as stress- levels in heat-shocked and control rice seedlings. In associated protein 104 or SAPI04 (Pareek et al 1995). general also, high temperature induced alterations in The relative intensity of accumulation of SAPI04 protein ABA levels are poorly understood as compared to water, appeared to be as follows: heat>salinity>air dry- cold and salinity stresses. ing > cold (Pareek et al 1995). Cross-induction of specific Hsps by stress conditions other than high temperature 3.7 OsHsp]04 is a developmentally regulated stress has been documented in several studies (Vierling 1991; protein Sanchez et al 1992; Almoguera et al 1993; Walther-Larsen Pattern of Hsp synthesis is reported to be different in et al t993; Singla et al 1997a) and OsHspl04 appears tissues representing different developmental stages to be a member of this category. (Almoguera et al ~993). For example, Structures such as dried pollen and seeds which are highly thermotolerant are known to contain high constitutive levels of Hsps 3.5 OsHspl04 accumulates in rice when irrigation is withheld in field-grown plants (Abernathy et al 1989; Almoguera and Jordano 1992; DeRocher and Vierling I994). Different organs of the Since most studies on the accumulation of stress proteins field grown mature rice plant were found to contain have been c~irried out with laboratory-grown plants, differential levels of OsHspl04 protein under unindueed whether or riot stress proteins accumulate in response and induced conditions (Singla I996; Singla et at 1998). to natural conditions of stress is still a debatable issue More importantly, it was found that upper portions of the culm as well as grains contain high uninduced levels (Hernandez and Vierling 1993). It was, therefore, worth- while to analyse OsHspl04 in field-grown rice plants. of this protein. Seeds of 7", aestivum, S. bicolor and Z It was found that OsHspl04 accumulated in shoot tissues mays also contained high uninduced levels while those when 15 day old rice seedlings were subjected to water of Brassica showed extremely low levels or no accu- stress by withholding irrigation for 8 days under field mulation at all. High levels of OsHsp90 proteins in upper culm and grains has also been noted previously conditions (Pareek et al 1995). These observations in our laboratory (Pareek et al 1997a). Such a kind of indicate that OsHspl04 protein might be naturally accu- pattern for high molecular weight Hsps with respect to mulating in field-grown rice pIants under stress conditions. culm and grain tissues has not been noted earlier in any Accumulation of Hsps has previously been reported in other study. It should be interesting to analyse the field-grown cotton (Burke et al 1985) and wheat plants (Nguyen et al 1994). Induction of hsp mRNA has been sensitivity of these portions of the rice plant to high temperature induced cellular damage and the induction shown in response to drought stress (imposed by with- drawing water for irrigation) in field-grown soybean, of thermotolerance. thus indicating a close relation of high temperature With 3.8 OsHspl04 is embryogenesis-related and drought stress in field (Kimpet and Key I985). it is disappears from the seeds during seed germination possible that accumulation of OsHspl04 in our experiment might as well be due to drought and temperature inter- During the course of embryo development under natural actions. conditions, uninduced levels of OsHspI04 protein were Plant HsplO0 family with special reference to rice 341 appreciably high at all stages of embryo maturation, shoots of young rice seedlings (Singla and Grover 1993). indicating that accumulation of OsHspl04 protein is Importantly, while seedlings showed positive cross embryogenesis related (Singla 1996). Accumulation of reaction of rice 110 kDa protein with respect to high osmotin, late embryogenesis abundant (LEA) proteins, temperature and ABA inducibility, mature leaves ABA responsive 16 kDa (RAB) protein as well as de- (obtained from 90 day old field grown plants and heat hydrins is known to he high during embryo development shocked at 45~ 6 h) do not appear to accumulate this as wen as in response to stress conditions (Coca et al protein (Singla and Grover 1993). 1994). OsHspl04 appears to be a new component in the spectrum of stress proteins known in this regard. 3.12 OsHspllO is induced by diverse abiotic stresses What happens to stress proteins which are present in uninduced seeds, during the germination process? Recent OsHsplIO was three times more strongly expressed in studies have shown that seed-localized stress proteins salinity stress as compared to high temperature stress in are degraded during the early phase of seed germination young rice seedlings (Singla et al I997a). Low but (Coca et at 1994). Likewise, rice Hsp104 was present significant induction of rice HspI10 protein was noted in seeds in detectable amounts till 48 h of the germination against water stress as welt as low temperature stress. process but disappeared thereafter (Singla 1996; Singla Taken together, these data impIy that rice Hsp110 protein et al 1998). is also a stress-associated protein (SAP).

3.9 OsHspl04 is localized in vascular bundles in 3.13 Tissue distribution of OsHspllO shoots and in embryos in dry seeds Low molecular weight (LMW)-Hsps in sunflower are The upper culm portion of the rice plant showed con- mainly distributed in the cotyledon, hypocotyI and radicle spicuously higher constitutive levels of OsHsp 110 which of dry seeds (Coca et al 1994). In the embryo, LMW-Hsps was only marginally aItered in response to HS. Consti- were localized in the parenchyma and provascular tissues. tutive amount of this protein was marginal in the lower Tissue print immunoblotting analysis of OsHspl04 culm and in this case, it increased remarkably in response revealed that these proteins are mainly associated with to HS. Significantly high constitutive levels of this the stelar tissue of various leaves as well as in the protein were also noted in grains which declined appre- central meristematic tissues of the stem apex in the ciably in response to HS (Singla et al 1997a). Uninduced heat-shocked shoots of the 10 day old seedlings (Singla levels of this protein were high in developing embryos i996; Singla et al 1998). In the germinating seeds, high at all the stages of their maturation. It has recently been levels of this protein were noted in the embryo. So far, shown that uninduced levels of Hsp0 are appreciably this analysis has been carried out in response to high high in whole grain, lemma and palea (pooled together) temperature stress. It remains to be determined how other and upper culm tissues (Pareek et al 1997a). It appears stress conditions (drought, eold and salinity) modulate that Hspll0, 104 and 90 are co-regulated to a large the tissue distribution of HspI04. extent with respect to their spatial distribution.

3.10 OsHspllO is immunologically related to yeast 3.14 Conservation of OsHspllO in different wild Hspt04 protein rices

Yeast Hspl04 is shown to have immunological kinship Hspll0 protein was detected in mature leaves in almost with a polypeptide of 100 kDa in E. cofi and a polypeptide all wild rices (Singla etal 1997a). However, a conspicuous of 1 I0 kDa in HeLa cells (Sanchez and Lindquist 1990; decline in OsHspll0 in response to HS was noted in Parsell et at 1991). From the stained SDS-gel, we noted topmost leaves of O. alta, O. longistaminata, O. matam- that yeast Hspl04 and rice HspI04 are at comparable puzhaensis, O. minuta, O. punctata and O. rufipogon. positions (Singla aad Grover 1993). When rice proteins On the other hand, levels of this protein remained either (from heat-shocked shoots) were probed with anti yeast the same or were marginalIy altered in response to HS Hspl04 antibodies on Western blots, a ll0kDa high in topmost leaves of O. eichengeri, O. glumaepatula, O. temperature-induced rice protein showed a positive cross- grandigtumis, O. latifolia, O. meridionalis, O. meyeriana reaction, indicating that rice plant indeed has a homologue and O. nivara. At yet another extreme, the topmost leaf to yeast HSP I04 protein (Singla and Grover 1993; of O. australiensis accumulated low levels of this protein Singla et af 1997a). in response to HS as compared to control. Anti yeast Hspl04 antibodies showed no cross-reaction to O. off/- 3.11 OsHsp If0 is ABA inducible cinalis proteins, indicating that either the levels of OsHspll0 are too low or it is absent in this cultivar OsHspll0 protein was found to be ABA inducible in (Singla et al 1997a). 342 Sneh Lata Singla, Ashwani Pareek and Anil Grover

4. Materials and methods remove the non-p~oteinaceous contaminants. The preci- pitated proteins were dissolved in Laemmli buffer In the present study, we analysed the extent of conser- (Laemmli 1970). For isolation of proteins from E. coli, vation of rice OsHspl04 in different wild rice types as cells were pelleted (3000 g, 5 min, 4~ after the stress well as in E. coli cells. Seeds of 15 different wild rices treatment was over. The pellet thus obtained was sus- [namely O. alta (IRGC 105143), O. austra-liensis (IRGC pended in Laemmli buffer. For denaturation of proteins, 105273), O. eichingeri (IRGC 105414), O. gIumaepatula samples were boiled at 100~ for 5 rain in a water bath. (IRGC 100184), O. grandiglumis (IRGC 105560), O. The solubilized proteins were recovered by centrifuga- latifolia ('IRGC 105133), O. longistaminata (IRGC tion (15,000 g, 15 rain, 20~ and an aliquot of super- 104977), O. malampuzhaensis (IRGC 105329), O. natant was used for protein quantification. Protein meridionalis (IRGC 101411), O. meyeriana (IRGC quantification, gel electrophoresis (7.5% uniform acry- 104990), O. rnin~ta (IRGC 105126), O. nivara ORGC lamide concentration) and Western blotting were per- I05410), O. officinalis (IRGC 105220), O. punctata formed as described earIier (Singla and Grover I993; (IRGC 105137), O. rufipogon (IRGC 105325)] were Pareek et al I995; Pareek et al 1997a; Singla et al grown as described earlier (Pareek et al 1997a; Singla 1997a). Anti-rabbit horseradish peroxidase-linked secon- et al 1997a). Dehusked seeds of these rices were initially dary antibodies were employed for the detection of germinated on a moist filter paper and 5 to 8 day old antigen-antibody complex. seedlings were transferred to earthern pots under natural day/night conditions (at Indian Agricultural Research 5. Results and discussion Institute, New .Delhi). Segments from the topmost leaf of vegetative (just prior to flowering) plants in each The relative profile of accumulation of rice Hspl04 in case were placed in a beaker containing distilled water the topmost leaves of fifteen different wild rice species and subjected to HS (45~ 6 h) (Pareek et at 1997a). at the comparable growth stage (i.e., vegetative stage Following HS, the tissues were stored in liquid nitrogen just prior to flowering) was examined in this study. till further use. Western blotting employiog anti rice HspI04 antibodies Single colony of E. coli [strain BL2I(DE3)pLysE] was showed that the significant levels of OsHspl04 were inoculated in 5 ml L-broth (1 g tryptone, 0-Sg yeast accumulated in response to HS in different wild rices extract and 1 g NaCI per 100ml, pH 7) containing (figure 1). Generally, 9the uninduced as well as the appropriate antibiotic and inoculated for overnight at HS-induced levels of OsHspl04 in these genotypes were 37~ with vigorous shaking (Sambrook et al 1989). The comparable amongst wiId rices as well as with respect mid log phase cultures of E. coii were subjected to high to comparison with cultivated O. sativa. In case of O. temperature stress by incubating the culture flasks at 42 mafampuzhaensis, higher uninduced amounts of and 48~ for 1 h in a water bath. OsHspl04 were noticed while all other rice types were Soluble proteins from various rice tissues were found to have negligible amounts of this protein under extracted as described earlier (Pareek et al 1995, 1997a). uninduced state. It is to be noted that these wild rices These proteins were precipitated using 8 volumes of are endemic to varied ecosystems. Detailed correlative chilled acetone containing 10ram 2-mercaptoethanol to studies on the stress response of wild rices and OsHspl04

, ./e e / O" O"LI " O'/o/ ' O" O' O' O" O" O' 0 0 C HS C HS C HS C HSC HS C HS C HS C I~ C AS C HS,C H5 C HS C HS C AS C HS HS SAP -,,-404 i

Figure 1. Pattern of OsHspl04 accumulation in topmost leaf of various wild rice species in response to heat shock as detected by Western blotting using anti rice Hspl04 antibodies. Heae'Shocked sample of the cultivated rice (O. sativa cv Pusa Basmati 1) is shown on the right side. Four gg total soluble proteins Were loaded in each lane. Arrow marks the position of HsptSAP104. C, control (28~ HS, heat shock (45~ 6 h). Plant Hsp]O0 family with special reference to rice 343 accumulation may provide useful information on the 9OsHspl04 and OsHspll0 show differential accumula- cellular role(s) of this protein. tion with respect to high temperature and salinity Further, we checked for the cross-reaction of anti rice (Pareek et al 1995; Singla et al 1997a). Hspl04 antibodies with proteins of E. coll. An immuno- 9OsHspl04 protein is accumulated in leaves of mature logical homologue of OsHspl04 was found to accumulate O. sativa plants in response to high temperature stress in response to HS in E. coil cells too (figure 2). The whereas, OsHsp110 declined in comparable tissues molecular mass of OsHspl04 homologue protein was 97 under similar conditions (Singla et al 1997a). kDa in E. coll. In control cells (37~ the levels of this homologue were low which showed a significant Possible kinship between OsHspt 04 and OsHsp110 pro- rise in response to HS (48~ 1 h). teins is indicated by following observations: These analyses indicated that conservation of OsHspl04 9Amino acid sequence of OsHspl04 tryptic peptides extends from cultivated rice plant to wild rice species show to an extent, homology to Arabidopsis AtHspl01 and to microbial systems and in this respect, Hspl04 is and Gfycine max GmHspi01 proteins which are homo- Similar to other established Hsps (Pareek et al t997a; logues to yeast Hspl04 protein (see Singla et af 1998 Singla et af 1997a,b). for data on amino acid sequence comparisons). We have presented information on OsHspl04 and OsHspl10 is immunologically homologous to yeast OsHspI10 proteins in relation to yeast Hspl04 protein HspI04 (Singla and Grover 1993). in this paper. We have highlighted that (i) OsHspI04 9OsHspl04 and OsHspll0 show similar patterns with is a major stress-inducible protein in rice and its homo- respect to spatial distribution to some an extent (Singla Iogues are detected in a number of plant and microbial 1996; Singla et al 1997a). species, (ii) yeast Hspl04 is the highest molecular weight Hsp in yeast and its homologues are known in microbial Put together, we suggest that OsHspl04 and OsHsplI0 and animal cells and (iii) OsHspl I0 is an immunological are likely to be the memberslisoforms of the rice Hspt00 homoIogue of the yeast Hspl04. We shall now attempt family which are differentially synthesized and regulated. to integrate information on these proteins. Following points indicate that OsHsp104 and OsHsp 110 6.. Concluding remarks proteins are probably different: Rice cells synthesize and accumulate predominant 9OsHspl04 and OsHspll0 are of different molecular amounts of Hspl04. These cells also accumulate Hspll0 weights. When anti OsHspl04 and anti yeast Hspl04 to an extent. Both of these proteins are accumulated in antibodies were pooled together, two distinctive bands response to a host of abiotic stress conditions (salinity, corresponding to 104 and 1.10kDa were noted in water stress and low temperature), apart from high Western blots (Pareek et al 1995). temperature. Considering that (i) these proteins are accumulated concomitant to acquisition of induced thermotolerance, (ii) these are present constitutively in high amounts in seeds which are relatively more thermo- tolerant and (iii) synthesis of these proteins is highly ~ E. coH 0. sativa conserved as their immunological homologues were detected in several plant and microbial species (including wheat, sorghum, brassica, pea, Neurospora, E. coli), it appears that rice Hspl00 proteins constitute an important component in the cascade of events associated with the response of rice plants to abiotic stress conditions. The precise cellular role(s) of rice Hspl00 family members remains to be identified. From the current work on Hsp90 (Pareek et al I998) and Hspl00 (this study) family of rice carried in our [aboratory, we find that these high molecular weight proteins share several common properties with respect to their expression char- Figure 2, Western detection of OsHspl04 homologue in E. acterisitics. coil cells using anti rice HspI04 antibodies, in response to high temperature stress (42 ~ and 48~C). Each lane was loaded Acknowledgements with 50/.tg proteins. Heat shock sample from O. sativa (45~ 8 h) is also shown for comparison o1' OsHspl04 position. The molecular mass (kDa) of standard proteins are shown towards Financial assistance from the Departme.nt..of Science and the left side. Arrow marks the position of Hsp]04 in rice while Technology, New Delhi (SP/SO/A-04t93), the Rockefeller its homologue in E. cofi is marked with an arrowhead. Foundation, USA (95001#350) and the Department of 344 Sneh Lata Singla, Ashwani Pareek and Anil Grover

Biotechnology (DBT), New Delhi (BTtABI05/02/91) is Key J L, Lin C-Y and Chen Y M 1981 Heat shock pro- gratefully acknowledged. SLS is thankful to the University reins of higher plants; P~vc. Nail Acad. Set USA 78 3526- 3530 Grant Commission and DBT and AP is thankful to the Khush G S and Toenniessen G H 1991 Rice biotechnology Council of Scientific and Industrial Research, New Delhi (International Rice Research Institute, Manila; CAB Interna- for the financial support. tional, UK) Kimpel J A and Key J L 1985 Presence of heat shock mRNAs References in field grown soybeans; Plant PhysioL 79 672-678 Kimura Y, Matsumoto S and Yahara I 1994 Temperature sensitive Abemathy R, Thiel D S, Petersen N S and Helm K 1989 mutants of hsp 82 of the budding yeast Saccharomyces Thermotolerance is developmentally dependent in germinating cereviseae; MoL Gen. 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Pareek A, Singla S L and Graver A 1998 Plant HSP 90 family L. to high temperature, Ph.D thesis, University of Delhi, with special reference to rice; J. BioscL 23 361-367 Delhi Parsell D A and LindquiSt S 1993 The function ofheat,shock Sing[a S L, Pareek A and Graver A 1997a Yeast HSP 104 proteins in stress tolerance : Degradation and reactivation of homologue rice HSP 110 is developmentally- and stress- damaged proteins; Annu. Rev. Genet. 27 437-496 regulated; Plant ScL 125 211-219 Parsell D A, Kowal A S, Singer M A and Lindquist S 1994 S~ng]a S L, Pareek A and Graver A 1997b High temperature; Protein disaggregation mediated by heat shock protein HSP in Plant ecophysiology (ed.) M N V Prasad (New York: John I04; Natui'e (London) 372 475-478 Wiley) pp 10t-127 Parsell D A, Sanchez Y, Stitzel J D and Lindquist S 1991 SingIa S L, Pareek A, Kush A K and Graver A 1998 Distribution HspI04 is a highly conserved protein with two essential patterns of 1041d:)a stress-associated protein in rice; Plant nucleotide binding sites; Nature (London) 353 270-273 Mol. Biol. (in press) Riabowol K T, Mizzen L A and Welch W J 1988 Heat shock Sinibaldi R M and Turpen T 1985 A heat shock protein is is lethal to fibr6bfasts microinjected with antibodies against encoded within mitochondria of higher plants; J. Biol, Chem. hsp 70; Science 242 433-436 260 15382-15385 Sambrook J T, Ffitsch E F and Maniatis T 1989 Molecular Somers D J, Giroux R W and Filion W G 199I The expression cloning: A laboratory manual, Second edition (New York: of temperature-stress proteins in a desert cactus (Opuntia ficus Cold Spring Harbor Laboratory) indica), Genome 34 940-943 Sanchez Y and Lindquist S L 1990 Hsp104 is required for Tissieres A, Mitchell H K and Tmcy U M t974 Protein synthesis induced thermotolerance; Science 248 11 ] 2-1115 in salivary glands of D. metanogaster. Relation to Chromosome Sanchez Y, Taulien J, Borkovich K A and Lindquist S I992 puffs; 3". Mol, Biol. 84 389-398 HspI04 is required for tolerance to many forms of stress; Vierling E 1991 The roles of heat shock proteins in plants; EMBO J. 11 2357-2364 Annu. Rev. Plant PhysioL Ptant~Ylol. Biol. 42 579-620 Satake T and Yoshida S 1978 High temperature induced sterility Walther-Larsen H, Brandt J, Collinge D B and Thordal-Chdstensen in indica rice at flowering; Jpt~ J. Crop Sci. 447 6-17 H 1993 A pathogen-induced gene of barley encodes a HSP 90 Schirmer E C, Lindquist S and Vierling E 1994 An Arabidopsis homologue showing striking similarity to vertebrate forms resident heat shock protein complements a thermotolerance defect in in the endoplasmic reticulum; Plant Mot BioL 21 1097-1108 yeast; Plant Cell 6 1899-1909 Widawsky D A and O'Toole J C 1990 Prioritizing the rice Singla S L and Graver A ]993 Antibodies raised against yeast biotechnology research agenda for eastern htdia (The Rocke- HSP 104 cross-react with a heat- and abscisic acid-regulated feller Foundation, USA) pp 86 polypeptide in rice; Plant MoL Biol. 22 t177-1t80 Yoshida S 1977 Rice; in Ecophysiotogy of tropical crops (eds) Singla S L and Graver A 1994 Detection and quantitation of P T Alvin and T T Kozlowski (New York: Academic Press) a rapidly accumulating and predominant I04kDa heat shock pp 57-87 polypeptide in rice; Plant ScL 97 23-30 Yoshida S, Satake T and Mackill D S 1981 High temperature Singla S L 1996 Molecular characterization of high molecular stress in rice (IRRI Research Paper Series No. 67, IRRI, weight stress proteins associated with response of 01yza sativa Manila, Philippines) pp 1-15