Sneh Lata Singla

Immunological evidence for accumulation of two high-molecular-weight (104 and 90 kDa) HSPs in response to different stresses in rice and in response to high temperature stress in diverse plant genera

Rice seedlings accumulate stainable amounts of the 104 and 90 kDa polypeptides in response to high temperature stress. We have purified and raised highly specific polyclonal antisera against both of these polypeptides. In western blotting experiments, we find that these proteins are accumulated to different extents in rice seedlings subjected to salinity (NaCl), water stress, low-temperature stress and exogenous abscisic acid application. These proteins also accumulated when rice seedlings grown in pots under natural conditions were subjected to water stress by withholding watering. Seedlings of Triticum aestivum, Sorghum bicolor, Pisum sativum, Zea mays, Brassica juncea and mycelium of Neurospora crassa showed accumulation of the immunological homologues of both the 104 and the 90 kDa polypeptides, in response to high-temperature stress. We have earlier shown that shoots of rice seedlings exposed to heat shock accumulate a 110 kDa polypeptide which is an immunological homologue of the yeast HSP 104 (Singla and Grover, Plant Mol Biol 22: 1177–1180, 1993). Employing anti-rice HSP 104 antibodies and anti-yeast HSP 104 antibodies together, we provide evidence that rice HSP 104 is different from the earlier characterized rice HSP 110.

Detection and quantitation of a rapidly accumulating and predominant 104 kDa heat shock polypeptide in rice

Abstract When 5-day-old rice seedlings were subjected to a high temperature stress (45°C), the shoots as well as roots synthesized and accumulated a 104kDa polypeptide within 1–2 h of the imposition of the stress. The higher levelsof this polypeptide persisted for nearly 16 h when the 45°C temperature stress was continued for 24 h or up to 4 h when seedlings were brought to recovery at 28°C after 4 h of 45°C temperature stress. The leaves obtained from mature plants also accumulated the same polypeptide in response to temperature stress. Exposure of seedlings to salinity, desiccation, cold, wounding or exogenous abscisic acid application had no effect on the accumulation of this polypeptide. Importanly, this polypeptide made nearly 0.4% of the total soluble protein fraction. High temperature induced accumulation of this polypeptide was noted to be conserved in three indica and three japonica cultivars Oryza sativa tested in this study.

Distribution patterns of 104 kDa stress-associated protein in rice

A 104 kDa protein (SAP 104) accumulates in rice seedlings in response to several abiotic stress conditions and immunological homologues of rice SAP 104 have been detected in several monocot and dicot species, as also Neurospora crassa, a fungus. In this report, we show that the amino acid sequence of a tryptic peptide generated from purified SAP 104 bears significant homology with an ATP-binding domain of the HSP 100 family proteins of Arabidopsis thaliana and Glycine max. It is further shown that differential uninduced and induced (by high-temperature stress) levels of this protein are accumulated in various organs of the mature rice plant grown under field conditions. Significant uninduced levels of this protein were in particular found in developing and mature rice grains. Seeds/grains of several other plant genera (i.e. Triticum aestivum, Zea mays, Brassica juncea) were also found to contain high uninduced levels of SAP 104. Importantly, the levels of uninduced SAP 104 in rice grains were found to decline during the seed germination phase: after two days of germination, this protein was undetectable in tissues representing pooled sample of seeds and just-emerged seedlings. Tissue print-immunoblotting analysis has indicated that in seeds high levels of this protein are specifically present in the embryo portion.

Antibodies raised against yeast HSP 104 cross-react with a heat-and abscisic acid-regulated polypeptide in rice

Antibodies raised against yeast heat shock protein (HSP) 104 recognized a heat-inducible polypeptide with a molecular mass of 110 kDa in shoot tissue of young rice seedlings. Root tissue of the same age showed no immuno-reaction with yeast HSP 104 antibodies. The 110 kDa polypeptide of rice was also shown to be abscisic acid-inducible in young seedlings. Though this polypeptide was seen to be constitutively present in the flag leaf of 90-day-old field-grown plant, it was not much affected by either heat shock or abscisic acid in this case.

Plant Hsp100 family with special reference to rice

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 Hsp100 family have been directly implicated in induction of thermotolerance in microbial and animal cells. Yeast cells harbouring defectivehsp104 gene do not show thermotolerance under conditions in which the normal cells do. Several plant species have been shown to synthesize Hsps in the range of 100 kDa. Rice Hsp104 (OsHsp104) is rapidly and predominantly accumulated in heat-shocked cells. Western blotting analysis show that anti rice Hsp104 antibodies (generated against purified Hsp104 protein from cultivated riceOryza sativa L.) cross-react with the same-sized high temperature inducible protein in 15 different wild rices. It was further found that anti rice Hsp104 antibodies also cross-react with a major high temperature regulated protein ofEscherichia coli. We have previously shown that a 110 kDa stress regulated protein in rice (OsHsp110) is immunologically related to yeast Hsp104 protein. In this paper, we present a comparative account of characteristics of the OsHsp104 and OsHsp110 proteins.

Distribution patterns of HSP 90 protein in rice

Rice seedlings exposed to high and low temperatures, salinity and water stress accumulate 87 and 85 kDa stress-associated-proteins (SAP), collectively referred to as SAP 90. In the present study, we provide evidence that SAP 87 of rice is a member of the eukaryotic stress 90/HSP 90 family. Further, high uninduced levels of rice SAP/HSP 90 exist in lemma, palea and culm tissues. Dry seeds of rice, wheat, maize and sorghum also contain high uninduced amounts of this protein. Seed-derived callus tissue show high constitutive levels of this protein as well. When seed and callus tissues were subjected to heat shock, the levels of this protein declined. Tissue print-immunoblot analysis using anti SAP 90 antibodies revealed higher accumulation of SAP/HSP 90 in the vascular bundles/procambial cells and in the outermost cell layers of various leaves as well as in the meristematic cells of the stem apex. In seeds, this protein was most abundant in the seed coat and whole embryo.

Yeast HSP104 homologue rice HSP110 is developmentally- and stress-regulated

HSP104 plays a critical role in the development of thermotolerance in yeast cells. Rice (Oryza sativa L.) HSP110 (OsHSP110) is previously shown to be an immunological homologue of yeast HSP104. This protein accumulates in rice seedlings in response to heat shock. However, no such high temperature-induced accumulation of OsHSP110 was found in the topmost leaf of 90-day-old (just prior to flowering) plants of the cultivated species of rice (Oryza sativa L.) in our earlier study. In this paper, we show that, at the comparable growth stage, leaves of O. australiensis accumulated this protein to a marginal extent in response to heat shock, while levels of HSP110 were either markedly declined or remained unaltered in 14 other wild rice species. Further, different organs of the mature cultivated rice plant accumulated differential levels of OsHSP110, constitutively as well as in response to heat shock. In particular, upper portions of culm, grains and developing embryos showed significantly high constitutive levels of OsHSP110. Tissue print immunolocalization studies showed that OsHSP110 is distributed in the vascular bundles in the shoot tissues and in seeds it is specifically localized in the seec coat (outermost layer) only. Importantly, OsHSP110 accumulated in shoots of rice seedlings in response to salinity, desiccation and low temperature stress also; furthermore, salinity stress (NaCl) caused nearly a three-fold higher accumulation of this protein than high temperature stress.

Evidence for accumulation of a 55 kDa stress-related protein in rice and several other plant genera

Rice cells synthesize a 87 kDa protein (SAP 87) which cross-reacts with anti Neurospora crassa HSP 80 antibodies. Apart from SAP 87, we show that a polypeptide of 55 kDa of rice cross-reacts with these antibodies. This 55 kDa polypeptide is accumulated in response to high and low temperatures, salinity and desiccation stress conditions in shoots of rice seedlings. Apart from rice, Triticum aestivum, Sorghum bicolor, Pisum sativum, Zea mays and Brassica juncea seedlings also showed accumulation of the 55 kDa polypeptide in response to high temperature stress on Western blots. Importantly, anti human HSP 56 antibodies showed cross-reaction with a major high temperature induced 55 kDa protein of rice that matched in position to the 55 kDa protein which cross-reacted with anti N. crassa HSP 80 antibodies.