Germin-Like Polypeptides Increase in Barley Roots during Salt Stress

Abstract

The 26 kilodalton, isoelectric point 6.3 and 6.5 (Gsl and Gs2) polypeptides that increase in barley (Hordeum vulgare L.) roots during salt stress were isolated and identified. Both Gsl and Gs2 had high sequence similarity to germin, a protein that increases significantly in germinating wheat seeds. Like germin, Gsl and Gs2 were resistant to proteases and were glycosylated. lmmunoblots were probed with antibodies to Gs1 and Gs2 to determine the distribution of these polypeptides among organs and cell-free fractions. Gsl and Gs2 were present in roots and coleoptiles, but absent from leaves. In roots, Gsl and Gs2 were present in the mature region, but not the tip. Gsl and Gs2 increased in roots, but decreased in coleoptiles in response to salt stress. Gsl and Gs2 were distributed among the soluble, microsomal, and cell wall fractions of roots, but the majority of Gsl and Gs2 was present in the soluble fraction. Although Gsl and Gs2 were heat stable, their synthesis was not affected by abscisic acid treatment. Gs2 accumulated during abscisic acid treatment, whereas Gsl did not. However, a 25.5 kilodalton, isoelectric point 6.1 polypeptide that was immunologically related to Gsl did accumulate with abscisic acid treatment.Salt stress alters the levels of a number of polypeptides and translatable mRNAs in barley roots (10, 1 1, 13-15). The most obvious changes in protein synthesis are increases in polypeptides with molecular masses of 26 and 27 kD and pIs2 of 6.3 and 6.5 (10,11,13,14). Evidence from studies on length of NaCl treatment, concentration of NaCl, recovery from salt stress, effect of other stresses, and analysis of different cvs indicates that these polypeptides have a specific role in barley plants during salt stress. The increase in the synthesis of the pl 6.3 and 6.5 polypeptides occurs within a short time after exposure of plants to salt (1 1, 27) and at NaCl concentrations as low as 50 mM (13). The synthesis of the pI 6.3 and 6.5 polypeptides decreases when salt-treated seedlings are transferred to nutrient solution without NaCl (13). The increase in the synthesis of the pl 6.3 and 6.5 polypeptides is a specific response to salt stress; synthesis does not increase in response to water deficit (14) or heat shock (10,13 synthesis of these polypeptides increases in CM 72, a salttolerant cv, but not in Prato, a salt-sensitive cv (1 1).The pl 6.3 and 6.5 polypeptides were isolated using preparative two dimensional gels to obtain clues to their identities and functions. N-terminal amino acid sequences were determined for the pl 6.3 and 6.5 polypeptides, and sequence comparisons were used to identify these polypeptides. Antibodies were raised against the pl 6.3 and 6.5 polypeptides and used to probe blots of polyacrylamide gels to determine their distribution among barley organs and cell-free fractions. Because the levels of osmotin (28, 31), rab2 1 (24), and salT (1) respond to salt stress and ABA, the effect of ABA on the levels of the pl 6.3 and 6.5 polypeptides was also examined. MATERIALS AND ...

Tissue-Specific Expression of Germin-Like Oxalate Oxidase during Development and Fungal Infection of Barley Seedlings

“…In young seedlings (3 d of growth) the enzyme is localized in the mature region of the root epidermis as well as in the coleorhiza, in accordance with the work of Hurkman et al (1991). However, after 10 d of growth the activity was found only in the coleorhiza and was not detectable in roots.…”

“…It has been shown that several germin isoforms are induced during the development and germination of wheat embryos (Grzelczak and Lane, 1984;Grzelczak et al, 1985;Lane et al, 1987Lane et al, , 1992, as well as in roots of salt-stressed barley plants (Hurkman et al, 1991(Hurkman et al, , 1994. Since only the wheat germin isoform G has been shown to be an oxalate oxidase (Lane et al, 1993), it remains unclear whether all these germin isoforms possess oxalate oxidase activity.…”

“…Although germin was initially discovered in cereals by Lane and co-workers, a cDNA sequence showing a significant homology with germin has been isolated from Arabidopsis thaliana (Hofte et al, 1993;Delseny et al, 1994), suggesting that germin homologs exist in dicotyledons. Recently, two polypeptides, Gsl and Gs2, induced during salt stress of a salt-tolerant barley cultivar, were identified as germin-like proteins by amino acid sequencing (Hurkman et al, 1991(Hurkman et al, , 1994, suggesting that members of the germin family may also have a protective function during osmotic stress. Moreover, studies of changes of protein synthesis during salt stress of the halophyte Mesembryanthemum c ystallinum led to the cloning of a cDNA induced by salt stress that has sequence similarity to germin (Michalowski and Bohnert, 1992;Andolfatto et al, 1994).…”

Tobacco Nectarin I

“…Numerous functions have been proposed for GLPs, including desiccation and hydration (39), restructuring of cell walls (13), salt and heavy metal response (40), and plant defenses (15,41,42). Because GLPs represent a large family of extracellular proteins, some of which have superoxide dismutase activity, we propose a novel function for these proteins in mediating the oxidative burst during the wound response.…”

Molecular Cloning of Abscisic Acid-Responsive mRNAs Expressed during the Induction of Freezing Tolerance in Bromegrass (Bromus inermis Leyss) Suspension Culture

“…Wheat germin is expressed in partially hydrated (60% water) germinating embryos (Lane et al, 1991). The function of this protein is not known; however, a polypeptide from barley that is specifically salt-stress inducible (Hurkman et al, 1991) has a high sequence similarity to it. Germin also has moderate amino acid similarity to spherulins of Physanum polycephalum, proteins expressed if plasmodia are subjected to environmental stresses, including osmotic and temperature extremes (Lane et al, 1991).…”