Differential expression of aspartic and cysteine proteinases, glutamine synthetase, and a stress protein, HVA1, in germinating barley

Abstract

Nitrogen is considered as a rate-limiting factor in plant growth. In barley (Hordeum vulgare, L.) seed, most of the nitrogen is deposited in storage proteins, which are remobilized for the growing seedling during germination. Proteolytic enzymes are of central importance in the hydrolysis of the reserve proteins. The main transport form of nitrogen in cereal seeds is glutamine, which is synthesized by glutamine synthetase. An ABA-responsive protein, HVAl, is induced during seed maturation but also due to desiccation stress during germination. In the present study, the expression of two different acid proteases, an aspartic and a cysteine proteinase, together with glutamine synthetase and a stress protein, HVAl, was studied by immuno-microscopy during barley germination. Aspartic proteinase is already present in the mature barley seed. During germination, it was gradually expressed in all tissues of the seed, excluding the dead starchy endosperm. Subcellulary, aspartic proteinase was localized in the protein storage vacuoles. Tissue distribution and the amount of the two molecular forms of the enzyme varied, suggesting different physiological roles for the two heterodimers. Contrary to the case of aspartic proteinase, the synthesis and secretion of the 30 kD cysteine proteinase was started in the scutellar epithelium after the beginning of germination, and continued in the aleurone layer. The enzyme was found to be secreted via the Golgi complex and the putative secretory vesicles to the cell exterior. Glutamine synthetase (GS) was expressed as a cytosolic isozyme in the scutellum and the aleurone layer of germinating barley seed. At the beginning of germination, GS was likely to be expressed as a homo-octamer, and later as different hetero-octamers. Under the stress conditions, a barley LEA3 protein, HVAl, was strongly induced, although it was present in the mature seed and declined gradually during germination. Although HVAl protein has no putative signal peptide, it was localized both in the cytoplasm and protein storage vacuoles, suggesting the presence of a direct uptake mechanism. HV Al might be degraded by the aspartic proteinase, as they were colocalized in the protein storage vacuoles. The exact function of HVAl remains an open question.