DE-NOVO SYNTHESIS AND ACCUMULATION OF APOPLASTIC PROTEINS IN LEAVES OF HEAVY METAL-EXPOSED BARLEY SEEDLINGS

The leaf apoplast is a site of preferential accumulation of heavy meta ls in the shoot when barley seedlings are grown in the presence of cad mium, nickel or zinc in hydroponic medium. It was shown recently that apoplastic protein content increased concomitantly in plants grown in the presence of high Zn (Brune et al. 1994, Journal Experimental Botan y 45, 1189), Here it is demonstrated that apoplastic proteins increase to an even greater extent in Ni-treated plants, and that the response is intermediate for Cd-treated plants, The paper focuses on possible causes for the increase in apoplastic proteins, (1) Synthesis of cell wall proteins was studied by in vivo pulse labelling of leaf proteins with S-35 methionine followed by extraction of extracellular polypepti des, Depending on growth conditions and leaf age, 1--6% of the protein synthesized de novo in the leaf was allocated to the extractable prot ein fraction of the leaf apoplast, Under control conditions, labelling reached its maximum after 2 h; labelling continued to rise in Ni-trea ted plants, where the maximum level of final labelling was reached onl y after several hours, Labelling decreased with leaf age and increased proportionally with Ni concentration in the nutrient solution, (2) Au tolysis experiments on extracted apoplastic proteins and in situ diges tion of infiltrated ovalbumin revealed a low proteolytic activity in t he apoplast which was not altered in heavy metal-treated leaves, (3) F ollowing a sudden application of Ni to the growth medium, protein cont ent and peroxidase activity increased with a 48 h delay and approached the level obtained for continuously Ni-treated plants, even in previo nsly fully expanded leaf tissue. It is concluded that altered cell wal l structure is probably not involved in increasing the extraction yiel d of apoplastic proteins, (4) The increase in extractable apoplastic p roteins was also not due to inhibited ionical or covalent immobilizati on of proteins in the cell wall matrix of heavy metal-challenged plant s, (5) Northern blotting revealed heavy metal-induced upregulation of mRNA levels of selected apoplastic proteins. Taken together, the data provide evidence for a transcriptional or translational level of regul ation as the main cause of the strong response of apoplastic proteins to heavy metal stress.