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 metals in the shoot when barley seedlings are grown in the presence of cadmium, 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 Botany 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 35S methionine followed by extraction of extracellular polypeptides. Depending on growth conditions and leaf age, 1–6% of the protein synthesized de novo in the leaf was allocated to the extractable protein fraction of the leaf apoplast. Under control conditions, labelling reached its maximum after 2 h; labelling continued to rise in Ni-treated plants, where the maximum level of final labelling was reached only after several hours. Labelling decreased with leaf age and increased proportionally with Ni concentration in the nutrient solution. (2) Autolysis experiments on extracted apoplastic proteins and in situ digestion of infiltrated ovalbumin revealed a low proteolytic activity in the apoplast which was not altered in heavy metal-treated leaves. (3) Following a sudden application of Ni to the growth medium, protein content and peroxidase activity increased with a 48 h delay and approached the level obtained for continuously Ni-treated plants, even in previously fully expanded leaf tissue. It is concluded that altered cell wall structure is probably not involved in increasing the extraction yield of apoplastic proteins. (4) The increase in extractable apoplastic proteins was also not due to inhibited ionical or covalent immobilization of proteins in the cell wall matrix of heavy metal-challenged plants. (5) Northern blotting revealed heavy metalinduced upregulation of mRNA levels of selected apoplastic proteins. Taken together, the data provide evidence for a transcriptional or translational level of regulation as the main cause of the strong response of apoplastic proteins to heavy metal stress.