The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley leaves

The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) leaves has been studied. In some dicotyledonous plant species, including tomato, exposure to chemical stress leads to the denovo synthesis of intercellular proteins known as pathogenesis-related proteins which have been implicated to be part of a defence mechanism. In barley, however, no such changes in the polypeptide composition of the intercellular fluid could be detected. On the other hand, similar stress conditions induce in barley a strong accumulation of mRNA encoding leaf-specific thionins. These barley thionins represent a novel class of cell-wall proteins toxic to phytopathogenic fungi and are possibly involved in the defence mechanism. These proteins could not be detected in tomato plants. In contrast to the pathogenesis-related proteins of dicotyledonous plants, the leaf-specific thionins of barley are not present in the intercellular fluid of leaves. These results indicate that barley may have evolved a different mechanism to cope with the presence of stress.Abbreviations PAGE polyacrylamide gel electrophoresis - PR pathogenesis-related - SDS sodium dodecyl sulfate     

Nucleotide sequence of a cDNA coding for the NADPH-protochlorophyllide oxidoreductase (PCR) of barley (Hordeum vulgare L.) and its expression in Escherichia coli

Summary The primary structure of the NADPH-protochlorophyllide oxidoreductase of barley has been deduced from the nucleotide sequence of a cloned full-length cDNA. This cDNA hybridizes to a 1.7 kb RNA whose steady-state level in dark-grown seedlings is drastically reduced upon illumination. The predicted amino acid sequence (388 residues in length) includes a transit peptide of 74 amino acids whose end point has been delimited by sequencing the N-terminus of the mature protein. Expression of the cDNA inEscherichia coli leads to the synthesis of an enzymatically active precursor of the NADPH-protochlorophyllide oxidoreductase. Activity of this protein in bacterial lysates is completely dependent on the presence of NADPH and protochlorophyllide and requires light.     

Isolation and characterization of cDNAs coding for leaf-specific thionins closely related to the endosperm-specific hordothionin of barley (Hordeum vulgare L.)

Summary In etiolated barley seedlings a highly abundant mRNA encoding a 15 000 M_r polypeptide is present whose concentration rapidly declines upon illumination. The amino acid sequence of the 15 000 M_r polypeptide has been deduced from cDNA sequences and the polypeptide has been identified as a high-molecular-weight thionin precursor. Closely related thionins, most of them highly toxic, have been described previously in several higher plants. In cereals the occurrence of these thionins has been thought to be confined to the seeds. Our present data demonstrate that, in additon to endosperm-specific hordothionin mRNA, a closely related but distinct second group of thionin mRNAs is present in the barley leaf during early seedling development. Since the appearance of the bordothionin mRNA is not controlled by light, two different gene families for thionins exist whose expression seems to be under a different mode of developmental control.Dedicated to Prof. W. Halbsguth on the occasion of his 75th birthday     

Evidence for a general light-dependent negative control of NADPH-protochlorophyllide oxidoreductase in angiosperms

The effect of light on NADPH-protochlorophyllide oxidoreductase and its mRNA has been studied in five different species of dicotyledonous plants, bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), tomato (Lycopersicon esculentum Mill.), sunflower (Helianthus annuns L.) and mustard (Sinapis alba L.), and in two monocotyledonous plant species, maize (Zea mays L.) and barley (Hordeum vulgare L.). In all these species, illumination of etiolated seedlings led to a rapid decline of both the activity and the content of the enzyme protein. These results indicate that there may be a general light-dependent regulation of the enzyme common to higher plants.Abbreviation Pchlide reductase NADPH-protochlorophyllide oxidoreductase Didicated to Professor H. Mohr on the occasion of his 60th birthdayWe are grateful to P. Westhoff and H. Schrubar (Botanisches Institut, Universität Düsseldorf, FRG) for making available to us their data on the Pchlide reductase of Sorghum bicolor prior to publication and for sending us seeds of this plant species. This work has been supported by the Deutsche Forschungsgemeinschaft.     

Phosphorylation of neuromodulin (GAP-43) by casein kinase II. Identification of phosphorylation sites and regulation by calmodulin.

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin-binding protein believed to play a role in regulation of neurite outgrowth and neuroplasticity. Neuromodulin is phosphorylated by protein kinase C, and this phosphorylation prevents calmodulin from binding to neuromodulin (Alexander, K. A., Cimler, B. M., Meier, K. E. & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). The only other protein kinase known to phosphorylate neuromodulin is casein kinase II (Pisano, M. R., Hegazy, M. G., Reimann, E. M. & Dokas, L. A. (1988) Biochem. Biophys. Res. Commun. 155, 1207-1212). Phosphoamino acid analyses revealed that casein kinase II modified serine and threonine residues in both native bovine and recombinant mouse neuromodulin. Two serines located in the C-terminal end of neuromodulin, Ser-192 and Ser-193, were identified as the major casein kinase II phosphorylation sites. Thr-88, Thr-89, or Thr-95 were identified as minor casein kinase II phosphorylation sites. Phosphorylation by casein kinase II did not affect the ability of neuromodulin to bind to calmodulin-Sepharose. However, calmodulin did inhibit the phosphorylation of neuromodulin by casein kinase II with a Ki of 1-2 microM. Calmodulin inhibition of casein kinase II phosphorylation was due to calmodulin binding to neuromodulin rather than to the protein kinase. These data suggest that the minimal secondary and tertiary structure exhibited by neuromodulin may be sufficient to juxtapose its calmodulin-binding domain, located at the N-terminal end, with the neuromodulin casein kinase II phosphorylation sites at the C-terminal end of the protein. We propose that calmodulin regulates casein kinase II phosphorylation of neuromodulin by binding to neuromodulin and sterically hindering the interaction of casein kinase II with its phosphorylation sites on neuromodulin.     

The proteolytic degradation in vitro of the NADPH-protochlorophyllide oxidoreductase of barley (Hordeum vulgare L.)

A cell-free membrane system has been developed from isolated barley etioplasts which displays a highly selective decrease of the NADPH-protochlorophyllide oxidoreductase in vitro which is indistinguishable from that observed previously in the intact plant. The rapid breakdown of the enzyme protein in vitro is caused by a membrane-bound proteolytic activity. The protease is essentially independent of pH in the physiological pH range of 6 to 8.5. The optimum temperature for the reaction is approximately 40 degrees C. In the presence of excessive protochlorophyllide the enzyme is no longer degraded or inactivated during illumination of dark-grown plants. In the isolated membrane fraction protochlorophyllide also enhances the stability of the enzyme, a similar effect is exerted by NADPH but not by NADH. The results suggest that the inactivation of the NADPH-protochlorophyllide oxidoreductase is influenced by the interaction of the enzyme with protochlorophyllide and NADPH. In the absence of these two components the enzyme becomes susceptible to proteolytic degradation.