William F. Ettinger

Targeting of glyoxysomal proteins to peroxisomes in leaves and roots of a higher plant.

Higher plants possess several classes of peroxisomes that are present at distinct developmental stages and serve different metabolic roles. To investigate the cellular processes that regulate developmental transitions of peroxisomal function, we analyzed the targeting of glyoxysomal proteins to leaf-type and root peroxisomes. We transferred genes encoding the glyoxysome-specific enzymes isocitrate lyase (IL) and malate synthase into Arabidopsis plants and showed, in cell fractionation and immunogold localization experiments, that the glyoxysomal proteins were imported into leaf-type and root peroxisomes. We next defined the sequences that target IL to peroxisomes and asked whether the same targeting determinant is recognized by different classes of the organelle. By localizing deletion and fusion derivatives of IL, we showed that the polypeptides carboxyl terminus is both necessary for its transport to peroxisomes and sufficient to redirect a passenger protein from the cytosol to both glyoxysomes and leaf-type peroxisomes. Thus, glyoxysomal proteins are transported into several classes of peroxisomes using a common targeting determinant, suggesting that protein import does not play a regulatory role in determining a peroxisomes function. Rather, the specific metabolic role of a peroxisome appears to be determined primarily by processes that regulate the synthesis and/or stability of its constituent proteins. These processes are specified by the differentiated state of the cells in which the organelles are found.

Assembly of Newly Imported Oxygen-Evolving Complex Subunits in Isolated Chloroplasts: Sites of Assembly and Mechanism of Binding.

We have examined the assembly of the nuclear-encoded subunits of the oxygen-evolving complex (OEC) after their import into isolated intact chloroplasts. We showed that all three subunits examined (OE33, OE23, and OE17) partition between the thylakoid lumen and a site on the inner surface of the thylakoid membrane after import in a homologous system (e.g., pea or spinach subunits into pea or spinach chloroplasts, respectively). Although some interspecies protein import experiments resulted in OEC subunit binding, maize OE17 did not bind thylakoid membranes in chloroplasts isolated from peas. Newly imported OE33 and OE23 were washed from the membranes at the same concentrations of urea and NaCl as the native, indigenous proteins; this observation suggests that the former subunits are bound productively within the OEC. Inhibition of neither chloroplast protein synthesis nor light- or ATP-dependent energization of the thylakoid membrane significantly affected these assembly reactions, and we present evidence suggesting that incoming subunits actively displace those already bound to the thylakoid membrane. Transport of OE33 took place primarily in the stromal-exposed membranes and proceeded through a protease-sensitive, mature intermediate. Initial binding of OE33 to the thylakoid membrane occurred primarily in the stromal-exposed membranes, from where it migrated with measurable kinetics to the granal region. In contrast, OE23 assembly occurred in the granal membrane regions. This information is incorporated into a model of the stepwise assembly of oxygen-evolving photosystem II.

Translational or post-translational processes affect differentially the accumulation of isocitrate lyase and malate synthase proteins and enzyme activities in embryos and seedlings of Brassica napus

We have analyzed the accumulation of the glyoxylate cycle enzymes isocitrate lyase and malate synthase in embryos and seedlings of Brassica napus L. The two enzyme activities and proteins begin to accumulate during late embryogeny, reach maximal levels in seedlings, and are not detected in young leaves of mature plants. We showed previously that mRNAs encoding the two enzymes exhibit similar qualitative patterns of accumulation during development and that the two mRNAs accumulate to different levels in both embryos and seedlings (L. Comai et al., 1989, Plant Cell 1, 293-300). In this report, we show that the relative accumulation of the proteins and activities do not correspond to these mRNA levels. In embryos and seedlings, the specific activities of isocitrate lyase and malate synthase are approximately constant. By contrast, the ratio of malate synthase protein to mRNA is 14-fold higher than that of isocitrate lyase. Differences in the translational efficiencies of the two mRNAs in vitro do not appear to account for the discrepancy between mRNA and protein levels. Our results suggest that translational and/or post-translational processes affect differentially the accumulation of the proteins.

Cuticular Lipids in Plant-microbe Interactions

Cuticle constitutes the boundary between higher plants and their environment. Therefore, this layer might be expected to play an important role in the interaction of the plant with environmental factors. The plant cuticle is composed almost entirely of lipids and the role of some of these lipids in the interaction between plants and microbes has become clear in the recent years. In this brief review, we shall confine our discussion to two specific examples of such interactions: a detrimental one with pathogenic fungi and a beneficial one with phyllospheric bacteria which might provide fixed nitrogen in return for the use of some of the cuticular components as the carbon source. In this context, we will deal only with the role of the insoluble 1ipid-derived polymer, cutin, but not the role of soluble waxes that are always constituents of the cuticle.

Cutinase and Pectinase in Host-Pathogen and Plant-Bacterial Interaction

The aerial parts of plants are covered by the cuticle which forms the boundary layer at which microbes come into contact with the plant. The cuticle is composed of an insoluble structural polymer, cutin, which is embedded in a complex mixture of soluble lipids collectively called wax (1,2). Cutin is composed of interesterified hydroxy and hydroxyepoxy fatty acids primarily derived from palmitic, oleic, and linoleic acids (Fig. 1). The component fatty acids are held together by ester bonds between the primary as well as secondary hydroxy groups and the carboxyls. The cutin polymer constitutes the major physical barrier to penetration of fungi into the aerial parts of plants (3). The cutin barrier is attached to a pectinaceous polymeric layer which may also serve as a barrier to penetration by pathogens. In this paper, we will briefly review progress recently made in our understanding of the role of fungal cutinases and pectinases in the interaction between pathogenic fungi and their host plants. We also describe a recent discovery that a phyllospheric bacterium which cohabits with an apparently nitrogen-fixing bacterium generates an extra-cellular cutinase and thus can provide a carbon source while receiving fixed N2 from the cohabiting partner.