No abstract

Plants accumulate a family of hydrophobic polymers known as polyprenols, yet how they are synthesized, where they reside in the cell, and what role they serve is largely unknown. Using Arabidopsis thaliana as a model, we present evidence for the involvement of a plastidial cis-prenyltransferase (AtCPT7) in polyprenol synthesis. Gene inactivation and RNAi-mediated knockdown of AtCPT7 eliminated leaf polyprenols, while its overexpression increased their content. Complementation tests in the polyprenol-deficient yeast Δrer2 mutant and enzyme assays with recombinant AtCPT7 confirmed that the enzyme synthesizes polyprenols of ;55 carbons in length using geranylgeranyl diphosphate (GGPP) and isopentenyl diphosphate as substrates. Immunodetection and in vivo localization of AtCPT7 fluorescent protein fusions showed that AtCPT7 resides in the stroma of mesophyll chloroplasts. The enzymatic products of AtCPT7 accumulate in thylakoid membranes, and in their absence, thylakoids adopt an increasingly "fluid membrane" state. Chlorophyll fluorescence measurements from the leaves of polyprenol-deficient plants revealed impaired photosystem II operating efficiency, and their thylakoids exhibited a decreased rate of electron transport. These results establish that (1) plastidial AtCPT7 extends the length of GGPP to ;55 carbons, which then accumulate in thylakoid membranes; and (2) these polyprenols influence photosynthetic performance through their modulation of thylakoid membrane dynamics.

Infection with avirulent pathogens, tobacco mosaic virus (TMV) or Pseudomonas syringae pv. tabaci induced accumulation of polyisoprenoid alcohols, solanesol and a family of polyprenols [from polyprenol composed of 14 isoprene units (Pren-14) to -18, with Pren-16 dominating] in the leaves of resistant tobacco plants Nicotiana tabacum cv. Samsun NN. Upon TMV infection, solanesol content was increased seven- and eight-fold in the inoculated and upper leaves, respectively, while polyprenol content was increased 2.5- and 2-fold in the inoculated and upper leaves, respectively, on the seventh day post-infection. Accumulation of polyisoprenoid alcohols was also stimulated by exogenously applied hydrogen peroxide but not by exogenous salicylic acid (SA). On the contrary, neither inoculation of the leaves of susceptible tobacco plants nor wounding of tobacco leaves caused an increase in polyisoprenoid content. Taken together, these results indicate that polyisoprenoid alcohols might be involved in plant resistance against pathogens. A putative role of accumulated polyisoprenoids in plant response to pathogen attack is discussed. Similarly, the content of plastoquinone (PQ) was increased two-fold in TMV-inoculated and upper leaves of resistant plants. Accumulation of PQ was also stimulated by hydrogen peroxide, bacteria (P. syringae) and SA. The role of PQ in antioxidant defense in cellular membranous compartments is discussed in the context of the enzymatic antioxidant machinery activated in tobacco leaves subjected to viral infection. Elevated activity of several antioxidant enzymes (ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and superoxide dismutase, especially the CuZn superoxide dismutase isoform) and high, but transient elevation of catalase was found in inoculated leaves of resistant tobacco plants but not in susceptible plants.

SUMMARYDolichol plays an indispensable role in the N-glycosylation of eukaryotic proteins. As proteins enter the secretory pathway they are decorated by a 'glycan', which is preassembled onto a membrane-anchored dolichol molecule embedded within the endoplasmic reticulum (ER). Genetic and biochemical evidence in yeast and animals indicate that a cis-prenyltransferase (CPT) is required for dolichol synthesis, but also point to other factor(s) that could be involved. In this study, RNAi-mediated suppression of one member of the tomato CPT family (SlCPT3) resulted in a~60% decrease in dolichol content. We further show that the involvement of SlCPT3 in dolichol biosynthesis requires the participation of a distantly related partner protein, designated as CPT-binding protein (SlCPTBP), which is a close homolog of the human Nogo-B receptor. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that SlCPT3 and its partner protein interact in vivo and that both SlCPT3 and SlCPTBP are required to complement the growth defects and dolichol deficiency of the yeast dolichol mutant, rer2Δ. Co-expression of SlCPT3 and SlCPTBP in yeast and in E. coli confirmed that dolichol synthase activity strictly requires both proteins. Finally, organelle isolation and in vivo localization of fluorescent protein fusions showed that both SlCPT3 and SlCPTBP localize to the ER, the site of dolichol accumulation and synthesis in eukaryotes.

In this work we provide evidence for the potential presence of a potassium channel in skeletal muscle mitochondria. In isolated rat skeletal muscle mitochondria, Ca(2+) was able to depolarize the mitochondrial inner membrane and stimulate respiration in a strictly potassium-dependent manner. These potassium-specific effects of Ca(2+) were completely abolished by 200 nM charybdotoxin or 50 nM iberiotoxin, which are well-known inhibitors of large conductance, calcium-activated potassium channels (BK(Ca) channel). Furthermore, NS1619, a BK(Ca)-channel opener, mimicked the potassium-specific effects of calcium on respiration and mitochondrial membrane potential. In agreement with these functional data, light and electron microscopy, planar lipid bilayer reconstruction and immunological studies identified the BK(Ca) channel to be preferentially located in the inner mitochondrial membrane of rat skeletal muscle fibers. We propose that activation of mitochondrial K(+) transport by opening of the BK(Ca) channel may be important for myoprotection since the channel opener NS1619 protected the myoblast cell line C2C12 against oxidative injury.