A role of Toc33 in the protochlorophyllide-dependent plastid import pathway of NADPH:proto¬chlorophyllide oxidoreductase (POR) A

NADPH: protochlorophyllide oxido reductase (POR) A is a key enzyme of chlorophyll biosynthesis in angiosperms. It is nucleus-encoded, synthesized as a larger precursor in the cytosol and imported into the plastids in a substrate-dependent manner. Plastid envelope membrane proteins, called protochlorophyllide dependent translocon proteins, Ptcs, have been identified that interact with pPORA during import. Amongthem are a 16-kDa ortholog of the previously characterized outer envelope protein Oep16 (named Ptc16) and a33-kDa protein (Ptc33) related to the GTP-binding proteins Toc33 and Toc34 of Arabidopsis. In the present work, we studied the interactions and roles of Ptc16 and Ptc33 during pPORA import. Radio labeled Ptc16/Oep16 was synthesized from a corresponding cDNA and imported into isolated Arabidopsis plastids. Crosslinking experiments revealed that import of35S-Oep16/Ptc16 is stimulated by GTP.35S-Oep16/Ptc16forms larger complexes with Toc33 but not Toc34. Plastids of the ppi1 mutant of Arabidopsis lacking Toc33, were unable to import pPORA in darkness but imported the small subunit precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSSU), precursor ferredoxin (pFd) as well as pPORB which is a close relative of pPORA. In white light, partial suppressions of pSSU, pFd and pPORB import were observed. Our results unveil a hitherto unrecognized role of Toc33 in pPORA import and suggest photo oxidative membrane damage, induced by excess Pchlide accumulating in ppi1 chloroplasts because of the lack of pPORA import, to be the cause of the general drop of protein import.

Transcriptional changes and oxidative stress associated with the synergistic interaction between potato virus X and potato virus Y and their relationship with symptom expression.

Many virus diseases of economic importance to agriculture result from mixtures of different pathogens invading the host at a given time. This contrasts with the relatively scarce studies available on the molecular events associated with virus---host interactions in mixed infections. Compared with single infections, co-infection of Nicotiana benthamiana with Potato virus X (PVX) and Potato virus Y (PVY) resulted in increased systemic symptoms (synergism) that led to necrosis of the newly emerging leaves and death of the plant. A comparative transcriptional analysis was undertaken to identify quantitative and qualitative differences in gene expression during this synergistic infection and correlate these changes with the severe symptoms it caused. Global transcription profiles of doubly infected leaves were compared with those from singly infected leaves using gene ontology enrichment analysis and metabolic pathway annotator software. Functional gene categories altered by the double infection comprise suites of genes regulated coordinately, which are associated with chloroplast functions (downregulated), protein synthesis and degradation (upregulated), carbohydrate metabolism (upregulated), and response to biotic stimulus and stress (upregulated). The expressions of reactive oxygen species?generating enzymes as well as several mitogen-activated protein kinases were also significantly induced. Accordingly, synergistic infection induced a severe oxidative stress in N. benthamiana leaves, as judged by increases in lipid peroxidation and by the generation of superoxide radicals in chloroplasts, which correlated with the misregulation of antioxidative genes in microarray data. Interestingly, expression of genes encoding oxylipin biosynthesis was uniquely upregulated by the synergistic infection. Virus-induced gene silencing of ?-dioxygenase1 delayed cell death during PVX?PVY infection.