Glucosinolate biosynthesis in Eruca sativa.

Glucosinolates (GSLs) are a highly important group of secondary metabolites in the Caparalles order, both due to their significance in plant-biome interactions and to their chemoprotective properties. This study identified genes involved in all steps of aliphatic and indolic GSL biosynthesis in Eruca sativa, a cultivated plant closely related to Arabidopsis thaliana with agronomic and nutritional value. The impact of nitrogen (N) and sulfur (S) availability on GSL biosynthetic pathways at a transcriptional level, and on the final GSL content of plant leaf and root tissues, was investigated. N and S supply had a significant and interactive effect on the GSL content of leaves, in a structure-specific and tissue-dependent manner; the metabolites levels were significantly correlated with the relative expression of the genes involved in their biosynthesis. A more complex effect was observed in roots, where aliphatic and indolic GSLs and related biosynthetic genes responded differently to the various nutritional treatments suggesting that nitrogen and sulfur availability are important factors that control plant GSL content at a transcriptional level. The biological activity of extracts derived from these plants grown under the specific nutritional schemes was examined. N and S availability were found to significantly affect the cytotoxicity of E. sativa extracts on human cancer cells, supporting the notion that carefully designed nutritional schemes can promote the accumulation of chemoprotective substances in edible plants.

A metabolic gene cluster in Lotus japonicus discloses novel enzyme functions and products in triterpene biosynthesis.

Genes for triterpene biosynthetic pathways exist as metabolic gene clusters in oat and Arabidopsis thaliana plants. We characterized the presence of an analogous gene cluster in the model legume Lotus japonicus. In the genomic regions flanking the oxidosqualene cyclase AMY2 gene, genes for two different classes of cytochrome P450 and a gene predicted to encode a reductase were identified. Functional characterization of the cluster genes was pursued by heterologous expression in Nicotiana benthamiana. The gene expression pattern was studied under different developmental and environmental conditions. The physiological role of the gene cluster in nodulation and plant development was studied in knockdown experiments. A novel triterpene structure, dihydrolupeol, was produced by AMY2. A new plant cytochrome P450, CYP71D353, which catalyses the formation of 20-hydroxybetulinic acid in a sequential three-step oxidation of 20-hydroxylupeol was characterized. The genes within the cluster are highly co-expressed during root and nodule development, in hormone-treated plants and under various environmental stresses. A transcriptional gene silencing mechanism that appears to be involved in the regulation of the cluster genes was also revealed. A tightly co-regulated cluster of functionally related genes is involved in legume triterpene biosynthesis, with a possible role in plant development.

Pepino mosaic virus capsid protein interacts with a tomato heat shock protein cognate 70.

Plant viral capsid proteins (CP) can be involved in virus movement, replication and symptom development as a result of their interaction with host factors. The identification of such interactions may thus provide information about viral pathogenesis. In this study, Pepino mosaic virus (PepMV) CP was used as bait to screen a tomato (Solanum lycopersicum) cDNA library for potential interactors in yeast. Of seven independent interacting clones, six were predicted to encode the C-termini of the heat shock cognate 70 (Hsc70) proteins. Three full length tomato Hsc70s (named Hsc70.1, .2, .3) were used to confirm the interaction in the yeast two hybrid assay and bimolecular fluorescent complementation (BiFC) in planta. The PepMV CP-Hsc70 interaction was confirmed only in the case of Hsc70.3 for both assays. In BiFC, the interaction was visualized in the cytoplasm and nucleus of agroinfiltrated Nicotiana benthamiana epidermal cells. During PepMV infection, Hsc70.3 mRNA levels were induced and protein accumulation increased at 48 and 72 h post inoculation. In transmission electron microscopy using immunogold labelling techniques, Hsc70 was detected to co-localize with virions in the phloem of PepMV-infected tomato leaves. These observations, together with the co-purification of Hsc70 with PepMV virions further support the notion of a PepMV CP/Hsc70 interaction during virus infection.

Characterization of two novel nodule-enhanced α-type carbonic anhydrases from Lotus japonicus.

Two cDNA clones coding for α-type carbonic anhydrases (CA; EC 4.2.1.1) in the nitrogen-fixing nodules of the model legume Lotus japonicus were identified. Functionality of the full-length proteins was confirmed by heterologous expression in Escherichia coli and purification of the encoded polypeptides. The developmental expression pattern of LjCAA1 and LjCAA2 revealed that both genes code for nodule enhanced carbonic anhydrase isoforms, which are induced early during nodule development. The genes were slightly to moderately down-regulated in ineffective nodules formed by mutant Mesorhizobium loti strains, indicating that these genes may also be involved in biochemical and physiological processes not directly linked to nitrogen fixation/assimilation. The spatial expression profiling revealed that both genes were expressed in nodule inner cortical cells, vascular bundles and central tissue. These results are discussed in the context of the possible roles of CA in nodule carbon dioxide (CO(2)) metabolism.