The Crotalaria juncea metal transporter CjNRAMP1 has a high Fe uptake activity, even in an environment with high Cd contamination.

Large quantities of Fe and Cd accumulate in the leaves of the metal-accumulating leguminous plant, Crotalaria juncea. A member of the metal transporter NRAMP family was cloned from C. juncea. The amino acid sequence of this clone, designated CjNRAMP1, was similar to the sequence of Arabidopsis AtNRAMP1, which is involved in Fe and Cd transport. Organ-specific analysis showed that CjNRAMP1 mRNA was expressed mainly in the leaves of C. juncea plants, as well as in stems and roots. Use of green fluorescent protein fused to CjNRAMP1 suggested its localization to the plasma membranes of plant cells. Complementation experiments using yeast strains with impaired metal transport systems showed that CjNRAMP1 transported both Fe and Cd in an inward direction within the cells. Transgenic Arabidopsis plants overexpressing CjNRAMP1 showed high tolerance to Cd, with Cd translocation from roots to leaves being substantially greater in transgenic than in wild-type plants. Overexpression of CjNRAMP1 resulted in a greater accumulation of Fe in shoots and roots, suggesting that CjNRAMP1 recognizes Fe and Cd as substrates and that the high Cd tolerance of CjNRAMP1 is due to its strong Fe uptake activity, even in the presence of high Cd concentrations in the rhizosphere.

Characterization of endo-1,3-1,4-ß-glucanases in GH family 12 from Magnaporthe oryzae.

We have cloned three putative endoglucanase cDNAs, designated MoCel12A, MoCel12B, and MoCel12C, from Magnaporthe oryzae. The deduced peptide sequences of both MoCel12A and MoCel12B contain secretion signal peptides and a catalytic core domain that classify them into GH subfamily 12-1. In contrast, the deduced peptide sequence of MoCel12C consists of a signal peptide, a catalytic core domain, and a fungal-type carbohydrate binding module belonging to GH subfamily 12-2. Although most GH family 12 endoglucanases hydrolyze ß-1,4-glucans such as carboxymethylcellulose or phosphoric acid-swollen cellulose, MoCel12A that was prepared by overexpression in M. oryzae and Brevibacillus choshinensis hydrolyzed specifically 1,3-1,4-ß-glucans, such as barley ß-glucan and lichenan. The specific activity of MoCel12A overexpressed in M. oryzae was about 20 times higher than that prepared from B. choshinensis. Furthermore, MoCel12B prepared by overexpression in B. choshinensis also revealed preferential hydrolysis of endo-1,3-1,4-ß-glucans with limited hydrolysis on carboxymethylcellulose. In comparison with MoCel12A, the activity of MoCel12B was more stable under alkaline conditions. Levels of mRNA encoding MoCel12A were constitutively high during infection and spore formation. The overexpression and disruption of the MoCel12A gene did not affect germination, appressorium formation, or invasion rate; however, M. oryzae overexpressing MoCel12A produced larger numbers of spores than the wild type or a mutant in which the MoCel12A gene was disrupted. These results suggest that MoCel12A functions in part to hydrolyze 1,3-1,4-ß-glucan during infection and spore formation.