Modification of substrate specificity in single point mutants of Agrobacterium tumefaciens type II NADH dehydrogenase.

Type II NADH dehydrogenases (NDH-2) are monomeric flavoenzymes catalyzing electron transfer from NADH to quinones. While most NDH-2 preferentially oxidize NADH, some of these enzymes have been reported to efficiently oxidize NADPH. With the aim to modify the NADPH vs NADH specificity of the relatively NADH specific Agrobacterium tumefaciens NDH-2, two conserved residues (E and A) of the substrate binding domain were, respectively, mutated to Q and S. We show that when E was replaced by Q at position 203 the enzyme was able to oxidize NADPH as efficiently as NADH. Growth on a minimal medium of an Escherichia coli double mutant lacking both NDH-1 and NDH-2 was restored more efficiently when mutated proteins able to oxidize NADPH were expressed. The biotechnological interest of expressing such modified enzymes in photosynthetic organisms is discussed.

Chloroplast targeting of phytochelatin synthase in Arabidopsis: effects on heavy metal tolerance and accumulation.

The enzymatically synthesized thiol peptide phytochelatin (PC) plays a central role in heavy metal tolerance and detoxification in plants. In response to heavy metal exposure, the constitutively expressed phytochelatin synthase enzyme (PCS) is activated leading to synthesis of PCs in the cytosol. Recent attempts to increase plant metal accumulation and tolerance reported that PCS over-expression in transgenic plants paradoxically induced cadmium hypersensitivity. In the present paper, we investigate the possibility of synthesizing PCs in plastids by over-expressing a plastid targeted phytochelatin synthase (PCS). Plastids represent a relatively important cellular volume and offer the advantage of containing glutathione, the precursor of PC synthesis. Using a constitutive CaMV 35S promoter and a RbcS transit peptide, we successfully addressed AtPCS1 to chloroplasts, significant PCS activity being measured in this compartment in two independent transgenic lines. A substantial increase in the PC content and a decrease in the glutathione pool were observed in response to cadmium exposure, when compared to wild-type plants. While over-expressing AtPCS1 in the cytosol importantly decreased cadmium tolerance, both cadmium tolerance and accumulation of plants expressing plastidial AtPCS1 were not significantly affected compared to wild-type. Interestingly, targeting AtPCS1 to chloroplasts induced a marked sensitivity to arsenic while plants over-expressing AtPCS1 in the cytoplasm were more tolerant to this metalloid. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.

Molecular characterization of CDSP 34, a chloroplastic protein induced by water deficit in Solanum tuberosum L. plants, and regulation of CDSP 34 expression by ABA and high illumination.

We previously described the accumulation of a 34 kDa thylakoid protein, named CDSP 34 for chloroplastic drought-induced stress protein, in Solanum tuberosum plants subjected to water deficit. A full-length CDSP 34 cDNA has been isolated and we report here that mature CDSP 34 is highly similar to two chromoplastic proteins, fibrillin from Capsicum annuum and CHRC (for chromoplast protein C) from Cucumis sativus, components of carotenoid-accumulating structures. Northern and Western analyses showed that both CDSP 34 transcript and protein accumulated from early stages of water deficit. In water-stressed tomato plants, similar increases in the CDSP 34-related transcript amount were noticed in wild-type and ABA-deficient flacca mutant, but protein accumulation was observed only in wild-type, suggesting a posttranscriptional role of ABA in CDSP 34 synthesis regulation. Substantial increases in CDSP 34 transcript and protein abundances were also observed in potato plants subjected to high illumination. The CDSP 34 protein is proposed to play a structural role in stabilizing stromal lamellae thylakoids upon osmotic or oxidative stress.