Dominique Rumeau

New Subunits NDH-M, -N, and -O, Encoded by Nuclear Genes, Are Essential for Plastid Ndh Complex Functioning in Higher Plants

In higher plants, the Ndh complex reduces plastoquinones and is involved in cyclic electron flow around photosystem I, supplying extra-ATP for photosynthesis, particularly under environmental stress conditions. Based on plastid genome sequences, the Ndh complex would contain 11 subunits (NDH-A to -K), but homologies with bacterial complex indicate the probable existence of additional subunits. To identify missing subunits, tobacco (Nicotiana tabacum) NDH-H was His tagged at its N terminus using plastid transformation. A functional Ndh subcomplex was purified by Ni2+ affinity chromatography and its subunit composition analyzed by mass spectrometry. Five plastid encoded subunits (NDH-A, -H, -I, -J, and -K) were identified as well as three new subunits (NDH-M, -N, and -O) homologous to cyanobacterial and higher plant proteins. Arabidopsis thaliana mutants missing one of these new subunits lack a functional Ndh complex, and NDH-M and NDH-N are not detected in a tobacco transformant lacking the Ndh complex. We discuss the involvement of these three nuclear-encoded subunits in the functional integrity of the plastidial complex.

An abscisic acid-independent oxylipin pathway controls stomatal closure and immune defense in Arabidopsis.

In Arabidopsis the stomatal defense response, a feature of the innate immunity in plants, involves oxylipin-mediated mechanisms that are independent of the phytohormone abscisic acid.

Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO2 to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated Mr of 30000. The chloroplastic isoenzyme (Mr 220000) is also an octamer composed of two different subunits with Mr estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the Mr-27 000 subunit was three amino acids shorter than that of the Mr-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl−, I−, N3-and NO3-) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.

An Orange Ripening Mutant Links Plastid NAD(P)H Dehydrogenase Complex Activity to Central and Specialized Metabolism during Tomato Fruit Maturation

In this study, the characterization of OrrDs, a dominant transposon-tagged tomato mutant deficient in the NDH-M subunit, provided evidence that the plastid NAD(P)H dehydrogenase (NDH) complex is essential for the fruit ripening process and related metabolism. In higher plants, the plastidial NADH dehydrogenase (Ndh) complex supports nonphotochemical electron fluxes from stromal electron donors to plastoquinones. Ndh functions in chloroplasts are not clearly established; however, its activity was linked to the prevention of the overreduction of stroma, especially under stress conditions. Here, we show by the characterization of OrrDs, a dominant transposon-tagged tomato (Solanum lycopersicum) mutant deficient in the NDH-M subunit, that this complex is also essential for the fruit ripening process. Alteration to the NDH complex in fruit changed the climacteric, ripening-associated metabolites and transcripts as well as fruit shelf life. Metabolic processes in chromoplasts of ripening tomato fruit were affected in OrrDs, as mutant fruit were yellow-orange and accumulated substantially less total carotenoids, mainly β-carotene and lutein. The changes in carotenoids were largely influenced by environmental conditions and accompanied by modifications in levels of other fruit antioxidants, namely, flavonoids and tocopherols. In contrast with the pigmentation phenotype in mature mutant fruit, OrrDs leaves and green fruits did not display a visible phenotype but exhibited reduced Ndh complex quantity and activity. This study therefore paves the way for further studies on the role of electron transport and redox reactions in the regulation of fruit ripening and its associated metabolism.

Thioredoxin m4 Controls Photosynthetic Alternative Electron Pathways in Arabidopsis

In addition to the linear electron flow, a cyclic electron flow (CEF) around photosystem I occurs in chloroplasts. In CEF, electrons flow back from the donor site of photosystem I to the plastoquinone pool via two main routes: one that involves the Proton Gradient Regulation5 (PGR5)/PGRL1 complex (PGR) and one that is dependent of the NADH dehydrogenase-like complex. While the importance of CEF in photosynthesis and photoprotection has been clearly established, little is known about its regulation. We worked on the assumption of a redox regulation and surveyed the putative role of chloroplastic thioredoxins (TRX). Using Arabidopsis (Arabidopsis thaliana) mutants lacking different TRX isoforms, we demonstrated in vivo that TRXm4 specifically plays a role in the down-regulation of the NADH dehydrogenase-like complex-dependent plastoquinone reduction pathway. This result was confirmed in tobacco (Nicotiana tabacum) plants overexpressing the TRXm4 orthologous gene. In vitro assays performed with isolated chloroplasts and purified TRXm4 indicated that TRXm4 negatively controls the PGR pathway as well. The physiological significance of this regulation was investigated under steady-state photosynthesis and in the pgr5 mutant background. Lack of TRXm4 reversed the growth phenotype of the pgr5 mutant, but it did not compensate for the impaired photosynthesis and photoinhibition sensitivity. This suggests that the physiological role of TRXm4 occurs in vivo via a mechanism distinct from direct up-regulation of CEF.