Person: Veramendi Charola, Jon
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Veramendi Charola
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Jon
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AgronomĆa, BiotecnologĆa y AlimentaciĆ³n
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IMAB. Research Institute for Multidisciplinary Applied Biology
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0000-0002-3214-213X
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539
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Publication Open Access New in vivo approach to broaden the thioredoxin family interactome in chloroplasts(MDPI, 2022) AncĆn RĆpodas, MarĆa; FernĆ”ndez Irigoyen, JoaquĆn; SantamarĆa MartĆnez, Enrique; Larraya Reta, Luis MarĆa; FernĆ”ndez San MillĆ”n, Alicia; Veramendi Charola, Jon; FarrĆ”n Blanch, Inmaculada; Ciencias de la Salud; Osasun Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMABPost-translational redox modifications provide an important mechanism for the control of major cellular processes. Thioredoxins (Trxs), which are key actors in this regulatory mechanism, are ubiquitous proteins that catalyse thiol-disulfide exchange reactions. In chloroplasts, Trx f, Trx m and NADPH-dependent Trx reductase C (NTRC) have been identified as transmitters of the redox signal by transferring electrons to downstream target enzymes. The number of characterised Trx targets has greatly increased in the last few years, but most of them were determined using in vitro procedures lacking isoform specificity. With this background, we have developed a new in vivo approach based on the overexpression of His-tagged single-cysteine mutants of Trx f, Trx m or NTRC into Nicotiana benthamiana plants. The over-expressed mutated Trxs, capable of forming a stable mixed disulfide bond with target proteins in plants, were immobilised on affinity columns packed with Ni-NTA agarose, and the covalently linked targets were eluted with dithiothreitol and identified by mass spectrometry-based proteomics. The in vivo approach allowed identification of 6, 9 and 42 new potential targets for Trx f, Trx m and NTRC, respectively, and an apparent specificity between NTRC and Trxs was achieved. Functional analysis showed that these targets are involved in several cellular processes.Publication Open Access NTRC and thioredoxin f overexpression differentially induces starch accumulation in tobacco leaves(MDPI, 2019) AncĆn RĆpodas, MarĆa; Larraya Reta, Luis MarĆa; FernĆ”ndez San MillĆ”n, Alicia; Veramendi Charola, Jon; Burch Smith, Tessa; FarrĆ”n Blanch, Inmaculada; Institute for Multidisciplinary Research in Applied Biology - IMABThioredoxin (Trx) f and NADPH-dependent Trx reductase C (NTRC) have both been proposed as major redox regulators of starch metabolism in chloroplasts. However, little is known regarding the specific role of each protein in this complex mechanism. To shed light on this point, tobacco plants that were genetically engineered to overexpress the NTRC protein from the chloroplast genome were obtained and compared to previously generated Trx f-overexpressing transplastomic plants. Likewise, we investigated the impact of NTRC and Trx f deficiency on starch metabolism by generating Nicotiana benthamiana plants that were silenced for each gene. Our results demonstrated that NTRC overexpression induced enhanced starch accumulation in tobacco leaves, as occurred with Trx f. However, only Trx f silencing leads to a significant decrease in the leaf starch content. Quantitative analysis of enzyme activities related to starch synthesis and degradation were determined in all of the genotypes. Zymographic analyses were additionally performed to compare the amylolytic enzyme profiles of both transplastomic tobacco plants. Our findings indicated that NTRC overexpression promotes the accumulation of transitory leaf starch as a consequence of a diminished starch turnover during the dark period, which seems to be related to a significant reductive activation of ADP-glucose pyrophosphorylase and/or a deactivation of a putative debranching enzyme. On the other hand, increased starch content in Trx f-overexpressing plants was connected to an increase in the capacity of soluble starch synthases during the light period. Taken together, these results suggest that NTRC and the ferredoxin/Trx system play distinct roles in starch turnover.Publication Open Access Overexpression of thioredoxin m in chloroplasts alters carbon and nitrogen partitioning in tobacco(Oxford University Press, 2021) AncĆn RĆpodas, MarĆa; Larraya Reta, Luis MarĆa; Florez-Sarasa, Igor; BĆ©nard, Camille; FernĆ”ndez San MillĆ”n, Alicia; Veramendi Charola, Jon; Gibon, Yves; Fernie, Alisdair R.; Aranjuelo Michelena, Iker; FarrĆ”n Blanch, Inmaculada; Agronomia, Bioteknologia eta Elikadura; Institute for Multidisciplinary Research in Applied Biology - IMAB; AgronomĆa, BiotecnologĆa y AlimentaciĆ³nIn plants, there is a complex interaction between carbon (C) and nitrogen (N) metabolism, and its coordination is fundamental for plant growth and development. Here, we studied the influence of thioredoxin (Trx) m on C and N partitioning using tobacco plants overexpressing Trx m from the chloroplast genome. The transgenic plants showed altered metabolism of C (lower leaf starch and soluble sugar accumulation) and N (with higher amounts of amino acids and soluble protein), which pointed to an activation of N metabolism at the expense of carbohydrates. To further delineate the effect of Trx m overexpression, metabolomic and enzymatic analyses were performed on these plants. These results showed an up-regulation of the glutamine synthetase-glutamate synthase pathway; specifically tobacco plants overexpressing Trx m displayed increased activity and stability of glutamine synthetase. Moreover, higher photorespiration and nitrate accumulation were observed in these plants relative to untransformed control plants, indicating that overexpression of Trx m favors the photorespiratory N cycle rather than primary nitrate assimilation. Taken together, our results reveal the importance of Trx m as a molecular mediator of N metabolism in plant chloroplasts.