Open Access
Overexpression of plastidial thioredoxin f leads to enhanced starch accumulation in tobacco leaves
(Wiley, 2013) Sanz Barrio, Ruth; Corral-Martínez, Patricia; Ancín Rípodas, María; Seguí-Simarro, José M.; Farrán Blanch, Inmaculada; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, IIM10865.RI1
Starch, the most abundant storage carbohydrate in plants, has been a major feedstock for first‐generation biofuels. Growing fuel demands require, however, that the starch yields of energy crops be improved. Leaf starch is synthesised during the day and degraded at night to power nonphotosynthetic metabolism. Redox regulation has been associated with the coordination of the enzymes involved in starch metabolism, but neither the signals nor mechanisms that regulate this metabolism are entirely clear. In this work, the thioredoxin (Trx) f and m genes, which code for key enzymes in plastid redox regulation, were overexpressed from the plastid genome. Tobacco plants overexpressing Trx f, but not Trx m, showed an increase of up to 700% in leaf starch accumulation, accompanied by an increase in leaf sugars, specific leaf weight (SLW), and leaf biomass yield. To test the potential of these plants as a nonfood energy crop, tobacco leaves overexpressing Trx f were subjected to enzymatic hydrolysis, and around a 500% increase in the release of fermentable sugars was recorded. The results show that Trx f is a more effective regulator of photosynthetic carbon metabolism in planta than Trx m. The overexpression of Trx f might therefore provide a means of increasing the carbohydrate content of plants destined for use in biofuel production. It might also provide a means of improving the nutritional properties of staple food crops.
Open Access
New in vivo approach to broaden the thioredoxin family interactome in chloroplasts
(MDPI, 2022) Ancín Rípodas, María; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Larraya Reta, Luis María; Fernández San Millán, Alicia; Veramendi Charola, Jon; Farrán Blanch, Inmaculada; Ciencias de la Salud; Osasun Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMAB
Post-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.
Open Access
NTRC and thioredoxin f overexpression differentially induces starch accumulation in tobacco leaves
(MDPI, 2019) Ancín Rípodas, María; Larraya Reta, Luis María; Fernández San Millán, Alicia; Veramendi Charola, Jon; Burch Smith, Tessa; Farrán Blanch, Inmaculada; Institute for Multidisciplinary Research in Applied Biology - IMAB
Thioredoxin (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.
Open Access
High-density seedling expression system for the production of bioactive human cardiotrophin-1, a potential therapeutic cytokine, in transgenic tobacco chloroplasts
(Wiley, 2008) Farrán Blanch, Inmaculada; Río-Manterola, Francisco; Íñiguez, María; Gárate, Sonia; Prieto, Jesús; Mingo Castel, Ángel; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, “Formación de Tecnólogos” 055/01/11. M.N.C
Histidine‐tagged human cardiotrophin‐1 (hCT‐1), a recently discovered cytokine with excellent therapeutic potential, was expressed in tobacco chloroplasts under the transcriptional and translational control of two different promoters (rrn and psbA) and 5′‐untranslated regions (5′‐UTRs) (psbA and phage T7 gene 10). The psbA 5′‐UTR promotes recombinant hCT‐1 (rhCT‐1) accumulation in chloroplasts at higher levels (eight‐fold) than those obtained for the phage T7 gene 10 5′‐UTR, regardless of the promoter used, indicating that the correct choice of translational control element is most important for protein production in chloroplasts. The maximum level of rhCT‐1 achieved was 1.14 mg/g fresh weight (equivalent to 5% of total soluble protein) with the psbA promoter and 5′‐UTR in young leaves harvested after 32 h of continuous light, although the bioactivity was significantly lower (~35%) than that of commercial hCT‐1. However, harvesting in the dark or after 12 h of light did not result in a significant decrease in the bioactivity of rhCT‐1, suggesting that 32 h of over‐lighting affects the biological activity of rhCT‐1. Because high levels of rhCT‐1 accumulation took place mainly in young leaves, it is proposed that seedlings should be used in a ‘closed system’ unit, yielding up to 3.2 kg per year of rhCT‐1. This amount would be sufficient to meet the estimated annual worldwide needs of hCT‐1 for liver transplantation surgery in a cost‐effective manner. Furthermore, our strategy is an environmentally friendly method for the production of plant‐based biopharmaceuticals.
Open Access
Overexpression of thioredoxin m in chloroplasts alters carbon and nitrogen partitioning in tobacco
(Oxford University Press, 2021) Ancín Rípodas, María; Larraya Reta, Luis María; Florez-Sarasa, Igor; Bénard, Camille; Fernández San Millán, Alicia; Veramendi Charola, Jon; Gibon, Yves; Fernie, Alisdair R.; Aranjuelo Michelena, Iker; Farrán Blanch, Inmaculada; Agronomia, Bioteknologia eta Elikadura; Institute for Multidisciplinary Research in Applied Biology - IMAB; Agronomía, Biotecnología y Alimentación
In 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.