Open Access
Physiological performance of transplastomic tobacco plants overexpressing aquaporin AQP1 in chloroplast membranes
(Oxford University Press, 2018) Fernández San Millán, Alicia; Aranjuelo Michelena, Iker; Ancín Rípodas, María; Larraya Reta, Luis María; Farrán Blanch, Inmaculada; Veramendi Charola, Jon; Agronomia, Bioteknologia eta Elikadura; Agronomía, Biotecnología y Alimentación; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The leaf mesophyll CO2 conductance and the concentration of CO2 within the chloroplast are major factors affecting photosynthetic performance. Previous studies have shown that the aquaporin NtAQP1 (which localizes to the plasma membrane and chloroplast inner envelope membrane) is involved in CO2 permeability in the chloroplast. Levels of NtAQP1 in plants genetically engineered to overexpress the protein correlated positively with leaf mesophyll CO2 conductance and photosynthetic rate. In these studies, the nuclear transformation method used led to changes in NtAQP1 levels in the plasma membrane and the chloroplast inner envelope membrane. In the present work, NtAQP1 levels were increased up to 16-fold in the chloroplast membranes alone by the overexpression of NtAQP1 from the plastid genome. Despite the high NtAQP1 levels achieved, transplastomic plants showed lower photosynthetic rates than wild-type plants. This result was associated with lower Rubisco maximum carboxylation rate and ribulose 1,5-bisphosphate regeneration. Transplastomic plants showed reduced mesophyll CO2 conductance but no changes in chloroplast CO2 concentration. The absence of differences in chloroplast CO2 concentration was associated with the lower CO2 fixation activity of the transplastomic plants. These findings suggest that non-functional pores of recombinant NtAQP1 may be produced in the chloroplast inner envelope membrane.
Open Access
Papel de las tiorredoxinas plastidiales en la regulación del metabolismo del cloroplasto
(2019) Ancín Rípodas, María; Farrán Blanch, Inmaculada; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura
Carbon (C) and nitrogen (N) are two essential nutrients required for plant growth and development. The cellular metabolism of both elements is tightly coordinated and subjected to a complex regulation. In this regard, the redox status operates as a major integrator of cellular metabolism, involving post-translational modifications of target enzymes by thiol-disulfide exchanges exerted by thioredoxins (Trxs). In plant chloroplasts two Trx systems function in parallel: (i) the ferredoxin (Fd)-Trx system that depends on photosynthetically reduced Fd to supply electrons to the Fd-Trx reductase, which in turn reduces Trxs; (ii) the NADPH dependent Trx reductase type C (NTRC) that contains both a Trx-reductase and a Trx domain in a single polypeptide. NTRC is reduced by NADPH, which in addition to the light reactions, is also produced in the oxidative pentose phosphate pathway in darkness. This PhD thesis has been focused on the regulatory role of plastid Trx f, m and NTRC in chloroplast metabolism.
Open Access
Overexpression of thioredoxin m in tobacco chloroplasts inhibits the protein kinase STN7 and alters photosynthetic performance
(Oxford University Press, 2019) Ancín Rípodas, María; Fernández San Millán, Alicia; Larraya Reta, Luis María; Morales Iribas, Fermín; Veramendi Charola, Jon; Aranjuelo Michelena, Iker; Farrán Blanch, Inmaculada; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The activity of the protein kinase STN7, involved in phosphorylation of the light-harvesting complex II (LHCII) proteins, has been reported as being co-operatively regulated by the redox state of the plastoquinone pool and the ferredoxin–thioredoxin (Trx) system. The present study aims to investigate the role of plastid Trxs in STN7 regulation and their impact on photosynthesis. For this purpose, tobacco plants overexpressing Trx f or m from the plastid genome were characterized, demonstrating that only Trx m overexpression was associated with a complete loss of LHCII phosphorylation that did not correlate with decreased STN7 levels. The absence of phosphorylation in Trx m-overexpressing plants impeded migration of LHCII from PSII to PSI, with the concomitant loss of PSI–LHCII complex formation. Consequently, the thylakoid ultrastructure was altered, showing reduced grana stacking. Moreover, the electron transport rate was negatively affected, showing an impact on energy-demanding processes such as the Rubisco maximum carboxylation capacity and ribulose 1,5-bisphosphate regeneration rate values, which caused a strong depletion in net photosynthetic rates. Finally, tobacco plants overexpressing a Trx m mutant lacking the reactive redox site showed equivalent physiological performance to the wild type, indicating that the overexpressed Trx m deactivates STN7 in a redox-dependent way.
Open Access
Photosynthetic metabolism under stressful growth conditions as a bases for crop breeding and yield improvement
(MDPI, 2020) Morales Iribas, Fermín; Ancín Rípodas, María; Fakhet, Dorra; González Torralba, Jon; Gámez Guzmán, Angie Lorena; Seminario Huárriz, Amaia; Soba Hidalgo, David; Ben Mariem, Sinda; Garriga, Miguel; Aranjuelo Michelena, Iker; Agronomia, Bioteknologia eta Elikadura; Institute for Multidisciplinary Research in Applied Biology - IMAB; Agronomía, Biotecnología y Alimentación
Increased periods of water shortage and higher temperatures, together with a reduction in nutrient availability, have been proposed as major factors that negatively impact plant development. Photosynthetic CO2 assimilation is the basis of crop production for animal and human food, and for this reason, it has been selected as a primary target for crop phenotyping/breeding studies. Within this context, knowledge of the mechanisms involved in the response and acclimation of photosynthetic CO2 assimilation to multiple changing environmental conditions (including nutrients, water availability, and rising temperature) is a matter of great concern for the understanding of plant behavior under stress conditions, and for the development of new strategies and tools for enhancing plant growth in the future. The current review aims to analyze, from a multi-perspective approach (ranging across breeding, gas exchange, genomics, etc.) the impact of changing environmental conditions on the performance of the photosynthetic apparatus and, consequently, plant growth.
Open Access
Optimizing oilseed rape growth: exploring the effect of foliar biostimulants on the interplay among metabolism, phenology, and yield
(Wiley, 2024-10-03) Ancín Rípodas, María; Soba Hidalgo, David; Picazo Rodríguez, Pedro Javier; Gámez Guzmán, Angie Lorena; Le Page, Jean-François; Houdusse, Diane; Aranjuelo Michelena, Iker; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura
The current agricultural system is in search of new strategies to achieve a more sustainable production while keeping or even increasing crop yield and quality. In this scenario, the application of biostimulants constitutes a potent solution. In the current study, the impact of a blue-green microalgal extract (MB) and a pig tissue hydrolysate (PTH) on rapeseed plants' development was characterized. Obtained results revealed a positive effect on yield parameters of plants treated with MB and, especially, PTH; this was associated to an improvement on the photosynthetic performance. Moreover, this study remarked the effects of biostimulants on plant phenology through their pivotal role in modulating developmental processes. More specifically, proteomic, metabolomic, and hormone content analyses revealed distinct alterations associated with the acceleration of phenology induced by biostimulant application. Additionally, some antioxidant enzymes and stress-related compounds were up-regulated upon MB and PTH treatments, indicating enhanced plant defense mechanisms in response to accelerated phenological transitions. Such findings highlight the intricate interplay between biostimulants and plant physiology, wherein biostimulants orchestrate rapid developmental changes, ultimately influencing growth dynamics. Altogether, the current study reveals that the application of both MB and PTH biostimulants promoted rapeseed plant phenology and productivity associated with an improvement in the photosynthetic machinery while boosting other physiological and molecular mechanisms.
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
Successful biocontrol of Pichia spp. strains against Botrytis cinerea infection in apple fruit: unraveling protection mechanisms from proteomic insights
(Elseiver, 2024-05-25) Fernández San Millán, Alicia; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Larraya Reta, Luis María; Ancín Rípodas, María; Farrán Blanch, Inmaculada; Veramendi Charola, Jon; Institute for Multidisciplinary Research in Applied Biology - IMAB; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Botrytis cinerea causes major crop losses, especially under postharvest conditions. We have found that Pichia fermentans Pf-31 and Pichia terricola Pt-36 are two promising yeast strains that are able to efficiently control B. cinerea infection in apples. This effect is most pronounced when the yeasts are applied as live cells, although dead cells or culture filtrates also show some degree of control. Both strains arrest spore germination, inhibit mycelial growth, strongly attach to hyphae and promote their own proliferation in the fruit when B. cinerea is present, probably due to preferential colonization of apple wounds. Indeed, this metabolism enhancement was corroborated by a proteomic analysis, which revealed the differentially accumulated yeast proteins that contribute towards this antagonistic behavior. Besides the boost in proteins involved in energetic metabolism, other changes in proteins related to cell envelope composition are implicated in the biocontrol abilities of both strains, and this might be to facilitate hyphal adhesion or biofilm formation. The results of this study are of great value because they promote a deep understanding of the proteins that undergo changes during yeast antagonistic interactions, but also because they provide new insights into the proteomes of non-Saccharomyces yeasts, which have not been previously described.
Open Access
Does the response of Rubisco and photosynthesis to elevated [CO2] change with unfavourable environmental conditions?
(Oxford University Press, 2024-09-12) Ancín Rípodas, María; Gámez Guzmán, Angie Lorena; Jáuregui Mosquera, Iván; Galmes, J.; Sharwood, R. E.; Erice, G.; Ainsworth, E. A.; Tissue, D. T.; Sanz-Sáez, A.; Aranjuelo Michelena, Iker; Ciencias; Zientziak; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura
Climate change due to anthropogenic CO2 emissions affects plant performance globally. To improve crop resilience, we need to understand the effects of elevated CO2 concentration (e[CO2]) on CO2 assimilation and Rubisco biochemistry. However, the interactive effects of e[CO2] and abiotic stress are especially unclear. This study examined the CO2 effect on photosynthetic capacity under different water availability and temperature conditions in 42 different crop species, varying in functional group, photosynthetic pathway, and phenological stage. We analysed close to 3000 data points extracted from 120 published papers. For C-3 species, e[CO2] increased net photosynthesis and intercellular [CO2], while reducing stomatal conductance and transpiration. Maximum carboxylation rate and Rubisco in vitro extractable maximal activity and content also decreased with e[CO2] in C-3 species, while C-4 crops are less responsive to e[CO2]. The interaction with drought and/or heat stress did not significantly alter these photosynthetic responses, indicating that the photosynthetic capacity of stressed plants responded to e[CO2]. Moreover, e[CO2] had a strong effect on the photosynthetic capacity of grasses mainly in the final stages of development. This study provides insight into the intricate interactions within the plant photosynthetic apparatus under the influence of climate change, enhancing the understanding of mechanisms governing plant responses to environmental parameters.
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
Plant growth-promoting traits of yeasts isolated from Spanish vineyards: benefits for seedling development
(Elsevier, 2020) Fernández San Millán, Alicia; Farrán Blanch, Inmaculada; Larraya Reta, Luis María; Ancín Rípodas, María; Arregui Odériz, Luis Miguel; Veramendi Charola, Jon; Institute for Multidisciplinary Research in Applied Biology - IMAB; Gobierno de Navarra / Nafarroako Gobernua
It is known that some microorganisms can enhance plant development. However, the use of yeasts as growth-promoting agents has been poorly investigated. The aim of this study was the characterisation of a collection of 69 yeast strains isolated from Spanish vineyards. Phytobeneficial attributes such as solubilisation of nutrients, synthesis of active biomolecules and cell wall-degrading enzyme production were analysed. Strains that revealed multiple growth-promoting characteristics were identified. The in vitro co-culture of Nicotiana benthamiana with yeast isolates showed enhancement of plant growth in 10 strains (up to 5-fold higher shoot dry weight in the case of Hyphopichiapseudoburtonii Hp-54), indicating a beneficial direct yeast-plant interaction. In addition, 18 out of the 69 strains increased dry weight and the number of roots per seedling when tobacco seeds were inoculated. Two of these, Pichia dianae Pd-2 and Meyerozymaguilliermondii Mg-11, also increased the chlorophyll content. The results in tobacco were mostly reproduced in lettuce with these two strains, which demonstrates that the effect of the yeast-plant interaction is not species-specific. In addition, the yeast collection was evaluated in maize seedlings grown in soil in a phytotron. Three isolates (Debaryomyces hansenii Dh-67, Lachancea thermotolerans Lt-69 and Saccharomyces cerevisiae Sc-6) promoted seedling development (increases of 10 % in dry weight and chlorophyll content). In conclusion, our data confirm that several yeast strains can promote plant growth and could be considered for the development of biological fertiliser treatments.