NADPH Thioredoxin Reductase C and Thioredoxins Act Concertedly in Seedling Development

Thiol-dependent redox regulation of enzyme activity plays a central role in the rapid acclimation of chloroplast metabolism to ever-fluctuating light availability. This regulatory mechanism relies on ferredoxin reduced by the photosynthetic electron transport chain, which fuels reducing power to thioredoxins (Trxs) via a ferredoxin-dependent Trx reductase. In addition, chloroplasts harbor an NADPH-dependent Trx reductase, which has a joint Trx domain at the carboxyl terminus, termed NTRC. Thus, a relevant issue concerning chloroplast function is to establish the relationship between these two redox systems and its impact on plant development. To address this issue, we generated Arabidopsis (Arabidopsis thaliana) mutants combining the deficiency of NTRC with those of Trxs f, which participate in metabolic redox regulation, and that of Trx x, which has antioxidant function. The ntrc-trxf1f2 and, to a lower extent, ntrc-trxx mutants showed severe growth-retarded phenotypes, decreased photosynthesis performance, and almost abolished light-dependent reduction of fructose-1,6-bisphosphatase. Moreover, the combined deficiency of both redox systems provokes aberrant chloroplast ultrastructure. Remarkably, both the ntrc-trxf1f2 and ntrc-trxx mutants showed high mortality at the seedling stage, which was overcome by the addition of an exogenous carbon source. Based on these results, we propose that NTRC plays a pivotal role in chloroplast redox regulation, being necessary for the activity of diverse Trxs with unrelated functions. The interaction between the two thiol redox systems is indispensable to sustain photosynthesis performed by cotyledons chloroplasts, which is essential for early plant development.España Mineco BIO2013-43556-PEspaña, Junta de Andalucía CVI-591

Disruption of both chloroplastic and cytosolic FBPase genes results in a dwarf phenotype and important starch and metabolite changes in Arabidopsis thaliana

In this study, evidence is provided for the role of fructose-1,6-bisphosphatases (FBPases) in plant development and carbohydrate synthesis and distribution by analysing two Arabidopsis thaliana T-DNA knockout mutant lines, cyfbp and cfbp1, and one double mutant cyfbp cfbp1 which affect each FBPase isoform, cytosolic and chloroplastic, respectively. cyFBP is involved in sucrose synthesis, whilst cFBP1 is a key enzyme in the Calvin–Benson cycle. In addition to the smaller rosette size and lower rate of photosynthesis, the lack of cFBP1 in the mutants cfbp1 and cyfbp cfbp1 leads to a lower content of soluble sugars, less starch accumulation, and a greater superoxide dismutase (SOD) activity. The mutants also had some developmental alterations, including stomatal opening defects and increased numbers of root vascular layers. Complementation also confirmed that the mutant phenotypes were caused by disruption of the cFBP1 gene. cyfbp mutant plants without cyFBP showed a higher starch content in the chloroplasts, but this did not greatly affect the phenotype. Notably, the sucrose content in cyfbp was close to that found in the wild type. The cyfbp cfbp1 double mutant displayed features of both parental lines but had the cfbp1 phenotype. All the mutants accumulated fructose-1,6-bisphosphate and triose-phosphate during the light period. These results prove that while the lack of cFBP1 induces important changes in a wide range of metabolites such as amino acids, sugars, and organic acids, the lack of cyFBP activity in Arabidopsis essentially provokes a carbon metabolism imbalance which does not compromise the viability of the double mutant cyfbp cfbp1.España, Ministerio de Economía y Competitividad BIO2009-07297España, Ministerio de Economía y Competitividad BIO2012-33292Junta de Andalucía P07-CVI-279

Disruption of both chloroplastic and cytosolic FBPase genes results in a dwarf phenotype and important starch and metabolite changes in Arabidopsis thaliana

In this study, evidence is provided for the role of fructose-1,6-bisphosphatases (FBPases) in plant development and carbohydrate synthesis and distribution by analysing two Arabidopsis thaliana T-DNA knockout mutant lines, cyfbp and cfbp1, and one double mutant cyfbp cfbp1 which affect each FBPase isoform, cytosolic and chloroplastic, respectively. cyFBP is involved in sucrose synthesis, whilst cFBP1 is a key enzyme in the Calvin–Benson cycle. In addition to the smaller rosette size and lower rate of photosynthesis, the lack of cFBP1 in the mutants cfbp1 and cyfbp cfbp1 leads to a lower content of soluble sugars, less starch accumulation, and a greater superoxide dismutase (SOD) activity. The mutants also had some developmental alterations, including stomatal opening defects and increased numbers of root vascular layers. Complementation also confirmed that the mutant phenotypes were caused by disruption of the cFBP1 gene. cyfbp mutant plants without cyFBP showed a higher starch content in the chloroplasts, but this did not greatly affect the phenotype. Notably, the sucrose content in cyfbp was close to that found in the wild type. The cyfbp cfbp1 double mutant displayed features of both parental lines but had the cfbp1 phenotype. All the mutants accumulated fructose-1,6-bisphosphate and triose-phosphate during the light period. These results prove that while the lack of cFBP1 induces important changes in a wide range of metabolites such as amino acids, sugars, and organic acids, the lack of cyFBP activity in Arabidopsis essentially provokes a carbon metabolism imbalance which does not compromise the viability of the double mutant cyfbp cfbp1.España, Ministerio de Economía y Competitividad BIO2009-07297España, Ministerio de Economía y Competitividad BIO2012-33292Junta de Andalucía P07-CVI-279

Tobacco plastidial thioredoxins as modulators of recombinant protein production in transgenic chloroplasts

Thioredoxins (Trxs) are small ubiquitous disulphide proteins widely known to enhance expression and solubility of recombinant proteins in microbial expression systems. Given the common evolutionary heritage of chloroplasts and bacteria, we attempted to analyse whether plastid Trxs could also act as modulators of recombinant protein expression in transgenic chloroplasts. For that purpose, two tobacco Trxs (m and f) with different phylogenetic origins were assessed. Using plastid transformation, we assayed two strategies: the fusion and the co-expression of Trxs with human serum albumin (HSA), which was previously observed to form large protein bodies in tobacco chloroplasts. Our results indicate that both Trxs behave similarly as regards HSA accumulation, although they act differently when fused or co-expressed with HSA. Trxs-HSA fusions markedly increased the final yield of HSA (up to 26% of total protein) when compared with control lines that only expressed HSA; this increase was mainly caused by higher HSA stability of the fused proteins. However, the fusion strategy failed to prevent the formation of protein bodies within chloroplasts. On the other hand, the co-expression constructs gave rise to an absence of large protein bodies although no more soluble HSA was accumulated. In these plants, electron micrographs showed HSA and Trxs co-localization in small protein bodies with fibrillar texture, suggesting a possible influence of Trxs on HSA solubilization. Moreover, the in vitro chaperone activity of Trx m and f was demonstrated, which supports the hypothesis of a direct relationship between Trx presence and HSA aggregates solubilization in plants co-expressing both proteins. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.Authors wish to thank MJ Villafranca for excellent plant care and cultivation. This work was supported by grants Res. 17/2004 and IIM10865.RI1 (Proyecto EUROINNOVA) from Gobierno de Navarra (Spain). RSB and PCM were supported by predoctoral fellowships from CSIC and Generalitat Valenciana, respectively. AFS was supported by a postdoctoral fellowship from Public University of Navarra.Sanz-Barrio, R.; Fernandez-San Millan, A.; Corral Martínez, P.; Seguí-Simarro, JM.; Farran, I. (2011). Tobacco plastidial thioredoxins as modulators of recombinant protein production in transgenic chloroplasts. Plant Biotechnology Journal. 9(6):639-650. https://doi.org/10.1111/j.1467-7652.2011.00608.xS6396509

Simultaneous stimulation of sedoheptulose 1,7‐bisphosphatase, fructose 1,6‐bisphophate aldolase and the photorespiratory glycine decarboxylase‐H protein increases CO2 assimilation, vegetative biomass and seed yield in Arabidopsis

In this paper we have altered the levels of three different enzymes involved in the Calvin Benson cycle and photorespiratory pathway. We have generated transgenic Arabidopsis plants with altered combinations of sedoheptulose 1,7-bisphosphatase (SBPase), fructose 1,6-bisphophate aldolase (FBPA) and the glycine decarboxylase H-protein (GDC-H) gene identified as targets to improve photosynthesis based on previous studies. Here, we show that increasing the levels of the three corresponding proteins, either independently or in combination, significantly increases the quantum efficiency of PSII. Furthermore, photosynthetic measurements demonstrated an increase in the maximum efficiency of CO2 fixation in lines over-expressing SBPase and FBPA. Moreover, the co-expression of GDC-H with SBPase and FBPA resulted in a cumulative positive impact on leaf area and biomass. Finally, further analysis of transgenic lines revealed a cumulative increase of seed yield in SFH lines grown in high light. These results demonstrate the potential of multigene-stacking for improving the productivity of food and energy crops

Circadian regulation of chloroplastic f and m thioredoxins through control of the CCA1 transcription factor

Chloroplastic thioredoxins f and m (TRX f and TRX m) mediate light regulation of carbon metabolism through the activation of Calvin cycle enzymes. The role of TRX f and m in the activation of Calvin cycle enzymes is best known among the TRX family. However, the discoveries of new potential targets extend the functions of chloroplastic TRXs to other processes in non-photosynthetic tissues. As occurs with numerous chloroplast proteins, their expression comes under light regulation. Here, the focus is on the light regulation of TRX f and TRX m in pea and Arabidopsis during the day/night cycle that is maintained during the subjective night. In pea (Pisum sativum), TRX f and TRX m1 expression is shown to be governed by a circadian oscillation exerted at both the transcriptional and protein levels. Binding shift assays indicate that this control probably involves the interaction of the CCA1 transcription factor and an evening element (EE) located in the PsTRX f and PsTRX m1 promoters. In Arabidopsis, among the multigene family of TRX f and TRX m, AtTRX f2 and AtTRX m2 mRNA showed similar circadian oscillatory regulation, suggesting that such regulation is conserved in plants. However, this oscillation was disrupted in plants overexpressing CCA1 (cca1-ox) or repressing CCA1 and LHY (cca1-lhy). The physiological role of the oscillatory regulation of chloroplastic TRX f and TRX m in plants during the day/night cycle is discussed

Characterization of the Arabidopsis thaliana 2-Cys peroxiredoxin interactome

This document is the Accepted Manuscript of the following article: Delphine Cerveau, et al, ‘Characterization of the Arabidopsis thaliana 2-Cys peroxiredoxin interactome’, Plant Science, Vol. 252, pp. 30-41, July 2016, doi: https://doi.org/10.1016/j.plantsci.2016.07.003. This manuscript version is distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License CC BY NC-ND 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.Peroxiredoxins are ubiquitous thiol-dependent peroxidases for which chaperone and signaling roles havebeen reported in various types of organisms in recent years. In plants, the peroxidase function of thetwo typical plastidial 2-Cys peroxiredoxins (2-Cys PRX A and B) has been highlighted while the otherfunctions, particularly in ROS-dependent signaling pathways, are still elusive notably due to the lack ofknowledge of interacting partners. Using an ex vivo approach based on co-immunoprecipitation of leafextracts from Arabidopsis thaliana wild-type and mutant plants lacking 2-Cys PRX expression followedby mass spectrometry-based proteomics, 158 proteins were found associated with 2-Cys PRXs. Alreadyknown partners like thioredoxin-related electron donors (Chloroplastic Drought-induced Stress Proteinof 32 kDa, Atypical Cysteine Histidine-rich Thioredoxin 2) and enzymes involved in chlorophyll synthe-sis (Protochlorophyllide OxidoReductase B) or carbon metabolism (Fructose-1,6-BisPhosphatase) wereidentified, validating the relevance of the approach. Bioinformatic and bibliographic analyses allowedthe functional classification of the identified proteins and revealed that more than 40% are localized inplastids. The possible roles of plant 2-Cys PRXs in redox signaling pathways are discussed in relation withthe functions of the potential partners notably those involved in redox homeostasis, carbon and aminoacid metabolisms as well as chlorophyll biosynthesis.Peer reviewe