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

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

Functional analysis of the pathways for 2-Cys peroxiredoxin reduction in Arabidopsis thaliana chloroplasts

Photosynthesis is a process that inevitably produces reactive oxygen species, such as hydrogen peroxide, which is reduced by chloroplast-localized detoxification mechanisms one of which involves 2-Cys peroxiredoxins (2-Cys Prxs). Arabidopsis chloroplasts contain two very similar 2-Cys Prxs (denoted A and B). These enzymes are reduced by two pathways: NADPH thioredoxin reductase C (NTRC), which uses NADPH as source of reducing power; and plastidial thioredoxins (Trxs) coupled to photosynthetically reduced ferredoxin of which Trx x is the most efficient reductant in vitro. With the aim of establishing the functional relationship between NTRC, Trx x, and 2-Cys Prxs in vivo, an Arabidopsis Trx x knock-out mutant has been identified and a double mutant (denoted Δ2cp) with <5% of 2-Cys Prx content has been generated. The phenotypes of the three mutants, ntrc, trxx, and Δ2cp, were compared under standard growth conditions and in response to continuous light or prolonged darkness and oxidative stress. Though all mutants showed altered redox homeostasis, no difference was observed in response to oxidative stress treatment. Moreover, the redox status of the 2-Cys Prx was imbalanced in the ntrc mutant but not in the trxx mutant. These results show that NTRC is the most relevant pathway for chloroplast 2-Cys Prx reduction in vivo, but the antioxidant function of this system is not essential. The deficiency of NTRC caused a more severe phenotype than the deficiency of Trx x or 2-Cys Prxs as determined by growth, pigment content, CO2 fixation, and Fv/Fm, indicating additional functions of NTRC

Biosíntesis fotoinducida de la fructosa-1, 6-bisfosfatasa cloroplastídica

Reducción altaLa fructuosa-1,6-bisfosfatasa fotosintética es una enzima clave del ciclo de Benson-Calvin, ciclo fundamental en la fotosíntesis de las plantas. El trabajo ha consistido en el estudio de la síntesis de la fructosa-1,6-bisfosfatsa fotosintética por la luz, y en el análisis y lugar de la codificación y síntesis de la enzima en hojas de guisante (pisum sativum var. Lincoln). De los resultados obtenidos se llegaron a las siguientes conclusiones: 1.-la luz induce la síntesis de la fructosa-1,6-bisfosfatasa fotosintética proporcionalmente a la intensidad lumínica en las primeras 15 horas de iluminación. A mas altos flujos lumínicos este incremento se prolonga hasta 50h. 2.-la síntesis de la fbpasa esta activada por longitudes de onda del azul lejano, por lo que se deduce una regulación por fotoreceptores de tipo flavínico, y no por fitocromo. 3.-la vida media de la fbpasa resulta ser de 20 horas. El nivel máximo de enzima tiene lugar a los 4 días. 4.-la fbpasa es codificada en el núcleo, apareciendo en el citoplasma como precursor de 59kd de pm. Este se identifica con anticuerpos contra la proteína madura. 5.-desde el citoplasma el precursor de la fbpasa pasa al cloroplasto perdiendo un péptido de transito formado aproximadamente por 100 aminoácidos. 6.-un pulso de 35s metionina in vivo demuestra una coexistencia de la forma precursora y madura de la fbpasa a las 4 horas de iluminación. Con posterioridad solo se identifica la forma madura del enzima.Univ. de Granada, Fctd. de Farmacia. Leída 198

Proteomic Analyses of Thioredoxins f and m Arabidopsis thaliana Mutants Indicate Specific Functions for These Proteins in Plants

A large number of plastidial thioredoxins (TRX) are present in chloroplast and the specificity versus the redundancy of their functions is currently under discussion. Several results have highlighted the fact that each TRX has a specific target protein and thus a specific function. In this study we have found that in vitro activation of the fructose-1,6-bisphosphatase (FBPase) enzyme is more efficient when f1 and f2 type thioredoxins (TRXs) are used, whilst the m3 type TRX did not have any effect. In addition, we have carried out a two-dimensional electrophoresis-gel to obtain the protein profiling analyses of the trxf1, f2, m1, m2, m3 and m4 Arabidopsis mutants. The results revealed quantitative alteration of 86 proteins and demonstrated that the lack of both the f and m type thioredoxins have diverse effects on the proteome. Interestingly, 68% of the differentially expressed proteins in trxf1 and trxf2 mutants were downregulated, whilst 75% were upregulated in trxm1, trxm2, trxm3 and trxm4 lines. The lack of TRX f1 provoked a higher number of down regulated proteins. The contrary occurred when TRX m4 was absent. Most of the differentially expressed proteins fell into the categories of metabolic processes, the Calvin–Benson cycle, photosynthesis, response to stress, hormone signalling and protein turnover. Photosynthesis, the Calvin–Benson cycle and carbon metabolism are the most affected processes. Notably, a significant set of proteins related to the answer to stress situations and hormone signalling were affected. Despite some studies being necessary to find specific target proteins, these results show signs that are suggest that the f and m type plastidial TRXs most likely have some additional specific functionThis work has been funded by research project BIO2009-07297, BIO2015-65272 from the Spanish Ministry of Science and Innovation and the European Fund for Regional Development, project P07-CVI-2795 and BIO 154 from the Andalusian Regional Government, Spain, and project BIO2012-33292, from the Spanish Ministry of Economy and CompetitivenessWe acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe

Expression of the chloroplast thioredoxins f and m is linked to short-term changes in the sugar and thiol status in leaves of Pisum sativum

Thioredoxins (TRXs) f and m are key components in the light regulation of photosynthetic metabolism via thioldithiol modulation in chloroplasts of leaves; however, little is known about the factors modulating the expression of these proteins. To investigate the effect of sugars as photosynthetic products on the expression of PsTRX f and m1 genes, sucrose and glucose were externally supplied to pea plants during the day. There was an increase in the mRNA levels of PsTRX f and m1 genes in response mainly to glucose. When leaf discs were incubated for up to 4h in the dark, glucose also led to an increase in both mRNA and protein levels of TRXs f and m, while sucrose had no substantial effect. Expression of PsDOF7, a carbon metabolism-related transcription factor gene, was also induced by glucose. ProteinDNA interaction showed that PsDOF7 binds specifically to the DOF core located in PsTRX f and m1 gene promoters. Transient expression in agroinfiltrated pea leaves demonstrated that PsDOF7 activated transcription of both promoters. The incubation of leaf discs in dithiotreitol (DTT) to increase the redox status led to a marked increase in the mRNA and protein levels of both TRXs within 4h. The increase in TRX protein levels occurred after 1h DTT feeding, implying a rapid effect of the thiol status on TRX f and m1 protein turnover rates, while transcriptional regulation took 3h to proceed. These results show that the protein levels of both TRXs are under short-term control of the sugar and thiol status in plants.Fil: de Dios Barajas López, Juan. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; EspañaFil: Tezycka, Justyna. Ludwig Maximilians Universitat; AlemaniaFil: Travaglia, Claudia Noemi. Universidad Nacional de Río Cuarto; Argentina. Centro de Investigaciones Cientificas ; Facultad de Ciencias Exactas Fisicas y Naturales ; Universidad Nacional de Cordoba;Fil: Serrato, Antonio Jesús. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; EspañaFil: Chueca, Ana. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; EspañaFil: Thormählen, Ina. Ludwig Maximilians Universitat; AlemaniaFil: Sahrawy, Mariam. Ludwig Maximilians Universitat; AlemaniaFil: Sahrawy, Mariam. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; Españ

Comprehensive expression analyses of plastidial thioredoxins of Arabidopsis thaliana indicate a main role of thioredoxin m2 in roots

Thioredoxins (TRXs) f and m are redox proteins that regulate key chloroplast processes. The existence of several isoforms of TRXs f and m indicates that these redox players have followed a specialization process throughout evolution. Current research efforts are focused on discerning the signalling role of the different TRX types and their isoforms in chloroplasts. Nonetheless, little is known about their function in non-photosynthetic plastids. For this purpose, we have carried out comprehensive expression analyses by using Arabidopsis thaliana TRXf (f1 and f2) and TRXm (m1, m2, m3 and m4) genes translationally fused to the green fluorescence protein (GFP). These analyses showed that TRX m has different localisation patterns inside chloroplasts, together with a putative dual subcellular localisation of TRX f1. Apart from mesophyll cells, these TRXs were also observed in reproductive organs, stomatal guard cells and roots. We also investigated whether photosynthesis, stomatal density and aperture or root structure were affected in the TRXs f and m loss-of-function Arabidopsis mutants. Remarkably, we immunodetected TRX m2 and the Calvin–Benson cycle fructose-1,6-bisphosphatase (cFBP1) in roots. After carrying out in vitro redox activation assays of cFBP1 by plastid TRXs, we propose that cFBP1 might be activated by TRX m2 in root plastids.This work has been funded by research projects BIO2009-07297 and BIO2015-65272 from the Spanish Ministry of Science and Innovation and the European Fund for Regional Development; project P07-CVI-2795 and BIO 154 from the Andalusian Regional Government, Spain; project BIO2012-33292 from the Spanish Ministry of Economy and Competitiveness; and by Grant PGC2018-096851-B-C21, from FEDER/Spanish Ministry of Science, Innovation and University: AEI, Spain