Redox dynamics in seeds of Acer spp: unraveling adaptation strategies of different seed categories.

Background: Seeds of woody plant species, such as those in the Acer genus like Norway maple (Acer platanoides L.) and sycamore (Acer pseudoplatanus L.), exhibit unique physiological traits and responses to environmental stress. Thioredoxins (Trxs) play a central role in the redox regulation of cells, interacting with other redox-active proteins such as peroxiredoxins (Prxs), and contributing to plant growth, development, and responses to biotic and abiotic stresses. However, there is limited understanding of potential variations in this system between seeds categorized as recalcitrant and orthodox, which could provide insights into adaptive strategies. Methods: Using proteomic analysis and DDA methods we investigated the Trx-h1 target proteins in seed axes. We complemented the results of the proteomic analysis with gene expression analysis of the Trx-h1, 1-Cys-Prx, and TrxR NTRA genes in the embryonic axes of maturing, mature, and stored seeds from two Acer species. Results and discussion: The expression of Trx-h1 and TrxR NTRA throughout seed maturation in both species was low. The expression of 1-Cys-Prx remained relatively stable throughout seed maturation. In stored seeds, the expression levels were minimal, with slightly higher levels in sycamore seeds, which may confirm that recalcitrant seeds remain metabolically active during storage. A library of 289 proteins interacting with Trx-h1 was constructed, comprising 68 from Norway maple and 221 from sycamore, with distinct profiles in each seed category. Recalcitrant seed axes displayed a wide array of metabolic, stress response, and signaling proteins, suggesting sustained metabolic activity during storage and the need to address oxidative stress. Conversely, the orthodox seed axes presented a protein profile, reflecting efficient metabolic shutdown, which contributes to their extended viability. The results of the study provide new insights into seed viability and storage longevity mechanisms. They enhance the understanding of seed biology and lay the foundation for further evolutionary research on seeds of different categories.

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.

The role of NADPH oxidases in regulating leaf gas exchange and ion homeostasis in Arabidopsis plants under cadmium stress.

NADPH oxidase, an enzyme associated with the plasma membrane, constitutes one of the main sources of reactive oxygen species (ROS) which regulate different developmental and adaptive responses in plants. In this work, the involvement of NADPH oxidases in the regulation of photosynthesis and cell ionic homeostasis in response to short cadmium exposure was compared between wild type (WT) and three RBOHs (Respiratory Burst Oxidase Homologues) Arabidopsis mutants (AtrbohC, AtrbohD, and AtrbohF). Plants were grown under hydroponic conditions and supplemented with 50 µM CdCl2 for 24 h. Cadmium treatment differentially affected photosynthesis, stomatal conductance, transpiration, and antioxidative responses in WT and Atrbohs mutants. The loss of function of RBOH isoforms resulted in higher Cd2+ influx, mainly in the elongation zone of roots, which was more evident in AtrbohD and AtrbohF mutants. In the mature zone, the highest Cd2+ influx was observed in rbohC mutant. The lack of functional RBOH isoforms also resulted in altered patterns of net K+ transport across cellular membranes, both in the root epidermis and leaf mesophyll. The analysis of expression of metal transporters by qPCR demonstrated that a loss of functional RBOH isoforms has altered transcript levels for metal NRAMP3, NRAMP6 and IRT1 and the K+ transporters outward-rectifying K+ efflux GORK channel, while RBOHD specifically regulated transcripts for high-affinity K+ transporters KUP8 and HAK5, and IRT1 and RBOHD and F regulated the transcription factors TGA3 and TGA10. It is concluded that RBOH-dependent H2O2 regulation of ion homeostasis and Cd is a highly complex process involving multilevel regulation from transpirational water flow to transcriptional and posttranslational modifications of K/metals transporters.

Arabidopsis fibrillin 1-2 subfamily members exert their functions via specific protein-protein interactions.

Fibrillins (FBNs) are plastidial proteins found in photosynthetic organisms from cyanobacteria to higher plants. The function of most FBNs remains unknown. Here, we focused on members of the FBN subgroup comprising FBN1a, FBN1b, and FBN2. We show that these three polypeptides interact between each other, potentially forming a network around the plastoglobule surface. Both FBN2 and FBN1s interact with allene oxide synthase, and the elimination of any of these FBNs results in a delay in jasmonate-mediated anthocyanin accumulation in response to a combination of moderate high light and low temperature. Mutations in the genes encoding FBN1s or FBN2 also affect the protection of PSII under the combination of these stresses. Fully developed leaves of these mutants have lower maximum quantum efficiency of PSII (Fv/Fm) and higher oxidative stress than wild-type plants. These effects are additive, and the fbn1a-1b-2 triple mutant shows a stronger decrease in Fv/Fm and a greater increase in oxidative stress than fbn1a-1b or fbn2 mutants. Co-immunoprecipitation analysis indicated that FBN2 also interacts with other proteins involved in different metabolic processes. We propose that these fibrillins facilitate accurate positioning of different proteins involved in distinct metabolic processes, and that their elimination leads to dysfunction of those proteins.

Current Knowledge on Mechanisms Preventing Photosynthesis Redox Imbalance in Plants.

Photosynthesis includes a set of redox reactions that are the source of reducing power and energy for the assimilation of inorganic carbon, nitrogen and sulphur, thus generating organic compounds, and oxygen, which supports life on Earth. As sessile organisms, plants have to face continuous changes in environmental conditions and need to adjust the photosynthetic electron transport to prevent the accumulation of damaging oxygen by-products. The balance between photosynthetic cyclic and linear electron flows allows for the maintenance of a proper NADPH/ATP ratio that is adapted to the plant's needs. In addition, different mechanisms to dissipate excess energy operate in plants to protect and optimise photosynthesis under adverse conditions. Recent reports show an important role of redox-based dithiol-disulphide interchanges, mediated both by classical and atypical chloroplast thioredoxins (TRXs), in the control of these photoprotective mechanisms. Moreover, membrane-anchored TRX-like proteins, such as HCF164, which transfer electrons from stromal TRXs to the thylakoid lumen, play a key role in the regulation of lumenal targets depending on the stromal redox poise. Interestingly, not all photoprotective players were reported to be under the control of TRXs. In this review, we discuss recent findings regarding the mechanisms that allow an appropriate electron flux to avoid the detrimental consequences of photosynthesis redox imbalances.

Thioredoxins m are major players in the multifaceted light-adaptive response in Arabidopsis thaliana.

Thioredoxins (TRXs) are well-known redox signalling players, which carry out post-translational modifications in target proteins. Chloroplast TRXs are divided into different types and have central roles in light energy uptake and the regulation of primary metabolism. The isoforms TRX m1, m2, and m4 from Arabidopsis thaliana are considered functionally related. Knowing their key position in the hub of plant metabolism, we hypothesized that the impairment of the TRX m signalling would not only have harmful consequences on chloroplast metabolism but also at different levels of plant development. To uncover the physiological and developmental processes that depend on TRX m signalling, we carried out a comprehensive study of Arabidopsis single, double, and triple mutants defective in the TRX m1, m2, and m4 proteins. As light and redox signalling are closely linked, we investigated the response to high light (HL) of the plants that are gradually compromised in TRX m signalling. We provide experimental evidence relating the lack of TRX m and the appearance of novel phenotypic features concerning mesophyll structure, stomata biogenesis, and stomatal conductance. We also report new data indicating that the isoforms of TRX m fine-tune the response to HL, including the accumulation of the protective pigment anthocyanin. These results reveal novel signalling functions for the TRX m and underline their importance for plant growth and fulfilment of the acclimation/response to HL conditions.

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 functions.

CRISPR/Cas9 mutations in the rice Waxy/GBSSI gene induce allele-specific and zygosity-dependent feedback effects on endosperm starch biosynthesis.

KEY MESSAGE: Induced mutations in the waxy locus in rice endosperm did not abolish GBSS activity completely. Compensatory mechanisms in endosperm and leaves caused a major reprogramming of the starch biosynthetic machinery. The mutation of genes in the starch biosynthesis pathway has a profound effect on starch quality and quantity and is an important target for plant breeders. Mutations in endosperm starch biosynthetic genes may impact starch metabolism in vegetative tissues such as leaves in unexpected ways due to the complex feedback mechanisms regulating the pathway. Surprisingly this aspect of global starch metabolism has received little attention. We used CRISPR/Cas9 to introduce mutations affecting the Waxy (Wx) locus encoding granule-bound starch synthase I (GBSSI) in rice endosperm. Our specific objective was to develop a mechanistic understanding of how the endogenous starch biosynthetic machinery might be affected at the transcriptional level following the targeted knock out of GBSSI in the endosperm. We found that the mutations reduced but did not abolish GBSS activity in seeds due to partial compensation caused by the upregulation of GBSSII. The GBSS activity in the mutants was 61-71% of wild-type levels, similarly to two irradiation mutants, but the amylose content declined to 8-12% in heterozygous seeds and to as low as 5% in homozygous seeds, accompanied by abnormal cellular organization in the aleurone layer and amorphous starch grain structures. Expression of many other starch biosynthetic genes was modulated in seeds and leaves. This modulation of gene expression resulted in changes in AGPase and sucrose synthase activity that explained the corresponding levels of starch and soluble sugars.

Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum.

Redox regulation is of great importance in chloroplasts. Many chloroplast enzymes, such as those belonging to the Calvin-Benson cycle (CBC), have conserved regulatory cysteines which form inhibitory disulphide bridges when physiological conditions become unfavourable. Amongst these enzymes, cFBP1, the CBC fructose-1,6-bisphosphatase (FBPase) isoform, is well known to be redox activated by thioredoxin f through the reduction of a disulphide bridge involving Cys153 and Cys173. Moreover, data obtained during recent years point to S-nitrosylation as another redox post-translational modification putatively regulating an increasing number of plant enzymes, including cFBP1. In this study we have shown that the Pisum sativum cFBP1 can be efficiently S-nitrosylated by GSNO and SNAP, triggering the formation of the regulatory disulphide. Using in vivo experiments with P. sativum we have established that cFBP1 S-nitrosylation only occurs during the light period and we have elucidated by activity assays with Cys-to-Ser mutants that this enzyme may be inactivated through the S-nitrosylation of Cys153. Finally, in the light of the new data, we have proposed an extended redox-regulation model by integrating the S-nitrosylation and the TRX f-mediated regulation of cFBP1.

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.

Transcriptomic and proteomic approach to identify differentially expressed genes and proteins in Arabidopsis thaliana mutants lacking chloroplastic 1 and cytosolic FBPases reveals several levels of metabolic regulation.

BACKGROUND: During the photosynthesis, two isoforms of the fructose-1,6-bisphosphatase (FBPase), the chloroplastidial (cFBP1) and the cytosolic (cyFBP), catalyse the first irreversible step during the conversion of triose phosphates (TP) to starch or sucrose, respectively. Deficiency in cyFBP and cFBP1 isoforms provokes an imbalance of the starch/sucrose ratio, causing a dramatic effect on plant development when the plastidial enzyme is lacking. RESULTS: We study the correlation between the transcriptome and proteome profile in rosettes and roots when cFBP1 or cyFBP genes are disrupted in Arabidopsis thaliana knock-out mutants. By using a 70-mer oligonucleotide microarray representing the genome of Arabidopsis we were able to identify 1067 and 1243 genes whose expressions are altered in the rosettes and roots of the cfbp1 mutant respectively; whilst in rosettes and roots of cyfbp mutant 1068 and 1079 genes are being up- or down-regulated respectively. Quantitative real-time PCR validated 100% of a set of 14 selected genes differentially expressed according to our microarray analysis. Two-dimensional (2-D) gel electrophoresis-based proteomic analysis revealed quantitative differences in 36 and 26 proteins regulated in rosettes and roots of cfbp1, respectively, whereas the 18 and 48 others were regulated in rosettes and roots of cyfbp mutant, respectively. The genes differentially expressed and the proteins more or less abundant revealed changes in protein metabolism, RNA regulation, cell signalling and organization, carbon metabolism, redox regulation, and transport together with biotic and abiotic stress. Notably, a significant set (25%) of the proteins identified were also found to be regulated at a transcriptional level. CONCLUSION: This transcriptomic and proteomic analysis is the first comprehensive and comparative study of the gene/protein re-adjustment that occurs in photosynthetic and non-photosynthetic organs of Arabidopsis mutants lacking FBPase isoforms.

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.

Plastid thioredoxins: a "one-for-all" redox-signaling system in plants.

The sessile nature of plants forces them to face an ever-changing environment instead of escape from hostile conditions as animals do. In order to overcome this survival challenge, a fine monitoring and controlling of the status of the photosynthetic electron transport chain and the general metabolism is vital for these organisms. Frequently, evolutionary plant adaptation has consisted in the appearance of multigenic families, comprising an array of enzymes, structural components, or sensing, and signaling elements, in numerous occasions with highly conserved primary sequences that sometimes make it difficult to discern between redundancy and specificity among the members of a same family. However, all this gene diversity is aimed to sort environment-derived plant signals to efficiently channel the external incoming information inducing a right physiological answer. Oxygenic photosynthesis is a powerful source of reactive oxygen species (ROS), molecules with a dual oxidative/signaling nature. In response to ROS, one of the most frequent post-translational modifications occurring in redox signaling proteins is the formation of disulfide bridges (from Cys oxidation). This review is focused on the role of plastid thioredoxins (pTRXs), proteins containing two Cys in their active site and largely known as part of the plant redox-signaling network. Several pTRXs types have been described so far, namely, TRX f, m, x, y, and z. In recent years, improvements in proteomic techniques and the study of loss-of-function mutants have enabled us to grasp the importance of TRXs for the plastid physiology. We will analyze the specific signaling function of each TRX type and discuss about the emerging role in non-photosynthetic plastids of these redox switchers.

Loss of starch granule initiation has a deleterious effect on the growth of arabidopsis plants due to an accumulation of ADP-glucose.

STARCH SYNTHASE4 (SS4) is required for proper starch granule initiation in Arabidopsis (Arabidopsis thaliana), although SS3 can partially replace its function. Unlike other starch-deficient mutants, ss4 and ss3/ss4 mutants grow poorly even under long-day conditions. They have less chlorophyll and carotenoids than the wild type and lower maximal rates of photosynthesis. There is evidence of photooxidative damage of the photosynthetic apparatus in the mutants from chlorophyll a fluorescence parameters and their high levels of malondialdehyde. Metabolite profiling revealed that ss3/ss4 accumulates over 170 times more ADP-glucose (Glc) than wild-type plants. Restricting ADP-Glc synthesis, by introducing mutations in the plastidial phosphoglucomutase (pgm1) or the small subunit of ADP-Glc pyrophosphorylase (aps1), largely restored photosynthetic capacity and growth in pgm1/ss3/ss4 and aps1/ss3/ss4 triple mutants. It is proposed that the accumulation of ADP-Glc in the ss3/ss4 mutant sequesters a large part of the plastidial pools of adenine nucleotides, which limits photophosphorylation, leading to photooxidative stress, causing the chlorotic and stunted growth phenotypes of the plants.

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 thiol-dithiol 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. Protein-DNA 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.

Plastid thioredoxins f and m are related to the developing and salinity response of post-germinating seeds of Pisum sativum.

Plastid thioredoxins (TRXs) f and m have long been considered to regulate almost exclusively photosynthesis-related processes. Nonetheless, some years ago, we found that type-f and m TRXs were also present in non-photosynthetic organs such as roots and flowers of adult pea plants. In the present work, using pea seedlings 2-5 days old, we have determined the mRNA expression profile of the plastid PsTRX f, m1, and m2, together with the ferredoxin NADP reductase (FNR). Our results show that these TRX isoforms are expressed in cotyledons, underlying similar expression levels in roots for PsTRX m2. We have also noted plastid TRX expression in cotyledons of etiolated seedlings of Arabidopsis thaliana lines carrying constructs corresponding to PsTRX f and m1 promoters fused to the reporter gene GUS, pointing to a role in reserve mobilization. Furthermore, the response of plastid TRXs to NaCl and their capacity in restoring the growth of a TRX-deficient yeast under saline conditions suggest a role in the tolerance to salinity. We propose that these redox enzymes take part of the reserve mobilization in seedling cotyledons and we suggest additional physiological functions of PsTRX m2 in roots and PsTRX m1 in the salinity-stress response during germination.

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 chi 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 chi knock-out mutant has been identified and a double mutant (denoted Delta 2cp) with <5% of 2-Cys Prx content has been generated. The phenotypes of the three mutants, ntrc, trxx, and Delta 2cp, 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 chi or 2-Cys Prxs as determined by growth, pigment content, CO(2) fixation, and F(v)/F(m), indicating additional functions of NTRC.

Evidence of non-functional redundancy between two pea h-type thioredoxins by specificity and stability studies.

The largest group of plant thioredoxins (TRXs) consists of the so-called h-type; their great number raises questions about their specific or redundant roles in plant cells. Pisum sativum thioredoxin h1 (PsTRXh1) and Pisum sativum thioredoxin h2 (PsTRXh2) are both h-type TRXs from pea (Pisum sativum) previously identified and biochemically characterized. While both are involved in redox regulation and show a high-sequence identity (60%), they display different behavior during in vitro and in vivo assays. In this work, we show that these two proteins display different specificity in the capturing of protein targets in vitro, by the use of a new stringent method. PsTRXh2 interacted with classical antioxidant proteins, whereas PsTRXh1 showed a completely different pattern of targeted proteins, and was able to capture a transcription factor. We also showed that the two proteins display very different thermal and chemical stabilities. We suggest that the differences in thermal and chemical stability point to a distinct and characteristic pattern of protein specificity.