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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.

An antioxidant redox system in the nucleus of wheat seed cells suffering oxidative stress

Cereal seed cells contain different mechanisms for protection against the oxidative stress that occurs during maturation and germination. One such mechanism is based on the antioxidant activity of a 1-Cys peroxiredoxin (1-Cys Prx) localized in the nuclei of aleurone and scutellum cells. However, nothing is known about the mechanism of activation of this enzyme. Here, we describe the pattern of localization of NADPH thioredoxin reductase (NTR) in developing and germinating wheat seeds using an immunocytochemical analysis. The presence of NTR in transfer cells, vascular tissue, developing embryo and root meristematic cells, agrees with the localization of thioredoxin h (Trx h), and supports the important function of the NTR/Trx system in cell proliferation and communication. Interestingly, NTR is found in the nuclei of seed cells suffering oxidative stress, thus showing co-localization with Trx h and 1-Cys Prx. To test whether the NTR/Trx system serves as a reductant of the 1-Cys Prx, we cloned a full-length cDNA encoding 1-Cys Prx from wheat, and expressed the recombinant protein in Escherichia coli. Using the purified components, we show NTR-dependent activity of the 1-Cys Prx. Mutants of the 1-Cys Prx allowed us to demonstrate that the peroxidatic residue of the wheat enzyme is Cys46, which is overoxidized in vitro under oxidant conditions. Analysis of extracts from developing and germinating seeds confirmed 1-Cys Prx overoxidation in vivo. Based on these results, we propose that NADPH is the source of the reducing power to regenerate 1-Cys Prx in the nuclei of seed cells suffering oxidative stress, in a process that is catalyzed by NTR.

New insights into plant isoprenoid metabolism.

Isoprenoids are a hugely diverse family of compounds derived from the C5 precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP).Although all free-living organisms synthesize isoprenoids,they are particularly abundant and diverse in plants,with tens of thousands structures known to date.The highest variety of plant isoprenoids are specialized (secondary) metabolites that participate in the interaction of plants with their environment.These include pigments,volatiles,and defense compounds,some of which have applications in industry and agriculture.For example,isoprenoid drugs are used against cancer (taxol) or malaria (artemisin).But plants also synthesize isoprenoids with essential (primary) functions in respiration (ubiquinone),photosynthesis (carotenoids,chlorophylls,tocopherols,phylIoquinones,plastoquinone),membrane architecture (sterols),and growth regulation (brassinosteroids,cytokinins,gibberellins,abscisic acid,strigolactones).Despite their economic importance and biological relevance,our knowledge of the core pathways for the production of the universal isoprenoid precursors IPP and DMAPP in plant cells remained incomplete until the mid-1990s.Impressive progress in the last decade has resulted in the complete elucidation of several isoprenoid pathways,the identification of regulatory mechanisms,the discovery of new functions and properties of specific isoprenoids,and the successful manipulation of isoprenoid biosynthesis in a number of metabolic engineering approaches.In this update article,we will discuss some of the most recent advances in the plant isoprenoid field,focusing on the pathways supplying the C5 precursors in plant cells and the novel insights into regulatory matters.

Metabolic flux analysis of plastidic isoprenoid biosynthesis in poplar leaves emitting and nonemitting isoprene.

Isoprene biosynthesis demands a huge carbon flux through the plastidic isoprenoid pathway, and the concentration of its immediate precursor modulates this flux. The plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway is one of the most important pathways in plants and produces a large variety of essential isoprenoids. Its regulation, however, is still not well understood. Using the stable isotope 13C-labeling technique, we analyzed the carbon fluxes through the MEP pathway and into the major plastidic isoprenoid products in isoprene-emitting and transgenic isoprene-nonemitting (NE) gray poplar (Populus × canescens). We assessed the dependence on temperature, light intensity, and atmospheric [CO2]. Isoprene biosynthesis was by far (99%) the main carbon sink of MEP pathway intermediates in mature gray poplar leaves, and its production required severalfold higher carbon fluxes compared with NE leaves with almost zero isoprene emission. To compensate for the much lower demand for carbon, NE leaves drastically reduced the overall carbon flux within the MEP pathway. Feedback inhibition of 1-deoxy-d-xylulose-5-phosphate synthase activity by accumulated plastidic dimethylallyl diphosphate almost completely explained this reduction in carbon flux. Our data demonstrate that short-term biochemical feedback regulation of 1-deoxy-d-xylulose-5-phosphate synthase activity by plastidic dimethylallyl diphosphate is an important regulatory mechanism of the MEP pathway. Despite being relieved from the large carbon demand of isoprene biosynthesis, NE plants redirected only approximately 0.5% of this saved carbon toward essential nonvolatile isoprenoids, i.e. β-carotene and lutein, most probably to compensate for the absence of isoprene and its antioxidant properties.

NADPH Thioredoxin Reductase C Controls the Redox Status of Chloroplast 2-Cys Peroxiredoxins in Arabidopsis thaliana

Chloroplast 2-Cys peroxiredoxins (2-Cys Prxs) are efficiently reduced by NADPH Thioredoxin reductase C (NTRC). To investigate the effect of light/darkness on NTRC function, the content of abundant plastidial enzymes, Rubisco, glutamine synthetase (GS), and 2-Cys Prxs was analyzed during two consecutive days in Arabidopsis wild-type and ntrc mutant plants. No significant difference of the content of these proteins was observed during the day or the night in wild-type and mutant plants. NTRC deficiency caused a lower content of fully reduced 2-Cys Prxs, which was undetectable in darkness, suggesting that NTRC is the most important pathway for 2-Cys Prx reduction, probably the only one during the night. Arabidopsis contains two plastidial 2-Cys Prxs, A and B, for which T-DNA insertion lines were characterized showing the same phenotype as wild-type plants. Two-dimensional gel analysis of leaf extracts from these mutants allowed the identification of basic and acidic isoforms of 2-Cys Prx A and B. In-vitro assays and mass spectrometry analysis showed that the acidic isoform of both proteins is produced by overoxidation of the peroxidatic Cys residue to sulfinic acid. 2-Cys Prx overoxidation was lower in the NTRC mutant. These results show the important function of NTRC to maintain the redox equilibrium of chloroplast 2-Cys Prxs.

NTRC new ways of using NADPH in the chloroplast

Despite being the primary source of energy in the biosphere, photosynthesis is a process that inevitably produces reactive oxygen species. Chloroplasts are a major source of hydrogen peroxide production in plant cells; therefore, different systems for peroxide reduction, such as ascorbate peroxidase and peroxiredoxins (Prxs), are found in this organelle. Most of the reducing power required for hydrogen peroxide reduction by these systems is provided by Fd reduced by the photosynthetic electron transport chain; hence, the function of these systems is highly dependent on light. Recently, it was described a novel plastidial enzyme, stated NTRC, formed by a thioredoxin reductase (NTR) domain at the N-terminus and a thioredoxin (Trx) domain at the C-terminus. NTRC is able to conjugate both NTR and Trx activities to efficiently reduce 2-Cys Prx using NADPH as a source of reducing power. Based on these results, it was proposed that NTRC is a new pathway to transfer reducing power to the chloroplast detoxification system, allowing the use of NADPH, besides reduced Fd, for such function. In this article, the most important features of NTRC are summarized and the implications of this novel activity in the context of chloroplast protection against oxidative damage are discussed.

Arabidopsis J-Protein J20 Delivers the First Enzyme of the Plastidial Isoprenoid Pathway to Protein Quality Control

Protein quality control mechanisms rely on chaperones and proteases to maintain cell proteins in working conditions. This study reports the identification of a J-protein cochaperone that binds to inactive forms of a plastidial enzyme required for plant photosynthesis and development, targeting them for either proper folding or degradation in the chloroplast. Plastids provide plants with metabolic pathways that are unique among eukaryotes, including the methylerythritol 4-phosphate pathway for the production of isoprenoids essential for photosynthesis and plant growth. Here, we show that the first enzyme of the pathway, deoxyxylulose 5-phosphate synthase (DXS), interacts with the J-protein J20 in Arabidopsis thaliana. J-proteins typically act as adaptors that provide substrate specificity to heat shock protein 70 (Hsp70), a molecular chaperone. Immunoprecipitation experiments showed that J20 and DXS are found together in vivo and confirmed the presence of Hsp70 chaperones in DXS complexes. Mutants defective in J20 activity accumulated significantly increased levels of DXS protein (but no transcripts) and displayed reduced levels of DXS enzyme activity, indicating that loss of J20 function causes posttranscriptional accumulation of DXS in an inactive form. Furthermore, J20 promotes degradation of DXS following a heat shock. Together, our data indicate that J20 might identify unfolded or misfolded (damaged) forms of DXS and target them to the Hsp70 system for proper folding under normal conditions or degradation upon stress.

Immunocytochemical localization of Pisum sativum TRXs f and m in non-photosynthetic tissues

Plants are the organisms containing the most complex multigenic family for thioredoxins (TRX). Several types of TRXs are targeted to chloroplasts, which have been classified into four subgroups: m, f, x, and y. Among them, TRXs f and m were the first plastidial TRXs characterized, and their function as redox modulators of enzymes involved in carbon assimilation in the chloroplast has been well-established. Both TRXs, f and m, were named according to their ability to reduce plastidial fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase (MDH), respectively. Evidence is presented here based on the immunocytochemistry of the localization of f and m-type TRXs from Pisum sativum in non-photosynthetic tissues. Both TRXs showed a different spatial pattern. Whilst PsTRXm was localized to vascular tissues of all the organs analysed (leaves, stems, and roots), PsTRXf was localized to more specific cells next to xylem vessels and vascular cambium. Heterologous complementation analysis of the yeast mutant EMY63, deficient in both yeast TRXs, by the pea plastidial TRXs suggests that PsTRXm, but not PsTRXf, is involved in the mechanism of reactive oxygen species (ROS) detoxification. In agreement with this function, the PsTRXm gene was induced in roots of pea plants in response to hydrogen peroxide.

Tomato fruit carotenoid biosynthesis is adjusted to actual ripening progression by a light‐dependent mechanism

Carotenoids are isoprenoid compounds that are essential for plants to protect the photosynthetic apparatus against excess light. They also function as health-promoting natural pigments that provide colors to ripe fruit, promoting seed dispersal by animals. Work in Arabidopsis thaliana unveiled that transcription factors of the phytochrome-interacting factor (PIF) family regulate carotenoid gene expression in response to environmental signals (i.e. light and temperature), including those created when sunlight reflects from or passes though nearby vegetation or canopy (referred to as shade). Here we show that PIFs use a virtually identical mechanism to modulate carotenoid biosynthesis during fruit ripening in tomato (Solanum lycopersicum). However, instead of integrating environmental information, PIF-mediated signaling pathways appear to fulfill a completely new function in the fruit. As tomatoes ripen, they turn from green to red due to chlorophyll breakdown and carotenoid accumulation. When sunlight passes through the flesh of green fruit, a self-shading effect within the tissue maintains high levels of PIFs that directly repress the master gene of the fruit carotenoid pathway, preventing undue production of carotenoids. This effect is attenuated as chlorophyll degrades, causing degradation of PIF proteins and boosting carotenoid biosynthesis as ripening progresses. Thus, shade signaling components may have been co-opted in tomato fruit to provide information on the actual stage of ripening (based on the pigment profile of the fruit at each moment) and thus finely coordinate fruit color change. We show how this mechanism may be manipulated to obtain carotenoid-enriched fruits.

Mathematical modelling of the diurnal regulation of the MEP pathway in Arabidopsis

Isoprenoid molecules are essential elements of plant metabolism. Many important plant isoprenoids, such as chlorophylls, carotenoids, tocopherols, prenylated quinones and hormones are synthesised in chloroplasts via the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Here we develop a mathematical model of diurnal regulation of the MEP pathway in Arabidopsis thaliana. We used both experimental and theoretical approaches to integrate mechanisms potentially involved in the diurnal control of the pathway. Our data show that flux through the MEP pathway is accelerated in light due to the photosynthesis-dependent supply of metabolic substrates of the pathway and the transcriptional regulation of key biosynthetic genes by the circadian clock. We also demonstrate that feedback regulation of both the activity and the abundance of the first enzyme of the MEP pathway (1-deoxy-D-xylulose 5-phosphate synthase, DXS) by pathway products stabilizes the flux against changes in substrate supply and adjusts the flux according to product demand under normal growth conditions. These data illustrate the central relevance of photosynthesis, the circadian clock and feedback control of DXS for the diurnal regulation of the MEP pathway.