Functional division of f-type and m-type thioredoxins to regulate the Calvin cycle and cyclic electron transport around photosystem I

Redox regulation of chloroplast proteins is necessary to adjust photosynthetic performance with changes in light. The thioredoxin (Trx) system plays a central role in this process. Chloroplast-localized classical Trx is a small redox-active protein that regulates many target proteins by reducing their disulfide bonds in a light-dependent manner. Arabidopsis thaliana mutants lacking f-type Trx (trx f1f2) or m-type Trx (trx m124-2) have been reported to show delayed reduction of Calvin cycle enzymes. As a result, the trx m124-2 mutant exhibits growth defects. Here, we characterized a quintuple mutant lacking both Trx f and Trx m to investigate the functional complementarity of Trx f and Trx m. The trx f1f2 m124-2 quintuple mutant was newly obtained by crossing, and is analyzed here for the first time. The growth defects of the trx m124-2 mutant were not enhanced by the lack of Trx f. In contrast, deficiencies of both Trxs additively suppressed the reduction of Calvin cycle enzymes, resulting in a further delay in the initiation of photosynthesis. Trx f appeared to be necessary for the rapid activation of the Calvin cycle during the early induction of photosynthesis. To perform effective photosynthesis, plants seem to use both Trxs in a coordinated manner to activate carbon fixation reactions. In contrast, the PROTON GRADIENT REGULATION 5 (PGR5)-dependent cyclic electron transport around photosystem I was regulated by Trx m, but not by Trx f. Lack of Trx f did not affect the activity and regulation of the PGR5-dependent pathway. Trx f may have a higher specificity for target proteins, whereas Trx m has a variety of target proteins to regulate overall photosynthesis and other metabolic reactions in the chloroplasts.

     

Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins

The activity of the molecular motor enzyme, chloroplast ATP synthase, is regulated in a redox-dependent manner. The γ subunit, CF₁-γ, is the central shaft of this enzyme complex and possesses the redox-active cysteine pair, which is reduced by thioredoxin (Trx). In light conditions, Trx transfers the reducing equivalent obtained from the photosynthetic electron transfer system to the CF₁-γ. Previous studies showed that the light-dependent reduction of CF₁-γ is more rapid than those of other Trx target proteins in the stroma. Although there are multiple Trx isoforms in chloroplasts, it is not well understood as to which chloroplast Trx isoform primarily contributes to the reduction of CF₁-γ, especially under physiological conditions. We therefore performed direct assessment of the CF₁-γ reduction capacity of each of the Trx isoforms. The kinetic analysis of the reduction process showed no significant difference in the reduction efficiency between two major chloroplast Trxs, namely Trx-f and Trx-m. Based on the thorough analyses of the CF₁-γ redox dynamics in Arabidopsis thaliana Trx mutant plants, we found that lack of Trx-f or Trx-m had no significant impact on the in vivo light-dependent reduction of CF₁-γ. The results showed that CF₁-γ can accept the reducing power from both Trx-f and Trx-m in chloroplasts.     

Hybrids from pea chloroplast thioredoxins f and m: physicochemical and kinetic characteristics

Two hybrid thioredoxins (Trx) have been constructed from cDNA clones coding for pea chloroplast Trxs m and f. The splitting point was the AvaII site situated between the two cysteines of the regulatory cluster. One hybrid, Trx m/f, was purified from Escherichia coli-expressed cell lysates as a high yielding 12 kDa protein. Western blot analysis showed a positive reaction with antibodies against pea Trxs m and f and, like the parenteral pea Trx m, displayed an acidic pI (5.0) and a high thermal stability. In contrast, the opposite hybrid Trx f/m appeared in E. coli lysates as inclusion bodies, where it was detected by Western blot against pea Trx f antibodies as a 40 kDa protein. Trx f/m was very unstable, sensitive to heat denaturation, and could not be purified. Trx m/f showed a higher affinity for pea chloroplast fructose-1,6-bisphosphatase (FBPase) and a smaller Trx/FBPase saturation ratio than both parenterals; however, the FBPase catalytic rate was lower than that with Trxs m and f. Surprisingly, the hybrid Trx m/f appeared to be incompetent in the activation of pea NADP-malate dehydrogenase. Computer-assisted models of pea Trxs m and f, and of the chimeric Trx m/f, showed a change in the orientation of the α₄-helix in the hybrid, which could explain the kinetic modifications with respect to Trxs m and f. We conclude that the stability of Trxs lies on the N-side of the regulatory cluster, and is associated with the acidic character of this fragment and, as a consequence, with the acidic pI of the whole molecule. In contrast, the ability of FBPase binding and enzyme catalysis depends on the structure on the C-side of the regulatory cysteines.     

Ferredoxin/thioredoxin system plays an important role in the chloroplastic NADP status of Arabidopsis

NADP is a key electron carrier for a broad spectrum of redox reactions, including photosynthesis. Hence, chloroplastic NADP status, as represented by redox status (ratio of NADPH to NADP⁺) and pool size (sum of NADPH and NADP⁺), is critical for homeostasis in photosynthetic cells. However, the mechanisms and molecules that regulate NADP status in chloroplasts remain largely unknown. We have now characterized an Arabidopsis mutant with imbalanced NADP status (inap1), which exhibits a high NADPH/NADP⁺ ratio and large NADP pool size. inap1 is a point mutation in At2g04700, which encodes the catalytic subunit of ferredoxin/thioredoxin reductase. Upon illumination, inap1 demonstrated earlier increases in NADP pool size than the wild type did. The mutated enzyme was also found in vitro to inefficiently reduce m‐type thioredoxin, which activates Calvin cycle enzymes, and NADP‐dependent malate dehydrogenase to export reducing power to the cytosol. Accordingly, Calvin cycle metabolites and amino acids diminished in inap1 plants. In addition, inap1 plants barely activate NADP‐malate dehydrogenase, and have an altered redox balance between the chloroplast and cytosol, resulting in inefficient nitrate reduction. Finally, mutants deficient in m‐type thioredoxin exhibited similar light‐dependent NADP dynamics as inap1. Collectively, the data suggest that defects in ferredoxin/thioredoxin reductase and m‐type thioredoxin decrease the consumption of NADPH, leading to a high NADPH/NADP⁺ ratio and large NADP pool size. The data also suggest that the fate of NADPH is an important influence on NADP pool size.     

Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function

The light‐dependent regulation of stromal enzymes by thioredoxin (Trx)‐catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that Trx targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Trx‐mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis. However, the extent of thiol‐dependent redox regulation in the thylakoid lumen has not been previously systematically explored. In this study, we addressed Trx‐linked redox control in the chloroplast lumen of Arabidopsis thaliana. Using complementary proteomics approaches, we identified 19 Trx target proteins, thus covering more than 40% of the currently known lumenal chloroplast proteome. We show that the redox state of thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II. Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin de‐epoxidase, which participates in thermal dissipation of excess absorbed light. Our results indicate that redox‐controlled reactions in the chloroplast lumen play essential roles in the function of photosystem II and the regulation of adaptation to light intensity.     

Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts

Redox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions. Most notably, by combining the discrimination of thiol status with mass spectrometry and activity measurement, we identified an uncharacterized aspect of the reductive activation of NADP-malate dehydrogenase; two redox-active Cys pairs harbored in this enzyme were reduced via distinct utilization of Trxs even within a single polypeptide. In our in vitro assays, Trx-f was effective in reducing all thiol-modulated enzymes analyzed here. We then investigated the in vivo physiological relevance of these in vitro findings, using Arabidopsis wild-type and Trx-f-deficient plants. Photoreduction of fructose-1,6-bisphosphatase was partially impaired in Trx-f-deficient plants, but the global impact of Trx-f deficiency on the redox behaviors of thiol-modulated enzymes was not as striking as expected from the in vitro data. Our results provide support for the in vivo functionality of the Trx system and also highlight the complexity and plasticity of the chloroplast redox network.     

Compartmentalisation of [FeFe]‐hydrogenase maturation in Chlamydomonas reinhardtii

Molecular hydrogen (H₂) can be produced in green microalgae by [FeFe]‐hydrogenases as a direct product of photosynthesis. The Chlamydomonas reinhardtii hydrogenase HYDA1 contains a catalytic site comprising a classic [4Fe4S] cluster linked to a unique 2Fe sub‐cluster. From in vitro studies it appears that the [4Fe4S] cluster is incorporated first by the housekeeping FeS cluster assembly machinery, followed by the 2Fe sub‐cluster, whose biosynthesis requires the specific maturases HYDEF and HYDG. To investigate the maturation process in vivo, we expressed HYDA1 from the C. reinhardtii chloroplast and nuclear genomes (with and without a chloroplast transit peptide) in a hydrogenase‐deficient mutant strain, and examined the cellular enzymatic hydrogenase activity, as well as in vivo H₂ production. The transformants expressing HYDA1 from the chloroplast genome displayed levels of H₂ production comparable to the wild type, as did the transformants expressing full‐length HYDA1 from the nuclear genome. In contrast, cells equipped with cytoplasm‐targeted HYDA1 produced inactive enzyme, which could only be activated in vitro after reconstitution of the [4Fe4S] cluster. This indicates that the HYDA1 FeS cluster can only be built by the chloroplastic FeS cluster assembly machinery. Further, the expression of a bacterial hydrogenase gene, CPI, from the C. reinhardtii chloroplast genome resulted in H₂‐producing strains, demonstrating that a hydrogenase with a very different structure can fulfil the role of HYDA1 in vivo and that overexpression of foreign hydrogenases in C. reinhardtii is possible. All chloroplast transformants were stable and no toxic effects were seen from HYDA1 or CPI expression.     

A protein with an inactive pterin‐4a‐carbinolamine dehydratase domain is required for Rubisco biogenesis in plants

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin–Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co‐factor of hepatocyte nuclear factor 1 (DCoH)/pterin‐4α‐carbinolamine dehydratases (PCD)‐like protein is the causative mutation in a seedling‐lethal, Rubisco‐deficient mutant named Rubisco accumulation factor 2 (raf2‐1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high‐molecular weight complex, the formation of which requires a specific chaperonin 60‐kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross‐linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co‐immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co‐immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins.     

Aminooxy‐naphthylpropionic acid and its derivatives are inhibitors of auxin biosynthesis targeting l‐tryptophan aminotransferase: structure–activity relationships

We previously reported l ‐α‐aminooxy‐phenylpropionic acid (AOPP) to be an inhibitor of auxin biosynthesis, but its precise molecular target was not identified. In this study we found that AOPP targets TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1). We then synthesized 14 novel compounds derived from AOPP to study the structure–activity relationships of TAA1 inhibitors in vitro. The aminooxy and carboxy groups of the compounds were essential for inhibition of TAA1 in vitro. Docking simulation analysis revealed that the inhibitory activity of the compounds was correlated with their binding energy with TAA1. These active compounds reduced the endogenous indole‐3‐acetic acid (IAA) content upon application to Arabidopsis seedlings. Among the compounds, we selected 2‐(aminooxy)‐3‐(naphthalen‐2‐yl)propanoic acid (KOK1169/AONP) and analyzed its activities in vitro and in vivo. Arabidopsis seedlings treated with KOK1169 showed typical auxin‐deficient phenotypes, which were reversed by exogenous IAA. In vitro and in vivo experiments indicated that KOK1169 is more specific for TAA1 than other enzymes, such as phenylalanine ammonia‐lyase. We further tested 41 novel compounds with aminooxy and carboxy groups to which we added protection groups to increase their calculated hydrophobicity. Most of these compounds decreased the endogenous auxin level to a greater degree than the original compounds, and resulted in a maximum reduction of about 90% in the endogenous IAA level in Arabidopsis seedlings. We conclude that the newly developed compounds constitute a class of inhibitors of TAA1. We designated them ‘pyruvamine’.     

Using fluorescence lifetime microscopy to study the subcellular localization of anthocyanins

Anthocyanins are flavonoid pigments that accumulate in most seed plants. They are synthesized in the cytoplasm but accumulate inside the vacuoles. Anthocyanins are pigmented at the lower vacuolar pH, but in the cytoplasm they can be visualized based on their fluorescence properties. Thus, anthocyanins provide an ideal system for the development of new methods to investigate cytoplasmic pools and association with other molecular components. We have analyzed the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), in both in vitro and in vivo conditions, using wild‐type and mutant Arabidopsis thaliana seedlings. Within plant cells, the amplitude‐weighted mean fluorescence lifetime (τ_m) correlated with distinct subcellular localizations of anthocyanins. The vacuolar pool of anthocyanins exhibited shorter τ_m than the cytoplasmic pool. Consistently, lowering the pH of anthocyanins in solution shortened their fluorescence decay. We propose that FLIM is a useful tool for understanding the trafficking of anthocyanins and, potentially, for estimating vacuolar pH inside intact plant cells.     

Small‐molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function

Auxin is essential for plant growth and development, this makes it difficult to study the biological function of auxin using auxin‐deficient mutants. Chemical genetics have the potential to overcome this difficulty by temporally reducing the auxin function using inhibitors. Recently, the indole‐3‐pyruvate (IPyA) pathway was suggested to be a major biosynthesis pathway in Arabidopsis thaliana L. for indole‐3‐acetic acid (IAA), the most common member of the auxin family. In this pathway, YUCCA, a flavin‐containing monooxygenase (YUC), catalyzes the last step of conversion from IPyA to IAA. In this study, we screened effective inhibitors, 4‐biphenylboronic acid (BBo) and 4‐phenoxyphenylboronic acid (PPBo), which target YUC. These compounds inhibited the activity of recombinant YUC in vitro, reduced endogenous IAA content, and inhibited primary root elongation and lateral root formation in wild‐type Arabidopsis seedlings. Co‐treatment with IAA reduced the inhibitory effects. Kinetic studies of BBo and PPBo showed that they are competitive inhibitors of the substrate IPyA. Inhibition constants (K_i) of BBo and PPBo were 67 and 56 nm , respectively. In addition, PPBo did not interfere with the auxin response of auxin‐marker genes when it was co‐treated with IAA, suggesting that PPBo is not an inhibitor of auxin sensing or signaling. We propose that these compounds are a class of auxin biosynthesis inhibitors that target YUC. These small molecules are powerful tools for the chemical genetic analysis of auxin function.     

Redox regulation of chloroplastic glucose-6-phosphate dehydrogenase: A new role for f-type thioredoxin

Glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme of the oxidative pentose phosphate pathway supplying reducing power (as NADPH) in non-photosynthesizing cells. We have examined in detail the redox regulation of the plastidial isoform predominantly present in Arabidopsis green tissues (AtG6PDH1) and found that its oxidative activation is strictly dependent on plastidial thioredoxins (Trxs) that show differential efficiencies. Light/dark modulation of AtG6PDH1 was reproduced in vitro in a reconstituted ferredoxin/Trx system using f-type Trx allowing to propose a new function for this Trx isoform co-ordinating both reductive (Calvin cycle) and oxidative pentose phosphate pathways.     

The macrocyclizing protease butelase 1 remains autocatalytic and reveals the structural basis for ligase activity

Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform maturation of seed storage protein upon cleavage‐dependent autoactivation in the low‐pH environment of storage vacuoles. The AEPs have attracted attention for their macrocyclization reactions, and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP, we show that butelase 1 retains cleavage functions in vitro and in vivo. The in vivo rescue was incomplete, consistent with some trade‐off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEPs, suggesting the many differences in its peptide‐binding region are the source of its efficient macrocyclization. All considered, it seems that either butelase 1 has not fully specialized or a requirement for autocatalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs.     

Single cystathionine β-synthase domain-containing proteins modulate development by regulating the thioredoxin system in Arabidopsis

Plant thioredoxins (Trxs) participate in two redox systems found in different cellular compartments: the NADP-Trx system (NTS) in the cytosol and mitochondria and the ferredoxin-Trx system (FTS) in the chloroplast, where they function as redox regulators by regulating the activity of various target enzymes. The identities of the master regulators that maintain cellular homeostasis and modulate timed development through redox regulating systems have remained completely unknown. Here, we show that proteins consisting of a single cystathionine β-synthase (CBS) domain pair stabilize cellular redox homeostasis and modulate plant development via regulation of Trx systems by sensing changes in adenosine-containing ligands. We identified two CBS domain-containing proteins in Arabidopsis thaliana, CBSX1 and CBSX2, which are localized to the chloroplast, where they activate all four Trxs in the FTS. CBSX3 was found to regulate mitochondrial Trx members in the NTS. CBSX1 directly regulates Trxs and thereby controls H(2)O(2) levels and regulates lignin polymerization in the anther endothecium. It also affects plant growth by regulating photosynthesis-related [corrected] enzymes, such as malate dehydrogenase, via homeostatic regulation of Trxs. Based on our findings, we suggest that the CBSX proteins (or a CBS pair) are ubiquitous redox regulators that regulate Trxs in the FTS and NTS to modulate development and maintain homeostasis under conditions that are threatening to the cell.     

Subdiffraction‐resolution live‐cell imaging for visualizing thylakoid membranes

The chloroplast is the chlorophyll‐containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live‐cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three‐dimensional structured illumination microscopy (3D‐SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D‐SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild‐type and mutant strains. Using 3D‐SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D‐SIM. This study demonstrates that 3D‐SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.     

GTSF‐1 is required for formation of a functional RNA‐dependent RNA Polymerase complex in Caenorhabditis elegans

Argonaute proteins and their associated small RNAs (sRNAs) are evolutionarily conserved regulators of gene expression. Gametocyte‐specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured C‐terminal tail, are conserved in animals and have been shown to interact with Piwi clade Argonautes, thereby assisting their activity. We identified the Caenorhabditis elegans Gtsf1 homolog, named it gtsf‐1 and characterized it in the context of the sRNA pathways of C. elegans. We report that GTSF‐1 is not required for Piwi‐mediated gene silencing. Instead, gtsf‐1 mutants show a striking depletion of 26G‐RNAs, a class of endogenous sRNAs, fully phenocopying rrf‐3 mutants. We show, both in vivo and in vitro, that GTSF‐1 interacts with RRF‐3 via its CHHC zinc fingers. Furthermore, we demonstrate that GTSF‐1 is required for the assembly of a larger RRF‐3 and DCR‐1‐containing complex (ERIC), thereby allowing for 26G‐RNA generation. We propose that GTSF‐1 homologs may act to drive the assembly of larger complexes that act in sRNA production and/or in imposing sRNA‐mediated silencing activities.     

In vivo detection of protein cysteine sulfenylation in plastids

Protein cysteine thiols are post‐translationally modified under oxidative stress conditions. Illuminated chloroplasts are one of the important sources of hydrogen peroxide (H₂O₂) and are highly sensitive to environmental stimuli, yet a comprehensive view of the oxidation‐sensitive chloroplast proteome is still missing. By targeting the sulfenic acid YAP1C‐trapping technology to the plastids of light‐grown Arabidopsis cells, we identified 132 putatively sulfenylated plastid proteins upon H₂O₂ pulse treatment. Almost half of the sulfenylated proteins are enzymes of the amino acid metabolism. Using metabolomics, we observed a reversible decrease in the levels of the amino acids Ala, Asn, Cys, Gln, Glu, His, Ile, Leu, Lys, Phe, Ser, Thr and Val after H₂O₂ treatment, which is in line with an anticipated decrease in the levels of the glycolysis and tricarboxylic acid metabolites. Through the identification of an organelle‐tailored proteome, we demonstrated that the subcellular targeting of the YAP1C probe enables us to study in vivo cysteine sulfenylation at the organellar level. All in all, the identification of these oxidation events in plastids revealed that several enzymes of the amino acid metabolism rapidly undergo cysteine oxidation upon oxidative stress.