Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 27
Filter
1.
Plant Physiol ; 193(4): 2498-2512, 2023 Nov 22.
Article in English | MEDLINE | ID: mdl-37606239

ABSTRACT

Plants cope with sudden increases in light intensity through various photoprotective mechanisms. Redox regulation by thioredoxin (Trx) systems also contributes to this process. Whereas the functions of f- and m-type Trxs in response to such fluctuating light conditions have been extensively investigated, those of x- and y-type Trxs are largely unknown. Here, we analyzed the trx x single, trx y1 trx y2 double, and trx x trx y1 trx y2 triple mutants in Arabidopsis (Arabidopsis thaliana). A detailed analysis of photosynthesis revealed changes in photosystem I (PSI) parameters under low light in trx x and trx x trx y1 trx y2. The electron acceptor side of PSI was more reduced in these mutants than in the wild type. This mutant phenotype was more pronounced under fluctuating light conditions. During both low- and high-light phases, the PSI acceptor side was largely limited in trx x and trx x trx y1 trx y2. After fluctuating light treatment, we observed more severe PSI photoinhibition in trx x and trx x trx y1 trx y2 than in the wild type. Furthermore, when grown under fluctuating light conditions, trx x and trx x trx y1 trx y2 plants showed impaired growth and decreased level of PSI subunits. These results suggest that Trx x and Trx y prevent redox imbalance on the PSI acceptor side, which is required to protect PSI from photoinhibition, especially under fluctuating light. We also propose that Trx x and Trx y contribute to maintaining the redox balance even under constant low-light conditions to prepare for sudden increases in light intensity.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Photosystem I Protein Complex/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Oxidation-Reduction , Photosynthesis , Arabidopsis/physiology , Light , Thioredoxins/genetics , Thioredoxins/metabolism
2.
Plant Cell ; 32(12): 3866-3883, 2020 12.
Article in English | MEDLINE | ID: mdl-33037145

ABSTRACT

In addition to linear electron transport, photosystem I cyclic electron transport (PSI-CET) contributes to photosynthesis and photoprotection. In Arabidopsis (Arabidopsis thaliana), PSI-CET consists of two partially redundant pathways, one of which is the PROTON GRADIENT REGULATION5 (PGR5)/PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1 (PGRL1)-dependent pathway. Although the physiological significance of PSI-CET is widely recognized, the regulatory mechanism behind these pathways remains largely unknown. Here, we report on the regulation of the PGR5/PGRL1-dependent pathway by the m-type thioredoxins (Trx m). Genetic and phenotypic characterizations of multiple mutants indicated the physiological interaction between Trx m and the PGR5/PGRL1-dependent pathway in vivo. Using purified Trx proteins and ruptured chloroplasts, in vitro, we showed that the reduced form of Trx m specifically decreased the PGR5/PGRL1-dependent plastoquinone reduction. In planta, Trx m4 directly interacted with PGRL1 via disulfide complex formation. Analysis of the transgenic plants expressing PGRL1 Cys variants demonstrated that Cys-123 of PGRL1 is required for Trx m4-PGRL1 complex formation. Furthermore, the Trx m4-PGRL1 complex was transiently dissociated during the induction of photosynthesis. We propose that Trx m directly regulates the PGR5/PGRL1-dependent pathway by complex formation with PGRL1.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Chloroplast Thioredoxins/metabolism , Membrane Proteins/metabolism , Photosynthetic Reaction Center Complex Proteins/metabolism , Plastoquinone/metabolism , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Chloroplast Thioredoxins/genetics , Chloroplasts/metabolism , Disulfides/metabolism , Electron Transport , Membrane Proteins/genetics , Photosynthesis , Photosynthetic Reaction Center Complex Proteins/genetics , Photosystem I Protein Complex/metabolism , Plants, Genetically Modified
3.
Plant Cell Physiol ; 63(1): 92-103, 2022 Jan 25.
Article in English | MEDLINE | ID: mdl-34623443

ABSTRACT

Light-dependent activation of chloroplast enzymes is required for the rapid induction of photosynthesis after a shift from dark to light. The thioredoxin (Trx) system plays a central role in this process. In chloroplasts, the Trx system consists of two pathways: the ferredoxin (Fd)/Trx pathway and the nicotinamide adenine dinucleotide phosphate (NADPH)-Trx reductase C (NTRC) pathway. In Arabidopsis (Arabidopsis thaliana) mutants defective in either pathway, the photoreduction of thiol enzymes was impaired, resulting in decreased carbon fixation. The close relationship between the Fd/Trx pathway and proton gradient regulation 5 (PGR5)-dependent photosystem I cyclic electron transport (PSI CET) in the induction of photosynthesis was recently elucidated. However, how the PGR5-dependent pathway is involved in the NTRC pathway is unclear, although NTRC has been suggested to physically interact with PGR5. In this study, we analyzed Arabidopsis mutants lacking either the PGR5 or the chloroplast NADH dehydrogenase-like complex (NDH)-dependent PSI CET pathway in the ntrc mutant background. The ntrc pgr5 double mutant suppressed both the growth defects and the high non-photochemical quenching phenotype of the ntrc mutant when grown under long-day conditions. By contrast, the inactivation of NDH activity with the chlororespiratory reduction 2-2 mutant failed to suppress either phenotype. We discovered that the phenotypic rescue of ntrc by pgr5 is caused by the partial restoration of Trx-dependent reduction of thiol enzymes. These results suggest that electron partitioning to the PGR5-dependent pathway and the Trx system needs to be properly regulated for the activation of the Calvin-Benson-Bassham cycle enzymes during the induction of photosynthesis.


Subject(s)
Arabidopsis/growth & development , Arabidopsis/genetics , Arabidopsis/metabolism , Chloroplasts/metabolism , Metabolic Networks and Pathways/radiation effects , Oxidation-Reduction/radiation effects , Thioredoxin-Disulfide Reductase/metabolism , Adaptation, Ocular/genetics , Adaptation, Ocular/physiology , Dark Adaptation/genetics , Dark Adaptation/physiology , Gene Expression Regulation, Plant , Genes, Plant , Genetic Variation , Genotype , Metabolic Networks and Pathways/genetics , Mutation , Photosynthesis/physiology , Thioredoxin-Disulfide Reductase/genetics
4.
J Plant Res ; 135(4): 543-553, 2022 Jul.
Article in English | MEDLINE | ID: mdl-35325335

ABSTRACT

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.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Photosynthetic Reaction Center Complex Proteins , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Chloroplasts/metabolism , Electron Transport , Oxidation-Reduction , Photosynthesis , Photosynthetic Reaction Center Complex Proteins/metabolism , Photosystem I Protein Complex/metabolism , Thioredoxins/genetics , Thioredoxins/metabolism
5.
Plant Physiol ; 184(3): 1291-1302, 2020 11.
Article in English | MEDLINE | ID: mdl-32917772

ABSTRACT

In response to light, plants efficiently induce photosynthesis. Light activation of thiol enzymes by the thioredoxin (Trx) systems and cyclic electron transport by the PROTON GRADIENT REGULATION5 (PGR5)-dependent pathway contribute substantially to regulation of photosynthesis. Arabidopsis (Arabidopsis thaliana) mutants lacking f-type Trxs (trx f1f2) show delayed activation of carbon assimilation due to impaired photoreduction of Calvin-Benson cycle enzymes. To further study regulatory mechanisms that contribute to efficiency during the induction of photosynthesis, we analyzed the contributions of PSI donor- and acceptor-side regulation in the trx f1f2 mutant background. The cytochrome b 6 f complex is involved in PSI donor-side regulation, whereas PGR5-dependent PSI cyclic electron transport is required for both donor and acceptor functions. Introduction of the pgr1 mutation, which is conditionally defective in cytochrome b 6 f complex activity, into the trx f1f2 mutant background did not further affect the induction of photosynthesis, but the combined deficiency of Trx f and PGR5 severely impaired photosynthesis and suppressed plant growth under long-day conditions. In the pgr5 trx f1f2 mutant, the acceptor-side of PSI was almost completely reduced, and quantum yields of PSII and PSI hardly increased during the induction of photosynthesis. We also compared the photoreduction of thiol enzymes between the trx f1f2 and pgr5 trxf1f2 mutants. The pgr5 mutation did not result in further impaired photoreduction of Calvin-Benson cycle enzymes or ATP synthase in the trx f1f2 mutant background. These results indicated that acceptor-side limitations in the pgr5 trx f1f2 mutant suppress photosynthesis initiation, suggesting that PGR5 is required for efficient photosynthesis induction.


Subject(s)
Arabidopsis/growth & development , Arabidopsis/genetics , Arabidopsis/metabolism , Chloroplast Thioredoxins/metabolism , Electron Transport/physiology , Photosynthetic Reaction Center Complex Proteins/metabolism , Photosystem I Protein Complex/metabolism , Gene Expression Regulation, Plant , Genes, Plant , Genetic Variation , Genotype , Mutation
7.
Plant J ; 84(5): 900-13, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26468055

ABSTRACT

Thioredoxins (Trxs) regulate the activity of various chloroplastic proteins in a light-dependent manner. Five types of Trxs function in different physiological processes in the chloroplast of Arabidopsis thaliana. Previous in vitro experiments have suggested that the f-type Trx (Trx f) is the main redox regulator of chloroplast enzymes, including Calvin cycle enzymes. To investigate the in vivo contribution of each Trx isoform to the redox regulatory system, we first quantified the protein concentration of each Trx isoform in the chloroplast stroma. The m-type Trx (Trx m), which consists of four isoforms, was the most abundant type. Next, we analyzed several Arabidopsis Trx-m-deficient mutants to elucidate the physiological role of Trx m in vivo. Deficiency of Trx m impaired plant growth and decreased the CO2 assimilation rate. We also determined the redox state of Trx target enzymes to examine their photo-reduction, which is essential for enzyme activation. In the Trx-m-deficient mutants, the reduction level of fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase was lower than that in the wild type. Inconsistently with the historical view, our in vivo study suggested that Trx m plays a more important role than Trx f in the activation of Calvin cycle enzymes.


Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/metabolism , Chloroplast Thioredoxins/physiology , Chloroplasts/metabolism , Photosynthesis , Adenosine Triphosphate/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/metabolism , Carbon Dioxide/metabolism , Chloroplast Thioredoxins/antagonists & inhibitors , Chloroplast Thioredoxins/metabolism , Enzyme Activation , Mutation , Oxidation-Reduction , Protein Isoforms/metabolism , Protein Isoforms/physiology , RNA Interference
9.
Protein Expr Purif ; 121: 46-51, 2016 May.
Article in English | MEDLINE | ID: mdl-26773743

ABSTRACT

Thioredoxins (Trxs) regulate the activity of target proteins in the chloroplast redox regulatory system. In vivo, a disulfide bond within Trxs is reduced by photochemically generated electrons via ferredoxin (Fd) and ferredoxin-thioredoxin reductase (FTR: EC 1.8.7.2). FTR is an αß-heterodimer, and the ß-subunit has a 4Fe-4S cluster that is indispensable for the electron transfer from Fd to Trxs. Reconstitution of the light-dependent Fd/Trx system, including FTR, is required for the biochemical characterization of the Trx-dependent reduction pathway in the chloroplasts. In this study, we generated functional FTR by simultaneously expressing FTR-α and -ß subunits under the control of tandem T7 promoters in Escherichia coli, and purifying the resulting FTR complex protein. The purified FTR complex exhibited spectroscopic absorption at 410 nm, indicating that it contained the Fe-S cluster. Modification of the expression system and simplification of the purification steps resulted in improved FTR complex yields compared to those obtained in previous studies. Furthermore, the light-dependent Trx-reduction system was reconstituted by using Fd, the purified FTR, and intact thylakoids.


Subject(s)
Chloroplast Thioredoxins/genetics , Ferredoxins/genetics , Iron-Sulfur Proteins/biosynthesis , Oxidoreductases/biosynthesis , Chloroplast Thioredoxins/chemistry , Chloroplast Thioredoxins/metabolism , Chloroplasts/chemistry , Chloroplasts/metabolism , Electron Transport , Ferredoxins/chemistry , Ferredoxins/metabolism , Iron-Sulfur Proteins/genetics , Light , Oxidation-Reduction , Oxidoreductases/genetics , Photosynthesis , Spinacia oleracea/enzymology
10.
Protein Expr Purif ; 118: 77-82, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26494602

ABSTRACT

Specific antibodies are a reliable tool to examine protein expression patterns and to determine the protein localizations within cells. Generally, recombinant proteins are used as antigens for specific antibody production. However, recombinant proteins from mammals and plants are often overexpressed as insoluble inclusion bodies in Escherichia coli. Solubilization of these inclusion bodies is desirable because soluble antigens are more suitable for injection into animals to be immunized. Furthermore, highly purified proteins are also required for specific antibody production. Plastidic acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) from Arabidopsis thaliana, which catalyzes the formation of malonyl-CoA from acetyl-CoA in chloroplasts, formed inclusion bodies when the recombinant protein was overexpressed in E. coli. To obtain the purified protein to use as an antigen, we applied preparative disk gel electrophoresis for protein purification from inclusion bodies. This method is suitable for antigen preparation from inclusion bodies because the purified protein is recovered as a soluble fraction in electrode running buffer containing 0.1% sodium dodecyl sulfate that can be directly injected into immune animals, and it can be used for large-scale antigen preparation (several tens of milligrams).


Subject(s)
Acetyl-CoA Carboxylase/isolation & purification , Antigens, Plant/isolation & purification , Arabidopsis Proteins/isolation & purification , Arabidopsis/enzymology , Electrophoresis, Polyacrylamide Gel/methods , Inclusion Bodies/chemistry , Acetyl-CoA Carboxylase/genetics , Acetyl-CoA Carboxylase/metabolism , Antigens, Plant/genetics , Antigens, Plant/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Inclusion Bodies/genetics , Inclusion Bodies/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
11.
Anal Biochem ; 486: 51-3, 2015 Oct 01.
Article in English | MEDLINE | ID: mdl-26133399

ABSTRACT

Seamless ligation cloning extract (SLiCE) is a simple and efficient method for DNA cloning without the use of restriction enzymes. Instead, SLiCE uses homologous recombination activities from Escherichia coli cell lysates. To date, SLiCE preparation has been performed using an expensive commercially available lytic reagent. To expand the utility of the SLiCE method, we evaluated different methods for SLiCE preparation that avoid using this reagent. Consequently, cell extracts prepared with buffers containing Triton X-100, which is a common and low-cost nonionic detergent, exhibited sufficient cloning activity for seamless gene incorporation into a vector.


Subject(s)
Cloning, Molecular/methods , Escherichia coli/genetics , DNA, Bacterial/genetics , Escherichia coli/cytology
12.
Protein Expr Purif ; 101: 152-6, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25017253

ABSTRACT

Plant redox-related proteins were overexpressed using a genetic codon substitution downstream of the translation initiation codon. This method significantly improved recombinant protein expression levels of Arabidopsis chloroplastic thioredoxins and cytosolic nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (E.C. 1.8.1.9) in Escherichia coli. Using these proteins, the in vitro chloroplastic thioredoxins-reduction system was reconstituted in an NADPH-dependent manner. This system could convert the five classes of chloroplastic Arabidopsis thioredoxins and two chloroplastic Spinach thioredoxins to their reduced forms, independent of dithiothreitol and the photosynthetic electron transport system.


Subject(s)
Arabidopsis/enzymology , Chloroplast Thioredoxins/genetics , Spinacia oleracea/enzymology , Thioredoxin-Disulfide Reductase/genetics , Amino Acid Sequence , Arabidopsis/chemistry , Base Sequence , Chloroplast Thioredoxins/biosynthesis , Chloroplast Thioredoxins/chemistry , Electron Transport/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Oxidation-Reduction , Photosynthesis/physiology , Plant Proteins/genetics , Recombinant Proteins/genetics , Thioredoxin-Disulfide Reductase/biosynthesis , Thioredoxin-Disulfide Reductase/chemistry
13.
Plant Cell Physiol ; 54(9): 1525-34, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23872270

ABSTRACT

In Arabidopsis thaliana, the main route of cyclic electron transport around PSI is sensitive to antimycin A, but the site of inhibition has not been clarified. We discovered that ferredoxin-dependent plastoquinone reduction in ruptured chloroplasts was less sensitive to antimycin A in Arabidopsis that overaccumulated PGR5 (PROTON GRADIENT REGULATION 5) originating from Pinus taeda (PtPGR5) than that in the wild type. Consistent with this in vitro observation, infiltration of antimycin A reduced PSII yields and the non-photochemical quenching (NPQ) of Chl fluorescence in wild-type leaves but not in leaves accumulating PtPGR5. There are eight amino acid differences between PGR5 of Arabidopsis (AtPGR5) and PtPGR5 in their mature forms. To determine the site conferring antimycin A resistance, a series of AtPGR5 and PtPGR5 variants was introduced into the Arabidopsis pgr5 mutant. We determined that the presence of lysine rather than valine at the third amino acid position was necessary and sufficient for resistance to antimycin A. High levels of resistance to antimycin A required overaccumulation of PtPGR5 in ruptured chloroplasts, suggesting that PtPGR5 is partly resistant to antimycin A. In contrast, PSII yield was almost fully resistant to antimycin A in leaves accumulating endogenous levels of PtPGR5 or AtPGR5 V3K that had lysine instead of valine at the third position. NPQ was also dramatically recovered in leaves of these lines. These results imply that partial recovery of PSI cyclic electron transport is sufficient for maintaining redox homeostasis in photosynthesis. Our discovery suggests that antimycin A inhibits the function of PGR5 or proteins localized close to PGR5.


Subject(s)
Amino Acid Substitution , Antimycin A/pharmacology , Arabidopsis Proteins/genetics , Drug Resistance/genetics , Photosynthetic Reaction Center Complex Proteins/genetics , Photosystem I Protein Complex/genetics , Amino Acid Sequence , Antifungal Agents/pharmacology , Arabidopsis Proteins/metabolism , Chlorophyll/metabolism , Chloroplasts/drug effects , Chloroplasts/genetics , Chloroplasts/metabolism , Electron Transport/drug effects , Genetic Complementation Test , Immunoblotting , Molecular Sequence Data , Mutation , Photosynthetic Reaction Center Complex Proteins/metabolism , Photosystem I Protein Complex/metabolism , Photosystem II Protein Complex/genetics , Photosystem II Protein Complex/metabolism , Pinus taeda/genetics , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified , Sequence Homology, Amino Acid
14.
Plant Cell Physiol ; 53(12): 2117-26, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23161858

ABSTRACT

The PGR5 (PROTON GRADIENT REGULATION 5) gene that is required for PSI cyclic electron transport in Arabidopsis was knocked down in rice (Oryza sativa). In three PGR5 knockdown (KD) lines, the PGR5 protein level was reduced to 5-8% of that in the wild type, resulting in a 50% reduction in PGRL1 (PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE 1) protein levels. In ruptured chloroplasts, ferredoxin-dependent plastoquinone reduction activity was partially impaired; the phenotype was mimicked by addition of antimycin A to wild-type chloroplasts. As occurred in the Arabidopsis pgr5 mutant, non-photochemical quenching of Chl fluorescence (NPQ) induction was impaired in the leaves, but the electron transport rate (ETR) was only mildly affected at high light intensity. The P700(+) level was reduced even at low light intensity, suggesting that the PGR5 function was severely disturbed as in the Arabidopsis pgr5 mutant and that the other alternative routes of electrons could not compensate the stromal redox balance. The amplitude of the light-dark electrochromic shift (ECS) signal (ECSt), which reflects the total size of the proton motive force in steady-state photosynthesis, was reduced by 13-25% at approximately the growth light intensity. The CO(2) fixation rate was only slightly reduced in the PGR5 KD lines. Despite the drastic reduction in NPQ and P700(+) levels, total biomass was only slightly reduced in PGR5 KD lines grown at 370 µmol photons m(-2) s(-1). These results suggest that CO(2) fixation and growth rate are very robust in the face of alterations in the fundamental reactions of photosynthesis under constant light conditions in rice.


Subject(s)
Carbon Dioxide/metabolism , Oryza/metabolism , Photosystem I Protein Complex/metabolism , Plant Proteins/metabolism , Antimycin A/pharmacology , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Biomass , Chlorophyll/metabolism , Chloroplasts/drug effects , Chloroplasts/metabolism , Chloroplasts/radiation effects , Electron Transport/radiation effects , Ferredoxins/metabolism , Gene Knockdown Techniques , Homeostasis/radiation effects , Light , Oryza/genetics , Oryza/growth & development , Oryza/radiation effects , Oxidation-Reduction/radiation effects , Phenotype , Photosynthesis/physiology , Photosynthetic Reaction Center Complex Proteins/genetics , Photosynthetic Reaction Center Complex Proteins/metabolism , Photosystem I Protein Complex/radiation effects , Plant Leaves/genetics , Plant Leaves/growth & development , Plant Leaves/metabolism , Plant Leaves/radiation effects , Plant Proteins/genetics , Plant Transpiration/radiation effects , Plants, Genetically Modified , Plastoquinone/metabolism
15.
Plant J ; 63(3): 458-68, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20497376

ABSTRACT

In addition to linear electron transport from water to NADP(+) , alternative electron transport pathways are believed to regulate photosynthesis. In the two routes of photosystem I (PSI) cyclic electron transport, electrons are recycled from the stromal reducing pool to plastoquinone (PQ), generating additional ΔpH (proton gradient across thylakoid membranes). Plastid terminal oxidase (PTOX) accepts electrons from PQ and transfers them to oxygen to produce water. Although both electron transport pathways share the PQ pool, it is unclear whether they interact in vivo. To investigate the physiological link between PSI cyclic electron transport-dependent PQ reduction and PTOX-dependent PQ oxidation, we characterized mutants defective in both functions. Impairment of PSI cyclic electron transport suppressed leaf variegation in the Arabidopsis immutans (im) mutant, which is defective in PTOX. The im variegation was more effectively suppressed in the pgr5 mutant, which is defective in the main pathway of PSI cyclic electron transport, than in the crr2-2 mutant, which is defective in the minor pathway. In contrast to this chloroplast development phenotype, the im defect alleviated the growth phenotype of the crr2-2 pgr5 double mutant. This was accompanied by partial suppression of stromal over-reduction and restricted linear electron transport. We discuss the function of the alternative electron transport pathways in both chloroplast development and photosynthesis in mature leaves.


Subject(s)
Arabidopsis/physiology , Plastoquinone/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Electron Transport , Mutation , Oxidation-Reduction , Photosynthesis , Plant Leaves/metabolism , Plant Leaves/physiology
16.
Front Plant Sci ; 12: 530376, 2021.
Article in English | MEDLINE | ID: mdl-33664754

ABSTRACT

The chloroplast-localized cystathionine ß-synthase X (CBSX) proteins CBSX1 and CBSX2 have been proposed as modulators of thioredoxins (Trxs). In this study, the contribution of CBSX proteins to the redox regulation of thiol enzymes in the chloroplast Trx system was evaluated both in vitro and in vivo. The in vitro biochemical studies evaluated whether CBSX proteins alter the specificities of classical chloroplastic Trx f and Trx m for their target proteins. However, addition of CBSX proteins did not alter the specificities of Trx f and Trx m for disulfide bond reduction of the photosynthesis-related major thiol enzymes, FBPase, SBPase, and NADP-MDH. In vivo analysis showed that CBSX-deficient mutants grew similarly to wild type plants under continuous normal light conditions and that CBSX deficiency did not affect photo-reduction of photosynthesis-related thiol enzymes by Trx system at several light intensities. Although CBSX proteins have been suggested as modulators in the chloroplast Trx system, our results did not support this model, at least in the cases of FBPase, SBPase, and NADP-MDH in leaves. However, fresh weights of the cbsx2 mutants were decreased under short day. Since Trxs regulate many proteins participating in various metabolic reactions in the chloroplast, CBSX proteins may function to regulate other chloroplast Trx target proteins, or serve as modulators in non-photosynthetic plastids of flowers. As a next stage, further investigations are required to understand the modulation of Trx-dependent redox regulation by plastidal CBSX proteins.

17.
iScience ; 24(2): 102059, 2021 Feb 19.
Article in English | MEDLINE | ID: mdl-33554065

ABSTRACT

In natural habitats, plants have developed sophisticated regulatory mechanisms to optimize the photosynthetic electron transfer rate at the maximum efficiency and cope with the changing environments. Maintaining proper P700 oxidation at photosystem I (PSI) is the common denominator for most regulatory processes of photosynthetic electron transfers. However, the molecular complexes and cofactors involved in these processes and their function(s) have not been fully clarified. Here, we identified a redox-active chloroplast protein, the triplet-cysteine repeat protein (TCR). TCR shared similar expression profiles with known photosynthetic regulators and contained two triplet-cysteine motifs (CxxxCxxxC). Biochemical analysis indicated that TCR localizes in chloroplasts and has a [3Fe-4S] cluster. Loss of TCR limited the electron sink downstream of PSI during dark-to-light transition. Arabidopsis pgr5-tcr double mutant reduced growth significantly and showed unusual oxidation and reduction of plastoquinone pool. These results indicated that TCR is involved in electron flow(s) downstream of PSI, contributing to P700 oxidation.

18.
Biochim Biophys Acta Bioenerg ; 1861(11): 148261, 2020 11 01.
Article in English | MEDLINE | ID: mdl-32659266

ABSTRACT

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.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Chloroplast Proton-Translocating ATPases/chemistry , Chloroplast Proton-Translocating ATPases/metabolism , Chloroplast Thioredoxins/metabolism , Chloroplasts/metabolism , Photosynthesis , Electron Transport , Kinetics , Oxidation-Reduction , Protein Isoforms
19.
Plants (Basel) ; 9(10)2020 Oct 15.
Article in English | MEDLINE | ID: mdl-33076473

ABSTRACT

Plants have a high regeneration capacity and some plant species can regenerate clone plants, called plantlets, from detached vegetative organs. We previously outlined the molecular mechanisms underlying plantlet regeneration from Rorippa aquatica (Brassicaceae) leaf explants. However, the fundamental difference between the plant species that can and cannot regenerate plantlets from vegetative organs remains unclear. Here, we hypothesized that the viability of leaf explants is a key factor affecting the regeneration capacity of R. aquatica. To test this hypothesis, the viability of R. aquatica and Arabidopsis thaliana leaf explants were compared, with respect to the maintenance of photosynthetic activity, senescence, and immune response. Time-course analyses of photosynthetic activity revealed that R. aquatica leaf explants can survive longer than those of A. thaliana. Endogenous abscisic acid (ABA) and jasmonic acid (JA) were found at low levels in leaf explant of R. aquatica. Time-course transcriptome analysis of R. aquatica and A. thaliana leaf explants suggested that senescence was suppressed at the transcriptional level in R. aquatica. Application of exogenous ABA reduced the efficiency of plantlet regeneration. Overall, our results propose that in nature, plant species that can regenerate in nature can survive for a long time.

20.
Plant Cell Physiol ; 49(5): 825-34, 2008 May.
Article in English | MEDLINE | ID: mdl-18388110

ABSTRACT

PSI cyclic electron transport is essential for photosynthesis and photoprotection. In higher plants, the antimycin A-sensitive pathway is the main route of electrons in PSI cyclic electron transport. Although a small thylakoid protein, PGR5 (PROTON GRADIENT REGULATION 5), is essential for this pathway, its function is still unclear, and there are numerous debates on the rate of electron transport in vivo and its regulation. To assess how PGR5-dependent PSI cyclic electron transport is regulated in vivo, we characterized its activity in ruptured chloroplasts isolated from Arabidopsis thaliana. The activity of ferredoxin (Fd)-dependent plastoquinone (PQ) reduction in the dark is impaired in the pgr5 mutant. Alkalinization of the reaction medium enhanced the activity of Fd-dependent PQ reduction in the wild type. Even weak actinic light (AL) illumination also markedly activated PGR5-dependent PSI cyclic electron transport in ruptured chloroplasts. Even in the presence of linear electron transport [11 mumol O2 (mg Chl)(-1) h(-1)], PGR5-dependent PSI electron transport was detected as a difference in Chl fluorescence levels in ruptured chloroplasts. In the wild type, PGR5-dependent PSI cyclic electron transport competed with NADP+ photoreduction. These results suggest that the rate of PGR5-dependent PSI cyclic electron transport is high enough to balance the production ratio of ATP and NADPH during steady-state photosynthesis, consistently with the pgr5 mutant phenotype. Our results also suggest that the activity of PGR5-dependent PSI cyclic electron transport is regulated by the redox state of the NADPH pool.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Chloroplasts/metabolism , Photosynthetic Reaction Center Complex Proteins/metabolism , Photosystem I Protein Complex/metabolism , Chloroplasts/radiation effects , Electron Transport/radiation effects , Hydrogen-Ion Concentration/radiation effects , Light , NADP/metabolism , Oxygen/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL