Interplay of starch debranching enzyme and its inhibitor is mediated by Redox-Activated SPL transcription factor.

The germination process is of central importance across the cultivated species involving several key enzymes for mobilization of stored food reserves. Pullulanase (PUL), a starch-debranching enzyme, plays an important role in mobilizing stored endosperm food reserves during germination. Pullulanase inhibitor (PULI) hinders PUL's activity through an unknown mechanism. Barley has one PUL and two PULI genes. During the time-dependent processes of seed germination, only PULI-1 expression shows an antagonistic relationship with that of PUL. Our data have indicated that the expression of PULI-1 is modulated by SPL (Squamosa-promoter-binding Protein Like) transcription factors, known to be targeted by miR156. We show that the binding of recombinant HvSPL3 protein to the PULI-1 promoter occurs under reducing, but not under oxidizing conditions. Replacement of Cys residues with threonine in HvSPL3 abolishes the binding, indicating an essential role of the redox state in the expression of PULI. Our findings may have important implications for the industrial use of starch.

Unexpected diversity of ferredoxin-dependent thioredoxin reductases in cyanobacteria.

Thioredoxin reductases control the redox state of thioredoxins (Trxs)-ubiquitous proteins that regulate a spectrum of enzymes by dithiol-disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. We have recently found that some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack NTR and FTR but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeply-rooted thioredoxin reductase or DTR), whose electron donor remained undefined. Here, we demonstrate that Fdx functions in this capacity and report the crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. Thereby, our data demonstrate that this cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. Our experiments further show that the redox-sensitive peptide CP12 is modulated in vitro by the FFTR/Trx system, demonstrating that FFTR functionally substitutes for FTR in light-linked enzyme regulation in Gloeobacter. Altogether, we demonstrate the FFTR is spread within the cyanobacteria phylum and propose that, by substituting for FTR, it connects the reduction of target proteins to photosynthesis. Besides, the results indicate that FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments.

Evolution of the thioredoxin system as a step enabling adaptation to oxidative stress.

Thioredoxins (Trxs) are low-molecular-weight proteins that participate in the reduction of target enzymes. Trxs contain a redox-active disulfide bond, in the form of a WCGPC amino acid sequence motif, that enables them to perform dithiol-disulfide exchange reactions with oxidized protein substrates. Widely distributed across the three domains of life, Trxs form an evolutionarily conserved family of ancient origin. Thioredoxin reductases (TRs) are enzymes that reduce Trxs. According to their evolutionary history, TRs have diverged, thereby leading to the emergence of variants of the enzyme that in combination with different types of Trxs meet the needs of the cell. In addition to participating in the regulation of metabolism and defense against oxidative stress, Trxs respond to environmental signals-an ability that developed early in evolution. Redox regulation of proteins targeted by Trx is accomplished with a pair of redox-active cysteines located in strategic positions on the polypeptide chain to enable reversible oxidative changes that result in structural and functional modifications target proteins. In this review, we present a general overview of the thioredoxin system and describe recent structural studies on the diversity of its components.

Ferredoxin-linked flavoenzyme defines a family of pyridine nucleotide-independent thioredoxin reductases.

Ferredoxin-dependent thioredoxin reductase was identified 35 y ago in the fermentative bacterium Clostridium pasteurianum [Hammel KE, Cornwell KL, Buchanan BB (1983) Proc Natl Acad Sci USA 80:3681-3685]. The enzyme, a flavoprotein, was strictly dependent on ferredoxin as reductant and was inactive with either NADPH or NADH. This early work has not been further pursued. We have recently reinvestigated the problem and confirmed that the enzyme, here designated ferredoxin-dependent flavin thioredoxin reductase (FFTR), is a flavoprotein. The enzyme differs from ferredoxin-thioredoxin reductase (FTR), which has a signature [4Fe-4S] cluster, but shows structural similarities to NADP-dependent thioredoxin reductase (NTR). Comparative amino acid sequence analysis showed that FFTR is present in a number of clostridial species, some of which lack both FTR and an archetypal NTR. We have isolated, crystallized, and determined the structural properties of FFTR from a member of this group, Clostridium acetobutylicum, both alone and in complex with Trx. The structures showed an elongated FFTR homodimer, each monomer comprising two Rossmann domains and a noncovalently bound FAD cofactor that exposes the isoalloxazine ring to the solvent. The FFTR structures revealed an alternative domain organization compared with NTR that enables the enzyme to accommodate Fdx rather than NADPH. The results suggest that FFTR exists in a range of conformations with varying degrees of domain separation in solution and that the stacking between the two redox-active groups for the transfer of reducing equivalents results in a profound structural reorganization. A mechanism in accord with the findings is proposed.

Crystal Structure of the Apo-Form of NADPH-Dependent Thioredoxin Reductase from a Methane-Producing Archaeon.

The redox regulation of proteins via reversible dithiol/disulfide exchange reactions involves the thioredoxin system, which is composed of a reductant, a thioredoxin reductase (TR), and thioredoxin (Trx). In the pyridine nucleotide-dependent Trx reduction pathway, reducing equivalents, typically from reduced nicotinamide adenine dinucleotide phosphate (NADPH), are transferred from NADPH-TR (NTR) to Trx and, in turn, to target proteins, thus resulting in the reversible modification of the structural and functional properties of the targets. NTR enzymes contain three functional sites: an NADPH binding pocket, a non-covalently bound flavin cofactor, and a redox-active disulfide in the form of CxxC. With the aim of increasing our knowledge of the thioredoxin system in archaea, we here report the high-resolution crystal structure of NTR from the methane-generating organism Methanosarcina mazei strain Gö1 (MmNTR) at 2.6 Å resolution. Based on the crystals presently described, MmNTR assumes an overall fold that is nearly identical to the archetypal fold of authentic NTRs; however, surprisingly, we observed no electron density for flavin adenine dinucleotide (FAD) despite the well-defined and conserved FAD-binding cavity in the folded module. Remarkably, the dimers of the apo-protein within the crystal were different from those observed by small angle X-ray scattering (SAXS) for the holo-protein, suggesting that the binding of the flavin cofactor does not require major protein structural rearrangements. Rather, binding results in the stabilization of essential parts of the structure, such as those involved in dimer stabilization. Altogether, this structure represents the example of an apo-form of an NTR that yields important insight into the effects of the cofactor on protein folding.

Unprecedented pathway of reducing equivalents in a diflavin-linked disulfide oxidoreductase.

Flavoproteins participate in a wide variety of physiologically relevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FAD molecules per monomer in redox communication with an active disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein "DDOR" (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-based transfer of reducing equivalents in bacterial membranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.

The Arnon-Buchanan cycle: a retrospective, 1966-2016.

For the first decade following its description in 1954, the Calvin-Benson cycle was considered the sole pathway of autotrophic CO2 assimilation. In the early 1960s, experiments with fermentative bacteria uncovered reactions that challenged this concept. Ferredoxin was found to donate electrons directly for the reductive fixation of CO2 into alpha-keto acids via reactions considered irreversible. Thus, pyruvate and alpha-ketoglutarate could be synthesized from CO2, reduced ferredoxin and acetyl-CoA or succinyl-CoA, respectively. This work opened the door to the discovery that reduced ferredoxin could drive the Krebs citric acid cycle in reverse, converting the pathway from its historical role in carbohydrate breakdown to one fixing CO2. Originally uncovered in photosynthetic green sulfur bacteria, the Arnon-Buchanan cycle has since been divorced from light and shown to function in a variety of anaerobic chemoautotrophs. In this retrospective, colleagues who worked on the cycle at its inception in 1966 and those presently working in the field trace its development from a controversial reception to its present-day inclusion in textbooks. This pathway is now well established in major groups of chemoautotrophic bacteria, instead of the Calvin-Benson cycle, and is increasingly referred to as the Arnon-Buchanan cycle. In this retrospective, separate sections have been written by the authors indicated. Bob Buchanan wrote the abstract and the concluding comments.

The Path to Thioredoxin and Redox Regulation Beyond Chloroplasts.

Once the ferredoxin/thioredoxin system was established as a mechanism linking light to the post-translational regulation of chloroplast enzymes, I considered that plants might harbor a light-independent mechanism utilizing this same enzyme chemistry based on thiol-disulfide redox transitions. After reflection, it occurred to me that such a mechanism could be fundamental to seeds of cereals that undergo dramatic change following exposure to oxygen during maturation and drying. The pursuit of this idea led to the discovery of a family of extraplastidic thioredoxins, designated the h-type, that resemble animal and bacterial counterparts in undergoing enzymatic reduction with NADPH. Current evidence suggests that h-type thioredoxins function broadly throughout the plant. Here I describe how the thioredoxin h field developed, its current status and potential for contributing material benefits to society.

Redox-dependent interaction between thaumatin-like protein and ß-glucan influences malting quality of barley.

Barley is the cornerstone of the malting and brewing industry. It is known that 250 quantitative trait loci (QTLs) of the grain are associated with 19 malting-quality phenotypes. However, only a few of the contributing genetic components have been identified. One of these, on chromosome 4H, contains a major malting QTL, QTL2, located near the telomeric region that accounts, respectively, for 28.9% and 37.6% of the variation in the ß-glucan and extract fractions of malt. In the current study, we dissected the QTL2 region using an expression- and microsynteny-based approach. From a set of 22 expressed sequence tags expressed in seeds at the malting stage, we identified a candidate gene, TLP8 (thaumatin-like protein 8), which was differentially expressed and influenced malting quality. Transcript abundance and protein profiles of TLP8 were studied in different malt and feed varieties using quantitative PCR, immunoblotting, and enzyme-linked immunosorbent assay (ELISA). The experiments demonstrated that TLP8 binds to insoluble (1, 3, 1, 4)-ß-D glucan in grain extracts, thereby facilitating the removal of this undesirable polysaccharide during malting. Further, the binding of TLP8 to ß-glucan was dependent on redox. These findings represent a stride forward in our understanding of the malting process and provide a foundation for future improvements in the final beer-making process.

Chloroplast FBPase and SBPase are thioredoxin-linked enzymes with similar architecture but different evolutionary histories.

The Calvin-Benson cycle of carbon dioxide fixation in chloroplasts is controlled by light-dependent redox reactions that target specific enzymes. Of the regulatory members of the cycle, our knowledge of sedoheptulose-1,7-bisphosphatase (SBPase) is particularly scanty, despite growing evidence for its importance and link to plant productivity. To help fill this gap, we have purified, crystallized, and characterized the recombinant form of the enzyme together with the better studied fructose-1,6-bisphosphatase (FBPase), in both cases from the moss Physcomitrella patens (Pp). Overall, the moss enzymes resembled their counterparts from seed plants, including oligomeric organization-PpSBPase is a dimer, and PpFBPase is a tetramer. The two phosphatases showed striking structural homology to each other, differing primarily in their solvent-exposed surface areas in a manner accounting for their specificity for seven-carbon (sedoheptulose) and six-carbon (fructose) sugar bisphosphate substrates. The two enzymes had a similar redox potential for their regulatory redox-active disulfides (-310 mV for PpSBPase vs. -290 mV for PpFBPase), requirement for Mg(2+) and thioredoxin (TRX) specificity (TRX f > TRX m). Previously known to differ in the position and sequence of their regulatory cysteines, the enzymes unexpectedly showed unique evolutionary histories. The FBPase gene originated in bacteria in conjunction with the endosymbiotic event giving rise to mitochondria, whereas SBPase arose from an archaeal gene resident in the eukaryotic host. These findings raise the question of how enzymes with such different evolutionary origins achieved structural similarity and adapted to control by the same light-dependent photosynthetic mechanism-namely ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxin.

The Path to Thioredoxin and Redox Regulation in Chloroplasts.

After a brief discussion of my graduate work at Duke University, I describe a series of investigations on redox proteins at the University of California, Berkeley. Starting with ferredoxin from fermentative bacteria, the Berkeley research fostered experiments that uncovered a pathway for fixing CO2 in bacterial photosynthesis. The carbon work, in turn, opened new vistas, including the discovery that thioredoxin functions universally in regulating the Calvin-Benson cycle in oxygenic photosynthesis. These experiments, which took place over a 50-year period, led to the formulation of a set of biological principles and set the stage for research demonstrating a role for redox in the regulation of previously unrecognized processes extending far beyond photosynthesis.

Seed thioredoxin h.

Thioredoxins are nearly ubiquitous disulfide reductases involved in a wide range of biochemical pathways in various biological systems, and also implicated in numerous biotechnological applications. Plants uniquely synthesize an array of thioredoxins targeted to different cell compartments, for example chloroplastic f- and m-type thioredoxins involved in regulation of the Calvin-Benson cycle. The cytosolic h-type thioredoxins act as key regulators of seed germination and are recycled by NADPH-dependent thioredoxin reductase. The present review on thioredoxin h systems in plant seeds focuses on occurrence, reaction mechanisms, specificity, target protein identification, three-dimensional structure and various applications. The aim is to provide a general background as well as an update covering the most recent findings. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

The carbon (formerly dark) reactions of photosynthesis.

In this brief account, I describe the background for dividing photosynthesis into "light" and "dark" reactions and show how this concept changed to "light" and "carbon" reactions as science in the field advanced.

Andrew A. Benson, 1917-2015.

UNLABELLED: On January 16, 2015, Professor Andrew Alm Benson, one of the leading plant biochemists of the twentieth century, died in La Jolla, California, at the age of 97; he was born on September 24, 1917. Benson was known especially for his pioneering studies on photosynthesis (CO2 assimilation, carbon reduction cycle) and plant lipids (phospholipid phosphatidyl glycerol; and the sulfolipid, sulfoquinovosyl diglyceride). A photograph of Benson is shown in Fig. 1. Fig. 1 Photograph of Andrew A. Benson. SOURCE: Annual Review of Plant Biology, Vol. 53, 2002, published with permission.

Thioredoxin, a master regulator of the tricarboxylic acid cycle in plant mitochondria.

Plant mitochondria have a fully operational tricarboxylic acid (TCA) cycle that plays a central role in generating ATP and providing carbon skeletons for a range of biosynthetic processes in both heterotrophic and photosynthetic tissues. The cycle enzyme-encoding genes have been well characterized in terms of transcriptional and effector-mediated regulation and have also been subjected to reverse genetic analysis. However, despite this wealth of attention, a central question remains unanswered: "What regulates flux through this pathway in vivo?" Previous proteomic experiments with Arabidopsis discussed below have revealed that a number of mitochondrial enzymes, including members of the TCA cycle and affiliated pathways, harbor thioredoxin (TRX)-binding sites and are potentially redox-regulated. We have followed up on this possibility and found TRX to be a redox-sensitive mediator of TCA cycle flux. In this investigation, we first characterized, at the enzyme and metabolite levels, mutants of the mitochondrial TRX pathway in Arabidopsis: the NADP-TRX reductase a and b double mutant (ntra ntrb) and the mitochondrially located thioredoxin o1 (trxo1) mutant. These studies were followed by a comparative evaluation of the redistribution of isotopes when (13)C-glucose, (13)C-malate, or (13)C-pyruvate was provided as a substrate to leaves of mutant or WT plants. In a complementary approach, we evaluated the in vitro activities of a range of TCA cycle and associated enzymes under varying redox states. The combined dataset suggests that TRX may deactivate both mitochondrial succinate dehydrogenase and fumarase and activate the cytosolic ATP-citrate lyase in vivo, acting as a direct regulator of carbon flow through the TCA cycle and providing a mechanism for the coordination of cellular function.

PSB27: A thylakoid protein enabling Arabidopsis to adapt to changing light intensity.

In earlier studies we have identified FKBP20-2 and CYP38 as soluble proteins of the chloroplast thylakoid lumen that are required for the formation of photosystem II supercomplexes (PSII SCs). Subsequent work has identified another potential candidate functional in SC formation (PSB27). We have followed up on this possibility and isolated mutants defective in the PSB27 gene. In addition to lack of PSII SCs, mutant plants were severely stunted when cultivated with light of variable intensity. The stunted growth was associated with lower PSII efficiency and defective starch accumulation. In response to high light exposure, the mutant plants also displayed enhanced ROS production, leading to decreased biosynthesis of anthocyanin. Unexpectedly, we detected a second defect in the mutant, namely in CP26, an antenna protein known to be required for the formation of PSII SCs that has been linked to state transitions. Lack of PSII SCs was found to be independent of PSB27, but was due to a mutation in the previously described cp26 gene that we found had no effect on light adaptation. The present results suggest that PSII SCs, despite being required for state transitions, are not associated with acclimation to changing light intensity. Our results are consistent with the conclusion that PSB27 plays an essential role in enabling plants to adapt to fluctuating light intensity through a mechanism distinct from photosystem II supercomplexes and state transitions.

Andrew Benson honored on birthday № 97.

We present a brief account of the 97th birthday celebration of Andrew A. Benson, a scientific legend who is known, among other contributions, for his pioneering work on the path of carbon in photosynthesis (the Calvin-Benson cycle).

Thioredoxin targets fundamental processes in a methane-producing archaeon, Methanocaldococcus jannaschii.

Thioredoxin (Trx), a small redox protein, controls multiple processes in eukaryotes and bacteria by changing the thiol redox status of selected proteins. The function of Trx in archaea is, however, unexplored. To help fill this gap, we have investigated this aspect in methanarchaea--strict anaerobes that produce methane, a fuel and greenhouse gas. Bioinformatic analyses suggested that Trx is nearly universal in methanogens. Ancient methanogens that produce methane almost exclusively from H2 plus CO2 carried approximately two Trx homologs, whereas nutritionally versatile members possessed four to eight. Due to its simplicity, we studied the Trx system of Methanocaldococcus jannaschii--a deeply rooted hyperthermophilic methanogen growing only on H2 plus CO2. The organism carried two Trx homologs, canonical Trx1 that reduced insulin and accepted electrons from Escherichia coli thioredoxin reductase and atypical Trx2. Proteomic analyses with air-oxidized extracts treated with reduced Trx1 revealed 152 potential targets representing a range of processes--including methanogenesis, biosynthesis, transcription, translation, and oxidative response. In enzyme assays, Trx1 activated two selected targets following partial deactivation by O2, validating proteomics observations: methylenetetrahydromethanopterin dehydrogenase, a methanogenesis enzyme, and sulfite reductase, a detoxification enzyme. The results suggest that Trx assists methanogens in combating oxidative stress and synchronizing metabolic activities with availability of reductant, making it a critical factor in the global carbon cycle and methane emission. Because methanogenesis developed before the oxygenation of Earth, it seems possible that Trx functioned originally in metabolic regulation independently of O2, thus raising the question whether a complex biological system of this type evolved at least 2.5 billion years ago.

Evolutionary development of redox regulation in chloroplasts.

SIGNIFICANCE: The post-translational modification of thiol groups stands out as a key strategy that cells employ for metabolic regulation and adaptation to changing environmental conditions. Nowhere is this more evident than in chloroplasts-the O2-evolving photosynthetic organelles of plant cells that are fitted with multiple redox systems, including the thioredoxin (Trx) family of oxidoreductases functional in the reversible modification of regulatory thiols of proteins in all types of cells. The best understood member of this family in chloroplasts is the ferredoxin-linked thioredoxin system (FTS) by which proteins are modified via light-dependent disulfide/dithiol (S-S/2SH) transitions. RECENT ADVANCES: Discovered in the reductive activation of enzymes of the Calvin-Benson cycle in illuminated chloroplast preparations, recent studies have extended the role of the FTS far beyond its original boundaries to include a spectrum of cellular processes. Together with the NADP-linked thioredoxin reductase C-type (NTRC) and glutathione/glutaredoxin systems, the FTS also plays a central role in the response of chloroplasts to different types of stress. CRITICAL ISSUES: The comparisons of redox regulatory networks functional in chloroplasts of land plants with those of cyanobacteria-prokaryotes considered to be the ancestors of chloroplasts-and different types of algae summarized in this review have provided new insight into the evolutionary development of redox regulation, starting with the simplest O2-evolving organisms. FUTURE DIRECTIONS: The evolutionary appearance, mode of action, and specificity of the redox regulatory systems functional in chloroplasts, as well as the types of redox modification operating under diverse environmental conditions stand out as areas for future study.