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.

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.

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.

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.

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 non-covalently 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.

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 FADmolecules 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.

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.

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.

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 β-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)-β-D glucan in grain extracts, thereby facilitating the removal of this undesirable polysaccharide during malting. Further, the binding of TLP8 to β-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.

Thioredoxins (Trxs) are key components of the redox system that regulates the activity of a spectrum of target proteins through dithiol-disulfide exchange reactions. Trxs are reduced by members of the Trx reductase (TR) family (Jacquot et al., 2009Jacquot J.-P. Eklund H. Rouhier N. Schürmann P. Structural and evolutionary aspects of thioredoxin reductases in photosynthetic organisms.Trends Plant Sci. 2009; 14: 336-343Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). NADP-dependent thioredoxin reductases (NTRs), the most common type, belong to the family of dimeric pyridine nucleotide disulfide oxidoreductase flavoproteins that use NADPH as the source of reducing equivalents. In oxyphotosynthetic organisms, in particular, NTRs coexist with the ferredoxin/thioredoxin system (FTS), composed of ferredoxin (Fdx), ferredoxin:thioredoxin reductase (FTR), and a Trx. FTRs convert the electron signal obtained from photoreduced Fdx to a thiol signal via a 4Fe-4S center and a redox-active disulfide catalytic center. FTR, in turn, reduces Trx. In cyanobacteria and chloroplasts, the FTS is closely associated with the regulation of enzymes of the Calvin-Benson cycle and associated processes; e.g., the oxidative pentose phosphate pathway (Balsera et al., 2014Balsera M. Uberegui E. Schürmann P. Buchanan B.B. Evolutionary development of redox regulation in chloroplasts.Antioxid. Redox Signal. 2014; 21: 1327-1355Crossref PubMed Scopus (70) Google Scholar). In contrast to other oxygenic photosynthetic organisms, the ancient cyanobacterium Gloeobacter and the ocean-dwelling green oxyphotobacteria Prochlorococcus lack an FTR gene. This observation raises the question of how these photosynthetic organisms link the Calvin-Benson cycle and related metabolic processes to light and other changing environmental conditions. To gain information on this point, we have conducted a comparative analysis of NTR-like TR protein sequences and identified an enzyme common to Gloeobacter and green oxyphotobacteria that possibly functions in this connection. The enzyme, provisionally named DTR for deeply rooted bacterial thioredoxin reductase, shows high similarity to NTR but exhibits unique structural features (Figure 1). The enzyme is present in other bacteria, including organisms within the deeply rooted bacterial lineages as well as marine algae (Supplemental Table 1 and Supplemental Figure 1). A multiple protein sequence alignment showed that Gloeobacter and Prochlorococcus DTRs harbor the conserved FAD-binding motif and the two active-site Cys residues typical of authentic NTRs (Figure 1A). Major differences were noted, however, in the amino acids responsible for pyridine nucleotide binding (GxGxxA/G and HRRxxR) (Figure 1A). These included substitutions of crucial residues for coordination to the pyrophosphate group and the absence of specific positively charged amino acids needed for electrostatic interactions with the 2′-phosphate group of the adenosine. Additional variations included a C-terminal extension with a conserved aromatic residue and a fused N-terminal redoxin domain that is restricted to a few bacteria (gray box; Supplemental Figure 1). The visible-UV absorption spectrum of purified Gloeobacter DTR (GvDTR) showed features typical of a flavoprotein, including absorption maxima at 391 and 459 nm (continuous black line, Figure 1B) (Prongay and Williams, 1992Prongay A.J. Williams C.H. Oxidation-reduction properties of Escherichia coli thioredoxin reductase altered at each active site cysteine residue.J. Biol. Chem. 1992; 267: 25181-25188Abstract Full Text PDF PubMed Google Scholar). In contrast to NTRs, the Gloeobacter flavoprotein failed to show activity in the assay of the enzyme in which the oxidation of NAD(P)H is coupled to the reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (Figure 1C) (Holmgren and Björnstedt, 1995Holmgren A. Björnstedt M. Thioredoxin and thioredoxin reductase.Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (816) Google Scholar). The apparent lack of activity could be due to either of two reasons. One, NAD(P)H reduces FAD, but electrons are not further shuttled to its homolog Trx and thus to DTNB; nonetheless, the activity of Gloeobacter Trx in the coupled assay was demonstrated with Escherichia coli NTR (Figure 1C). Alternatively, the flavoprotein does not function with NAD(P)H. To explore the second possibility, we applied isothermal titration calorimetry (ITC) as the most direct approach for assessing protein-ligand binding. For these experiments, we included 3-acetylpyridine adenine dinucleotide phosphate (AADP), a non-hydrolyzable analog of NADPH, that binds tightly to NAD(P)-dependent enzymes. In contrast to E. coli NTR (EcNTR), which binds NADP+ and AADP+ with a Kd in the micromolar range, the Gloeobacter protein failed to bind either ligand in vitro (Figure 1D and Supplemental Figure 3). The lack of affinity for pyridine nucleotides resembles TR from the archeon Thermoplasma acidophilum (TaTR; Hernandez et al., 2008Hernandez H. Jaquez O. Hamill M. Elliott S. Drennan C. Thioredoxin reductase from Thermoplasma acidophilum: a new twist on redox regulation.Biochemistry. 2008; 47: 9728-9737Crossref PubMed Scopus (21) Google Scholar), although structural differences were detected between the two proteins. GvDTR displayed a modified GxGxxA/G structural pattern for nucleotide binding (Supplemental Figure 4, red box), and contained a C-terminal extension missing in TaTR (Supplemental Figure 4, green box). To confirm that the Gloeobacter flavoprotein fulfills the function of a TR, we determined whether it could reduce Trx using dithionite as an artificial electron donor. Trx m (also known as TrxA) is the only canonical Trx form in Gloeobacter and Prochlorococcus (Florencio et al., 2006Florencio F. Pérez-Pérez M. López-Maury L. Mata-Cabana A. Lindahl M. The diversity and complexity of the cyanobacterial thioredoxin systems.Photosynth. Res. 2006; 89: 157-171Crossref PubMed Scopus (64) Google Scholar, Balsera et al., 2014Balsera M. Uberegui E. Schürmann P. Buchanan B.B. Evolutionary development of redox regulation in chloroplasts.Antioxid. Redox Signal. 2014; 21: 1327-1355Crossref PubMed Scopus (70) Google Scholar). We observed that dithionite was able to slowly reduce the flavoenzyme anaerobically as measured by a decrease in absorbance at 459 nm (Figure 1B). Partial reoxidation of the enzyme was observed immediately after adding Gloeobacter Trx as seen by recovery of the main features of the visible-UV spectrum. These results provide evidence that the Gloeobacter flavoprotein has TR activity and functions independently of pyridine nucleotides. To gain information on the molecular properties of DTR, we determined a high-resolution structure of the Gloeobacter protein by X-ray crystallography at 1.9 Å resolution (PDB: 5J60; Supplemental Table 2). The crystal structure revealed that GvDTR is a homodimer and that each monomer adopts a typical NTR fold (Figure 1E). Despite the high similarity between the FAD- and pseudo-NADPH-binding domains with those of authentic NTRs, the relative orientation of the domains in the crystal structure differed from the flavin-oxidizing (FO) and flavin-reducing (FR) conformations found in NTRs (Lennon and Williams, 1997Lennon B.W. Williams C.H. Reductive half-reaction of thioredoxin reductase from Escherichia coli.Biochemistry. 1997; 36: 9464-9477Crossref PubMed Scopus (53) Google Scholar) (Supplemental Figure 5). No direct interaction was observed between amino acids of the pseudo-NADPH-binding domains, as detected between the two corresponding domains in the NTR dimer both in FO and FR conformations, suggesting that the two functional domains in DTRs are flexible relative to each other. FAD in GvDTR was found to be oxidized based on the planar conformation and the yellow color of the crystal. The redox-active Cys residues were reduced and distant from the re face of the isoalloxazine ring instrumental for electron transfer (Figure 1E). Coordination to the FAD cofactor is well conserved compared with NTRs of known 3D structure (Supplemental Figure 6). However, analysis of the NADPH-binding pocket revealed significant structural deviations from NTRs (Supplemental Figure 7). One of the most striking variations is related to the substitution of the second strictly conserved Gly in the GxGxxA/G motif in EcNTR by an Asn in GvDTR (Figure 1A, red box). GvDTR structure showed that the substrate binding pocket is blocked by the side chain carboxamide of Asn (N154; Supplemental Figure 7E), supporting the finding that the enzyme does not bind pyridine nucleotides. Remarkably, the aromatic side chain of a tryptophan (W315) in one monomer stacks over the central portion of the FAD isoalloxazine ring of the second monomer on its re face (Figure 1E). This position is generally occupied by either the pyridine nucleotide or the disulfide bridge in NTRs. The tryptophan position is highly conserved in the protein sequences of the DTR family, including early branching bacteria, cyanobacteria, and marine algae, replaced by a tyrosine residue only in firmicutes and chlorobi (Supplemental Figure 1), and suggests a potential physiological role for this residue. As displayed here, the C-terminal tail has not previously been reported and is a unique feature of DTR. Relative to WT, deletion of the C-terminal motif in Gloeobacter DTR (GvDTR_Δt) was accompanied by ca. 7–9 nm shift in the flavin absorption maxima in the blue region of the spectrum (Figure 1B, lower panel). More interestingly, removal of the C-terminal tail increased the rate of flavin reduction by dithionite (∼10-fold) to levels comparable with those with the E. coli enzyme (Figure 1B). Collectively, these results suggest that the unique C-terminal module on DTR has a direct influence on enzyme activity and might constitute a mechanism of regulation not previously reported. The C-terminal extension of DTR resembles the C-terminal subdomain of certain ferredoxin-NADP reductase enzymes (FNRs) in which an aromatic residue protrudes over the re side of FAD, thereby requiring its displacement for a productive interaction between the pyridine nucleotide and FAD (Ceccarelli et al., 2004Ceccarelli E.A. Arakaki A.K. Cortez N. Carrillo N. Functional plasticity and catalytic efficiency in plant and bacterial ferredoxin-NADP(H) reductases.Biochim. Biophys. Acta. 2004; 1698: 155-165Crossref PubMed Scopus (127) Google Scholar). Among the different types of FNRs, structural similarities were detected between DTR and TR-type FNRs. These FNRs harbor conserved FAD and NADPH-binding motifs but lack the redox-active Cys. An aromatic residue in a short α helix at the C terminus protrudes over the re side of FAD (Supplemental Figure 8). Mutational studies in TR-type FNR enzymes have shown that replacement of the aromatic residue on the re face of the flavin perturbs binding with its redox partner (Seo et al., 2014Seo D. Asano T. Komori H. Sakurai T. Role of the C-terminal extension stacked on the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group in ferredoxin-NADP+ oxidoreductase from Bacillus subtilis.Plant Physiol. Biochem. 2014; 81: 143-148Crossref PubMed Scopus (11) Google Scholar). Following a molecular mechanism similar to these FNRs, displacement of the C-terminal extension of DTR would allow access of the redox disulfide to the re face of FAD likely in a regulatory manner. As for NTRs, conformational flexibility is expected to play an important role in the mechanism of enzyme action. Thus, DTR in solution may exist in equilibrium between the structure observed in the crystal and other configurations in which the donor molecule (and/or the disulfide) is in close contact with the flavin group. In light of the high structural similarity with NTRs and the finding that DTR lacks the archetypal NADPH-binding site, the physiological donor is thought to be a cofactor acting similar to pyridine nucleotides. Other types of molecules such as small redox carrier proteins can, however, not be excluded. To this point, a ferredoxin-linked flavoprotein enzyme that reduces Trx has been isolated from clostridia (Hammel et al., 1983Hammel K.E. Cornwell K.L. Buchanan B.B. Ferredoxin/flavoprotein-linked pathway for the reduction of thioredoxin.Proc. Natl. Acad. Sci. USA. 1983; 80: 3681-3685Crossref PubMed Scopus (29) Google Scholar). However, only identification of the physiological electron donor will clarify the catalytic mechanism of enzyme reduction and its physiological importance in the Trx system. In the present study, we have provided evidence for the diversity of Trx systems in bacteria and for variation in the ability of the reductases to deliver electrons to Trx. Our results support the view that the redox systems consist of structural modules and regulatory elements that different organisms have conveniently combined during evolution to create proteins capable of adapting to specific metabolic and environmental situations. They also provide a structural foundation for future exploration of the mechanism of action and open the door to understanding the evolutionary development of different flavin reductases in relation to particular metabolic demands. B.B.B. acknowledges support from an Alexander von Humboldt Research Award that catalyzed the launching of this project at the Ludwig-Maximilians-Universität in Munich. R.M.B. is supported by a Ramón y Cajal contract from the Spanish Ministerio de Economía y Competitividad. L.L.-M. is supported by a postdoctoral contract from Universidad de Sevilla.

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 reversible oxidation of cysteine (Cys) residues is commonly used to regulate a range of cellular processes throughout biology. Modification of regulatory Cys alters protein solubility and activity and, under control conditions, ultimately results in the fine-tuning of processes in response to metabolic demands. In oxyphotosynthetic organisms, the reversible modification of protein thiols is a major mechanism for direct regulation of chloroplast enzymes in a light-dependent manner and in response to different types of stress. The main redox regulatory system in chloroplasts is the ferredoxin-dependent thioredoxin system (FTS) which coordinates metabolic pathways of oxygenic photosynthesis via thioredoxin-mediated dithiol/ disulfide (SH/S-S) transitions. NADP-linked thioredoxin reductase C and the glutathione/ glutaredoxin systems complement the FTS in maintaining redox balance under certain environmental conditions.

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.

Rubén M. Buey, Sergio Galindo-Trigo, Luis López-Maury, Adrián VelázquezCampoy, José Luis Revuelta, F. Javier Florencio, José M. de Pereda, Peter Schürmann, Bob B. Buchanan, Monica Balsera Dept. Microbiología y Genética. Universidad de Salamanca, Salamanca, Spain; Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Salamanca, Spain; Instituto de Bioquímica Vegetal y Fotosíntesis (CSIC-Universidad de Sevilla), Sevilla, Spain; Institute of Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, Zaragoza, Spain; Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain; Laboratoire de Biologie Moléculaire et Cellulaire, Université de Neuchâtel, Neuchâtel, Switzerland; Department of Plant & Microbial Biology, University of California, Berkeley, USA; Corresponding author: monica.balsera@csic.es

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.

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).

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.

Photograph of Andrew A. Benson. Source: Annual Review of Plant Biology, Vol. 53, 2002, published with permission