Dark complexes of the Calvin-Benson cycle in a physiological perspective.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) are two enzymes of the Calvin Benson cycle that stand out for some peculiar properties they have in common: (i) they both use the products of light reactions for catalysis (NADPH for GAPDH, ATP for PRK), (ii) they are both light-regulated through thioredoxins and (iii) they are both involved in the formation of regulatory supramolecular complexes in the dark or low photosynthetic conditions, with or without the regulatory protein CP12. In the complexes, enzymes are transiently inactivated but ready to recover full activity after complex dissociation. Fully active GAPDH and PRK are in large excess for the functioning of the Calvin-Benson cycle, but they can limit the cycle upon complex formation. Complex dissociation contributes to photosynthetic induction. CP12 also controls PRK concentration in model photosynthetic organisms like Arabidopsis thaliana and Chlamydomonas reinhardtii. The review combines in vivo and in vitro data into an integrated physiological view of the role of GAPDH and PRK dark complexes in the regulation of photosynthesis.

Tonoplast cytochrome b561 is a transmembrane ascorbate-dependent monodehydroascorbate reductase: functional characterization of electron currents in plant vacuoles.

Ascorbate (Asc) is a major redox buffer of plant cells, whose antioxidant activity depends on the ratio with its one-electron oxidation product monodehydroascorbate (MDHA). The cytoplasm contains millimolar concentrations of Asc and soluble enzymes that can regenerate Asc from MDHA or fully oxidized dehydroascorbate. Also, vacuoles contain Asc, but no soluble Asc-regenerating enzymes. Here, we show that vacuoles isolated from Arabidopsis mesophyll cells contain a tonoplast electron transport system that works as a reversible, Asc-dependent transmembrane MDHA oxidoreductase. Electron currents were measured by patch-clamp on isolated vacuoles and found to depend on the availability of Asc (electron donor) and ferricyanide or MDHA (electron acceptors) on opposite sides of the tonoplast. Electron currents were catalyzed by cytochrome b561 isoform A (CYB561A), a tonoplast redox protein with cytoplasmic and luminal Asc binding sites. The Km for Asc of the luminal (4.5 mM) and cytoplasmic site (51 mM) reflected the physiological Asc concentrations in these compartments. The maximal current amplitude was similar in both directions. Mutant plants with impaired CYB561A expression showed no detectable trans-tonoplast electron currents and strong accumulation of leaf anthocyanins under excessive illumination, suggesting a redox-modulation exerted by CYB561A on the typical anthocyanin response to high-light stress.

Conformational Disorder Analysis of the Conditionally Disordered Protein CP12 from Arabidopsis thaliana in Its Different Redox States.

CP12 is a redox-dependent conditionally disordered protein universally distributed in oxygenic photosynthetic organisms. It is primarily known as a light-dependent redox switch regulating the reductive step of the metabolic phase of photosynthesis. In the present study, a small angle X-ray scattering (SAXS) analysis of recombinant Arabidopsis CP12 (AtCP12) in a reduced and oxidized form confirmed the highly disordered nature of this regulatory protein. However, it clearly pointed out a decrease in the average size and a lower level of conformational disorder upon oxidation. We compared the experimental data with the theoretical profiles of pools of conformers generated with different assumptions and show that the reduced form is fully disordered, whereas the oxidized form is better described by conformers comprising both the circular motif around the C-terminal disulfide bond detected in previous structural analysis and the N-terminal disulfide bond. Despite the fact that disulfide bridges are usually thought to confer rigidity to protein structures, in the oxidized AtCP12, their presence coexists with a disordered nature. Our results rule out the existence of significant amounts of structured and compact conformations of free AtCP12 in a solution, even in its oxidized form, thereby highlighting the importance of recruiting partner proteins to complete its structured final folding.

Crystal structure of chloroplastic thioredoxin z defines a type-specific target recognition.

Thioredoxins (TRXs) are ubiquitous disulfide oxidoreductases structured according to a highly conserved fold. TRXs are involved in a myriad of different processes through a common chemical mechanism. Plant TRXs evolved into seven types with diverse subcellular localization and distinct protein target selectivity. Five TRX types coexist in the chloroplast, with yet scarcely described specificities. We solved the crystal structure of a chloroplastic z-type TRX, revealing a conserved TRX fold with an original electrostatic surface potential surrounding the redox site. This recognition surface is distinct from all other known TRX types from plant and non-plant sources and is exclusively conserved in plant z-type TRXs. We show that this electronegative surface endows thioredoxin z (TRXz) with a capacity to activate the photosynthetic Calvin-Benson cycle enzyme phosphoribulokinase. The distinct electronegative surface of TRXz thereby extends the repertoire of TRX-target recognitions.

The circadian night depression of photosynthesis analyzed in a herb, Pulmonaria vallarsae. Day/night quantitative relationships.

Although many photosynthesis related processes are known to be controlled by the circadian system, consequent changes in photosynthetic activities are poorly understood. Photosynthesis was investigated during the daily cycle by chlorophyll fluorescence using a PAM fluorometer in Pulmonaria vallarsae subsp. apennina, an understory herb. A standard test consists of a light induction pretreatment followed by light response curve (LRC). Comparison of the major diagnostic parameters collected during day and night showed a nocturnal drop of photosynthetic responses, more evident in water-limited plants and consisting of: (i) strong reduction of flash-induced fluorescence peaks (FIP), maximum linear electron transport rate (Jmax, ETREM) and effective PSII quantum yield (ΦPSII); (ii) strong enhancement of nonphotochemical quenching (NPQ) and (iii) little or no change in photochemical quenching qP, maximum quantum yield of linear electron transport (Φ), and shape of LRC (θ). A remarkable feature of day/night LRCs at moderate to high irradiance was their linear-parallel course in double-reciprocal plots. Photosynthesis was also monitored in plants subjected to 2-3 days of continuous darkness ("long night"). In such conditions, plants exhibited high but declining peaks of photosynthetic activity during subjective days and a low, constant value with elevated NPQ during subjective night tests. The photosynthetic parameters recorded in subjective days in artificial darkness resembled those under natural day conditions. On the basis of the evidence, we suggest a circadian component and a biochemical feedback inhibition to explain the night depression of photosynthesis in P. vallarsae.

Arabidopsis and Chlamydomonas phosphoribulokinase crystal structures complete the redox structural proteome of the Calvin-Benson cycle.

In land plants and algae, the Calvin-Benson (CB) cycle takes place in the chloroplast, a specialized organelle in which photosynthesis occurs. Thioredoxins (TRXs) are small ubiquitous proteins, known to harmonize the two stages of photosynthesis through a thiol-based mechanism. Among the 11 enzymes of the CB cycle, the TRX target phosphoribulokinase (PRK) has yet to be characterized at the atomic scale. To accomplish this goal, we determined the crystal structures of PRK from two model species: the green alga Chlamydomonas reinhardtii (CrPRK) and the land plant Arabidopsis thaliana (AtPRK). PRK is an elongated homodimer characterized by a large central ß-sheet of 18 strands, extending between two catalytic sites positioned at its edges. The electrostatic surface potential of the catalytic cavity has both a positive region suitable for binding the phosphate groups of substrates and an exposed negative region to attract positively charged TRX-f. In the catalytic cavity, the regulatory cysteines are 13 Å apart and connected by a flexible region exclusive to photosynthetic eukaryotes-the clamp loop-which is believed to be essential for oxidation-induced structural rearrangements. Structural comparisons with prokaryotic and evolutionarily older PRKs revealed that both AtPRK and CrPRK have a strongly reduced dimer interface and an increased number of random-coiled regions, suggesting that a general loss in structural rigidity correlates with gains in TRX sensitivity during the molecular evolution of PRKs in eukaryotes.

Combining mutations at genes encoding key enzymes involved in starch synthesis affects the amylose content, carbohydrate allocation and hardness in the wheat grain.

Modifications to the composition of starch, the major component of wheat flour, can have a profound effect on the nutritional and technological characteristics of the flour's end products. The starch synthesized in the grain of conventional wheats (Triticum aestivum) is a 3:1 mixture of the two polysaccharides amylopectin and amylose. Altering the activity of certain key starch synthesis enzymes (GBSSI, SSIIa and SBEIIa) has succeeded in generating starches containing a different polysaccharide ratio. Here, mutagenesis, followed by a conventional marker-assisted breeding exercise, has been used to generate three mutant lines that produce starch with an amylose contents of 0%, 46% and 79%. The direct and pleiotropic effects of the multiple mutation lines were identified at both the biochemical and molecular levels. Both the structure and composition of the starch were materially altered, changes which affected the functionality of the starch. An analysis of sugar and nonstarch polysaccharide content in the endosperm suggested an impact of the mutations on the carbon allocation process, suggesting the existence of cross-talk between the starch and carbohydrate synthesis pathways.

Role of the NAD(P)H quinone oxidoreductase NQR and the cytochrome b AIR12 in controlling superoxide generation at the plasma membrane.

MAIN CONCLUSION: The quinone reductase NQR and the b-type cytochrome AIR12 of the plasma membrane are important for the control of reactive oxygen species in the apoplast. AIR12 and NQR are two proteins attached to the plant plasma membrane which may be important for generating and controlling levels of reactive oxygen species in the apoplast. AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b. The NADPH quinone oxidoreductase NQR is a two-electron-transferring flavoenzyme that contributes to the generation of O 2•- in isolated plasma membranes. A. thaliana double knockout plants of both NQR and AIR12 generated more O 2•- and germinated faster than the single mutant affected in AIR12. To test whether NQR and AIR12 are able to interact functionally, recombinant purified proteins were added to plasma membranes isolated from soybean hypocotyls. In vitro NADH-dependent O 2•- production at the plasma membrane in the presence of NQR was reduced upon addition of AIR12. Electron donation from semi-reduced menadione to AIR12 was shown to take place. Biochemical analysis showed that purified plasma membrane from soybean hypocotyls or roots contained phylloquinone and menaquinone-4 as redox carriers. This is the first report on the occurrence of menaquinone-4 in eukaryotic photosynthetic organisms. We propose that NQR and AIR12 interact via the quinone, allowing an electron transfer from cytosolic NAD(P)H to apoplastic monodehydroascorbate and control thereby the level of reactive oxygen production and the redox state of the apoplast.

The analysis of the different functions of starch-phosphorylating enzymes during the development of Arabidopsis thaliana plants discloses an unexpected role for the cytosolic isoform GWD2.

The genome of Arabidopsis thaliana encodes three glucan, water dikinases. Glucan, water dikinase 1 (GWD1; EC 2.7.9.4) and phosphoglucan, water dikinase (PWD; EC 2.7.9.5) are chloroplastic enzymes, while glucan, water dikinase 2 (GWD2) is cytosolic. Both GWDs and PWD catalyze the addition of phosphate groups to amylopectin chains at the surface of starch granules, changing its physicochemical properties. As a result, GWD1 and PWD have a positive effect on transitory starch degradation at night. Because of its cytosolic localization, GWD2 does not have the same effect. Single T-DNA mutants of either GWD1 or PWD or GWD2 have been analyzed during the entire life cycle of A. thaliana. We report that the three dikinases are all important for proper seed development. Seeds from gwd2 mutants are shrunken, with the epidermal cells of the seed coat irregularly shaped. Moreover, gwd2 seeds contain a lower lipid to protein ratio and are impaired in germination. Similar seed phenotypes were observed in pwd and gwd1 mutants, except for the normal morphology of epidermal cells in gwd1 seed coats. The gwd1, pwd and gwd2 mutants were also very similar in growth and flowering time when grown under continuous light and all three behaved differently from wild-type plants. Besides pinpointing a novel role of GWD2 and PWD in seed development, this analysis suggests that the phenotypic features of the dikinase mutants in A. thaliana cannot be explained solely in terms of defects in leaf starch degradation at night.

Direct Recording of Trans-Plasma Membrane Electron Currents Mediated by a Member of the Cytochrome b561 Family of Soybean.

Trans-plasma membrane electron transfer is achieved by b-type cytochromes of different families, and plays a fundamental role in diverse cellular processes involving two interacting redox couples that are physically separated by a phospholipid bilayer, such as iron uptake and redox signaling. Despite their importance, no direct recordings of trans-plasma membrane electron currents have been described in plants. In this work, we provide robust electrophysiological evidence of trans-plasma membrane electron flow mediated by a soybean (Glycine max) cytochrome b561 associated with a dopamine ß-monooxygenase redox domain (CYBDOM), which localizes to the plasma membrane in transgenic Arabidopsis (Arabidopsis thaliana) plants and CYBDOM complementary RNA-injected Xenopus laevis oocytes. In oocytes, two-electrode voltage clamp experiments showed that CYBDOM-mediated currents were activated by extracellular electron acceptors in a concentration- and type-specific manner. Current amplitudes were voltage dependent, strongly potentiated in oocytes preinjected with ascorbate (the canonical electron donor for cytochrome b561), and abolished by mutating a highly conserved His residue (H292L) predicted to coordinate the cytoplasmic heme b group. We believe that this unique approach opens new perspectives in plant transmembrane electron transport and beyond.

ß-amylase 1 (BAM1) degrades transitory starch to sustain proline biosynthesis during drought stress.

During photosynthesis of higher plants, absorbed light energy is converted into chemical energy that, in part, is accumulated in the form of transitory starch within chloroplasts. In the following night, transitory starch is mobilized to sustain the heterotrophic metabolism of the plant. ß-amylases are glucan hydrolases that cleave α-1,4-glycosidic bonds of starch and release maltose units from the non-reducing end of the polysaccharide chain. In Arabidopsis, nocturnal degradation of transitory starch involves mainly ß-amylase-3 (BAM3). A second ß-amylase isoform, ß-amylase-1 (BAM1), is involved in diurnal starch degradation in guard cells, a process that sustains stomata opening. However, BAM1 also contributes to diurnal starch turnover in mesophyll cells under osmotic stress. With the aim of dissecting the role of ß-amylases in osmotic stress responses in Arabidopsis, mutant plants lacking either BAM1 or BAM3 were subject to a mild (150mM mannitol) and prolonged (up to one week) osmotic stress. We show here that leaves of osmotically-stressed bam1 plants accumulated more starch and fewer soluble sugars than both wild-type and bam3 plants during the day. Moreover, bam1 mutants were impaired in proline accumulation and suffered from stronger lipid peroxidation, compared with both wild-type and bam3 plants. Taken together, these data strongly suggest that carbon skeletons deriving from BAM1 diurnal degradation of transitory starch support the biosynthesis of proline required to face the osmotic stress. We propose the transitory-starch/proline interplay as an interesting trait to be tackled by breeding technologies aimingto improve drought tolerance in relevant crops.

FeON-FeOFF: the Helicobacter pylori Fur regulator commutates iron-responsive transcription by discriminative readout of opposed DNA grooves.

Most transcriptional regulators bind nucleotide motifs in the major groove, although some are able to recognize molecular determinants conferred by the minor groove of DNA. Here we report a transcriptional commutator switch that exploits the alternative readout of grooves to mediate opposite output regulation for the same input signal. This mechanism accounts for the ability of the Helicobacter pylori Fur regulator to repress the expression of both iron-inducible and iron-repressible genes. When iron is scarce, Fur binds to DNA as a dimer, through the readout of thymine pairs in the major groove, repressing iron-inducible transcription (FeON). Conversely, on iron-repressible elements the metal ion acts as corepressor, inducing Fur multimerization with consequent minor groove readout of AT-rich inverted repeats (FeOFF). Our results provide first evidence for a novel regulatory paradigm, in which the discriminative readout of DNA grooves enables to toggle between the repression of genes in a mutually exclusive manner.

Unravelling the shape and structural assembly of the photosynthetic GAPDH-CP12-PRK complex from Arabidopsis thaliana by small-angle X-ray scattering analysis.

Oxygenic photosynthetic organisms produce sugars through the Calvin-Benson cycle, a metabolism that is tightly linked to the light reactions of photosynthesis and is regulated by different mechanisms, including the formation of protein complexes. Two enzymes of the cycle, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), form a supramolecular complex with the regulatory protein CP12 with the formula (GAPDH-CP122-PRK)2, in which both enzyme activities are transiently inhibited during the night. Small-angle X-ray scattering analysis performed on both the GAPDH-CP12-PRK complex and its components, GAPDH-CP12 and PRK, from Arabidopsis thaliana showed that (i) PRK has an elongated, bent and screwed shape, (ii) the oxidized N-terminal region of CP12 that is not embedded in the GAPDH-CP12 complex prefers a compact conformation and (iii) the interaction of PRK with the N-terminal region of CP12 favours the approach of two GAPDH tetramers. The interaction between the GAPDH tetramers may contribute to the overall stabilization of the GAPDH-CP12-PRK complex, the structure of which is presented here for the first time.

Arabidopsis thaliana AMY3 is a unique redox-regulated chloroplastic α-amylase.

α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. In vascular plants, α-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for ß-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp(666)), identified as the catalytic nucleophile in other plant α-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a ß-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and ß-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant α-amylases, with a pH optimum of 7.5-8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys(499) and Cys(587) is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast.

Insights in progressive myoclonus epilepsy: HSP70 promotes cystatin B polymerization.

Cystatin B (CSTB) is an anti-protease frequently mutated in progressive myoclonus epilepsy (EPM1), a devastating degenerative disease. This work shows that rat CSTB is an unstable protein that undergoes structural changes following the interaction with a chaperone, either prokaryotic or eukaryotic. Both the prokaryotic DnaK and eukaryotic HSP70 promote CSTB polymerization. Denaturated CSTB is polymerized by the chaperone alone. Native CSTB monomers are more stable than denatured monomers and require Cu(2+) for chaperone-dependent polymerization. Cu(2+) interacts with at least two conserved histidines, at positions 72 and 95 modifying the structure of native monomeric CSTB. Subsequently, CSTB becomes unstable and readily responds to the addition of DnaK or HSP70, generating polymers. This reaction depends strictly on the presence of this divalent metal ion and on the presence of one cysteine in the protein chain. The cysteine deletion mutant does not polymerize. We propose that Cu(2+) modifies the redox environment of the protein, allowing the oxidation of the cysteine residue of CSTB that triggers polymerization. These polymers are sensitive to reducing agents while polymers obtained from denatured CSTB monomers are DTT resistant. We propose that the Cu(2+)/HSP70 dependent polymers are physiological and functional in eukaryotic cells. Furthermore, while monomeric CSTB has anti-protease function, it seems likely that polymeric CSTB fulfils different function(s).

Influence of the extraction method on functional properties of commercial snail secretion filtrates.

Snail secretion is a complex mixture of several components, including proteins, glycoproteins, mucopolysaccharides and smaller molecules. Its growing use in nutraceutical, cosmetic and biomedical applications, as well as a component of edible and green packaging to replace chemical plasticizer, implies more affordable and sustainable extraction methods. We chose four extracts obtained from Cornu aspersum snails, different by origin, extraction medium (namely, citric acid, lactic acid or none) and additives and we performed a series of characterizations including the SDS-page, the measure of pH and density, the evaluation of dry matter and of protein content, supported by structural determinations by means of UV-visible and infrared spectroscopy, X-Rays diffraction and thermogravimetric measurements. Biological assays comprising evaluation of cytotoxicity and antibacterial activity were also carried out. All the tests were performed both on the as received snail filtrates and on the samples after proper dialysis to remove preservatives added by manufacturers. The obtained results put into evidence that the properties and composition of the final extract are strongly influenced by the collection method, that can be relevant for the proper use of snail filtrate in specific applications.

Control of aragonite deposition in colonial corals by intra-skeletal macromolecules.

Scleractinian coral skeletons are composed mainly of aragonite in which a small percentage of organic matrix (OM) molecules is entrapped. It is well known that in corals the mineral deposition occurs in a biological confined nucleation site, but it is still unclear to what extent the calcification is controlled by OM molecules. Hence, the shape, size and organization of skeletal crystals from the fiber level through the colony architecture, were also attributed to factors as diverse as nucleation site mineral supersaturation and environmental factors in the habitat. In this work the OMs were extracted from the skeleton of three colonial corals, Acropora digitifera, Lophelia pertusa and Montipora caliculata. A. digitifera has a higher calcification rate than the other two species. OM molecules were characterized and their CaCO3 mineralization activity was evaluated by experiments of overgrowth on coral skeletons and of precipitation from solutions containing OM soluble and insoluble fractions and magnesium ions. The precipitates were characterized by spectroscopic and microscopic techniques. The results showed that the OM molecules of the three coral share similar features, but differ from those associated with mollusk shells. However, A. digitifera OM shows peculiarities from those from L. pertusa and M. caliculata. The CaCO3 overgrowth and precipitation experiments confirm the singularity of A. digitifera OM molecules as mineralizers. Moreover, their comparison indicates that only specific molecules are involved in the polymorphism control and suggests that when the whole extracted materials are used the OM's main effect is on the control of particles' shape and morphology.

Conformational selection and folding-upon-binding of intrinsically disordered protein CP12 regulate photosynthetic enzymes assembly.

Carbon assimilation in plants is regulated by the reduction of specific protein disulfides by light and their re-oxidation in the dark. The redox switch CP12 is an intrinsically disordered protein that can form two disulfide bridges. In the dark oxidized CP12 forms an inactive supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase, two enzymes of the carbon assimilation cycle. Here we show that binding of CP12 to GAPDH, the first step of ternary complex formation, follows an integrated mechanism that combines conformational selection with induced folding steps. Initially, a CP12 conformation characterized by a circular structural motif including the C-terminal disulfide is selected by GAPDH. Subsequently, the induced folding of the flexible C-terminal tail of CP12 in the active site of GAPDH stabilizes the binary complex. Formation of several hydrogen bonds compensates the entropic cost of CP12 fixation and terminates the interaction mechanism that contributes to carbon assimilation control.

Proline, Cysteine and Branched-Chain Amino Acids in Abiotic Stress Response of Land Plants and Microalgae.

Proteinogenic amino acids are the building blocks of protein, and plants synthesize all of them. In addition to their importance in plant growth and development, growing evidence underlines the central role played by amino acids and their derivatives in regulating several pathways involved in biotic and abiotic stress responses. In the present review, we illustrate (i) the role of amino acids as an energy source capable of replacing sugars as electron donors to the mitochondrial electron transport chain and (ii) the role of amino acids as precursors of osmolytes as well as (iii) precursors of secondary metabolites. Among the amino acids involved in drought stress response, proline and cysteine play a special role. Besides the large proline accumulation occurring in response to drought stress, proline can export reducing equivalents to sink tissues and organs, and the production of H2S deriving from the metabolism of cysteine can mediate post-translational modifications that target protein cysteines themselves. Although our general understanding of microalgae stress physiology is still fragmentary, a general overview of how unicellular photosynthetic organisms deal with salt stress is also provided because of the growing interest in microalgae in applied sciences.

Arabidopsis thaliana Sucrose Phosphate Synthase A2 Affects Carbon Partitioning and Drought Response.

Sucrose is essential for plants for several reasons: It is a source of energy, a signaling molecule, and a source of carbon skeletons. Sucrose phosphate synthase (SPS) catalyzes the conversion of uridine diphosphate glucose and fructose-6-phosphate to sucrose-6-phosphate, which is rapidly dephosphorylated by sucrose phosphatase. SPS is critical in the accumulation of sucrose because it catalyzes an irreversible reaction. In Arabidopsis thaliana, SPSs form a gene family of four members, whose specific functions are not clear yet. In the present work, the role of SPSA2 was investigated in Arabidopsis under both control and drought stress conditions. In seeds and seedlings, major phenotypic traits were not different in wild-type compared with spsa2 knockout plants. By contrast, 35-day-old plants showed some differences in metabolites and enzyme activities even under control conditions. In response to drought, SPSA2 was transcriptionally activated, and the divergences between the two genotypes were higher, with spsa2 showing reduced proline accumulation and increased lipid peroxidation. Total soluble sugars and fructose concentrations were about halved compared with wild-type plants, and the plastid component of the oxidative pentose phosphate pathway was activated. Unlike previous reports, our results support the involvement of SPSA2 in both carbon partitioning and drought response.