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In many biogenic materials, chitin chains are assembled in fibrils that are wrapped by a protein fold. In them, the mechanical properties are supposed to be related to intra- and inter- interactions among chitin and proteins. This hypothesis has been poorly investigated. Here, this research theme is studied using the pen of Loligo vulgaris as a model material of chitin-protein composites. Chemical treatments were used to change the interactions involving only the proteic phase, through unfolding and/or degradation processes. Successively, structural and mechanical parameters were examined using spectroscopy, microscopy, X-ray diffractometry, and tensile tests. The data analysis showed that chemical treatments did not modify the structure of the chitin matrix. This allowed to derive from the mechanical test analysis the following conclusions: (i) the maximum stress (σ max) relies on the presence of the disulfide bonds; (ii) the Young's modulus (E) relies on the overall correct folding of the proteins; (iii) the whole removal of proteins induces a decrease of E (> 90%) and σ max (> 80%), and an increase in the maximum elongation. These observations indicate that in the chitin matrix the proteins act as a strengthener, which efficacy is controlled by the presence of disulfide bridges. This reinforcement links the chitin fibrils avoiding them to slide one on the other and maximizing their resistance and stiffness. In conclusion, this knowledge can explain the physio-chemical properties of other biogenic polymeric composites and inspire the design of new materials. To date, no study has addressed on how proteins influence chitin-composite material's mechanical properties. Here we show that the Young's modulus and the maximum stress mainly rely on protein disulfide bonds, the inter-proteins ones and those controlling the folding of chitin-binding domains. The removal of protein matrix induce a reduction of Young's modulus and maximum stress, leaving the chitin matrix structurally unaltered. The measure of the maximum elongation shows that the chitin fibrils slide on each other only after removing the protein matrix. In conclusion, this research shows that the proteins act as a stiff matrix reinforced by di-sulfide bridges that link crystalline chitin fibrils avoiding them to slide one on the other. Image, graphical abstract [ABSTRACT FROM AUTHOR]

Since spring ephemerals are credited to be all “sun” species with unusually elevate photosynthesis, in contrast to shade-tolerant trees and understory geophytes with a long aboveground cycle, we examined the photosynthetic efficiency of 6 woody species, 9 long-cycle geophytes, and 8 spring ephemeral geophytes using blue flashes of increasing energy with the Imaging PAM fluorometer. Several parameters were obtained: quantum yield of electron transport (ΦETR) or of PSII (ΦPSII), maximum measured photosynthesis rate (ETR hv ), maximum extrapolated rate of photosynthesis (ETR em ), half-saturating photon flux density (K PAR ), and in some cases photochemical (qP) and non-photochemical quenching (NPQ). Results confirm the ecological consistency of the three plant groups, with internal differences. Woody species have low ETR em and K PAR values with good ΦETR; long-cycle herbs have low ETR em and ΦETR and moderate K PAR values; spring ephemerals have elevate ΦETR, ETR em and K PAR values. The mean ETR em of ephemerals of 91 μmol m −2 s −1 exceeds that of long-cycle herbs 2.9-fold and woody species 4.8-fold, and corresponds to 19 μmol CO 2 m −2 s −1 by assuming an ETR/ΦCO 2 ratio of 4.7. Highest photosynthesis rates and K PAR were exhibited by five ephemerals ( Eranthis, Erythronium, Narcissus, Scilla, Tulipa ) with peak ETR em values equivalent to ∼40 μmol CO 2 m −2 s −1 or ∼60 μmol CO 2 (g Chl) −1 s −1 (“sun” species). According to a new, fluorescence based heliophily index, all trees and five long-cycle herbs were definitely “shade” species, while four long-cycle herbs and three ephemerals were intermediate shade-tolerant. [ABSTRACT FROM AUTHOR]

In rice, maize and barley, the lack of Isoamylase 1 activity materially affects the composition of endosperm starch. Here, the effect of this deficiency in durum wheat has been characterized, using transgenic lines in which Isa1 was knocked down via RNAi. Transcriptional profiling confirmed the partial down-regulation of Isa1 and revealed a pleiotropic effect on the level of transcription of genes encoding other isoamylases, pullulanase and sucrose synthase. The polysaccharide content of the transgenic endosperms was different from that of the wild type in a number of ways, including a reduction in the content of starch and a moderate enhancement of both phytoglycogen and β-glucan. Some alterations were also induced in the distribution of amylopectin chain length and amylopectin fine structure. The amylopectin present in the transgenic endosperms was more readily hydrolyzable after a treatment with hydrochloric acid, which disrupted its semi-crystalline structure. The conclusion was that in durum wheat, Isoamylase 1 is important for both the synthesis of amylopectin and for determining its internal structure. [ABSTRACT FROM AUTHOR]

The benzothiadiazole derivative acibenzolar-S-methyl (BTH; Bion, Syngenta), a well known chemical inducer of systemic acquired resistance (SAR), was tested for its ability to protect pear (Pyrus communis cv. Abate Fetel) from fire blight following inoculation with Erwinia amylovora. Two-year-old pear plants sprayed with BTH 10 days before inoculation were partially protected from fire blight, both in terms of incidence (-33%) and severity (-37%). The effect of BTH proved to be systemic. However, a significant portion (28%) of BTH-treated plants showing little or no symptoms 6 months after inoculation still harboured viable E. amylovora cells, as judged by PCR-based detection. Salicylic acid, known to be physiologically implicated in the onset of SAR, gave similar protection as BTH when sprayed 24 h before inoculation.In several plants species systemic acquired resistance has been shown to correlate with the induced expression of a defined set of genes coding for pathogenesis-related (PR) proteins. However, the expression of a member of the PR-1 family in pear plants treated with BTH and salicylic acid and subsequently inoculated with E. amylovora was found to be constitutive and unaffected by treatments. These results therefore suggest that molecules other than PR-1 may be important in the BTH-induced systemic resistance of pear against E. amylovora. [Copyright &y& Elsevier]

Starch is one of the major sinks of fixed carbon in photosynthetic tissues of higher plants. Carbon fixation and the synthesis of primary starch occur during the day in the chloroplast stroma, whereas starch degradation typically occurs during the following night to fuel the whole plant with energy and carbon in the absence of photosynthesis. Redox-based regulatory systems play a central role in the modulation of several chloroplastic pathways. Reversible oxidations of cysteine residues are post-translational modifications that orchestrate the precise functioning of chloroplast pathways together with changes in pH, Mg 2+ and concentrations of metabolic intermediates. Leaf starch metabolism has been intensively studied. The enzymes involved in starch synthesis and degradation have been identified and characterized. However, the redox control of the enzymes responsible for starch degradation at night remains elusive, and their response to redox transitions conflicts with the timing of the physiological events. Most of the enzymes of starch degradation are activated by reducing conditions, characteristic of daytime. Thus, redox control may have only a minor role during starch degradation at night, but could become relevant for daily stomatal opening in guard cells or in the re-allocation of fixed carbon in mesophyll cells in response to stress conditions. [ABSTRACT FROM AUTHOR]

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-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp666), 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 starchwith a β-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and β-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 β-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 Cys499 and Cys587 is central to this regulation. This work provides new insights into how-amylase activity may be regulated in the chloroplast. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Oxygenic phototrophs use the Calvin–Benson cycle to fix CO 2 during photosynthesis. In the dark, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), two enzymes of the Calvin–Benson cycle, form an inactive complex with the regulatory protein CP12, mainly under the control of thioredoxins and pyridine nucleotides. In the light, complex dissociation allows GAPDH and PRK reactivation. The GAPDH/CP12/PRK complex is conserved from cyanobacteria to angiosperms and coexists in land plants with an autoassembling GAPDH that is analogously regulated. With the recently described 3D structures of PRK and GAPDH/CP12/PRK, the structural proteome of this ubiquitous regulatory system has been completed. This outcome opens a new avenue for understanding the regulatory potential of photosynthetic carbon fixation by laying the foundation for its knowledge-based manipulation. Two enzymes of the Calvin–Benson cycle, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), together with the regulatory protein CP12, can assemble into an inactive multimeric complex. With the recent characterization of the structures of free PRK and GAPDH/CP12/PRK ternary complexes, the hierarchical process of protein assembly can be described at molecular definition. CP12 complexes are conserved in oxygenic phototrophs, but land plants also contain an autoassembling GAPDH isoform, evolutionarily derived from CP12. Both types of complexes form in the dark and dissociate in light, mainly under the control of thioredoxins and pyridine nucleotides. CP12 is a major light/dark regulator of the Calvin–Benson cycle in cyanobacteria and contributes to the more sophisticated regulation of the cycle in land plants, where dark complexes may play an additional role in protecting enzymes from proteolysis. [ABSTRACT FROM AUTHOR]

AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b . Recombinant AIR12 from Arabidopsis accepted electrons from ascorbate or superoxide, and donated electrons to either monodehydroascorbate or oxygen. AIR12 was found associated in vivo to the plasma membrane. Though linked to the membrane by a glycophosphatidylinositol anchor, AIR12 is a hydrophilic and glycosylated protein predicted to be fully exposed to the apoplast. The expression pattern of AIR12 in Arabidopsis is developmentally regulated and correlated to sites of controlled cell separation ( e.g. micropilar endosperm during germination, epidermal cells surrounding the emerging lateral root) and cells around wounds. Arabidopsis (Landsberg erecta-0) mutants with altered levels of AIR12 did not show any obvious phenotype. However, AIR12-overexpressing plants accumulated ROS (superoxide, hydrogen peroxide) and lipid peroxides in leaves, indicating that AIR12 may alter the redox state of the apoplast under particular conditions. On the other hand, AIR12-knock out plants displayed a strongly decreased susceptibility to Botrytis cinerea infection, which in turn induced AIR12 expression in susceptible wild type plants. Altogether, the results suggest that AIR12 plays a role in the regulation of the apoplastic redox state and in the response to necrotrophic pathogens. Possible relationships between these functions are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) are two energy-consuming enzymes of the Calvin–Benson cycle, whose regulation is crucial for the global balance of the photosynthetic process under different environmental conditions. In oxygen phototrophs, GAPDH and PRK regulation involves the redox-sensitive protein CP12. In the dark, oxidized chloroplast thioredoxins trigger the formation of a GAPDH/CP12/PRK complex in which both enzyme activities are down-regulated. In this report, we show that free GAPDH (A4-isoform) and PRK are also inhibited by oxidants like H2O2, GSSG and GSNO. Both in the land plant Arabidopsis thaliana and in the green microalga Chlamydomonas reinhardtii, both enzymes can be glutathionylated as shown by biotinylated-GSSG assay and MALDI-ToF mass spectrometry. CP12 is not glutathionylated but homodisulfides are formed upon oxidant treatments. In Arabidopsis but not in Chlamydomonas, the interaction between oxidized CP12 and GAPDH provides full protection from oxidative damage. In both organisms, preformed GAPDH/CP12/PRK complexes are protected from GSSG or GSNO oxidation, and in Arabidopsis also from H2O2 treatment. Overall, the results suggest that the role of CP12 in oxygen phototrophs needs to be extended beyond light/dark regulation, and include protection of enzymes belonging to Calvin–Benson cycle from oxidative stress. [Copyright &y& Elsevier]

Abstract: In oxygenic photosynthetic organisms, the activities of two Calvin cycle enzymes (glyceraldehyde-3-phosphate dehydrogenase, GAPDH and phosphoribulokinase, PRK) are regulated by CP12-mediated complex formation. The Arabidopsis genome contains three genes encoding different CP12 isoforms (CP12-1, At2g47400; CP12-2, At3g62410 and CP12-3, At1g76560), all plastid-targeted, as demonstrated by localization in the chloroplast stroma of CP12 precursor sequences fused with the green fluorescence protein (GFP). The disorder predictor PONDR® classified Arabidopsis CP12s as largely disordered proteins, and circular dichroism spectra confirmed these predictions. Based on sequence similarity, 66 CP12s from different organisms were identified and clustered in six types, with CP12-1 and -2 grouping together with other largely disordered sequences (Type I), while a lower level of disorder was predicted within the cluster including CP12-3 (Type II). The three Arabidopsis CP12 isoforms were expressed as mature recombinant forms and purified to homogeneity. Redox titrations demonstrated that the four conserved cysteines of each CP12 isoform could form two internal disulfide bridges with different midpoint redox potentials (E m,7.9 −326mV and −350mV in both CP12-1 and CP12-2; E m,7.9 −332mV and −373mV in CP12-3). In agreement with their similar redox properties, all CP12 isoforms formed, in vitro, a supramolecular complex with GAPDH and PRK, with comparable inhibitory effects on both enzyme activities. In order to test whether CP12 isoforms might have broader regulatory functions than regulating Calvin cycle enzymes, CP12 proteins were analyzed for their capacity to bind plastidial glycolytic GAPDH (GapCp). To this purpose, the mature form of Arabidopsis GapCp2 was cloned, expressed in recombinant form and purified to homogeneity. However, contrary to expectations, no CP12 isoform was able to bind GapCp2 under any of the conditions tested. [Copyright &y& Elsevier]

CP12 is a protein of 8.7 kDa that contributes to Calvin cycle regulation by acting as a scaffold element in the formation of a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) in photosynthetic organisms. NMR studies of recombinant CP12 (isoform 2) of Arabidopsis thaliana show that CP12-2 is poorly structured. CP12-2 is monomeric in solution and contains four cysteines, which can form two intramolecular disulfides with midpoint redox potentials of -326 and -352 mV, respectively, at pH 7.9. Site-specific mutants indicate that the C-terminal disulfide is involved in the interaction between CP12-2 and GAPDH (isoform A4), whereas the N-terminal disulfide is involved in the interaction between this binary complex and PRK. In the presence of NAD, oxidized CP12-2 interacts with A4-GAPDH (KD = 0.18 μM) to form a binary complex of 170 kDa with (A4-GAPDH)-(CP12-2)2 stoichiometry, as determined by isothermal titration calorimetry and multiangle light scattering analysis. PRK is a dimer and by interacting with this binary complex (KD = 0.17 μM) leads to a 498-kDa ternary complex constituted by two binary complexes and two PRK dimers, i.e. ((A4-GAPDH)-(CP12-2)2-(PRK))2. Thermodynamic parameters indicate that assembly of both binary and ternary complexes is exoergonic although penalized by a decrease in entropy that suggests an induced folding of CP12-2 upon binding to partner proteins. The redox dependence of events leading to supramolecular complexes is consistent with a role of CP12 in coordinating the reversible inactivation of chloroplast enzymes A4-GAPDH and PRK during darkness in photosynthetic tissues. [ABSTRACT FROM AUTHOR]