Starch is a polymer of D-glucose that plants accumulate as semi-crystalline and osmotically inert granules. Besides being the major energy storage in plants, starch is of primary importance also in human and animal diet, industry and biofuels production. Two kinds of starch, structurally indistinguishable, but different for location and rates of synthesis and degradation are found in plants: secondary starch (characterized by long term-accumulation and found in storage organs) and transitory starch (mainly located in chloroplasts, produced during the day and degraded the subsequent night to meet the energy demand of the plant). Due to its structure, several enzymes are required in starch biosynthesis and degradation, controlling distinct features of starch granules and conferring different physical-chemical properties. Here, the TILLING approach was used on the barley TILLMore population to identify new alleles in five genes related to secondary starch metabolism and known to be expressed in barley seeds. Moreover, the role in Arabidopsis development of the (phospho)glucan, water dikinase proteins (GWD1, GWD2, PWD), known to be involved in nighttime transitory starch degradation, was investigated. Other enzymes, such as β-amylase 1 (BAM1) and α-amylase 3 (AMY3), were demonstrated to be involved in diurnal transitory starch degradation in specialized cells or under stress conditions. Here, we demonstrated that carbon skeletons deriving from BAM1 diurnal degradation of transitory starch support the biosynthesis of proline, a compatible solute, required to face osmotic stress. Moreover, the behaviour of Arabidopsis BAM1 and AMY3 enzymes under oxidative treatments and the possible role of glutathionylation, a redox post-translational modification occurring mainly under stress conditions and promoted by ROS, were investigated. AtBAM1 and AtAMY3 were found to be sensitive to oxidants and glutathionylated, with a modulatory and protective effect on protein activity.

In photosynthetic organisms the redox-dependent modification of thiols belonging to enzymes of the photosynthetic cycle, plays a prominent role in the regulation of metabolism and signalling. An important regulatory mechanism is represented by light that acts through a ferredoxin-thioredoxin system. The system permits the reduction/oxidation of disulfide bridges of the target enzymes. Moreover, cysteines residues can undergo other modifications such as glutathionylation and nitrosylation. Proteomic studies have allowed the identification of new putative targets of redox modifications, including Calvin Benson cycle enzymes. The aim of the studies was to investigate whether phosphoglycerate kinase, triose phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii undergo redox modifications. All enzymes and their mutants (i.e. cysteines variants) were purified and the treatments with alkylating and oxidative agents have permitted to confirm the presence of reactive cysteine(s). The sensitivity of recombinant proteins to redox modifications and the cysteine(s) involved were analyzed by biochemical approcheas. The structural features were analyzed, and the crystallography structure of CrTPI and CrGPA were solved. The three enzymes result all redox regulated although with different biochemical features. The CrPGK contains two cysteines sensitive to redox treatments, although the inhibitor effects of these modifications are different, indeed glutathionylation slightly affected the enzymatic activity compare to nitrosylation. Moreover, the crystallographic structure of CrTPI was determined at a resolution of 1.1.Å, showing a homodimeric conformation containing the typical α/β- barrel fold. No evidence for CrTPI Trx-dependent regulation was obtained but was found to undergo glutathionylation and nitrosylation with a moderate down-regulation on activity. Furthermore, the CrGAPA shows an extreme sensitivity to oxidant molecules and the crystallographic structure of CrGAPA was determined at a resolution of 1.8.Å, confirming the tetrameric fold of the enzyme. the results suggest that redox modifications could constitute a mechanism for the regulation of the Calvin-Benson cycle under oxidative stress conditions.

No description available.

No description available.

The aim of the present study is understanding the properties of a new group of redox proteins having in common a DOMON-type domain with characteristics of cytochromes b. The superfamily of proteins containing a DOMON of this type includes a few protein families. With the aim of better characterizing this new protein family, the present work addresses both a CyDOM protein (a cytochrome b561) and a protein only comprised of DOMON(AIR12), both of plant origin. Apoplastic ascorbate can be regenerated from monodehydroascorbate by a trans-plasma membrane redox system which uses cytosolic ascorbate as a reductant and comprises a high potential cytochrome b. We identified the major plasma membrane (PM) ascorbate-reducible b-type cytochrome of bean (Phaseolus vulgaris) and soybean (Glycine max) hypocotyls as orthologs of Arabidopsis auxin-responsive gene air12. The protein, which is glycosylated and glycosylphosphatidylinositol-anchored to the external side of the PM in vivo, was expressed in Pichia pastoris in a recombinant form, lacking the glycosylphosphatidylinositol-modification signal, and purified from the culture medium. Recombinant AIR12 is a soluble protein predicted to fold into a β-sandwich domain and belonging to the DOMON superfamily. It is shown to be a b-type cytochrome with a symmetrical α-band at 561 nm, to be fully reduced by ascorbate and fully oxidized by monodehydroascorbate. Redox potentiometry suggests that AIR12 binds two high-potential hemes (Em,7 +135 and +236 mV). Phylogenetic analyses reveal that the auxin-responsive genes AIR12 constitute a new family of plasma membrane b-type cytochromes specific to flowering plants. Although AIR12 is one of the few redox proteins of the PM characterized to date, the role of AIR12 in trans-PM electron transfer would imply interaction with other partners which are still to be identified. Another part of the present project was aimed at understanding of a soybean protein comprised of a DOMON fused with a well-defined b561 cytochrome domain (CyDOM). Various bioinformatic approaches show this protein to be composed of an N-terminal DOMON followed by b561 domain. The latter contains five transmembrane helices featuring highly conserved histidines, which might bind haem groups. The CyDOM has been cloned and expressed in the yeast Pichia pastoris, and spectroscopic analyses have been accomplished on solubilized yeast membranes. CyDOM clearly reveal the properties of b-type cytochrome. The results highlight the fact that CyDOM is clearly able to lead an electron flux through the plasmamembrane. Voltage clamp experiments demonstrate that Xenopus laevis oocytes transformed with CyDOM of soybean exhibit negative electrical currents in presence of an external electron acceptor. Analogous investigations were carried out with SDR2, a CyDOM of Drosophila melanogaster which shows an electron transport capacity even higher than plant CyDOM. As quoted above, these data reinforce those obtained in plant CyDOM on the one hand, and on the other hand allow to attribute to SDR2-like proteins the properties assigned to CyDOM. Was expressed in Regenerated tobacco roots, transiently transformed with infected a with chimeral construct GFP: CyDOM (by A. rhizogenes infection) reveals a plasmamembrane localization of CyDOM both in epidermal cells of the elongation zone of roots and in root hairs. In conclusion. Although the data presented here await to be expanded and in part clarified, it is safe to say they open a new perspective about the role of this group of proteins. The biological relevance of the functional and physiological implications of DOMON redox domains seems noteworthy, and it can but increase with future advances in research. Beyond the very finding, however interesting in itself, of DOMON domains as extracellular cytochromes, the present study testifies to the fact that cytochrome proteins containing DOMON domains of the type of “CyDOM” can transfer electrons through membranes and may represent the most important redox component of the plasmamembrane as yet discovered.

No description available.

The DOMON domain is a domain widespread in nature, predicted to fold in a β-sandwich structure. In plants, AIR12 is constituted by a single DOMON domain located in the apoplastic space and is GPI-modified for anchoring to the plasma membrane. Arabidopsis thaliana AIR12 has been heterologously expressed as a recombinant protein (recAtAIR12) in Pichia pastoris. Spectrophotometrical analysis of the purified protein showed that recAtAir12 is a cytochrome b. RecAtAIR12 is highly glycosylated, it is reduced by ascorbate, superoxide and naftoquinones, oxidised by monodehydroascorbate and oxygen and insensitive to hydrogen peroxide. The addition of recAtAIR12 to permeabilized plasma membranes containing NADH, FeEDTA and menadione, caused a statistically significant increase in hydroxyl radicals as detected by electron paramagnetic resonance. In these conditions, recAtAIR12 has thus a pro-oxidant role. Interestingly, AIR12 is related to the cytochrome domain of cellobiose dehydrogenase which is involved in lignin degradation, possibly via reactive oxygen species (ROS) production. In Arabidopsis the Air12 promoter is specifically activated at sites where cell separations occur and ROS, including •OH, are involved in cell wall modifications. air12 knock-out plants infected with Botrytis cinerea are more resistant than wild-type and air12 complemented plants. Also during B. cinerea infection, cell wall modifications and ROS are involved. Our results thus suggest that AIR12 could be involved in cell wall modifying reactions by interacting with ROS and ascorbate. CyDOMs are plasma membrane redox proteins of plants that are predicted to contain an apoplastic DOMON fused with a transmembrane cytochrome b561 domain. CyDOMs have never been purified nor characterised. The trans-membrane portion of a soybean CyDOM was expressed in E. coli but purification could not be achieved. The DOMON domain was expressed in P. pastoris and shown to be itself a cytochrome b that could be reduced by ascorbate.

Il mais è uno dei cereali maggiormente diffusi perché utilizzato in ambito alimentare umano e animale, per la produzione di materiale biodegradabile e di bioetanolo. I processi fisiologici che coordinano la crescita della cariosside vengono regolati principalmente dall’auxina che agisce a livello trascrizionale e post-traduzionale. Attraverso analisi comparative tra il mutante de18 (defective endosperm 18), deficitario nella produzione di acido indolo-3-acetico (IAA), e del suo corrispettivo wild-type, è stato possibile individuare i geni coinvolti nella determinazione delle dimensioni della cariosside. In particolare, sono state effettuate analisi morfologiche e di quantificazione dell’amido su cariossidi de18 e wild type negli stadi precoci di sviluppo. E’ stato inoltre allestito un esperimento di RNA sequencing sull’endosperma dei due genotipi a 8 e 12 DAP (Days After Pollination). L’analisi dei geni differenzialmente espressi attraverso la classificazione GO (Gene Ontology) ha permesso di studiare l’effetto della carenza di auxina sull’espressione genica. Nel mutante si riscontra l’attivazione tardiva della sintesi dell’amido e l’incremento delle proteine di riserva. Inoltre, la carenza di auxina determina una riduzione dell’attività mitotica ed endoreduplicativa, confermata dalla repressione dell’attività di geni legati al ciclo cellulare. / Maize is one of the world’s leading cereal grains due to its diverse functionality as a food source for both humans and animals, as well as a source of raw materials and biofuel. The physiological processes responsible for the growth of the kernel are regulated mainly by auxin acting at the transcriptional and post-translational level. Through comparative analysis between mutant de18 (defective endosperm 18), defective in indol-3-acetic acid (IAA) production, and its wild-type B37, it has been possible to identify the genes involved in the determination of the final seed size. Morphological analysis and quantification of starch were done on seed mutant and wild-type in the early stages of their development. Finally, an RNA sequencing analysis was carried out on mutant and wild-type endosperm at 8 and 12 DAP (Days After Pollination) and differentially expressed genes were classified by Gene Ontology. Down-regulation of genes related to sugar metabolism suggested a delayed activation of starch biosynthesis. This finding was confirmed by the determination of starch content that was lower in the mutant endosperms respect to the normal in the early stages of gran filling (12 and 16 DAP). The reduced auxin level affected the mitotic and endoreduplication activities as suggested by the repression of genes involved in the cell cycle.

Fusarium verticillioides è responsabile della fusariosi della spiga in mais e della contaminazione della granella con micotossine. Sono state individuate le regioni geniche e i geni candidati per la resistenza a Fusarium dal confronto tra una linea di mais resistente (CO441) e una suscettibile (CO354), impiegando tre diversi approcci: analisi QTL, analisi trascrittomica (RNASeq) e analisi metabolomica. 184 famiglie F2:3 (CO441xCO354) sono state valutate in due diversi ambienti nell’anno 2011 e inoculate artificialmente con due diverse tecniche (forchetta e stuzzicadente). E’ stata rilevata una significativa variazione genotipica in risposta all’infezione. Sulla base di una mappa preliminare di linkage molecolare contenente 74 marcatori microsatelliti polimorfici, sono stati determinati 8 QTLs comuni alla resistenza alla fusariosi della spiga e alla riduzione della contaminazione da fumonisine. Sono stati considerati geni candidati per la resistenza i geni differenzialmente espressi, risultanti dall’ RNASeq, in semi di mais CO441 prima e 72 ore dopo l’infezione. I metaboliti putativi correlati alla resistenza sono stati rilevati tramite high resolution LC-MS in entrambe le linee di mais. I geni candidati e i metaboliti mappano in pathway coinvolti nei meccanismi di difesa: fenilalanina, tirosina e triptofano biosintesi, fenilpropanoidi e flavonoidi biosintesi, metabolismo dell’acido linoleico e α-linolenico. Abbondanti trascritti derivano dalla biosintesi dei terpenoidi e dei diterpenoidi. Nei geni candidati verranno ricercati polimorfismi fra le due linee di mais e che andranno ad arricchire la mappa di linkage molecolare. / Fusarium verticillioides is responsible for Fusarium ear rot in maize and mycotoxin contamination of grain. Genomic regions and candidate genes for resistance to Fusarium were detected through the comparison of resistant (CO441) and susceptible (CO354) maize lines, by following three different approaches: Quantitative Trait Locus (QTL), transcriptomic (RNASeq) and metabolomic analyses. 184 F2:3 families (CO441xCO354) were evaluated in two different environments in 2011 and artificially infected with two side-needle inoculation methods (pin-bar and toothpick). Significant genotypic variation in response to infection was detected. On the basis of a genetic draft map containing 74 polymorphic SSRs markers, 8 common QTLs for Fusarium ear rot resistance and fumonisin contamination reduction were revealed. Candidate genes for resistance resulted from differentially expressed genes before and 72 hours post infection of CO441 kernels through RNASeq technology. Putative metabolites related to resistance were detected through high resolution LC-MS in both maize lines. Candidate genes and metabolites mapped in pathways involved in defense mechanism: phenylalanine, tyrosine and tryptophan biosynthesis, phenylpropanoid and flavonoid biosynthesis, linoleic and α-linolenic acid metabolism. Abundant genic transcripts derived from terpenoid and diterpenoid biosynthesis. Candidate genes will be screened for polymorphisms between the two maize lines in order to enrich the linkage map.

le antocianine sono una classe di metaboliti secondari nelle piante, appartenenti ai flavonoidi, categoria di molecole antiossidanti utili per la salute di piante, animali e umana. Sono ampiamente studiati nelle Leguminosae poiché implicati in numerose funzioni fisiologiche. In questa tesi, due wild-types e cinque mutanti di Medicago truncatula, coinvolti nel metabolismo dei flavonoidi, sono stati caratterizzati a livello fenotipico, fisiologico e molecolare. La principale differenza tra i wild-type e i mutanti è la riduzione del contenuto di antocianine nelle foglie. In seguito all'esposizione alle radiazioni UV-B, è stata analizzata la loro risposta, evidenziando un diverso comportamento. Inoltre, il metabolismo delle antocianine è stato studiato anche a livello di regolazione genica e un nuovo gene myb è stato isolato e caratterizzato, risultando più espresso nei tessuti che accumulano antocianine. / Anthocyanins are secondary metabolites in plants. They belong to a class of flavonoids synthesized via the phenylpropanoid pathway and they are antioxidant molecules important for plant, animal and human health. Flavonoids are widely studied in legume because they are implicated in several biological and physiological functions. In this thesis seven genotypes (two wild-types and five indipendent mutants) of Medicago truncatula (the model legume plant) affected in flavonoid metabolism have been characterized at phenotypic, physiological and molecular level. The main difference between wild-types and mutants, is the reduction in anthocyanin content in leaves. The anthocyanin accumulation during the leaf life, the flowering time and the fruit formation have been registered and compared. The two wild-types contain 6 μg cyanidin.mg-1 of fresh weight, while in the mutants the anthocyanin amount ranged from 0.12 μg cyanidin mg-1 to 2 μg cyanidin mg-1 of fresh weight. After HPLC-DAD-MS/MS analysis, the main anthocyanin present in the two wild-types is the cyanidin-3-O feruloyl. The Expression profile of genes codifying enzymes and transcriptional factor involved in flavonoid biosynthesis has been investigated. RT-PCR and qPCR results show different possible pathways of reduction of anthocyanins in the five mutants. These mutants, have been exposed to UV-B radiations and their response has been investigated measuring the chlorophyll fluorescence parameters (Fv/Fm, qP and NPQ) in untreated plants, during treatment (after 4hrs and 14hrs of treatment) and in the recovering phase. All genotypes, regardless of the anthocyanins amount, showed a decrease of the photosyntetic efficiency after 14hrs of treatment. This indicates a marginal role of these pigments in the oxidative damages protection. Mutants do not response in the same manner to the UV—B exposure and the anthocyanin amount does not increase equally in all genotypes. The anthocyanin metabolism is studied also at the gene regulation level. A novel Myb gene (MtMYBA) involved in anthocyanin pathway has been isolated and characterized. This gene is more expressed in tissues which accumulate anthocyanins in M. truncatula plants. The functional analysis has been investigated overexpressing this Myb gene under the control of 35S promoter. This construct has been used to transform Arabisopsis thaliana and Medicago truncatula plants. In addition, the MtMYBA promoter has been cloned in a plasmids containing GUS and GFP reporter genes.