Marina Tucci

Novel Hydroxycinnamoyl-Coenzyme A Quinate Transferase Genes from Artichoke Are Involved in the Synthesis of Chlorogenic Acid

Artichoke (Cynara cardunculus subsp. scolymus) extracts have high antioxidant capacity, due primarily to flavonoids and phenolic acids, particularly chlorogenic acid (5-caffeoylquinic acid [CGA]), dicaffeoylquinic acids, and caffeic acid, which are abundant in flower bracts and bioavailable to humans in the diet. The synthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl-coenzyme A transferases are important enzymes in these pathways. Here, we report on the isolation and characterization of two novel genes both encoding hydroxycinnamoyl-coenzyme A quinate transferases (HQT) from artichoke. The recombinant proteins (HQT1 and HQT2) were assayed after expression in Escherichia coli, and both showed higher affinity for quinate over shikimate. Their preferences for acyl donors, caffeoyl-coenzyme A or p-coumaroyl-coenzyme A, were examined. Modeling and docking analyses were used to propose possible pockets and residues involved in determining substrate specificities in the HQT enzyme family. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT1 might be more directly associated with CGA content. Transient and stable expression of HQT1 in Nicotiana resulted in a higher production of CGA and cynarin (1,3-dicaffeoylquinic acid). These findings suggest that several isoforms of HQT contribute to the synthesis of CGA in artichoke according to physiological needs and possibly following various metabolic routes.

Pathogen-responsive wheat PR4 genes are induced by activators of systemic acquired resistance and wounding

Abstract The expression of Triticum aestivum PR4 genes is regulated in response to infection with Fusarium culmorum , a widespread soil-borne pathogen that causes severe damages in crops through foot rot. The induction of PR4 transcripts in wheat coleoptils and roots is correlated with the expression of the corresponding proteins that are expressed only in the infected tissues. Wheat PR4 genes and proteins are also inducible upon treatments with systemic acquired resistance (SAR) chemical inducers such as salicylic acid (SA), benzo (1,2,3) thiodiazole-7-carbothioic acid S-methyl ester (BTH) and methyl jasmonate (MeJA) indicating that activation of PR4 genes follows both SA- and JA-dependent defence response pathways. Moreover, wheat PR4 genes are induced in response to wounding in young shoots and roots as well as in mature leaves suggesting that they can be considered useful markers of plant defence response, although SAR has not been conclusively demonstrated in wheat. Wheat PR1 and PR5 genes used for comparison did not respond to either SAR activators or pathogen attack.

Comparative Analysis of Short- and Long-Term Changes in Gene Expression Caused by Low Water Potential in Potato (Solanum tuberosum) Cell-Suspension Cultures

To dissect the cellular response to water stress and compare changes induced as a generalized response with those involved in tolerance/acclimation mechanisms, we analyzed changes in two-dimensional electrophoretic patterns of in vivo [35S]methionine-labeled polypeptides of cultured potato (Solanum tuberosum) cells after gradual and long exposure to polyethylene glycol (PEG)-mediated low water potential versus those induced in cells abruptly exposed to the same stress intensity. Protein synthesis was not inhibited by gradual stress imposition, and the expression of 17 proteins was induced in adapted cells. Some polypeptides were inducible under mild stress conditions (5% PEG) and accumulated further when cells were exposed to a higher stress intensity (10 and 20% PEG). The synthesis of another set of polypeptides was up-regulated only when more severe water-stress conditions were applied, suggesting that plant cells were able to monitor different levels of stress intensity and modulate gene expression accordingly. In contrast, in potato cells abruptly exposed to 20% PEG, protein synthesis was strongly inhibited. Nevertheless, a large set of polypeptides was identified whose expression was increased. Most of these polypeptides were not induced in adapted cells, but many of them were common to those observed in abscisic acid (ABA)-treated cells. These data, along with the finding that cellular ABA content increased in PEG-shocked cells but not in PEG-adapted cells, suggested that this hormone is mainly involved in the rapid response to stress rather than long-term adaptation. A further group of proteins included those induced after long exposure to both water stress and shock. Western blot analysis revealed that osmotin was one protein belonging to this common group. This class may represent induced proteins that accumulate specifically in response to low water potential and that are putatively involved in the maintenance of cellular homeostasis under prolonged stress.

Isolation and Characterisation of Wheat cDNA Clones Encoding PR4 Proteins

Two cDNA clones encoding the previously characterised PR4 proteins wheatwin 1 and wheatwin2 from wheat (Triticum aestivum cv. S. Pastore) have been identified and named wPR4a and wPR4b, respectively. The clones have been isolated by screening a cDNA library with a specific cDNA probe obtained by RT-PCR. The wPR4a and wPR4b cDNAs contain open reading frames of 441 and 447 bp that encode for wheatwin1 and wheatwin2, respectively.

Molecular and functional analysis of new members of the wheat PR4 gene family

Abstract Five new genes belonging to the pathogenesis-related (PR) 4 family have been cloned and characterised in Triticum aestivum. Two full-length genes, named wPR4e and wPR4f-b, were isolated by library screening, demonstrating the presence of a small intron only in wPR4f-b. Two other PR4 genes (wPR4f-a and wPR4f-c) were isolated by PCR, showing very high sequence identity with wPR4f-b and constituting a new sub-family. Transcription start analysis was performed by RLM-RACE, leading to the isolation of a fifth gene, named wPR4g, that is highly homologous to wPR4e; both encode putative vacuolar PR4 proteins (Wheatwin7 and Wheatwin5, respectively). wPR4e and wPR4f sub-family genes are induced by F. culmorum infection, by chemicals that lead to systemic acquired resistance and by wounding, showing different spatial and temporal induction pathways. In silico analysis of the 5′ untranslated regions of wPR4e and wPR4f-b revealed the presence of several abiotic and biotic stress-responsive elements. wPR4e and wPR4f-b putative promoters were fused to the β-glucuronidase (GUS) reporter gene, and transient and stable expression assays demonstrated that both are able to drive expression of GUS. Characterisation of these new PR4 genes and particularly of their 5′ untranslated regions, as well as the determination of their expression patterns, will contribute to our understanding of the responsiveness of this gene family to various stress conditions and of its role in plant defence.

Phenylpropanoids Accumulation in Eggplant Fruit: Characterization of Biosynthetic Genes and Regulation by a MYB Transcription Factor

Phenylpropanoids are major secondary metabolites in eggplant (Solanum melongena) fruits. Chlorogenic acid (CGA) accounts for 70–90% of total phenolics in flesh tissues, while anthocyanins are mainly present in the fruit skin. As a contribution to the understanding of the peculiar accumulation of these health-promoting metabolites in eggplant, we report on metabolite abundance, regulation of CGA and anthocyanin biosynthesis, and characterization of candidate CGA biosynthetic genes in S. melongena. Higher contents of CGA, Delphinidin 3-rutinoside, and rutin were found in eggplant fruits compared to other tissues, associated to an elevated transcript abundance of structural genes such as PAL, HQT, DFR, and ANS, suggesting that active in situ biosynthesis contributes to anthocyanin and CGA accumulation in fruit tissues. Putative orthologs of the two CGA biosynthetic genes PAL and HQT, as well as a variant of a MYB1 transcription factor showing identity with group six MYBs, were isolated from an Occidental S. melongena traditional variety and demonstrated to differ from published sequences from Asiatic varieties. In silico analysis of the isolated SmPAL1, SmHQT1, SmANS, and SmMyb1 promoters revealed the presence of several Myb regulatory elements for the biosynthetic genes and unique elements for the TF, suggesting its involvement in other physiological roles beside phenylpropanoid biosynthesis regulation. Transient overexpression in Nicotiana benthamiana leaves of SmMyb1 and of a C-terminal SmMyb1 truncated form (SmMyb1Δ9) resulted in anthocyanin accumulation only of SmMyb1 agro-infiltrated leaves. A yeast two-hybrid assay confirmed the interaction of both SmMyb1 and SmMyb1Δ9 with an anthocyanin-related potato bHLH1 TF. Interestingly, a doubled amount of CGA was detected in both SmMyb1 and SmMyb1Δ9 agro-infiltrated leaves, thus suggesting that the N-terminal region of SmMyb1 is sufficient to activate its synthesis. These data suggest that a deletion of the C-terminal region of SmMyb1 does not limit its capability to regulate CGA accumulation, but impairs anthocyanin biosynthesis. To our knowledge, this is the first study reporting a functional elucidation of the role of the C-term conserved domain in MYB activator proteins.

Suppression Subtractive Hybridization analysis provides new insights into the tomato (Solanum lycopersicum L.) response to the plant probiotic microorganism Trichoderma longibrachiatum MK1

Trichoderma species include widespread rhizosphere-colonising fungi that may establish an opportunistic interaction with the plant, resulting in growth promotion and/or increased tolerance to biotic and abiotic stresses. For this reason, Trichoderma-based formulations are largely used in agriculture to improve yield while reducing the application of agro-chemicals. By using the Suppression Subtractive Hybridization method, we identified molecular mechanisms activated during the in vitro interaction between tomato (Solanum lycopersicum L.) and the selected strain MK1 of Trichoderma longibrachiatum, and which may participate in the stimulation of plant growth and systemic resistance. Screening and sequence analysis of the subtractive library resulted in forty unique transcripts. Their annotation in functional categories revealed enrichment in cell defence/stress and primary metabolism categories, while secondary metabolism and transport were less represented. Increased transcription of genes involved in defence, cell wall reinforcement and signalling of reactive oxygen species suggests that improved plant pathogen resistance induced by T. longibrachiatum MK1 in tomato may occur through stimulation of the above mechanisms. The array of activated defence-related genes indicates that different signalling pathways, beside the jasmonate/ethylene-dependent one, collaborate to fine-tune the plant response. Our results also suggest that the growth stimulation effect of MK1 on tomato may involve a set of genes controlling protein synthesis and turnover as well as energy metabolism and photosynthesis. Transcriptional profiling of several defence-related genes at different time points of the tomato-Trichoderma interaction, and after subsequent inoculation with the pathogen Botrytis cinerea, provided novel information on genes that may specifically modulate the tomato response to T. longibrachiatum, B. cinerea or both.

Insights in the Fruit Flesh Browning Mechanisms in Solanum melongena Genetic Lines with Opposite Postcut Behavior.

Color, taste, flavor, nutritional value, and shelf life are important factors determining quality and healthiness of food and vegetables. These factors are strongly affected by browning processes, occurring after fruit or vegetable cutting. Characterization of ten eggplant genotypes for chlorogenic acid (CGA) content, total phenols (TP), polyphenoloxidase (PPO) activity, and browning tendency corroborated a lack of significant correlations between biochemical factors and fruit flesh browning. Further in-depth molecular and biochemical analyses of two divergent eggplant genetic lines, AM199 (high browning) and AM086 (low browning), within 30 min from cutting, highlighted differences in the physiological mechanisms underlying the browning process. qRT-PCR analysis revealed distinct activation mechanisms of CGA biosynthetic and PPO genes in the two genetic lines. Metabolic data on CGA, sugars, and ascorbic acid contents confirmed that their different browning tendency matched with different metabolic responses to cutting. Our findings suggest that the complex mechanism of flesh browning in the two eggplant genetic lines might be mediated by multiple specific factors.

Isolation and functional characterization of a novel gene coding for flavonoid 3′-hydroxylase from globe artichoke

Globe artichoke (Cynara cardunculus L. var. scolymus) is rich in flavonoids which contribute to its health-promoting properties. With the aim of understanding the genetic control of flavonoid accumulation in artichoke, we isolated an artichoke full-length cDNA sequence encoding flavonoid 3′-hydroxylase (F3′H), a major enzyme of the flavonoid hydroxylation pattern. In silico studies confirmed that the deduced amino acid sequence of CcF3′H is highly similar to F3′Hs isolated from other Asteraceae. The Northern blot analysis demonstrated that CcF3′H was highly expressed in leaves and in specific parts of the heads. Its expression differed slightly among artichoke cultivars. The overexpression of CcF3′H in tobacco plants led to the accumulation of flavonoids and to an increase of flower colour intensity, thus identifying CcF3′H as promising candidate for genetic engineering. CcF3′H represents the first structural gene of the flavonoid biosynthesis isolated from C. cardunculus, and its characterization sheds light on the accumulation of flavonoids.

Regulation of Gene Expression during Cellular Adaptation to Water Stress

Gradual adaptation to water stress was accomplished by a set of metabolic adjustments, including proline accumulation, synthesis ofde novo proteins, and changes in gene expression. The ability of the adapted cells to maintain the cellular and subcellular membrane integrity under conditions of severe water stress was found to be associated with a reduced level of unsaturation of fatty acids of membrane lipids. By cloning two different desaturase genes (Δ9-stearoyl-ACP-desaturase and a Δ12-oleoyl-desaturase gene, respectively) it was possible to establish that the reduced level of unsaturation observed in the adapted cells was related to the down-regulation of this class of gene.