Monica De Palma

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.

Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance

The possibility of altering the unsaturation level of fatty acids in plant lipids by genetic transformation has implications for the stress tolerance of higher plants as well as for their nutritional value and industrial utilisation. While the integration and expression of transgenes in the plastome has several potential advantages over nuclear transformation, very few attempts have been made to manipulate fatty acid biosynthesis using plastid transformation. We produced transplastomic tobacco plants that express a Δ9 desaturase gene from either the wild potato species Solanum commersonii or the cyanobacterium Anacystis nidulans, using PEG-mediated DNA uptake by protoplasts. Incorporation of chloroplast antibiotic-insensitive point mutations in the transforming DNA was used to select transformants. The presence of the transcript and the Δ9 desaturase protein in transplastomic plants was confirmed by northern and western blot analyses. In comparison with control plants, transplastomic plants showed altered fatty acid profiles and an increase in their unsaturation level both in leaves and seeds. The two transgenes produced comparable results. The results obtained demonstrate the feasibility of using plastid transformation to engineer lipid metabolic pathways in both vegetative and reproductive tissues and suggest an increase of cold tolerance in transplastomic plants showing altered leaf fatty acid profiles. This is the first example of transplastomic plants expressing an agronomically relevant gene produced with the “binding-type” vectors, which do not contain a heterologous marker gene. In fact, the transplastomic plants expressing the S. commersonii gene contain only plant-derived sequences, a clear attraction from a public acceptability perspective.

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.

Tolerance to abiotic stresses in potato plants: a molecular approach

SummaryContinuing study of the potato plants response to stressful conditions has led to the identification of a large number of plant genes whose expression, is regulated by external stimuli. Stress-induced genes can be broadly divided into functional or regulatory genes. To the first category belong genes encoding proteins or enzymes of plant metabolic pathway, of molecules involved in repairing cellular damages and/or indispensable for restoring a new cellular homeostasis compatible with the external conditions. The other class includes genes primarily involved in the perception and/or intracellular transduction of the stress signal, such as kinases, phosphatases or transcription, factors. The research objectives in the field of plant stress tolerance has recently evolved from a mere cloning and description of stress-induced genes to the design of the best strategy of producing transgenic plants tolerant to environmental constraints. It is well known that stress tolerance is a complex trait, requiring the coordinated regulation of a network of genes that act synergistically and additively. At best, manipulation of one single down-stream gene may contribute only partially to the tolerance of the transgenic plants. Recent studies have shown that it is feasible to regulate the level of expression of many down-stream stress-induced genes in a coordinated fashion by regulating the expression of genes encoding transcription factors able to bind DNA motifs in the promoter of stress-induced genes. However, the constitutive high level of expression of transcription factors often causes detrimental phenotypic effects. This drawback could be bypassed by putting genes for transcription factors under the control of inducible promoters. In this way, endogenous tolerance genes are activated only when the stress event occurs, minimizing the negative pleiotropic effect. Novel technology (reverse genetics, DNA microarrays, mRNA differential display, T-DNA tagging, complementation and over-expression of plant cDNA in yeast as model for cellular stress tolerance), improvement of genetic transformation techniques (multiple gene transfer, gene targeting by homologous recombination) as well as a better efficiency of foreign gene expression (discovery of plant promoters with cell-specific, tissue-specific, developmental stage-specific, and/or inducible patterns of expression) will give a tremendous impulse to produce stress tolerant commercial cultivars of the main crops through genetic engineering.

Whole-genome re-sequencing of two Italian tomato landraces reveals sequence variations in genes associated with stress tolerance, fruit quality and long shelf-life traits

Abstract Tomato is a high value crop and the primary model for fleshy fruit development and ripening. Breeding priorities include increased fruit quality, shelf life and tolerance to stresses. To contribute towards this goal, we re-sequenced the genomes of Corbarino (COR) and Lucariello (LUC) landraces, which both possess the traits of plant adaptation to water deficit, prolonged fruit shelf-life and good fruit quality. Through the newly developed pipeline Reconstructor, we generated the genome sequences of COR and LUC using datasets of 65.8 M and 56.4 M of 30–150 bp paired-end reads, respectively. New contigs including reads that could not be mapped to the tomato reference genome were assembled, and a total of 43, 054 and 44, 579 gene loci were annotated in COR and LUC. Both genomes showed novel regions with similarity to Solanum pimpinellifolium and Solanum pennellii. In addition to small deletions and insertions, 2, 000 and 1, 700 single nucleotide polymorphisms (SNPs) could exert potentially disruptive effects on 1, 371 and 1, 201 genes in COR and LUC, respectively. A detailed survey of the SNPs occurring in fruit quality, shelf life and stress tolerance related-genes identified several candidates of potential relevance. Variations in ethylene response components may concur in determining peculiar phenotypes of COR and LUC.