Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp.

Proanthocyanidins (PAs), or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila) to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR) to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (-) catechin/g FW and 228.5 nmol (-) epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA) method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce "bloat-safe" plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass) are discussed.

Unraveling Salt Tolerance Mechanisms in Halophytes: A Comparative Study on Four Mediterranean Limonium Species with Different Geographic Distribution Patterns.

We have performed an extensive study on the responses to salt stress in four related Limonium halophytes with different geographic distribution patterns, during seed germination and early vegetative growth. The aims of the work were twofold: to establish the basis for the different chorology of these species, and to identify relevant mechanisms of salt tolerance dependent on the control of ion transport and osmolyte accumulation. Seeds were germinated in vitro, in the presence of increasing NaCl concentrations, and subjected to "recovery of germination" tests; germination percentages and velocity were determined to establish the relative tolerance and competitiveness of the four Limonium taxa. Salt treatments were also applied to young plants, by 1-month irrigation with NaCl up to 800 mM; then, growth parameters, levels of monovalent and divalent ions (in roots and leaves), and leaf contents of photosynthetic pigments and common osmolytes were determined in control and stressed plants of the four species. Seed germination is the most salt-sensitive developmental phase in Limonium. The different germination behavior of the investigated species appears to be responsible for their geographical range size: L. narbonense and L. virgatum, widespread throughout the Mediterranean, are the most tolerant and the most competitive at higher soil salinities; the endemic L. santapolense and L. girardianum are the most sensitive and more competitive only at lower salinities. During early vegetative growth, all taxa showed a strong tolerance to salt stress, although slightly higher in L. virgatum and L. santapolense. Salt tolerance is based on the efficient transport of Na+ and Cl- to the leaves and on the accumulation of fructose and proline for osmotic adjustment. Despite some species-specific quantitative differences, the accumulation patterns of the different ions were similar in all species, not explaining differences in tolerance, except for the apparent activation of K+ transport to the leaves at high external salinity, observed only in the most tolerant L. narbonense and L. virgatum. This specific response may be therefore relevant for salt tolerance in Limonium. The ecological implications of these results, which can contribute to a more efficient management of salt marshes conservation/regeneration programs, are also discussed.

Drought Tolerance in Pinus halepensis Seed Sources As Identified by Distinctive Physiological and Molecular Markers.

Drought is one of the main constraints determining forest species growth, survival and productivity, and therefore one of the main limitations for reforestation or afforestation. The aim of this study is to characterize the drought response at the physiological and molecular level of different Pinus halepensis (common name Aleppo pine) seed sources, previously characterized in field trials as drought-sensitive or drought-tolerant. This approach aims to identify different traits capable of predicting the ability of formerly uncharacterized seedlings to cope with drought stress. Gas-exchange, water potential, photosynthetic pigments, soluble sugars, free amino acids, glutathione and proteomic analyses were carried out on control and drought-stressed seedlings in greenhouse conditions. Gas-exchange determinations were also assessed in field-planted seedlings in order to validate the greenhouse experimental conditions. Drought-tolerant seed sources presented higher values of photosynthetic rates, water use efficiency, photosynthetic pigments and soluble carbohydrates concentrations. We observed the same pattern of variation of photosynthesis rate and maximal efficiency of PSII in field. Interestingly drought-tolerant seed sources exhibited increased levels of glutathione, methionine and cysteine. The proteomic profile of drought tolerant seedlings identified two heat shock proteins and an enzyme related to methionine biosynthesis that were not present in drought sensitive seedlings, pointing to the synthesis of sulfur amino acids as a limiting factor for drought tolerance in Pinus halepensis. Our results established physiological and molecular traits useful as distinctive markers to predict drought tolerance in Pinus halepensis provenances that could be reliably used in reforestation programs in drought prone areas.

Diversity and relationships in key traits for functional and apparent quality in a collection of eggplant: fruit phenolics content, antioxidant activity, polyphenol oxidase activity, and browning.

Eggplant (Solanum melongena) varieties with increased levels of phenolics in the fruit present enhanced functional quality, but may display greater fruit flesh browning. We evaluated 18 eggplant accessions for fruit total phenolics content, chlorogenic acid content, DPPH scavenging activity, polyphenol oxidase (PPO) activity, liquid extract browning, and fruit flesh browning. For all the traits we found a high diversity, with differences among accessions of up to 3.36-fold for fruit flesh browning. Variation in total content in phenolics and in chlorogenic acid content accounted only for 18.9% and 6.0% in the variation in fruit flesh browning, and PPO activity was not significantly correlated with fruit flesh browning. Liquid extract browning was highly correlated with chlorogenic acid content (r = 0.852). Principal components analysis (PCA) identified four groups of accessions with different profiles for the traits studied. Results suggest that it is possible to develop new eggplant varieties with improved functional and apparent quality.

A noncoding plant pathogen provokes both transcriptional and posttranscriptional alterations in tomato.

Viroids are single-stranded, circular, noncoding RNAs that infect plants, causing devastating diseases. In this work, we employed 2D DIGE, followed by MS identification, to analyze the response of tomato plants infected by Citrus exocortis viroid (CEVd). Among the differentially expressed proteins detected, 45 were successfully identified and classified into different functional categories. Validation results by RT-PCR allowed us to classify the proteins into two expression groups. First group included genes with changes at the transcriptional level upon CEVd infection, such as an endochitinase, a ß-glucanase, and pathogenesis-related proteins, PR10 and P69G. All these defense proteins were also induced by gentisic acid, a pathogen-induced signal in compatible interactions. The second group of proteins showed no changes at the transcriptional level and included several ribosomal proteins and translation factors, such as the elongation factors 1 and 2 and the translation initiation factor 5-alpha. These results were validated by 2D Western blot, and possible PTMs caused by CEVd infection were detected. Moreover, an interaction between eukaryotic elongation factor 1 and CEVd was observed by 2D Northwestern. The present study provides new protein-related information on the mechanisms of plant resistance to pathogens.

Shared and novel molecular responses of mandarin to drought.

Drought is the most important stress experienced by citrus crops. A citrus cDNA microarray of about 6.000 genes has been utilized to identify transcriptomic responses of mandarin to water stress. As observed in other plant species challenged with drought stress, key genes for lysine catabolism, proline and raffinose synthesis, hydrogen peroxide reduction, vacuolar malate transport, RCI2 proteolipids and defence proteins such as osmotin, dehydrins and heat-shock proteins are induced in mandarin. Also, some aquaporin genes are repressed. The osmolyte raffinose could be detected in stressed roots while the dehydrin COR15 protein only accumulated in stressed leaves but not in roots. Novel drought responses in mandarin include the induction of genes encoding a new miraculin isoform, chloroplast beta-carotene hydroxylase, oleoyl desaturase, ribosomal protein RPS13A and protein kinase CTR1. These results suggest that drought tolerance in citrus may benefit from inhibition of proteolysis, activation of zeaxanthin and linolenoyl synthesis, reinforcement of ribosomal structure and down-regulation of the ethylene response.

The lithium tolerance of the Arabidopsis cat2 mutant reveals a cross-talk between oxidative stress and ethylene.

In order to investigate the effects of a permanent increase in cellular H(2)O(2) on cation homeostasis we have studied a T-DNA insertion mutant of the Arabidopsis CATALASE 2 gene. This mutant (cat2-1) exhibits 20% of wild-type leaf catalase activity and accumulates more H(2)O(2) than the wild type under normal growth conditions. In addition to reduced size, a pale green color and great reduction in secondary roots, the cat2-1 mutant exhibited increased sensitivity to H(2)O(2), NaCl, norspermidine, high light and cold stress. On the other hand, the germination of the cat2-1 mutant is more tolerant to lithium than the wild type. This novel phenotype cannot be explained by changes in lithium transport. Actually, the uptake of lithium (and of other toxic cations such as sodium and norspermidine) is increased in the cat2-1 mutant while K(+) levels were decreased. The lithium tolerance of this mutant seems to result both from insensitivity to the inhibitory ethylene induced by this cation and a reduced capability for ethylene production. Accordingly, induction by ethylene of responsive genes such as PR4 and EBP/ERF72 is decreased in cat2-1. Mutants insensitive to ethylene such as etr1-1 and ein3-3 are lithium tolerant, and inhibition of ethylene biosynthesis with 2-aminoisobutyrate protects against lithium toxicity. Microarray analysis of gene expression indicates that the expression of genes related to cation transport and ethylene synthesis and perception was not altered in the cat2-1 mutant, suggesting that H(2)O(2) modulates these processes at the protein level. These results uncover a cross-talk between oxidative stress, cation homeostasis and ethylene.

An Arabidopsis quiescin-sulfhydryl oxidase regulates cation homeostasis at the root symplast-xylem interface.

A genetic screen of Arabidopsis 'activation-tagging' mutant collection based on tolerance to norspermidine resulted in a dominant mutant (par1-1D) with increased expression of the QSO2 gene (At1g15020), encoding a member of the quiescin-sulfhydryl oxidase (QSO) family. The par1-1D mutant and transgenic plants overexpressing QSO2 cDNA grow better than wild-type Arabidopsis in media with toxic cations (polyamines, Li(+) and Na(+)) or reduced K(+) concentrations. This correlates with a decrease in the accumulation of toxic cations and an increase in the accumulation of K(+) in xylem sap and shoots. Conversely, three independent loss-of-function mutants of QSO2 exhibit phenotypes opposite to those of par1-1D. QSO2 is mostly expressed in roots and is upregulated by K(+) starvation. A QSO2Colon, two colonsGFP fusion ectopically expressed in leaf epidermis localized at the cell wall. The recombinant QSO2 protein, produced in yeast in secreted form, exhibits disulfhydryl oxidase activity. A plausible mechanism of QSO2 action consists on the activation of root systems loading K(+) into xylem, but different from the SKOR channel, which is not required for QSO2 action. These results uncover QSOs as novel regulators of ion homeostasis.

Accumulation of gentisic acid as associated with systemic infections but not with the hypersensitive response in plant-pathogen interactions.

In the present work we have studied the accumulation of gentisic acid (2,5-dihydroxybenzoic acid, a metabolic derivative of salicylic acid, SA) in the plant-pathogen systems, Cucumis sativus and Gynura aurantiaca, infected with either prunus necrotic ringspot virus (PNRSV) or the exocortis viroid (CEVd), respectively. Both pathogens produced systemic infections and accumulated large amounts of the intermediary signal molecule gentisic acid as ascertained by electrospray ionization mass spectrometry (ESI-MS) coupled on line with high performance liquid chromatography (HPLC). The compound was found mostly in a conjugated (beta-glucoside) form. Gentisic acid has also been found to accumulate (although at lower levels) in cucumber inoculated with low doses of Pseudomonas syringae pv. tomato, producing a nonnecrotic reaction. In contrast, when cucumber was inoculated with high doses of this pathogen, a hypersensitive reaction occurred, but no gentisic-acid signal was induced. This is consistent with our results supporting the idea that gentisic-acid signaling may be restricted to nonnecrotizing reactions of the host plant (Bellés et al. in Mol Plant-Microbe Interact 12:227-235, 1999). In cucumber and Gynura plants, the activity of gentisic acid as inducing signal was different to that of SA, thus confirming the data found for tomato. Exogenously supplied gentisic acid was able to induce peroxidase activity in both Gynura and cucumber plants in a similar way as SA or pathogens. However, gentisic-acid treatments strongly induced polyphenol oxidase activity in cucumber, whereas pathogen infection or SA treatment resulted in a lower induction of this enzyme. Nevertheless, gentisic acid did not induce other defensive proteins which are induced by SA in these plants. This indicates that gentisic acid could act as an additional signal to SA for the activation of plant defenses in cucumber and Gynura plants.

Lithium treatment induces a hypersensitive-like response in tobacco.

Treatment of tobacco ( Nicotiana tabacum L.) plants with lithium induces the formation of necrotic lesions and leaf curling as in the case of incompatible pathogen interactions. Further similarities at the molecular level include accumulation of ethylene and of salicylic and gentisic acids, and induced expression of pathogenesis-related PR-P, PR5 and PR1 genes. With the exception of PR1 induction, lithium produced the same effects in transgenic tobacco plants that do not accumulate salicylate because of overexpression of the bacterial hydroxylase gene nahG. On the other hand, inhibition of ethylene biosynthesis with aminoethoxyvinylglycine prevented lithium-induced cell death and PR5 expression. These results suggest that lithium triggers a hypersensitive-like response where ethylene signalling is essential.

Yeast inositol mono- and trisphosphate levels are modulated by inositol monophosphatase activity and nutrients.

Yeast lithium-sensitive inositol monophosphatase (IMPase) is encoded by a non-essential gene pair (IMP1 and IMP2). Inhibition of IMPase with either Li(+) or Na(+) or a double null mutation imp1 imp2 causes increased levels of inositol monophosphates and reduced level of inositol 1,4,5-trisphosphate. Overexpression of the IMP2 gene has the opposite effects and these results suggest that IMPase activity is limiting for the inositol cycle. Addition of ammonium to cells starved for this nutrient results in a decrease of inositol monophosphates and an increase of inositol 1,4,5-triphosphate, pointing to simultaneous regulation of both inositol 1,4,5-triphosphate production and IMPase activity.

The short-chain alcohol dehydrogenase ABA2 catalyzes the conversion of xanthoxin to abscisic aldehyde.

Mutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified during the course of two independent screenings and named salt resistant (sre) and salobreño (sañ). The sre and sañ mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay. Complementation analyses revealed that sre1-1, sre1-2, sañ3-1, and sañ3-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene. A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of four new aba2 alleles. The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD- or NADP-dependent oxidoreductases. Recombinant ABA2 protein produced in Escherichia coli exhibits a K(m) value for xanthoxin of 19 micro M and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC-mass spectrometry. The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress. The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin-->abscisic aldehyde-->ABA transition as the last steps of the major ABA biosynthetic pathway.

Comparative physiology of salt tolerance in Candida tropicalis and Saccharomyces cerevisiae.

The salt tolerance of the respiratory yeast Candida tropicalis and the fermentative yeast Saccharomyces cerevisiae have been compared in glucose media. C. tropicalis showed a better adaptation to Na+ and Li+ and maintained higher intracellular K+:Na+ and K+:Li+ ratios than S. cerevisiae. However, C. tropicalis showed a poorer adaptation to osmotic stress (produced by KCl and sorbitol) and exhibited reduced glycerol production as compared to S. cerevisiae. In media with the non-repressing sugar galactose as carbon source, S. cerevisiae exhibited reduced glycerol production and increased sensitivity to osmotic stress. Under these conditions, S. cerevisiae, but not C. tropicalis, utilized trehalose as a more important osmolyte than glycerol. These results suggest that the relative tolerance of yeast to the osmotic and cation toxicities of NaCl, and the underlying relative capabilities for osmolyte synthesis and cation transport, are modulated by the general catabolite control exerted by glucose.