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

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Agricultural and Food Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work seehttp://dx.doi.org/10.1021/jf402429kEggplant (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.This project has been funded by Universitat Politecnica de Valencia through the grants SP20120681 and PAID-06-11 Nr. 2082, and by Ministerio de Economia y Competitividad Grant AGL2012-34213 (jointly funded by FEDER).Plazas Ávila, MDLO.; López Gresa, MP.; Vilanova Navarro, S.; Torres Vidal, C.; Hurtado Ricart, M.; Gramazio, P.; Andújar Pérez, I.... (2013). 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. Journal of Agricultural and Food Chemistry. 61(37):8871-8879. https://doi.org/10.1021/jf402429kS88718879613

Effects of Salt Stress on Three Ecologically Distinct Plantago Species

Comparative studies on the responses to salt stress of taxonomically related taxa should help to elucidate relevant mechanisms of stress tolerance in plants. We have applied this strategy to three Plantago species adapted to different natural habitats, P. crassifolia and P. coronopus both halophytes and P. major, considered as salt-sensitive since it is never found in natural saline habitats. Growth inhibition measurements in controlled salt treatments indicated, however, that P. major is quite resistant to salt stress, although less than its halophytic congeners. The contents of monovalent ions and specific osmolytes were determined in plant leaves after four-week salt treatments. Salt-treated plants of the three taxa accumulated Na+ and Cl- in response to increasing external NaCl concentrations, to a lesser extent in P. major than in the halophytes; the latter species also showed higher ion contents in the non-stressed plants. In the halophytes, K+ concentration decreased at moderate salinity levels, to increase again under high salt conditions, whereas in P. major K+ contents were reduced only above 400 mM NaCl. Sorbitol contents augmented in all plants, roughly in parallel with increasing salinity, but the relative increments and the absolute values reached did not differ much in the three taxa. On the contrary, a strong (relative) accumulation of proline in response to high salt concentrations (600 800 mM NaCl) was observed in the halophytes, but not in P. major. These results indicate that the responses to salt stress triggered specifically in the halophytes, and therefore the most relevant for tolerance in the genus Plantago are: a higher efficiency in the transport of toxic ions to the leaves, the capacity to use inorganic ions as osmotica, even under low salinity conditions, and the activation, in response to very high salt concentrations, of proline accumulation and K+ transport to the leaves of the plants.MAH was a recipient of an Erasmus Mundus pre-doctoral scholarship financed by the European Commission (Welcome Consortium). AP acknowledges the Erasmus mobility programme for funding her stay in Valencia to carry out her Master Thesis.Al Hassan, M.; Pacurar, AM.; López Gresa, MP.; Donat Torres, MDP.; Llinares Palacios, JV.; Boscaiu Neagu, MT.; Vicente Meana, Ó. (2016). Effects of Salt Stress on Three Ecologically Distinct Plantago Species. PLoS ONE. 11(8):1-21. doi:10.1371/journal.pone.0160236S12111

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

[EN] 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 "bloatsafe" plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass) are discussed.This work was supported by grants BIO2012-39849-C02-01 and BIO2016-75485-R from the Spanish Ministry of Economy and Competitiveness (MINECO) (http://www.idi.mineco.gob.es/portal/site/MICINN) to LAC and a fellowship of the JAE-CSIC program to SF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Fresquet-Corrales, S.; Roque Mesa, EM.; Sarrión-Perdigones, A.; Rochina, M.; López-Gresa, MP.; Díaz-Mula, HM.; Belles Albert, JM.... (2017). Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp. PLoS ONE. 12(9). https://doi.org/10.1371/journal.pone.0184839Se018483912

SlS5H silencing reveals specific pathogen-triggered salicylic acid metabolism in tomato

Abstract Background Salicylic acid (SA) is a major plant hormone that mediates the defence pathway against pathogens. SA accumulates in highly variable amounts depending on the plant-pathogen system, and several enzyme activities participate in the restoration of its levels. Gentisic acid (GA) is the product of the 5-hydroxylation of SA, which is catalysed by S5H, an enzyme activity regarded as a major player in SA homeostasis. GA accumulates at high levels in tomato plants infected by Citrus Exocortis Viroid (CEVd), and to a lesser extend upon Pseudomonas syringae DC3000 pv. tomato (Pst) infection. Results We have studied the induction of tomato SlS5H gene by different pathogens, and its expression correlates with the accumulation of GA. Transient over-expression of SlS5H in Nicotiana benthamiana confirmed that SA is processed by SlS5H in vivo. SlS5H-silenced tomato plants were generated, displaying a smaller size and early senescence, together with hypersusceptibility to the necrotrophic fungus Botrytis cinerea. In contrast, these transgenic lines exhibited an increased defence response and resistance to both CEVd and Pst infections. Alternative SA processing appears to occur for each specific pathogenic interaction to cope with SA levels. In SlS5H-silenced plants infected with CEVd, glycosylated SA was the most discriminant metabolite found. Instead, in Pst-infected transgenic plants, SA appeared to be rerouted to other phenolics such as feruloyldopamine, feruloylquinic acid, feruloylgalactarate and 2-hydroxyglutarate. Conclusion Using SlS5H-silenced plants as a tool to unbalance SA levels, we have studied the re-routing of SA upon CEVd and Pst infections and found that, despite the common origin and role for SA in plant pathogenesis, there appear to be different pathogen-specific, alternate homeostasis pathways

Additional file 1 of SlS5H silencing reveals specific pathogen-triggered salicylic acid metabolism in tomato

Additional file 1: Figure S1. Phylogenetic analysis ofAtS5H orthologs in tomato. The box in the phylogenetic tree highlights AtS5H from Arabidopsis thaliana (At5g24530) and its closest homolog in tomato (Solyc03g080190). The multiple alignment was made using ClustalW and the dendrogram was built using the MegAlign program from the Lasergene package (DNASTAR, Madison, Wisconsin, USA). Figure S2. SA-induced expression of SlS5H in wild type (WT) tomato plants.SlS5H (A) and PR1(B) expression of tomato plants treated with 2 mM of SA (SA) or water (MOCK) by stem feeding at 0, 0.5, 1, 6, 24 and 48 h post-treatment. The qRT-PCR values were normalized with the level of expression of the actin gene. The expression levels correspond to the mean ± the standard error of a representative experiment (n = 3). Significant differences between mock and infected or treated plants at different time points are represented by different letters when p-value < 0.05. No statistical differences were observed regarding SlS5H gene expression. Figure S3. S5H in vivo activity in Nicotiana benthamiana plants. (A) SDS-PAGE (left panel) and western blot analysis (right panel) of N. benthamiana plants agroinoculated with pGWB8 empty vector (C) or pGWB8-SlS5H (S5H). (B) Diagram of the cloning cassette. Panels on the right show the nanomoles of SA (C) and GA (D) per gram of fresh weight in Nicotiana benthamiana leaves embedded with SA and agroinoculated with the construction pGWB8-S5H, compared with its control (plasmid pGWB8 without insert). The results correspond to a representative experiment (n = 3). Student’s t-statistic analysis shows the mean ± standard deviation since p-value < 0.001 in free (**) and total (**’) SA accumulation. No statistical differences were observed for GA accumulation. 2,3-DHBA was not detected. Figure S4. Gene expression analysis of wild type (WT) and RNAi_SlS5H(lines 14 and 16) transgenic tomato plants, mock-inoculated (MOCK) and inoculated with CEVd (CEVd). DCL1(A), DCL2(B), RDR1(C) and TCI21(D) gene expression was analyzed 3 weeks after viroid infection. The qRT-PCR values were normalized with the level of expression of the actin gene. The expression levels correspond to the mean ± the standard error of a representative experiment (n = 3). The significant differences between different genotypes and infected or mock-inoculated plants are represented by different letters since p-value < 0.05. Figure S5. Score plot of PCA based on whole range of on the whole array of the mass spectra within a m/z range from 100 to 1500 using unit variance (UV) scaling method of methanolic extracts from tomato leaves. (A) CEVd infected plants at 3 wpi, green: wild type (WT); light purple: RNAi_SlS5H 14; dark purple: RNAi_SlS5H 16; (B) Pst infected plants at 24 hpi, yellow: wild type (WT); orange: RNAi_SlS5H 14; brown: RNAi_SlS5H 16. Figure S6. Analysis of phenotypic differences between WT andRNAi_S5H transgenic lines 14 and 16. Differences related to weight (A), internode length (B), conductivity (C) and chlorophyll content (D) in WT and RNAi_SlS5H 14 and 16 transgenic plants were measured 10 weeks after germination. Bars represent the mean ± the standard deviation of a representative experiment (n = 6

Expression analyses of transgenes and key genes involved in the anthocyanin biosynthetic pathway in transgenic <i>Nicotiana tabacum</i> leaves.

<p><b>(A)</b> qRT–PCR analysis of <i>AmRosea1</i>, <i>AmDelila</i>, <i>MtANR</i> and <i>MtLAR</i> transgenes in transformed leaves of <i>N</i>. <i>tabacum</i>. Error bars correspond to the standard deviation of three replicates. The expression value of <i>AmRosea1</i> in plant Nt#5 was set to 1.00 and the expression levels of the rest of transgenes were plotted relative to this value. To normalize the samples the constitutive <i>NtACT8</i> gene was used. <b>(B)</b> RT-PCR expression analysis of key genes involved in the anthocyanin pathway in <i>N</i>. <i>tabacum</i> Nt#6 and Nt#7 transgenic plants. PCR results were obtained after 30 amplification cycles for all genes and 25 cycles for the housekeeping <i>NtACT8</i> gene.</p

Anthocyanin and proanthocyanidin biosynthetic pathways and multigenic construct assembly strategy.

<p><b>(A)</b> Schematic representation of the biosynthetic pathways for anthocyanins and proanthocyanidins. Abbreviations: chalcone synthase (CHS); chalcone isomerase (CHI); dihydroflavonol reductase (DFR); flavanone 3-hydroxylase (F3H); flavonol synthase (FLS); leucoanthocyanidin reductase (LAR); anthocyanidin synthase (ANS); anthocyanidin reductase (ANR); uridine diphosphate glucose-flavonoid 3-<i>O</i>-glucosyl transferase (UFGT). Purple colored arrows represent the catalytic steps that are upregulated by the overexpression of the <i>A</i>. <i>majus</i> transcription factors <i>Rosea1 and Delila</i>. The yellow highlighted area indicates the catalytic steps that are overexpressed in this work by the <i>M</i>. <i>truncatula</i> genes introduced in our multigenic construct, whose catalytic steps are highlighted in darker yellow and blue. <b>(B)</b> Premade GBParts, Modules and vectors used in this work. This includes the parts pCaMV35S promoter (GB0030), pTNos (GB0035), three vectors of the pGreenII-based pDGB1 series (Alfa1, Alfa2 and Omega2), one vector of the pCAMBIA pDGB2 series (Omega1) and two preassembled modules that were previously tested by the GB2.0 developers. The first module (GB0129) expresses the two <i>A</i>. <i>majus</i> transcriptional factors <i>Rosea1</i> and <i>Delila</i> that under the control of the CaMV35S promoter. The second module (GB0235) is the hygromycin resistant cassette that is used to select the transformed plants in the stable transformation process. CaMV35S is the Cauliflower Mosaic Virus 35S Promoter; TNos is the Nopaline synthase terminator; PNos is the Nopaline synthase promoter; K^R and S^R stand for bacterial kanamycin and spectinomycin resistance cassettes; LB and RB represent the Left and Right Borders of the T-DNA. <b>(C)</b> GoldenBraid 2.0 multigenic construct <i>AmRosea1</i>:<i>AmDelila</i>:<i>MtANR</i>:<i>MtLAR</i> generated in this work. The multigenic construct was generated in five steps that include the assembly of the <i>MtANR</i> and <i>MtLAR</i> transcriptional units from its basic parts (Assemblies 1 and 2), the combination of these transcriptional units in a single vector (Assembly 3), the later addition of the <i>A</i>. <i>majus</i> transcriptional factors to the <i>M</i>. <i>truncatula</i> genes (Assembly 4) and finally the incorporation of the hygromycin resistance cassette to generate the multigenic construct that is used in all the experiments of this work. <i>MtANR</i> is the <i>M</i>. <i>truncatula</i> anthocyanidin reductase gene; <i>MtLAR</i> is the <i>M</i>. <i>truncatula</i> leucoanthocyanidin reductase gene.</p

Phenotypes of the <i>AmRosea1</i>:<i>AmDelila</i>:<i>MtANR</i>:<i>MtLAR</i> transgenic tobacco plants.

<p><b>(A)</b> Some of the <i>in vitro</i> regenerated transgenic calli showed an intense purple pigmentation due to accumulation of anthocyanins. <b>(B)</b> <i>In vitro</i> regenerated control plantlet. <b>(C)</b> <i>In vitro</i> regenerated transgenic tobacco plant showing intense purple color in all tissues. <b>(D)</b> Control plant after acclimation in the greenhouse. <b>(E)</b> Transgenic tobacco plant Nt#7 with intense purple pigmentation after acclimation in the greenhouse. <b>(F)</b> Detail of a leaf from the transgenic plant Nt#7 showing intense purple pigmentation in the abaxial side and vascular bundles. <b>(G)</b> Detail of a leaf from the trasngenic plant Nt#5 showing only small patches of purple pigmentation. <b>(H)</b> Entire and disected flower from a control (left) and transgenic plant Nt#7 (right). <b>(I)</b> Carpel and stamens from a disected flower of a control (left) and the Nt#7 transgenic plant (right).</p

Relationship between purple phenotype, transgene expression and PAs production in three T1 <i>Nicotiana tabacum</i> plants.

<p><b>A-D.</b> Different leaf coloured phenotypes in the T1 of <i>N</i>. <i>tabacum</i> transgenic plants Nt#6.1 (green), Nt#6.8 (purple spots), Nt#6.11 (purple patches) and Nt#7.6 (full purple). <b>E.</b> In the lineage of plants Nt#6 and Nt#7 we analyzed by semi-qRT-PCR three plants with the complete set of transgenes showing a weak (Nt#6.8), a middle (Nt#6.11) and a strong purple phenotype (Nt#7.6). In the three plants the four transgenes were properly expressed. To normalize the samples the constituve <i>NtACT8</i> gene was used. <b>F.</b> The Nt#6.11, Nt#6.8 and Nt#7.6 transgenic plants produced PAs as demonstrated by HPLC-MS analysis of leaf extracts when compared with the WT.</p