Identification of stress associated microRNAs in Solanum lycopersicum by high-throughput Sequencing

Tomato (Solanum lycopersicum) is one of the most important crops around the world and also a model plant to study response to stress. High-throughput sequencing was used to analyse the microRNA (miRNA) profile of tomato plants undergoing five biotic and abiotic stress conditions (drought, heat, P. syringae infection, B. cinerea infection, and herbivore insect attack with Leptinotarsa decemlineata larvae) and one chemical treatment with a plant defence inducer, hexanoic acid. We identified 104 conserved miRNAs belonging to 37 families and we predicted 61 novel tomato miRNAs. Among those 165 miRNAs, 41 were stress-responsive. Reverse transcription quantitative PCR (RT-qPCR) was used to validate high-throughput expression analysis data, confirming the expression profiles of 10 out of 11 randomly selected miRNAs. Most of the differentially expressed miRNAs were stress-specific, except for sly-miR167c-3p upregulated in B. cinerea and P. syringae infection, sly-newmiR26-3p upregulated in drought and Hx treatment samples, and sly-newmiR33-3p, sly-newmiR6-3p and sly-newmiR8-3p differentially expressed both in biotic and abiotic stresses. From mature miRNAs sequences of the 41 stress-responsive miRNAs 279 targets were predicted. An inverse correlation between the expression profiles of 4 selected miRNAs (sly-miR171a, sly-miR172c, sly-newmiR22-3p and sly-miR167c-3p) and their target genes (Kinesin, PPR, GRAS40, ABC transporter, GDP and RLP1) was confirmed by RT-qPCR. Altogether, our analysis of miRNAs in different biotic and abiotic stress conditions highlight the interest to understand the functional role of miRNAs in tomato stress response as well as their putative targets which could help to elucidate plants molecular and physiological adaptation to stress

Hormonal regulation of tomato gibberellin 20-oxidase1 expressed in Arabidopsis

Gibberellin 20-oxidases, enzymes of gibberellin (GA) biosynthesis, play an important role in (GA) homeostasis. To investigate the regulation of tomato SlGA20ox1 expression a genomic clone was isolated, its promoter transcriptionally fused to the GUS reporter gene and the construct used to transform Arabidopsis. Expression was found in diverse vegetative (leaves and roots) and reproductive (flowers) organs. GUS staining was also localized in the columella of secondary roots. GA negative feed-back regulation of SlGA20ox1:GUS was shown to be active both in tomato and in transformed Arabidopsis. Auxin (indol-3-acetic acid, 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid), triiodobenzoic acid (an inhibitor of auxin transport) and benzyladenine (a cytokinin) treatment induced SlGA20ox1:GUS expression associated with increased auxin content and/or signalling , detected using DR5:GUS expression as a marker. Interestingly, SlGA20ox:GUS expression was induced by auxin and root excision in the hypocotyl, an organ not showing GUS staining in control seedlings. In etiolated seedlings, SlGA20ox1:GUS expression occurred in the elongating hypocotyl region of etiolated seedlings and was down-regulated upon transfer to light associated with decrease of growth rate elongation. Our results show that feed-back, auxin and light regulation of SlGA20ox1 expression depends on DNA elements contained within the first 834 bp of the 5´upstream promoter region. Putative DNA regulatory sequences involved in negative feed-back regulation and auxin response were identified in that promoter.This work was supported by Ministerio de Educación y Ciencia of Spain (grants BIO2003-00151 and BIO2006-13437 to J.L.G.-M.) and Consellería de Agricultura de la Generalitat Valenciana (fellowship to E. M.)Peer reviewe

Reducing sampling costs in multivariate SPC with a double-dimension T-2 control chart

[EN] In some real situations there is the need of controlling p variables of a multivariate process, where p1 out of these p variables are easy and inexpensive to monitor, while the p(2)=p-p(1) remaining variables are difficult and/or expensive to measure. However, this set of p(2) variables is important to quickly detect the process shifts. This paper develops a control chart based on the T-2 statistic where normally only the set of p1 variables is monitored, and only when the T-2 value falls in a warning area the rest of variables (p(2)) are measured and combined with the sample values from the p(1) variables, in order to obtain a new T-2 statistic. This new chart is the double dimension T-2 (DDT2) control chart. The ARL of the DDT2 chart is obtained and the chart's parameters are optimized using genetic algorithms with the aim of maximizing the performance in detecting a given process shift. The optimized DDT2 chart is compared against the standard T-2 chart when all the variables are monitored. The results show that the DDT2 clearly outperforms T-2 chart in terms of cost, and in some cases even detects process shifts faster than the latter. In addition, friendly software has been developed with the objective of promoting the real application of this new control chart.S90104144

The variable sample size variable dimension T 2 control chart

In this paper, we describe the development of the variable dimension and variable sample size T2 control chart (VSSVDT2), which is an enhancement of the variable dimension T2 chart (VDT2). In the VDT2 control chart, the number of variables that are measured to compute the T2 statistic is made variable. Some of the variables are easy or inexpensive to measure and are always monitored. The variables that are more difficult or expensive to measure are measured only when the T 2 value from the previous sample exceeds a specified threshold. The VDT2 control chart performs well for moderate and large shifts in the mean vector. However, its performance for small shifts is poor. To improve the charts performance in detecting such shifts, we propose the application of the variable sample size technique to the VDT2 control chart, resulting in the VSSVDT2 control chart. To promote the use of the VSSVDT 2 chart, a user-friendly software has been developed, which the final user can use to find the best parameters of the VSSVDT2 chart for a particular process. © 2013 Taylor and Francis.This work was supported by the CNPq (the Brazilian Council for Scientific and Technological Development), project number 307453/2011-1, and the SENESCYT-Ecuador (National Secretary of Higher Education, Science, Technology and Innovation of Ecuador). The authors are grateful to the referees for their comments, which led to significant improvement of the paper.Aparisi García, FJ.; Kahn Epprecht, E.; Carrión García, A.; Ruiz Rivero, OJ. (2014). The variable sample size variable dimension T 2 control chart. International Journal of Production Research. 52(2):368-383. https://doi.org/10.1080/00207543.2013.826832S36838352

Characterization of the procera Tomato Mutant Shows Novel Functions of the SlDELLA Protein in the Control of Flower Morphology, Cell Division and Expansion, and the Auxin-Signaling Pathway during Fruit-Set and Development

procera (pro) is a tall tomato (Solanum lycopersicum) mutant carrying a point mutation in the GRAS region of the gene encoding SlDELLA, a repressor in the gibberellin (GA) signaling pathway. Consistent with the SlDELLA loss of function, pro plants display a GA-constitutive response phenotype, mimicking wild-type plants treated with GA(3). The ovaries from both nonemasculated and emasculated pro flowers had very strong parthenocarpic capacity, associated with enhanced growth of preanthesis ovaries due to more and larger cells. pro parthenocarpy is facultative because seeded fruits were obtained by manual pollination. Most pro pistils had exserted stigmas, thus preventing self-pollination, similar to wild-type pistils treated with GA(3) or auxins. However, Style2.1, a gene responsible for long styles in noncultivated tomato, may not control the enhanced style elongation of pro pistils, because its expression was not higher in pro styles and did not increase upon GA(3) application. Interestingly, a high percentage of pro flowers had meristic alterations, with one additional petal, sepal, stamen, and carpel at each of the four whorls, respectively, thus unveiling a role of SlDELLA in flower organ development. Microarray analysis showed significant changes in the transcriptome of preanthesis pro ovaries compared with the wild type, indicating that the molecular mechanism underlying the parthenocarpic capacity of pro is complex and that it is mainly associated with changes in the expression of genes involved in GA and auxin pathways. Interestingly, it was found that GA activity modulates the expression of cell division and expansion genes and an auxin signaling gene (tomato AUXIN RESPONSE FACTOR7) during fruit-set.Peer reviewe

Gibberellin Regulation of Fruit Set and Growth in Tomato1[W]

The role of gibberellins (GAs) in tomato (Solanum lycopersicum) fruit development was investigated. Two different inhibitors of GA biosynthesis (LAB 198999 and paclobutrazol) decreased fruit growth and fruit set, an effect reversed by GA3 application. LAB 198999 reduced GA1 and GA8 content, but increased that of their precursors GA53, GA44, GA19, and GA20 in pollinated fruits. This supports the hypothesis that GA1 is the active GA for tomato fruit growth. Unpollinated ovaries developed parthenocarpically in response to GA3 > GA1 = GA4 > GA20, but not to GA19, suggesting that GA 20-oxidase activity was limiting in unpollinated ovaries. This was confirmed by analyzing the effect of pollination on transcript levels of SlCPS, SlGA20ox1, -2, and -3, and SlGA3ox1 and -2, encoding enzymes of GA biosynthesis. Pollination increased transcript content of SlGA20ox1, -2, and -3, and SlCPS, but not of SlGA3ox1 and -2. To investigate whether pollination also altered GA inactivation, full-length cDNA clones of genes encoding enzymes catalyzing GA 2-oxidases (SlGA2ox1, -2, -3, -4, and -5) were isolated and characterized. Transcript levels of these genes did not decrease early after pollination (5-d-old fruits), but transcript content reduction of all of them, mainly of SlGA2ox2, was found later (from 10 d after anthesis). We conclude that pollination mediates fruit set by activating GA biosynthesis mainly through up-regulation of GA20ox. Finally, the phylogenetic reconstruction of the GA2ox family clearly showed the existence of three gene subfamilies, and the phylogenetic position of SlGA2ox1, -2, -3, -4, and -5 was established

The characterization of transgenic tomato overexpressing gibberellin 20-oxidase reveals induction of parthenocarpic fruit growth, higher yield, and alteration of the gibberellin biosynthetic pathway

Fruit-set and growth in tomato depend on the action of gibberellins (GAs). To evaluate the role of the GA biosynthetic enzyme GA 20-oxidase (GA20ox) in that process, the citrus gene CcGA20ox1 was overexpressed in tomato (Solanum lycopersicum L.) cv Micro-Tom. The transformed plants were taller, had non-serrated leaves, and some flowers displayed a protruding stigma due to a longer style, thus preventing self-pollination, similar to GA(3)-treated plants. Flowering was delayed compared with wild-type (WT) plants. Both yield and number of fruits per plant, some of them seedless, were higher in the transgenic plants. The Brix index value of fruit juice was also higher due to elevated citric acid content, but not glucose or fructose content. When emasculated, 1430% of ovaries from transgenic flowers developed parthenocarpically, whereas no parthenocarpy was found in emasculated WT flowers. The presence of early-13-hydroxylation and non-13-hydroxylation GA pathways was demonstrated in the shoot and fruit of Micro-Tom, as well as in two tall tomato cultivars (Ailsa Craig and UC-82). The transgenic plants had altered GA profiles containing higher concentrations of GA(4), from the non-13-hydroxylation pathway, which is generally a minor active GA in tomato. The effect of GA(4) application in enhancing stem growth and parthenocarpic fruit development was proportional to dose, with the same activity as GA(1). The results support the contention that GA20ox overexpression diverts GA metabolism from the early-13-hydroxylation pathway to the non-13-hydroxylation pathway. This led to enhanced GA(4) synthesis and higher yield, although the increase in GA(4) content in the ovary was not sufficient to induce full parthenocarpy.Ministerio de Ciencia y Tecnologia of Spain BIO2006-13437 BIO2009-07968 Ramon y Cajal grant Biotechnology and Biological Sciences Research Council of the UKPeer reviewe

Role of gibberellins during arbuscular mycorrhizal formation in tomato: New insights revealed by endogenous quantification and genetic analysis of their metabolism in mycorrhizal roots

© 2014 Scandinavian Plant Physiology Society. Gibberellins (GAs) are key regulators of plant growth and development and recent studies suggest also a role during arbuscular mycorrhizal (AM) formation. Here, complementary approaches have been used to obtain a clearer picture that correlates AM fungal development inside roots with GA metabolism. An extensive analysis of genes associated with GA metabolism as well as a quantification of GA content in roots was made. Application of GA3 and its biosynthesis inhibitor prohexadione calcium (PrCa) combined with a GA-constitutive response mutant (procera) were used to determine whether fungal colonization is altered by the level of these hormones or by changes in the GA-signaling pathway. The increased levels of specific GAs from the 13-hydroxylation pathway in mycorrhizal roots correlate closely with the increased expression of genes coding enzymes from the GA biosynthetic trail. The imbalance of GAs in tomato roots caused by exogenous applications of GA3 or PrCa affects arbuscules in both negative and positive ways, respectively. In addition, procera plants were adversely affected by the mycorrhization process. Our findings demonstrate that an imbalance in favor of an increased amount of GAs negatively affects the frequency of mycorrhization and particularly the arbuscular abundance in tomato mycorrhizal roots and the results point out that AM formation is associated with a change in the 13-hydroxylation pathway of GAs.We wish to thank the Tomato Genet- ics Resource Centre (TGRC) of the University of California for providing tomato seeds. We would also like to thank Isabel Tamayo for her help with the plant experiments and Michael O’Shea for proof-reading the document. Financial support for this study was provided by grants from the Comisión Interministerial de Ciencia y Teconlogía (CICYT) and Fondos Europeos de Desarrollo Regional (FEDER) through the Ministerio de Economía y Competitividad in Spain (AGL2008-00742; AGL2011-25930) as well as the Junta de Andalucía (Research Group BIO 260). JA Martín Rodríguez was supported by fellowships under the FPU-MICINN program. The authors also acknowledge financial support of Ministry of Education, Youth and Sports of the Czech Republic (LK21306), EU funding from the Operational Program Research and Development for Innovations (ED0007/01/01) and Internal Grant Agency of Palack y University (PrF_2013_023).Peer Reviewe

Inhibition of Auxin Transport from the Ovary or from the Apical Shoot Induces Parthenocarpic Fruit-Set in Tomato Mediated by Gibberellins

Fruit-set in tomato (Solanum lycopersicum L.) depends on gibberellins and auxin (GAs). Here we show, using the cv MicroTom, that application of N-1- naphthylphthalamic acid (NPA) (an inhibitor of auxin transport) to unpollinated ovaries induced parthenocarpic fruit-set, associated with an increase of indole-3-acetic acid (IAA) content, and that this effect was negated by paclobutrazol (PAC; an inhibitor of GA biosynthesis). NPA-induced ovaries contained higher content of GA1 (an active GA) and transcripts of GA biosynthetic genes (SlCPS, SlGA20ox1 and -2). Interestingly, application of NPA to pollinated ovaries prevented their growth, potentially due to supraoptimal IAA accumulation. Plant decapitation, and inhibition of auxin transport by NPA from the apical shoot, also induced parthenocarpic fruit growth of unpollinated ovaries. Application of IAA to the severed stump negated the plant decapitation effect, indicating that the apical shoot prevents unpollinated ovary growth through IAA transport. Parthenocarpic fruit growth induced by plant decapitation was associated with high levels of GA1 and was counteracted by PAC treatment. Plant decapitation also produced changes in transcript levels of genes encoding enzymes of GA biosynthesis (SlCPS and SlGA20ox1) in the ovary, quite similar to those found in NPA-induced fruits. All these results suggest that auxin can have opposing effects on fruit-set, either inducing (when accumulated in the ovary) or repressing (when transported from the apical shoot) that process, and that GAs act as mediators in both cases. The effect of NPA application and decapitation on fruit-set induction was also observed in MicroTom lines bearing introgressed D (DWARF) and Sp (SELT23 PRUNING) wild type alleles.Ministerio de Educación y Ciencia of 19 Spain (BIO2006-13437).Peer reviewe