Insights into the origin of the invasive populations of Trioza erytreae in Europe using microsatellite markers and mtDNA barcoding approaches.

The African citrus psyllid Trioza erytreae is one of the major threats to citrus industry as the vector of the incurable disease known as huanglongbing (HLB) or citrus greening. The psyllid invaded the northwest of the Iberian Peninsula 6 years ago. The invasion alarmed citrus growers in the Mediterranean basin, the largest citrus producing area in Europe, which is still free of HLB. Before our study, no research had been carried out on the genetic diversity of T. erytreae populations that have invaded the Iberian Peninsula and the archipelagos of the Macaronesia (Madeira and the Canary Islands). In this study, combining microsatellites markers and mtDNA barcoding analysis, we characterize the genetic diversity, structure and maternal relationship of these new invasive populations of T. erytreae and those from Africa. Our results suggest that the outbreaks of T. erytreae in the Iberian Peninsula may have derived from the Canary Islands. The populations of T. erytreae that invaded Macaronesia and the Iberian Peninsula are likely to have originated from southern Africa. We anticipate our results to be a starting point for tracking the spread of this invasive pest outside of Africa and to be important for optimizing contingency and eradication plans in newly invaded and free areas.

Regulation of ovule initiation by gibberellins and brassinosteroids in tomato and Arabidopsis: two plant species, two molecular mechanisms.

Ovule primordia formation is a complex developmental process with a strong impact on the production of seeds. In Arabidopsis this process is controlled by a gene network, including components of the signalling pathways of auxin, brassinosteroids (BRs) and cytokinins. Recently, we have shown that gibberellins (GAs) also play an important role in ovule primordia initiation, inhibiting ovule formation in both Arabidopsis and tomato. Here we reveal that BRs also participate in the control of ovule initiation in tomato, by promoting an increase on ovule primordia formation. Moreover, molecular and genetic analyses of the co-regulation by GAs and BRs of the control of ovule initiation indicate that two different mechanisms occur in tomato and Arabidopsis. In tomato, GAs act downstream of BRs. BRs regulate ovule number through the downregulation of GA biosynthesis, which provokes stabilization of DELLA proteins that will finally promote ovule primordia initiation. In contrast, in Arabidopsis both GAs and BRs regulate ovule number independently of the activity levels of the other hormone. Taken together, our data strongly suggest that different molecular mechanisms could operate in different plant species to regulate identical developmental processes even, as for ovule primordia initiation, if the same set of hormones trigger similar responses, adding a new level of complexity.

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.

The Altered Expression of microRNA408 Influences the Arabidopsis Response to Iron Deficiency.

MicroRNAs contribute to the adaptation of plants to varying environmental conditions by affecting systemic mineral nutrient homeostasis. Copper and iron deficiencies antagonistically control the expression of Arabidopsis thaliana microRNA408 (miR408), which post-transcriptionally regulates laccase-like multicopper oxidase family members LAC3, LAC12, and LAC13. In this work, we used miR408 T-DNA insertion mutants (408-KO1 and 408-KO2) and a previously characterized transgenic line overexpressing miR408 (35S:408-14) to explore how miR408 influences copper- and iron-dependent metabolism. We observed that the altered expression of miR408 diminished plant performance and the activation of the iron-regulated genes under iron-deficient conditions. Consistently with the low expression of the miR408-target laccases, we showed that the vascular bundle lignification of the 35S:408-14 plants diminished. The decrease in the phenoloxidase and ferroxidase activities exhibited by wild-type plants under iron deficiency did not occur in the 408-KO1 plants, probably due to the higher expression of laccases. Finally, we observed that the hydrogen peroxide levels under iron starvation were altered in both the 408-KO1 and 35S:408-14 lines. Taken together, these results suggest that Arabidopsis plants with modified miR408 levels undergo multiple deregulations under iron-deficient conditions.

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

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.

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 GA3. 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 GA3 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 GA3 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.

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, 14-30% 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.

Analysis of the developmental roles of the Arabidopsis gibberellin 20-oxidases demonstrates that GA20ox1, -2, and -3 are the dominant paralogs.

Gibberellin (GA) biosynthesis is necessary for normal plant development, with later GA biosynthetic stages being governed by multigene families. Arabidopsis thaliana contains five GA 20-oxidase (GA20ox) genes, and past work has demonstrated the importance of GA20ox1 and -2 for growth and fertility. Here, we show through systematic mutant analysis that GA20ox1, -2, and -3 are the dominant paralogs; their absence results in severe dwarfism and almost complete loss of fertility. In vitro analysis revealed that GA20ox4 has full GA20ox activity, but GA20ox5 catalyzes only the first two reactions of the sequence by which GA(12) is converted to GA(9). GA20ox3 functions almost entirely redundantly with GA20ox1 and -2 at most developmental stages, including the floral transition, while GA20ox4 and -5 have very minor roles. These results are supported by analysis of the gene expression patterns in promoter:ß-glucuronidase reporter lines. We demonstrate that fertility is highly sensitive to GA concentration, that GA20ox1, -2, and -3 have significant effects on floral organ growth and anther development, and that both GA deficiency and overdose impact on fertility. Loss of GA20ox activity causes anther developmental arrest, with the tapetum failing to degrade. Some phenotypic recovery of late flowers in GA-deficient mutants, including ga1-3, indicated the involvement of non-GA pathways in floral development.

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 signaling, 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 834bp of the 5' upstream promoter region. Putative DNA regulatory sequences involved in negative feed-back regulation and auxin response were identified in that promoter.

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) depends on gibberellins and auxins (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 (an inhibitor of GA biosynthesis). NPA-induced ovaries contained higher content of GA(1) (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 GA(1) and was counteracted by paclobutrazol 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 DWARF and SELF-PRUNING wild-type alleles.

Characterization of hemizygous deletions in citrus using array-comparative genomic hybridization and microsynteny comparisons with the poplar genome.

BACKGROUND: Many fruit-tree species, including relevant Citrus spp varieties exhibit a reproductive biology that impairs breeding and strongly constrains genetic improvements. In citrus, juvenility increases the generation time while sexual sterility, inbreeding depression and self-incompatibility prevent the production of homozygous cultivars. Genomic technology may provide citrus researchers with a new set of tools to address these various restrictions. In this work, we report a valuable genomics-based protocol for the structural analysis of deletion mutations on an heterozygous background. RESULTS: Two independent fast neutron mutants of self-incompatible clementine (Citrus clementina Hort. Ex Tan. cv. Clemenules) were the subject of the study. Both mutants, named 39B3 and 39E7, were expected to carry DNA deletions in hemizygous dosage. Array-based Comparative Genomic Hybridization (array-CGH) using a Citrus cDNA microarray allowed the identification of underrepresented genes in these two mutants. Subsequent comparison of citrus deleted genes with annotated plant genomes, especially poplar, made possible to predict the presence of a large deletion in 39B3 of about 700 kb and at least two deletions of approximately 100 and 500 kb in 39E7. The deletion in 39B3 was further characterized by PCR on available Citrus BACs, which helped us to build a partial physical map of the deletion. Among the deleted genes, ClpC-like gene coding for a putative subunit of a multifunctional chloroplastic protease involved in the regulation of chlorophyll b synthesis was directly related to the mutated phenotype since the mutant showed a reduced chlorophyll a/b ratio in green tissues. CONCLUSION: In this work, we report the use of array-CGH for the successful identification of genes included in a hemizygous deletion induced by fast neutron irradiation on Citrus clementina. The study of gene content and order into the 39B3 deletion also led to the unexpected conclusion that microsynteny and local gene colinearity in this species were higher with Populus trichocarpa than with the phylogenetically closer Arabidopsis thaliana. This work corroborates the potential of Citrus genomic resources to assist mutagenesis-based approaches for functional genetics, structural studies and comparative genomics, and hence to facilitate citrus variety improvement.

Auxin-induced fruit-set in tomato is mediated in part by gibberellins.

Tomato (Solanum lycopersicum L.) fruit-set and growth depend on gibberellins (GAs). Auxins, another kind of hormone, can also induce parthenocarpic fruit growth in tomato, although their possible interaction with GAs is unknown. We showed that fruit development induced by the auxins indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid (2,4-D) were significantly reduced by the simultaneous application of inhibitors of GA biosynthesis, and that this effect was reversed by the application of GA(3). This suggested that the effect of auxin was mediated by GA. Parthenocarpic fruits induced by 2,4-D had higher levels of the active GA(1), its precursors and metabolites, than unpollinated non-treated ovaries, but similar levels as those found in pollinated ovaries. Application experiments of radioactive-labelled GAs to unpollinated ovaries showed than 2,4-D altered GA metabolism (both biosynthesis and catabolism) in vivo. Transcript levels of genes encoding copalyldiphosphate synthase (SlCPS), SlGA20ox1, SlGA20ox2 and SlGA20ox3, and SlGA3ox1 were higher in unpollinated ovaries treated with 2,4-D. In contrast, transcript levels of SlGA2ox2 (out of the five SlGA2ox genes known to encode this kind of GA-inactivating enzyme) were lower in ovaries treated with 2,4-D. Our results support the idea that auxins induce fruit-set and growth in tomato, at least partially, by enhancing GA biosynthesis (GA 20-oxidase, GA 3-oxidase and CPS), and probably by decreasing GA inactivation (GA2ox2) activity, thereby leading to higher levels of GA(1). The expression of diverse Aux/indole-3-acetic acid (IAA) and auxin response factors, which may be involved in this effect of auxin, was also altered in 2,4-D-induced ovaries.

The gibberellin biosynthetic genes AtGA20ox1 and AtGA20ox2 act, partially redundantly, to promote growth and development throughout the Arabidopsis life cycle.

The activity of the gibberellin (GA) biosynthetic enzymes GA 20-oxidases (GA20ox) is of particular importance in determining GA concentration in many plant species. In Arabidopsis these enzymes are encoded by a family of five genes: AtGA20ox1-AtGA20ox5. Transcript analysis indicated that they have different expression patterns and may thus participate differentially in GA-regulated developmental processes. We have used reverse genetics to determine the physiological roles of AtGA20ox1 and AtGA20ox2, the most highly expressed GA20ox genes during vegetative and early reproductive development. AtGA20ox1 and AtGA20ox2 act redundantly to promote hypocotyl and internode elongation, flowering time, elongation of anther filaments, the number of seeds that develop per silique and elongation of siliques, with AtGA20ox1 making the greater contribution to internode and filament elongation, and AtGA20ox2 making the greater contribution to flowering time and silique length. Pollination of the double mutant with wild-type pollen indicated that the GA promoting silique elongation is of maternal origin. The ga20ox2 phenotype revealed that GA promotes the number of stem internodes that elongate upon bolting, and does so independently of its effect on internode elongation. Comparison of the phenotype of the double mutant with that of the highly GA-deficient ga1-3 mutant indicates that other GA20ox genes contribute to all the developmental processes examined, and, in some cases such as root growth and leaf expansion, make major contributions, as these processes were unaffected in the double mutant. In addition, the effects of the mutations are mitigated by the homeostatic mechanism that acts on expression of other GA dioxygenase and GID1 receptor genes.

Gibberellin regulation of fruit set and growth in tomato.

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 GA(3) application. LAB 198999 reduced GA(1) and GA(8) content, but increased that of their precursors GA(53), GA(44), GA(19), and GA(20) in pollinated fruits. This supports the hypothesis that GA(1) is the active GA for tomato fruit growth. Unpollinated ovaries developed parthenocarpically in response to GA(3) > GA(1) = GA(4) > GA(20), but not to GA(19), 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.

Conserved MYC transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis.

Jasmonates (JA) are important regulators of plant defense responses that activate expression of many wound-induced genes including the tomato proteinase inhibitor II (pin2) and leucine aminopeptidase (LAP) genes. Elements required for JA induction of the LAP gene are all present in the -317 to -78 proximal promoter region. Using yeast one-hybrid screening, we have identified the bHLH-leu zipper JAMYC2 and JAMYC10 proteins, specifically recognizing a T/G-box AACGTG motif in this promoter fragment. Mutation of the G-box element decreases JA-responsive LAP promoter expression. Expression of JAMYC2 and JAMYC10 is induced by JA, with a kinetics that precedes that of the LAP or pin2 transcripts. JAMYC overexpression enhanced JA-induced expression of these defense genes in potato, but did not result in constitutive transcript accumulation. Using footprinting assays, an additional protected element was identified, located directly adjacent to the T/G-box motif. Mutation of this element abolishes JA response, showing that recognition of this duplicated element is also required for gene expression. Knockout mutants in the AtMYC2 homolog gene of Arabidopsis are insensitive to JA and exhibit a decreased activation of the JA-responsive genes AtVSP and JR1. Activation of the PDF1.2 and b-CHI, ethylene/JA-responsive genes, is, however, increased in these mutants. These results show that the JAMYC/AtMYC2 transcription factors function as members of a MYC-based regulatory system conserved in dicotyledonous plants with a key role in JA-induced defense gene activation.