Genetic and molecular mechanisms underlying the parthenocarpic fruit mutation in tomato.

Parthenocarpy allows fruit set independently of fertilization. In parthenocarpic-prone tomato genotypes, fruit set can be achieved under pollen-limiting environmental conditions and in sterile mutants. Parthenocarpy is also regarded as a quality-related trait, when seedlessness is associated with positive fruit quality aspects. Among the different sources of genetic parthenocarpy described in tomato, the parthenocarpic fruit (pat) mutation is of particular interest because of its strong expressivity, high fruit set, and enhanced fruit quality. The complexity of the pat "syndrome" associates a strong competence for parthenocarpy with a complex floral phenotype involving stamen and ovule developmental aberrations. To understand the genetic basis of the phenotype, we mapped the pat locus within a 0.19-cM window of Chr3, comprising nine coding loci. A non-tolerated missense mutation found in the 14th exon of Solyc03g120910, the tomato ortholog of the Arabidopsis HD-Zip III transcription factor HB15 (SlHB15), cosegregated with the pat phenotype. The role of SlHB15 in tomato reproductive development was supported by its expression in developing ovules. The link between pat and SlHB15 was validated by complementation and knock out experiments by co-suppression and CRISPR/Cas9 approaches. Comparing the phenotypes of pat and those of Arabidopsis HB15 mutants, we argued that the gene plays similar functions in species with fleshy and dry fruits, supporting a conserved mechanism of fruit set regulation in plants.

Novel phenotypes related to the breeding of purple-fruited tomatoes and effect of peel extracts on human cancer cell proliferation.

The production of anthocyanins in the tomato (Solanum lycopersicum L.) fruit is normally absent or poor, but a number of mutants or introgression lines are known to increase anthocyanin levels in vegetative and reproductive tissues. Through conventional breeding, a genetic combination was obtained with the remarkable phenotype of a deep purple fruit pigmentation, due to an accumulation of anthocyanins on the peel. Such a genotype was named Sun Black (SB) as a consequence of its sensitivity to light induction. When characterized for morpho-agronomic traits, SB plants showed increased fertility. Purple fruits displayed an arrangement of the epicarp cells different from normal tomatoes, a feature that could account for different mechanical properties and shelf-life potential. The SB genotype and, to a lesser extent, its single mutant parents showed the capacity to accumulate anthocyanins in the seedling root when grown under light. This phenotype, which was greatly improved by the addition of sucrose to the germination medium, proved to be useful as selection index and gave new insights for in vitro production of anthocyanin extracts. To assess the nutraceutical potential of purple tomatoes, we tested the activity of SB skin extracts on the proliferation of two human cancer cells lines. Cell proliferation was significantly inhibited by SB extract in a dose-dependent manner. When the bioactivity of SB extracts was compared with that of other anthocyanin-containing fruits or vegetables, a significant "Extract*Line" interaction was evidenced, suggesting a crucial role for the extract composition in terms of anthocyanidins and other eventual cell growth-inhibiting compounds.

Constitutive co-suppression of the GA 20-oxidase1 gene in tomato leads to severe defects in vegetative and reproductive development.

To dissect the role of gibberellins in tomato development, we have constitutively down-regulated the gene GA 20-oxidase1 (GA20ox1). Plants co-suppressed for GA20ox1 (referred to as CO-6 plants) showed vegetative defects typical of GA deficiency such as darker and mis-shaped leaves and dwarfism. CO-6 plants flowered as the controls, although their flowers had subtle defects in the pedicel and in organ insertion. Analysis of male development revealed defects before, during and after meiosis, and a final pollen viability of 22%. The development of female organs and gametes appeared normal. Pollination experiments indicated that the pollen produced by CO-6 plants was able to fertilize control ovaries, but the analysis of the progeny showed that the construct was not transmitted. Ovaries of CO-6 plants showed high fruit set and normal fruit development when pollinated with control pollen. However these fruits were completely seedless due to a stenospermocarpic behaviour that was evidenced by callose layering in the endothelium between 7 and 15 days after pollination. We conclude that GA20ox1 in tomato exerts specific developmental roles that are not redundantly shared with other members of this gene family. For reproductive male development, silencing of this gene is detrimental for pollen production and either gametophytically lethal or severely hampering seed germination. In the pistil, the co-suppression construct does not affect the progamic phase, nor fruit set and growth, but it interferes with seed development after fertilization leading to seed abortion.

Characterization of genes controlling stamen identity and development in a parthenocarpic tomato mutant indicates a role for the DEFICIENS ortholog in the control of fruit set.

The development of the ovary into a fruit depends on pollination and fertilization. It has been proposed that the restriction of ovary growth before pollination is because of the stamens acting as negative regulators. Accordingly, the silencing of genes responsible for stamen identity has been correlated with parthenocarpy in different species. The tomato (Solanum lycopersicum L.) parthenocarpic fruit (pat) mutation associates autonomous ovary development with homeotic transformation of the anthers and aberrancy of ovules in the ovary. In this study, we tested the hypothesis that stamen aberrations and parthenocarpy in pat are driven by cues coming from the altered expression of class B MADS box genes. The data showed that the Pat locus is not allelic to either of the two tomato mutations putatively involved in the B function, stamenless (sl)-2 and pistillate (pi) or to genes encoding class B transcription factors. Whereas pat pi double mutants were not recovered because of tight linkage, pat sl-2 double mutants showed mainly epistatic effects. The developmental regulation of the Sl DEFICIENS (DEF) gene in the wild-type (WT) at anthesis as well as its differential transcription in the pat ovary suggest that it plays a role in the control of ovary growth. Accordingly, when compared with the WT, the gene was also differentially expressed in the parthenocarpic fruit-2 (pat-2) mutant, that is not allelic to pat and has normal ovule development. Altogether the results indicate that in tomato SlDEF plays a role in the control of ovary growth and that the pat mutation is located upstream of this regulatory cascade.

Genetic diversity, structure and marker-trait associations in a collection of Italian tomato (Solanum lycopersicum L.) landraces.

The study of phenotypic and genetic diversity in landrace collections is important for germplasm conservation. In addition, the characterisation of very diversified materials with molecular markers offers a unique opportunity to define significant marker-trait associations of biological and agronomic interest. Here, 50 tomato landraces (mainly collected in central Italy), nine vintage and modern cultivars, and two wild outgroups were grown at two locations in central Italy and characterised for 15 morpho-physiological traits and 29 simple sequence repeat (SSR) loci. The markers were selected to include a group of loci in regions harbouring reported quantitative trait loci (QTLs) that affect fruit size and/or shape (Q-SSRs) and a group of markers that have not been mapped or shown to have a priori known linkage (NQ-SSRs). As revealed by univariate and multivariate analyses of morphological data, the landraces grouped according to vegetative and reproductive traits, with emphasis on fruit size, shape and final destination of the product. Compared to the low molecular polymorphism reported in tomato modern cultivars, our data reveal a high level of molecular diversity in landraces. Such diversity has allowed the inference of the existence of a genetic structure that was factored into the association analysis. As the proportion of significant associations is higher between the Q-SSR subset of markers and the subset of traits related to fruit size and shape than for all of the other combinations, we conclude that this approach is valid for establishing true-positive marker-trait relationships in tomato.

Tomato fruit set driven by pollination or by the parthenocarpic fruit allele are mediated by transcriptionally regulated gibberellin biosynthesis.

We investigated the role of gibberellins (GAs) in the phenotype of parthenocarpic fruit (pat), a recessive mutation conferring parthenocarpy in tomato (Solanum lycopersicum L.). Novel phenotypes that parallel those reported in plants repeatedly treated with gibberellic acid or having a GA-constitutive response indicate that the pat mutant probably expresses high levels of GA. The retained sensitivity to the GA-biosynthesis inhibitor paclobutrazol reveals that this condition is dependent on GA biosynthesis. Expression analysis of genes encoding key enzymes involved in GA biosynthesis shows that in normal tomato ovaries, the GA20ox1 transcript is in low copy number before anthesis and only pollination and fertilization increase its transcription levels and, thus, GA biosynthesis. In the unpollinated ovaries of the pat mutant, this mechanism is de-regulated and GA20ox1 is constitutively expressed, indicating that a high GA concentration could play a part in the parthenocarpic phenotype. The levels of endogenous GAs measured in the floral organs of the pat mutant support such a hypothesis. Collectively, the data indicate that transcriptional regulation of GA20ox1 mediates pollination-induced fruit set in tomato and that parthenocarpy in pat results from the mis-regulation of this mechanism. As genes involved in the control of GA synthesis (LeT6, LeT12 and LeCUC2) and response (SPY) are also altered in the pat ovary, it is suggested that the pat mutation affects a regulatory gene located upstream of the control of fruit set exerted by GAs.

Tomato transgenic lines and Tetranychus urticae: changes in plant suitability and susceptibility.

A critical aspect dealing with the use of transgenic plants is the global evaluation of their environmental impact. The polyphagous mite Tetranychus urticae can be considered a suitable species to investigate unpredictable and undesirable effects on phytophagous arthropods. Three tomato near isogenic lines, that is, the cv. Riogrande (RIG), the transgenic lines RC332 (containing the Gox gene and showing high glucose oxidase activity), and MS498 (containing the KTI3 gene and exhibiting a high trypsin inhibition) were used in laboratory and greenhouse trials. Trichomes and contents of C and N of the leaves, differences in development and oviposition of T. urticae and damage caused were evaluated for each line. The laboratory trials evidenced that (1) the intrinsic rate of increase of two strains of T. urticae (T from tomato, B from bindweed), reared on the lower surface of tomato leaflets, was significantly lower in RIG than in transgenic lines and doubling time ranged between 6.9 and 11.6 days in the first and between 3.9 and 5.3 days in the latter; (2) the glandular four-lobed trichomes were always higher in RIG than in other genotypes; (3) the N leaf content was from 1.3 to 1.9 fold lower and the C/N ratio from 1.3 to 1.9 fold higher in RIG than in other lines. The greenhouse experiment, that lasted over a month and was performed by inducing an initially equal infestation of strain T, evidenced: (1) no significant difference between plant lines in the final mite infestation (motile stages per plant), nevertheless an almost double number of spider mites was counted in RC332; (2) a significantly higher percentage of damaged leaves and a significant higher average damage index on RC332 than on RIG (79% and 2.3 in the former, and 62% and 2.1 in the latter, respectively), even if in both transgenics a higher level of the most severe damages and a shorter time to approach them were observed; (4) a comparable number of mites causing the same damage level in all genotypes and a strong linear relation between the first four levels of damage and mite infestation. Although in the laboratory studies both transgenic lines enhanced the T. urticae population increase, the glasshouse studies were not as conclusive and they only suggest the possibility of any real difference between the transgenic and non-transgenic genotypes.

A H2O2-forming peroxidase rather than a NAD(P)H-dependent O2•- synthase may be the major player in cell death responses controlled by the Pto-Fen complex following fenthion treatment.

Four tomato (Lycopersicon esculentum Mill.) near-isogenic lines were treated by foliar spraying with the insecticide fenthion. Two, Riogrande and Rimone, differed from each other only for the presence in the latter of the Fen gene, conferring propensity to develop foliar symptoms upon exposure to fenthion. The other two, namely RC332 and RC131, were the transgenic versions of Riogrande and Rimone, respectively, harbouring the Gox gene encoding for glucose oxidase of Aspergillus niger. The production of H2O2 as well as the activities of H+-ATPase, NAD(P)H-dependent superoxide synthase, catalase, peroxidase, and Cu,Zn-superoxide dismutase were evaluated in the foliar tissues up to 24 h after exposure to fenthion. The Fen gene conferred sensitivity to fenthion, regardless of the expression of a Gox transgene. A prolonged accumulation of H2O2 was observed in the leaves of Rimone and of RC131, which was instead transient in Riogrande and in RC332. In all the tomato lines, exposure to fenthion induced rapid but transient changes in the activities of most enzymes. The only exception was peroxidase activity in the leaves of Rimone and of RC131, which steadily increased until the end of the sampling period. It is suggested that the sensitivity of Rimone to fenthion might be due to the sustained activity of a H2O2-forming peroxidase.

The Cf-2 / Rcr3esc gene interaction in tomato (Lycopersicon esculentum) induces autonecrosis and triggers biochemical markers of oxidative burst at cellular level.

A tomato plant (Lycopersicon esculentum Mill.) with necrotic leaf spots mimicking disease lesions was singled out in progeny under selection in Moscow (breeding material of Ignatova Svetlana). The progeny from spontaneous selfing of such a plant (V20368), in the presence of increasing temperature and high light intensity, exhibited spontaneous necrotic lesions on the leaves, with acropetal progression (autonecrosis). A similar phenotype, described in 1948 by Langford, appeared to be associated with the Cf-2 resistance gene, introgressed from L. pimpinellifolium. Recently, Kruger et al. (2002) demonstrated that the Cf-2 effect depends on a second gene (Rcr3pim) encoding a cysteine protease, and that autonecrosis is activated by the contemporary presence of Cf-2 and the L. esculentum allele Rcr3esc. In this work we characterised the V20368 mutant and verified that autonecrosis is caused by the presence of an interaction between Cf-2 and Rcr3esc. When the environmental conditions are favourable, this interaction triggers an oxidative burst, as evidenced by a strong increase in H2O2 production and activities of catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7) and ATPase (EC 3.6.1.3). In addition, by grafting the necrotic mutant on the cv. Riogrande and vice versa, we proved that the necrotic phenotype is not associated with the movement of a signal molecule, since the autonecrosis was not transmitted across the grafting point. Finally, the interaction between Cf-2 and Rcr3esc appeared to lower the threshold of stress perception, as evidenced by an increased sensitivity to the insecticide Fenthion.