The 51 categories listed contained significant numbers of differentially expressed genes with a false discovery rate <0.27. For each functional category a colour between blue and red and the number of genes differentially regulated were assigned.Parthenocarpy is a desirable trait in many commercially grown fruit crops if undesirable changes to quality, flavour, or nutrition can be avoided. This work is the first to demonstrate the ability of INO to induce parthenocarpy and a significant reduction in the number of seeds. The INO gene is required for ovule development in Arabidopsis and expression is limited to the predictive initiation site and developing outer cell layer of the ovule outer integument . There are several lines of indirect evidence that suggest INO expression is ovule specific in tomato. The tomato INO promoter was fused to the Arabidopsis INO and green fluorescent protein coding regions . Expression was observed in only one cell layer in the outer integument. This was fully consistent with in situ hybridizations that showed this same expression pattern for the endogenous tomato INO gene . Although a thicker pericarp was observed in some parthenocarpic fruits, there were no significant changes to radial pericarp thickness in the four different types of transgenic tomatoes compared with wild-type tissues. Finally, the strongest piece of evidence that INO expression in tomato is ovule specific is the comparison with the corresponding expression of DefH9.

There was a strong overlap in the expression changes triggered by these two promoters, hydroponic dutch buckets with only a very few specific genes being expressed exclusively in either INO or DefH9 transgenic plants . Several transgenic plants were generated for each of the four possible promoter –gene combinations and evaluated for seedlessness. For both promoters, ;25–28% of transgenic tomato lines expressing iaaM lacked seeds; another 36–42% had only a few seeds . The remaining 33–36% had greatly diminished seed production. Of the transgenic plants expressing rolB, 20–33% lacked seeds, 40–45% had just a few seeds, and the remaining 22–40% had greatly diminished seed production . INO and DefH9 have similar effects when controlling expression of iaaM or rolB and produce similar proportions of seedless and reduced seed lines. Since several published works evaluated fruit quality and productivity of transgenic parthenocarpic fruits , the focus here was on fruit morphology and soluble solids content in order to measure differences between transgenic and wild-type fruits. MicroTom is a tomato cultivar suitable for analysis of fruit morphology but not fruit productivity. Only plants bearing completely parthenocarpic fruits were used for detailed transcriptome and metabolite analysis. Analysis was restricted to T0 plants as they produced very few seeds and the germination of these seeds was also low. Parthenocarpic fruits from plants transformed with DefH9-iaaM had reduced weight and equatorial diameter, but seeded and parthenocarpic INO-rolB fruits had increased polar and equatorial diameter. In DefH9-rolB plants, no clear relationship was observed between the presence of seeds and fruit diameter. No significant differences in soluble solids were found among wild-type and INO-iaaM, DefH9-iaaM, and INO-rolB fruits , but DefH9-rolB transgenic tomatoes with few seeds/fruit had more soluble solids than control fruit.

There were no significant differences among lines in the number of locules in individual fruits. Although differences were observed in diameters and weight between transgenic and wild-type fruits, no clear correlation between number of seeds, transgene, and fruit size was observed. Fruit growth is also controlled by the developing seeds, as parthenocarpic fruits are generally smaller than seeded fruits . The presence of seed-like structures that resemble pseudoembryos was observed. These were also found in auxin-induced fruit , and originate from divisions of cells of the inner integument . These seed-like structures in transgenic tomato fruits were hypothesized to substitute for the seeds in stimulating fruit growth . Carmi et al. found a positive correlation between rolB and soluble solids, which was directly affected by seedlessness. Tomatoes transformed with DefH9-iaaM had increased soluble solids, probably because seedless fruits reallocate assimilates from the seeds to the pericarp . Seedless fruit are more desirable than seeded: they are less acidic and have more soluble solids than seeded cultivars. DefH9-rolB lines with few seeds in the present study had increased soluble solids, but no significant changes were observed in the other transgenic lines.It is possible that the presence/absence of seeds could have significant effects on the surrounding carpel tissue. The Affymetrix tomato GeneChip was used to compare gene expression profiles of wild-type and transgenic fruits at the breaker stage and identify changes directly induced by transgene expression. At this stage, the formerly green MicroTom fruit had turned yellow, but not red. Although direct changes induced by genetic transformation in both the transcriptome and metabolome are likely to occur at earlier stages of development, it was expected to see longterm effects at the breaker stage, where the fruit has a clear and distinct phenotype that allows physiologically similar fruits to be compared.

At this very active physiological stage, many gene expression changes occur, emphasizing differences among transgenic and wild-type untransformed fruits. Using one-way ANOVA and multiple hypothesis testing, 1748 of 10 209 genes with significant variation were identified among wild-type and transgenic fruits. Thus, 83% of the genes analysed showed no significant differences in expression due to parthenocarpy. Pairwise comparison of the most differentially expressed genes showed 98 and 101 down-regulated genes , respectively, for rolB- and iaaM-transformed plants. There were also 13 and 21 upregulated genes , respectively, for iaaM- and rolB-transformed plants . Thus, only a small proportion of genes were regulated differently in all transgenic fruits than in wild-type fruits with seeds removed, and fewer genes were up-regulated than were down-regulated. Among promoters controlling the same transgene, there was much overlap between downregulated and up-regulated genes in DefH9- and INOtransformed plants, suggesting very similar effects of the two promoters on gene expression. This evidence strongly supports the hypothesis that the A. thaliana INO promoter drives ovule-specific expression in tomato. PCA was applied to 1748 genes with significant differences in expression . Principal component 1, accounting for 53% of the variance, was probably the result of genes affected by the transgene’s ovule-specific expression or by the absence of seeds. Principal component 2, accounting for 12.6% of the variance, represents gene expression modifications induced in different ways by iaaM and rolB ovule-specific expression, which act by different molecular mechanisms. iaaM encodes a tryptophanmonoxidase producing indolacetamide, which is converted either chemically or enzymatically to indole-3-acetic acid . RolB is a putative tyrosine phosphatase operating in auxin signalling . Despite extensive research, the actual function of the product of the rolB gene is still not clearly understood. Cluster analysis of individual genes confirmed the induction of six different gene expression patterns in transgenic lines . Genes in group 6 were up-regulated in at least one transgenic line compared with seeded or seedless wild type fruits. These genes do not have seed-specific expression, but are involved in pathways affected by the ovule specific expression of iaaM and rolB. Thus, they may play important roles in fruit quality and warrant further investigation. Functions of other genes up-regulated specifi- cally in iaaM-transformed or rolB-transformed fruits may be of interest to better characterize gene expression differences induced by ovule-specific expression of these genes. The roles of genes down-regulated in parthenocarpic transgenic fruits and those with seed-specific expression may also be of interest. Functional analysis of the significantly differently regulated genes was performed using MapMan software . GSEA was very useful for showing the most important changes taking place in seedless transgenic fruits . Among the 1748 differentially regulated genes, bato bucket those that followed a similar pattern in GSEA analysis and had similar functions were strongly supposed to be affected by the absence of seeds. Interestingly, one effect of the transgene modification was the up-regulation of lipid transfer proteins in all four different transgenic fruit types compared with wild-type fruits with seeds removed. LTPs and puroindolines can inhibit the growth of fungal pathogens in vitro and they are capable of synergistically enhancing the antimicrobial properties of other antimicrobial peptides such as defensins and thionins .

LTPsare also involved in the signalling of the defence mechanism of plants against their pathogens, recognizing membrane receptors involved in the transduction pathways of local defence responses . Although the accumulation of these proteins has to be confirmed, the upregulation of the transcript is intriguing for the possibility to increase the resistance of plants to biotic stresses. In this regard, over expression of these antimicrobial proteins induces significantly increased resistance of plants toward microbial pathogens . In addition, these antimicrobial proteins can help preserve fruit products. The two analyses were complementary and showed important similarities, providing a clearer picture of which gene categories were more affected by transgenic induction of parthenocarpy. The important conclusions of both analyses are outlined . Interestingly, iaaM-transformed fruits up-regulated genes involved in abiotic stress and three of the four types of transgenic fruits up-regulated genes involved in protein degradation. These effects are also important topics of further investigations. Among the down-regulated genes, short chain dehydrogenase genes and PHOR1 transcription factors were associated with rolB. Transgenic potato plants expressing an antisense PHOR1 construct had a semi-dwarf phenotype, displayed reduced response to GA application, and had more endogenous GAs than control plants , supporting the hypothesis that PHOR1 is a positive regulator of GA signalling . Another specific analysis of individual genes was performed by determining NCBI accession annotations of the 62 most differentially regulated genes with adjusted Pvalues <10 4 and clustering them into 19 functional categories . Among down-regulated genes, the functional categories most affected by gene expression changes were those involved in light reactions, transcription factors, and redox reactions , and flavonoid metabolism and storage proteins . Excluding genes with unknown function, these genes clustered primarily into the minor CHO metabolism, DNA synthesis, and transcription factor categories. In addition, several differentially regulated genes were involved in secondary metabolism and hormone metabolism. Another interesting category, transport/transporters, was represented by four differentially regulated genes: a cation exchanger, two sugar transporters, and nitrate transporter NRT1-3. Hormone metabolism genes such as those encoding Dwf1 , ERF/AP2 transcription factor, and allene oxide synthase were differentially regulated between transgenic and wild-type fruits. Interestingly, transgenic plants that overexpress dwarf4 in the brassinosteroidbiosynthesis pathway showed increased vegetative growth and seed yield, consistent with the result found here that a putative dwarf4 gene was highly down-regulated in transgenic seedless fruits. Although some transgenic tomatoes showed lower internodes, no clear correlation was observed between seedlessness and reduction of vegetative growth. MicroTom plants are naturally bushy and short, however, so any possible effect of rolB and iaaM ovule-specific expression on brassinosteroid biosynthetic genes and vegetative growth must be investigated in other tomato cultivars. Interestingly, the functional characterization showed that several ethylene- and IAA- associated genes were also down-regulated in transgenic parthenocarpic fruits , while others were up-regulated . Possible interactions between auxin and ethylene metabolism and perception are also of interest. EREBPs are both transcriptional activators and repressors in plants , and constitute a large gene family in tomato with important consequences for fruit softening and shelf life. Since some EREBPs induce ripening and others are repressed, Fei et al. proposed a model in which EREBPs dynamically regulate fruit ripening using antagonistic mechanisms. Among the IAA-responsive genes, down-regulation of an auxin-regulated protein in transgenic parthenocarpic fruits was confirmed using real-time RTPCR. This evidence agrees with previously published data that showed down-regulation of IAA-responsive genes associated with parthenocarpy such as the silencing of an auxin-responsive factor . Mutations in Arabidopsis ARF8, also referred to as Fruit Without Fertilization , cause fruit set in the absence of pollination and fertilization . Parthenocarpic fruits have been obtained through down regulation of IAA9, a tomato Aux/IAA family member . Recently it has been shown that auxins induce fruit set and growth in tomato, partially enhancing GA biosynthesis and decreasing GA inactivation, leading to more GA1 as observed in parthenocarpic fruits induced by 2,4-D. These conclusions were made after observation of more transcript for genes encoding copalyldiphosphate synthase , SlGA20ox1, SlGA20ox2, SlGA20ox3, and SlGA3ox1 in unpollinated ovaries treated with 2,4-D than in unpollinated untreated ovaries . Seedless fruit often has a longer shelf life than seeded fruit because seeds produce hormones such as ethylene that trigger senescence . Interestingly, the present data showed that IAA-responsive genes were down-regulated in all transgenic fruits compared with seedless or seeded wild-type fruits, implying that the ovule and ovary- driven expression of IAA and rolB induces down-regulation of other auxin-associated genes irrespective of seeds.