Functional analysis of MYB alleles from Solanum chilense and Solanum lycopersicum in controlling anthocyanin levels in heterologous tobacco plants.

Flavonoids are natural pigments occurring in plants and are present in fruits, leaves, stems, roots, and flowers. Tobacco plants transformed with an MYB regulatory gene from either Solanum chilense (Sc) or S. lycopersicum (Sl) demonstrate that ScANT1 induces a higher level of anthocyanin accumulation in comparison to SlANT1 and that this gene is sufficient to promote increased anthocyanin levels. We compared the aptitude of ScANT1 protein to induce anthocyanin accumulation to that of SlANT1 protein in tobacco plants. We also tested the effect of amino acid substitutions in ScANT1 and SlANT1. We examined these synthetic alleles' effect following the over-expression of additional anthocyanin synthesis regulators, such as the tomato bHLH (SlJAF13) protein. Our results show that the amino acid changes that differentiate ScANT1 from SlANT1 are the main contributors to the advantage that ScANT1 has over SlANT1 in anthocyanin accumulation per transcript unit. We further demonstrated that altering the amino acid composition of SlANT1 could increase anthocyanin accumulation, while reciprocally modifying ScANT1 lowers the anthocyanin level. These results confirm the increased anthocyanin level in tobacco is attributed to the amino acid differences between ScANT1 and SlANT1. We also show that the co-expression of SlJAF13 with SlANT1 in tobacco plants represses the anthocyanin production.

Natural genetic variation for expression of a SWEET transporter among wild species of Solanum lycopersicum (tomato) determines the hexose composition of ripening tomato fruit.

The sugar content of Solanum lycopersicum (tomato) fruit is a primary determinant of taste and quality. Cultivated tomato fruit are characterized by near-equimolar levels of the hexoses glucose and fructose, derived from the hydrolysis of translocated sucrose. As fructose is perceived as approximately twice as sweet as glucose, increasing its concentration at the expense of glucose can improve tomato fruit taste. Introgressions of the FgrH allele from the wild species Solanum habrochaites (LA1777) into cultivated tomato increased the fructose-to-glucose ratio of the ripe fruit by reducing glucose levels and concomitantly increasing fructose levels. In order to identify the function of the Fgr gene, we combined a fine-mapping strategy with RNAseq differential expression analysis of near-isogenic tomato lines. The results indicated that a SWEET protein was strongly upregulated in the lines with a high fructose-to-glucose ratio. Overexpressing the SWEET protein in transgenic tomato plants dramatically reduced the glucose levels and increased the fructose : glucose ratio in the developing fruit, thereby proving the function of the protein. The SWEET protein was localized to the plasma membrane and expression of the SlFgr gene in a yeast line lacking native hexose transporters complemented growth with glucose, but not with fructose. These results indicate that the SlFgr gene encodes a plasma membrane-localized glucose efflux transporter of the SWEET family, the overexpression of which reduces glucose levels and may allow for increased fructose levels. This article identifies the function of the tomato Fgr gene as a SWEET transporter, the upregulation of which leads to a modified sugar accumulation pattern in the fleshy fruit. The results point to the potential of the inedible wild species to improve fruit sugar accumulation via sugar transport mechanisms.

Using genomic analysis to identify tomato Tm-2 resistance-breaking mutations and their underlying evolutionary path in a new and emerging tobamovirus.

In September 2014, a new tobamovirus was discovered in Israel that was able to break Tm-2-mediated resistance in tomato that had lasted 55 years. The virus was isolated, and sequencing of its genome showed it to be tomato brown rugose fruit virus (ToBRFV), a new tobamovirus recently identified in Jordan. Previous studies on mutant viruses that cause resistance breaking, including Tm-2-mediated resistance, demonstrated that this phenotype had resulted from only a few mutations. Identification of important residues in resistance breakers is hindered by significant background variation, with 9-15% variability in the genomic sequences of known isolates. To understand the evolutionary path leading to the emergence of this resistance breaker, we performed a comprehensive phylogenetic analysis and genomic comparison of different tobamoviruses, followed by molecular modeling of the viral helicase. The phylogenetic location of the resistance-breaking genes was found to be among host-shifting clades, and this, together with the observation of a relatively low mutation rate, suggests that a host shift contributed to the emergence of this new virus. Our comparative genomic analysis identified twelve potential resistance-breaking mutations in the viral movement protein (MP), the primary target of the related Tm-2 resistance, and nine in its replicase. Finally, molecular modeling of the helicase enabled the identification of three additional potential resistance-breaking mutations.

A Novel Route Controlling Begomovirus Resistance by the Messenger RNA Surveillance Factor Pelota.

Tomato yellow leaf curl virus (TYLCV) is a devastating disease of tomato (Solanum lycopersicum) that can be effectively controlled by the deployment of resistant cultivars. The TYLCV-resistant line TY172 carries a major recessive locus for TYLCV resistance, designated ty-5, on chromosome 4. In this study, the association between 27 polymorphic DNA markers, spanning the ty-5 locus, and the resistance characteristics of individual plants inoculated with TYLCV in 51 segregating recombinant populations were analyzed. These analyses localized ty-5 into a 425 bp region containing two transversions: one in the first exon of a gene encoding the tomato homolog of the messenger RNA surveillance factor Pelota (Pelo), and a second in its proximal promoter. Analyses of susceptible and resistant lines revealed that the relative transcript level of the gene remained unchanged, regardless of whether the plants were infected with TYLCV or not. This suggests that the polymorphism discovered in the coding region of the gene controls the resistance. Silencing of Pelo in a susceptible line rendered the transgenic plants highly resistant, while in the resistant line TY172 had no effect on symptom development. In addition, over-expression of the susceptible allele of the gene in the resistant TY172 line rendered it susceptible, while over-expression of the resistant allele in susceptible plants had no effect. These results confirm that Pelo is the gene controlling resistance at the ty-5 locus. Pelo, implicated in the ribosome recycling-phase of protein synthesis, offers an alternative route to promote resistance to TYLCV and other viruses.

ANTHOCYANIN1 from Solanum chilense is more efficient in accumulating anthocyanin metabolites than its Solanum lycopersicum counterpart in association with the ANTHOCYANIN FRUIT phenotype of tomato.

Anthocyanins are flavonoid metabolites contributing attractive colors and antioxidant qualities to the human diet. Accordingly, there is a growing interest in developing crops enriched with these compounds. Fruits of the cultivated tomato, Solanum (S.) lycopersicum, do not normally produce high levels of anthocyanins. However, several wild tomato species yield anthocyanin-pigmented fruits, and this trait has been introgressed into the cultivated tomato. Two genes encoding homologous R2R3 MYB transcription factors, termed ANT1 and AN2, were previously genetically implicated in anthocyanin accumulation in tomato fruit peels of the ANTHOCYANIN FRUIT (AFT) genotype originating from S. chilense. Here we compared transgenic tomato plants constitutively over-expressing the S. lycopersicum (35S::ANT1 ( L ) ) or the S. chilense (35S::ANT1 ( C )) allele, and show that each displayed variable levels of purple pigmentation in vegetative as well as reproductive tissues. However, 35S::ANT1 ( C ) was significantly more efficient in producing anthocyanin pigments, attributed to its gene coding-sequence rather than to its transcript levels. These results expand the potential of enhancing anthocyanin levels through engineering coding-sequence polymorphisms in addition to the transcriptional alterations commonly used. In addition, a segregating population obtained from a recombinant genotype revealed that the native ANT1, and not AN2, is fully associated with the AFT phenotype and that ANT1 alone can generate the characteristic phenotype of anthocyanin accumulation in AFT fruits. Our results therefore provide further support to the hypothesis that ANT1 is the gene responsible for anthocyanin accumulation in fruits of the AFT genotype.

Expressing yeast SAMdc gene confers broad changes in gene expression and alters fatty acid composition in tomato fruit.

Tomato (Solanum lycopersicum) fruits expressing a yeast S-adenosyl methionine decarboxylase (ySAMdc) gene under control of a ripening-induced promoter show altered phytonutrient content and broad changes in gene expression. Genome-wide transcriptional alterations in pericarp tissues of the ySAMdc-expressing fruits are shown. Consistent with the ySAMdc expression pattern from the ripening-induced promoter, very minor transcriptional alterations were detected at the mature green developmental stage. At the breaker and red stages, altered levels of numerous transcripts were observed with a general tendency toward upregulation in the transgenic fruits. Ontological analysis of up- and downregulated transcript groups revealed various affected metabolic processes, mainly carbohydrate and amino acid metabolism, and protein synthesis, which appeared to be intensified in the ripening transgenic fruits. Other functional ontological categories of altered transcripts represented signal transduction, transcription regulation, RNA processing, molecular transport and stress response, as well as metabolism of lipids, glycans, xenobiotics, energy, cofactors and vitamins. In addition, transcript levels of genes encoding structural enzymes for several biosynthetic pathways showed strong correlations to levels of specific metabolites that displayed altered levels in transgenic fruits. Increased transcript levels of fatty acid biosynthesis enzymes were accompanied by a change in the fatty acid profile of transgenic fruits, most notably increasing ω-3 fatty acids at the expense of other lipids. Thus, SAMdc is a prime target in manipulating the nutritional value of tomato fruits. Combined with analyses of selected metabolites in the overripe fruits, a model of enhanced homeostasis of the pericarp tissue in the polyamine-accumulating tomatoes is proposed.

Identification and Mapping of Tomato Genome Loci Controlling Tolerance and Resistance to Tomato Brown Rugose Fruit Virus.

Tomato brown rugose fruit virus (ToBRFV) was identified in Israel during October 2014 in tomato plants (Solanum lycopersicum). These plants, carrying the durable resistance gene against tomato mosaic virus, Tm-22 , displayed severe disease symptoms and losses to fruit yield and quality. These plants were found infected with a tobamovirus similar to that discovered earlier in Jordan. This study was designed to screen and identify tomato genotypes resistant or tolerant to ToBRFV. The identified resistance and tolerance traits were further characterized virologically and genetically. Finally, DNA markers linked to genes controlling these traits were developed as tools to expedite resistance breeding. To achieve these objectives, 160 genotypes were screened, resulting in the identification of an unexpectedly high number of tolerant genotypes and a single genotype resistant to the virus. A selected tolerant genotype and the resistant genotype were further analyzed. Analysis of genetic inheritance revealed that a single recessive gene controls tolerance whereas at least two genes control resistance. Allelic test between the tolerant and the resistant genotype revealed that these two genotypes share a locus controlling tolerance, mapped to chromosome 11. This locus displayed a strong association with the tolerance trait, explaining nearly 91% of its variation in segregating populations. This same locus displayed a statistically significant association with symptom levels in segregating populations based on the resistant genotype. However, in these populations, the locus was able to explain only ~41% of the variation in symptom levels, confirming that additional loci are involved in the genetic control of the resistance trait in this genotype. A locus on chromosome 2, at the region of the Tm-1 gene, was finally found to interact with the locus discovered on chromosome 11 to control resistance.

Overexpression of UV-DAMAGED DNA BINDING PROTEIN 1 links plant development and phytonutrient accumulation in high pigment-1 tomato.

Fruits of tomato plants carrying the high pigment-1 mutations hp-1 and hp-1(w) are characterized by an increased number of plastids coupled with enhanced levels of functional metabolites. Unfortunately, hp-1 mutant plants are also typified by light-dependent retardation in seedling and whole-plant growth and development, which limits their cultivation. These mutations were mapped to the gene encoding UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1) and, recently, fruit-specific RNA interference studies have demonstrated an increased number of plastids and enhanced carotenoid accumulation in the transgenic tomato fruits. However, whole-plant overexpression of DDB1, required to substantiate its effects on seedling and plant development and to couple them with fruit phenotypes, has heretofore been unsuccessful. In this study, five transgenic lines constitutively overexpressing normal DDB1 in hp-1 mutant plants were analysed. Eleven-day-old seedlings, representing these lines, displayed up to approximately 73- and approximately 221-fold overexpression of the gene in hypocotyls and cotyledons, respectively. This overexpression resulted in statistically significant reversion to the non-mutant developmental phenotypes, including more than a full quantitative reversion. This reversion of phenotypes was generally accompanied by correlated responses in chlorophyll accumulation and altered expression of selected light signalling genes: PHYTOCHROME A, CRYPTOCHROME 1, ELONGATED HYPOCOTYL 5, and the gene encoding CHLOROPHYLL A/B-BINDING PROTEIN 4. Cumulatively, these results provide the missing link between DDB1 and its effects on tomato plant development.

Molecular dissection of Tomato leaf curl virus resistance in tomato line TY172 derived from Solanum peruvianum.

Tomato yellow leaf curl virus (TYLCV) is devastating to tomato (Solanum lycopersicum) crops and resistant cultivars are highly effective in controlling the disease. The breeding line TY172, originating from Solanum peruvianum, is highly resistant to TYLCV. To map quantitative trait loci (QTLs) controlling TYLCV resistance in TY172, appropriate segregating populations were analyzed using 69 polymorphic DNA markers spanning the entire tomato genome. Results show that TYLCV resistance in TY172 is controlled by a previously unknown major QTL, originating from the resistant line, and four additional minor QTLs. The major QTL, we term Ty-5, maps to chromosome 4 and accounts for 39.7-46.6% of the variation in symptom severity among segregating plants (LOD score 33-35). The minor QTLs, originated either from the resistant or susceptible parents, were mapped to chromosomes 1, 7, 9 and 11, and contributed 12% to the variation in symptom severity in addition to Ty-5.

Molecular aspects of Anthocyanin fruit tomato in relation to high pigment-1.

The tomato Anthocyanin fruit (Aft) genotype is characterized by purple color in skin and outer pericarp of its fruits due to higher levels of anthocyanins-flavonoid metabolites. Our objectives were to carry out metabolic and molecular characterization of this genotype, emphasizing its interaction with the high pigment-1 (hp-1) mutation, known to increase flavonoids in tomato fruits. These objectives fit the growing interest in developing tomato fruits with higher levels of functional metabolites. Our results show that 1) Aft fruits are also characterized by significantly higher levels of the flavonols quercetin and kaempferol, thus enhancing their functional value; 2) the tomato Anthocyanin1 (Ant1) gene, encoding a Myb transcription factor, displayed nucleotide and amino acid polymorphisms between the Aft genotype and cultivated genotypes; 3) a DNA marker based on Ant1 showed that the Aft trait is encoded by a single locus on chromosome 10 fully associated with Ant1; and 4) double homozygotes Aft/Aft hp-1/hp-1 plants displayed a more-than-additive effect on the production of fruit anthocyanidins and flavonols. This effect was manifested by approximately 5-, 19-, and 33-fold increase of petunidin, malvidin, and delphinidin, respectively, in the double mutants compared with the cumulative levels of their parental lines.

A New Israeli Tobamovirus Isolate Infects Tomato Plants Harboring Tm-22 Resistance Genes.

An outbreak of a new disease infecting tomatoes occurred in October-November 2014 at the Ohad village in Southern Israel. Symptomatic plants showed a mosaic pattern on leaves accompanied occasionally by narrowing of leaves and yellow spotted fruit. The disease spread mechanically and rapidly reminiscent of tobamovirus infection. Epidemiological studies showed the spread of the disease in various growing areas, in the South and towards the Southeast and Northern parts of the country within a year. Transmission electron microscope (TEM) analysis showed a single rod-like form characteristic to the Tobamovirus genus. We confirmed Koch's postulates for the disease followed by partial host range determination and revealed that tomato cultivars certified to harbor the Tm-22 resistance gene are susceptible to the new viral disease. We further characterized the viral source of the disease using a range of antisera for serological detection and analyzed various virus genera and families for cross-reactivity with the virus. In addition, next generation sequencing of total small RNA was performed on two cultivars grown in two different locations. In samples collected from commercial cultivars across Israel, we found a single virus that caused the disease. The complete genome sequence of the new Israeli tobamovirus showed high sequence identity to the Jordanian isolate of tomato brown rugose fruit virus.

Sucrose uptake, invertase localization and gene expression in developing fruit of Lycopersicon esculentum and the sucrose-accumulating Lycopersicon hirsutum.

By using immunolocalization and differential extraction methods we show that only apoplastic invertase, but not vacuolar invertase, was present in the mature, sucrose-accumulating L. hirsutum pericarp. In contrast, in the hexose-accumulating L. esculentum fruit, both the apoplastic and vacuolar invertase activities and protein content increase in the mature fruit. Quantitative expression studies of the soluble invertase gene (TIV1) and the apoplastic invertase genes (LINs) showed that only TIV1 gene expression could account for the species and developmental differences of both soluble and insoluble enzyme activity of the pericarp. The expression of the LIN genes encoding for apoplastic tomato invertases was unrelated to the differences in bound enzyme activity and could not account for the rise in bound invertase activity in the mature L. esculentum fruit. Evidence is presented that the bound invertase activity of tomato fruit is also the TIV1 gene product. The presence of apoplastic invertase in the mature sucrose-accumulating L. hirsutum fruit suggests a hydrolysis-resynthesis mechanism of sucrose uptake. In order to test this hypothesis, we studied short- and long-term uptakes of asymmetrically labelled 3H-fructosyl-sucrose accompanied by compartmental analysis of the sugars in attached whole fruits of L. hirsutum and L. esculentum. The results indicate that hydrolysis-resynthesis is slow in the sucrose-accumulating fruit but is not an integral part of an uptake and compartmentation mechanism.

Using genetically modified tomato crop plants with purple leaves for absolute weed/crop classification.

BACKGROUND: Weed/crop classification is considered the main problem in developing precise weed-management methodologies, because both crops and weeds share similar hues. Great effort has been invested in the development of classification models, most based on expensive sensors and complicated algorithms. However, satisfactory results are not consistently obtained due to imaging conditions in the field. RESULTS: We report on an innovative approach that combines advances in genetic engineering and robust image-processing methods to detect weeds and distinguish them from crop plants by manipulating the crop's leaf color. We demonstrate this on genetically modified tomato (germplasm AN-113) which expresses a purple leaf color. An autonomous weed/crop classification is performed using an invariant-hue transformation that is applied to images acquired by a standard consumer camera (visible wavelength) and handles variations in illumination intensities. CONCLUSION: The integration of these methodologies is simple and effective, and classification results were accurate and stable under a wide range of imaging conditions. Using this approach, we simplify the most complicated stage in image-based weed/crop classification models.

Optical Absorption and Visible Photoluminescence from Thin Films of Silicon Phthalocyanine Derivatives.

The interest of microelectronics industry in new organic compounds for the manufacture of luminescent devices has increased substantially in the last decade. In this paper, we carried out a study of the usage feasibility of three organic bidentate ligands (2,6-dihydroxyanthraquinone, anthraflavic acid and potassium derivative salt of anthraflavic acid) for the synthesis of an organic semiconductor based in silicon phthalocyanines (SiPcs). We report the visible photoluminescence (PL) at room temperature obtained from thermal-evaporated thin films of these new materials. The surface morphology of these films was analyzed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). AFM indicated that the thermal evaporation technique is an excellent resource in order to obtain low thin film roughness when depositing these kinds of compounds. Fourier transform infrared spectroscopy (FTIR) spectroscopy was employed to investigate possible changes in the intra-molecular bonds and to identify any evidence of crystallinity in the powder compounds and in the thin films after their deposition. FTIR showed that there was not any important change in the samples after the thermal deposition. The absorption coefficient (α) in the absorption region reveals non-direct transitions. Furthermore, the PL of all the investigated samples were observed with the naked eye in a bright background and also measured by a spectrofluorometer. The normalized PL spectra showed a Stokes shift ≈ 0.6 eV in two of our three samples, and no PL emission in the last one. Those results indicate that the Vis PL comes from a recombination of charge carriers between conduction band and valence band preceded by a non-radiative relaxation in the conduction band tails.

Light signaling genes and their manipulation towards modulation of phytonutrient content in tomato fruits.

Due to its economic importance, ease of genetic manipulation, cultivation and processing, the tomato plant has been a target for increasing and diversifying content of fruit phytonutrients by transgenic and non-transgenic approaches. The tomato high pigment (hp) mutations exemplify the latter alternative and due to their positive effect on fruit lycopene content, they were introgressed into elite tomato germplasm for cost effective extraction of this important carotenoid. Interestingly, hp mutant fruits are also characterized by higher fruit levels of other functional metabolites, phenotypes caused by mutations in central genes regulating light signal-transduction. This gene identification suggests that modulation of light signaling machinery in plants may be highly effective towards manipulation of fruit phytonutrients but has never been thoroughly reviewed. This review therefore summarizes the progress which has been made on this valuable approach, emphasizing the consequences of transgenic modulation of light signaling components on the functional properties of the tomato fruit.

Characterization of the intense pigment tomato genotype emphasizing targeted fruit metabolites and chloroplast biogenesis.

The tomato INTENSE PIGMENT (IP) genotype is characterized by intense visual pigmentation of unripe and ripe fruits, not thoroughly analyzed thus far. This study was therefore designed to analyze key morphologic, metabolomic, and photomorphogenic phenotypes of this genotype in comparison to its near-isogenic normal counterpart and to evaluate its significance relative to other tomato mutants known for increased fruit pigmentation. The IP genotype produced smaller and darker red fruits, and a substantially increased chloroplast biogenesis was found in its green fruit and leaf tissues. Ripe-red fruits of the IP genotype produced 34-38% more soluble solids and up to 62.6% more carotenoids, but no differences were found in the concentration of flavonoid compounds in its peel tissue. The IP genotype was found to display a greater hypocotyl inhibition response to blue and yellow light, but a more prominent and novel response to total darkness. As a whole, the IP genotype exhibited highly desirable traits, making it a valuable genotype for tomato breeders attempting to introduce functional and taste qualities into tomato fruits.

A mutation in the tomato DDB1 gene affects cell and chloroplast compartment size and CDT1 transcript.

Fruits harvested from tomato (Solanum lycopersicum) plants carrying mutations at the DAMAGED DNA BINDING PROTEIN 1 gene (SlDDB1; hp-1 and hp-1(w)) are characterized by significantly elevated levels of lycopene and several other phytonutrients. We hypothesize that the pleotropic effect on mutant Slddb1 are some general function of DDB1 protein on cell growth. The main objective of this research was to carry out functional analysis of the mutant SlDDB1 alleles both in-planta and ex-planta in comparison to their normal counterparts. Our major results indicate that: mutant Slddb1 seedlings exhibited delayed growth and smaller cell size, greater chloroplast density, smaller chloroplasts and higher concentration of chlorophyll.Cotyledons cells of Slddb1 mutant also displayed abnormal stomatal pattern, reduced content of CDT1 transcript and altered response to cytokinin. Some of these observations were previously described to be connected to defects in cell cycle control. Our results, coupled with former studies, also suggest that the CDD complex (composed of DDB1, DET1 and COP10) mediate the effect of light and cytokinin activity by possibly regulating the replication licensing factor CDT1 thus affecting both cell size and plastid multiplication.

Susceptibility of the tomato mutant high pigment-2dg (hp-2dg) to Orobanche spp. infection.

The consumption of natural products with potential health benefits has been continuously growing, and enhanced pigmentation is of major economic importance in fruits and vegetables. The tomato hp-2 ( dg ) is an important mutant line that has been introgressed into commercial tomato cultivars marketed as lycopene rich tomatoes (LRT) because of their enhanced fruit pigmentation, attributed to higher levels of carotenoids, including lycopene. Strigolactones are signaling compounds that mediate host finding in root parasitic plants and are biosynthetically derived from carotenoids. Considering the high carotenoid content of the hp-2 ( dg ) mutant, we studied its susceptibility to the root parasite Orobanche. In a field experiment, the average number of Orobanche aegyptiaca plants growing on hp-2 ( dg ) was surprisingly significantly reduced compared with its isogenic wild-type counterpart. In vitro assays and LC-MS/MS analysis showed that this reduction was associated with a lower production of strigolactones, which apparently renders the high-carotenoid hp-2 ( dg ) mutant less susceptible to Orobanche.

Transcriptional profiling of high pigment-2dg tomato mutant links early fruit plastid biogenesis with its overproduction of phytonutrients.

Phenotypes of the tomato (Solanum lycopersicum) high pigment-2(dg) (hp-2(dg)) and hp-2(j) mutants are caused by lesions in the gene encoding DEETIOLATED1, a negative regulator of light signaling. Homozygous hp-2(dg) and hp-2(j) plants display a plethora of distinctive developmental and metabolic phenotypes in comparison to their normal isogenic counterparts. These mutants are, however, best known for the increased levels of carotenoids, primarily lycopene, and other plastid-accumulating functional metabolites. In this study we analyzed the transcriptional alterations in mature-green, breaker, and early red fruits of hp-2(dg)/hp-2(dg) plants in relation to their normal counterparts using microarray technology. Results show that a large portion of the genes that are affected by hp-2(dg) mutation display a tendency for up- rather than down-regulation. Ontology assignment of these differentially regulated transcripts revealed a consistent up-regulation of transcripts related to chloroplast biogenesis and photosynthesis in hp-2(dg) mutants throughout fruit ripening. A tendency of up-regulation was also observed in structural genes involved in phytonutrient biosynthesis. However, this up-regulation was not as consistent, positioning plastid biogenesis as an important determinant of phytonutrient overproduction in hp-2(dg) and possibly other hp mutant fruits. Microscopic observations revealed a highly significant increase in chloroplast size and number in pericarp cells of mature-green hp-2(dg)/hp-2(dg) and hp-2(j)/hp-2(j) fruits in comparison to their normal counterparts. This increase could be observed from early stages of fruit development. Therefore, the molecular trigger that drives phytonutrient overproduction in hp-2(dg) and hp-2(j) mutant fruits should be initially traced at these early stages.

Temporally extended gene expression of the ADP-Glc pyrophosphorylase large subunit (AgpL1) leads to increased enzyme activity in developing tomato fruit.

Tomato plants (Solanum lycopersicum) harboring the allele for the AGPase large subunit (AgpL1) derived from the wild species Solanum habrochaites (AgpL1 ( H )) are characterized by higher AGPase activity and increased starch content in the immature fruit, as well as higher soluble solids in the mature fruit following the breakdown of the transient starch, as compared to fruits from plants harboring the cultivated tomato allele (AgpL1 ( E )). Comparisons of AGPase subunit gene expression and protein levels during fruit development indicate that the increase in AGPase activity correlates with a prolonged expression of the AgpL1 gene in the AgpL1 ( H ) high starch line, leading to an extended presence of the L1 protein. The S1 (small subunit) protein also remained for an extended period of fruit development in the AgpL1 ( H ) fruit, linked to the presence of the L1 protein. There were no discernible differences between the kinetic characteristics of the partially purified AGPase-L1(E) and AGPase-L1(H) enzymes. The results indicate that the increased activity of AGPase in the AgpL1 ( H ) tomatoes is due to the extended expression of the regulatory L1 and to the subsequent stability of the heterotetramer in the presence of the L1 protein, implying a role for the large subunit not only in the allosteric control of AGPase activity but also in the stability of the AGPase L1-S1 heterotetramer. The introgression line of S. lycopersicum containing the wild species AgpL1 ( H ) allele is a novel example of transgressive heterosis in which the hybrid multimeric enzyme shows higher activity due to a modulated temporal expression of one of the subunits.