Ilan Levin

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-2dg (hp-2dg) and hp-2j mutants are caused by lesions in the gene encoding DEETIOLATED1, a negative regulator of light signaling. Homozygous hp-2dg and hp-2j 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-2dg/hp-2dg plants in relation to their normal counterparts using microarray technology. Results show that a large portion of the genes that are affected by hp-2dg 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-2dg 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-2dg 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-2dg/hp-2dg and hp-2j/hp-2j 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-2dg and hp-2j mutant fruits should be initially traced at these early stages.

The tomato homolog of the gene encoding UV-damaged DNA binding protein 1 (DDB1) underlined as the gene that causes the high pigment-1 mutant phenotype.

A tomato EST sequence, highly homologous to the human and Arabidopsis thaliana UV-damaged DNA binding protein 1 (DDB1), was mapped to the centromeric region of the tomato chromosome 2. This region was previously shown to harbor the HP-1 gene, encoding the high pigment-1 (hp-1) and the high pigment-1w (hp-1w) mutant phenotypes. Recent results also show that the A. thaliana DDB1 protein interacts both genetically and biochemically with the protein encoded by DEETIOLATED1, a gene carrying three tomato mutations that are in many respects isophenotypic to hp-1: high pigment-2 (hp-2), high pigment-2j (hp-2j) and dark green (dg). The entire coding region of the DDB1 gene was sequenced in an hp-1 mutant and its near-isogenic normal plant in the cv. Ailsa Craig background, and also in an hp-1w mutant and its isogenic normal plant in the GT breeding line background. Sequence analysis revealed a single A931-to-T931 base transversion in the coding sequence of the DDB1 gene in the hp-1 mutant plants. This transversion results in the substitution of the conserved asparagine at position 311 to a tyrosine residue. In the hp-1w mutant, on the other hand, a single G2392-to-A2392 transition was observed, resulting in the substitution of the conserved glutamic acid at position 798 to a lysine residue. The single nucleotide polymorphism that differentiates hp-1 mutant and normal plants in the cv. Ailsa Craig background was used to design a pyrosequencing genotyping system. Analysis of a resource F2 population segregating for the hp-1 mutation revealed a very strong linkage association between the DDB1 locus and the photomorphogenic response of the seedlings, measured as hypocotyl length (25

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.

The tomato dark green mutation is a novel allele of the tomato homolog of the DEETIOLATED1 gene.

Abstract.A comprehensive, multi-generation, allele test, carried out in this study, suggests that the tomato mutations dark-green (dg) and high pigment 2j (hp-2j) are allelic. The hp-2j mutant is caused by a mutation in the tomato homolog of the DEETIOLATED1 (DET1) gene, involved in the signal transduction cascade of light perception and morphogenesis. This suggestion is in agreement with the exaggerated photomorphogenic de-etiolation response of homozygous dg mutants grown under modulated light conditions. Sequence analysis of the DET1 gene was carried out in dg mutants representing two different lines, and revealed a single A-to-T base transversion in the second exon of the DET1 gene in comparison with the normal wild-type sequence. This transversion results in a conserved Asparagine34-to-Isoleucine34 amino-acid substitution, and eliminates a recognition site for the AclI restriction endonuclease, present in the wild-type and in the other currently known tomato mutants at the DET1 locus. This polymorphism was used to develop a PCR-based DNA marker, which enables an early genotypic selection for breeding lycopene-rich tomatoes. Using this marker and sequence analysis we demonstrate that an identical base transversion also exists in dg mutants of the cultivar Manapal, in which the natural dg mutation was originally discovered. A linkage analysis, carried out in a F2 population, shows a very strong linkage association between the DET1 locus of dg mutant plants and the photomorphogenic response of the seedlings, measured as hypocotyl length (12 < LOD Score < 13, R2 = 51.1%). The results presented in this study strongly support the hypothesis that the tomato dg mutation is a novel allele of the tomato homolog of the DET1 gene.

Fgr, a major locus that modulates the fructose to glucose ratio in mature tomato fruits

Abstract A genetic trait determining the ratio of fructose to glucose in mature tomato fruits is described. A backcross breeding program based on the interspecific cross of Lycopersiconhirsutum and L. esculentum yielded stable genotypes with a high ratio of fructose to glucose (>1.5:1) compared with the approximately equimolar ratios found in L. esculentum. Two inter-simple- sequence repeat (ISSR) DNA sequences, highly associated (20 <LOD score <21) with the trait, were identified. The markers were found to be less associated with either glucose or fructose levels individually (2 <LOD score <3) and were statistically unlinked to total sugars and total soluble solids (TSS). These two ISSR bands segregated in a dominant fashion and were found to be allelic to each other, one associated in coupling and the other in repulsion with the trait of high fructose to glucose ratio. Both ISSR markers were mapped to the centromeric region of tomato chromosome 4. Quantitative analysis of the identified locus, based on data from segregating F2, BC and F3 populations from the cross between genotypes having high and low fructose to glucose ratios, suggested that the L. hirsutum-derived allele (FgrH), which increases the fructose to glucose ratio, is partially dominant. FgrH leads to an increase in fructose levels and a subsequent decrease in glucose levels, with no effect on total hexose levels. Accordingly, we conclude that the Fgr locus modulates the partitioning of hexose sugars between fructose and glucose, with no effect on total sugars or TSS.

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.

ADPglucose pyrophosphorylase activity and starch accumulation in immature tomato fruit: the effect of a Lycopersicon hirsutum-derived introgression encoding for the large subunit

Abstract A breeding line of tomato with an increased level of starch in the immature fruit, and a concomitantly increased soluble sugar level in the mature fruit, was developed from an interspecific cross with Lycopersicon hirsutum. A comparison of in vitro activities of enzymes of the sucrose-to-starch metabolic pathway indicated that only the activity of ADPglucose pyrophosphorylase was significantly increased in the high starch line, compared to the Lycopersicon esculentum cultivar, M-82. Polymerase chain reaction (PCR) markers were developed to distinguish between the L. esculentum and L. hirsutum alleles for each of the four known genes of ADPglucose pyrophosphorylase in tomato (LS1, LS2, LS3, SS). Analysis of the high starch line showed that it carries the LS1 allele from L. hirsutum (LS1H). In segregating populations from the cross between the high starch line and M-82 the LS1H allele was correlated with increases in ADPglucose pyrophosphorylase activity and starch level in the immature fruit and soluble solids content in the mature fruit. The LS1H allele was cloned and sequenced and a comparison with the consensus sequence of the L. esculentum LS1E allele shows a three amino acid duplication of Glu-Lys-Lys near the N-terminal, and three additional amino acid changes. The four genes were mapped to different chromosomal segments, using Lycopersicon pennellii introgression lines, which is further evidence that these are distinct ADPglucose pyrophosphorylase genes in tomato. These results suggest that ADPglucose pyrophosphorylase activity is limiting to starch synthesis in developing fruits; furthermore, there exists natural genetic variability within the Lycopersicon genus that may be utilized to increase enzyme activity and starch accumulation in the cultivated fruit. Furthermore, we proposes a possible gene function for a hitherto described QTL for fruit Brix levels.

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.

High pigment tomato mutants—more than just lycopene (a review)

Fruit constitutes a major component of our diet, providing fiber, vitamins, minerals, and many phytonutrients that promote good health. Fleshy fruits such as tomatoes already contain high levels of several of these ingredients. Nevertheless, efforts have been invested in increasing and diversifying the content of phytonutrients, such as carotenoids and flavonoids, in tomato fruits. These efforts rely on transgenic approaches, and the use of single-point mutations and/or quantitative trait loci affecting levels of these phytonutrients. The tomato high pigment (hp) mutations are a good example of the latter alternative. Due to their impact on fruit lycopene content, hp mutations were already introgressed into elite tomato germplasm. Interestingly, plants carrying these mutations are also characterized by higher levels of other health-promoting metabolites, such as flavonoids and vitamins. These mutations were initially marked as lesions in structural genes of the carotenoid biosynthetic pathway. However, recent studies have shown that they represent mutations in two regulatory genes active in light signal transduction, also known as photomorphogenesis. This gene-identification has created a conceptual link between photomorphogenesis and biosynthesis of fruit phytonutrients, and suggests that manipulation of the light signal transduction machinery in plants may be an effective approach towards practical manipulation of fruit phytonutrients.

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 (AgpL1H) 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 (AgpL1E). 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 AgpL1H 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 AgpL1H fruit, linked to the presence of the L1 protein. There were no discernible differences between the kinetic characteristics of the partially purified AGPase-L1E and AGPase-L1H enzymes. The results indicate that the increased activity of AGPase in the AgpL1H 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 AgpL1H 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.