A Chimeric TGA Repressor Slows Down Fruit Maturation and Ripening in Tomato.

The bZIP transcription factor (TF) SlTGA2.2 was previously highlighted as a possible hub in a network regulating fruit growth and transition to ripening (maturation phase). It belongs to a clade of TFs well known for their involvement in the regulation of the salicylic acid-dependent systemic acquired resistance. To investigate if this TGA TF plays a role in tomato fruit growth and maturation, we took advantage of the fruit-specific SlPPC2 promoter (PPC2pro) to target the expression of a SlTGA2.2-SRDX chimeric repressor in a developmental window restricted to early fruit growth and maturation. Here, we show that this SlTGA2.2-SRDX repressor alters early fruit development and metabolism, including chloroplast number and structure, considerably extends the time necessary to reach the mature green stage and slows down fruit ripening. RNA sequencing and plant hormone analyses reveal that PPC2pro:SlTGA2.2-SRDX fruits are maintained in an immature stage as long as PPC2pro is active, through early modifications of plant hormonal signaling and down-regulation of MADS-RIN and NAC-NOR ripening regulators. Once PPC2pro becomes inactive and therefore SlTGA2.2-SRDX expression is reduced, ripening can proceed, albeit at a slower pace than normal. Altogether, this work emphasizes the developmental continuum between fruit growth, maturation and ripening and provides a useful tool to alter and study the molecular bases of tomato fruit transition to ripening.

The conserved brassinosteroid-related transcription factor BIM1a negatively regulates fruit growth in tomato.

Brassinosteroids (BRs) are steroid hormones that play key roles in plant development and defense. Our goal is to harness the extensive knowledge of the Arabidopsis BR signaling network to improve productivity in crop species. This first requires identifying components of the conserved network and their function in the target species. Here, we investigated the function of SlBIM1a, the closest tomato homolog of AtBIM1, which is highly expressed in fruit. SlBIM1a-overexpressing lines displayed severe plant and fruit dwarfism, and histological characterization of different transgenic lines revealed that SlBIM1a expression negatively correlated with fruit pericarp cell size, resulting in fruit size modifications. These growth phenotypes were in contrast to those found in Arabidopsis, and this was confirmed by the reciprocal ectopic expression of SlBIM1a/b in Arabidopsis and of AtBIM1 in tomato. These results determined that BIM1 function depends more on the recipient species than on its primary sequence. Yeast two-hybrid interaction studies and transcriptomic analyses of SlBIM1a-overexpressing fruit further suggested that SlBIM1a acts through its interaction with SlBZH1 to govern the transcriptional regulation of growth-related BR target genes. Together, these results suggest that SlBIM1a is a negative regulator of pericarp cell expansion, possibly at the crossroads with auxin and light signaling.

Root-knot nematode genetic diversity associated with host compatibility to sweetpotato cultivars.

Plant parasitic root-knot nematodes (RKN) such as Meloidogyne incognita cause significant crop losses worldwide. Although RKN are polyphagous, with wide host ranges, races with differing host compatibilities have evolved. Associations between genotype and infection phenotype in M. incognita have not yet been discovered. In this study, 48 M. incognita isolates were collected from geographically diverse fields in Japan and their genomes sequenced. The isolates exhibited various infection compatibilities to five sweetpotato (SP) cultivars and were assigned to SP races. Genome-wide association analysis identified 743 SNPs affecting gene coding sequences, a large number of which (575) were located on a single 1 Mb region. To examine how this polymorphic region evolved, nucleotide diversity (Pi) was scanned at the whole genome scale. The SNP-rich 1 Mb region exhibited high Pi values and was clearly associated with the SP races. SP1 and 2 races showed high Pi values in this region whereas the Pi values of SP3, 4, and 6 were low. Principal component analysis of isolates from this study and globally collected isolates showed selective divergence in this 1 Mb region. Our results suggest for the first time that the host could be a key determining factor stimulating the genomic divergence of M. incognita.

Plant Genome DataBase Japan (PGDBj).

A portal website that integrates a variety of information related to genomes of model and crop plants from databases (DBs) and the literature was generated. This website, named the Plant Genome DataBase Japan (PGDBj, http://pgdbj. jp/en/ ), is comprised of three component DBs and a cross-search engine which provides a seamless search over their contents. One of the three component DBs is the Ortholog DB, which provides gene cluster information based on the amino acid sequence similarity. Over 1,000,000 amino acid sequences of 40 Viridiplantae species were collected from the public DNA DBs, and plant genome DBs such as TAIR and RAP-DB were subjected to reciprocal BLAST searches for clustering. Another component DB is the Plant Resource DB for genomic- and bio-resources. This DB also integrates the SABRE DB, which provides cDNA and genome sequence resources maintained in the RIKEN BioResource Center and National BioResource Projects Japan. The third component DB of PGDBj is the DNA Marker DB, which manually or automatically collects curated information on DNA markers, quantitative trait loci (QTL), and related genetic linkage maps, from the literature and external DBs. By combining these component DBs and a cross-search engine, PGDBj serves as a useful platform to study genetic systems for both fundamental and applied researches for a wide range of plant species.

Genome sequence and analysis of the Japanese morning glory Ipomoea nil.

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88 Mb (contig N50 of 1.87 Mb), covering 98% of the 750 Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil, and analysing the dwarf gene, CONTRACTED, located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families.

Pleurochrysome: A Web Database of Pleurochrysis Transcripts and Orthologs Among Heterogeneous Algae.

Pleurochrysis is a coccolithophorid genus, which belongs to the Coccolithales in the Haptophyta. The genus has been used extensively for biological research, together with Emiliania in the Isochrysidales, to understand distinctive features between the two coccolithophorid-including orders. However, molecular biological research on Pleurochrysis such as elucidation of the molecular mechanism behind coccolith formation has not made great progress at least in part because of lack of comprehensive gene information. To provide such information to the research community, we built an open web database, the Pleurochrysome (http://bioinf.mind.meiji.ac.jp/phapt/), which currently stores 9,023 unique gene sequences (designated as UNIGENEs) assembled from expressed sequence tag sequences of P. haptonemofera as core information. The UNIGENEs were annotated with gene sequences sharing significant homology, conserved domains, Gene Ontology, KEGG Orthology, predicted subcellular localization, open reading frames and orthologous relationship with genes of 10 other algal species, a cyanobacterium and the yeast Saccharomyces cerevisiae. This sequence and annotation information can be easily accessed via several search functions. Besides fundamental functions such as BLAST and keyword searches, this database also offers search functions to explore orthologous genes in the 12 organisms and to seek novel genes. The Pleurochrysome will promote molecular biological and phylogenetic research on coccolithophorids and other haptophytes by helping scientists mine data from the primary transcriptome of P. haptonemofera.

The bifunctional plant receptor, OsCERK1, regulates both chitin-triggered immunity and arbuscular mycorrhizal symbiosis in rice.

Plants are constantly exposed to threats from pathogenic microbes and thus developed an innate immune system to protect themselves. On the other hand, many plants also have the ability to establish endosymbiosis with beneficial microbes such as arbuscular mycorrhizal (AM) fungi or rhizobial bacteria, which improves the growth of host plants. How plants evolved these systems managing such opposite plant-microbe interactions is unclear. We show here that knockout (KO) mutants of OsCERK1, a rice receptor kinase essential for chitin signaling, were impaired not only for chitin-triggered defense responses but also for AM symbiosis, indicating the bifunctionality of OsCERK1 in defense and symbiosis. On the other hand, a KO mutant of OsCEBiP, which forms a receptor complex with OsCERK1 and is essential for chitin-triggered immunity, established mycorrhizal symbiosis normally. Therefore, OsCERK1 but not chitin-triggered immunity is required for AM symbiosis. Furthermore, experiments with chimeric receptors showed that the kinase domains of OsCERK1 and homologs from non-leguminous, mycorrhizal plants could trigger nodulation signaling in legume-rhizobium interactions as the kinase domain of Nod factor receptor1 (NFR1), which is essential for triggering the nodulation program in leguminous plants, did. Because leguminous plants are believed to have developed the rhizobial symbiosis on the basis of AM symbiosis, our results suggest that the symbiotic function of ancestral CERK1 in AM symbiosis enabled the molecular evolution to leguminous NFR1 and resulted in the establishment of legume-rhizobia symbiosis. These results also suggest that OsCERK1 and homologs serve as a molecular switch that activates defense or symbiotic responses depending on the infecting microbes.

Identification of the carotenoid modifying gene PALE YELLOW PETAL 1 as an essential factor in xanthophyll esterification and yellow flower pigmentation in tomato (Solanum lycopersicum).

Xanthophylls, the pigments responsible for yellow to red coloration, are naturally occurring carotenoid compounds in many colored tissues of plants. These pigments are esterified within the chromoplast; however, little is known about the mechanisms underlying their accumulation in flower organs. In this study, we characterized two allelic tomato (Solanum lycopersicum L.) mutants, pale yellow petal (pyp) 1-1 and pyp1-2, that have reduced yellow color intensity in the petals and anthers due to loss-of-function mutations. Carotenoid analyses showed that the yellow flower organs of wild-type tomato contained high levels of xanthophylls that largely consisted of neoxanthin and violaxanthin esterified with myristic and/or palmitic acids. Functional disruption of PYP1 resulted in loss of xanthophyll esters, which was associated with a reduction in the total carotenoid content and disruption of normal chromoplast development. These findings suggest that xanthophyll esterification promotes the sequestration of carotenoids in the chromoplast and that accumulation of these esters is important for normal chromoplast development. Next-generation sequencing coupled with map-based positional cloning identified the mutant alleles responsible for the pyp1 phenotype. PYP1 most likely encodes a carotenoid modifying protein that plays a vital role in the production of xanthophyll esters in tomato anthers and petals. Our results provide insight into the molecular mechanism underlying the production of xanthophyll esters in higher plants, thereby shedding light on a longstanding mystery.

A novel tomato mutant, Solanum lycopersicum elongated fruit1 (Slelf1), exhibits an elongated fruit shape caused by increased cell layers in the proximal region of the ovary.

Genes controlling fruit morphology offer important insights into patterns and mechanisms determining organ shape and size. In cultivated tomato (Solanum lycopersicum L.), a variety of fruit shapes are displayed, including round-, bell pepper-, pear-, and elongate-shaped forms. In this study, we characterized a tomato mutant possessing elongated fruit morphology by histologically analyzing its fruit structure and genetically analyzing and mapping the genetic locus. The mutant line, Solanum lycopersicum elongated fruit 1 (Slelf1), was selected in a previous study from an ethylmethane sulfonate-mutagenized population generated in the background of Micro-Tom, a dwarf and rapid-growth variety. Histological analysis of the Slelf1 mutant revealed dramatically increased elongation of ovary and fruit. Until 6 days before flowering, ovaries were round and they began to elongate afterward. We also determined pericarp thickness and the number of cell layers in three designated fruit regions. We found that mesocarp thickness, as well as the number of cell layers, was increased in the proximal region of immature green fruits, making this the key sector of fruit elongation. Using 262 F2 individuals derived from a cross between Slelf1 and the cultivar Ailsa Craig, we constructed a genetic map, simple sequence repeat (SSR), cleaved amplified polymorphism sequence (CAPS), and derived CAPS (dCAPS) markers and mapped to the 12 tomato chromosomes. Genetic mapping placed the candidate gene locus within a 0.2 Mbp interval on the long arm of chromosome 8 and was likely different from previously known loci affecting fruit shape.

Plant Genome DataBase Japan (PGDBj): a portal website for the integration of plant genome-related databases.

The Plant Genome DataBase Japan (PGDBj, http://pgdbj.jp/?ln=en) is a portal website that aims to integrate plant genome-related information from databases (DBs) and the literature. The PGDBj is comprised of three component DBs and a cross-search engine, which provides a seamless search over the contents of the DBs. The three DBs are as follows. (i) The Ortholog DB, providing gene cluster information based on the amino acid sequence similarity. Over 500,000 amino acid sequences of 20 Viridiplantae species were subjected to reciprocal BLAST searches and clustered. Sequences from plant genome DBs (e.g. TAIR10 and RAP-DB) were also included in the cluster with a direct link to the original DB. (ii) The Plant Resource DB, integrating the SABRE DB, which provides cDNA and genome sequence resources accumulated and maintained in the RIKEN BioResource Center and National BioResource Projects. (iii) The DNA Marker DB, providing manually or automatically curated information of DNA markers, quantitative trait loci and related linkage maps, from the literature and external DBs. As the PGDBj targets various plant species, including model plants, algae, and crops important as food, fodder and biofuel, researchers in the field of basic biology as well as a wide range of agronomic fields are encouraged to perform searches using DNA sequences, gene names, traits and phenotypes of interest. The PGDBj will return the search results from the component DBs and various types of linked external DBs.

Suppression of γ-aminobutyric acid (GABA) transaminases induces prominent GABA accumulation, dwarfism and infertility in the tomato (Solanum lycopersicum L.).

Tomatoes accumulate γ-aminobutyric acid (GABA) at high levels in the immature fruits. GABA is rapidly converted to succinate during fruit ripening through the activities of GABA transaminase (GABA-T) and succinate semialdehyde dehydrogenase (SSADH). Although three genes encoding GABA-T and both pyruvate- and α-ketoglutarate-dependent GABA-T activities have been detected in tomato fruits, the mechanism underlying the GABA-T-mediated conversion of GABA has not been fully understood. In this work, we conducted loss-of-function analyses utilizing RNA interference (RNAi) transgenic plants with suppressed pyruvate- and glyoxylate-dependent GABA-T gene expression to clarify which GABA-T isoforms are essential for its function. The RNAi plants with suppressed SlGABA-T gene expression, particularly SlGABA-T1, showed severe dwarfism and infertility. SlGABA-T1 expression was inversely associated with GABA levels in the fruit at the red ripe stage. The GABA contents in 35S::SlGABA-T1(RNAi) lines were 1.3-2.0 times and 6.8-9.2 times higher in mature green and red ripe fruits, respectively, than the contents in wild-type fruits. In addition, SlGABA-T1 expression was strongly suppressed in the GABA-accumulating lines. These results indicate that pyruvate- and glyoxylate-dependent GABA-T is the essential isoform for GABA metabolism in tomato plants and that GABA-T1 primarily contributes to GABA reduction in the ripening fruits.

Mapping of Micro-Tom BAC-End Sequences to the Reference Tomato Genome Reveals Possible Genome Rearrangements and Polymorphisms.

A total of 93,682 BAC-end sequences (BESs) were generated from a dwarf model tomato, cv. Micro-Tom. After removing repetitive sequences, the BESs were similarity searched against the reference tomato genome of a standard cultivar, "Heinz 1706." By referring to the "Heinz 1706" physical map and by eliminating redundant or nonsignificant hits, 28,804 "unique pair ends" and 8,263 "unique ends" were selected to construct hypothetical BAC contigs. The total physical length of the BAC contigs was 495, 833, 423 bp, covering 65.3% of the entire genome. The average coverage of euchromatin and heterochromatin was 58.9% and 67.3%, respectively. From this analysis, two possible genome rearrangements were identified: one in chromosome 2 (inversion) and the other in chromosome 3 (inversion and translocation). Polymorphisms (SNPs and Indels) between the two cultivars were identified from the BLAST alignments. As a result, 171,792 polymorphisms were mapped on 12 chromosomes. Among these, 30,930 polymorphisms were found in euchromatin (1 per 3,565 bp) and 140,862 were found in heterochromatin (1 per 2,737 bp). The average polymorphism density in the genome was 1 polymorphism per 2,886 bp. To facilitate the use of these data in Micro-Tom research, the BAC contig and polymorphism information are available in the TOMATOMICS database.

Availability of Micro-Tom mutant library combined with TILLING in molecular breeding of tomato fruit shelf-life.

Novel mutant alleles of an ethylene receptor Solanum lycopersicum ETHYLENE RESPONSE1 (SlETR1) gene, Sletr1-1 and Sletr1-2, were isolated from the Micro-Tom mutant library by TILLING in our previous study. They displayed different levels of impaired fruit ripening phenotype, suggesting that these alleles could be a valuable breeding material for improving shelf life of tomato fruit. To conduct practical use of the Sletr1 alleles in tomato breeding, genetic complementation analysis by transformation of genes carrying each allele is required. In this study, we generated and characterized transgenic lines over-expressing Sletr1-1 and Sletr1-2. All transgenic lines displayed ethylene insensitive phenotype and ripening inhibition, indicating that Sletr1-1 and Sletr1-2 associate with the ethylene insensitive phenotype. The level of ethylene sensitivity in the seedling was different between Sletr1-1 and Sletr1-2 transgenic lines, whereas no apparent difference was observed in fruit ripening phenotype. These results suggested that it is difficult to fine-tune the extent of ripening by transgenic approach even if the weaker allele (Sletr1-2) was used. Our present and previous studies indicate that the Micro-Tom mutant library combined with TILLING could be an efficient tool for exploring genetic variations of important agronomic traits in tomato breeding.

A novel class of sticky peel and light green mutations causes cuticle deficiency in leaves and fruits of tomato (Solanum lycopersicum).

The plant cuticle consists of aliphatic wax and cutin, and covers all the aerial tissues, conferring resistance to both biotic and abiotic stresses. In this study, we performed phenotypic characterizations of tomato mutants having both sticky peel (pe) and light green (lg) mutations. Our genetic analysis showed that these two mutations are tightly linked and behave like a monogenic recessive mutation. The double mutant (pe lg) produced glossy soft fruits with light green leaves, most likely due to defects in cuticle formation. Cytological analysis revealed that the thickness of the fruit cuticle layer was dramatically reduced in the pe lg mutant. The epidermal cells of the leaves were also deformed in the pe lg mutant, suggesting that leaf cuticle formation was also disrupted in the mutant. Consistent with this, transmission electron microscopic analysis showed that the electron density of the cuticle layer of the adaxial surface of the leaf was reduced in the pe lg mutant compared to WT, suggesting that there are changes in cuticle structure and/or composition in the pe lg mutant. Both physiological analysis to measure the rate of transpiration, and staining of the fruits and leaves with toluidine blue, revealed that water permeability was enhanced in the pe lg mutant, consistent with the reduced thickness of its cuticle layer. Taken together the preliminary analyses of the cuticle components, the PE LG is most likely involved in proper cuticle formation.

Development of gene-based markers and construction of an integrated linkage map in eggplant by using Solanum orthologous (SOL) gene sets.

We constructed an integrated DNA marker linkage map of eggplant (Solanum melongena L.) using DNA marker segregation data sets obtained from two independent intraspecific F(2) populations. The linkage map consisted of 12 linkage groups and encompassed 1,285.5 cM in total. We mapped 952 DNA markers, including 313 genomic SSR markers developed by random sequencing of simple sequence repeat (SSR)-enriched genomic libraries, and 623 single-nucleotide polymorphisms (SNP) and insertion/deletion polymorphisms (InDels) found in eggplant-expressed sequence tags (ESTs) and related genomic sequences [introns and untranslated regions (UTRs)]. Because of their co-dominant inheritance and their highly polymorphic and multi-allelic nature, the SSR markers may be more versatile than the SNP and InDel markers for map-based genetic analysis of any traits of interest using segregating populations derived from any intraspecific crosses of practical breeding materials. However, we found that the distribution of microsatellites in the genome was biased to some extent, and therefore a considerable part of the eggplant genome was first detected when gene-derived SNP and InDel markers were mapped. Of the 623 SNP and InDel markers mapped onto the eggplant integrated map, 469 were derived from eggplant unigenes contained within Solanum orthologous (SOL) gene sets (i.e., sets of orthologous unigenes from eggplant, tomato, and potato). Out of the 469 markers, 326 could also be mapped onto the tomato map. These common markers will be informative landmarks for the transfer of tomato's more saturated genomic information to eggplant and will also provide comparative information on the genome organization of the two solanaceous species. The data are available from the DNA marker database of vegetables, VegMarks (http://vegmarks.nivot.affrc.go.jp).

Characterisation of 13 glutamate receptor-like genes encoded in the tomato genome by structure, phylogeny and expression profiles.

Glutamate receptor-like genes (GLRs) are intimately associated with plant development, defence responses and signalling pathways. Structural and expression analyses of SlGLRs were performed to better characterise their roles in fruit development and metabolism. Utilising recently released tomato genomic sequence data, 15 GLRs were identified in the tomato genome (SlGLRs). Thirteen of these genes were represented by full-length sequences. Phylogenetic analysis of the SlGLRs and the AtGLRs indicates the occurrence of a tomato-specific clade (Clade I) that may have diverged prior to the evolving of other clades. Among the Clade II genes, five (SlGLR2.1, SlGLR2.2, SlGLR2.3, SlGLR2.4, and SlGLR2.5) were located proximally on chromosome 6, indicating possible gene duplication events. The expression level of four of these genes was low in all analysed samples. However, SlGLR2.2 expression level was notably higher, indicating that this gene may be functionally important. The results of this study may provide clues to the functions of the SlGLRs and enable future detailed characterisations of each gene.

Tomato TILLING technology: development of a reverse genetics tool for the efficient isolation of mutants from Micro-Tom mutant libraries.

To accelerate functional genomic research in tomato, we developed a Micro-Tom TILLING (Targeting Induced Local Lesions In Genomes) platform. DNA pools were constructed from 3,052 ethyl methanesulfonate (EMS) mutant lines treated with 0.5 or 1.0% EMS. The mutation frequency was calculated by screening 10 genes. The 0.5% EMS population had a mild mutation frequency of one mutation per 1,710 kb, whereas the 1.0% EMS population had a frequency of one mutation per 737 kb, a frequency suitable for producing an allelic series of mutations in the target genes. The overall mutation frequency was one mutation per 1,237 kb, which affected an average of three alleles per kilobase screened. To assess whether a Micro-Tom TILLING platform could be used for efficient mutant isolation, six ethylene receptor genes in tomato (SlETR1-SlETR6) were screened. Two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) that resulted in reduced ethylene responses were identified, indicating that our Micro-Tom TILLING platform provides a powerful tool for the rapid detection of mutations in an EMS mutant library. This work provides a practical and publicly accessible tool for the study of fruit biology and for obtaining novel genetic material that can be used to improve important agronomic traits in tomato.

Genetic suppression analysis in novel vacuolar processing enzymes reveals their roles in controlling sugar accumulation in tomato fruits.

In plant cells, many vacuolar proteins are synthesized as precursors in the endoplasmic reticulum and are subsequently transported to the vacuole. These precursors are subject to post-translational modifications to allow the active mature forms to be produced. Vacuolar processing enzyme (VPE) has been identified as a family of cysteine proteases involved in protein maturation in the vacuole. In this study, novel VPE genes were isolated from tomato (Solanum lycopersicum), and they were designated SlVPE1-SlVPE5. Phylogenic analysis suggested that SlVPE1 and SlVPE2 were categorized as the seed coat type, SlVPE4 was categorized as the seed type, and both SlVPE3 and SlVPE5 were categorized as the vegetative type. Expression analysis demonstrated that these genes were expressed during fruit development, and that their expression profiles agreed with this classification. High VPE enzyme activity was observed during tomato fruit development; the enzyme activity was correlated with the SlVPE mRNA levels, indicating that the SlVPE encoded active VPE proteins. The total sugar content was higher in RNA interference (RNAi) lines compared with the control plants, suggesting negative roles for SlVPE in sugar accumulation. The quantitative expression analysis of each SlVPE gene in the RNAi lines suggested that the suppression of SlVPE5 probably had the strongest effect on the sugar accumulation observed. The suppression of SlVPE did not influence the total amino acid content, suggesting that the molecular targets of SlVPE were mainly involved in sugar accumulation.

TOMATOMA: a novel tomato mutant database distributing Micro-Tom mutant collections.

The tomato is an excellent model for studies of plants bearing berry-type fruits and for experimental studies of the Solanaceae family of plants due to its conserved genetic organization. In this study, a comprehensive mutant tomato population was generated in the background of Micro-Tom, a dwarf, rapid-growth variety. In this and previous studies, a family including 8,598 and 6,422 M(2) mutagenized lines was produced by ethylmethane sulfonate (EMS) mutagenesis and γ-ray irradiation, and this study developed and investigated these M(2) plants for alteration of visible phenotypes. A total of 9,183 independent M(2) families comprising 91,830 M(2) plants were inspected for phenotypic alteration, and 1,048 individual mutants were isolated. Subsequently, the observed mutant phenotypes were classified into 15 major categories and 48 subcategories. Overall, 1,819 phenotypic categories were found in 1,048 mutants. Of these mutants, 549 were pleiotropic, whereas 499 were non-pleiotropic. Multiple different mutant alleles per locus were found in the mutant libraries, suggesting that the mutagenized populations were nearly saturated. Additionally, genetic analysis of backcrosses indicated the successful inheritance of the mutations in BC(1)F(2) populations, confirming the reproducibility in the morphological phenotyping of the M(2) plants. To integrate and manage the visible phenotypes of mutants and other associated data, we developed the in silico database TOMATOMA, a relational system interfacing modules between mutant line names and phenotypic categories. TOMATOMA is a freely accessible database, and these mutant recourses are available through the TOMATOMA (http://tomatoma.nbrp.jp/index.jsp).

Enhanced polyamine accumulation alters carotenoid metabolism at the transcriptional level in tomato fruit over-expressing spermidine synthase.

Polyamines are involved in crucial plant physiological events, but their roles in fruit development remain unclear. We generated transgenic tomato plants that show a 1.5- to 2-fold increase in polyamine content by over-expressing the spermidine synthase gene, which encodes a key enzyme for polyamine biosynthesis. Pericarp-columella and placental tissue from transgenic tomato fruits were subjected to (1)H-nuclear magnetic resonance (NMR) for untargeted metabolic profiling and high-performance liquid chromatography-diode array detection for carotenoid profiling to determine the effects of high levels of polyamine accumulation on tomato fruit metabolism. A principal component analysis of the quantitative (1)H NMR data from immature green to red ripe fruit showed a clear discrimination between developmental stages, especially during ripening. Quantification of 37 metabolites in pericarp-columella and 41 metabolites in placenta tissues revealed distinct metabolic profiles between the wild type and transgenic lines, particularly at the late ripening stages. Notably, the transgenic tomato fruits also showed an increase in carotenoid accumulation, especially in lycopene (1.3- to 2.2-fold), and increased ethylene production (1.2- to 1.6-fold) compared to wild-type fruits. Genes responsible for lycopene biosynthesis, including phytoene synthase, phytoene desaturase, and deoxy-d-xylulose 5-phosphate synthase, were significantly up-regulated in ripe transgenic fruits, whereas genes involved in lycopene degradation, including lycopene-epsilon cyclase and lycopene beta cyclase, were down-regulated in the transgenic fruits compared to the wild type. These results suggest that a high level of accumulation of polyamines in the tomato regulates the steady-state level of transcription of genes responsible for the lycopene metabolic pathway, which results in a higher accumulation of lycopene in the fruit.