Class II knotted-like homeodomain protein SlKN5 with BEL1-like homeodomain proteins suppresses fruit greening in tomato fruit.

Knotted1-like homeodomain (KNOX) proteins are essential in regulating plant organ differentiation. Land plants, including tomato (Solanum lycopersicum), have two classes of the KNOX protein family, namely, class I (KNOX I) and class II KNOX (KNOX II). While tomato KNOX I proteins are known to stimulate chloroplast development in fruit, affecting fruit coloration, the role of KNOX II proteins in this context remains unclear. In this study, we employ CRISPR/Cas9 to generate knockout mutants of the KNOX II member, SlKN5. These mutants display increased leaf complexity, a phenotype commonly associated with reduced KNOX II activity, as well as enhanced accumulation of chloroplasts and chlorophylls in smaller cells within young, unripe fruit. RNA-seq data analyses indicate that SlKN5 suppresses the transcriptions of genes involved in chloroplast biogenesis, chlorophyll biosynthesis, and gibberellin catabolism. Furthermore, protein-protein interaction assays reveal that SlKN5 physically interacts with three transcriptional repressors from the BLH1-clade of BEL1-like homeodomain (BLH) protein family, SlBLH4, SlBLH5, and SlBLH7, with SlBLH7 showing the strongest interaction. CRISPR/Cas9-mediated knockout of these SlBLH genes confirmed their overlapping roles in suppressing chloroplast biogenesis, chlorophyll biosynthesis, and lycopene cyclization. Transient assays further demonstrate that the SlKN5-SlBLH7 interaction enhances binding capacity to regulatory regions of key chloroplast- and chlorophyll-related genes, including SlAPRR2-like1, SlCAB-1C, and SlGUN4. Collectively, our findings elucidate that the KNOX II SlKN5-SlBLH regulatory modules serve to inhibit fruit greening and subsequently promote lycopene accumulation, thereby fine-tuning the color transition from immature green fruit to mature red fruit.

Genome-wide cis-decoding for expression design in tomato using cistrome data and explainable deep learning.

In the evolutionary history of plants, variation in cis-regulatory elements (CREs) resulting in diversification of gene expression has played a central role in driving the evolution of lineage-specific traits. However, it is difficult to predict expression behaviors from CRE patterns to properly harness them, mainly because the biological processes are complex. In this study, we used cistrome datasets and explainable convolutional neural network (CNN) frameworks to predict genome-wide expression patterns in tomato (Solanum lycopersicum) fruit from the DNA sequences in gene regulatory regions. By fixing the effects of trans-acting factors using single cell-type spatiotemporal transcriptome data for the response variables, we developed a prediction model for crucial expression patterns in the initiation of tomato fruit ripening. Feature visualization of the CNNs identified nucleotide residues critical to the objective expression pattern in each gene, and their effects were validated experimentally in ripening tomato fruit. This cis-decoding framework will not only contribute to the understanding of the regulatory networks derived from CREs and transcription factor interactions, but also provides a flexible means of designing alleles for optimized expression.

Molecular, hormonal, and metabolic mechanisms of fruit set, the ovary-to-fruit transition, in horticultural crops.

Fruit set is the process by which the ovary develops into a fruit and is an important factor in determining fruit yield. Fruit set is induced by two hormones, auxin and gibberellin, and the activation of their signaling pathways, partly by suppressing various negative regulators. Many studies have investigated the structural changes and gene networks in the ovary during fruit set, revealing the cytological and molecular mechanisms. In tomato (Solanum lycopersicum), SlIAA9 and SlDELLA/PROCERA act as auxin and gibberellin signaling repressors, respectively, and are important regulators of the activity of transcription factors and downstream gene expression involved in fruit set. Upon pollination, SlIAA9 and SlDELLA are degraded, which subsequently activates downstream cascades and mainly contributes to active cell division and cell elongation, respectively, in ovaries during fruit setting. According to current knowledge, the gibberellin pathway functions as the most downstream signal in fruit set induction, and therefore its role in fruit set has been extensively explored. Furthermore, multi-omics analysis has revealed the detailed dynamics of gene expression and metabolites downstream of gibberellins, highlighting the rapid activation of central carbon metabolism. This review will outline the relevant mechanisms at the molecular and metabolic levels during fruit set, particularly focusing on tomato.

Fruit setting rewires central metabolism via gibberellin cascades.

Fruit set is the process whereby ovaries develop into fruits after pollination and fertilization. The process is induced by the phytohormone gibberellin (GA) in tomatoes, as determined by the constitutive GA response mutant procera However, the role of GA on the metabolic behavior in fruit-setting ovaries remains largely unknown. This study explored the biochemical mechanisms of fruit set using a network analysis of integrated transcriptome, proteome, metabolome, and enzyme activity data. Our results revealed that fruit set involves the activation of central carbon metabolism, with increased hexoses, hexose phosphates, and downstream metabolites, including intermediates and derivatives of glycolysis, the tricarboxylic acid cycle, and associated organic and amino acids. The network analysis also identified the transcriptional hub gene SlHB15A, that coordinated metabolic activation. Furthermore, a kinetic model of sucrose metabolism predicted that the sucrose cycle had high activity levels in unpollinated ovaries, whereas it was shut down when sugars rapidly accumulated in vacuoles in fruit-setting ovaries, in a time-dependent manner via tonoplastic sugar carriers. Moreover, fruit set at least partly required the activity of fructokinase, which may pull fructose out of the vacuole, and this could feed the downstream pathways. Collectively, our results indicate that GA cascades enhance sink capacities, by up-regulating central metabolic enzyme capacities at both transcriptional and posttranscriptional levels. This leads to increased sucrose uptake and carbon fluxes for the production of the constituents of biomass and energy that are essential for rapid ovary growth during the initiation of fruit set.

Overproduction of ascorbic acid impairs pollen fertility in tomato.

Ascorbate is a major antioxidant buffer in plants. Several approaches have been used to increase the ascorbate content of fruits and vegetables. Here, we combined forward genetics with mapping-by-sequencing approaches using an ethyl methanesulfonate (EMS)-mutagenized Micro-Tom population to identify putative regulators underlying a high-ascorbate phenotype in tomato fruits. Among the ascorbate-enriched mutants, the family with the highest fruit ascorbate level (P17C5, up to 5-fold wild-type level) had strongly impaired flower development and produced seedless fruit. Genetic characterization was performed by outcrossing P17C5 with cv. M82. We identified the mutation responsible for the ascorbate-enriched trait in a cis-acting upstream open reading frame (uORF) involved in the downstream regulation of GDP-l-galactose phosphorylase (GGP). Using a specific CRISPR strategy, we generated uORF-GGP1 mutants and confirmed the ascorbate-enriched phenotype. We further investigated the impact of the ascorbate-enriched trait in tomato plants by phenotyping the original P17C5 EMS mutant, the population of outcrossed P17C5 × M82 plants, and the CRISPR-mutated line. These studies revealed that high ascorbate content is linked to impaired floral organ architecture, particularly anther and pollen development, leading to male sterility. RNA-seq analysis suggested that uORF-GGP1 acts as a regulator of ascorbate synthesis that maintains redox homeostasis to allow appropriate plant development.

Efficient base editing in tomato using a highly expressed transient system.

KEY MESSAGE: Base editing in tomatoes was achieved by transient expression. The Solanaceae plants, particularly the tomato (Solanum lycopersicum), is of huge economic value worldwide. The tomato is a unique model plant for studying the functions of genes related to fruit ripening. Deeper understanding of tomatoes is of great importance for both plant research and the economy. Genome editing technology, such as CRISPR/Cas9, has been used for functional genetic research. However, some challenges, such as low transformation efficiency, remain with this technology. Moreover, the foreign Cas9 and gRNA expression cassettes must be removed to obtain null-segregants In this study, we used a high-level transient expression system to improve the base editing technology. A high-level transient expression system has been established previously using geminiviral replication and a double terminator. The pBYR2HS vector was used for this transient expression system. nCas9-CDA and sgRNA-SlHWS were introduced into this vector, and the protein and RNA were then transiently expressed in tomato tissues by agroinfiltration. The homozygous mutant produced by base editing was obtained in the next generation with an efficiency of about 18%. nCas9-free next-generation plants were 71%. All the homozygous base-edited plants in next generation are nCas9-free. These findings show that the high-level transient expression system is useful for base editing in tomatoes.

JRE4 is a master transcriptional regulator of defense-related steroidal glycoalkaloids in tomato.

Steroidal glycoalkaloids (SGAs) are specialized anti-nutritional metabolites that accumulate in Solanum lycopersicum (tomato) and Solanum tuberosum (potato). A series of SGA biosynthetic genes is known to be upregulated in Solanaceae species by jasmonate-responsive Ethylene Response Factor transcription factors, including JRE4 (otherwise known as GAME9), but the exact regulatory significance in planta of each factor has remained unaddressed. Here, via TILLING-based screening of an EMS-mutagenized tomato population, we isolated a JRE4 loss-of-function line that carries an amino acid residue missense change in a region of the protein important for DNA binding. In this jre4 mutant, we observed downregulated expression of SGA biosynthetic genes and decreased SGA accumulation. Moreover, JRE4 overexpression stimulated SGA production. Further characterization of jre4 plants revealed their increased susceptibility to the generalist herbivore Spodoptera litura larvae. This susceptibility illustrates that herbivory resistance is dependent on JRE4-mediated defense responses, which include SGA accumulation. Ethylene treatment attenuated the jasmonate-mediated JRE4 expression induction and downstream SGA biosynthesis in tomato leaves and hairy roots. Overall, this study indicated that JRE4 functions as a primary master regulator of SGA biosynthesis, and thereby contributes toward plant defense against chewing insects.

Aberrant Stamen Development is Associated with Parthenocarpic Fruit Set Through Up-Regulation of Gibberellin Biosynthesis in Tomato.

Parthenocarpy, a process in which fruit set occurs without fertilization, leads to the production of seedless fruit. A number of floral homeotic mutants with abnormal stamen development exhibit parthenocarpic fruit set. Flower development is thought to repress ovary growth before anthesis. However, the mechanism of parthenocarpic fruit development caused by aberrant flower formation is poorly understood. To investigate the molecular mechanism of parthenocarpic fruit development in floral homeotic mutants, we performed functional analysis of Tomato APETALA3 (TAP3) by loss-of-function approaches. Organ-specific promoter was used to induce organ-specific loss of function in stamen and ovary/fruit. We observed increased cell expansion in tap3 mutants and TAP3-RNAi lines during parthenocarpic fruit growth. These were predominantly accompanied by the up-regulation of GA biosynthesis genes, including SlGA20ox1, SlGA20ox2, and SlGA20ox3, as well as reduced expression of the GA-inactivating gene SlGA2ox1 and the auxin signaling gene SlARF7 involved in a crosstalk between GA and auxin. These transcriptional profiles are in agreement with the GA levels in these lines. These results suggest that stamen development negatively regulates fruit set by repressing the GA biosynthesis.

Agrobacterium tumefaciens Enhances Biosynthesis of Two Distinct Auxins in the Formation of Crown Galls.

The plant pathogen Agrobacterium tumefaciens infects plants and introduces the transferred-DNA (T-DNA) region of the Ti-plasmid into nuclear DNA of host plants to induce the formation of tumors (crown galls). The T-DNA region carries iaaM and iaaH genes for synthesis of the plant hormone auxin, indole-3-acetic acid (IAA). It has been demonstrated that the iaaM gene encodes a tryptophan 2-monooxygenase which catalyzes the conversion of tryptophan to indole-3-acetamide (IAM), and the iaaH gene encodes an amidase for subsequent conversion of IAM to IAA. In this article, we demonstrate that A. tumefaciens enhances the production of both IAA and phenylacetic acid (PAA), another auxin which does not show polar transport characteristics, in the formation of crown galls. Using liquid chromatography-tandem mass spectroscopy, we found that the endogenous levels of phenylacetamide (PAM) and PAA metabolites, as well as IAM and IAA metabolites, are remarkably increased in crown galls formed on the stem of tomato plants, implying that two distinct auxins are simultaneously synthesized via the IaaM-IaaH pathway. Moreover, we found that the induction of the iaaM gene dramatically elevated the levels of PAM, PAA and its metabolites, along with IAM, IAA and its metabolites, in Arabidopsis and barley. From these results, we conclude that A. tumefaciens enhances biosynthesis of two distinct auxins in the formation of crown galls.

Evidence of the functional role of the ethylene receptor genes SlETR4 and SlETR5 in ethylene signal transduction in tomato.

Ethylene receptors are key factors for ethylene signal transduction. In tomato, six ethylene receptor genes (SlETR1-SlETR6) have been identified. Mutations in different ethylene receptor genes result in different phenotypes that are useful for elucidating the roles of each gene. In this study, we screened mutants of two ethylene receptor genes, SLETR4 and SLETR5, from a Micro-Tom mutant library generated by TILLING. We identified two ethylene receptor mutants with altered phenotypes and named them Sletr4-1 and Sletr5-1. Sletr4-1 has a mutation between the transmembrane and GAF domains, while Sletr5-1 has a mutation within the GAF domain. Sletr4-1 showed increased hypocotyl and root lengths, compared to those of wild type plants, under ethylene exposure. Moreover, the fruit shelf life of this mutant was extended, titratable acidity was increased and total soluble solids were decreased, suggesting a reduced ethylene sensitivity. In contrast, in the absence of exogenous ethylene, the hypocotyl and root lengths of Sletr5-1 were shorter than those of the wild type, and the fruit shelf life was shorter, suggesting that these mutants have increased ethylene sensitivity. Gene expression analysis showed that SlNR was up-regulated in the Sletr5-1 mutant line, in contrast to the down-regulation observed in the Sletr4-1 mutant line, while the down-regulation of SlCTR1, SlEIN2, SlEIL1, SlEIL3, and SlERF.E4 was observed in Sletr4-1 mutant allele, suggesting that these two ethylene receptors have functional roles in ethylene signalling and demonstrating, for the first time, a function of the GAF domain of ethylene receptors. These results suggest that the Sletr4-1 and Sletr5-1 mutants are useful for elucidating the complex mechanisms of ethylene signalling through the analysis of ethylene receptors in tomato.

SlLAX1 is Required for Normal Leaf Development Mediated by Balanced Adaxial and Abaxial Pavement Cell Growth in Tomato.

Leaves are the major plant organs with a primary function for photosynthesis. Auxin controls various aspects of plant growth and development, including leaf initiation, expansion and differentiation. Unique and intriguing auxin features include its polar transport, which is mainly controlled by the AUX1/LAX and PIN gene families as influx and efflux carriers, respectively. The role of AUX1/LAX genes in root development is well documented, but the role of these genes in leaf morphogenesis remains unclear. Moreover, most studies have been conducted in the plant model Arabidopsis thaliana, while studies in tomato are still scarce. In this study, we isolated six lines of the allelic curly leaf phenotype 'curl' mutants from a γ-ray and EMS (ethyl methanesulfonate) mutagenized population. Using a map-based cloning strategy combined with exome sequencing, we observed that a mutation occurred in the SlLAX1 gene (Solyc09g014380), which is homologous to an Arabidopsis auxin influx carrier gene, AUX1 (AtAUX1). Characterization of six alleles of single curl mutants revealed the pivotal role of SlLAX1 in controlling tomato leaf flatness by balancing adaxial and abaxial pavement cell growth, which has not been reported in tomato. Using TILLING (Targeting Induced Local Lesions IN Genome) technology, we isolated an additional mutant allele of the SlLAX1 gene and this mutant showed a curled leaf phenotype similar to other curl mutants, suggesting that Solyc09g014380 is responsible for the curl phenotype. These results showed that SlLAX1 is required for normal leaf development mediated by balanced adaxial and abaxial pavement cell growth in tomato.

Low Infection of Phelipanche aegyptiaca in Micro-Tom Mutants Deficient in CAROTENOIDCLEAVAGE DIOXYGENASE 8.

Strigolactones (SLs), a group of plant hormones, induce germination of root-parasitic plants and inhibit shoot branching in many plants. Shoot branching is an important trait that affects the number and quality of flowers and fruits. Root-parasitic plants, such as Phelipanche spp., infect tomato roots and cause economic damage in Europe and North Africa-hence why resistant tomato cultivars are needed. In this study, we found carotenoid cleavage dioxygenase 8-defective mutants of Micro-Tom tomato (slccd8) by the "targeting induced local lesions in genomes" (TILLING) method. The mutants showed excess branching, which was suppressed by exogenously applied SL. Grafting shoot scions of the slccd8 mutants onto wild-type (WT) rootstocks restored normal branching in the scions. The levels of endogenous orobanchol and solanacol in WT were enough detectable, whereas that in the slccd8 mutants were below the detection limit of quantification analysis. Accordingly, root exudates of the slccd8 mutants hardly stimulated seed germination of root parasitic plants. In addition, SL deficiency did not critically affect the fruit traits of Micro-Tom. Using a rhizotron system, we also found that Phelipanche aegyptiaca infection was lower in the slccd8 mutants than in wild-type Micro-Tom because of the low germination. We propose that the slccd8 mutants might be useful as new tomato lines resistant to P. aegyptiaca.

SEXUAL STERILITY is Essential for Both Male and Female Gametogenesis in Tomato.

Gametogenesis is a key step in the production of ovules or pollen in higher plants. The molecular aspects of gametogenesis are well characterized in the model plant Arabidopsis; however, little information is known in tomato, which is a model plant for fleshy fruit development. In this study, we characterized a tomato (Solanum lycopersicum L.) γ-ray mutant, sexual sterility (Slses), that exhibited both male and female sterility. Morphological analysis revealed that the Slses mutant forms incomplete ovules and wilted anthers devoid of pollen grains at the anthesis stage. Genetic and next-generation sequencing analyses revealed that the Slses mutant carried a 13 bp deletion within the first exon of a homolog of SPOROCYTELESS/NOZZLE (SPL/NZZ), which plays an important role in gametogenesis in Arabidopsis. Complementation analysis in which the complete SlSES genomic region was introduced into the Slses mutant fully restored normal phenotypes, demonstrating that Solyc07g063670 is responsible for the Slses mutation. SlSES probably act as a transcriptional repressor because of an EAR motif at the C-terminal region. Gene expression levels of WUSCHEL (SlWUS) and INNER NO OUTER (SlINO), both of which are required for ovule development, were dramatically reduced in the early stages of pistil development in the Slses mutant, suggesting a positive regulatory role for SlSES in the transcription of gametogenesis genes and differences in the regulation of INO (SlINO) and integument development by SPL/NZZ (SLSES) between Arabidopsis and tomato. Taken together, our results indicate that SlSES is a novel tomato gametogenesis gene essential for both male and female gametogenesis.

Activating glutamate decarboxylase activity by removing the autoinhibitory domain leads to hyper γ-aminobutyric acid (GABA) accumulation in tomato fruit.

KEY MESSAGE: The C-terminal extension region of SlGAD3 is likely involved in autoinhibition, and removing this domain increases GABA levels in tomato fruits. γ-Aminobutyric acid (GABA) is a ubiquitous non-protein amino acid with several health-promoting benefits. In many plants including tomato, GABA is synthesized via decarboxylation of glutamate in a reaction catalyzed by glutamate decarboxylase (GAD), which generally contains a C-terminal autoinhibitory domain. We previously generated transgenic tomato plants in which tomato GAD3 (SlGAD3) was expressed using the 35S promoter/NOS terminator expression cassette (35S-SlGAD3-NOS), yielding a four- to fivefold increase in GABA levels in red-ripe fruits compared to the control. In this study, to further increase GABA accumulation in tomato fruits, we expressed SlGAD3 with (SlGAD3 OX ) or without (SlGAD3ΔC OX ) a putative autoinhibitory domain in tomato using the fruit ripening-specific E8 promoter and the Arabidopsis heat shock protein 18.2 (HSP) terminator. Although the GABA levels in SlGAD3 OX fruits were equivalent to those in 35S-SlGAD3-NOS fruits, GABA levels in SlGAD3ΔC OX fruits increased by 11- to 18-fold compared to control plants, indicating that removing the autoinhibitory domain increases GABA biosynthesis activity. Furthermore, the increased GABA levels were accompanied by a drastic reduction in glutamate and aspartate levels, indicating that enhanced GABA biosynthesis affects amino acid metabolism in ripe-fruits. Moreover, SlGAD3ΔC OX fruits exhibited an orange-ripe phenotype, which was associated with reduced levels of both carotenoid and mRNA transcripts of ethylene-responsive carotenogenic genes, suggesting that over activation of GAD influences ethylene sensitivity. Our strategy utilizing the E8 promoter and HSP terminator expression cassette, together with SlGAD3 C-terminal deletion, would facilitate the production of tomato fruits with increased GABA levels.

Ethylene suppresses tomato (Solanum lycopersicum) fruit set through modification of gibberellin metabolism.

Fruit set in angiosperms marks the transition from flowering to fruit production and a commitment to seed dispersal. Studies with Solanum lycopersicum (tomato) fruit have shown that pollination and subsequent fertilization induce the biosynthesis of several hormones, including auxin and gibberellins (GAs), which stimulate fruit set. Circumstantial evidence suggests that the gaseous hormone ethylene may also influence fruit set, but this has yet to be substantiated with molecular or mechanistic data. Here, we examined fruit set at the biochemical and genetic levels, using hormone and inhibitor treatments, and mutants that affect auxin or ethylene signaling. The expression of system-1 ethylene biosynthetic genes and the production of ethylene decreased during pollination-dependent fruit set in wild-type tomato and during pollination-independent fruit set in the auxin hypersensitive mutant iaa9-3. Blocking ethylene perception in emasculated flowers, using either the ethylene-insensitive Sletr1-1 mutation or 1-methylcyclopropene (1-MCP), resulted in elongated parthenocarpic fruit and increased cell expansion, whereas simultaneous treatment with the GA biosynthesis inhibitor paclobutrazol (PAC) inhibited parthenocarpy. Additionally, the application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to pollinated ovaries reduced fruit set. Furthermore, Sletr1-1 parthenocarpic fruits did not exhibit increased auxin accumulation, but rather had elevated levels of bioactive GAs, most likely reflecting an increase in transcripts encoding the GA-biosynthetic enzyme SlGA20ox3, as well as a reduction in the levels of transcripts encoding the GA-inactivating enzymes SlGA2ox4 and SlGA2ox5. Taken together, our results suggest that ethylene plays a role in tomato fruit set by suppressing GA metabolism.

TOMATOMA Update: Phenotypic and Metabolite Information in the Micro-Tom Mutant Resource.

TOMATOMA (http://tomatoma.nbrp.jp/) is a tomato mutant database providing visible phenotypic data of tomato mutant lines generated by ethylmethane sulfonate (EMS) treatment or γ-ray irradiation in the genetic background of Micro-Tom, a small and rapidly growing variety. To increase mutation efficiency further, mutagenized M3 seeds were subjected to a second round of EMS treatment; M3M1 populations were generated. These plants were self-pollinated, and 4,952 lines of M3M2 mutagenized seeds were generated. We checked for visible phenotypes in the M3M2 plants, and 618 mutant lines with 1,194 phenotypic categories were identified. In addition to the phenotypic information, we investigated Brix values and carotenoid contents in the fruits of individual mutants. Of 466 samples from 171 mutant lines, Brix values and carotenoid contents were between 3.2% and 11.6% and 6.9 and 37.3 µg g(-1) FW, respectively. This metabolite information concerning the mutant fruits would be useful in breeding programs as well as for the elucidation of metabolic regulation. Researchers are able to browse and search this phenotypic and metabolite information and order seeds of individual mutants via TOMATOMA. Our new Micro-Tom double-mutagenized populations and the metabolic information could provide a valuable genetic toolkit to accelerate tomato research and potential breeding programs.

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.

Tomato Glutamate Decarboxylase Genes SlGAD2 and SlGAD3 Play Key Roles in Regulating γ-Aminobutyric Acid Levels in Tomato (Solanum lycopersicum).

Tomato (Solanum lycopersicum) can accumulate relatively high levels of γ-aminobutyric acid (GABA) during fruit development. However, the molecular mechanism underlying GABA accumulation and its physiological function in tomato fruits remain elusive. We previously identified three tomato genes (SlGAD1, SlGAD2 and SlGAD3) encoding glutamate decarboxylase (GAD), likely the key enzyme for GABA biosynthesis in tomato fruits. In this study, we generated transgenic tomato plants in which each SlGAD was suppressed and those in which all three SlGADs were simultaneously suppressed. A significant decrease in GABA levels, i.e. 50-81% compared with wild-type (WT) levels, was observed in mature green (MG) fruits of the SlGAD2-suppressed lines, while a more drastic reduction (up to <10% of WT levels) was observed in the SlGAD3- and triple SlGAD-suppressed lines. These findings suggest that both SlGAD2 and SlGAD3 expression are crucial for GABA biosynthesis in tomato fruits. The importance of SlGAD3 expression was also confirmed by generating transgenic tomato plants that over-expressed SlGAD3. The MG and red fruits of the over-expressing transgenic lines contained higher levels of GABA (2.7- to 5.2-fold) than those of the WT. We also determined that strong down-regulation of the SlGADs had little effect on overall plant growth, fruit development or primary fruit metabolism under normal growth conditions.

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.