Introgression of a dominant phototropin1 mutant enhances carotenoids and boosts flavour-related volatiles in genome-edited tomato RIN mutants.

The tomato (Solanum lycopersicum) ripening inhibitor (rin) mutation is known to completely repress fruit ripening. The heterozygous (RIN/rin) fruits have extended shelf life, ripen normally, but have inferior taste/flavour. To address this, we used genome editing to generate newer alleles of RIN (rinCR ) by targeting the K-domain. Unlike previously reported CRISPR alleles, the rinCR alleles displayed delayed onset of ripening, suggesting that the mutated K-domain represses the onset of ripening. The rinCR fruits had extended shelf life and accumulated carotenoids at an intermediate level between rin and progenitor line. Besides, the metabolites and hormonal levels in rinCR fruits were more akin to rin. To overcome the negative attributes of rin, we crossed the rinCR alleles with Nps1, a dominant-negative phototropin1 mutant, which enhances carotenoid levels in tomato fruits. The resulting Nps1/rinCR hybrids had extended shelf life and 4.4-7.1-fold higher carotenoid levels than the wild-type parent. The metabolome of Nps1/rinCR fruits revealed higher sucrose, malate, and volatiles associated with tomato taste and flavour. Notably, the boosted volatiles in Nps1/rinCR were only observed in fruits bearing the homozygous Nps1 mutation. The Nps1 introgression into tomato provides a promising strategy for developing cultivars with extended shelf life, improved taste, and flavour.

Seeing the unseen: a trifoliate (MYB117) mutant allele fortifies folate and carotenoids in tomato fruits.

In tomato (Solanum lycopersicum), mutations in the gene encoding the R2R3-MYB117 transcription factor elicit trifoliate leaves and initiate the formation of axillary meristems; however, their effects on fruit ripening remain unexplored. The fruits of a new trifoliate (tf) mutant (tf-5) were firmer and had higher °Brix values and higher folate and carotenoid contents. The transcriptome, proteome, and metabolome profiling of tf-5 reflected a broad-spectrum change in cellular homeostasis. The tf-5 allele enhanced the fruit firmness by suppressing cell wall softening-related proteins. tf-5 fruit displayed a substantial increase in amino acids, particularly γ-aminobutyric acid, with a parallel reduction in aminoacyl-tRNA synthases. The increased lipoxygenase protein and transcript levels seemingly elevated jasmonic acid levels. In addition, increased abscisic acid hydrolase transcript levels coupled with reduced precursor supply lowered abscisic acid levels. The upregulation of carotenoids was mediated by modulation of methylerythreitol and plastoquinone pathways and increased the levels of carotenoid isomerization proteins. The upregulation of folate in tf-5 was connoted by the increase in the precursor p-aminobenzoic acid and transcript levels of several folate biosynthesis genes. The reduction in pterin-6-carboxylate levels and γ-glutamyl hydrolase activity indicated that reduced folate degradation in tf-5 increased folate levels. Our study delineates that in addition to leaf development, MYB117 also influences fruit metabolism. The tf-5 allele can be used to increase γ-aminobutyric acid, carotenoid, and folate levels in tomato.

Mutations in tomato 1-aminocyclopropane carboxylic acid synthase2 uncover its role in development beside fruit ripening.

The role of ethylene in plant development is mostly inferred from its exogenous application. The usage of mutants affecting ethylene biosynthesis proffers a better alternative to decipher its role. In tomato (Solanum lycopersicum), 1-aminocyclopropane carboxylic acid synthase2 (ACS2) is a key enzyme regulating ripening-specific ethylene biosynthesis. We characterised two contrasting acs2 mutants; acs2-1 overproduces ethylene, has higher ACS activity, and has increased protein levels, while acs2-2 is an ethylene underproducer, displays lower ACS activity, and has lower protein levels than wild type. Consistent with high/low ethylene emission, the mutants show opposite phenotypes, physiological responses, and metabolomic profiles compared with the wild type. The acs2-1 mutant shows early seed germination, faster leaf senescence, and accelerated fruit ripening. Conversely, acs2-2 has delayed seed germination, slower leaf senescence, and prolonged fruit ripening. The phytohormone profiles of mutants were mostly opposite in the leaves and fruits. The faster/slower senescence of acs2-1/acs2-2 leaves correlated with the endogenous ethylene/zeatin ratio. The genetic analysis showed that the metabolite profiles of respective mutants co-segregated with the homozygous mutant progeny. Our results uncover that besides ripening, ACS2 participates in the vegetative and reproductive development of tomato. The distinct influence of ethylene on phytohormone profiles indicates the intertwining of ethylene action with other phytohormones in regulating plant development.

The new kid on the block: a dominant-negative mutation of phototropin1 enhances carotenoid content in tomato fruits.

Phototropins, the UVA-blue light photoreceptors, endow plants to detect the direction of light and optimize photosynthesis by regulating positioning of chloroplasts and stomatal gas exchange. Little is known about their functions in other developmental responses. A tomato Non-phototropic seedling1 (Nps1) mutant, bearing an Arg495His substitution in the vicinity of LOV2 domain in phototropin1, dominant-negatively blocks phototropin1 responses. The fruits of Nps1 mutant were enriched in carotenoids, particularly lycopene, compared with its parent, Ailsa Craig. On the contrary, CRISPR/CAS9-edited loss of function phototropin1 mutants displayed subdued carotenoids compared with the parent. The enrichment of carotenoids in Nps1 fruits is genetically linked with the mutation and exerted in a dominant-negative fashion. Nps1 also altered volatile profiles with high levels of lycopene-derived 6-methyl 5-hepten2-one. The transcript levels of several MEP and carotenogenesis pathway genes were upregulated in Nps1. Nps1 fruits showed altered hormonal profiles with subdued ethylene emission and reduced respiration. Proteome profiles showed a causal link between higher carotenogenesis and increased levels of protein protection machinery, which may stabilize proteins contributing to MEP and carotenogenesis pathways. The enhancement of carotenoid content by Nps1 in a dominant-negative fashion offers a potential tool for high lycopene-bearing hybrid tomatoes.

Reanalysis of genome sequences of tomato accessions and its wild relatives: development of Tomato Genomic Variation (TGV) database integrating SNPs and INDELs polymorphisms.

MOTIVATION: Facilitated by technological advances and expeditious decrease in the sequencing costs, whole-genome sequencing is increasingly implemented to uncover variations in cultivars/accessions of many crop plants. In tomato (Solanum lycopersicum), the availability of the genome sequence, followed by the resequencing of tomato cultivars and its wild relatives, has provided a prodigious resource for the improvement of traits. A high-quality genome resequencing of 84 tomato accessions and wild relatives generated a dataset that can be used as a resource to identify agronomically important alleles across the genome. Converting this dataset into a searchable database, including information about the influence of single-nucleotide polymorphisms (SNPs) on protein function, provides valuable information about the genetic variations. The database will assist in searching for functional variants of a gene for introgression into tomato cultivars. RESULTS: A recent release of better-quality tomato genome reference assembly SL3.0, and new annotation ITAG3.2 of SL3.0, dropped 3857 genes, added 4900 novel genes and updated 20 766 genes. Using the above version, we remapped the data from the tomato lines resequenced under the '100 tomato genome resequencing project' on new tomato genome assembly SL3.0 and made an online searchable Tomato Genomic Variations (TGVs) database. The TGV contains information about SNPs and insertion/deletion events and expands it by functional annotation of variants with new ITAG3.2 using SIFT4G software. This database with search function assists in inferring the influence of SNPs on the function of a target gene. This database can be used for selecting SNPs, which can be potentially deployed for improving tomato traits. AVAILABILITY AND IMPLEMENTATION: TGV is freely available at http://psd.uohyd.ac.in/tgv.

Next-generation sequencing (NGS)-based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population.

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next-generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty-five genes belonging to carotenoids and folate metabolism were PCR-amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600-bp amplicons were directly sequenced in a non-overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128-fold pooling. An evaluation of six different software programs (camba, crisp, gatk unified genotyper, lofreq, snver and vipr) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.

Green-fruited Solanum habrochaites lacks fruit-specific carotenogenesis due to metabolic and structural blocks.

Members of the tomato clade exhibit a wide diversity in fruit color, but the mechanisms governing inter-species diversity of coloration are largely unknown. The carotenoid profiles, carotenogenic gene expression and proteome profiles of green-fruited Solanum habrochaites (SH), orange-fruited S. galapagense, and red-fruited S. pimpinellifolium were compared with cultivated tomato [S. lycopersicum cv. Ailsa Craig (SL)] to decipher the molecular basis of coloration diversity. Green-fruited SH, though it showed normal expression of chromoplast-specific phytoene synthase1 and lycopene ß-cyclase genes akin to orange/red-fruited species, failed to accumulate lycopene and ß-carotene. The SH phytoene synthase1 cDNA encoded an enzymatically active protein, whereas the lycopene ß-cyclase cDNA was barely active. Consistent with its green-fruited nature, SH's fruits retained chloroplast structure and PSII activity, and had impaired chlorophyll degradation with high pheophorbide a levels. Comparison of the fruit proteomes with SL revealed retention of the proteome complement related to photosynthesis in SH. Targeted peptide monitoring revealed a low abundance of key carotenogenic and sequestration proteins in SH compared with tomato. The green-fruitedness of SH appears to stem from blocks at several critical steps regulating fruit-specific carotenogenesis namely the absence of chloroplast to chromoplast transformation, block in carotenoid biosynthesis, and a dearth of carotenoid sequestering proteins.

A dominant mutation in the light-oxygen and voltage2 domain vicinity impairs phototropin1 signaling in tomato.

In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movement and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning responses. Here, we report the isolation and characterization of a Nonphototropic seedling1 (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses, including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark-grown seedlings of the Nps1 mutant, phot1 protein accumulates at a highly reduced level relative to the wild type and lacks BL-induced autophosphorylation. The mutant harbors a single glycine-1484-to-alanine transition in the Hinge1 region of a phot1 homolog, resulting in an arginine-to-histidine substitution (R495H) in a highly conserved A'α helix proximal to the light-oxygen and voltage2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of the A'α helix region in phototropic signaling of tomato.

Chromoplast-specific carotenoid-associated protein appears to be important for enhanced accumulation of carotenoids in hp1 tomato fruits.

Tomato (Solanum lycopersicum) high-pigment mutants with lesions in diverse loci such as DNA Damage-Binding Protein1 (high pigment1 [hp1]), Deetiolated1 (hp2), Zeaxanthin Epoxidase (hp3), and Intense pigment (Ip; gene product unknown) exhibit increased accumulation of fruit carotenoids coupled with an increase in chloroplast number and size. However, little is known about the underlying mechanisms exaggerating the carotenoid accumulation and the chloroplast number in these mutants. A comparison of proteome profiles from the outer pericarp of hp1 mutant and wild-type (cv Ailsa Craig) fruits at different developmental stages revealed at least 72 differentially expressed proteins during ripening. Hierarchical clustering grouped these proteins into three clusters. We found an increased abundance of chromoplast-specific carotenoid-associated protein (CHRC) in hp1 fruits at red-ripe stage that is also reflected in its transcript level. Western blotting using CHRC polyclonal antibody from bell pepper (Capsicum annuum) revealed a 2-fold increase in the abundance of CHRC protein in the red-ripe stage of hp1 fruits compared with the wild type. CHRC levels in hp2 were found to be similar to that of hp1, whereas hp3 and Ip showed intermediate levels to those in hp1, hp2, and wild-type fruits. Both CHRC and carotenoids were present in the isolated plastoglobules. Overall, our results suggest that loss of function of DDB1, DET1, Zeaxanthin Epoxidase, and Ip up-regulates CHRC levels. Increase in CHRC levels may contribute to the enhanced carotenoid content in these high-pigment fruits by assisting in the sequestration and stabilization of carotenoids.

Complex and shifting interactions of phytochromes regulate fruit development in tomato.

Tomato fruit ripening is a complex metabolic process regulated by a genetical hierarchy. A subset of this process is also modulated by light signalling, as mutants encoding negative regulators of phytochrome signal transduction show higher accumulation of carotenoids. In tomato, phytochromes are encoded by a multi-gene family, namely PHYA, PHYB1, PHYB2, PHYE and PHYF; however, their contribution to fruit development and ripening has not been examined. Using single phytochrome mutants phyA, phyB1 and phyB2 and multiple mutants phyAB1, phyB1B2 and phyAB1B2, we compared the on-vine transitory phases of ripening until fruit abscission. The phyAB1B2 mutant showed accelerated transitions during ripening, with shortest time to fruit abscission. Comparison of transition intervals in mutants indicated a phase-specific influence of different phytochrome species either singly or in combination on the ripening process. Examination of off-vine ripened fruits indicated that ripening-specific carotenoid accumulation was not obligatorily dependent upon light and even dark-incubated fruits accumulated carotenoids. The accumulation of transcripts and carotenoids in off-vine and on-vine ripened mutant fruits indicated a complex and shifting phase-dependent modulation by phytochromes. Our results indicate that, in addition to regulating carotenoid levels in tomato fruits, phytochromes also regulate the time required for phase transitions during ripening.

Identification of Volatiles in Tomato Fruit Using Headspace Solid-Phase-Micro-Extraction (HS-SPME) Coupled with Gas Chromatography-Mass Spectrometry (GC-MS).

Plant volatile organic compounds (VOCs) are organic chemicals that plants release as part of their natural biological processes. Various plant tissues produce VOCs, including leaves, stems, flowers, and roots. VOCs are essential in plant communication, defense against pests and pathogens, aroma and flavor, and attracting pollinators. The study of plant volatiles has become an increasingly important area of research in recent years, as scientists have recognized these compounds' important roles in plant physiology. As a result, there has been a growing interest in developing methods for collecting and analyzing plant VOCs. HS-SPME-GC-MS (headspace solid-phase microextraction-gas chromatography-mass spectrometry) is commonly used for plant volatile analysis due to its high sensitivity and selectivity. This chapter describes an efficient method for extracting and identifying volatile compounds by HS-SPME coupled with GC-MS in tomato fruits.

A Comprehensive Protocol for Assembly of Multiple gRNAs into a Direct Vector for Genome Editing in Tomato.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas 9 (CRISPR-associated protein 9) is a robust DNA-encoded, RNA-mediated sequence-specific nuclease system widely used for genome editing of various plants. Although there are many reports on the assembly of gRNAs and plant transformation, there is no single resource for the complete gene editing methodology in tomato. This chapter provides a comprehensive protocol for designing gRNAs, their assembly into the vector, plant transformation, and final mutant analysis in tomato.

Reduced γ-glutamyl hydrolase activity likely contributes to high folate levels in Periyakulam-1 tomato.

Tomato cultivars show wide variation in nutraceutical folate in ripe fruits, yet the loci regulating folate levels in fruits remain unexplored. To decipher regulatory points, we compared two contrasting tomato cultivars: Periyakulam-1 (PKM-1) with high folate and Arka Vikas (AV) with low folate. The progression of ripening in PKM-1 was nearly similar to AV but had substantially lower ethylene emission. In parallel, the levels of phytohormones salicylic acid, ABA, and jasmonic acid were substantially lower than AV. The fruits of PKM-1 were metabolically distinct from AV, with upregulation of several amino acids. Consistent with higher °Brix, the red ripe fruits also showed upregulation of sugars and sugar-derived metabolites. In parallel with higher folate, PKM-1 fruits also had higher carotenoid levels, especially lycopene and ß-carotene. The proteome analysis showed upregulation of carotenoid sequestration and folate metabolism-related proteins in PKM-1. The deglutamylation pathway mediated by γ-glutamyl hydrolase (GGH) was substantially reduced in PKM-1 at the red-ripe stage. The red-ripe fruits had reduced transcript levels of GGHs and lower GGH activity than AV. Conversely, the percent polyglutamylation of folate was much higher in PKM-1. Our analysis indicates the regulation of GGH activity as a potential target to elevate folate levels in tomato fruits.

Augmenting tomato functional genomics with a genome-wide induced genetic variation resource.

Induced mutations accelerate crop improvement by providing novel disease resistance and yield alleles. However, the alleles with no perceptible phenotype but have an altered function remain hidden in mutagenized plants. The whole-genome sequencing (WGS) of mutagenized individuals uncovers the complete spectrum of mutations in the genome. Genome-wide induced mutation resources can improve the targeted breeding of tomatoes and facilitate functional genomics. In this study, we sequenced 132 doubly ethyl methanesulfonate (EMS)-mutagenized lines of tomato and detected approximately 41 million novel mutations and 5.5 million short InDels not present in the parental cultivar. Approximately 97% of the genome had mutations, including the genes, promoters, UTRs, and introns. More than one-third of genes in the mutagenized population had one or more deleterious mutations predicted by Sorting Intolerant From Tolerant (SIFT). Nearly one-fourth of deleterious genes mapped on tomato metabolic pathways modulate multiple pathway steps. In addition to the reported GC>AT transition bias for EMS, our population also had a substantial number of AT>GC transitions. Comparing mutation frequency among synonymous codons revealed that the most preferred codon is the least mutagenic toward EMS. The validation of a potato leaf-like mutation, reduction in carotenoids in ζ-carotene isomerase mutant fruits, and chloroplast relocation loss in phototropin1 mutant validated the mutation discovery pipeline. Our database makes a large repertoire of mutations accessible to functional genomics studies and breeding of tomatoes.

Tomato root penetration in soil requires a coaction between ethylene and auxin signaling.

During seed germination, emerging roots display positive gravitropism and penetrate into the soil for nutrition and anchorage. Tomato (Solanum lycopersicum) seeds germinated in the presence of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, failed to insert roots into Soilrite and grew in the air, forming loops. Time-lapse video imaging showed that 1-MCP-grown root tips retained positive gravitropism and made contact with the surface of Soilrite but failed to penetrate into the Soilrite. Time-course studies revealed that the effect of 1-MCP was most prominent when seed imbibition and germination were carried out in the continual presence of 1-MCP. Conversely, 1-MCP was ineffective when applied postgermination after penetration of roots in the Soilrite. Furthermore, treatment with 1-MCP caused a reduction in DR5::ß-glucuronidase auxin-reporter activity and modified the expression of SlIAA3 and SlIAA9 transcripts, indicating interference with auxin signaling. The reduced ethylene perception mutant, Never-ripe, displayed decreased ability for root penetration, and the enhanced polar auxin transport mutant, polycotyledon, showed a nearly normal root penetration in the presence of 1-MCP, which could be reversed by application of auxin transport inhibitors. Our results indicate that during tomato seed germination, a coaction between ethylene and auxin is required for root penetration into the soil.

Phytoene synthase 2 in tomato fruits remains functional and contributes to abscisic acid formation.

In ripening tomato fruits, the leaf-specific carotenoids biosynthesis mediated by phytoene synthase 2 (PSY2) is replaced by a fruit-specific pathway by the expression of two chromoplast-specific genes: phytoene synthase 1 (PSY1) and lycopene-ß-cyclase (CYCB). Though both PSY1 and PSY2 genes express in tomato fruits, the functional role of PSY2 is not known. To decipher whether PSY2-mediated carotenogenesis operates in ripening fruits, we blocked the in vivo activity of lycopene-ß-cyclases in fruits of several carotenoids and ripening mutants by CPTA (2-(4-Chlorophenylthio)triethylamine hydrochloride), an inhibitor of lycopene-ß-cyclases. The CPTA-treatment induced accumulation of lycopene in leaves, immature-green and ripening fruits. Even in psy1 mutants V7 and r that are deficient in fruit-specific carotenoid biosynthesis, CPTA triggered lycopene accumulation but lowered the abscisic acid level. Differing from fruit-specific carotenogenesis, CPTA-treated V7 and r mutant fruits accumulated lycopene but not phytoene and phytofluene. The lack of phytoene and phytofluene accumulation was reminiscent of PSY2-mediated leaf-like carotenogenesis, where phytoene and phytofluene accumulation is never seen. The lycopene accumulation was associated with the partial transformation of chloroplasts to chromoplasts bearing thread-like structures. Our study uncovers the operation of a parallel carotenogenesis pathway mediated by PSY2 that provides precursors for abscisic acid biosynthesis in ripening tomato fruits.

iTRAQ-based proteome profiling revealed the role of Phytochrome A in regulating primary metabolism in tomato seedling.

In plants, during growth and development, photoreceptors monitor fluctuations in their environment and adjust their metabolism as a strategy of surveillance. Phytochromes (Phys) play an essential role in plant growth and development, from germination to fruit development. FR-light (FR) insensitive mutant (fri) carries a recessive mutation in Phytochrome A and is characterized by the failure to de-etiolate in continuous FR. Here we used iTRAQ-based quantitative proteomics along with metabolomics to unravel the role of Phytochrome A in regulating central metabolism in tomato seedlings grown under FR. Our results indicate that Phytochrome A has a predominant role in FR-mediated establishment of the mature seedling proteome. Further, we observed temporal regulation in the expression of several of the late response proteins associated with central metabolism. The proteomics investigations identified a decreased abundance of enzymes involved in photosynthesis and carbon fixation in the mutant. Profound accumulation of storage proteins in the mutant ascertained the possible conversion of sugars into storage material instead of being used or the retention of an earlier profile associated with the mature embryo. The enhanced accumulation of organic sugars in the seedlings indicates the absence of photomorphogenesis in the mutant.

A Low-Cost High-Throughput Method for Plant Genomic DNA Isolation.

Many of the functional genomics methods require isolation of genomic DNA from large population of plants. The selection of DNA isolation protocols depends on several factors such as choice of starting material, ease of handling, time and labor required for isolation, the final quantity as well as the quality of genomic DNA. We outline here a high-throughput method of DNA extraction from different plant species including cereal crops. The protocol can be used for extraction of DNA in single tubes as well as for large formats in 96-well plates. The protocol includes steps for eliminating interfering secondary products such as phenolics. This protocol can be applied for high-throughput isolation of DNA for varied applications such as TILLING, mapping, fingerprinting, etc. as a cost-effective protocol compared to commercial kits.

Is naphthylphthalamic acid a specific phytotropin? It elevates ethylene and alters metabolic homeostasis in tomato.

In higher plants, phytohormone indole-3-acetic acid is characteristically transported from the apex towards the base of the plant, termed as polar auxin transport (PAT). Among the inhibitors blocking PAT, N-1-naphthylphthalamic acid (NPA) that targets ABCB transporters is most commonly used. NPA-treated light-grown Arabidopsis seedlings show severe inhibition of hypocotyl and root elongation. In light-grown tomato seedlings, NPA inhibited root growth, but contrary to Arabidopsis stimulated hypocotyl elongation. The NPA-stimulation of hypocotyl elongation was milder in blue, red, and far-red light-grown seedlings. The NPA-treatment stimulated emission of ethylene from the seedlings. The scrubbing of ethylene by mercuric perchlorate reduced NPA-stimulated hypocotyl elongation. NPA action on hypocotyl elongation was antagonized by 1-methylcyclopropene, an inhibitor of ethylene action. NPA-treated seedlings had reduced levels of indole-3-butyric acid and higher levels of zeatin in the shoots. NPA did not alter indole-3-acetic levels in shoots. The analysis of metabolic networks indicated that NPA-treatment induced moderate shifts in the networks compared to exogenous ethylene that induced a drastic shift in metabolic networks. Our results indicate that in addition to ethylene, NPA-stimulated hypocotyl elongation in tomato may also involve zeatin and indole-3- butyric acid. Our results indicate that NPA-mediated physiological responses may vary in a species-specific fashion.