Deregulation of ζ-carotene desaturase in Arabidopsis and tomato exposes a unique carotenoid-derived redundant regulation of floral meristem identity and function.

A level of redundancy and interplay among the transcriptional regulators of floral development safeguards a plant's reproductive success and ensures crop production. In the present study, an additional layer of complexity in the regulation of floral meristem (FM) identity and flower development is elucidated linking carotenoid biosynthesis and metabolism to the regulation of determinate flowering. The accumulation and subsequent cleavage of a diverse array of ζ-carotenes in the chloroplast biogenesis 5 (clb5) mutant of Arabidopsis results in the reprogramming of meristematic gene regulatory networks establishing FM identity mirroring that of the FM identity master regulator, APETALA1 (AP1). The immediate transition to floral development in clb5 requires long photoperiods in a GIGANTEA-independent manner, whereas AP1 is essential for the floral organ development of clb5. The elucidation of this link between carotenoid metabolism and floral development translates to tomato exposing a regulation of FM identity redundant to and initiated by AP1 and proposed to be dependent on the E class floral initiation and organ identity regulator, SEPALLATA3 (SEP3).

Deconvoluting apocarotenoid-mediated retrograde signaling networks regulating plastid translation and leaf development.

Signals originating within plastids modulate organelle differentiation by transcriptionally regulating nuclear-encoded genes. These retrograde signals are also integral regulators of plant development, including leaf morphology. The clb5 mutant displays severe leaf morphology defects due to Apocarotenoid Signal 1 (ACS1) accumulation in the developmentally arrested plastid. Transcriptomic analysis of clb5 validates that ACS1 accumulation deregulates hundreds of nuclear genes, including the suppression of most genes encoding plastid ribosomal proteins. Herein, we order the molecular events causing the leaf phenotype associated with the accumulation of ACS1, which includes two consecutive retrograde signaling cascades. Firstly, ACS1 originating in the plastid drives inhibition of plastid translation (IPT) via nuclear transcriptome remodeling of chlororibosomal proteins, requiring light as an essential component. Subsequently, IPT results in leaf morphological defects via a GUN1-dependent pathway shared with seedlings undergoing chemical IPT treatments and is restricted to an early window of the leaf development. Collectively, this work advances our understanding of the complexity within plastid retrograde signaling exemplified by sequential signal exchange and consequences that in a particular temporal and spatial context contribute to the modulation of leaf development.

The role of carotenoids as a source of retrograde signals: impact on plant development and stress responses.

Communication from plastids to the nucleus via retrograde signal cascades is essential to modulate nuclear gene expression, impacting plant development and environmental responses. Recently, a new class of plastid retrograde signals has emerged, consisting of acyclic and cyclic carotenoids and/or their degradation products, apocarotenoids. Although the biochemical identity of many of the apocarotenoid signals is still under current investigation, the examples described herein demonstrate the central roles that these carotenoid-derived signals play in ensuring plant development and survival. We present recent advances in the discovery of apocarotenoid signals and their role in various plant developmental transitions and environmental stress responses. Moreover, we highlight the emerging data exposing the highly complex signal transduction pathways underlying plastid to nucleus apocarotenoid retrograde signaling cascades. Altogether, this review summarizes the central role of the carotenoid pathway as a major source of retrograde signals in plants.

A GDSL Esterase/Lipase Catalyzes the Esterification of Lutein in Bread Wheat.

Xanthophylls are a class of carotenoids that are important micronutrients for humans. They are often found esterified with fatty acids in fruits, vegetables, and certain grains, including bread wheat (Triticum aestivum). Esterification promotes the sequestration and accumulation of carotenoids, thereby enhancing stability, particularly in tissues such as in harvested wheat grain. Here, we report on a plant xanthophyll acyltransferase (XAT) that is both necessary and sufficient for xanthophyll esterification in bread wheat grain. XAT contains a canonical Gly-Asp-Ser-Leu (GDSL) motif and is encoded by a member of the GDSL esterase/lipase gene family. Genetic evidence from allelic variants of wheat and transgenic rice (Oryza sativa) calli demonstrated that XAT catalyzes the formation of xanthophyll esters. XAT has broad substrate specificity and can esterify lutein, ß-cryptoxanthin, and zeaxanthin using multiple acyl donors, yet it has a preference for triacylglycerides, indicating that the enzyme acts via transesterification. A conserved amino acid, Ser-37, is required for activity. Despite xanthophylls being synthesized in plastids, XAT accumulated in the apoplast. Based on analysis of substrate preferences and xanthophyll ester formation in vitro and in vivo using xanthophyll-accumulating rice callus, we propose that disintegration of the cellular structure during wheat grain desiccation facilitates access to lutein-promoting transesterification.plantcell;31/12/3092/FX1F1fx1.

Manipulation of ZDS in tomato exposes carotenoid- and ABA-specific effects on fruit development and ripening.

Spontaneous mutations in fruit-specific carotenoid biosynthetic genes of tomato (Solanum lycopersicum) have led to improved understanding of ripening-associated carotenogenesis. Here, we confirm that ZDS is encoded by a single gene in tomato transcriptionally regulated by ripening transcription factors RIN, NOR and ethylene. Manipulation of ZDS was achieved through transgenic repression and heterologous over-expression in tomato. CaMV 35S-driven RNAi repression inhibited carotenoid biosynthesis in all aerial tissues examined resulting in elevated levels of ζ-carotene isomers and upstream carotenoids, while downstream all trans-lycopene and subsequent photoprotective carotenes and xanthophylls were diminished. Consequently, immature fruit displayed photo-bleaching consistent with reduced levels of the photoprotective carotenes and developmental phenotypes related to a reduction in the carotenoid-derived phytohormone abscisic acid (ABA). ZDS-repressed ripe fruit was devoid of the characteristic red carotenoid, all trans-lycopene and displayed brilliant yellow pigmentation due to elevated 9,9' di-cis-ζ-carotene. Over-expression of the Arabidopsis thaliana ZDS (AtZDS) gene bypassed endogenous co-suppression and revealed ZDS as an additional bottleneck in ripening-associated carotenogenesis of tomato. Quantitation of carotenoids in addition to multiple ripening parameters in ZDS-altered lines and ABA-deficient fruit-specific carotenoid mutants was used to separate phenotypic consequences of ABA from other effects of ZDS manipulation and reveal a unique and dynamic ζ-carotene isomer profile in ripe fruit.

Volatile apocarotenoid discovery and quantification in Arabidopsis thaliana: optimized sensitive analysis via HS-SPME-GC/MS.

INTRODUCTION: In the field of carotenoid metabolism researchers' focus has been directed recently toward the discovery and quantification of carotenoid cleavage products (i.e. apocarotenoids, excluding the well-studied carotenoid-derived hormones abscisic acid and strigolactones), due to their emerging roles as putative signaling molecules. Gas chromatography mass spectrometry (GC/MS) and sample preparation via headspace solid phase micro-extraction (HS-SPME) are widely used analytical techniques for broad untargeted metabolomics studies and until now, no optimized quantitative targeted HS-SPME-GC/MS method has been developed specifically for volatile apocarotenoids (VAs) in planta. OBJECTIVES: Optimization and subsequent validation of the HS-SPME technique for extracting and quantifying volatile apocarotenoids in planta. METHODS: Factors considered during method optimization were HS-SPME parameters; vial storage conditions; different adsorbent SPME fibre coating chemistries; plant tissue matrix effects; and fresh tissues to be analyzed. RESULTS: Mean linear regression in planta calibration correlation coefficients (R2) for VAs was 0.974. The resultant method mean limits of detection (LOD) and lower limits of quantification (LLOQ) for VAs using in planta standard additions were 0.384 ± 0.139 and 0.640 ± 0.231 µg/L, respectively. VAs remained stable at elevated SPME incubation temperatures, with no observable effects of thermal and photo-stereoisomerisation and oxidation. The bipolar 50/30 µm divinylbenzene/carboxen on polydimethylsiloxane (PDMS/DVB/CAR) was identified as the optimal fibre for broad molecular weight range VA analysis. CONCLUSIONS: An optimized HS-SPME-GC/MS method for VA detection and quantification was validated in vitro and in planta: based on biological replicates and stringent QA/QC approaches, thereby providing robust detection and quantification of VAs across a broad range of Arabidopsis tissues, fifteen of which were identified for the first time in Arabidopsis.

AtPDS overexpression in tomato: exposing unique patterns of carotenoid self-regulation and an alternative strategy for the enhancement of fruit carotenoid content.

The regulation of plant carotenogenesis is an active research area for both biological discovery and practical implementation. In tomato (Solanum lycopersicum), we demonstrate additional bottlenecks exist in the poly-cis-transformation of phytoene to lycopene in the context of ripening-induced PSY1 expression and activity and reveal phytoene desaturase (PDS), as a target for manipulation towards elevated lycopene content in maturing tomato fruit. Overexpression of Arabidopsis PDS, AtPDS, elevated PDS transcript abundance in all aerial tissues resulting in both altered carotenoid accumulation and associated pathway gene expression in a tissue-specific manner. Significant increases in downstream carotenoids (all-trans-lycopene and ß-carotene) and minimal changes in carotenogenic gene expression (carotenoid isomerase-like 1, CRTIL1) suggest overexpression of heterologous AtPDS in tomato circumvents endogenous regulatory mechanism observed with previous strategies. In transgenic leaves, depletion of the PDS substrate, phytoene, was accompanied by minor, but significant increases in xanthophyll production. Alterations in the leaf carotenogenic transcript profile, including the upstream MEP pathway, were observed revealing unique feedback and feedforward regulatory mechanisms in response to AtPDS overexpression. AtPDS overexpression in the background of the tangerine (carotenoid isomerase, CRTISO) mutant exposes its potential in elevating downstream cis-lycopene accumulation in ripe tomato fruit, as cis-lycopene is more bioavailable yet less abundant than all-trans-lycopene in the wild-type control. In summary, we demonstrate the limitation of PDS in ripening fruit, its utility in modifying carotenoid profiles towards improved quality, and reveal novel carotenoid pathway feedback regulation.

Loss of plastoglobule kinases ABC1K1 and ABC1K3 causes conditional degreening, modified prenyl-lipids, and recruitment of the jasmonic acid pathway.

Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the double mutant (k1 k3) displayed rapid chlorosis upon high light stress. Also, k1 k3 showed a slower, but irreversible, senescence-like phenotype during moderate light stress that was phenocopied by drought and nitrogen limitation, but not cold stress. This senescence-like phenotype involved degradation of the photosystem II core and upregulation of chlorophyll degradation. The senescence-like phenotype was independent of the EXECUTER pathway that mediates genetically controlled cell death from the chloroplast and correlated with increased levels of the singlet oxygen-derived carotenoid ß-cyclocitral, a retrograde plastid signal. Total PG volume increased during light stress in wild type and k1 k3 plants, but with different size distributions. Isolated PGs from k1 k3 showed a modified prenyl-lipid composition, suggesting reduced activity of PG-localized tocopherol cyclase (VTE1), and was consistent with loss of carotenoid cleavage dioxygenase 4. Plastid jasmonate biosynthesis enzymes were recruited to the k1 k3 PGs but not wild-type PGs, while pheophytinase, which is involved in chlorophyll degradation, was induced in k1 k3 and not wild-type plants and was localized to PGs. Thus, the ABC1K1/3 complex contributes to PG function in prenyl-lipid metabolism, stress response, and thylakoid remodeling.

Molecular and genetic regulation of fruit ripening.

Fleshy fruit undergo a novel developmental program that ends in the irreversible process of ripening and eventual tissue senescence. During this maturation process, fruit undergo numerous physiological, biochemical and structural alterations, making them more attractive to seed dispersal organisms. In addition, advanced or over-ripening and senescence, especially through tissue softening and eventual decay, render fruit susceptible to invasion by opportunistic pathogens. While ripening and senescence are often used interchangeably, the specific metabolic activities of each would suggest that ripening is a distinct process of fleshy fruits that precedes and may predispose the fruit to subsequent senescence.

Altered chloroplast development and delayed fruit ripening caused by mutations in a zinc metalloprotease at the lutescent2 locus of tomato.

The chloroplast is the site of photosynthesis in higher plants but also functions as the center of synthesis for primary and specialized metabolites including amino acids, fatty acids, starch, and diverse isoprenoids. Mutants that disrupt aspects of chloroplast function represent valuable tools for defining structural and biochemical regulation of the chloroplast and its interplay with whole-plant structure and function. The lutescent1 (l1) and l2 mutants of tomato (Solanum lycopersicum) possess a range of chlorophyll-deficient phenotypes including reduced rates of chlorophyll synthesis during deetiolation and enhanced rates of chlorophyll loss in leaves and fruits as they age, particularly in response to high-light stress and darkness. In addition, the onset of fruit ripening is delayed in lutescent mutants by approximately 1 week although once ripening is initiated they ripen at a normal rate and accumulation of carotenoids is not impaired. The l2 locus was mapped to the long arm of chromosome 10 and positional cloning revealed the existence of a premature stop codon in a chloroplast-targeted zinc metalloprotease of the M50 family that is homologous to the Arabidopsis (Arabidopsis thaliana) gene ETHYLENE-DEPENDENT GRAVITROPISM DEFICIENT AND YELLOW-GREEN1. Screening of tomato germplasm identified two additional l2 mutant alleles. This study suggests a role for the chloroplast in mediating the onset of fruit ripening in tomato and indicates that chromoplast development in fruit does not depend on functional chloroplasts.

Integrative transcript and metabolite analysis of nutritionally enhanced DE-ETIOLATED1 downregulated tomato fruit.

Fruit-specific downregulation of the DE-ETIOLATED1 (DET1) gene product results in tomato fruits (Solanum lycopersicum) containing enhanced nutritional antioxidants, with no detrimental effects on yield. In an attempt to further our understanding of how modulation of this gene leads to improved quality traits, detailed targeted and multilevel omic characterization has been performed. Metabolite profiling revealed quantitative increases in carotenoid, tocopherol, phenylpropanoids, flavonoids, and anthocyanidins. Qualitative differences could also be identified within the phenolics, including unique formation in fruit pericarp tissues. These changes resulted in increased total antioxidant content both in the polar and nonpolar fractions. Increased transcription of key biosynthetic genes is a likely mechanism producing elevated phenolic-based metabolites. By contrast, high levels of isoprenoids do not appear to result from transcriptional regulation but are more likely related to plastid-based parameters, such as increased plastid volume per cell. Parallel metabolomic and transcriptomic analyses reveal the widespread effects of DET1 downregulation on diverse sectors of metabolism and sites of synthesis. Correlation analysis of transcripts and metabolites independently indicated strong coresponses within and between related pathways/processes. Interestingly, despite the fact that secondary metabolites were the most severely affected in ripe tomato fruit, our integrative analyses suggest that the coordinated activation of core metabolic processes in cell types amenable to plastid biogenesis is the main effect of DET1 loss of function.

Detection and analysis of novel and known plant volatile apocarotenoids.

As the climate becomes increasingly unpredictable due to global warming, plants will encounter a greater challenge to adapt to their hostile environment (e.g., drought, heat, pollution). Volatile apocarotenoids (VAs) are an integral part of this necessary adaptation. VAs are involved in diverse plant life processes such as defense against biotic or abiotic stresses and regulate various aspects of plant development. The discovery of new VAs will help enhance abiotic and biotic stress tolerance, optimize biomass and crop yield, improve root development to better search for nutrients and promote symbiotic associations. This chapter describes an optimized method, HeadSpace Solid-Phase MicroExtraction (HS-SPME) coupled to Gas Chromatography-Mass Spectrometry (GC/MS), for the sensitive, reproducible, accurate, and high-throughput detection and quantification of novel and known VAs. Further optimization of this method can be performed by (1) adapting optimal growth conditions for your plants, (2) identifying the correct SPME fiber coating chemistry for the VAs of interest, (3) adapting optimal sample HS-SPME extraction temperature and time, and the desorption time in the GC inlet, (4) identifying the correct GC column and applying the optimal GC/MS parameters for good chromatographic baseline separation of the VAs, mass spectral matching and retention index (RI) validation, and (5) performing suitable quantification and statistical analyses. With this optimized and validated analytical technique, we detected and quantified 28 VAs; 20 of these were identified for the first time in Arabidopsis.

Synthesis and Function of Apocarotenoid Signals in Plants.

In plants, carotenoids are essential for photosynthesis and photoprotection. However, carotenoids are not the end products of the pathway; apocarotenoids are produced by carotenoid cleavage dioxygenases (CCDs) or non-enzymatic processes. Apocarotenoids are more soluble or volatile than carotenoids but they are not simply breakdown products, as there can be modifications post-cleavage and their functions include hormones, volatiles, and signals. Evidence is emerging for a class of apocarotenoids, here referred to as apocarotenoid signals (ACSs), that have regulatory roles throughout plant development beyond those ascribed to abscisic acid (ABA) and strigolactone (SL). In this context we review studies of carotenoid feedback regulation, chloroplast biogenesis, stress signaling, and leaf and root development providing evidence that apocarotenoids may fine-tune plant development and responses to environmental stimuli.

More than meets the eye: from carotenoid biosynthesis, to new insights into apocarotenoid signaling.

Carotenoids are a class of isoprenoids synthesized almost exclusively in plants involved in a myriad of roles including the provision of flower and fruit pigmentation for the attraction of pollinators and seed dispersing organisms. While carotenoids are essential throughout plant development, they are also extremely important in human diets providing necessary nutrition and aiding in the prevention of various cancers, age-related diseases and macular degeneration. Utilization of multiple plant models systems (i.e. Arabidopsis; maize; and tomato) has provided a comprehensive framework detailing the regulation of carotenogenesis throughout plant development covering all levels of genetic regulation from epigenetic to post-translational modifications. That said, the understanding of how carotenoids self-regulate remains fragmented. Recent reports demonstrate the potential influence of carotenoid-cleavage products (apocarotenoids) as signaling molecules regulating carotenoid biosynthesis in addition to various aspects of plants development (i.e. leaf and root development). This review highlights recent advances in carotenogenic regulation and insights into potential roles of novel apocarotenoids in plants.

Developmental and feedforward control of the expression of folate biosynthesis genes in tomato fruit.

Little is known about how plants regulate their folate content, including whether the expression of folate biosynthesis genes is orchestrated during development or modulated by folate levels. Nor is much known about how folate levels impact the expression of other genes. These points were addressed using wild-type tomato fruit and fruit engineered for high folate content. In wild-type fruit, the expression of genes specifying early steps in folate biosynthesis declined during development but that of other genes did not. In engineered fruit overexpressing foreign GTP cyclohydrolase I and aminodeoxychorismate synthase genes, the expression of the respective endogenous genes did not change, but that of three downstream pathway genes-aminodeoxychorismate lyase, dihydroneopterin aldolase, and mitochondrial folylpolyglutamate synthase-respectively increased by up to 7.8-, 2.8-, and 1.7-fold, apparently in response to the build-up of specific folate pathway metabolites. These results indicate that, in fruit, certain folate pathway genes are developmentally regulated and that certain others are subject to feedforward control by pathway intermediates. Microarray analysis showed that only 14 other transcripts (of 11 000 surveyed) increased in abundance by two-fold or more in high-folate fruit, demonstrating that the induction of folate pathway genes is relatively specific.

Tomato gamma-glutamylhydrolases: expression, characterization, and evidence for heterodimer formation.

Folates typically have gamma-linked polyglutamyl tails that make them better enzyme substrates and worse transport substrates than the unglutamylated forms. The tail can be shortened or removed by the vacuolar enzyme gamma-glutamyl hydrolase (GGH). It is known that GGH is active only as a dimer and that plants can have several GGH genes whose homodimeric products differ functionally. However, it is not known whether GGH dimers dissociate under in vivo conditions, whether heterodimers form, or how heterodimerization impacts enzyme activity. These issues were explored using the GGH system of tomato (Solanum lycopersicum). Tomato has three GGH genes that, like those in other eudicots, apparently diverged recently. LeGGH1 and LeGGH2 are expressed in fruit and all other organs, whereas LeGGH3 is expressed mainly in flower buds. LeGGH1 and LeGGH2 homodimers differ in bond cleavage preference; the LeGGH3 homodimer is catalytically inactive. Homodimers did not dissociate in physiological conditions. When coexpressed in Escherichia coli, LeGGH1 and LeGGH2 formed heterodimers with an intermediate bond cleavage preference, whereas LeGGH3 formed heterodimers with LeGGH1 or LeGGH2 that had one-half the activity of the matching homodimer. E. coli cells expressing LeGGH2 showed approximately 85% reduction in folate polyglutamates, but cells expressing LeGGH3 did not, confirming that LeGGH2 can function in vivo and LeGGH3 cannot. The formation of LeGGH1-LeGGH2 heterodimers was demonstrated in planta using bimolecular fluorescence complementation. Plant GGH heterodimers thus appear to form wherever different GGH genes are expressed simultaneously and to have catalytic characteristics midway between those of the corresponding homodimers.

Amino acid substitutions in homologs of the STAY-GREEN protein are responsible for the green-flesh and chlorophyll retainer mutations of tomato and pepper.

Color changes often accompany the onset of ripening, leading to brightly colored fruits that serve as attractants to seed-dispersing organisms. In many fruits, including tomato (Solanum lycopersicum) and pepper (Capsicum annuum), there is a sharp decrease in chlorophyll content and a concomitant increase in the synthesis of carotenoids as a result of the conversion of chloroplasts into chromoplasts. The green-flesh (gf) and chlorophyll retainer (cl) mutations of tomato and pepper, respectively, are inhibited in their ability to degrade chlorophyll during ripening, leading to the production of ripe fruits characterized by both chlorophyll and carotenoid accumulation and are thus brown in color. Using a positional cloning approach, we have identified a point mutation at the gf locus that causes an amino acid substitution in an invariant residue of a tomato homolog of the STAY-GREEN (SGR) protein of rice (Oryza sativa). Similarly, the cl mutation also carries an amino acid substitution at an invariant residue in a pepper homolog of SGR. Both GF and CL expression are highly induced at the onset of fruit ripening, coincident with the ripening-associated decline in chlorophyll. Phylogenetic analysis indicates that there are two distinct groups of SGR proteins in plants. The SGR subfamily is required for chlorophyll degradation and operates through an unknown mechanism. A second subfamily, which we have termed SGR-like, has an as-yet undefined function.

Transcriptomic and reverse genetic analyses of branched-chain fatty acid and acyl sugar production in Solanum pennellii and Nicotiana benthamiana.

Acyl sugars containing branched-chain fatty acids (BCFAs) are exuded by glandular trichomes of many species in Solanaceae, having an important defensive role against insects. From isotope-feeding studies, two modes of BCFA elongation have been proposed: (1) fatty acid synthase-mediated two-carbon elongation in the high acyl sugar-producing tomato species Solanum pennellii and Datura metel; and (2) alpha-keto acid elongation-mediated one-carbon increments in several tobacco (Nicotiana) species and a Petunia species. To investigate the molecular mechanisms underlying BCFAs and acyl sugar production in trichomes, we have taken a comparative genomic approach to identify critical enzymatic steps followed by gene silencing and metabolite analysis in S. pennellii and Nicotiana benthamiana. Our study verified the existence of distinct mechanisms of acyl sugar synthesis in Solanaceae. From microarray analyses, genes associated with alpha-keto acid elongation were found to be among the most strongly expressed in N. benthamiana trichomes only, supporting this model in tobacco species. Genes encoding components of the branched-chain keto-acid dehydrogenase complex were expressed at particularly high levels in trichomes of both species, and we show using virus-induced gene silencing that they are required for BCFA production in both cases and for acyl sugar synthesis in N. benthamiana. Functional analysis by down-regulation of specific KAS I genes and cerulenin inhibition indicated the involvement of the fatty acid synthase complex in BCFA production in S. pennellii. In summary, our study highlights both conserved and divergent mechanisms in the production of important defense compounds in Solanaceae and defines potential targets for engineering acyl sugar production in plants for improved pest tolerance.

Ethylene insensitivity conferred by the Green-ripe and Never-ripe 2 ripening mutants of tomato.

The ripening of a fleshy fruit represents the summation of an array of biochemical processes that are regulated by interactions between developmental programs and environmental inputs. Analysis of tomato (Solanum lycopersicum) mutants and inhibitor studies indicate that ethylene is necessary for full development of the ripening program of climacteric fruit such as tomato, yet ethylene alone is not sufficient. This suggests that an interaction between ethylene and nonethylene (or developmental) pathways mediates ripening. In this study, we have examined the physiological basis for ripening inhibition of the dominant Green-ripe (Gr) and Never-ripe 2 (Nr-2) mutants of tomato. Our data suggest that this inhibition is due to ethylene insensitivity in mutant fruit. Further investigation of ethylene responses in Gr and Nr-2 plants also revealed weak ethylene insensitivity during floral senescence and abscission and, during inhibition of root elongation, a phenotype associated with the triple response. However, ethylene-induced inhibition of hypocotyl elongation and petiole epinasty are normal in Gr and Nr-2, suggesting that these loci regulate a subset of ethylene responses. We have mapped both dominant mutations to a 2-cM overlapping region of the long arm of chromosome 1 of tomato, a region not previously linked to any known ethylene signaling loci. The phenotypic similarity and overlapping map location of these mutations suggest Gr and Nr-2 may be allelic and may possibly encode a novel component of the ethylene response pathway.