Search

Summary Differential display was used to isolate early ethylene-regulated genes from late immature green tomato fruit in order to obtain a broader understanding of the molecular basis by which ethylene coordinates the ripening process. Nineteen novel ethylene-responsive (ER) cDNA clones were isolated that fell into three classes: (i) ethylene up-regulated (ii) ethylene down-regulated, and (iii) transiently induced. Expression analysis revealed that ethylene-dependent changes in mRNA accumulation occurred rapidly (15 min) for most of the ER clones. The predicted proteins encoded by the ER genes are putatively involved in processes as diverse as primary metabolism, hormone signalling and stress responses. Although a number of the isolated ER clones correspond to genes already documented in other species, their responsiveness to ethylene is described here for the first time. Among the ER clones sharing high homology with regulatory genes, ER43 , a putative GTP-binding protein, and ER50 , a CTR1 -like clone, are potentially involved in signal transduction. ER24 is homologous to the multi-protein bridging factor MBF1 involved in transcriptional activation, and finally, two clones are homologous to genes involved in post-transcriptional regulation: ER49 , a putative translational elongation factor, and ER68 , a mRNA helicase-like gene. Six ER clones correspond to as yet unidentified genes. The expression studies indicated that all the ER genes are ripening-regulated, and, depending on the clone, show changes in transcript accumulation either at the breaker, turning, or red stage. Analysis of transcript accumulation in different organs indicated a strong bias towards expression in the fruit for many of the clones. The potential roles for some of the ER clones in propagating the ethylene response and regulating fruit ripening are discussed. [ABSTRACT FROM AUTHOR]

Summary Following differential screening of gene expression during tomato fruit development, we isolated developmentally regulated (DR) clones, including several putative transcription factors. Based on sequence homology, DR1, DR3, DR4 and DR8 are members of the Aux/IAA family, and DR12 belongs to the auxin response factor (ARF) family of transcription factors. Importantly, mRNA accumulation for the Aux/IAA -like genes was regulated by ethylene in tomato fruit but not in the leaves, indicating that these putative auxin response components also participate to the ethylene-dependent regulation of gene expression in a tissue-specific manner. The functional significance of DR12 , the ARF -like gene, was investigated by cellular biology and reverse genetics approaches. Heterologous protein targeting studies, carried out using a DR12–GFP gene fusion construct, revealed specific nuclear localization of the DR12-encoded protein, in accordance with its putative function as a transcriptional regulator. Transgenic plants over- and under-expressing DR12 were generated in order to explore the physiological role of the gene. Both antisense and sense co-suppressed DR12-inhibited lines displayed a pleiotropic phenotype that included dark-green immature fruit, unusual cell division in the fruit pericarp, blotchy ripening, enhanced fruit firmness, upward curling leaves and increased hypocotyl and cotyledon growth. While a perturbation of the response to auxin may explain some of the phenotypes, surprisingly, the expression of members of four classes of early auxin-regulated genes was unaffected in the DR12-inhibited plants. The involvement of this ARF-like encoded protein in mediating the auxin response is discussed along with the possibility that it might affect responsiveness to other phytohormones in the tomato. [ABSTRACT FROM AUTHOR]

Summary:Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl aldehyde, is a toxin produced byEutypa lata,the causal agent of eutypa dieback of grapevines. It has previously been demonstrated that tolerance of some cultivars to this disease was correlated with their capacity to convert eutypine to the corresponding alcohol, eutypinol, which lacks phytotoxicity. We have thus purified to homogeneity a protein fromVigna radiatathat exhibited eutypine-reducing activity and have isolated the corresponding cDNA. This encodes an NADPH-dependent reductase of 36 kDa that we have namedVigna radiataeutypine-reducing enzyme (VR-ERE), based on the capacity of a recombinant form of the protein to reduce eutypine into eutypinol. The strongest homologies (86.8%) of VR-ERE at the amino acid level were found with CPRD14, a drought-inducible gene of unknown function, isolated fromVigna unguiculataand with an aromatic alcohol dehydrogenase (71.7%) fromEucalyptus gunnii. Biochemical characterization of VR-ERE revealed that a variety of compounds containing an aldehyde group can act as substrates. However, the highest affinity was observed with 3-substituted benzaldehydes. Expression of aVR-EREtransgene inVitis viniferacells culturedin vitroconferred resistance to the toxin. This discovery opens up new biotechnological approaches for the generation of grapevines resistant to eutypa dieback.. [ABSTRACT FROM AUTHOR]

Tomato is a model for fruit development and ripening. The isolation of intact plastids from this organism is therefore important for metabolic and proteomic analyses. Pepper, a species from the same family, is also of interest since it allows isolation of intact chromoplasts in large amounts. Here, we provide a detailed protocol for the isolation of tomato plastids at three fruit developmental stages, namely, nascent chromoplasts from the mature green stage, chromoplasts from an intermediate stage, and fully differentiated red chromoplasts. The method relies on sucrose density gradient centrifugations. It yields high purity organelles suitable for proteome analyses. Enzymatic and microscopy assays are summarized to assess purity and intactness. A method is also described for subfractionation of pepper chromoplast lipoprotein structures.

Ethylene Response Factors (ERFs) are downstream components of the ethylene signal transduction pathway, although their role in ethylene-dependent developmental processes remains poorly understood. As the ethylene-inducible tomato Sl-ERF.B3 has been shown previously to display a strong binding affinity to GCC-box-containing promoters, its physiological significance was addressed here by a reverse genetics approach. However, classical up- and down-regulation strategies failed to give clear clues to its roles in planta, probably due to functional redundancy among ERF family members. Expression of a dominant repressor ERF.B3-SRDX version of Sl-ERF.B3 in the tomato resulted in pleiotropic ethylene responses and vegetative and reproductive growth phenotypes. The dominant repressor etiolated seedlings displayed partial constitutive ethylene response in the absence of ethylene and adult plants exhibited typical ethylene-related alterations such as leaf epinasty, premature flower senescence and accelerated fruit abscission. The multiple symptoms related to enhanced ethylene sensitivity correlated with the altered expression of ethylene biosynthesis and signaling genes and suggested the involvement of Sl-ERF.B3 in a feedback mechanism that regulates components of ethylene production and response. Moreover, Sl-ERF.B3 was shown to modulate the transcription of a set of ERFs and revealed the existence of a complex network interconnecting different ERF genes. Overall, the study indicated that Sl-ERF.B3 had a critical role in the regulation of multiple genes and identified a number of ERFs among its primary targets, consistent with the pleiotropic phenotypes displayed by the dominant repression lines., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

Successful completion of fruit developmental programs depends on the interplay between multiple phytohormones. However, besides ethylene, the impact of other hormones on fruit quality traits remains elusive. A previous study has shown that down-regulation of SlARF4, a member of the tomato (Solanum lycopersicum) auxin response factor (ARF) gene family, results in a dark-green fruit phenotype with increased chloroplasts (Jones et al., 2002). This study further examines the role of this auxin transcriptional regulator during tomato fruit development at the level of transcripts, enzyme activities, and metabolites. It is noteworthy that the dark-green phenotype of antisense SlARF4-suppressed lines is restricted to fruit, suggesting that SlARF4 controls chlorophyll accumulation specifically in this organ. The SlARF4 underexpressing lines accumulate more starch at early stages of fruit development and display enhanced chlorophyll content and photochemical efficiency, which is consistent with the idea that fruit photosynthetic activity accounts for the elevated starch levels. SlARF4 expression is high in pericarp tissues of immature fruit and then undergoes a dramatic decline at the onset of ripening concomitant with the increase in sugar content. The higher starch content in developing fruits of SlARF4 down-regulated lines correlates with the up-regulation of genes and enzyme activities involved in starch biosynthesis, suggesting their negative regulation by SlARF4. Altogether, the data uncover the involvement of ARFs in the control of sugar content, an essential feature of fruit quality, and provide insight into the link between auxin signaling, chloroplastic activity, and sugar metabolism in developing fruit.

Background: Mature-fruit abscission (MFA) in fleshy-fruit is a genetically controlled process with mechanisms that, contrary to immature-fruit abscission, has not been fully characterized. Here, we use pyrosequencing to characterize the transcriptomes of melon abscission zone (AZ) at three stages during AZ-cell separation in order to understand MFA control at an early stage of AZ-activation., Principal Findings: The results show that by early induction of MFA, the melon AZ exhibits major gene induction, while by late induction of MFA, melon AZ shows major gene repression. Although some genes displayed similar regulation in both early and late induction of abscission, such as EXT1-EXT4, EGase1, IAA2, ERF1, AP2D15, FLC, MADS2, ERAF17, SAP5 and SCL13 genes, the majority had different expression patterns. This implies that time-specific events occur during MFA, and emphasizes the value of characterizing multiple time-specific abscission transcriptomes. Analysis of gene-expression from these AZs reveal that a sequential induction of cell-wall-degrading genes is associated with the upregulation of genes involved in endo and exocytosis, and a shift in plant-hormone metabolism and signaling genes during MFA. This is accompanied by transcriptional activity of small-GTPases and synthaxins together with tubulins, dynamins, V-type ATPases and kinesin-like proteins potentially involved in MFA signaling. Early events are potentially controlled by down-regulation of MADS-box, AP2/ERF and Aux/IAA transcription-factors, and up-regulation of homeobox, zinc finger, bZIP, and WRKY transcription-factors, while late events may be controlled by up-regulation of MYB transcription-factors., Significance: Overall, the data provide a comprehensive view on MFA in fleshy-fruit, identifying candidate genes and pathways associated with early induction of MFA. Our comprehensive gene-expression profile will be very useful for elucidating gene regulatory networks of the MFA in fleshy-fruit.

A tomato short-chain dehydrogenase-reductase (SlscADH1) is preferentially expressed in fruit with a maximum expression at the breaker stage while expression in roots, stems, leaves and flowers is very weak. It represents a potential candidate for the formation of aroma volatiles by interconverting alcohols and aldehydes. The SlscADH1 recombinant protein produced in Escherichia coli exhibited dehydrogenase-reductase activity towards several volatile compounds present in tomato flavour with a strong preference for the NAD/NADH co-factors. The strongest activity was observed for the reduction of hexanal (K(m)=0.175mM) and phenylacetaldehyde (K(m)=0.375mM) in the presence of NADH. The oxidation process of hexanol and 1-phenylethanol was much less efficient (K(m)s of 2.9 and 23.0mM, respectively), indicating that the enzyme preferentially acts as a reductase. However activity was observed only for hexanal, phenylacetaldehyde, (E)-2-hexenal and acetaldehyde and the corresponding alcohols. No activity could be detected for other aroma volatiles important for tomato flavour, such as methyl-butanol/methyl-butanal, 5-methyl-6-hepten-2-one/5-methyl-6-hepten-2-ol, citronellal/citronellol, neral/nerol, geraniol. In order to assess the function of the SlscADH1 gene, transgenic plants have been generated using the technique of RNA interference (RNAi). Constitutive down-regulation using the 35S promoter resulted in the generation of dwarf plants, indicating that the SlscADH1 gene, although weakly expressed in vegetative tissues, had a function in regulating plant development. Fruit-specific down-regulation using the 2A11 promoter had no morphogenetic effect and did not alter the aldehyde/alcohol balance of the volatiles compounds produced by the fruit. Nevertheless, SlscADH1-inhibited fruit unexpectedly accumulated higher concentrations of C5 and C6 volatile compounds of the lipoxygenase pathway, possibly as an indirect effect of the suppression of SlscADH1 on the catabolism of phospholipids and/or integrity of membranes., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Background: The phytohormone ethylene is involved in a wide range of developmental processes and in mediating plant responses to biotic and abiotic stresses. Ethylene signalling acts via a linear transduction pathway leading to the activation of Ethylene Response Factor genes (ERF) which represent one of the largest gene families of plant transcription factors. How an apparently simple signalling pathway can account for the complex and widely diverse plant responses to ethylene remains yet an unanswered question. Building on the recent release of the complete tomato genome sequence, the present study aims at gaining better insight on distinctive features among ERF proteins., Results: A set of 28 cDNA clones encoding ERFs in the tomato (Solanum lycopersicon) were isolated and shown to fall into nine distinct subclasses characterised by specific conserved motifs most of which with unknown function. In addition of being able to regulate the transcriptional activity of GCC-box containing promoters, tomato ERFs are also shown to be active on promoters lacking this canonical ethylene-responsive-element. Moreover, the data reveal that ERF affinity to the GCC-box depends on the nucleotide environment surrounding this cis-acting element. Site-directed mutagenesis revealed that the nature of the flanking nucleotides can either enhance or reduce the binding affinity, thus conferring the binding specificity of various ERFs to target promoters.Based on their expression pattern, ERF genes can be clustered in two main clades given their preferential expression in reproductive or vegetative tissues. The regulation of several tomato ERF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signalling pathways of the two hormones., Conclusions: The data reveal that regions flanking the core GCC-box sequence are part of the discrimination mechanism by which ERFs selectively bind to their target promoters. ERF tissue-specific expression combined to their responsiveness to both ethylene and auxin bring some insight on the complexity and fine regulation mechanisms involving these transcriptional mediators. All together the data support the hypothesis that ERFs are the main component enabling ethylene to regulate a wide range of physiological processes in a highly specific and coordinated manner.

A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato (Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration) and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome.

Background and Aims: There are several studies suggesting that tomato (Solanum lycopersicum) chromoplasts arise from chloroplasts, but there is still no report showing the fluorescence of both chlorophylls and carotenoids in an intermediate plastid, and no video showing this transition phase., Methods: Pigment fluorescence within individual plastids, isolated from tomato fruit using sucrose gradients, was observed at different ripening stages, and an in situ real-time recording of pigment fluorescence was performed on live tomato fruit slices., Key Results: At the mature green and red stages, homogenous fractions of chloroplasts and chromoplasts were obtained, respectively. At the breaker stage, spectral confocal microscopy showed that intermediate plastids contained both chlorophylls and carotenoids. Furthermore, an in situ real-time recording (a) showed that the chloroplast to chromoplast transition was synchronous for all plastids of a single cell; and (b) confirmed that all chromoplasts derived from pre-existing chloroplasts., Conclusions: These results give details of the early steps of tomato chromoplast biogenesis from chloroplasts, with the formation of intermediate plastids containing both carotenoids and chlorophylls. They provide information at the sub-cellular level on the synchronism of plastid transition and pigment changes.

Chromoplasts are carotenoid-accumulating plastids conferring color to many flowers and fruits as well as to some tubers and roots. Chromoplast differentiation proceeds from preexisting plastids, most often chloroplasts. One of the most prominent changes is remodeling of the internal membrane system associated with the formation of carotenoid-accumulating structures. During the differentiation process the plastid genome is essentially stable and transcriptional activity is restricted. The buildup of the chromoplast for specific metabolic characteristics is essentially dependent upon the transcriptional activity of the nucleus. Important progress has been made in terms of mediation of the chloroplast-to-chromoplast transition with the discovery of the crucial role of the Or gene. In this article we review recent developments in the structural, biochemical and molecular aspects of chromoplast differentiation and also consider the reverse differentiation of chromoplasts into chloroplast-like structures during the regreening process occurring in some fruit. Future perspectives toward a full understanding of chromoplast differentiation include in-depth knowledge of the changes occurring in the plastidial proteome during chromoplastogenesis, elucidation of the role of hormones and the search for signals that govern the dialog between the nuclear and the chromoplastic genome.

Chromoplasts are non-photosynthetic specialized plastids that are important in ripening tomato fruit (Solanum lycopersicum) since, among other functions, they are the site of accumulation of coloured compounds. Analysis of the proteome of red fruit chromoplasts revealed the presence of 988 proteins corresponding to 802 Arabidopsis unigenes, among which 209 had not been listed so far in plastidial databanks. These data revealed several features of the chromoplast. Proteins of lipid metabolism and trafficking were well represented, including all the proteins of the lipoxygenase pathway required for the synthesis of lipid-derived aroma volatiles. Proteins involved in starch synthesis co-existed with several starch-degrading proteins and starch excess proteins. Chromoplasts lacked proteins of the chlorophyll biosynthesis branch and contained proteins involved in chlorophyll degradation. None of the proteins involved in the thylakoid transport machinery were discovered. Surprisingly, chromoplasts contain the entire set of Calvin cycle proteins including Rubisco, as well as the oxidative pentose phosphate pathway (OxPPP). The present proteomic analysis, combined with available physiological data, provides new insights into the metabolic characteristics of the tomato chromoplast and enriches our knowledge of non-photosynthetic plastids.

Mountain papaya ( Vasconcellea pubescens ) is a climacteric fruit that develops a strong and characteristic aroma during ripening. Esters are the main volatile compounds produced by the fruit, and most of them are dependent on ethylene. As esters are synthesized through alcohol acyltransferases (AAT), a full-length cDNA (VpAAT1) was isolated that displayed the characteristic motifs of most plant acyltransferases. The full-length cDNA sequence was cloned and expressed in yeasts, obtaining a functional enzyme with high AAT activity toward the formation of benzyl acetate. The transcript accumulation pattern provided by qPCR analysis showed that the VpAAT1 gene is expressed exclusively in fruit tissues and that a high level of transcripts is accumulated during ripening. The increase in VpAAT1 transcripts in fruit is coincident with the increase in AAT activity; transcript accumulation is induced by ethylene, and it is avoided by 1-methylcyclopropene (1-MCP) treatment. The data indicate that VpAAT1 is involved in aroma formation and that ethylene plays a major role in regulating its expression.

Indole Acetic Acid 9 (IAA9) is a negative auxin response regulator belonging to the Aux/IAA transcription factor gene family whose downregulation triggers fruit set before pollination, thus giving rise to parthenocarpy. In situ hybridization experiments revealed that a tissue-specific gradient of IAA9 expression is established during flower development, the release of which upon pollination triggers the initiation of fruit development. Comparative transcriptome and targeted metabolome analysis uncovered important features of the molecular events underlying pollination-induced and pollination-independent fruit set. Comprehensive transcriptomic profiling identified a high number of genes common to both types of fruit set, among which only a small subset are dependent on IAA9 regulation. The fine-tuning of Aux/IAA and ARF genes and the downregulation of TAG1 and TAGL6 MADS box genes are instrumental in triggering the fruit set program. Auxin and ethylene emerged as the most active signaling hormones involved in the flower-to-fruit transition. However, while these hormones affected only a small number of transcriptional events, dramatic shifts were observed at the metabolic and developmental levels. The activation of photosynthesis and sucrose metabolism-related genes is an integral regulatory component of fruit set process. The combined results allow a far greater comprehension of the regulatory and metabolic events controlling early fruit development both in the presence and absence of pollination/fertilization.

Whereas the interplay of multiple hormones is essential for most plant developmental processes, the key integrating molecular players remain largely undiscovered or uncharacterized. It is shown here that a member of the tomato auxin/indole-3-acetic acid (Aux/IAA) gene family, Sl-IAA3, intersects the auxin and ethylene signal transduction pathways. Aux/IAA genes encode short-lived transcriptional regulators central to the control of auxin responses. Their functions have been defined primarily by dominant, gain-of-function mutant alleles in Arabidopsis. The Sl-IAA3 gene encodes a nuclear-targeted protein that can repress transcription from auxin-responsive promoters. Sl-IAA3 expression is auxin and ethylene dependent, is regulated on a tight tissue-specific basis, and is associated with tissues undergoing differential growth such as in epinastic petioles and apical hook. Antisense down-regulation of Sl-IAA3 results in auxin and ethylene-related phenotypes, including altered apical dominance, lower auxin sensitivity, exaggerated apical hook curvature in the dark and reduced petiole epinasty in the light. The results provide novel insights into the roles of Aux/IAAs and position the Sl-IAA3 protein at the crossroads of auxin and ethylene signalling in tomato.

Alcohol acyltransferases (AAT) play a key role in the biosynthesis of ester aroma volatiles in fruit. Three ripening-specific recombinant AATs of cantaloupe Charentais melon fruit (Cm-AAT1, Cm-AAT3, and Cm-AAT4) are capable of synthesizing thioether esters with Cm-AAT1 being by far the most active. All proteins, as well as AAT(s) extracted from melon fruit, are active as tetramers of around 200 kDa. Kinetic analysis demonstrated that CoA-SH, a product of the reaction, is an activator at low concentrations and an inhibitor at higher concentrations. This was confirmed by the addition of phosphotransacetylase at various concentrations, capable of modulating the level of CoA-SH in the reaction medium. Site-directed mutagenesis of some amino acids that were specific to the Cm-AAT sequences into amino acids that were consensus to other characterized AATs greatly affected the selectivity of the original protein and the number of esters produced.

Cell wall disassembly in ripening fruit is highly complex, involving the dismantling of multiple polysaccharide networks by diverse families of wall-modifying proteins. While it has been reported in several species that multiple members of each such family are expressed in the same fruit tissue, it is not clear whether this reflects functional redundancy, with protein isozymes from a single enzyme class performing similar roles and contributing equally to wall degradation, or whether they have discrete functions, with some isoforms playing a predominant role. Experiments reported here sought to distinguish between cell wall-related processes in ripening melon that were softening-associated and softening-independent. Cell wall polysaccharide depolymerization and the expression of wall metabolism-related genes were examined in transgenic melon (Cucumis melo var. cantalupensis Naud.) fruit with suppressed expression of the 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene and fruits treated with ethylene and 1-methylcyclopropene (1-MCP). Softening was completely inhibited in the transgenic fruit but was restored by treatment with exogenous ethylene. Moreover, post-harvest application of 1-MCP after the onset of ripening completely halted subsequent softening, suggesting that melon fruit softening is ethylene-dependent. Size exclusion chromatography of cell wall polysaccharides, from the transgenic fruits, with or without exogenous ethylene, indicated that the depolymerization of both pectins and xyloglucans was also ethylene dependent. However, northern analyses of a diverse range of cell wall-related genes, including those for polygalacturonases, xyloglucan endotransglucosylase/hydrolases, expansin, and beta-galactosidases, identified specific genes within single families that could be categorized as ethylene-dependent, ethylene-independent, or partially ethylene-dependent. These results support the hypothesis that while individual cell wall-modifying proteins from each family contribute to cell wall disassembly that accompanies fruit softening, other closely related family members are regulated in an ethylene-independent manner and apparently do not directly participate in fruit softening.

Ethylene response factors (ERFs) are plant transcriptional regulators mediating ethylene-dependent gene expression via binding to the GCC motif found in the promoter region of ethylene-regulated genes. We report here on the structural and functional characterization of the tomato Sl-ERF2 gene that belongs to a distinct class of the large ERF gene family. Both spliced and unspliced versions of Sl-ERF2 transcripts were amplified from RNA samples and the search in the public tomato expressed sequence tag (EST) database confirmed the existence of the two transcript species in a number of cDNA libraries. The unspliced transcript contains two open reading frames yielding two hypothetical proteins, a small highly truncated version lacking the APETALA2 domain and a bigger protein lacking the N-terminal MCGGAAI(I)/(L) consensus peptide specific to ERF members from subfamily IV. Nevertheless, functional Sl-ERF2 protein may only derive from spliced transcripts since, depending on the tissue, the level of the spliced transcript is much higher than that of the unspliced transcript. Sl-ERF2 is expressed in all plant tissues tested, though its transcript accumulates preferentially in germinating seeds and ripening fruit. Overexpression of the Sl-ERF2 gene in transgenic tomato lines results in premature seed germination and enhanced hook formation of dark-grown seedlings, which is indicative of increased ethylene sensitivity. The expression of the mannanase2 gene is upregulated in Sl-ERF2-overexpressing seeds, suggesting that Sl-ERF2 stimulates seed germination through the induction of the mannanase2 gene. It is noteworthy that the exaggerated hook phenotype is abolished when ethylene perception is blocked, strongly suggesting that Sl-ERF2 requires other ethylene-dependent components to impact the hook formation process.

Alcohol dehydrogenases (ADH) participate in the biosynthetic pathway of aroma volatiles in fruit by interconverting aldehydes to alcohols and providing substrates for the formation of esters. Two highly divergent ADH genes (15% identity at the amino acid level) of Cantaloupe Charentais melon (Cucumis melo var. Cantalupensis) have been isolated. Cm-ADH1 belongs to the medium-chain zinc-binding type of ADHs and is highly similar to all ADH genes expressed in fruit isolated so far. Cm-ADH2 belongs to the short-chain type of ADHs. The two encoded proteins are enzymatically active upon expression in yeast. Cm-ADH1 has strong preference for NAPDH as a co-factor, whereas Cm-ADH2 preferentially uses NADH. Both Cm-ADH proteins are much more active as reductases with K (m)s 10-20 times lower for the conversion of aldehydes to alcohols than for the dehydrogenation of alcohols to aldehydes. They both show strong preference for aliphatic aldehydes but Cm-ADH1 is capable of reducing branched aldehydes such as 3-methylbutyraldehyde, whereas Cm-ADH2 cannot. Both Cm-ADH genes are expressed specifically in fruit and up-regulated during ripening. Gene expression as well as total ADH activity are strongly inhibited in antisense ACC oxidase melons and in melon fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. These data suggest that each of the Cm-ADH protein plays a specific role in the regulation of aroma biosynthesis in melon fruit.

Auxin/indole-3-acetic acid (Aux/IAA) proteins are transcriptional regulators that mediate many aspects of plant responses to auxin. While functions of most Aux/IAAs have been defined mainly by gain-of-function mutant alleles in Arabidopsis thaliana, phenotypes associated with loss-of-function mutations have been scarce and subtle. We report here that the downregulation of IAA9, a tomato (Solanum lycopersicum) gene from a distinct subfamily of Aux/IAA genes, results in a pleiotropic phenotype, consistent with its ubiquitous expression pattern. IAA9-inhibited lines have simple leaves instead of wild-type compound leaves, and fruit development is triggered before fertilization, giving rise to parthenocarpy. This indicates that IAA9 is a key mediator of leaf morphogenesis and fruit set. In addition, antisense plants displayed auxin-related growth alterations, including enhanced hypocotyl/stem elongation, increased leaf vascularization, and reduced apical dominance. Auxin dose-response assays revealed that IAA9 downregulated lines were hypersensitive to auxin, although the only early auxin-responsive gene that was found to be upregulated in the antisense lines was IAA3. The activity of the IAA3 promoter was stimulated in the IAA9 antisense genetic background, indicating that IAA9 acts in planta as a transcriptional repressor of auxin signaling. While no mutation in any member of subfamily IV has been reported to date, the phenotypes associated with the downregulation of IAA9 reveal distinct and novel roles for members of the Aux/IAA gene family.

Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon.

A cDNA clone (LeCRK1), encoding a novel isoform of calcium-dependent protein kinase (CDPK), was isolated by screening a tomato (Lycopersicon esculentum) cDNA library. The protein derived from the full-length sequence indicated that it belongs to the family of CDPK-related kinases (CRKs) and the predicted amino acid sequence shows a modular organization of the protein consisting of different characteristic domains. The kinase domain of LeCRK1 shares a high degree of similarity with the catalytic domain of CDPKs. In contrast to canonical members of the family, LeCRK1 has a degenerate sequence in the C-terminal calmodulin-like domain. LeCRK1 protein was shown to be a functional kinase, but, consistent with the lack of calcium-binding activity, its autophosphorylation activity did not require calcium. LeCRK1 harbours an amphiphilic amino acid region revealed to be a functional calmodulin-binding site by in vitro assay. A putative myristoylation/palmitoylation sequence has been identified at the N-terminus. Expressing an LeCRK1::GFP fusion protein in the protoplast resulted in its targeting to the plasma membrane. Site-directed mutagenesis of critical amino acids of the myristoylation/palmitoylation consensus sites led to the accumulation of the mutated protein in the cytoplasm, suggesting that the native protein is anchored to the plasma membrane by acylated residues. Expression studies revealed significant accumulation of LeCRK1 transcripts during fruit ripening, although transcripts were also detected in stem, leaf, and flower. LeCRK1 mRNA level in leaves was slightly induced by ethylene and salicylic acid, and upon mechanical wounding and cold treatment. It is noteworthy that LeCRK1 mRNAs were undetectable in different tomato-ripening natural mutants such as NR, Rin, and Nor, suggesting a role in the ripening process.

Ethylene-induced ripening in tomato (Lycopersicon esculentum) resulted in the accumulation of a transcript designated LeEF-Ts(mt) that encodes a protein with significant homology to bacterial Ts translational elongation factor (EF-Ts). Transient expression in tobacco and sunflower protoplasts of full-length and truncated LeEF-Ts(mt)-GFP fusion constructs and confocal microscopy observations clearly demonstrated the targeting of LeEF-Ts(mt) to mitochondria and not to chloroplasts and the requirement for a signal peptide for the proper sorting of the protein. Escherichia coli recombinant LeEF-Ts(mt) co-eluted from Ni-NTA resins with a protein corresponding to the molecular weight of the elongation factor EF-Tu of E. coli, indicating an interaction with bacterial EF-Tu. Increasing the GDP concentration in the extraction buffer reduced the amount of EF-Tu in the purified LeEF-Ts(mt) fraction. The purified LeEF-Ts(mt) stimulated the poly(U)-directed polymerization of phenylalanine 10-fold in the presence of EF-Tu. Furthermore, LeEF-Ts(mt) was capable of catalysing the nucleotide exchange reaction with E. coli EF-Tu. Altogether, these data demonstrate that LeEF-Ts(mt) encodes a functional mitochondrial EF-Ts. LeEF-Ts(mt) represents the first mitochondrial elongation factor to be isolated and functionally characterized in higher plants.

Four new members of the ERF (ethylene-response factor) family of plant-specific DNA-binding (GCC box) factors were isolated from tomato fruit (LeERF1-4). Phylogenetic analysis indicated that LeERF2 belongs to a new ERF class, characterized by a conserved N-terminal signature sequence. Expression patterns and cis/trans binding affinities differed between the LeERFs. Combining experimental data and modeled three-dimensional analysis, it was shown that binding affinity of the LeERFs was affected by both the variation of nucleotides surrounding the DNA cis-element sequence and the nature of critical amino acid residues within the ERF domain.

LeCTR1 was initially isolated by both differential display reverse transcriptase-polymerase chain reaction screening for tomato (Lycopersicon esculentum) fruit ethylene-inducible genes and through homology with the Arabidopsis CTR1 cDNA. LeCTR1 shares strong nucleotide sequence homology with Arabidopsis CTR1, a gene acting downstream of the ethylene receptor and showing similarity to the Raf family of serine/threonine protein kinases. The length of the LeCTR1 transcribed region from ATG to stop codon (12,000 bp) is more than twice that of Arabidopsis CTR1 (4,700 bp). Structural analysis reveals perfect conservation of both the number and position of introns and exons in LeCTR1 and Arabidopsis CTR1. The introns in LeCTR1 are much longer, however. To address whether this structural conservation is indicative of functional conservation of the corresponding proteins, we expressed LeCTR1 in the Arabidopsis ctr1-1 (constitutive triple response 1) mutant under the direction of the 35S promoter. Our data clearly show that ectopic expression of LeCTR1 in the Arabidopsis ctr1-1 mutant can restore normal ethylene signaling. The recovery of normal ethylene sensitivity upon heterologous expression of LeCTR1 was also confirmed by restored glucose sensitivity absent in the Arabidopsis ctr1-1 mutant. Expression studies confirm ethylene responsiveness of LeCTR1 in various tissues, including ripening fruit, and may suggest the evolution of alternate regulatory mechanisms in tomato versus Arabidopsis.

Two genes (CM-AAT1 and CM-AAT2) with strong sequence homology (87% identity at the protein level) putatively involved in the formation of aroma volatile esters have been isolated from Charentais melon fruit. They belong to a large and highly divergent family of multifunctional plant acyl-transferases and show at most 21% identity to the only other fruit acyl-transferase characterized so far in strawberry. RT-PCR studies indicated that both genes were specifically expressed in fruit at increasing rates in the early and mid phases of ripening. Expression was severely reduced in ethylene-suppressed antisense ACC oxidase (AS) fruit and in wild-type (WT) fruit treated with the ethylene antagonist 1-MCP. Cloning of the two genes in yeast revealed that the CM-AAT1 protein exhibited alcohol acyl-transferase activity while no such activity could be detected for CM-AAT2 despite the strong homology between the two sequences. CM-AAT1 was capable of producing esters from a wide range of combinations of alcohols and acyl-CoAs. The higher the carbon chain of aliphatic alcohols, the higher the activity. Branched alcohols were esterified at differential rates depending on the position of the methyl group and the nature of the acyl donor. Phenyl and benzoyl alcohols were also good substrates, but activity varied with the position and size of the aromatic residue. The cis/trans configuration influenced activity either positively (2-hexenol) or negatively (3-hexenol). Because ripening melons evolve the whole range of esters generated by the recombinant CM-AAT1 protein, we conclude that CM-AAT1 plays a major role in aroma volatiles formation in the melon.

Fruit ripening and abscission are associated with an ethylene burst in several melon (Cucumis melo) genotypes. In cantaloupe as in other climacteric fruit, exogenous ethylene can prematurely induce abscission, ethylene production, and ripening. Melon genotypes without fruit abscission or without ethylene burst also exist and are, therefore, non-climacteric. In the nonabscising melon fruit PI 161375, exogenous ethylene failed to stimulate abscission, loss of firmness, ethylene production, and expression of all target genes tested. However, the PI 161375 etiolated seedlings displayed the usual ethylene-induced triple response. Genetic analysis on a population of recombinant cantaloupe Charentais x PI 161375 inbred lines in segregation for fruit abscission and ethylene production indicated that both characters are controlled by two independent loci, abscission layer (Al)-3 and Al-4. The non-climacteric phenotype in fruit tissues is attributable to ethylene insensitivity conferred by the recessive allelic forms from PI 161375. Five candidate genes (two ACO, two ACS, and ERS) that were localized on the melon genetic map did not exhibit colocalization with Al-3 or Al-4.

Compared to other melon types, Cantaloupe Charentais melons are highly aromatic with a major contribution to the aroma being made by aliphatic and branched esters. Using a transgenic line in which the synthesis of the plant hormone ethylene has been considerably lowered by antisense ACC oxidase mRNA (AS), the aliphatic ester pathway steps at which ethylene exerts its regulatory role were found. The data show that the production of aliphatic esters such as hexyl and butyl acetate was blocked in AS fruit and could be reversed by ethylene. Using fruit discs incubated in the presence of various precursors, the steps at which ester formation was inhibited in AS fruit was shown to be the reduction of fatty acids and aldehydes, the last step of acetyl transfer to alcohols being unaffected. However, treating AS fruit with the ethylene antagonist 1-methylcyclopropene resulted in about 50% inhibition of acetyl transfer activity, indicating that this portion of activity was ethylene-dependent and this was supported by the low residual ethylene concentration of AS fruit discs (around 2 microl l(-1)). In conclusion, the reduction of fatty acids and aldehydes appears essentially to be ethylene-dependent, whilst the last step of alcohol acetylation has ethylene-dependent and ethylene-independent components, probably corresponding to differentially regulated alcohol acetyltransferases.