Various tomato cultivars display contrasting morphological and molecular responses to a chronic heat stress.

Climate change is one of the biggest threats that human society currently needs to face. Heat waves associated with global warming negatively affect plant growth and development and will increase in intensity and frequency in the coming years. Tomato is one of the most produced and consumed fruit in the world but remarkable yield losses occur every year due to the sensitivity of many cultivars to heat stress (HS). New insights into how tomato plants are responding to HS will contribute to the development of cultivars with high yields under harsh temperature conditions. In this study, the analysis of microsporogenesis and pollen germination rate of eleven tomato cultivars after exposure to a chronic HS revealed differences between genotypes. Pollen development was either delayed and/or desynchronized by HS depending on the cultivar considered. In addition, except for two, pollen germination was abolished by HS in all cultivars. The transcriptome of floral buds at two developmental stages (tetrad and pollen floral buds) of five cultivars revealed common and specific molecular responses implemented by tomato cultivars to cope with chronic HS. These data provide valuable insights into the diversity of the genetic response of floral buds from different cultivars to HS and may contribute to the development of future climate resilient tomato varieties.

Identification and genetic characterization of a gibberellin 2-oxidase gene that controls tree stature and reproductive growth in plum.

Several dwarf plum genotypes (Prunus salicina L.), due to deficiency of unknown gibberellin (GA) signalling, were identified. A cDNA encoding GA 2-oxidase (PslGA2ox), the major gibberellin catabolic enzyme in plants, was cloned and used to screen the GA-deficient hybrids. This resulted in the identification of a dwarf plum hybrid, designated as DGO24, that exhibits a markedly elevated PslGA2ox signal. Grafting 'Early Golden' (EG), a commercial plum cultivar, on DGO24 (EG/D) enhanced PslGA2ox accumulation in the scion part and generated trees of compact stature. Assessment of active GAs in such trees revealed that DGO24 and EG/D accumulated relatively much lower quantities of main bioactive GAs (GA(1) and GA(4)) than control trees (EG/M). Moreover, the physiological function of PslGA2ox was studied by determining the molecular and developmental consequences due to ectopic expression in Arabidopsis. Among several lines, two groups of homozygous transgenics that exhibited contrasting phenotypes were identified. Group-1 displayed a dwarf growth pattern typical of mutants with a GA deficiency including smaller leaves, shorter stems, and delay in the development of reproductive events. In contrast, Group-2 exhibited a 'GA overdose' phenotype as all the plants showed elongated growth, a typical response to GA application, even under limited GA conditions, potentially due to co-suppression of closely related Arabidopsis homologous. The studies reveal the possibility of utilizing PslGA2ox as a marker for developing size-controlling rootstocks in Prunus.

Expression of auxin-binding protein1 during plum fruit ontogeny supports the potential role of auxin in initiating and enhancing climacteric ripening.

Auxin-binding protein1 (ABP1) is an active element involved in auxin signaling and plays critical roles in auxin-mediated plant development. Here, we report the isolation and characterization of a putative sequence from Prunus salicina L., designated PslABP1. The expected protein exhibits a similar molecular structure to that of well-characterized maize-ABP1; however, PslABP1 displays more sequence polarity in the active-binding site due to substitution of some crucial amino-acid residues predicted to be involved in auxin-binding. Further, PslABP1 expression was assessed throughout fruit ontogeny to determine its role in fruit development. Comparing the expression data with the physiological aspects that characterize fruit-development stages indicates that PslABP1 up-regulation is usually associated with the signature events that are triggered in an auxin-dependent manner such as floral induction, fruit initiation, embryogenesis, and cell division and elongation. However, the diversity in PslABP1 expression profile during the ripening process of early and late plum cultivars seems to be due to the variability of endogenous auxin levels among the two cultivars, which consequently can change the levels of autocatalytic ethylene available for the fruit to co-ordinate ripening. The effect of auxin on stimulating ethylene production and in regulating PslABP1 was investigated. Our data suggest that auxin is involved in the transition of the mature green fruit into the ripening phase and in enhancing the ripening process in both auxin- and ethylene-dependent manners thereafter.

Regulation of two germin-like protein genes during plum fruit development.

Germin-like proteins (GLPs) have several proposed roles in plant development and defence. Two novel genes (Ps-GLP1 and 2) encoding germin-like protein were isolated from plum (Prunus salicina). Their regulation was studied throughout fruit development and during ripening of early and late cultivars. These two genes exhibited similar expression patterns throughout the various stages of fruit development excluding two important stages, pit hardening (S2) and fruit ripening (S4). During fruit development until the ripening phase, the accumulation of both Ps-GLPs is related to the evolution of auxin. However, during the S2 stage only Ps-GLP1 is induced and this could putatively be in a H(2)O(2)-dependent manner. On the other hand, the diversity in the Ps-GLPs accumulation profile during the ripening process seems to be putatively due to the variability of endogenous auxin levels among the two plum cultivars, which consequently change the levels of autocatalytic ethylene available for the fruit to co-ordinate ripening. The effect of auxin on stimulating ethylene production and in regulating Ps-GLPs transcripts was also investigated. These data, supported by their localization in the extracellular matrix, suggest that auxin is somehow involved in the regulation of both transcripts throughout fruit development and ripening.

Molecular characterization of seven genes encoding ethylene-responsive transcriptional factors during plum fruit development and ripening.

Seven ERF cDNAs were cloned from two Japanese plum (Prunus salicina L.) cultivars, 'Early Golden' (EG) and 'Shiro' (SH). Based on the sequence characterization, these Ps-ERFs could be classified into three of the four known ERF families. Their predicted amino acid sequences exhibited similarities to ERFs from other plant species. Functional nuclear localization signal analyses of two Ps-ERF proteins (Ps-ERF1a and -1b) were carried out using confocal microscopy. Expression analyses of Ps-ERF mRNAs were studied in the two plum cultivars in order to determine the role of this gene family in fruit development and ripening. The seven Ps-ERFs displayed differential expression pattern and levels throughout the various stages of flower and fruit development. The diversity in Ps-ERFs accumulation was largely due to the differences in their responses to the levels of ethylene production. However, other plant hormones such as cytokinin and auxin, which accumulate strongly throughout the various developmental stages, also influence the Ps-ERFs expression. The effect of the plant hormones, gibberellin, cytokinin, auxin, and ethylene in regulating the different Ps-ERF transcripts was investigated. A model was proposed in which the role played by the plant hormone auxin is as important as that of ethylene in initiating and determining the date and rate of ripening in Japanese plums.

Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits.

The plant hormone ethylene plays a major role in the ripening of climacteric fruit. We have generated transgenic cantaloupe Charentais melons expressing an antisense ACC oxidase gene; ACC oxidase catalyzes the last step of ethylene biosynthesis. Ethylene production of transgenic fruit was < 1% of control untransformed fruit, and the ripening process was blocked both on and off the vine. The antisense phenotype could be reversed by exogenous ethylene treatment. Analysis of antisense ACC oxidase melons indicated that the ripening process includes ethylene-dependent and ethylene-independent pathways. Because the transgenic line we generated displays extended storage life and improved quality, it has a promising potential for commercial development.

Ethylene-regulated gene expression in tomato fruit: characterization of novel ethylene-responsive and ripening-related genes isolated by differential display

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.

Purification and characterization of a NADPH-dependent aldehyde reductase from mung bean that detoxifies eutypine, a toxin from eutypa lata1

Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a Km value of 6.3 &mgr;M for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification.

The Aux/IAA, Sl-IAA17 regulates quality parameters over tomato fruit development.

Auxin is known to be involved in all the stages of fruit development. Aux/IAAs are regulators of the auxin signaling at the transcription level. In a recent study, using RNAi strategy to limit the expression Sl-IAA17, it was shown that this tomato AuxIAA regulates fruit size mainly through altering the ploidy level of pericarp cells. Indeed, Sl-IAA17 down-regulated lines showed fruit with larger diameter, bigger volume and heavier weight than wild-type. The increase in fruit size was associated with thicker pericarp rather than larger locular spaces. The thicker pericarp was linked to larger cells harboring higher ploidy level, probably due to more active endoreduplication at the beginning of fruit development. The present report describes some additional phenotypes, not described in the initial article, among which are soluble solid content, juice pH, firmness, seed weight and fruit morphology.

Expression of an antisense 1-aminocyclopropane-1-carboxylate oxidase gene stimulates shoot regeneration in Cucumis melo.

The role of ethylene in shoot regeneration was investigated using transgenic Cucumis melo plants expressing an antisense 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene. ACC oxidase catalyses the last step of ethylene biosynthesis. Leaf and cotyledon explants from the transgenic plants exhibited low ACC oxidase activity and ethylene production, whereas the regeneration capacity of the tissues was greatly enhanced (3.5- and 2.8-fold, respectively) compared to untransformed control tissues. Addition of ethylene released by 50 or 100 µM 2-chloroethylphosphonic acid dramatically reduced the shoot regeneration rate of the transgenic tissues. The results clearly demonstrate that ethylene plays an important role in C. melo morphogenesis in vitro.

Subcellular localization of the sites of conversion of 1-aminocyclopropane-1-carboxylic acid into ethylene in plant cells.

The subcellular localization of the sites of 1-aminocyclopropane-1-carboxylic acid (ACC) conversion into ethylene was studied by comparing the specific radioactivity of ethylene evolved from the whole cells with that of intra- and extracellular pools of labelled ACC. We demonstrate that some cells cultured in vitro (Vitis vinifera L. cv. Muscat) or leaf tissues (Hordeum vulgare L. and Triticum aestivum L.) have two sites of ethylene production: (i) an external site, converting apoplastic ACC, located at the plasma membrane, and very sensitive to high osmotica and, (ii) an intracellular site, converting internal ACC and remaining unaffected even under severe plasmolysis. In other cells cultured in vitro (Vitis vinifera L. cv. Gamay) and pea leaves (Pisum sativum L.), only the intracellular site operates and ethylene production is almost unaffected by plasmolysis. Protoplasts obtained from plasmolysis-sensitive Muscat cells lose 97% of their capacity for ethylene production compared with the parent cell, while those from plasmolysisinsensitive Gamay cells retain up to 50%. Protoplasts from both Gamay and Muscat cells cultured for 8 d in vitro, recover the full capacity of ethylene production of the initial whole cells, whether or not they are allowed to reform their cell wall. Therefore, we exclude a cooperation between the cell wall and the plasma membrane in ethylene production.

Intracellular Sites of Synthesis and Storage of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Acer pseudoplatanus Cells.

Vacuoles were isolated from Acer pseudoplatanus cells that were incubated with [(14)C]1-aminocyclopropane-1-carboxylic acid (ACC). The kinetics of [(14)C]1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) formation are consistent with the interpretation that MACC is synthesized in the cytosol, transported through the tonoplast, and accumulated in the vacuole. Twenty hours after chasing the labeled ACC with unlabeled ACC and adding 1 millimolar unlabeled MACC, all the [(14)C]MACC synthesized is located in the vacuole. Whole cells preloaded with [(14)C]MACC and then submitted to a continuous washing out, readily release their cytosolic MACC until complete exhaustion. The half-time of MACC efflux from the cytosol, calculated by the technique of compartmental analysis, is about 22 minutes. In contrast, vacuolar MACC remains sequestered within the vacuole. The transport of labeled MACC into the vacuole is stimulated by the presence of unlabeled MACC in the suspension medium, probably as a result of a reduced efflux of the labeled MACC from the cytosol into the suspending medium.

Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast.

The ethylene-forming enzyme (EFE), which catalyzes the last step in the biosynthesis of the plant hormone ethylene, has never been purified and no molecular probes are available. Recently, a putative cDNA clone for tomato EFE (pTOM13) has been identified by inhibiting ethylene synthesis with an antisense gene expressed in transgenic plants. A direct test of its function has been made by expression of a pTOM13 gene in Saccharomyces cerevisiae. After cloning artefacts were discovered in the 5' region of the cDNA, a corrected cDNA (pRC13) was created by the fusion of the 5' end of a genomic clone to the 3' end of the cDNA and expressed in S. cerevisiae. Cultures of transformed yeast converted 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, whereas control cells did not. This EFE activity displays similar characteristics to EFE found in plant tissue: it converts the trans isomer of the ACC analogue 1-amino-2-ethylcyclopropane-1-carboxylic acid to 1-butene in preference to the cis isomer, and it is strongly inhibited by cobaltous ions and 1,10-phenanthroline. Furthermore, information gained from the activity of effectors on yeast EFE activity supports the hypothesis that EFE is one of a group of hydroxylase enzymes.

Carrier-Mediated Uptake of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Vacuoles Isolated from Catharanthus roseus Cells.

The uptake of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, into vacuoles isolated from Catharanthus roseus cells has been studied by silicone layer floatation filtering. The transport across the tonoplast of MACC is stimulated fourfold by 5 millimolar MgATP, has a K(m) of about 2 millimolar, an optimum pH around 7, and an optimum temperature at 30 degrees C. Several effectors known to inhibit ATPase (N,N'-dicyclohexylcarbodiimide) and to collapse the transtonoplastic H(+) electrochemical gradient (carbonylcyanide m-chlorophenylhydrazone, gramicidin, and benzylamine) all reduced MACC uptake. Abolishing the membrane potential with SCN(-) and valinomycin also greatly inhibited MACC transport. Our data demonstrate that MACC accumulates in the vacuole against a concentration gradient by means of a proton motive force generated by a tonoplastic ATPase. The involvement of a protein carrier is suggested by the strong inhibition of uptake by compounds known to block SH-, OH-, and NH(2)- groups. MACC uptake is antagonized competitively by malonyl-d-tryptophan, indicating that the carrier also accepts malonyl-d-amino acids. Neither the moities of these compounds taken separately [1-aminocyclopropane-1-carboxylic acid, malonate, d-tryptophan or d-phenylalanine] nor malate act as inhibitors of MACC transport. The absence of inhibition of malate uptake by MACC suggests that MACC and malate are taken up by two different carriers. We propose that the carrier identified here plays an important physiological role in withdrawing from the cytosol MACC and malonyl-d-amino acids generated under stress conditions.

Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato.

The tomato ACC oxidase gene family is comprised of three members designated AC01, AC02 and AC03. These are highly homologous throughout the protein coding regions but do show a degree of sequence divergence within the 3' untranslated regions. These regions have been cloned and used as gene-specific probes to analyse the differential expression of the tomato ACC oxidase gene family in various tissues at different stages of development. Results indicate that all three genes are transcriptionally active and display a high degree of inducibility in a number of organs at various stages of the life cycle. Both AC01 and Ac03 transcripts accumulate during the senescence of leaves, fruit and flowers. In addition, it appears that AC01 is wound-inducible in leaves. All three ACC oxidase genes are expressed during flower development, with each showing a temporally distinct pattern of accumulation. In addition, the ACC oxidase transcripts are also spatially regulated throughout flower development; AC01 is predominantly expressed in the petals and the stigma and style, AC02 expression is mainly restricted to tissues associated with the anther cone whereas AC03 transcripts accumulate in all of the floral organs examined apart from the sepals. ACC oxidase enzyme assays and Western blot analysis indicate that both enzyme activity and ACC oxidase protein increase with transcript abundance in several tissues. The physiological role of the differential expression of the ACC oxidase gene family, in relation to the regulation of ethylene synthesis, during these various developmental processes is discussed.

Immunocytolocalization of 1-aminocyclopropane-1-carboxylic acid oxidase in tomato and apple fruit.

The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethylene, has been studied in ripening fruits of tomato (Lycopersicum esculentum Mill.). Two types of antibody have been raised against (i) a synthetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overproduced in Escherichia coli expressing the pRC13 cDNA. Immunoblot analysis showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidase. Superimposition of Calcofluor white with immunofluorescence labeling, analysed by optical microscopy, indicated that ACC oxidase is located at the cell wall in the pericarp of breaker tomato and climacteric apple (Malus x domestica Borkh.) fruit. The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy. Transgenic tomato fruits in which ACC-oxidase gene expression was inhibited by an antisense gene exhibited a considerable reduction of labeling. Immunocytological controls made with pre-immune serum or with antibodies pre-absorbed on their corresponding antigens gave no staining. The discrepancy between these findings and the targeting of the protein predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed.

Vacuolar Release of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid, the Conjugated Form of the Ethylene Precursor.

The mechanisms underlying the vacuolar retention or release of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, has been studied in grape (Vitis vinifera) cells grown in vitro using the technique of compartmental analysis of radioisotope elution. Following its accumulation in the vacuole, M[2,3-(14)C]ACC could be released from cells when the vacuolar pH was artificially lowered by external buffers from its initial value of 6.2 to below the critical pH of 5.5. Successive release and retention of vacuolar MACC could be achieved by switching the vacuolar pH from values lower and higher than 5.5. The rate constant of efflux was highly correlated with the vacuolar pH. In plant tissues having low vacuolar pH under natural conditions, e.g. apple fruits (pH 4.2) and mung bean hypocotyls (pH 5.3), an efflux of M[2,3-(14)C]ACC also occurred. Its rate constant closely corresponded to the theorical values derived from the correlation established for grape cells. Evidence is presented that the efflux proceeded by passive lipophilic membrane diffusion only when MACC was in the protonated form. In contrast to other organic anions like malic acid, the mono and diionic species could not permeate the tonoplast, thus indicating the strict dependence of MACC retention upon the ionic status of the molecule and the absence of carrier-mediated efflux.

Iron: an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene.

The activity of the ethylene-forming enzyme (EFE) in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells was almost completely abolished within 10 min by 0.4 mM of the metal-chelating agent 1,10-phenanthroline. Subsequent addition of 0.4 mM FeSO4 immediately reversed this inhibition. A partial reversion was also obtained with 0.6 mM CuSO4 and ZnSO4, probably as a consequence of the release of iron ions from the 1,10-phenanthroline complex. The inhibition was not reversed by Mn(2+) or Mg(2+). Tomato cells starved of iron exhibited a very low EFE activity. Addition of Fe(2+) to these cells caused a rapid recovery of EFE while Cu(2+), Zn(2+) and other bivalent cations were ineffective. The recovery of EFE activity in iron-starved cells was insensitive to cycloheximide and therefore does not appear to require synthesis of new protein. The EFE activity in tomato cells was induced by an elicitor derived from yeast extract. Throughout the course of induction, EFE activity was blocked within 10-20 min by 1,10-phenanthroline, and the induced level was equally rapidly restored after addition of iron. We conclude that iron is an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in vivo.

Identification of transposon-like elements in non-coding regions of tomato ACC oxidase genes.

1-aminocyclopropane-1-carboxylate (ACC) oxidase, which catalyses the terminal step in ethylene biosynthesis, is encoded by a small multigene family in tomato that is differentially expressed in response to developmental and environmental cues. In this study we report the isolation and sequencing of approximately 2 kb of 5'-flanking sequence of three tomato ACC oxidase genes (LEACO1, LEACO2, LEACO3) and the occurrence of class I and class II mobile element-like insertions in promoter and intron regions of two of them. The LEA CO1 upstream region contains a 420-bp direct repeat which is present in multiple copies in the tomato genome and is very similar to sequences in the promoters of the tomato E4 and 2A11 genes. The region covering the repeats resembles the remnant of a retrotransposon. Two copies of a small transposable element, belonging to the Stowaway inverted repeat element family, have been found in the 5'-flanking sequence and the third intron of LEACO3.

Characterization of ripening-regulated cDNAs and their expression in ethylene-suppressed charentais melon fruit.

Charentais melons (Cucumis melo cv Reticulatus) are climacteric and undergo extremely rapid ripening. Sixteen cDNAs corresponding to mRNAs whose abundance is ripening regulated were isolated to characterize the changes in gene expression that accompany this very rapid ripening process. Sequence comparisons indicated that eight of these cDNA clones encoded proteins that have been previously characterized, with one corresponding to ACC (1-aminocyclopropane-1-carboxylic acid) oxidase, three to proteins associated with pathogen responses, two to proteins involved in sulfur amino acid biosynthesis, and two having significant homology to a seed storage protein or a yeast secretory protein. The remaining eight cDNA sequences did not reveal significant sequence similarities to previously characterized proteins. The majority of the 16 ripening-regulated cDNAs corresponded to mRNAs that were fruit specific, although three were expressed at low levels in vegetative tissues. When examined in transgenic antisense ACC oxidase melon fruit, three distinct patterns of mRNA accumulation were observed. One group of cDNAs corresponded to mRNAs whose abundance was reduced in transgenic fruit but inducible by ethylene treatment, indicating that these genes are directly regulated by ethylene. A second group of mRNAs was not significantly altered in the transgenic fruit and was unaffected by treatment with ethylene, indicating that these genes are regulated by ethylene-independent developmental cues. The third and largest group of cDNAs showed an unexpected pattern of expression, with levels of mRNA reduced in transgenic fruit and remaining low after exposure to ethylene. Regulation of this third group of genes thus appears to ethylene independent, but may be regulated by developmental cues that require ethylene at a certain stage in fruit development. The results confirm that both ethylene-dependent and ethylene-independent pathways of gene regulation coexist in climacteric fruit.