The influence of water on the nanomechanical behavior of the plant biopolyester cutin as studied by AFM and solid-state NMR

Atomic force microscopy and solid-state nuclear magnetic resonance have been used to investigate the effect of water absorption on the nanoscale elastic properties of the biopolyester, cutin, isolated from tomato fruit cuticle. Changes in the humidity and temperature at which fruits are grown or stored can affect the plant surface (cuticle) and modify its susceptibility to pathogenic attack by altering the cuticle's rheological properties. In this work, atomic force microscopy measurements of the surface mechanical properties of isolated plant cutin have been made as a first step to probing the impact of water uptake from the environment on surface flexibility. A dramatic decrease in surface elastic modulus (from approximately 32 to approximately 6 MPa) accompanies increases in water content as small as 2 wt %. Complementary solid-state nuclear magnetic resonance measurements reveal enhanced local mobility of the acyl chain segments with increasing water content, even at molecular sites remote from the covalent cross-links that are likely to play a crucial role in cutin's elastic properties.     

Biomechanics of isolated tomato (Solanum lycopersicum L.) fruit cuticles: the role of the cutin matrix and polysaccharides

The mechanical characteristics of the cuticular membrane (CM), a complex composite biopolymer basically composed of a cutin matrix, waxes, and hydrolysable polysaccharides, have been described previously. The biomechanical behaviour and quantitative contribution of cutin and polysaccharides have been investigated here using as experimental material mature green and red ripe tomato fruits. Treatment of isolated CM with anhydrous hydrogen fluoride in pyridine allowed the selective elimination of polysaccharides attached to or incrusted into the cutin matrix. Cutin samples showed a drastic decrease in elastic modulus and stiffness (up to 92%) compared with CM, which clearly indicates that polysaccharides incorporated into the cutin matrix are responsible for the elastic modulus, stiffness, and the linear elastic behaviour of the whole cuticle. Reciprocally, the viscoelastic behaviour of CM (low elastic modulus and high strain values) can be assigned to the cutin. These results applied both to mature green and red ripe CM. Cutin elastic modulus, independently of the degree of temperature and hydration, was always significantly higher for the ripe than for the green samples while strain was lower; the amount of phenolics in the cutin network are the main candidates to explain the increased rigidity from mature green to red ripe cutin. The polysaccharide families isolated from CM were pectin, hemicellulose, and cellulose, the main polymers associated with the plant cell wall. The three types of polysaccharides were present in similar amounts in CM from mature green and red ripe tomatoes. Physical techniques such as X-ray diffraction and Raman spectroscopy indicated that the polysaccharide fibres were mainly randomly oriented. A tomato fruit CM scenario at the supramolecular level that could explain the observed CM biomechanical properties is presented and discussed.     

Flavonoid biosynthesis in tomato fruit cuticles after in vivo incorporation of 3 H-phenylalanine precursor

Radiolabelling of epicuticular waxes and cutin of isolated tomato fruit cuticles were determined after fruit surface application of 3 H-phenylalanine precursor. During fruit ripening, the precursor is incorporated in different phenolic components: the flavanone naringenin was found to be the major compound in the epicuticular waxes, while the amount of the labelled flavonoid in the cutin matrix was progressively increased throughout fruit ripening. Confocal microscopy, together with experimental estimation of the mobility (diffusion coefficient, D ) and affinity (partition coefficient, K ) of the flavonoids naringenin and chalconaringenin for the different cuticular components, indicate that these compounds are extruded to the outer surface of tomato fruits, forming molecular clusters.     

Tomato GDSL1 Is Required for Cutin Deposition in the Fruit Cuticle

The plant cuticle consists of cutin, a polyester of glycerol, hydroxyl, and epoxy fatty acids, covered and filled by waxes. While the biosynthesis of cutin building blocks is well documented, the mechanisms underlining their extracellular deposition remain unknown. Among the proteins extracted from dewaxed tomato (Solanum lycopersicum) peels, we identified GDSL1, a member of the GDSL esterase/acylhydrolase family of plant proteins. GDSL1 is strongly expressed in the epidermis of growing fruit. In GDSL1-silenced tomato lines, we observed a significant reduction in fruit cuticle thickness and a decrease in cutin monomer content proportional to the level of GDSL1 silencing. A significant decrease of wax load was observed only for cuticles of the severely silenced transgenic line. Fourier transform infrared (FTIR) analysis of isolated cutins revealed a reduction in cutin density in silenced lines. Indeed, FTIR-attenuated total reflectance spectroscopy and atomic force microscopy imaging showed that drastic GDSL1 silencing leads to a reduction in ester bond cross-links and to the appearance of nanopores in tomato cutins. Furthermore, immunolabeling experiments attested that GDSL1 is essentially entrapped in the cuticle proper and cuticle layer. These results suggest that GDSL1 is specifically involved in the extracellular deposition of the cutin polyester in the tomato fruit cuticle.     

Rheological Properties of Enzymatically Isolated Tomato Fruit Cuticle

Rheological properties were determined for cuticular membranes (CMs) enzymatically isolated from mature tomato (Lycopersicon esculentum Mill. cv Pik Red) fruit. The cuticle responded as a viscoelastic polymer in stress-strain studies. Both CM and dewaxed CM expanded and became more elastic and susceptible to fracture when hydrated, suggesting that water plasticized the cuticle. Dewaxing of the CM caused similar changes in elasticity and fracturing, indicating that wax may serve as a supporting filler in the cutin matrix. Exposure of the cuticle to the surfactant Triton X-100 did not significantly affect its rheological properties.     

Evidence for cross-linking in tomato cutin using HR-MAS NMR spectroscopy

Cutin is a polyester biopolymer component of plant leaf and fruit cuticles, most often associated with waxes and cuticular polysaccharides, and sometimes with another aliphatic biopolymer called cutan. Insolubility of these cuticular biopolymers has made it difficult to apply traditional analytical techniques for structure determination, because most techniques providing molecular level details require solubility. By using the relatively new technique of one and two-dimensional high-resolution magic angle spinning (HR-MAS) NMR spectroscopy, with added information from solid-state 13C NMR spectroscopy, detailed through-bond connectivities and assignments are made for cutin from Lycopersicon esculentum (tomato) fruit. Based on the data obtained, tomato cutin is found to be predominantly an aliphatic polyester with some olefinic and aromatic moieties, consistent with previous studies that employed various degradative approaches. Aside from esters, there are free primary and secondary alcohol groups, as well as free fatty acids. A significant finding is the presence of alpha-branched fatty acids/esters. Mid-chain hydroxyls appear to be generally unesterified, but esters of mid-chain hydroxyls have been identified. The alpha-branched fatty acids/esters and esters of mid-chain hydroxyls could point towards cross-linking.     

Viscoelastic nature of isolated tomato (Solanum lycopersicum) fruit cuticles: a mathematical model

Viscoelastic behaviour of isolated tomato fruit cuticle (CM) is well known and extensively described. Temperature and hydration conditions modify the mechanical properties of CM. Mechanical data from previous transient‐creep analysis developed in tomato fruit cuticle under different temperature and hydration conditions have been used to propose a rheological model that describes the viscoelastic nature of CM. As a composite material, the biomechanical behaviour of the plant cuticle will depend not only on the mechanical characteristics of the individual components by themselves but also on the sum of them. Based on this previous information, we proposed a two‐element model to describe the experimental behaviour: an elastic hookean element connected in parallel to a viscous element or Voigt element that will describe the mechanical behaviour of the isolated CM and cutin under the studied conditions. The main parameters of the model, E₁ and E₂ will reflect the elastic and viscoelastic behaviour of the cuticle. Relationship between these physical parameters and the change in CM properties were discussed in order to elucidate the rheological processes taking place in CM. This model describes both the influence of temperature and hydration and the behaviour of the isolated cutin and the inferred contribution of the cuticle fraction of polysaccharides when the whole cuticle is tested.     

Subcellular localization of peroxidase in tomato fruit skin and the possible implications for the regulation of fruit growth

The cessation of tomato fruit growth has been associated with the appearance of three 'wall-bound' peroxidase isozymes in the skin of tomato fruit. However, the presence of these isozymes in the ionically eluted 'wall-bound' fraction may be an artefact of either non-specific binding of symplastic peroxidase to the cell wall, or isozymes bound to membranes included in the 'wall-bound' fraction. Therefore, subcellular localization of peroxidase in both immature and mature tomato fruit skins was studied. Immature fruits showed intense peroxidase activity associated with the tonoplast and pro-vacuolar membranes, but little or no activity associated with the cell wall. However, the presence of peroxidase activity within the cell wall of mature green fruits was confirmed. Furthermore, peroxidase activity was also observed associated with the plasma membrane and large vesicles allied to the plasma membrane. While cross-linking in cell wall components was previously assumed to be the mechanism by which peroxidase might control fruit growth, the incorporation of 'lignin-like' phenolics may also play a part. Isoelectric focusing (IEF) of both symplastic and apoplastic peroxidase extracted from immature and mature tomato fruit skin showed that all peroxidase isozymes present were highly anionic. In this current study, histochemical techniques are used to demonstrate a developmental increase in 'lignin-like' phenolics within the sub-cuticular cell walls of the fruit skin. The localization of peroxidase within tomato fruit skin is discussed in relation to its potential role in the regulation of tomato fruit growth.     

Differences in the initial events of infection of Botrytis cinerea strains isolated from tomato and grape

Various stages of the infection process among B. cinerea strains isolated from tomatoes or grapes, belonging to different genetic groups, were compared. It was found that strains of B. cinerea isolated from either grapes or tomatoes showed differences in adhesion patterns and in the percentage of germination on tomato cutin. In strains isolated from tomato the first stage of adhesion occurred faster than in strains isolated from grape. At the same time strains isolated from tomato showed a higher percentage of germination on tomato cutin than the other strains after 9 h of incubation. The production and isoenzymatic patterns of polygalacturonases, pectin methyl esterases, pectin lyases, p-nitrophenylbutyrate esterases and laccases by B. cinerea in solid-state fermentation also were analyzed. Correlation between the production of these enzymes and the origin of the strains was not found. On the other hand all strains produced different isoenzymes and a common pattern between the strains was not observed. The ability of B. cinerea strains to colonize tomato leaves also differs between the isolated strains obtained from grapes and tomato. Strains isolated from tomato were more virulent on tomato leaves than strains isolated from grapes.     

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.¹ A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer ''epicuticular'' layer as well as the blend consisting of the cutin biopolymer and ''intracuticular'' waxes.² Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Schäffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.^3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) ¹³C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS ¹³C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH₂O) chemical moieties.Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state ¹³C NMR reveals differences in the relative numbers of CHO and CH₂O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-μm length scale.     

Development of plant cuticles: occurrence and role of non-ester bonds in cutin of Clivia miniata Reg. leaves

The effect of BF₃-methanol treatment on the mass and fine structure of isolated Clivia leaf cuticles at different stages of development has been investigated. BF₃-methanol cleaves ester linkages in cutin; however, the cuticles are not completely depolymerized. With increasing age, the residue left after BF₃-methanol treatment increases in mass. In very young cuticles, 10% of the total cutin resisted BF₃-methanol and the fraction of nonester cutin increased up to 62% in mature leaves. Transmission electron microscopy shows that fine structure of the cuticle proper is severely distorted but not destroyed. The internal cuticular layer, which exhibits a heavy contrast when fixed with KMnO₄, is completely depolymerized, while the external cuticular layer is hardly affected. The results are discussed in relation to cuticle development and to the function of cuticles as transpiration resistances.Abbreviation CP cuticle proper - ECL external cuticular layer - E cutin ester bonded cutin - ICL internal cuticular layer - MX-membrane polymer matrix membrane - NE-cutin non-ester bonded cutin - TEM transmission electron microscopy     

Tomato Cutin Deficient 1 (CD1) and putative orthologs comprise an ancient family of cutin synthase‐like (CUS) proteins that are conserved among land plants

The aerial epidermis of all land plants is covered with a hydrophobic cuticle that provides essential protection from desiccation, and so its evolution is believed to have been prerequisite for terrestrial colonization. A major structural component of apparently all plant cuticles is cutin, a polyester of hydroxy fatty acids; however, despite its ubiquity, the details of cutin polymeric structure and the mechanisms of its formation and remodeling are not well understood. We recently reported that cutin polymerization in tomato (Solanum lycopersicum) fruit occurs via transesterification of hydroxyacylglycerol precursors, catalyzed by the GDSL‐motif lipase/hydrolase family protein (GDSL) Cutin Deficient 1 (CD1). Here, we present additional biochemical characterization of CD1 and putative orthologs from Arabidopsis thaliana and the moss Physcomitrella patens, which represent a distinct clade of cutin synthases within the large GDSL superfamily. We demonstrate that members of this ancient and conserved family of cutin synthase‐like (CUS) proteins act as polyester synthases with negligible hydrolytic activity. Moreover, solution‐state NMR analysis indicates that CD1 catalyzes the formation of primarily linear cutin oligomeric products in vitro. These results reveal a conserved mechanism of cutin polyester synthesis in land plants, and suggest that elaborations of the linear polymer, such as branching or cross‐linking, may require additional, as yet unknown, factors.     

Synthesis of Suberin during Wound-healing in Jade Leaves, Tomato Fruit, and Bean Pods

The structure and composition of the aliphatic monomers of the polymeric material deposited during wound-healing of tomato fruit, bean pods, and Jade leaves were examined. After removing the cuticle-containing layer of tissue, the wounds were healed for 14 days and the resulting surface layer was excised, lyophilized, solvent-extracted, and depolymerized by hydrogenolysis with LiAlH₄ or transesterified with BF₃ in methanol. The products obtained by the chemical depolymerization were subjected to thin layer chromatography and combined gas chromatography and mass spectrometry. The major aliphatic components isolated from the hydrogenolysate of the wound polymer produced by tomato fruit were hexadecane-1,16-diol and octadec-9-ene-1,18-diol, which were shown to be derived from a 1:1 mixture of ω-hydroxy and dicarboxylic acids of the appropriate chain length by LiAlH₄ reduction. Also identified in the wound polymer were long chain (>C₂₀) fatty acids and alcohols. This monomer composition is typical of suberin polymers and is in sharp contrast with that of the cutin of tomato fruit which contains dihydroxy C₁₆ acid as the major aliphatic component. The hydrogenolysis of the wound material from bean pods gave octadecene-1,18-diol as the major aliphatic component, and smaller amounts of hexadecane-1,16-diol and long chain alcohols. Similar treatment of the normal cuticular tissue of these pods gave hexadecane triol, as well as C₁₆ and C₁₈ alcohols. Hydrogenolysis of wound material from the Jade leaves gave octadecene-1,18-diol, C₁₆ and C₂₂ diols, as well as alcohols from C₁₆ to C₂₆, whereas similar treatment of the cutin-containing tissue from these leaves gave C₁₆ triol as the major aliphatic component. Thus, the major aliphatic monomers of the polymeric material deposited during the wound-healing of bean pods and Jade leaves are very similar to those of suberin, although the natural protective polymer of these tissues is cutin. From these results, it is concluded that suberization is a fundamental process involved in wound-healing in plants, irrespective of the chemical nature of the natural protective polymer of the tissue.     

Aliphatic composition of cutin from inner seed coat of apple

Lithium aluminum deuteride reduction released aliphatic monomers from the inner seed coat fraction but not from the outer seed coat fraction of mature apples. These monomers were identified by GC/MS and the results indicate that the inner coat of apple seed contains a cutin polymer with the major monomer acids being 18-hydroxyoctadec-9-enoic (31%), 9,10-epoxy-18-hydroxyoctadecanoic (28%) and 9,10,18-trihydroxyoctadecanoic (20 %). The monomer composition of this seed coat cuticular polymer was very similar in seeds taken from freshly harvested fruit and in those taken from fruit which had been stored at 4° for 6 months.     

Fruit softening and pectin disassembly: an overview of nanostructural pectin modifications assessed by atomic force microscopy

Background

One of the main factors that reduce fruit quality and lead to economically important losses is oversoftening. Textural changes during fruit ripening are mainly due to the dissolution of the middle lamella, the reduction of cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Pectins, major components of fruit cell walls, are extensively modified during ripening. These changes include solubilization, depolymerization and the loss of neutral side chains. Recent evidence in strawberry and apple, fruits with a soft or crisp texture at ripening, suggests that pectin disassembly is a key factor in textural changes. In both these fruits, softening was reduced as result of antisense downregulation of polygalacturonase genes. Changes in pectic polymer size, composition and structure have traditionally been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides, and studies focusing on modifications at the nanostructural level are scarce. Atomic force microscopy (AFM) allows the study of individual polymers at high magnification and with minimal sample preparation; however, AFM has rarely been employed to analyse pectin disassembly during fruit ripening.

Scope

In this review, the main features of the pectin disassembly process during fruit ripening are first discussed, and then the nanostructural characterization of fruit pectins by AFM and its relationship with texture and postharvest fruit shelf life is reviewed. In general, fruit pectins are visualized under AFM as linear chains, a few of which show long branches, and aggregates. Number- and weight-average values obtained from these images are in good agreement with chromatographic analyses. Most AFM studies indicate reductions in the length of individual pectin chains and the frequency of aggregates as the fruits ripen. Pectins extracted with sodium carbonate, supposedly located within the primary cell wall, are the most affected.     

Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss

Plant cuticles are broadly composed of two major components: polymeric cutin and a mixture of waxes, which infiltrate the cutin matrix and also accumulate on the surface, forming an epicuticular layer. Although cuticles are thought to play a number of important physiological roles, with the most important being to restrict water loss from aerial plant organs, the relative contributions of cutin and waxes to cuticle function are still not well understood. Tomato ( Solanum lycopersicum ) fruits provide an attractive experimental system to address this question as, unlike other model plants such as Arabidopsis, they have a relatively thick astomatous cuticle, providing a poreless uniform material that is easy to isolate and handle. We identified three tomato mutants, cutin deficient 1 ( cd1 ), cd2 and cd3 , the fruit cuticles of which have a dramatic (95–98%) reduction in cutin content and substantially altered, but distinctly different, architectures. This cutin deficiency resulted in an increase in cuticle surface stiffness, and in the proportions of both hydrophilic and multiply bonded polymeric constituents. Furthermore, our data suggested that there is no correlation between the amount of cutin and the permeability of the cuticle to water, but that cutin plays an important role in protecting tissues from microbial infection. The three cd mutations were mapped to different loci, and the cloning of CD2 revealed it to encode a homeodomain protein, which we propose acts as a key regulator of cutin biosynthesis in tomato fruit.     

Novel promoters that induce specific transgene expression during the green to ripening stages of tomato fruit development

Fruit-specific promoters have been used as genetic engineering tools for studies on molecular mechanism of fruit development and advance in fruit quality and additional value by increasing functional component. Especially fruit-ripening specific promoters have been well utilized and studied in tomato; however, few studies have reported the development of promoters that act at fruit developing stages such as immature green and mature green periods. In this study, we report novel promoters for gene expression during the green to ripening stages of tomato fruit development. Genes specifically expressed at tomato fruit were selected using microarray data. Subsequent to confirmation of the expression of the selected 12 genes, upstream DNA fragments of the genes LA22CD07, Les.3122.2.A1_a_at and LesAffx.6852.1.S1_at which specifically expressed at fruit were isolated from tomato genomic DNA as promoter regions. Isolated promoter regions were fused with the GUS gene and the resultant constructs were introduced into tomato by agrobacterium-mediated transformation for evaluation of promoter activity in tomato fruit. The two promoters of LA22CD07, and LesAffx.6852.1.S1_at showed strong activity in the fruit, weak activity in the flower and undetectable activity in other tissues. Unlike well-known fruit-ripening specific promoters, such as the E8 promoter, these promoters exhibited strong activity in green fruit in addition to red-ripening fruit, indicating that the promoters are suitable for transgene expression during green to ripening stages of tomato fruit development. KEY MESSAGE: Novel fruit-specific promoters have been identified and are suitable for transgene expression during green to ripening stages of tomato fruit development.     

Evidence for Covalently Attached p-Coumaric Acid and Ferulic Acid in Cutins and Suberins

p-Coumaric acid (4-hydroxycinnamic acid) and ferulic acid (4-hydroxy-3-methoxycinnamic acid) have been identified as constituents of cutin. Their reduction products were isolated from a phenolic fraction released from the cutin of the fruits of apple, peach, pear, and two varieties of tomato and apple leaf by treatment with LiAlH(4) or LiAlD(4). They were identified by combined gas chromatography and mass spectrometry. p-Coumaric acid was present in all samples of cutin (0.07-0.53% by weight), whereas only peach and pear cutin contained measurable amounts of ferulic acid (0.007% and 0.035%, respectively). Both p-coumaric acid and ferulic acid were identified to be constituents of the insoluble material recovered after partial hydrolysis (12-42% loss) of cutin in 1 m NaOH at 80 C. A significant part (48%) of the p-coumaric acid contained in tomato cutin was contained in the insoluble material recovered after partial degradation (7.4%) of this cutin with 0.01 m NaOH. These data indicate that these phenolic components are tightly (possibly covalently) bound to cutin. Similar analysis of the phenolic fractions from the suberins of potato, sweet potato, turnip, rutabaga, carrot, and red beet revealed that they contained only ferulic acid (0.05-0.22%). Ferulic acid was identified as a constituent of the insoluble material recovered after partial hydrolysis of potato and beet suberins (34% and 32% loss, respectively) in 1 m NaOH at 80 C. A major part (65%) of the ferulic acid contained in potato suberin was contained in the insoluble material recovered after partial (26.8% loss) degradation of this suberin with 0.01 m NaOH. Ferulic acid appears to be tightly (probably covalently) bound to suberin.     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate