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.     

The Solanum melongena COP1LIKE manipulates fruit ripening and flowering time in tomato (Solanum lycopersicum)

Plant Growth Regulation - The CONSTITUTIVE PHOTOMORPHOGENIC (COP) 1LIKE is a regulatory protein and repressor of photomorphogenesis; which control many processes of development in plants. Here, the...     

Phenolic constituents of tomato fruit cuticles

Chalconaringenin, naringenin, naringenin-7-glucoside, and m- and p-coumaric acids have been identified in the fruit cuticles of three tomato cultivars. The phenolic content of the cuticles increased substantially during fruit development, those from immature green and mature ripe fruits of cv Ailsa Craig yielding respectively 2.8 and 61 μg/cm² (representing 1.4 and 6% of the total membrane wt). Coumaric acids, present only in the ‘cutin-bound’ phenolics, increased from 2 to 24 μg/cm² during fruit development. Flavonoids, synthesized mainly during the climacteric, occurred free in the epicuticular (0.3–7.2 μg/cm²) and cuticular (0.7–5.7 μg/cm²) phenolics but the major part of this class of constituents in ripe fruit cuticles was also ‘bound’ to the cutin matrix (30–43 μg/cm²). The composition of the flavonoid fraction was controlled by the spectral quality of incident radiation, red light favouring the formation of chalconaringenin.     

Changes in distribution of cell wall polysaccharides in floral and fruit abscission zones during fruit development in tomato (Solanum lycopersicum)

After fruit development has been triggered by pollination, the abscission zone (AZ) in the pedicel strengthens its adhesion to keep the fruit attached. Unpollinated flowers are shed at their respective AZs, whereas an enlargement of the same tissue is observed in pollinated flowers. After the fruit has developed and is fully ripened, shedding occurs easily at the AZ, indicating an acceleration of abscission. Cell wall degradation and synthesis may play important roles in these processes; however, little is understood. In this report, we have visualized changes in polysaccharide distribution in the AZs of pollinated versus unpollinated flowers and in the ripened fruits using immunohistochemistry. During floral abscission, a large increase was observed in LM15 labeling of xyloglucan specifically at the AZ in the abscising pedicel. LM5 and LM6 labeling of galactan and arabinan, respectively, also increased—LM5 throughout the pedicel and LM6 at the basal side of the AZ. The results suggest that xyloglucan, pectic galactan and arabinan play key roles in the abscission process. During fruit abscission, unlike in floral abscission, no AZ-specific cell wall polysaccharide deposition was observed; however, high autofluorescence was seen in the AZ of over-ripe fruit pedicels, suggesting secondary cell wall synthesis and lignification of the AZ prior to fruit abscission.     

Metabolic profiling of leaves and fruit of wild species tomato: a survey of the Solanum lycopersicum complex

The domestication of the tomato Solanum lycopersicum and associated selective pressures eventually led to the large-fruited varieties cultivated today. S. lycopersicum varieties are generally red-fruited, but display considerable variance in fruit colour intensity, shape, and quality. The increase in productivity on cultivation is, however, somewhat offset by the narrowing of the crops genetic base which leads to increased susceptibility to biotic and abiotic stresses. Since S. lycopersicum can easily be crossed with its wild species relatives, this exotic germplasm can provide a valuable source for the improvement of agriculturally important traits. A GC-MS based survey is presented here of the relative metabolic levels of leaves and fruit of S. lycopersicum and five wild species of tomato that can be crossed with it (S. pimpinellifolium, S. neorickii, S. chmielewskii, S. habrochaites, and S. pennellii). Changes in metabolite contents were identified in the wild species that are potentially important with respect to stress responses, as well as in metabolites of nutritional importance. The significance of these changes is discussed with respect to the use of the various wild species for metabolic engineering within wide breeding strategies.     

Investigation of tomato (Solanum lycopersicum) aminotransferases involved in biosynthesis of branched-chain-amino-acids

This study presents evidence for the role of BCAT3 and BCAT4 proteins in the synthesis of branched-chain-amino-acids in tomato Solanum lycopersicum. BCAT3 and BCAT4 genes were located on tomato chromosomal map by RFLP method (restriction fragment length polymorphism). Using confocal microscopy it was shown that BCAT3-GFP and BCAT4-GFP fusion proteins were localised in chloroplasts. It was observed that these aminotransferase isoforms exhibited distinct kinetic properties and a differential expression pattern of mRNA levels in various tomato tissues.     

Metabolic engineering of flavonoids in tomato (Solanum lycopersicum): the potential for metabolomics

Flavonoids comprise a large and diverse group of polyphenolic plant secondary metabolites. In plants, flavonoids play important roles in many biological processes such as pigmentation of flowers, fruits and vegetables, plant-pathogen interactions, fertility and protection against UV light. Being natural plant compounds, flavonoids are an integral part of the human diet and there is increasing evidence that dietary polyphenols are likely candidates for the observed beneficial effects of a diet rich in fruits and vegetables on the prevention of several chronic diseases. Within the plant kingdom, and even within a single plant species, there is a large variation in the levels and composition of flavonoids. This variation is often due to specific mutations in flavonoid-related genes leading to quantitative and qualitative differences in metabolic profiles. The use of such specific flavonoid mutants with easily scorable, visible phenotypes has led to the isolation and characterisation of many structural and regulatory genes involved in the flavonoid biosynthetic pathway from different plant species. These genes have been used to engineer the flavonoid biosynthetic pathway in both model and crop plant species, not only from a fundamental perspective, but also in order to alter important agronomic traits, such as flower and fruit colour, resistance, nutritional value. This review describes the advances made in engineering the flavonoid pathway in tomato (Solanum lycopersicum). Three different approaches will be described; (I) Increasing endogenous tomato flavonoids using structural or regulatory genes; (II) Blocking specific steps in the flavonoid pathway by RNA interference strategies; and (III) Production of novel tomato flavonoids by introducing novel branches of the flavonoid pathway. Metabolite profiling is an essential tool to analyse the effects of pathway engineering approaches, not only to analyse the effect on the flavonoid composition itself, but also on other related or unrelated metabolic pathways. Metabolomics will therefore play an increasingly important role in revealing a more complete picture of metabolic perturbation and will provide additional novel insights into the effect of the introduced genes and the role of flavonoids in plant physiology and development.     

Salt-stress induced physiological and proteomic changes in tomato (Solanum lycopersicum) seedlings

Soil salinity is one of the major abiotic stress limiting crop productivity and the geographical distribution of many important crops worldwide. To gain a better understanding of the salinity stress responses at physiological and molecular level in cultivated tomato (Solanum lycopersicum. cv. Supermarmande), we carried out a comparative physiological and proteomic analysis. The tomato seedlings were cultivated using a hydroponic system in the controlled environment growth chamber. The salt stress (NaCl) was applied (0, 50, 100, 150 and 200?mM), and maintained for 14 days. Salt treatment induced a plant growth reduction estimated as fresh-dry weight. Photosynthetic pigments (chlorophyll a, b) content of NaCl-treated tomato plants was significantly decreased as the salinity level increased. Proline accumulation levels in leaf and root tissues increased significantly with increasing NaCl concentration. Relative electrolyte leakage known as an indicator of membrane damage caused by salt stress was increased proportionally according to the NaCl concentrations. Roots of control and salt-stressed plants were also sampled for phenol protein extraction. Proteins were separated by two-dimensional gel electrophoresis (2-DGE). Several proteins showed up- and downregulation during salt stress. MALDI-TOF/MS analysis and database searching of some of the identified proteins indicated that the proteins are known to be in a wide range of physiological processes, that is, energy metabolism, ROS (reactive oxygen species) scavenging and detoxification, protein translation, processing and degradation, signal transduction, hormone and amino acid metabolism, and cell wall modifications. All proteins might work cooperatively to reestablish cellular homeostasis under salt stress, water deficiency, and ionic toxicity.     

Relative humidity and temperature modify the mechanical properties of isolated tomato fruit cuticles

The mechanical properties of enzymatically isolated cuticular membrane (CM) from ripe tomato fruits were investigated at 10 to 45°C and relative humidity (RH) of 40 to wet. CM samples were stressed by uniaxial tension loads to determine their tensile modulus, E, breaking stress (strength), σ(max), and maximum elongation, ε(max). The CM stress-strain curves revealed a biphasic behavior when tested at RH values below wet conditions. In the first phase, CM responded to the loads by instantaneous extension with no further extension recorded until a further load was added: defined as pure elastic strain (E(e)). In the second phase, CM responded by instantaneous extension and by some additional time-dependent extension, defined as viscoelastic strain (E(v)). When CMs were submerged in aqueous solution (wet), the stress-strain curves were monophasic, with both elastic and viscoelastic strain. E(e) depended on RH and was higher than E(v), which was independent of RH. Temperature decreased E(e) and σ(max) of tomato fruit CM. Temperature response was not linear but consisted of two temperature-independent phases separated by a transition temperature. This transition zone has been related previously to the presence of a secondary phase transition in the cutin matrix of the tomato fruit CM.     

Identification and localization of a lipase-like acyltransferase in phenylpropanoid metabolism of tomato (Solanum lycopersicum)

We have isolated an enzyme classified as chlorogenate: glucarate caffeoyltransferase (CGT) from seedlings of tomato (Solanum lycopersicum) that catalyzes the formation of caffeoylglucarate and caffeoylgalactarate using chlorogenate (5-O-caffeoylquinate) as acyl donor. Peptide sequences obtained by trypsin digestion and spectrometric sequencing were used to isolate the SlCGT cDNA encoding a protein of 380 amino acids with a putative targeting signal of 24 amino acids indicating an entry of the SlCGT into the secretory pathway. Immunogold electron microscopy revealed the localization of the enzyme in the apoplastic space of tomato leaves. Southern blot analysis of genomic cDNA suggests that SlCGT is encoded by a single-copy gene. The SlCGT cDNA was functionally expressed in Nicotiana benthamiana leaves and proved to confer chlorogenate-dependent caffeoyltransferase activity in the presence of glucarate. Sequence comparison of the deduced amino acid sequence identified the protein unexpectedly as a GDSL lipase-like protein, representing a new member of the SGNH protein superfamily. Lipases of this family employ a catalytic triad of Ser-Asp-His with Ser as nucleophile of the GDSL motif. Site-directed mutagenesis of each residue of the assumed respective SlCGT catalytic triad, however, indicated that the catalytic triad of the GDSL lipase is not essential for SlCGT enzymatic activity. SlCGT is therefore the first example of a GDSL lipase-like protein that lost hydrolytic activity and has acquired a completely new function in plant metabolism, functioning in secondary metabolism as acyltransferase in synthesis of hydroxycinnamate esters by employing amino acid residues different from the lipase catalytic triad.     

Root-synthesized cytokinins improve shoot growth and fruit yield in salinized tomato (Solanum lycopersicum L.) plants

Salinity limits crop productivity, in part by decreasing shoot concentrations of the growth-promoting and senescence-delaying hormones cytokinins. Since constitutive cytokinin overproduction may have pleiotropic effects on plant development, two approaches assessed whether specific root-localized transgenic IPT (a key enzyme for cytokinin biosynthesis) gene expression could substantially improve tomato plant growth and yield under salinity: transient root IPT induction (HSP70::IPT) and grafting wild-type (WT) shoots onto a constitutive IPT-expressing rootstock (WT/35S::IPT). Transient root IPT induction increased root, xylem sap, and leaf bioactive cytokinin concentrations 2- to 3-fold without shoot IPT gene expression. Although IPT induction reduced root biomass (by 15%) in control (non-salinized) plants, in salinized plants (100?mM NaCl for 22?d), increased cytokinin concentrations delayed stomatal closure and leaf senescence and almost doubled shoot growth (compared with WT plants), with concomitant increases in the essential nutrient K(+) (20%) and decreases in the toxic ion Na(+) (by 30%) and abscisic acid (by 20-40%) concentrations in transpiring mature leaves. Similarly, WT/35S::IPT plants (scion/rootstock) grown with 75?mM NaCl for 90?d had higher fruit trans-zeatin concentrations (1.5- to 2-fold) and yielded 30% more than WT/non-transformed plants. Enhancing root cytokinin synthesis modified both shoot hormonal and ionic status, thus ameliorating salinity-induced decreases in growth and yield.