Developmental changes in tomato fruit composition in response to water deficit and salinity

Processing tomato ( Lycopersicon esculentum Mill. cv. UC82B) plants were subjected to moderate levels of water deficit and salinity (Na₂SO₄/CaCl₂) in sand culture. Fruit water content and the relative contributions of organic and inorganic constituents to fruit solute potential (_Ψ) and soluble solids content were determined throughout development. Fruit _Ψ averaged –0.63, –0.86 and –0.77 MPa in the control, salinity and water deficit plants, respectively. Reduced net water import and maintenance of solute accumulation, irrespective of water import, accounted for the reductions in _Ψ of stressed fruits. Mineral ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl⁻ and SO^2-₄) contributed –0.31 MPa to _Ψ in salinized fruit, compared with –0.19 MPa in control and water deficit treatments. Changes in net carbon accumulation were not observed among treatments, despite considerable differences in fruit K⁺ status. Starch accumulation in immature fruit was increased and hexose accumulation was decreased by both salinity and water deficit. Maximum starch levels were negatively correlated with total fruit _Ψ, but were independent of fruit K⁺. Organic acid levels were generally higher throughout development in salinized plants, relative to control plants, and correlated with increased inorganic cation rather than anion accumulation in these fruits.     

Expansion rate of young tomato fruit growing on plants at positive water potential

Changes in tomato fruit expansion rate and carbohydrate content have been assessed during treatments designed to alter the carbon import rate. Because fruit expansion is sensitive to plant water status, the relationship with carbon import is difficult to assess, and thus, the diameter growth rate of young fruit was measured on plants maintained at positive water potentials. The detached top metre of a tomato plant was supplied with water, through the cut stem base, at a pressure of 0.08 MPa. Developing fruit on the stem continued to grow at high rates for up to 2 d. Fruit diameter growth rate after plant detachment was directly proportional to temperature. Plants acclimated to different continuous irradiances for 5 d before detachment gave fruit growth rates after plant detachment which were directly proportional to the irradiance up to 7 MJ m⁻²d⁻¹ photosynthetically active radiation (PAR). In continuous darkness, fruit growth rate remained unchanged for 20 h and then declined to less than 40% of the original rate over the following 30 h. On re-exposure to light, about 5 h elapsed before fruit growth rate increased but the growth rate stabilized at approximately 50% of the rate in continuously illuminated plants. During darkness, both fruit starch and hexose content decreased in comparison to illuminated controls, but on re-illumination, carbohydrate content increased before carbon was allocated to structural growth. Heat-killing the phloem of the fruit pedicel caused an immediate, but temporary, cessation of growth. After a partial recovery, expansion growth continued, but more slowly than in untreated fruit and at steadily declining rates. Starch and hexose sugars were not used to provide substrates for growth and starch synthesis was maintained. Continuing cell expansion was assumed to have been supported by water import via the xylem. Thus, fruit expansion may be related to carbon accumulation in most circumstances, but the changing allocation of imported carbon to storage and cell expansion may modify this relationship.     

Water relations and growth of tomato fruit pericarp tissue

The water relations of young tomato fruit pericarp tissue were examined and related to tissue expansion. The relationship between bulk turgor pressure and tissue expansion (as change in fresh mass or length of tissue) was determined in slices of pericarp cut from young, growing fruit by incubation in different osmotic concentrations of polyethylene glycol 6000 or mannitol. The bulk turgor of this tissue was low (about 0.2 MPa), even in fruit from plants that were otherwise fully turgid, whether measured psychrometrically or by length change in osmotic solutions. The rate of tissue growth at maximum turgor was less than that at moderate turgor unless calcium was added to the incubation medium. However, added calcium also decreased the rate of growth at lower turgor pressures. Yield turgor was < 0.1 MPa, but it was increased by the addition of calcium ions. Electrolyte leakage from tissue was greatest at maximum turgor pressure but was decreased by the addition of calcium ions or osmoticum. Tissue growth was unaffected by a range of plant growth regulators (IAA, abscisic acid, benzyladenine and GA₃) but was inhibited, particularly at high turgor, by low concentrations of malic or citric acid. The low turgor pressure of pericarp tissue could be due to the presence of apoplastic solutes within the pericarp, and evidence for this is discussed. Thus, fruit tissue may be able to maintain optimal expansion rates only at moderate turgor and low calcium concentration.     

Peroxidase isozyme patterns in the skin of maturing tomato fruit

The cessation of tomato fruit growth is thought to be induced by an increase in the activity of enzymes which rigidify cell walls in the fruit skin. Peroxidase could catalyse such wall‐stiffening reactions, and marked rises in peroxidase activity were recently reported in skin cell walls towards fruit maturity. Peroxidase isoforms in the fruit are here analysed using native gel electrophoresis. New isoforms of apparent M_r 44, 48 and 53 kDa are shown to appear in cell walls of the fruit skin at around the time of cessation of growth. It is inferred that these isozymes are present in the cell wall in vivo. Fruit from a range of non‐ripening mutants were also examined. Some of these do not soften or ripen for many weeks after achieving their final size. The new isozymes were found in skin cell walls of mature fruit in each of these mutants, as in the wild‐type and commercial varieties. It is concluded that the late‐appearing isozymes are not associated with fruit ripening or softening, and are probably not ethylene‐induced. They may act to control fruit growth by cross‐linking wall polymers within the fruit skin, thus mechanically stiffening the walls and terminating growth.     

Sugar uptake by protoplasts isolated from tomato fruit tissues during various stages of fruit growth

The uptake of radioactive glucose and sucrose by protoplasts isolated from pericarp and placenta tissues of tomato ( Lycopersicon esculentum cv. Counter) fruit was investigated in relation to the dry matter accumulation rates of these tissues. Uptake of glucose by protoplasts isolated from pericarp tissue was highest in fruit of around 20 g fresh weight or 25 days after anthesis. Sucrose uptake by pericarp protoplasts was lower than that of glucose and did not show a peak of uptake. The maximum rate of glucose uptake by protoplasts from the pericarp was at the time when the tomato fruit was accumulating dry matter at the highest rate. Glucose uptake by placenta protoplasts was lower and at a similar level as sucrose.
Protoplast uptake of glucose, but not of sucrose, was partially inhibited by (1) p -chloromercuribenzene sulphonic acid, a sulphydryl group modifier; (2) erythrosin B, an H⁺-ATPase inhibitor; and (3) valinomycin, a K⁺-ionophore, suggesting that membrane transport of glucose by tomato fruit sink cells may be a carrier-mediated, energy-dependent process.
The main route of carbohydrate accumulation by tomato fruit during the period of rapid fruit growth may be by cleavage of sucrose by apoplastic acid invertase prior to hexose transport across the plasma membrane.     

Function of the polygalacturonase convertor in ripening tomato fruit

In extracts from pericarp tissue of ripening tomato ( Lycopersicon esculentum Mill. cv, Sonato) fruits, two isoenzymes of polygalacturonase (E.C. 3.2.1.15), PG1 and PG2, are usually found. Also in such extracts, or as part of PG1, a convertor (CV) occurs. Incubation of PG2 with this CV gives rise to PG1 or a different isoenzyme, PGx, that is also stable at 65°C but differs in _pH optimum and size from PG1. It appears that CV has two affinity sites that can bind with PG2 or with a polydextran. PG1 is an extraction artifact, consisting of one molecule of CV and two molecules of PG2. PGx is made up of one molecule of CV and one molecule of PG2. It is the CV part of PGx that binds to polydextrans such as Blue Dextran 2000, Sephadex G-100, and cell wall preparations. In this last form PGx is the physiologically active form of the enzyme, solubilizing demethylated pectin.
On Sephacryl S-300, CV appears to have a molecular weight of 81 kDa, but because of its heat stability and partial leakage through a 10 kDa cut-off membrane, it might be a much smaller, rod-like molecule. The polygalacturonase convertor might be a lectin without intrinsic enzyme activity, with a function to immobilize, stabilize and activate enzymic proteins in the cell wall.     

Internal carbon dioxide and ethylene levels in ripening tomato fruit attached to or detached from the plant

The carbon dioxide and ethylene concentrations in tomato fruit ( Lycopersicon esculentum cv. Castelmart) and their stage of ripeness (characteristic external color changes) were periodically measured in fruit attached to and detached from the plant. An external collection apparatus was attached to the surface of individual tomato fruit to permit non-destructive sampling of internal gases. The concentration of carbon dioxide and ethylene in the collection apparatus reached 95% of the concentration in the fruit after 8 h. Gas samples were collected every 24 h. A characteristic climacteric surge in carbon dioxide (2-fold) and ethylene (10-fold) concentration occurred coincident with ripening of detached tomato fruit. Fruit attached to the plant exhibited a climacteric rise in ethylene (20-fold) concentration during ripening, but only a linear increase in carbon dioxide concentration. The carbon dioxide concentration increases in attached fruit during ripening, but the increase is a continuation of the linear increase seen in both attached and detached fruit before ripening and does not exhibit the characteristic pattern normally associated with ripening climacteric fruit. In tomato fruit, it appears that a respiratory climacteric per se, which has been considered intrinsic to the ripening of certain fruit, may not be necessary for the ripening of "climacteric" fruit at all, but instead may be an artifact of using harvested fruit.     

Moderate water stress affects tomato leaf water relations in dependence on the nitrogen supply

The responses of water relations, stomatal conductance (g_s) and growth parameters of tomato (Lycopersicon esculentum Mill. cv. Royesta) plants to nitrogen fertilisation and drought were studied. The plants were subjected to a long-term, moderate and progressive water stress by adding 80 % of the water evapotranspirated by the plant the preceding day. Well-watered plants received 100 % of the water evapotranspirated. Two weeks before starting the drought period, the plants were fertilised with Hoagland’s solution with 14, 60 and 110 mM NO₃ ⁻ (N14, N60 and N110, respectively). Plants of the N110 treatment had the highest leaf area. However, g_s was higher for N60 plants and lower for N110 plants. At the end of the drought period, N60 plants showed the lowest values of water potential (Ψ_w) and osmotic potential (Ψ_s), and the highest values of pressure potential (Ψ_p). N60 plants showed the highest Ψ_s at maximum Ψ_p and the highest bulk modulus of elasticity.     

Glycosyl-linkage composition of tomato fruit cell wall hemicellulosic fractions during ripening

Hemicelluloses were extracted from isolated tomato ( Lycopersicon esculentum Mill. cv. Rutgers) pericarp cell wall material at 3 different stages of ripeness with 4 M and 8 M KOH. Little change in molecular weight or composition of 4 M KOH-extracted material was observed during ripening. However, the composition of 8 M KOH-extracted material changed, and a relative increase in polymers of < 40 kDa was observed during ripening. Changes in glycosyl linkage composition of the 8 M KOH hemicellulosic material were detected, including increases in 4-linked mannosyl, 4,6-linked mannosyl, and 4-linked glucosyl, and decreases in 5-linked arabinosyl residues in polymers of < 40 kDa, and decreases in terminal glocosyl residues in polymers of > 40 kDa. These data may indicate that de novo hemicellulose synthesis occurs throughout tomato fruit ripening, even at the red ripe stage.     

A model for an early stage of tomato fruit development: cell multiplication and cessation of the cell proliferative activity

Changes in cell number during the early period of tomato fruit development were analysed by means of a deterministic model of cell multiplication. The period commenced at the seed stage with one theoretical cell undergoing intensive cell division, and ended when the cell number became nearly constant. The model takes into consideration the proliferative activity of the fruit cell population which, a few days before flower anthesis, begins to decrease progressively after each mitotic cycle. Model parameters, namely the time at which proliferative activity diminishes, its rate of decrease and the length of the cell cycle, were estimated by fitting the model to observed cell population dynamics in tomato fruits growing in three different positions on the truss. It is hypothesized that the molecular mechanism responsible for the cessation of mitosis in growing fruits is associated with shortening of telomeric ends of nuclear DNA, as suggested previously for other growing cell populations.     

The distribution of xylem hydraulic resistance in the fruiting truss of tomato

The distribution of hydraulic resistances in xylem throughout the pathway leading to the tomato fruit was investigated. Previous work had indicated that there were large resistances within the supporting sections of this pathway (the peduncle and pedicel), perhaps associated with interruptions in the xylem. These high resistances are believed to impede calcium flux into the fruit and thus impair fruit development. It is shown here that fruit on intact plants do not shrink detectably during drought, even when the drought is sufficient to cause marked shrinkage of leaves and visible wilting of the shoot. In explants, it is possible to induce back‐flow from the fruit into the stem (probably via the xylem) but this flow is small and very slow. These observations support the view that there is a large hydraulic resistance in the pathway between fruit and stem. When pulses of water were made available within explants, by scorching of one leaflet, there was a rapid swelling of leaves and sepals. Such rapid fluxes indicate the presence of strong hydraulic (xylem) connections throughout the pathway between leaf and calyx. This shows that there are no significant hydraulic constrictions in the xylem proximal to the calyx. This finding is contrary to some previous conclusions but it is supported by experiments with dyes which showed continuous, functional xylem throughout the peduncle and pedicel. Calculations show that over 90% of the hydraulic resistance between stem and fruit must reside within the fruit pericarp. Implications for calcium nutrition are discussed.     

A cellular hypothesis for the induction of blossom-end rot in tomato fruit

BACKGROUND: The incidence of blossom-end rot (BER) is generally associated with a calcium (Ca) deficiency in the distal portion of tomato fruits. The visible symptom is a necrotic lesion, which is presumed to be a consequence of cell death and the subsequent leakage of solutes into the extracellular space. Environmental factors that affect either fruit cell expansion or Ca delivery to the distal portion of the fruit influence the occurrence of BER. However, since no absolute, critical fruit Ca concentration for the occurrence of BER has been identified, it is now important to define the role of Ca in fruit cell physiology and to seek the cause of BER at the cellular level. HYPOTHESIS: Here, it is suggested that BER is initiated by a cellular dysfunction in the distal portion of a young fruit during rapid cell expansion. It is proposed that insufficient Ca(2+) is available for critical apoplastic and cytoplasmic functions when the cellular Ca demand imposed by vacuolation exceeds the Ca delivery to an expanding cell. A local Ca deficiency, therefore, may result in aberrant intracellular Ca(2+) signals, a weakening of cell walls and a loss of cellular integrity. Ultimately it may lead to cell death and the visible symptoms of BER. Several experimental strategies are suggested to confirm the occurrence of aberrant Ca(2+) concentrations in cells contributing to BER. PERSPECTIVE: Many genetic and genomic resources are becoming available for tomato. Ultimately, these will allow genes affecting the occurrence of BER to be identified. Such knowledge will inform breeding strategies to eliminate BER. In the meanwhile, increasing the apoplastic Ca concentration in susceptible fruit tissue should provide a simple and reliable, practical solution for the prevention of BER in tomatoes. It is suggested that current horticultural practices, such as the manipulation of the mineral composition of the feed or the growth environment, are not completely effective in reducing BER because they affect apoplastic Ca concentration in fruit tissue indirectly. Therefore, spraying Ca directly onto young fruits is recommended for the prevention of BER.     

Differential expression of expansin gene family members during growth and ripening of tomato fruit

cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955–5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.     

Reduction, assimilation and transport of N in normal and gibberellin-deficient tomato plants

A fast-growing normal and a slow-growing gibberellin-deficient mutant of Lycopersicon esculentum (L.) Mill. cv. Moneymaker were used to test the hypothesis that slow-growing plants reduce NO₃^? in the root to a greater extent than do fast-growing plants. Plants that reduce NO₃^? in the root may grow more slowly due to the higher energetic and carbon costs associated with root-based NO₃^? reduction compared to photosynthetically driven shoot NO₃^? reduction. The plants were grown hydroponically with a complete nutrient solution containing 10 mM NO₃^? and the biomass production, gas exchange characteristics, root respiratory O₂ consumption, nitrate reductase activity and translocation of N in the xylem were measured. The gibberellin-deficient mutants accumulated more total N unit^?1 dry weight than did the faster-growing normal plants. There were no significant differences between the genotypes in the rates of photosynthesis expressed on a leaf dry weight basis. The plants differed in the proportion of photosynthetic carbon available to growth due to a greater proportion of daily photo-synthate production being consumed by respiration in the slow-growing genotype. This difference in allocation of carbon was associated with differences in the specific leaf area and specific root length. In addition, a greater leaf weight ratio in the fast-growing than in the slow-growing plants indicates a greater investment of carbon into biomass supporting photosynthetic production in the former. We did not find differences in the activity or distribution of nitrate reductase or in the N composition of the xylem sap between the genotypes. We thus conclude that the growth rate was determined by the efficiency of carbon partitioning and that the site of NO₃^? reduction and assimilation was not related to the growth rate of these plants.     

High hexokinase activity in tomato fruit perturbs carbon and energy metabolism and reduces fruit and seed size

Tomato (Lycopersicon esculentum var MP‐1) plants overexpressing Arabidopsis hexokinase 1 (AtHXK1) exhibited high hexokinase (HXK) activity in correlation with drastic phenotypic modifications in fruit. Transgenic fruit and seeds were reduced in size. Reduction in fruit size was due to decreased cell expansion, which could not be corrected by perfusion with sucrose (Suc). Neither could wild type (WT) fruit and seed size be obtained by grafting of transgenic flowers onto WT shoots. Starch and hexose contents were lower but organic and amino acids were higher in transgenic fruit. Lower respiratory rates measured in vitro accompanied by even lower ATP levels and ATP/ADP ratios indicated metabolic perturbations that may explain, in part, reduced fruit and seed size.     

Ion balance in tomato cultivars differing in salt tolerance. I. Sodium and potassium accumulation and fluxes under moderate salinity

The magnitude of sodium and potassium fluxes in Lycopersicon escutentum cuhivars Ace and Edkawi (Edkawi is considered more sait-tolerant I was evaluated in planls grown for 10 days in aerated Hoagland solution with the addition of 25 or 100 mM NaCl. Ion accumulatiun in different plant pans, ion concentration in xylem exudate. transpiration and membrane leakiness were measured. Both cultivars responded very similarly to these levels of salinity in terms of growth. No conspicuous differences in membrane leakiness were observed. Net uprake rates were calculated from ion contents data. Potassium uptake rates were lower in salinized planls than in controls, especially in cv. Aee. Potassium/sodium selectivity ratios were much higher in Edkawi than in Ace. and higher in shoot uptake rates than in xy lem exudate. This indicates that Edakw i has a higher capacity to retain potassium under salinity, a character which could contribute to its salt-tolerance.     

Protoplast hexose carrier activity is a determinate of genotypic difference in hexose storage in tomato fruit

Post-phloem sugar transport in developing tomato (Lycopersicon esculentum Mill. cv. Flora-Dade) fruit follows an apoplastic route during the rapid phase of sugar accumulation. The pathway is characterized by sugar retrieval by the storage parenchyma cells from the fruit apoplast. Two tomato genotypes differing in fruit hexose content were compared in terms of the transport and transfer processes controlling fruit sugar levels. The genotypic difference in fruit sugar content was independent of photoassimilate export from source leaves. Discs of pericarp tissue were cultured in a medium based on analyses of the fruit apoplastic sap. The cultured discs maintained a composition, a relative growth rate and a respiration rate similar to those of the pericarp tissue of intact fruit. Estimates of hexose fluxes into metabolic and storage pools suggested that membrane transport regulated the genotypic difference in hexose accumulation. Short-term [¹⁴C]hexose uptake experiments demonstrated a genotypic difference in V_max for glucose, fructose and 3-O-methyl-glucose, and this difference was abolished in the presence of the inhibitor p-chloromercuribenzenesulphonic acid (PCMBS). The results support the hypothesis that the activity of energized hexose carriers on the plasma membranes of storage parenchyma cells is a significant determinate of the genotypic difference in hexose accumulation.     

Regulation of tomato fruit growth by epidermal cell wall enzymes

Water relations of tomato fruit and the epidermal and pericarp activities of the putative cell wall loosening and tightening enzymes Xyloglucan endotransglycosylase (XET) and peroxidase were investigated, to determine whether tomato fruit growth is principally regulated in the epidermis or pericarp. Analysis of the fruit water relations and observation of the pattern of expansion of tomato fruit slices in vitro , has shown that the pericarp exerts tissue pressure on the epidermis in tomato fruit, suggesting that the rate of growth of tomato fruit is determined by the physical properties of the epidermal cell walls. The epidermal activities of XET and peroxidase were assayed throughout fruit development. Temporal changes in these enzyme activities were found to correspond well with putative cell wall loosening and stiffening during fruit development. XET activity was found to be proportional to the relative expansion rate of the fruit until growth ceased, and a peroxidase activity weakly bound to the epidermal cell wall appeared shortly before cessation of fruit expansion. No equivalent peroxidase activity was detected in pericarp tissue of any age. It is therefore plausible that the expansion of tomato fruit is regulated by the combined action of these enzyme activities in the fruit epidermis.     

Physiology and firmness determination of ripening tomato fruit

Tomato ( Lycopersicon esculentum Mill.) genotypes varying in intrinsic firmness were examined to determine the quantitative relationships between polygalacturonase (EC 3.2.1.15) activity, firmness and other ripening parameters including rate (days from mature-green to full red) and intensity (rate of ethylene production at climacteric peak) of ripening. Texture, respiration and ethylene production were monitored in the immature-green through the red (ripe) stages of development. Polygalacturonase activity was measured by direct assay of salt-extractable wall protein or by monitoring the release of pectins from isolated, enzymically active wall. In all fruit, polygalacturonase activity was highly correlated with pericarp softening, but only moderately correlated with softening of whole fruit (r = 0.920 and 0.757, respectively). Polygalacturonase activity was positively correlated with cell-wall autolytic activity in pink (r = 0.969) and red (r = 0.900) fruit. Firmer genotypes exhibited lower rates of respiration and ethylene production during ripening. Polygalacturonase activity in isolates prepared from fruit at the climacteric peak was positively correlated with ethylene production and respiration, and negatively correlated with days to ripening (r = 0.929, 0.805, and -0.791, respectively). The data demonstrate the importance of selecting the appropriate method of firmness determination and are consistent with the hypothesis that pectin fragments released by polygalacturonase contribute to the production of autocatalytic (system II) ethylene.     

Genetic engineering of parthenocarpic fruit development in tomato

Parthenocarpy was engineered in two genotypes of Lycopersicon esculentum Mill. by using the DefH9-iaaM chimeric gene. The parthenocarpic trait consists of fruit set and growth in the absence of fertilization. Seedless parthenocarpic fruits were obtained from emasculated flowers, and fruits with seeds from pollinated flowers. All parthenocarpic tomato plants analysed expressed the DefH9-iaaM gene during flower development. The fruit set percentage of emasculated transgenic flowers was similar to that of control plants. In 7 out of 8 independent transgenic plants, the fresh weight of fruits derived from pollinated or emasculated flowers did not significantly differ from that of fruits obtained by pollination of the control plants. The pH of the parthenocarpic fruit was generally unaffected and the soluble solid concentration was either unchanged or increased. Thus, the DefH9-iaaM gene is a genetic tool that might be used to improve tomato productivity.