Contrasted responses to carbohydrate limitation in tomato fruit at two stages of development

The metabolic consequences of long‐term carbohydrate depletion have been well documented in many sink organs but not extensively in fruit. Therefore, in the present study the response to sugar limitation in tomato fruit (Lycopersicon esculentum Mill.) was investigated at two developmental stages; during the cell division and cell expansion phases. First, the response in excised fruit cultured in vitro was characterized. Sugar depletion caused an arrest of growth and an exhaustion of carbon reserves. The proteins that were degraded and the nitrogen released was transiently stored as asparagine and glutamine in both developmental stages and also as γ ‐aminobutyric acid (GABA) in expanding fruit. Fruit at the cell division stage appeared to be more sensitive to sugar limitation. The response to sugar depletion was then characterized in fruit from plants submitted to extended darkness. In planta, the effects of sugar‐limitation were similar to those described in vitro but much more attenuated, especially in expanding fruit, which still accumulated dry matter. The expression of cell cycle genes, sugar‐ and nitrogen‐related genes was reduced by darkness. Only asparagine synthetase gene expression was induced in both dark‐treated fruit. Together the present data revealed that the effects of the carbon limitation are more pronounced in the youngest fruits as it is probably controlled by the relative sink strength of the fruit.     

Multiple forms of ADP‐glucose pyrophosphorylase from tomato leaf

ADP‐glucose pyrophosphorylase (AGP, EC 2.7.7.27) was partially purified from tomato ( Lycopersicon esculentum Mill. cv. Laura) leaf by a procedure previously used for purification of AGP from tomato pericarp. SDS‐PAGE and western blot analysis of the final preparation indicated that the leaf enzyme is composed of two subunits of 50 and 54 kDa. Two‐dimensional PAGE and western blot analysis of the same preparation, however, revealed at least six isoforms of the large subunit and two isoforms of the small subunit. The leaf AGP is very sensitive to regulation by 3‐phosphoglycerate and inorganic phosphate. Its properties are compared to those of AGP from tomato fruit.     

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.     

Time-course of tomato whole-plant respiration and fruit and stem growth during prolonged darkness in relation to carbohydrate reserves

To evaluate the relevance of a simple carbon balance model (Seginer et al., 1994, Scientia Horticulturae 60: 55-80) in source-limiting conditions, the dynamics of growth, respiration and carbohydrate reserves of tomato plants were observed in prolonged darkness. Four days prior to the experiments, plants were exposed to high or low light levels and CO(2) concentrations. The concentration of carbohydrates in vegetative organs was 30-50 % lower in plants that were exposed to low carbon assimilation conditions compared with those exposed to high carbon assimilation conditions. During prolonged darkness, plants with low carbohydrate reserves exhibited a lower whole-plant respiration rate, which decreased rapidly to almost zero after 24 h, and carbohydrate pools were almost exhausted in leaves, roots and flowers. In plants with high carbohydrate reserves, the whole-plant respiration rate was maintained for a longer period and carbohydrates remained available for at least 48 h in leaves and flowers. In contrast, fruits maintained fairly stable and identical concentrations of carbohydrates and the reduction in their rate of expansion was moderate irrespective of the pre-treatment carbon assimilation conditions. The time-course of asparagine and glutamine concentrations showed the occurrence of carbon stress in leaves and flowers. Estimation of source and sink activities indicated that even after low carbon assimilation, vegetative organs contained enough carbohydrates to support fruit growth provided their own growth stopped. The time of exhaustion of these carbohydrates corresponded grossly to the maintenance stage simulated by the model proposed by Seginer et al. (1994), thus validating the use of such a model for optimizing plant growth.     

The influence of plant age on wound induction of proteinase inhibitors in tomato

Proteinase inhibitors can be induced by wounding in shoots of tomato ( Lycopersicon esculentum [L.] Mill. cv. Moneymaker). These inhibitors are toxic to insects, but their ecological importance is not clear. Published work suggests that proteinase inhibitors may be wound-inducible in tomato only while the plants are young (less than 30 days). In the present investigation the influence of plant age on wound-inducible proteinase inhibitor was re-assessed using tomato plants grown in an outdoor polythene tunnel, with natural lighting and without supplementary heat. In contrast to previous findings, proteinase inhibitor was shown to be induced by wounding in plants of all ages. However, the systemic efficacy of wounds was much reduced in mature plants, possibly because such plants have outgrown the range of the wound-signalling system.     

Oligosaccharides that induce proteinase inhibitor activity in tomato plants cause depolarization of tomato leaf cells

Abstract. Two size ranges of oligosaccharide elicitors of pectic origin have been investigated for their effects on tomato plants. Both size ranges, with degrees of polymerization of 1–7 and 10–20 respectively, induced the accumulation of proteinase inhibitor (PI) activity in excised plants, and also induced changes in membrane potential of leaf mesophyll cells. The depolarizations were substantial, rapid, and reversible on removal of the elicitors. The effects are discussed in the context of early events in the signal transduction pathway linking oligosaccharides to changes in PI gene expression.     

An experimental assessment of the factors influencing Agrobacterium-mediated transformation in tomato

Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pTOK233, which contained the GUS reporter gene and a kanamycin-resistance gene nptII, was employed for optimizing the transformation efficiency evaluated by a GUS gene transient expression level. Eight factors including explant types, explant size and source, the concentration of cytokinin, inoculation time, pH of inoculation and cocultivation media, bacterial concentration, acetosyringone concentration, and cocultivation duration were investigated in detail. This optimized protocol was then adopted to obtain transgenic tomato plants resistant to cucumber mosaic virus (CMV) mediated by Agrobacterium tumefaciens, strain LBA4404, carrying a binary vector pR-ΔGDD containing the kanamy cin-resistance gene and CMV replicase gene with GDD deletion. The presence of the CMV-RNA2 gene was confirmed by genomic DNA Southern blot analysis in all transformants analyzed. Field spray test showed that the transgenic tomato plants were resistant to 100 mg/l kanamycin. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 280–284. The text was submitted by the authors in English.     

Use of lysophosphatidylethanolamine, a natural lipid, to retard tomato leaf and fruit senescence

We studied the influence of lysophosphatidylethanolamine (LPE) on the pattern and rate of ethylene production and respiration of tomato ( Lycopersicon esculentum cv. H7155) leaflets and fruit. Leaflets that had been senescing on the plant showed a climacteric-like rise in ethylene production but not in respiration rate which decreased continuously with leaf age. Detached leaflets had a climacteric-like pattern in respiration whether they were incubated in complete darkness or in light. Detached leaflets incubated in the dark had higher rates of ethylene production and CO₂ evolution than did light-incubated leaves. There was no change in the pattern of ethylene production or CO₂ evolution as a result of LPE treatment. However, LPE-treated attached and detached leaflets had consistently lower rates of CO₂ evolution. The reduction in CO₂ evolution by LPE was most pronounced at the climacteric-like peak of the detached leaves. LPE-treated leaflets had a higher chlorophyll content and fresh weight and lower electrolyte leakage than the control. LPE-treated fruits had lower rates of ethylene and CO₂ production than did the control. LPE-treated fruits also had higher pericarp firmness and lower electrolyte leakage than the control. The results of the present study provide evidence that LPE is able to retard senescence of attached leaves and detached leaves and tomato fruits. Several recent studies suggest that lysolipids can act in a specific manner as metabolic regulators. Our results suggest a specific role of lysolipid LPE in aging and senescence     

Salt stress increases ferredoxin-dependent glutamate synthase activity and protein level in the leaves of tomato

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) catalyzes an essential step in the pathway of glutamate biosynthesis. Exposing detached tomato ( Lycopersicon esculentum ) leaves for 6 h to 12 g l⁻¹ NaCl resulted in a significant two-fold increase in the activity of ferredoxin-dependent glutamate synthase extracted from the leaves. Western blot studies demonstrated that salt treatment also increased the ferredoxin-dependent glutamate synthase content of the leaves. A similar effect of salt on the concentration of this enzyme was found in the leaves of hydroponically-grown tomato plants. The induction of ferredoxin-dependent glutamate synthase under salt stress may provide the glutamate required for the proline synthesis which is a common response to salt stress.     

The role of endogenous gibberellin in seed and fruit development of tomato: Studies with a gibberellin-deficient mutant

The role of endogenous gibberellin (GA) in seed and fruit development was studied with the use of the GA-deficient ga-1 mutant of tomato ( Lycopersicon esculentum Mill. cv. Moneymaker). Flowers of the ga-1 mutant were abnormal and sterile, but parthenocarpic fruit development was observed occasionally on the dwarf plants. A single application of GA₄₊₇ restored the fertility of the mutant flowers and resulted in seed set. Development of GA-producing and GA-deficient seeds in GA-deficient fruits was compared by pollination of ga-1/ga-1 flowers with wild-type or ga-1 pollen, respectively. In ga-1/ga-1 seed dehydration started about 1 week earlier than in Ga-1/ga-1 seeds. Ultimate fresh and dry weights of mature Ga-1/ga-1 seeds were higher than those of ga-1/ga-1 seeds and showed negative correlations with the total number of seeds per fruit. Total content and composition of seed proteins were not influenced by the GA-deficiency. Germination of the mature seeds depended on embryonal GA synthesis and was not influenced by maternal GA production. Final fresh weight of the ga-1/ga-1 fruits was positively correlated with the number of seeds per fruit. In these fruits, the minimum number of seeds for growth above the parthenocarpic level was about 10 or 35 in the presence of Ga-1/ga-1 or ga-1/ga-1 seeds, respectively. Fruits containing GA-producing seeds reached a higher fresh weight than those containing GA-deficient seeds, and their ripening was delayed by one week. It is concluded that gibberellin is indispensable for the development of fertile flowers and for seed germination, but only promoting in later stages of fruit and seed development.     

Increase in leaf mass per area benefits plant growth at elevated CO2 concentration

An increase in leaf mass per area (MLA) of plants grown at elevated [CO2] is often accompanied by accumulation of non-structural carbohydrates, and has been considered to be a response resulting from source-sink imbalance. We hypothesized that the increase in MLA benefits plants by increasing the net assimilation rate through maintaining a high leaf nitrogen content per area (NLA). To test this hypothesis, Polygonum cuspidatum was grown at ambient (370 micro mol mol-1) and elevated (700 micro mol mol-1) [CO2] with three levels of N supply. Elevated [CO2] significantly increased MLA with smaller effects on NLA and leaf mass ratio (fLM). The effect of change in MLA on plant growth was investigated by the sensitivity analysis: MLA values observed at ambient and elevated [CO2] were substituted into a steady-state growth model to calculate the relative growth rate (R). At ambient [CO2], substitution of a high MLA (observed at elevated [CO2]) did not increase R, compared with R for a low MLA (observed at ambient [CO2]), whereas at elevated [CO2] the high MLA always increased R compared with R at the low MLA. These results suggest that the increase in MLA contributes to growth enhancement under elevated [CO2]. The optimal combination of fLM and MLA to maximize R was determined for different [CO2] and N availabilities. The optimal fLM was nearly constant, while the optimal MLA increased at elevated [CO2], and decreased at higher N availabilities. The changes in fLM of actual plants may compensate for the limited plasticity of MLA.     

Development of galactomannan-hydrolyzing activity in the micropylar endosperm tip of tomato seed prior to germination

Development of galactomannan-hydrolyzing activity, that is involved in the weakening of the mechanical restraint of the endosperm, was followed at pre-germinative stages in tomato ( Lycopersicon esculentum ) seed. Prior to germination the activity developed exclusively in the endosperm portion just adjacent to the radicle tip. In other parts of the endosperm, the activity developed only after germination occurred. Under the conditions where germination was suppressed (far-red light- or ABA-treatment). no activity was detected in the endosperm at the pre-germinative stages. Under the conditions where the inhibition of germination was alleviated (far-red + red or ABA + GA₃), the activity developed prior to germination in the endosperm part in front of the radicle tip. Thus, a clear parallel relationship was observed between germinability of the seed and the pre-germinative development of activity in the part of the endosperm portion adjacent to the radicle tip.     

Association of some plant defense enzyme activities with systemic resistance to early leaf blight and leaf spot induced in tomato plants by azoxystrobin and Pseudomonas fluorescens

Abstract

Azoxystrobin at three different concentrations, namely, 31.25, 62.50 and 125 g a.i. ha^?1 mancozeb (1 kg ha^?1) and Pseudomonas fluorescens (10 kg ha^?1) were evaluated for their efficacy in inducing defense enzymes in tomato against Alternaria solani and Septoria lycopersici. The activity of defense enzymes peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), β-1, 3 glucanase, chitinase, catalase and defense-inducing chemicals (total phenols) was found to be increased in azoxystrobin and P. fluorescens-treated tomato plants. The activity of these defense enzymes and chemicals was higher in azoxystrobin (125 g a.i. ha^?1) and P. fluorescens-treated tomato plants challenge inoculated with the pathogens compared to other treatments. Increased expression of specific isoforms of PO and PPO was also observed due to ISR induction.     

The wilty tomato mutants flacca and sitiens are impaired in the oxidation of ABA-aldehyde to ABA

Abstract. Deuterium-labelled ABA-aldehyde was fed to various tomato genotypes. Normal and notabilis mutant plants incorporated substantial amounts of the label into ABA. In contrast, two ABA-deficient mutants, flacca and sitiens , reduced ABA-aldehyde to a mixture of cis- and trans -ABA alcohol rather than oxidizing it to ABA. It was concluded that ABA-aldehyde is the immediate precursor of ABA in higher plants. It appears that the flacca and sitiens lesions both act to block the last step of the ABA biosynthetic pathway. The mutant gene loci are likely to be involved in coding for different sub-units of the same dehydrogenase enzyme.     

mRNA levels of four tomato (Lycopersicon esculentum Mill. L.) genes related to fluctuating plant and soil water status

Expression of the genes le20, ni3212, le25 and lcyp2 was quantified in relation to soil and plant water status for pot-grown tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) in a replicated trial in a partially environmentally controlled glasshouse. Leaf water potential, relative water content, stomatal conductance and gravimetric soil water content were measured at 0800,1200 and 1600 h on three days during the imposition of three different watering regimes. Paired leaf samples were analysed for mRNA. Plant-to-plant variability was quantified and partitioned. le20 mRNA was found in all leaves and responded both to short-term diurnal fluctuations in water status and to more severe stress, though with reduced sensitivity to the greatest stresses. An overnight decline in le20 mRNA was independent of soil or plant water status. The pattern of expression for ni3212 was similar though ni3212 mRNA was much more abundant and the overnight drop in expression was not significant. le25 mRNA was only detected where leaf water potential was below ?0.9 MPa and increased linearly with further decline in leaf water potential. lcyp2 was constitutively expressed with a diurnal pattern characterized by a sharp depression at 1600 h but with expression being enhanced by increasing water deficit at all times. The control and significance of the four genes are discussed in relation to the proposed function of their respective gene products.     

Growth rate, plant development and water relations of the ABA-deficient tomato mutant sitiens

Given the close relationship between a plant's growth rate and its pattern of biomass allocation and the effects of abscisic acid (ABA) on biomass allocation, we studied the influence of ABA on biomass allocation and growth rate of wildtype tomato ( Lycopersicon esculentum Mill. cv. Moneymaker) plants and their strongly ABA-deficient mutant sitiens. The relative growth rate of sitiens was 22% lower than that of the wildtype, as the result of a decreased specific leaf area. The net assimilation rate and the leaf weight ratio were not affected. The mutant showed a much higher transpiration rate and lower hydraulic conductance of the roots. These two factors resulted in sitiens having a significantly lower leaf water potential and turgor. resulting in reduced leaf expansion and, consequently, a lower specific leaf area relative to the wildtype. Addition of ABA to the sitiens roots resulted in phenotypic reversion to the wildtype. We conclude that the influence of ABA-deficiency on biomass allocation and relative growth rate is the result of altered water relations in the plants, rather than of a direct effect on sink strength of different plant organs.     

Endo-β-mannanases in the endosperm of germinated tomato seeds

Three forms of galactomannan-hydrolyzing enzymes (dyed galactomannan as substrate) were partially purified from germinated tomato [ Lycopersicon esculentum (L.) Mill.] seed. Two of the enzymes were of the same molecular mass, 38 kDa, as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), but the points of elution from a CM-Toyopearl column by a pH-gradient were different between the two (pH 5.15 and 5.45. respectively). The molecular mass of the third form was slightly less (37.5 kDa) than that of the other two. These 3 enzymes showed no α-galactosidase (EC 3.2.1.22) or β-mannosidase (EC 3.2.1.25) activity. Thin-layer chromatography (TLC) revealed that the products of the reaction were oligosaccharides and that free galactose and mannose were not released. These results indicate that the 3 galactomannan-hydrolyzing enzymes are endo-β-mannanases (EC 3.2.1.78). Polyclonal antibodies raised against the 37.5-kDa polypeptide cross-reacted with the two 38-kDa polypeptides, indicating that the 3 endo-β-mannanases are immunologically homologous. Activity staining and immunoblotting of native PAGE of endosperm extracts revealed that only two (38-kDa. elution point pH 5.15 and 37.5-kDa proteins) of the 3 forms were major endo-β-mannanases present in the endosperm of germinated tomato seeds.     

Galactomannan hydrolyzing activity develops during priming in the micropylar endosperm tip of tomato seeds

Development of galactomannan hydrolyzing activity was followed in seeds of tomato [ Lycopersicon esculentum (L.) Mill. cv. Toyonishiki] during priming and germination. The activity developed in seeds that were being primed in polyethylene glycol (-0.8 MPa). The activity was detected exclusively in the endosperm portion just adjacent to the radicle tip. Part of the activity remained active after desiccation of the primed seeds. After transfer to water, the activity in the primed seeds immediately began to increase, while in unprimed seeds the beginning of the increase in activity was delayed by about 1 day. In scanning electron microscopy, the inner surface of the cell walls of the micropylar endosperm portion appeared eroded in primed seeds that had been imbibed in water for 16 h (before germination), but not in unprimed seeds imbibed for the same period. These results support the hypothesis that galactomannan hydrolyzing enzyme, which is believed to be responsible for breakdown of tomato endosperm cell walls and hence for the weakening of mechanical restraint against radicle growth, may be involved in the improved germination of primed tomato seeds.     

Analysis of the tomato fruit growth response to temperature and plant fruit load in relation to cell division, cell expansion and DNA endoreduplication

BACKGROUND AND AIMS: To better understand the regulation of fruit growth in response to environmental factors, the effects of temperature and plant fruit load on cell number, cell size and DNA endoreduplication were analysed. METHODS: Plants were grown at 20/20 degrees C, 25/25 degrees C and 25/20 degrees C day/night temperatures, and inflorescences were pruned to two ('2F') or five ('5F') flowers. KEY RESULTS AND CONCLUSIONS: Despite a lower fruit growth rate at 20/20 degrees C, temperature did not affect final fruit size because of the compensation between cell number and size. The higher cell number at 20/20 degrees C (9.0 x 10(6) against 7.9 x 10(6) at 25/25 degrees C and 7.7 x 10(6) at 25/20 degrees C) resulted from an extended period of cell division, and the smaller cell size was due to a shorter period of expansion rather than a lower expansion rate. By contrast, the lower fruit growth rate and size of 5F fruits compared with 2F fruits resulted from the slow down of cell expansion, whereas the number of cells was hardly affected in the proximal fruit. However, within the inflorescence the decreasing gradient of fruit size from proximal to distal fruits was due to a decrease in cell number with similar cell size. Fruit size variations within each treatment were always positively correlated to variations in cell number, but not in cell size. Negative correlations between cell size and cell number suggested that cells of tomato pericarp can be seen as a population of competing sinks. Mean ploidy was slightly delayed and reduced in 5F fruits compared with 2F fruits. It was highest at 25/25 degrees C and lowest at 25/20 degrees C. Treatments did not affect ploidy and cell size in similar ways, but within each treatment, positive correlations existed between mean ploidy and cell size, though significant only in the 2F-25/20 treatment.