Suppression of Acid Invertase Activity by Antisense RNA Modifies the Sugar Composition of Tomato Fruit

cDNA for an acid invertase (EC 3.2.1.26 [EC] ) of tomato (Lycopersiconesculentum Mill.) fruit was introduced into tomato plants underthe control of the cauliflower mosaic virus 35S promoter inthe antisense orientation. The antisense gene effectively suppressedthe invertase activity in soluble and cell wall fractions fromripening fruits. The sucrose content of fruits of the transformantswas markedly increased, while the hexose content was reduced.These results indicate that acid invertase is one of main determinantsof the sugar composition of tomato fruit. The invertase activityin the cell wall fraction of the leaf tissues of the transformantswas not suppressed to the same extent as that in the solublefraction. Wounding of the control leaf tissues induced invertaseactivity in both soluble and cell wall fractions. The inductionof activity in the soluble fraction was suppressed by the antisensegene, while that in the cell wall fraction was unaffected. Thesefindings suggest that mRNA for some other invertase, in particular,the mRNA for a cell wall-bound invertase, was present in leaves. ¹Present address: Plant Breeding and Genetics Research Laboratory,Japan Tobacco Inc., 700 Higashibara, Toyoda, Iwata, Shizuoka,438 Japan. ²Present address: National Institute of Agrobiological Resources,Kannondai, Tsukuba, Ibaraki, 305 Japan.     

Regulatory Role of Cystathionine-γ-Synthase and de novo Synthesis of Methionine in Ethylene Production during tomato Fruit Ripening

The essential amino acid methionine is a substrate for the synthesis of S-adenosyl-methionine (SAM), that donates its methyl group to numerous methylation reactions, and from which polyamines and ethylene are generated. To study the regulatory role of methionine synthesis in tomato fruit ripening, which requires a sharp increase in ethylene production, we cloned a cDNA encoding cystathionine γ-synthase (CGS) from tomato and analysed its mRNA and protein levels during tomato fruit ripening. CGS mRNA and protein levels peaked at the “turning” stage and declined as the fruit ripened. Notably, the tomato CGS mRNA level in both leaves and fruit was negatively affected by methionine feeding, a regulation that Arabidopsis, but not potato CGS mRNA is subject to. A positive correlation was found between elevated ethylene production and increased CGS mRNA levels during the ethylene burst of the climacteric ripening of tomato fruit. In addition, wounding of pericarp from tomato fruit at the mature green stage stimulated both ethylene production and CGS mRNA level. Application of exogenous methionine to pericarp of mature green fruit increased ethylene evolution, suggesting that soluble methionine may be a rate limiting metabolite for ethylene synthesis. Moreover, treatment of mature green tomato fruit with the ethylene-releasing reagent Ethephon caused an induction of CGS mRNA level, indicating that CGS gene expression is regulated by ethylene. Taken together, these results imply that in addition to recycling of the methionine moieties via the Yang pathway, operating during synthesis of ethylene, de novo synthesis of methionine may be required when high rates of ethylene production are induced.     

Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening

To determine the relationship between invertase gene expression and glucose and fructose accumulation in ripening tomato fruit, fruit vacuolar invertase cDNA and genomic clones from the cultivated species, Lycopersicon esculentum cv. UC82B, and a wild species, Lycopersicon pimpinellifolium, were isolated and characterized. The coding sequences of all cDNA clones examined are identical. By comparison to the known amino acid sequence of mature L. esculentum fruit vacuolar invertase, a putative signal sequence and putative amino-terminal and carboxy-terminal propeptides were identified in the derived amino acid sequence. Of the residues 42% are identical with those of carrot cell wall invertase. A putative catalytic site and a five-residue motif found in carrot, yeast, and bacterial invertases are also present in the tomato sequence. Minor differences between the nucleotide sequences of the genomic clones from the two tomato species were found in one intron and in the putative regulatory region. The gene appears to be present in one copy per haploid genome. Northern analysis suggests a different temporal pattern of vacuolar invertase mRNA levels during fruit development in the two species, with the invertase mRNA appearing at an earlier stage of fruit development in the wild species. Nucleotide differences found in the putative regulatory regions may be involved in species differences in temporal regulation of this gene, which in turn may contribute to observed differences in hexose accumulation in ripening fruit.     

Intercellular Localization of Acid Invertase in Tomato Fruit and Molecular Cloning of a cDNA for the Enzyme

The localization of acid invertase (AI, EC 3.2.1.26 [EC] ) in tomatofruits was studied. AI was localized in the intercellular fraction(cell wall fraction). A cDNA encoding a wall-bound form of AIfrom tomato fruits was cloned and its nucleotide sequence wasdetermined. The cloned cDNA was 2363 base pairs long and containedan open reading frame of 1908 base pairs which encoded a polypeptideof 636 amino acids. RNA blot analysis indicated that the mRNAfor the acid invertase was about 2.5 kb in length. The levelsof the mRNA were low at the mature green stage but increasedduring ripening of fruit. (Received July 13, 1992; Accepted December 3, 1992)     

Sink Metabolism in Tomato Fruit : IV. Genetic and Biochemical Analysis of Sucrose Accumulation

Fruit of domesticated tomato (Lycopersicon esculentum) accumulate primarily glucose and fructose, whereas some wild tomato species, including Lycopersicon chmielewskii, accumulate sucrose. Genetic analysis of progeny resulting from a cross between L. chmielewskii and L. esculentum indicated that the sucrose-accumulating trait could be stably transferred and that the trait was controlled by the action of one or two recessive genes. Biochemical analysis of progeny resulting from this cross indicated that the sucrose-accumulating trait was associated with greatly reduced levels of acid invertase, but normal levels of sucrose synthase. Invertase from hexose-accumulating fruit was purified and could be resolved into three isoforms by chromatofocusing, each with isoelectric points between 5.1 and 5.5. The invertase isoforms showed identical polypeptide profiles on sodium dodecyl sulfate polyacrylamide gel electrophoresis, consisting of a primary 52 kilodalton polypeptide and two lower molecular mass polypeptides that appear to be degradation products of the 52 kilodalton polypeptide. The three invertase isoforms were indistinguishable based on pH, temperature, and substrate concentration dependence. Immunological detection of invertase indicated that the low level of invertase in sucrose-accumulating fruit was due to low levels of invertase protein rather than the presence of an invertase inhibitor. Based on comparison of genetic and biochemical data we speculate that a gene either encoding tomato fruit acid invertase or one required for its expression, plays an important role in determining sucrose accumulation.     

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.     

cDNA cloning and characterisation of novel ripening-related mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant

A cDNA library produced from mRNA isolated from the pericarp of wild-type tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig) at the first visible sign of fruit ripening was differentially screened to identify clones whose homologous mRNAs were present at reduced levels in fruit of the tomato ripening mutant, ripening inhibitor,rin. Five clones were isolated (pERT 1, 10, 13, 14, 15). Accumulation of mRNA homologous to each of these clones increased during the ripening of wild-type fruit and showed reduced accumulation in ripening rin fruit. The levels of three of them (homologous to ERT 1, 13 and 14) were increased by ethylene treatment of the mutant fruit. A further clone, ERT 16 was identified for a mRNA present at a high level in both normal and mutant fruit at early stages of ripening. Database searches revealed no significant homology to the DNA sequence of ERT 14 and 15; however, DNA and derived amino acid sequence of ERT 1 both contain regions of homology with several reported UDP-glucosyl and glucuronosyl transferases (UDPGT) and with a conserved UDPGT motif. A derived amino acid sequence from the ERT 10 cDNA contains a perfect match to a consensus sequence present in a number of dehydrogenases. The ERT 13 DNA sequence has homology with an mRNA present during potato tuberisation. The presence of these mRNAs in tomato fruit is unreported and their role in ripening is unknown. The ERT 16 DNA sequence has homology with a ripening/stress-related cDNA isolated from tomato fruit pericarp.     

Factors Affecting Calcium Transport and Basipetal IAA Movement in Tomato Fruit in Relation to Blossom-end Rot

Factors affecting the uptake and distribution of calcium (Ca)by detached tomato (Lycopersicon esculentum Mill.) fruit wereinvestigated in seven cultivars with different susceptibilitiesto blossom-end rot (BER), a physiological disorder caused byCa deficiency. Plants were grown with different levels of salinityin the root zone or under shade to induce BER. In addition,fruit grown at different salinities were treated with CME, aninhibitor of auxin transport to alter IAA movement. The basipetalmovement of indole-3-acetic acid (IAA) out of detached fruit(i.e. IAA efflux) was determined concurrently with ⁴⁵Ca uptaketo assess the possible involvement of IAA in Ca import or theincidence of BER. High salinity in the root zone during fruitdevelopment decreased both the uptake and distribution of ⁴³Cato the blossom-end of the detached fruit. Shading and the applicationof CME reduced ⁴⁵Ca uptake to a lesser extent. IAA efflux, however,was not consistently reduced by these treatments. Neither theuptake and transport of ⁴⁵Ca within, nor the efflux of IAA from,detached fruit was related to the cultivar susceptibility toBER. The proposed role of IAA on the uptake and distributionof Ca by tomato fruit is assessed. Key words: Tomato, calcium, IAA, blossom-end rot, salinity     

Starch synthesis in tomato remains constant throughout fruit development and is dependent on sucrose supply and sucrose synthase activity

Studies designed to investigate the cellular pathway of phloem unloading were conducted on two tomato lines with either high or low fruit invertase activities. Experiments were based on determination of the degree to which ³H label from [³H]-(fructosyl)-sucrose was randomized between fructose and glucose following exposure of excised fruit to a pulse of labelled sucrose delivered through pedicels. Fruit from the low invertase line harvested 10, 20 and 40 d after anthesis had similar sucrose uptake kinetics to the high invertase line. A positive correlation was found between sucrose synthase activity and sucrose uptake in both low and high invertase lines. In contrast, no correlation was observed between acid or neutral invertase activities and sucrose uptake. Within the putative apoplasmic sap collected from fruit, label in [³H]-(fructosyl)-sucrose was randomized between the free hexoses and sucrose hexose moieties. Label asymmetry was retained in sucrose on arrival within the tissues. Randomization patterns were similar in both the low and high acid invertase lines. These data support the view that sucrose imported into the fruit was not exposed to extracellular hydrolysis. This suggests that movement from the phloem is likely to occur predominantly through a symplastic pathway. About 25% of the sucrose taken up by the fruit was converted into starch regardless of fruit age, suggesting that starch turnover remains constant throughout fruit development and that starch synthesis was dependent on sucrose supply.     

Posttranslational Elevation of Cell Wall Invertase Activity by Silencing Its Inhibitor in Tomato Delays Leaf Senescence and Increases Seed Weight and Fruit Hexose Level

Invertase plays multiple pivotal roles in plant development. Thus, its activity must be tightly regulated in vivo. Emerging evidence suggests that a group of small proteins that inhibit invertase activity in vitro appears to exist in a wide variety of plants. However, little is known regarding their roles in planta. Here, we examined the function of INVINH1, a putative invertase inhibitor, in tomato (Solanum lycopersicum). Expression of a INVINH1:green fluorescent protein fusion revealed its apoplasmic localization. Ectopic overexpression of INVINH1 in Arabidopsis thaliana specifically reduced cell wall invertase activity. By contrast, silencing its expression in tomato significantly increased the activity of cell wall invertase without altering activities of cytoplasmic and vacuolar invertases. Elevation of cell wall invertase activity in RNA interference transgenic tomato led to (1) a prolonged leaf life span involving in a blockage of abscisic acid–induced senescence and (2) an increase in seed weight and fruit hexose level, which is likely achieved through enhanced sucrose hydrolysis in the apoplasm of the fruit vasculature. This assertion is based on (1) coexpression of INVINH1 and a fruit-specific cell wall invertase Lin5 in phloem parenchyma cells of young fruit, including the placenta regions connecting developing seeds; (2) a physical interaction between INVINH1 and Lin5 in vivo; and (3) a symplasmic discontinuity at the interface between placenta and seeds. Together, the results demonstrate that INVINH1 encodes a protein that specifically inhibits the activity of cell wall invertase and regulates leaf senescence and seed and fruit development in tomato by limiting the invertase activity in planta.     

Ripening‐induced acceleration of volatile aldehyde generation following tissue disruption in tomato fruit

Hexanal and cis-3-hexenal are principal flavor volatiles in ripe tomato fruit, but whether they accumulate during ripening or are formed upon maceration of the tissue has not been clarified. This has been addressed by measuring levels of these aldehydes in green and ripe fruit with discrimination between intrinsic aldehyde content and aldehyde generation following tissue disruption. Volatile sampling of tomato fruit homogenates was accomplished by purge/trapping, followed by thermal desorption on a gas chromatograph equipped with a mass selective detector. Incubation of some samples with alcohol dehydrogenase to convert the aldehydes to their respective alcohols permitted positive identification of the isomeric form of hexenal as cis-3-hexenal. Red and green tomato fruit homogenized in buffer with saturated CaCl₂ contained low (0.1-0.8 µg g^?1 fresh weight) levels of hexanal and cis-3-hexenal; thus there is minimal endogenous volatile content in intact fruit. Volatile levels increased rapidly, up to 10-fold, following homogenization of ripe tomato fruit in the absence of CaCl₂, and more modestly in corresponding green tomato fruit homogenates. Incubation with the appropriate lipoxygenase/hydroperoxide lyase substrate (linoleic acid for hexanal, linolenic acid for cis-3-hexenal) doubled the amount of volatile compound produced. Hexanal generation was suppressed in the presence of linolenic acid, suggesting that the enzyme complex has greater affinity for this substrate. As well, levels of cis-3-hexenal, but not hexanal, tended to decline within 30 min of homogenization, possibly reflecting a specific degradative process. The results collectively indicate that the contribution of six-carbon aldehydes to tomato fruit flavor is attributable to metabolism invoked following tissue disruption rather than within the intact fruit.     

Ripening-related occurrence of phosphoenolpyruvate carboxykinase in tomato fruit

Phosphoenolpyruvate carboxykinase (PEPCK) is present in ripening tomato fruits. A cDNA encoding PEPCK was identified from a PCR-based screen of a cDNA library from ripe tomato fruit. The sequence of the tomato PEPCK cDNA and a cloned portion of the genomic DNA shows that the complete cDNA sequence contains an open reading frame encoding a peptide of 662 amino acid residues in length and predicts a polypeptide with a molecular mass of 73.5 kDa, which corresponds to that detected by western blotting. Only one PEPCK gene was identified in the tomato genome. PEPCK is shown to be present in the pericarp of ripening tomato fruits by activity measurements, western blotting and mRNA analysis. PEPCK abundance and activity both increased during fruit ripening, from an undetectable amount in immature green fruit to a high amount in ripening fruit. PEPCK mRNA, protein and activity were also detected in germinating seeds and, in lower amounts, in roots and stems of tomato. The possible role of PEPCK in the pericarp of tomato fruit during ripening is discussed.     

A role for glutamate decarboxylase during tomato ripening: the characterisation of a cDNA encoding a putative glutamate decarboxylase with a calmodulin-binding site

A tomato fruit cDNA library was differentially screened to identify mRNAs present at higher levels in fruit of the tomato ripening mutant rin (ripening inhibitor). Complete sequencing of a unique clone ERT D1 revealed an open reading frame with homology to several glutamate decarboxylases. The deduced polypeptide sequence has 80% overall amino acid sequence similarity to a Petunia hybrida glutamate decarboxylase (petGAD) which carries a calmodulin-binding site at its carboxyl terminus and ERT D1 appears to have a similar domain. ERT D1 mRNA levels peaked at the first visible sign of fruit colour change during normal tomato ripening and then declined, whereas in fruit of the ripening impaired mutant, rin, accumulation of this mRNA continued until at least 14 days after the onset of ripening. This mRNA was present at much lower levels in other tissues, such as leaves, roots and stem, and was not increased by wounding. Possible roles for GAD, and its product -aminobutyric acid (GABA) in fruit, are discussed.     

Isolation and characterization of acid invertase cDNA clone in hot pepper (Capsicum annuum L.) fruits

An acid invertase (EC 3.2.1.26.) cDNA clone,CaAIV-18, was isolated from the red pericarp cDNA library of the hot pepper (Capsicum annuum L.) fruit. TheCaAIV-18 clone has 2223 nucleotides and one open reading frame encoding 641 amino acid residues. Analysis of deduced amino acid sequences reveals thatCaAIV-18 has a 24-amino acid transmembrane anchor region in its N-terminal, implying acid invertase in hot pepper may be localized in the membrane and not in the cytosol. This clone showed high homology to tomato acid invertase,Aiv1, in nucleotide and deduced amino acid sequences. In the Southern blot analysis, this clone proved to exist as single or low copy numbers on the genome of hot pepper. The clones had two well-conserved regions which appears in acid invertase of other plant species (eg. tomato,Arabidopsis, etc.) and yeasts. During fruit development,CaAIV-18 was expressed preferentially in the ripe red stage.     

Invertase Activity and its Relation to Hexose Accumulation in Potato Tubers

Hexose accumulation was shown to occur in freshly harvestedmature potato tubers (Solanum tuberosum L.) both after storageat 10 ?C and when subsequently transferred to low temperature(3 ?C) storage. In general, changes in hexoses and sucrose werefound to be related to changes in acid invertase activity. Totalacid invertase activity (i.e. assayed after destroying the endogenousinvertase inhibitor present in the extracts) generally reflectedsugar changes more closely than did basal activity (i.e. assayedwith the inhibitor present). There was no evidence of a specificalkaline invertase. A comparison of the temperature responsesof cultivar Record with that of two SCRI² clones demonstrateddistinct genotypic variation in the extent of hexose accumulation.However, these differences were not always reflected by genotypicdifferences in total invertase activity. Key words: Invertase inhibitor, glucose, fructose, sucrose     

Antisense acid invertase (TIV1) gene alters soluble sugar composition and size in transgenic tomato fruit.

Invertase (beta-fructosidase, EC 3.2.1.26) hydrolyzes sucrose to hexose sugars and thus plays a fundamental role in the energy requirements for plant growth and maintenance. Transgenic plants with altered extracellular acid invertase have highly disturbed growth habits. We investigated the role of intracellular soluble acid invertase in plant and fruit development. Transgenic tomato (Lycopersicon esculentum Mill.) plants expressing a constitutive antisense invertase transgene grew identically to wild-type plants. Several lines of transgenic fruit expressing a constitutive antisense invertase gene had increased sucrose and decreased hexose sugar concentrations. Each transgenic line with fruit that had increased sucrose concentrations also had greatly reduced levels of acid invertase in ripe fruit. Sucrose-accumulating fruit were approximately 30% smaller than control fruit, and this differential growth correlated with high rates of sugar accumulation during the last stage of development. These data suggest that soluble acid invertase controls sugar composition in tomato fruit and that this change in composition contributes to alterations in fruit size. In addition, sucrose-accumulating fruit have elevated rates of ethylene evolution relative to control fruit, perhaps as a result of the smaller fruit size of the sucrose-accumulating transgenic lines.     

Effects of Diurnal Changes in the Salinity of the Nutrient Solution on the Accumulation of Calcium by Tomato Fruit

Tomato fruit grown in diurnally fluctuating salinities (8 mScm^–1 during the day and 3 mS cm^–1 at night; 8/3mS cm^–1), accumulated the same amount of dry matter andmagnesium (Mg) as those in constant 3 or 8 mS cm^–1, butan intermediate amount of calcium (Ca). Raising the salinityof the nutrient solution by enriching with macronutrients orby adding NaCl had similar effects. The uptake of ⁴⁵Ca by tomato plants during the day was greaterthan at night and was reduced by salinity in both periods. Whilethe uptake of ⁴⁵Ca by 8/3 mS plants at night was similar tothat of 3 mS plants, the daily uptake was less than that in3 and 5.5 mS plants. The Ca content of tomato fruit increased with truss number at3 and 5.5 mS cm^–1 but not at 8/3 and 8 mS cm^–1.Within the same truss, the distal fruit had a lower Ca contentbut higher Mg content than the proximal fruit. The reductionin Ca content of the distal fruit at 8/3 mS cm^–1 was similarto that at 5.5 mS cm^–1. The Ca content of the tissue atthe distal end of the 8/3 mS fruit was lower than that of the5.5 mS fruit. Similarly, the distribution of ⁴⁵Ca to the distalhalf of the detached 8/3 mS fruit was less than that of 5.5mS fruit. A reduced uptake and inadequate distribution of Cato the truss and to the distal end of the 8/3 mS fruit werethe main causes of these differences. Lycopersicon esculentum(Mill.), tomato, fruit, calcium, magnesium, diurnal salinity