Reduction of allergenic proteins by the effect of the ripening inhibitor (rin) mutant gene in an F1 hybrid of the rin mutant tomato

The ripening inhibitor (rin) mutant tomato yields non-ripening fruit, and the rin hybrid fruit (RIN/rin) shows an intermediate phenotype between the wild and mutant fruit, that is, red-ripe and extended shelf life. We found by a microarray analysis that the genes encoding possible allergenic proteins were expressed at a significantly lower level in the rin hybrid fruit than in the wild-type fruit. These allergenic proteins, which were beta-fructofuranosidase and polygalacturonase 2A (PG-2A), were confirmed to accumulate at a lower level in the rin hybrid fruit than in the wild-type fruit. The immunoglobulin E (IgE) in serum from a tomato-allergic patient showed lower reactivity to the extract of the rin hybrid fruit than to that of the wild fruit. These results suggest that the rin gene has the potential to regulate allergen accumulation in tomato fruit.     

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.     

Ethylene receptor degradation controls the timing of ripening in tomato fruit

Fruit ripening in tomato requires the coordination of both developmental cues and the phytohormone ethylene. The multigene ethylene receptor family has been shown to negatively regulate ethylene signal transduction and suppress ethylene responses. Here we demonstrate that reduction in the levels of either of two family members, LeETR4 or LeETR6, causes an early-ripening phenotype. We provide evidence that the receptors are rapidly degraded in the presence of ethylene, and that degradation probably occurs through the 26S proteasome-dependent pathway. Ethylene exposure of immature fruits causes a reduction in the amount of receptor protein and earlier ripening. The results are consistent with a model in which receptor levels modulate timing of the onset of fruit ripening by measuring cumulative ethylene exposure.     

Ethylene regulation of fruit ripening: Molecular aspects

Progress in ethylene regulating fruit ripening concerning itsperception and signal transduction and expression of ACC synthaseand ACC oxidase genes is reviewed. ACC synthase and ACC oxidasehave been characterized and their genes cloned from various fruittissues. Both ACC synthase and ACC oxidase are encoded bymultigene families, and their activities are associated withfruit ripening. In climacteric fruit, the transition toautocatalytic ethylene production appears to be due to a seriesof events in which ACC sythase and ACC oxidase genes have beenexpressed developmentally. Differential expression of ACCsynthase and ACC oxidase gene family members is probably involvedin such a transition that ultimately controls the onset of fruitripening.In comparison to ACC synthase and ACC oxidase, less is knownabout ethylene perception and signal transduction because of thedifficulties in isolating and purifying ethylene receptors orethylene-binding proteins using biochemical methods. However, theidentification of the Nr tomato ripening mutant as anethylene receptor, the applications of new potent anti-ethylenecompounds and the generation of transgenic fruits with reducedethylene production have provided evidence that ethylenereceptors regulate a defined set of genes which are expressedduring fruit ripening. The properties and functions of ethylenereceptors, such as ETR1, are being elucidated.Application of molecular genetics, in combination withbiochemical approaches, will enable us to better understand theindividual steps leading from ethylene perception and signaltransduction and expression of ACC synthase and ACC oxidase genefamily member to the physiological responses.     

Polyuronide solubilization during ripening of normal and mutant tomato fruit

The amount and molecular size of soluble polyuronide extractable from ripening tomatoes is markedly affected by residual enzyme activity. The efficacy of phenol-acetic acid-water treatment to remove this residual activity is demonstrated. Data obtained using treated wall preparations confirms that there is an increase in soluble polyuronide during normal ripening and that this also occurs in the ‘Never-ripe’ mutant, and to a lesser degree in the ‘ripening-inhibitor’ mutant. However, changes in the molecular size of this polyuronide during normal ripening were not as extensive as previously reported and few changes were apparent in either of the mutants.Measurements were also made of polygalacturonase (EC 3.2.1.15) and pectinesterase (EC 3.1.1.11) activity during ripening. The level of polygalacturonase activity does not appear to correlate with the amount of soluble polyuronide released, but may be related to the extent of depolymerisation. No relationship was apparent between the level of pectinesterase and either soluble polyuronide released or depolymerization.     

1-Deoxy-D-xylulose 5-phosphate reductoisomerase and plastid isoprenoid biosynthesis during tomato fruit ripening

The recently discovered 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of plastid isoprenoids (including carotenoids) is not fully elucidated yet despite its central importance for plant life. It is known, however, that the first reaction completely specific to the pathway is the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) into MEP by the enzyme DXP reductoisomerase (DXR). We have identified a tomato cDNA encoding a protein with homology to DXR and in vivo activity, and show that the levels of the corresponding DXR mRNA and encoded protein in fruit tissues are similar before and during the massive accumulation of carotenoids characteristic of fruit ripening. The results are consistent with a non-limiting role of DXR, and support previous work proposing DXP synthase (DXS) as the first regulatory enzyme for plastid isoprenoid biosynthesis in tomato fruit. Inhibition of DXR activity by fosmidomycin showed that plastid isoprenoid biosynthesis is required for tomato fruit carotenogenesis but not for other ripening processes. In addition, dormancy was reduced in seeds from fosmidomycin-treated fruit but not in seeds from the tomato yellow ripe mutant (defective in phytoene synthase-1, PSY1), suggesting that the isoform PSY2 might channel the production of carotenoids for abscisic acid biosynthesis. Furthermore, the complete arrest of tomato seedling development using fosmidomycin confirms a key role of the MEP pathway in plant development.     

Enzyme activities in mitochondria isolated from ripening tomato fruit

Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.     

Ethylene evolution during ripening of detached tomato fruit: Its relation with polyamine metabolism

Ethylene and polyamine metabolism, both sharing a common precursor, S-adenosylmethionine (SAM), were investigated during detached tomato (Lycopersicon esculentum Mill. nothovar F1 Lorena) fruit ripening. Putrescine (PUT) was found to be the major polyamine in the fruits, always over 100 nmols/g FW, while spermidine (SPD) was between 7% and 3% of the level of PUT. Spermine (SPM) was not detected at any stage of ripening. The level of PUT and SPD, did not change significantly during ripening in spite of the almost continuous synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC), the ethylene precursor, and only at the last stage of ripening was a drastic decrease in SPD content observed. The results obtained show that the onset of ACC synthesis and its accumulation within the tissue is not a consequence of a decrease in SPD synthesis.     

Immunological properties of β-fructofuranosidase from ripening tomato fruit

The amount of tomato fruit β-fructofuranosidase extractable from the cell walls during ripening parallelled the changes in activity of the enzyme. Using the techniques of radioimmunoassay, double immunodiffusion analysis and immunotitration, no differences in immunological properties of β-fructofuranosidase between the various stages of fruit ripening were detected.     

Immunocytolocalization of polygalacturonase in ripening tomato fruit

Using tissue blotting and immunocytolocalization, we have investigated the appearance and accumulation of polygalacturonase (PG) during tomato (Lycopersicon esculentum Mill.) fruit ripening. Results show that PG first appears in the collumella region followed by sequential appearance in the exopericarp and endopericarp, respectively. Detectable levels of PG were not present in the locular material containing seeds. This result indicates that PG synthesis initiates at the central collumella region of tomato fruit during ripening.     

Abscisic acid biosynthesis and catabolism and their regulation roles in fruit ripening

Abscisic acid (ABA) plays a series of significant physiology roles in higher plants including but not limited to promote bud and seed dormancy, accelerate foliage fall, induce stomatal closure, inhibit growth and enhance resistance. Recently, it has been revealed that ABA also has an important regulator role in the growth, development and ripening of fruit. In higher plants ABA is produced from an indirect pathway from the cleavage products of carotenoids. The accumulation of endogenous ABA levels in plants is a dynamic balance controlled by the processes of biosynthesis and catabolism, through the regulation of key ABA biosynthetic gene and enzyme activities. It has been hypothesized that ABA levels could be part of the signal that trigger fruit ripening, and that ABA may play an important role in the regulation of ripening and senescence of both non-climacteric and climacteric fruit. The expensive costs of natural ABA and labile active ABA for its chemical synthesis limit its application in scientific research and agricultural production. These findings that ABA has various of important roles in the regulation of growth and development, quality formation, coloring and softening, ripening and senescence of fruit, are providing opportunities and challenges for Horticultural Science. This is to elucidate the specific mechanism of response and biosynthesis, signal transduction, and receptor recognition of ABA in fruit, employing comprehensive research methods, such as molecular biology, plant physiology and molecular genetics. Further and more in-depth research about ABA has a great, realistic significance for knowing the mechanisms behind the process of fruit ripening.     

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.     

Wound ethylene and 1-aminocyclopropane-1-carboxylate synthase in ripening tomato fruit

Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ACC-synthase activity were compared in intact and wounded tomato fruits (Lycopersicon esculentum Mill.) at different ripening stages. Freshly cut and wounded pericarp discs produced relatively little ethylene and had low levels of ACC and of ACC-synthase activity. The rate of ethylene synthesis, the level of ACC and the activity of ACC synthase all increased manyfold within 2 h after wounding. The rate of wound-ethylene formation and the activity of wound-induced ACC synthase were positively correlated with the rate of ethylene production in the intact fruit. When pericarp discs were incubated overnight, wound ethylene synthesis subsided, but the activity of ACC synthase remained high, and ACC accumulated, especially in discs from ripe fruits. In freshly harvested tomato fruits, the level of ACC and the activity of ACC synthase were higher in the inside parts of the fruit than in the pericarp. When wounded pericarp tissue of green tomato fruits was treated with cycloheximide, the activity of ACC synthase declined with an apparent half life of 30–40 in. The activity of ACC synthase in cycloheximide-treated, wounded pericarp of ripening tomatoes declined more slowly.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid