A role for jasmonates in climacteric fruit ripening

Jasmonates are a class of oxylipins that induce a wide variety of higher-plant responses. To determine if jasmonates play a role in the regulation of climacteric fruit ripening, the effects of exogenous jasmonates on ethylene biosynthesis and color, as well as the endogenous concentrations of jasmonates were determined during the onset of ripening of apple (Malus domestica Borkh. cv. Golden Delicious) and tomato (Lycopersicon esculentum Mill. cv. Cobra) fruit. Transient (12 h) treatment of pre-climacteric fruit discs with exogenous jasmonates at low concentration (1 or 10 μM) promoted ethylene biosynthesis and color change in a concentration-dependent fashion. Activities of both 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase and ACC synthase were stimulated by jasmonate treatments in this concentration range. The endogenous concentration of jasmonates increased transiently prior to the climacteric increase in ethylene biosynthesis during the onset of ripening of both apple and tomato fruit. The onset of tomato fruit ripening was also preceded by an increase in the percentage of the cis-isomer of jasmonic acid. Inhibition of ethylene action by diazocyclopentadiene negated the jasmonate-induced stimulation of ethylene biosynthesis, indicating jasmonates act at least in part via ethylene action. These results suggest jasmonates may play a role together with ethylene in regulating the early steps of climacteric fruit ripening. Received: 14 August 1997 / Accepted: 4 October 1997     

Regulation of climacteric respiration in ripening avocado fruit

Ripening of avocado fruit is associated with a dramatic increase in respiration. In vivo³¹P nuclear magnetic resonance spectroscopy revealed large increases in ATP levels accompanying the increase in respiration. Both glycolytic enzymes, phosphofructokinase, and pyrophosphate: fructose-6-phosphate phosphotransferase were present in avocado fruit with the latter activity being highly stimulated by fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate levels increased approximately 90% at the onset of ripening, suggesting that the respiratory increase in ripening avocado fruit may be regulated by the activation of pyrophosphate:fructose-6-phosphate phosphotransferase by an increase in fructose 2,6-bisphosphate.     

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.     

Molecular genetics of tomato fruit ripening

Tomato ripening is an excellent system for studying control of gene expression in plants. A multiplicity of well-defined biochemical and genetic changes occur in a precise sequence, regulated by a gaseous hormone. The generation of targeted mutations using sense and antisense genes provides a means of manipulating endogenous gene expression, both for answering fundamental questions and for crop improvement.     

Synthesis of polygalacturonase during tomato fruit ripening

The cell wall degrading enzyme polygalacturonase (E.C. 3.2.1.15) is not detectable in green tomatoes (Lycopersicon esculentum Mill). Activity appears at the onset of ripening and in ripe fruit it is one of the major cell-wall-bound proteins. Radioimmunoassay results, employing an antibody against purified polygalacturonase, suggest that during ripening the enzyme is synthesised de novo. Radioimmunoassay data also show that the low level of polygalacturonase in Never ripe mutants and the lack of activity in ripening inhibitor mutants can be correlated to the levels of immunologically detectable polygalacturonase protein.Abbreviations PG polygalacturonase - Nr Never ripe mutation - rin ripening inhibitor mutation     

Effect of salinity on tomato fruit ripening

Tomato (Lycopersicon esculentum Mill) plants from various cultivars growing on half-strength Hoagland solution were exposed at anthesis to 3 or 6 grams per liter NaCl. Salinity shortened the time of fruit development by 4 to 15%. Fruits of salt-treated plants were smaller and tasted better than did fruits of control plants. This result was obtained both for ripe fruits tested on the day of picking and for those picked at 100% development and allowed to ripen at room temperature for 9 days. Percentage of dry weight, total soluble solids, and titratable acidity; content of reducing sugars, Cl⁻, Na⁺, and various pericarp pigments; and electrical conductivity of the juice were higher in fruits of saline-treated plants than they were in those of control plants, while the pH was lower. Ethylene and CO₂ evolution rates during ripening; as well as the activities of pectin methyl esterase, polymethylgalacturonase, and polygalacturonase; were also higher in fruits of the saline-treated plants. The treatment with 6 grams per liter NaCl shortened the fruit shelf life considerably.     

Gentiobiose: a novel oligosaccharin in ripening tomato fruit

Two neutral disaccharides, gentiobiose [beta- D-Glc p-(1-->6)- D-Glc] and nigerose [alpha- D-Glc p-(1-->3)- D-Glc], were detected in tomato ( Lycopersicon esculentum Mill.) pericarp and locule. Gentiobiose was present in the locule of green fruit and ripe fruit at 0.88 and 5.8 micro mol (kg fresh weight)(-1), respectively. When vacuum-infiltrated into green tomato fruit, exogenous gentiobiose (50 or 200 micro g per fruit) hastened the initiation of ripening (as judged by colour change) by 1-3 days relative to fruit that were infiltrated with glucose or isomaltose. Nigerose plus gentiobiose was particularly effective, but nigerose alone had no significant effect. The endogenous disaccharides were found to be present in the apoplastic fluid of the fruit, compatible with a proposed intercellular signalling role. The origin and metabolic fate of the disaccharides were investigated. Phenolic esters of these disaccharides were not detectable in tomato fruit and it is therefore unlikely that the free disaccharides were formed from a pool of such esters. An alternative possible biosynthetic origin, via transglycosylation, is discussed. When [(14)C]gentiobiose was vacuum-infiltrated into unripe or ripe fruit, the disaccharide remained intact for at least 1 h but was largely degraded within 24 h. The results suggest that gentiobiose is a new, naturally occurring oligosaccharin with a rapid turnover rate.     

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.     

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.     

Behaviour of some mitochondrial enzymes in ripening tomato fruit

The activities of four mitochondrial enzymes were studied in four stages of ripening tomato fruit. The highest enzyme activity was recorded for malate dehydrogenase followed by cytochrome c oxidase. Succinate dehydrogenase and NADH oxidase levels were low and could only be determined in the green stage of the fruit. However, peaks of various enzyme activities coincided in identical mitochondrial fractions on the sucrose density gradient. Moreover, the levels of malate dehydrogenase and cytochrome c oxidase were constant during the ripening process while the other two enzymes, succinate dehydrogenase and NADH oxidase, declined. This might indicate that mitochondria retain some of their essential functions through the ripening process.     

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.     

A histidine decarboxylase-like mRNA is involved in tomato fruit ripening

DNA sequencing of a tomato ripening-related cDNA, TOM 92, revealed an open reading frame with homology to several pyridoxal 5-phosphate histidine decarboxylases, containing the conserved amino acid residues known to bind pyridoxal phosphate and -fluoromethylhistidine, an inhibitor of enzyme activity. TOM 92 mRNA accumulated during early fruit ripening and then declined. Fruit of the ripeningimpaired tomato mutant, ripening inhibitor (rin), did not accumulate TOM 92 mRNA, and its accumulation was not restored by treatment of fruit with ethylene. The TOM 92 mRNA was not detected in tomato leaves and unripe fruit.     

Lipids of ripening tomato fruit and its mitochondrial fraction

The lipid composition of tomato fruit and its mitochondrial fraction were examined at various stages of fruit ripeness. Phosphatidyl choline, phosphatidyl ethanolamine, monogalactosyl diglyceride, digalactosyl diglyceride and phosphatidyl inositol were found to be the major lipids of tomato pericarp at all stages of ripeness. Mitochondrial lipids resembled those of the parent tissue except for the absence of monogalactosyl diglyceride and a greater percentage of diphosphatidyl glycerol and phosphatidic acid. Changes in the lipid-protein ratio of mitochondria were noted with ripening.     

Ethylene production and peroxidase activity during tomato fruit ripening

Peroxidase activity and enzymic production of ethylene werestudied in tomato fruits (Lycopersicon esculentum MILL.) at3 ripeness stages. As the fruit ripens, one isoperoxidase disappears,and 3 new ones are formed. Activity of peroxidase and of ethylene-formingenzyme both increased 3 to 4 times as the fruit ripened. Histochemicalstaining showed that peroxidase is confined to the outermostand innermost layers of the pericarp, the placental tissue andvascular tissues; stained particles were neither mitochondrianor plastids. (Received October 27, 1969; )     

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.