Regulation of Ethylene Biosynthesis in Avocado Fruit during Ripening

Preclimacteric avocado (Persea americana Mill.) fruits produced very little ethylene and had only a trace amount of l-aminocyclopropane-1-carboxylic acid (ACC) and a very low activity of ACC synthase. In contrast, a significant amount of l-(malonylamino)cyclopropane-1-carboxylic acid (MACC) was detected during the preclimacteric stage. In harvested fruits, both ACC synthase activity and the level of ACC increased markedly during the climacteric rise reaching a peak shortly before the climacteric peak. The level of MACC also increased at the climacteric stage. Cycloheximide and cordycepin inhibited the synthesis of ACC synthase in discs excised from preclimacteric fruits. A low but measurable ethylene forming enzyme (EFE) activity was detected during the preclimacteric stage. During ripening, EFE activity increased only at the beginning of the climacteric rise. ACC synthase and EFE activities and the ACC level declined rapidly after the climacteric peak. Application of ACC to attached or detached fruits resulted in increased ethylene production and ripening of the fruits. Exogenous ethylene stimulated EFE activity in intact fruits prior to the increase in ethylene production. The data suggest that conversion of S-adenosylmethionine to ACC is the major factor limiting ethylene production during the preclimacteric stage. ACC synthase is first synthesized during ripening and this leads to the production of ethylene which in turn induces an additional increase in ACC synthase activity. Only when ethylene reaches a certain level does it induce increased EFE activity.     

Glucose Inhibits ACC Oxidase Activity and Ethylene Biosynthesis in Ripening Tomato Fruit

Experiments were carried out to evaluate the effect of glucose on ripening and ethylene biosynthesis in tomato fruit (Lycopersicon esculentum Mill.). Fruit at the light-red stage were vacuum infiltrated with glucose solutions post-harvest and changes in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC, ACC oxidase, and ethylene production monitored over time. ACC oxidase activity was also measured in pericarp discs from the same fruits that were treated either with glucose, fructose, mannose, or galactose. While control fruit displayed a typical peak of ethylene production, fruit treated with glucose did not. Glucose appeared to exert its effect on ethylene biosynthesis by suppressing ACC oxidase activity. Fructose, mannose, and galactose did not inhibit ACC oxidase activity in tomato pericarp discs. Glucose treatment inhibited ripening-associated colour development in whole fruit. The extent of inhibition of colour development was dependent upon the concentration of glucose. These results indicate that glucose may play an important role in ethylene-associated regulation of fruit ripening.     

Interrelationship of Polyamine and Ethylene Biosynthesis during Avocado Fruit Development and Ripening

Concentrations of polyamines (PA) and the activities of the PA-synthesizing enzymes ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) extracted from the mesocarp tissue of avocado (Persea americana Mill, cv `Simmonds') fruits at different stages of development were compared with DNA content and the activities of 5′-methylthioadenosine (MTA) nucleosidase and 5-methylthioribose (MTR) kinase. Putrescine, spermidine, and spermine were at their peak concentrations during the early stages of fruit development (362, 201, and 165 nanomoles per gram fresh weight, respectively, at 15 days from full bloom), then declined to 30% or less at full maturity. Agmatine showed only a slight change in concentration throughout the fruit development. The activity of ODC, which was low during flowering (8 nmoles per milligram protein per hour), increased more than threefold during the first 2 months then declined at the later stages of fruit development, while ADC activity showed only a slight increase. DNA content followed a similar pattern of change as that of PA and ODC. The decline in DNA and ODC activity suggest a lack of correlation between cell proliferation and PA at the later stages of the avocado fruit development. It is also possible that any cell division which may take place during the latter stages of the fruit development is not sufficient to alter the pattern of PA biosynthesis. MTA nucleosidase and MTR kinase activities increased during the first 15 days of fruit development followed by a slight decline at 60 and 90 days from full bloom. At 120 days (1 month before full maturity) both MTA nucleosidase and MTR kinase activities increased significantly. During maximum ethylene synthesis, MTA nucleosidase and MTR kinase activities were approximately fivefold and eightfold, respectively, higher than during maximum PA synthesis. The data indicate that the MTA molecules produced during PA and ethylene synthesis are actively metabolized to MTR and MTR-1-P, the two intermediates involved in the regeneration of S-adenosylmethionine from MTA. The data also suggest that the PA and ethylene biosynthetic pathways are not actively competing for the same substrates at any given stage of the avocado fruit development and ripening.     

5'-Methylthioadenosine Nucleosidase and 5-Methylthioribose Kinase Activities and Ethylene Production during Tomato Fruit Development and Ripening

5′-Methylthioadenosine (MTA) nucleosidase and 5-methylthioribose (MTR) kinase activities were measured in crude extracts of tomato fruits (Lycopersicon esculentum Mill cv Rutgers) during fruit development and ripening. The highest activity of MTA nucleosidase (1.2 nanomoles per milligram protein per minute) was observed in small green fruits. The activity decreased during ripening; at the overripe stage only 6.5% of the peak activity remained. MTR kinase activity was low at the small green stage and increased thereafter until it reached peak activity at the breaker stage (0.7 nanomoles per milligram protein per minute) followed by a sharp decline at the later stages of fruit ripening. 1-Amino-cyclopropane-1-carboxylic acid (ACC) levels peaked at the red stage, while ethylene reached its highest level at the light-red stage. Several analogs of MTA and MTR were tested as both enzyme and ethylene inhibitors. Of the MTA analogs examined for their ability to inhibit MTA nucleosidase, 5′-chloroformycin reduced enzyme activity 89%, whereas 5′-chloroadenosine, 5′-isobutylthioadenosine, 5′-isopropylthioadenosine, and 5′-ethylthioadenosine inhibited the reaction with MTA by about 40%. 5′-Chloroformycin and 5′-chloroadenosine inhibited ethylene production over a period of 24 hours by about 64 and 42%, respectively. Other analogs of MTA were not effective inhibitors of ethylene production, whereas aminoethoxyvinylglycine showed a 34% inhibition over the same period of time. Of the MTR analogs tested, 5-isobutylthioribose was the most effective inhibitor of both MTR-kinase (41%) and ethylene production (35%).     

AUXIN RESPONSE FACTOR 2 Intersects Hormonal Signals in the Regulation of Tomato Fruit Ripening

The involvement of ethylene in fruit ripening is well documented, though knowledge regarding the crosstalk between ethylene and other hormones in ripening is lacking. We discovered that AUXIN RESPONSE FACTOR 2A (ARF2A), a recognized auxin signaling component, functions in the control of ripening. ARF2A expression is ripening regulated and reduced in the rin, nor and nr ripening mutants. It is also responsive to exogenous application of ethylene, auxin and abscisic acid (ABA). Over-expressing ARF2A in tomato resulted in blotchy ripening in which certain fruit regions turn red and possess accelerated ripening. ARF2A over-expressing fruit displayed early ethylene emission and ethylene signaling inhibition delayed their ripening phenotype, suggesting ethylene dependency. Both green and red fruit regions showed the induction of ethylene signaling components and master regulators of ripening. Comprehensive hormone profiling revealed that altered ARF2A expression in fruit significantly modified abscisates, cytokinins and salicylic acid while gibberellic acid and auxin metabolites were unaffected. Silencing of ARF2A further validated these observations as reducing ARF2A expression let to retarded fruit ripening, parthenocarpy and a disturbed hormonal profile. Finally, we show that ARF2A both homodimerizes and interacts with the ABA STRESS RIPENING (ASR1) protein, suggesting that ASR1 might be linking ABA and ethylene-dependent ripening. These results revealed that ARF2A interconnects signals of ethylene and additional hormones to co-ordinate the capacity of fruit tissue to initiate the complex ripening process.     

Differential Expression of Genes Involved in the Biosynthesis and Perception of Ethylene during Ripening of Passion Fruit (Passiflora edulis Sims)

An increase in the enzyme activity of 1-aminocyclopropane-1-carboxylicacid (ACC) synthase and ACC oxidase induces the evolution ofethylene during the ripening of passion fruit. A much higherlevel of ethylene is produced in arils than in seeds or peelsduring ripening. The pattern of expression of two ACC synthasegenes (PE-ACS1 and PE-ACS2), one ACC oxidase gene (PE-ACO1),and two ethylene receptor genes (PE-ETR1 and PE-ERS1) revealedthat the expression of these genes is differentially regulated.Expression of PE-ACS1 and PE-ACO1 was enhanced during ripeningand after ethylene treatment. However, prominent expressionof PE-ACS1 was delayed compared to that of PE-ACO1. Much largerquantities of PE-ACS1 mRNA and PE-ACO1 mRNA were seen in arilsthan in seeds; this corresponds well with an increase in theamount of ethylene produced by the plant tissue itself. Thelevel of PE-ACS2 mRNA was detectable in arils of the preclimactericfruit, although it decreased during ripening. These resultssuggest that expression of PE-ACS1 and PE-ACO1 is required toincrease the activity of ethylene biosynthetic enzymes duringripening. The level of expression of PE-ETR1 and PE-ERS1 didnot significantly change over the course of ripening; however,the mRNA levels of PE-ETR1 and PE-ERS1 were much higher in arilsthan in seeds. ⁴Present address: Center forMolecular Genetics Research, Shizuoka University, Shizuoka, 422-8529 Japan.     

Carboxypeptidase Activity of Tomato Fruit during the Ripening Process and Some Enzymatic Properties

Carboxypeptidase activity of tomato fruit reached a maximum at an early period of ripening. During storage of the fruit at 25°C, the enzyme activity decreased, accompanied by a fall of the pH value of the sap.

The enzyme was apparently localized in the soluble fraction, as determined by differential centrifugation.

The enzyme was optimally active at pH 5.0 ~ 5.5, was most stable at pH 4.5 ~ 6.5, and was strongly inhibited by DFP and HgCl₂, but not by EDTA and 1,10-phenanthroIine. Z-dipeptides containing arginine, proline and several neutral amino acids were hydrolyzed by the enzyme.

The similarity of the enzymatic properties of the present enzyme to those of other plant carboxypeptidases and pig kidney cathepsin A is also discussed.     

The Synthesis of Ethylene in Melon Fruit during the Early Stage of Ripening

The levels of mRNA and polypeptide for a 1-aminocyclopropane-1-carboxylate(ACC) oxidase were studied to identify the tissues in whichthe synthesis of ethylene first occurs during the initial stageof ripening. RNA and immunoblot analysis showed that the levelsof the mRNA and polypeptide for ACC oxidase were very low inunripe fruit. They first became detectable in the placentaltissue at the pre-climacteric stage, and then their levels increasedin the mesocarp tissue during the climacteric increase in theproduction of ethylene. Two mRNAs for ACC synthase (transcribedfrom ME-ACS1 and ME-ACS2) were detected in the placental tissueand seeds at the pre-climacteric stage, but only the level ofME-ACS1 mRNA, which has been characterized as the mRNA for awound-inducible ACC synthase, increased in mesocarp, placentaltissues and seeds during ripening. The level of ME-ACS2 mRNAthat was isolated from etiolated seedlings of melon, did notchange markedly during ripening. These results suggest thatthe central region of melon fruit (placental tissue and seeds)plays a major role in the production of ethylene during theearly stage of ripening. ³These three authors made equal contribution to this study.     

Ethylene Production,Peroxidase Activity and the Change of Peroxidase Isozyme during the Ripening of Tomato Fruits

Climacteric rise, ethylene production, peroxidase activity and its isozyme and their interrelationships during the ripening of tomato fruits have been studied. It was found' that there was parallelism between ethylene production and climacteric rise. The climacteric rise of tomato fruits was hastened by ethylene applied at the mature green stage. The ethylene production was inhibited by low oxygen and high carbon dioxide partial pressure. The peroxidase activity in the tomato fruits appeared to be different at three stages, higher in the half red fruits and lower in both green mature and fully red fruits. This activity was increased by ethylene and decreased by lower partial pres- sure of oxygen. The peroxidase isozymes sppeared also different at different stages of ripening. There were 4 bands in young fruits, 3 in green mature fruits, 5 in half red fruits and 3 in fully red fruits. After the application of ethylene to the tomato fruits, there appear one new band of peroxidase isozyme.     

Changes in Structural Features of Free N-Glycan and Endoglycosidase Activity during Tomato Fruit Ripening

In developing plants, free N-glycans occur ubiquitously at micromolar concentrations. Such oligosaccharides have been proposed to be signaling molecules in plant development. As a part of a study to elucidate the physiological roles of de-N-glycosylation machinery involved in fruit ripening, we analyzed changes in the amounts and structural features of free N-glycans in tomato fruits at four ripening stages. The amount of high-mannose type free N-glycans increased significantly in accordance with fruit ripening, and the relative amounts of high-molecular size N-glycans, such as Man_8-9GlcNAc₁, became predominant. These observations suggest that the de-N-glycosylation machinery, including endo-β-N-acetylglucosaminidase (ENGase) activity, is stimulated in the later stages of fruit ripening. But contrary to expectation, we found that total ENGase activities in the tomato fruits did not vary significantly with the ripening process, suggesting that ENGase activity must be maintained at a certain level, and that the expression of α-mannosidase involved in the clearance of free N-glycans decreases during tomato fruit ripening.     

Application of a Rapid and Sensitive Method for Hormonal and Vitamin E Profiling Reveals Crucial Regulatory Mechanisms in Flower Senescence and Fruit Ripening

Knowledge of ripeness and regulation of postharvest processes is an important tool to prevent loss of commercial value in both fruit and cut flower markets. The joint analysis of hormones and vitamin E levels can reveal complex interactions between hormones and oxidative stress as key regulators of postharvest processes. Profiling of both groups of metabolic compounds was performed during the ripening of non-climacteric fruits (red raspberry, Rubus idaeus L.) and senescence of ethylene-insensitive flowers (Dutch Iris, Iris x hollandica L.). After an initial extraction of the sample, without further purification steps, the hormonal profile was analyzed by UPLC-MS/MS and vitamin E levels were measured by HPLC. This methodological approach was very fast and had enough sensitivity for the analysis of small samples. Raspberry fruit maturation was characterized by a decline of cytokinin levels [zeatin, zeatin riboside, 2-isopentenyl adenine, and isopentenyl adenosine (Z, ZR, 2-iP, and IPA, respectively)] and gibberellins (GA₁ in particular). Exogenous application of ABA prevented δ-tocopherol loss during fruit ripening. Iris floral senescence was also under strict hormonal control, also mediated by cytokinins and gibberellins. Z, ZR, 2-iP, GA₉, and GA₂₄ levels decreased in inner tepals, whereas the level of IPA decreased in style-merged-to-stigma tissues, thus suggesting tissue-specific roles for different hormones. α-Tocopherol levels decreased during senescence of inner tepals, hence suggesting enhanced oxidative stress. In conclusion, the rapid and sensitive hormonal and vitamin E profiling presented here can help in understanding the key physiological processes underlying fruit ripening and floral senescence.     

Effect of Heat Treatment on the Development of Polygalacturonase Activity in Tomato Fruit during Ripening

When mature green tomato fruits are stored at 22?C for 30 days,they ripen normally and soften, but if they are kept at 33?Cfor 15 days (heat treatment), then stored at 22?C they do notsoften. The effect of heat treatment on the development of polygalacturonase(PG, EC 3.2.1.15 [EC] ) activities in tomato fruits during storagetherefore was studied. When mature green tomato fruits werestored at 22?C, PG activity, which had not been detectable inthe fruits, appeared as the color changed and increased dramaticallythereafter. PG activity, however, did not appear during heattreatment. When heat-treated fruits were transferred to 22?C,PG activity appeared after a 6-day lag period and increasedduring the next 30 days at 22?C to about 15% of the value detectedin ripe tomato fruits. The PG in ripe tomato fruits was composed of two isoenzymesthat had different mol wts. A high molecular form (PG-1, molwt 100K) appeared during the early stage of ripening and a lowmolecular form (PG-2, mol wt 44K) a little later. PG-2 increasedvigorously during ripening and eventually accounted for mostof the enzyme activity in the ripe fruits. Only a single isoenzyme(Y-PG, mol wt 100K), however, was detected in heat-treated tomatofruits stored at 22?C for 30 days. PG-1 and Y-PG gave the samemol wt on Sephacryl S-200 gel nitration, but could be separatedby Toyopearl HW-55 F gel filtration. (Received October 31, 1983; Accepted February 20, 1984)