Enzymic hydrolysis of placental cell wall pectins and cell separation in watermelon (Citrullus lanatus) fruits exposed to ethylene

Watermelon [ Citrullus lanatus (Thunb.) Matsum and Nakai, cv. Charleston Gray] fruits were examined to determine the effect of ethylene on cell wall hydrolases. pectin degradation, and cell wall ultrastructure. Enzymic studies showed that activity of polygalacturonase (EC 3.2.1.15) increased in placental tissue following 1 day of ethylene treatment and was 10 times higher after 6 days of treatment. The increase in polygalacturonase activity was accompanied by the appearance in ethanol powders of low-molecular-weight pectic polymers and a decrease in total pectin. The enhanced enzyme activity and decrease in total pectins were observed only in fruits exposed to ethylene. Ultrastructural studies of ethylene-treated tissue revealed an early disintegration of the middle lamella. The onset of wall separation coincided with the first notable increase in polygalacturonase activity. Cell wall of untreated fruit showed no evidence of structural changes. The results indicate that initiation of enzymic activity and cell wall separation in response to ethylene are not characteristic phenomena of normal ripening and senescence in watermelon fruit.     

Dehiscence of Fruit in Oilseed Rape (Brassica nap us L.): II. THE ROLE OF CELL WALL DEGRADING ENZYMES AND ETHYLENE

Pod shatter in oilseed rape is accompanied by the degradationof the cell wall at the site of fruit dehiscence. Cell separationis preceded by an increase in the activity of the hydrolyticenzyme cellulase (ß 1,4-glucanase, E.C. 3.1.2.4 [EC] ),and this rise in enzyme activity is restricted to the dehiscencezone cells. In contrast, the activity of the cell wall degradingenzyme polygalacturonase (E.C. 3.2.1.15 [EC] ) exhibits no correlationeither temporally or spatially with pod dehiscence. An analysisof the ethylene production profile by intact pods during maturationhas revealed the existence of an ethylene climacteric and thisis temporally correlated with the tissue-specific increase incellulase activity. The major site of ethylene production bythe fruit has been identified to be the developing seed. Sincemaintenance of intact pods in ethylene accelerates both thesenescence and dehiscence of the tissue, it is possible thatthis gaseous regulator plays an important role in the processof pod shatter in vivo. Key words: Oilseed rape, Brassica napus, pod shatter, cellulase, polygalacturonase, ethylene     

Characterization of ethylene production in developing strawberry fruit

Ethylene production, ACC content, and ACC oxidase activity were determined in strawberry fruit harvested at different stages of development and in fruit harvested green and developed in vitro in solutions containing sucrose. In fruit harvested at progressive stages of development from green through full ripe, ethylene production and ACC oxidase activity decreased whereas ACC content increased between the white and pink stages. Fruit detached at the green stage and developed to full ripe by immersion of the cut pedicel in sucrose solutions exhibited an increase in ACC content, decreased ethylene production, and no change in ACC oxidase activity. Detached green fruit provided with sucrose containing 0.5 mM silver (STS) had elevated ethylene production and more ACC oxidase activity than did fruit incubated without the silver salt. Green fruit provided with sucrose containing 1 mM ACC showed markedly increased ACC content, ACC oxidase activity, and ethylene production. These increases were noted following 4 days incubation in ACC, and were more pronounced after 11 days, at which time fruit of all treatments had attained a full-ripe stage of development. Calyx tissue exhibited more ACC oxidase activity, less ACC content, and similar ethylene production compared with receptacle tissue. ACC synthase could not be detected in fruit harvested at different developmental stages or in fruit detached and developed in vitro.abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - HQS 8-hydroxyquinoline hemisulfate - SAM S-adenosyl methionine - STS silver thiosulfate     

Polygalacturonase activity and variation in ripening of papaya fruit with tissue depth and heat treatment

Effects of tissue position (viz. outer vs inner mesocarp) and heat treatment (48°C, 20 min) on variations in polygalacturonase (EC 3.2.1.15 and EC 3.2.1.67) activity and ripening of fruits of Carica papaya L. cv. Backcross Solo were investigated. Polygalacturonase activity increased during ripening concomitantly with an increase in tissue softness and soluble polyuronide level. Throughout ripening, inner mesocarp tissue was softer and contained higher polygalacturonase activity than outer mesocarp tissue. Titratable acidity as well as ß-galactosidase (EC 3.2.1.23) activity also increased during ripening; however, unlike polygalacturonase, their level or activity was lower in inner than in outer mesocarp. Ascorbic acid could partially account for the increase in titratable acidity during ripening but contributed very little to the differences in titratable acid levels between outer and inner mesocarp. Heat treatment had no effect on either fruit softness or titratable acidity, but it markedly reduced the increase in ascorbic acid and polygalacturonase activity during ripening. Ripening, as reflected by changes in tissue softness and polygalacturonase activity, progressed outwardly from the interior towards the exterior of the fruit. The effect of heat treatment in suppressing polygalacturonase activity was relatively greater in inner than in outer mesocarp, suggesting that sensitivity of the enzyme to heat treatment may vary with stage of ripeness of the tissue.     

一氧化氮处理对香蕉多聚半乳糖醛酸酶及MaPGs基因表达的影响

为了解多聚半乳糖醛酸酶（PG）在香蕉采后软化中的分子调控机制,采用40 μL/L NO熏蒸处理绿熟期的‘巴西’香蕉果实3 h后,在20℃和相对湿度为85%的条件下贮藏,研究NO对香蕉果实乙烯释放量、硬度、PG活性及MaPGs基因表达的影响。结果表明：NO处理降低了果实乙烯释放量,延缓了果实硬度的下降,抑制了PG的活性;降低了MaPG2、MaPG3和MaPG4基因的表达,延缓了香蕉果实的软化。     

Change in the level of 1-aminochloropropane-1-carbonic acid. Activity of a protein inhibitor of polygalacturonase, intensity of formation of oligouronides in apples during ripening and treatment with haloethane derivatives and aminoethoxyvinylglycine

We studied changes in the intensity of ethylene release and accumulation of 1-aminocyclopropane-1-carboxylic acid during ripening of two apple varieties characterized by various physiological states and treated with halothane derivatives and L-alpha-(2-aminoethoxyvinyl)-glycine. We observed changes in activity of the protein polygalacturonase inhibitor in the fruit tissue and accumulation of oligouronides. The data suggest that pretreatment with the inhibitor of 1-aminocyclopropane-1-carboxylic acid synthase affects ethylene release, accumulation of 1-aminocyclopropane-1-carboxylic acid, activity of the protein polygalacturonase inhibitor, and potential intensity of oligouronide formation in apple fruits and tissues.     

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.     

Cell wall-degrading enzymes and pectin solubility and depolymerization in immature and ripe watermelon (Citrullus lanatus) fruit in response to exogenous ethylene

Watermelon fruit have been shown to be extremely sensitive to exogenous ethylene, exhibiting acute symptoms of whole-fruit softening and placental-tissue water-soaking following short periods of exposure to the gas. This study addressed the firmness, specific activities of cell wall hydrolases, and solubility and molecular mass properties of polyuronides in placental tissue in response to treatment of intact fruit with ethylene. Watermelon fruit were harvested at the immature and full-ripe stages and exposed to 50 µl l⁻¹ ethylene for 6 days at 20°C. The firmness of placental tissue from ethylene-treated ripe and immature fruit decreased nearly 80% during 6 days of ethylene exposure, whereas the firmness of placental tissue from fruit maintained in air remained relatively constant up to day 3 and then decreased slightly (12%) during the following 3 days of storage. Although ethylene treatment in some cases influenced the levels of extractable placental-tissue polygalacturonase (EC 3.2.1.15), pectinmethylesterase (EC 3.2.1.11), and α -(EC 3.2.1.22) and β -galactosidase (EC 3.2.1.23) specific activities, these effects were not observed for fruit of both developmental stages and appeared not to be directly involved in the water–soaking syndrome. Symptoms of water-soaking were accompanied by increases in the levels of water- and CDTA ( trans -1,2-cyclohexanediamine- N,N,N',N' -tetraacetic acid)-soluble polyuronides and significant molecular mass downshifts in polyuronides in both immature and ripe watermelon fruit. Polyuronide depolymerization in ethylene-treated ripe fruit was extensive. The parallel trends of enzyme changes in ethylene- compared with air-treated fruit indicate that extractable enzyme levels are not associated with development of the water-soaking disorder. The potential involvement of membrane dysfunction in the water-soaking phenomenon is discussed.     

Flavor volatilcs, sugars and color development in ripening in vitro-cultured tomato fruit and calyx

Previous studies showed that the developmental program of calyces of a tomato cultivar ( Lycopersicon esculentum , cv. VFNT Cherry) changed in many aspects to that of fruit when cultured in vitro. The calyces turned red, produced ethylene, had increased tissue content of 1-aminocyclopropane-1-carboxylic acid, had increased levels of the mRNA of polygalacturonase and developed ultrastructural changes in their cell walls that were indistinguishable from those of ripe tomato fruit tissue. We report in the present study the synthesis of volatile flavor compounds, changes in sugar concentrations and color development in cultured calyces that are characteristic of ripening tomato fruit. These ripening parameters of in vitro-cultured tomato fruit were also compared to those of fruit grown in the greenhouse.     

Timing of ethylene and polygalacturonase synthesis in relation to the control of tomato fruit ripening

A critical role in the initiation of ripening has been proposed for pectolytic enzymes which are known to be involved in fruit softening. The hypothesis that tomato (Lycopersicon esculentum Mill.) ripening is controlled by the initial synthesis of the cell-wall-degrading enzyme polygalacturonase (EC 3.2.1.15), which subsequently liberates cell-wall-bound enzymes responsible for the initiation of ethylene synthesis and other ripening events, has been examined. A study of kinetics of ethylene evolution and polygalacturonase synthesis by individual fruits in a ripening series, employing an immunological method and protein purification to identify and measure polygalacturonase synthesis, showed that ethylene evolution preceded polygalacturonase synthesis by 20h. Exogenous ethylene stimulated the synthesis of polygalacturonase and other ripening events, when applied to mature green fruit, whereas the maintenance of fruits in a low ethylene environment delayed ripening and polygalacturonase synthesis. It is concluded that enhanced natural ethylene synthesis occurs prior to polygalacturonase production and that ethylene is responsible for triggering polygalacturonase synthesis indirectly. Possible mechanisms for ethylene action are discussed.     

The Tomato Fruit Cell Wall : II. Polyuronide Metabolism in a Nonsoftening Tomato Mutant

A nonsoftening tomato (Lycopersicon esculentum L.) variety, dg, was examined to assess the physiological basis for its inability to soften during ripening. Total uronic acid levels, 18 milligrams uronic acid/100 milligrams wall, and the extent of pectin esterification, 60 mole%, remained constant throughout fruit development in this mutant. The proportion of uronic acid susceptible to polygalacturonase in vitro also remained constant. Pretreatment of heat-inactivated dg fruit cell walls with tomato pectinmethylesterase enhances polygalacturonase susceptibility at all ripening stages. Pectinesterase activity of cell wall protein extracts from red ripe dg fruit was half that in extracts from analogous tissue of VF145B. Polygalacturonase activities of cell wall extracts, however, were similar in both varieties. Diffusion of uronic acid from tissue discs of both varieties increased beginning at the turning stage to a maximum of 2.0 milligrams uronic acid released/gram fresh weight at the ripe stage. The increased quantity of hydrolytic products released during ripening suggests the presence of in situ polygalacturonase activity. Low speed centrifugation was employed to induce efflux of uronide components from the cell wall tree space. In normal fruit, at the turning stage, 2.1 micrograms uronic acid/gram fresh weight was present in the eluant after 1 hour, and this value increased to a maximum of 8.2 micrograms uronic acid/gram fresh weight at the red ripe stage. However, centrifuge-aided extraction of hydrolytic products failed to provide evidence for in situ polygalacturonase activity in dg fruit. We conclude that pectinesterase and polygalacturonase enzymes are not active in situ during the ripening of dg fruit. This could account for the maintenance of firmness in ripe fruit tissue.     

Ethylene-independent and ethylene-dependent biochemical changes in ripening tomatoes

Fruits of Lycopersicon esculentum Mill cv Sonatine stored in 6% CO₂, 6% O₂, and 88% N₂ for 14 weeks at 12°C, exhibited a temporal separation of certain biochemical events associated with ripening.

The specific activity of two citric acid cycle enzymes, citrate synthase and malate dehydrogenase, fell substantially during the first 2 weeks of storage when changes in organic acid concentration also occurred. During this period, lycopene, polygalacturonase, and ethylene were undetectable.

When fruit were removed from store, ethylene was evolved and polygalacturonase and invertase activity were rapidly initiated as was synthesis of lycopene.

To determine whether the changes in organic acid metabolism were affected by ethylene, fruit was kept at 22°C in either a normal atmosphere or a normal atmosphere supplemented with 27 microliters per liter of ethylene, and it was shown that in both atmospheres similar quantitative changes to those described above occurred in the citric acid cycle enzymes specific activities before any detectable increase in the specific activities of invertase and polygalacturonase. These latter changes, together with pigment changes, occurred between 2 and 3 days earlier in fruit exposed to ethylene, compared with those kept in a normal atmosphere.

     

Ethylene biosynthesis in tissues of young and mature avocado fruits

Sitrit, Y., Blumenfeld, A. and Riov, J. 1987. Ethylene biosynthesis in tissues of young and mature avocado fruits.
Avocado (Persea americana Mill.) fruit tissues differ greatly in their capability to pro duce wound ethylene. In fruitlets, the endosperm lacks the ability to produce ethylene because no 1-aminocyclopropane-1-carboxylic acid (ACC) is synthesized and no activity of the ethylene-forming enzyme (EFE) is present. The cotyledons (embryo) do not produce significant amounts of ethylene at any of the developmental stages of the fruits, although in both young and mature fruits they contain a relatively high level of ACC synthase (EC 4.4.1.-) activity. Because of the very low EFE activity present in the cotyledons, most of the ACC formed in this tissue is conjugated. Of the various fruitlet tissues, the seed coat has the highest potential to produce ethylene. This is due to a high ACC synthase activity and particularly a high EFE activity. Also, the seed coat is very sensitive to the autocatalytic effect of ethylene. Fruitletpericarp possesses a lower potential to produce ethylene than the seed coat. Towardruit maturiy, the endosperm disappears and the seed coat shrivels and dies so that the pericarp and the cotyledons remain as the only active tissues in the mature fruit. At this stage, the pericarp is the only tissue producing ethylene. Mature precli macteric pericarp has a lower potential to produce ethylene than fruitlet pericarpThe role of ethylene in regulating various physiological processes at different stages of fruit maturation is discussed.     

果实多聚半乳糖醛酸酶分子生物学研究进展

多聚半乳糖醛酸酶（PG酶）是一种在植物细胞壁降解中起重要的作用的酶。作者介绍了PG酶在果实成熟软化中的作用。概述了PG酶基因及其表达调控,评述了乙烯对PG酶合成的影响。     

Products Released from Enzymically Active Cell Wall Stimulate Ethylene Production and Ripening in Preclimacteric Tomato (Lycopersicon esculentum Mill.) Fruit

Enzymically active cell wall from ripe tomato (Lycopersicon esculentum Mill.) fruit pericarp release uronic acids through the action of wall-bound polygalacturonase. The potential involvement of products of wall hydrolysis in the induction of ethylene synthesis during tomato ripening was investigated by vacuum infiltrating preclimacteric (green) fruit with solutions containing pectin fragments enzymically released from cell wall from ripe fruit. Ripening initiation was accelerated in pectin-infiltrated fruit compared to control (buffer-infiltrated) fruit as measured by initiation of climacteric CO₂ and ethylene production and appearance of red color. The response to infiltration was maximum at a concentration of 25 micrograms pectin per fruit; higher concentrations (up to 125 micrograms per fruit) had no additional effect. When products released from isolated cell wall from ripe pericarp were separated on Bio-Gel P-2 and specific size classes infiltrated into preclimacteric fruit, ripening-promotive activity was found only in the larger (degree of polymerization >8) fragments. Products released from pectin derived from preclimacteric pericarp upon treatment with polygalacturonase from ripe pericarp did not stimulate ripening when infiltrated into preclimacteric fruit.     

5'-Methylthioadenosine nucleosidase and 5-methylthioribose kinase activities in relation to stress-induced ethylene biosynthesis

The activities of 5'-methylthioadenosine (MTA) nucleosidase (EC 2.2.2.28) and 5-methylthioribose (MTR) kinase (EC 2.7.1.100) were related to changes in ethylene biosynthesis in tomato ( Lycopersicon esculentum Mill. cv. Rutgers) and cucumber ( Cucumis sativus Mill. cv. Poinsett 76) fruit following wounding and chemically induced stresses. Stress ethylene formation in wounded tomato and cucumber tissue continued to increase after wounding, reached its peak by 3h, and then declined. The activities of MTA nucleosidase and MTR kinase increased parallel to stress ethylene in both tissues. At peak ethylene formation, MTA and MTR kinase activities were 2- to 4-fold higher in wounded than in intact tissue. Wounded, mature-green tomato tissue treated with specific inhibitors of MTA nucleosidase and MTR kinase showed a significant reduction in the activities of these enzymes, which was concomitant with a decline in stress ethylene biosynthesis. When mature-green tomato discs were infiltrated with [¹⁴CH₃] MTA and wounded, radioactive MTR and methionine were formed. Incubation of mature-green tomato discs with Cu²⁺ and Li⁺ in the presence of kinetin increased ethylene biosynthesis. MTA nucleosidase activity was higher than that of the control in the presence of Cu²⁺ but not in the presence of Li⁺, while MTR kinase activity was lower than that of the control in both Cu²⁺ and Li⁺ treatments. Data indicate that MTA nucleosidase and MTR kinase are required for wound-induced ethylene biosynthesis but not for chemical stress-induced ethylene by Cu²⁺ or Li⁺ treatments.     

Changes in the Content of 1-Aminocyclopropane-1-carboxylic Acid, Activity of the Protein Polygalacturonase Inhibitor, and Intensity of Oligouronide Formation in Apples during Ripening and Treatment with Haloethane Derivatives or L-α-(2-Aminoethoxyvinyl)-glycine

We studied changes in the intensity of ethylene release and accumulation of 1-aminocyclopropane-1-carboxylic acid during ripening of two apple cultivars, differing in their physiological state, following treatment with haloethane derivatives or L--(2-aminoethoxyvinyl)-glycine. This changed both the activity of the protein polygalacturonase inhibitor in the fruit tissue and the accumulation of oligouronides. The data suggest that pretreatment with an inhibitor of 1-aminocyclopropane-1-carboxylic acid synthase affects ethylene release, accumulation of 1-aminocyclopropane-1-carboxylic acid, the activity of the protein polygalacturonase inhibitor, and the potential intensity of oligouronide formation in apple fruits and tissues.     

Postharvest Variation in Cellulase, Polygalacturonase, and Pectinmethylesterase in Avocado (Persea americana Mill, cv. Fuerte) Fruits in Relation to Respiration and Ethylene Production

Cellulase, polygalacturonase (PG), pectinmethylesterase (PME), respiration, and ethylene production were determined in single “Fuerte” avocado fruits from the day of harvest through the start of fruit breakdown. PME declined from its maximum value at the time of picking to a low level early in the climacteric. PG activity was not detectable in the preclimacteric stage, increased during the climacteric, and continued to increase during the postclimacteric phase to a level three times greater than when the fruit reached the edible soft stage. Cellulase activity was low in the preclimacteric fruit, started to increase just as respiration increased, and reached a level two times greater than at the edible soft stage. Cellulase activity started to increase 3 days before PG activity could be detected. Increased production of ethylene followed the increase in respiration and cellulase activity by about 1.5 days. These results indicate that a close relation exists between the rapid increase in the cell wall-depolymerizing enzymes and the rise in respiration and ethylene production and refocused attention on the role of the cell wall and the associated plasma membrane in the early events of fruit ripening.