Effects of Ethylene and 2,4-D on the Activity of Cellulase Isoenzymes in Abscission Zones of the Developing Orange Fruit

The role of ethylene and 2,4-D in the abscission process, and the induction of cellulase isoenzymes in the abscission zones of Citrus fruit at two physiological stages of fruit development, were studied using a new staining technique for the detection of cellulase isoenzymes in polyacrylamide gels following electrophoretic separation. Four to seven isoenzymes were detected in the shoot-peduncle (zone A) and peduncle-fruit (zone C) abscission zones; at least two of them could be detected at excision time, and of these at least one could not be connected with abscission. In the young fruit, ethylene enhanced and 2,4-D delayed both abscission and the formation of several isoenzymes. In the older fruit, ethylene enhanced and 2,4-D delayed the formation of isoenzymes at a time where no abscission occurred any more in zone A. A slower but significant increase in most of the isoenzyme activity detected was also observed in abscission zone A of untreated older fruit explants after excision. These results fully agree with those reported earlier in relation to total cellulase and polygalacturonase activity (Greenberg et al., Physiol. Plant. 34: 1, 1975) tested at the same stages of fruit development. It is suggested, that the generality of the concept that a rise in hydrolytic enzymes in the abscission zone is necessarily followed by separation of the organ should be re-evaluated.     

The Role of Cellulase and Polygalacturonase in Abscission of Young and Mature Shamouti Orange Fruits

During the first eight weeks after setting young citrus fruits gradually lose their ability to abscise at the abscission zone between the stem and the pedicel; in fruits older than eight weeks abscission occurs at the calyx area. The activity of cellulase and polygalacturonase in the two abscission zones was markedly increased before and during abscission, and was localized mainly in the abscission zone. Ethylene accelerated the increase in enzymic activity after an 8- to 10-h lag period; 2,4-D delayed abscission and enzymic activity when applied during the first 24 h after excision. During this period 2,4-D also partly suppressed the enhancing effect of ethylene. Early application of cyclo-heximide inhibited the formation of the enzymes and thus abscission was delayed to a certain extent. Although there are some indications that the relationship between enzymic activity and abscission is a complex one, the data presented indicate that cellulase and polygalacturonase play a significant role in abscission of citrus fruits at various developmental stages. Both enzymes act almost simultaneously and are equally controlled by ethylene and 2,4-D.     

Anatomical aspects of hormonal regulation of abscission in citrus – The shoot-peduncle abscission zone in the non-abscising stage

Although mature citrus fruits [ Citrus sinensis (L.) Osbeck cv. Shamouti] did not abscise at the peduncle-shoot abscission zone (AZ–A) when incubated in ethylene environment, abscission processes did occur in a limited number of cell layers situated in the inner bark, the starch sheath region, and in the pith of AZ–A. These processes were regulated by 2,4-D and ethylene treatments. Cells responding to the "separation processes", particularly in the ethylene treatment, underwent either (a) cell wall swelling, dissolving and breakdown, or (b) growth and expansion in a radial plane. Further away from the dissolving area, the response of some cells of the mid and outer bark took the form of divisions or growth in a circumferential plane, while other cells remained unchanged. Non-responding tissues of the outer bark formed a "sleeve" of undissolved cells, and the vascular cylinder produced no abscission in AZ–A. It is concluded that the partial cell wall dissolution in AZ–A explains the increased activity of cellulase and polygalacturonase in the non-abscising AZ–A of the mature fruit (Greenberg et al. 1975. Physiol. Plant. 37: 1–7).     

Endo‐1,4‐β‐glucanase gene expression and cell wall hydrolase activities during abscission in Valencia orange

The physiological and molecular events of ethylene‐induced abscission in mature fruit calyx, laminar and floral abscission zones of cv. Valencia orange were examined. Continuous exposure of fruit explants to 5 µl 1⁻¹ ethylene for 2 to 40 h resulted in marked increases in endo‐1,4‐β‐glucanase (cellulase) and polygalacturonase (PG) activities in calyx abscission zones. Two abscission‐related cellulases and one PG were found. The major peak of cellulase activity corresponded to a pI of 8.0 and molecular weight of 51 kDa, whereas the minor cellulase peak had a pI of 5.5. The abscission polygalacturonase had a pI of 5.5. Calyx abscission zone RNA was amplified with degenerate primers based on sequence of the purified Valencia orange calyx abscission cellulase, and cloned. The two partial cellulase cDNA clones were 59% identical at the nucleotide level. Genomic Southern analysis suggested that Valencia orange contained two groups of cellulase genes. A full‐length cDNA clone from each group was isolated from a cDNA library prepared from ethylene‐induced calyx abscission zone mRNA. Both genes were expressed in ethylene‐induced calyx, laminar and floral abscission zones, but were not expressed in non‐induced abscission zones or mature leaves treated with or without ethylene, young bark or young fruit of Valencia.     

Pectolytic and Cellulolytic Activity of Botrytis cinerea Pers. Related to Infection of Non-ripening Tomato Mutants

Enzymes of Botrytis cinerea were detected in vitro using various carbon sources. Pectin-pectate as a sole carbon source induced both polygalacturonase (PG) and pectin lyase (PL) activity, whereas carboxymethylcellulose served as an inducer for cellulase (C_x) activity. PG activity appeared earlier than C_x activity when induced by their respective sources. Both PG and PL activities were detected earlier and their level was higher on cell walls of the normal tomato fruit, than of the nor mutant, and in each case activity was higher on cell walls of the mature fruits than of the mature-green ones. Whereas relatively high rates of PG and PL activity were recorded on autoclaved tomato homogenate (TH) of both the normal and the nor fruits, only trace levels of PG activity were recorded on unautoclaved media, except for those prepared from ripe normal fruits, and no PL activity was detected on either of the unsterilized media. Botrytis-infection resulted in PG activity in the enzyme-less rin and nor mutant fruits at both stages of maturity and in the normal and hybrid fruits at their mature-green stage. In the ripe normal and hybrid fruits, infection increased the level of PG activity recorded prior to inoculation. An association was drawn between the low PG activity recorded in the nor mutant and its hybrid at initial stages of invasion and their resistance to infection. Following infection an increase in the level of C_x activity over that recorded in healthy fruits was found in all the tomato genotypes, whereas no PL was recorded in either healthy or infected fruits.     

葡萄贮藏过程中落粒与离区酶活性变化及植物生长调节物质的关系

研究了无核白葡萄(Vitis vinifera L.)采后贮藏过程中离区纤维素酶、果胶酯酶(Pectinesterase,PE)、多聚半乳糖醛酸酶(polygalacturonase,PG)、脂氧合酶(lipoxygenase,LOX)和过氧化物酶(peroxidase,POD)活性的变化与落粒的关系及植物生长调节物质对其的影响。结果表明,葡萄在贮藏过程中,伴随浆果落粒的增加,离区纤维素酶、PG、LOX、POD活性升高,PE活性下降。离区纤维素酶、PG、LOX等酶的活性与葡萄落粒程度之间呈显著正相关。外源ABA和CEPA处理能增强离区纤维素酶、PG、LOX活性,促进落粒;GA3,IAA处理则能抑制离区纤维素酶、PG、LOX活性,减轻落粒。ABA对落粒的促进效应及GA3对纤维素酶活性和落粒的抑制效应尤为明显,表明GA3与ABA比值在葡萄采后落粒过程中起重要的作用。     

三个脐橙品种果实主要细胞壁酶动态变化研究

以罗伯逊脐橙、丰脐、纽荷尔脐橙果实为试材,对其果皮和果肉的果胶甲酯酶(PE)、多聚半乳糖醛酸酶(PG)、β-葡萄糖苷酶和纤维素酶(CX)的动态变化进行研究。结果表明,三个品种PE活性均为果肉明显高于果皮,其中纽荷尔和罗脐分别在果实膨大期、转色期和成熟期出现三个峰值;PE活性变幅纽荷尔大于罗脐,丰脐变化较平稳。PG活性果肉略高果皮,且皆在转色期开始快速升高。首次对脐橙果实β-葡萄糖苷酶研究表明,该酶活性微弱,成熟期活性最高,纽荷尔高于丰脐和罗脐;动态变化中果皮和果肉无明显差异。酶活性高峰出现的时间为PE早于PG和β-葡萄糖苷酶;PE和PG是影响脐橙果实发育的主要酶,β-葡萄糖苷酶可能是PE和PG的补充。     

Is uronic acid oxidase involved in the hormonal regulation of abscission in explants of citrus leaves and fruits?

The role of uronic acid oxidase in abscission was studied in explants of citrus ( Citrus sinensis L. Osbeck; var. Shamouti) leaves and fruits. In leaf explants, activity of uronic acid oxidase prior to onset of abscission and the rate of abscission were markedly accelerated by ethylene and delayed by 2,4-dichlorophenoxyacetie acid. Similar results were obtained for uronic acid oxidase activity in the exocellular fraction of young fruit explants. In mature fruit explants, treated with ethylene, an immediate increase in activity was evidenty in the non-active shoot/peduncle abscission zone, whereas in the calyx abscission zone the rise in activity occurred after a prolonged exposure to ethylene, when most of the fruits had already abscised. Whenever ethylene enhanced uronic acid oxidase activity, 2,4-dichlorophenoxyacetic acid delayed it. A gradient of decreasing activity or uronic acid oxidase was recorded from both sides of the abscission zone in leaves and fruits toward the separation line, where activity was the lowest as compared with the activity found in adjacent tissues. It is suggested that uronic acid oxidase is involved in senescence and cell wall degradation. However, it is yet questionable whether this enzyme is directly related to the control mechanism of abscission.     

Effect of phytohormones on pectate lyase activity in ripening Musa acuminata.

A differential activity peak of pectate lyase (PEL) was observed during ripening of banana fruits (Musa acuminata Harichhal) receiving different hormone treatments. Exposure of fruits to 25 ppm ethylene for 24 h, as well as dipping of M. acuminata fruits in 1 mM 2,4-dichlorophenoxy acetic acid (2,4-D) for 4 h, hastened fruit ripening. Both PEL activity peak and climacteric peak were observed on the 4th and 10th days of treatment with ethylene and 2,4-D, respectively, compared to the 16th day in control fruits. Gibberellic acid (GA) treatment retarded fruit ripening and both PEL activity and climacteric peaks were observed on the 19th day. Treatment of fruits with ethylene or 2,4-D also advanced the appearance of a polygalacturonase (PG) peak and GA delayed its appearance, but the activity peaks always appeared in post-climacteric fruits, in contrast to PEL activity peaks coinciding with the respiratory peaks.     

Changes in Two Forms of Membrane-Associated Cellulase during Ethylene-Induced Abscission

Only one form of membrane-associated cellulase was found previously in the lower petiolar pulvinus of Phaseolus vulgaris (cv Red Kidney). The cellulase has an isoelectric point (pI) of 4.5 (DE Koehler, LN Lewis 1979 Plant Physiol 63: 677-679). This enzyme was detected in abscission zones collected before the onset of abscission (control tissue), and was thought to represent a pre-secretory form of another cellulase, the abscission cellulase, which has a basic pI and is secreted during abscission. We now show that this acidic, membrane-associated cellulase is a glycoprotein, tightly bound to the membrane, with maximum activity at pH 5.1, and that it is not immunologically related to the abscission cellulase. Furthermore, when bean explants are induced to abscise with ethylene, the activity of the acidic cellulase declines rapidly to 50% of control levels in the first day. When abscission is fully developed, the membranes contain a basic form of cellulase with a pI of 8.0 to 9.0 and only trace levels of the acidic cellulase. The basic form is not a high mannose glycoprotein; it has maximum activity in a broad pH range (4.0-8.0) and is antigenically related to the abscission cellulase, which is induced during abscission and transported to the cell wall. Antibody raised against the abscission cellulase recognized two proteins in a crude membrane fraction from abscising tissue. One of those proteins comigrated with the abscission cellulase, and the other was 1 to 2 kilodaltons larger. Thus, during abscission, the acidic membrane-associated cellulase rapidly declines before the appearance of the abscission cellulase. We conclude that there is no conversion from the acidic cellulase to the basic cellulase and suggest that the acidic and basic cellulase isoenzymes are proteins derived from two different genes.     

Compositional and enzymatic changes in guava (Psidium guajava L.) fruits during ripening

Changes in chemical composition and hydrolytic enzyme activities in guava fruits cv. Lucknow-49 have been reported at four different stages of maturity, viz., mature green (MG), color turning (CT), ripe (R) and over ripe (OR). Chlorophyll content decreased, while carotenoid content increased with advancement of ripening. Starch content decreased with concomitant increase in alcohol soluble sugars. The cell wall constituents viz., cellulose, hemicellulose, and lignin decreased up to R stage, while the pectin content decreased throughout up to OR stage. Among the cell wall hydrolyzing enzymes, polygalacturonase (PG) and cellulase exhibited progressive increase in activity throughout ripening, while pectin methyl esterase (PME) activity increased up to CT stage and then decreased up to OR stage. The maximum increase in the activities of cell wall hydrolysing enzymes was observed between MG and CT stages. The activities of starch hydrolyzing enzymes, α-amylase and β-amylase decreased significantly with advancement of ripening. These changes in the activities of hydrolyzing enzymes could be considered good indicators of ripening in guava.     

Differential ethylene-inducible expression of cellulase in pepper plants

Ethylene promotes the abscission of leaves and the ripening of fruits in pepper plants, and in both events an increase in cellulase activity is observed. However, two enzyme isoforms (pI 7.2 and 8.5, respectively) are differentially involved in the two physiological phenomena. The pI 8.5 form has been purified from ripe fruits. It is a glycoprotein with an apparent molecular mass of 54 kDa. Two short peptides were sequenced and a very high homology to a tomato cellulase was observed. Polyclonal antibodies, raised against the purified enzyme, have allowed us to demonstrate that the observed ethylene-induced increase in cellulase activity is paralleled by de novo synthesis of protein. Three cDNAs (CX1, CX2 and CX3), encoding different cellulases, were obtained and characterized and their expression investigated. Accumulation of all three mRNAs is induced by ethylene treatment, though to different levels. CX1 is mainly expressed in ripe fruits while CX2 is especially found in abscission zones. CX3 accumulates at very low levels in activated abscission zones. Comparisons with other known cellulases demonstrate clear heterogeneity within the higher plant cellulases. Differences in ethylene inducibility and molecular structure suggest different physiological roles for cellulase in pepper plants.This paper is dedicated to Prof. G. Dall'Olio on the occasion of his 70th birthday.     

Polygalacturonase and Cellulase Enzymes in the Normal Rutgers and Mutant rin Tomato Fruits and Their Relationship to the Respiratory Climacteric

Cell wall enzymes at different stages of fruit development were compared between the normal Rutgers and the isogenic nonripening rin tomato. In Rutgers, a detectable increase in polygalacturonase (PG) activity was observed 6 days prior to the respiratory climacteric (43 days postanthesis). The maximum increase in PG activity occurred after C₂H₂ and CO₂ production reached their peak. However, in the rin tomato, no change in PG activity was noted up to 100 days postanthesis. Cellulase activity increased in Rutgers fruits prior to the respiratory climacteric and continued to increase thereafter. Similar changes in cellulase activity were also observed in the nonclimacteric rin fruits. Short term ethylene treatment (2 days) of 36-day-old rin fruits increased cellulase activity, but had no effect on PG activity. Detectable changes in other parameters of ripening, such as chlorophyll loss and softening, also occurred prior to the respiratory climacteric. These results suggest that the failure of rin fruits to ripen is related to their low PG activity during maturity as compared with normal fruits.     

A simple and fast method for the determination of endo- and exo-cellulase activity in cellulase preparations using filter paper

This study describes a procedure for the selective determination of endo- (EG) and exo- (ExG) cellulase activities using filter paper as the sole substrate. The procedure is based on the enzymes mode of action whereby EG activity predominantly forms insoluble reducing sugars and ExG activity soluble reducing sugars. The procedure was developed using filter paper as substrate for hydrolysis with three cellulase preparations of Hypocrea jecorina containing either endoglucanase (EG), predominantly exoglucanase (ExG) or both endo- and exoglucanase activities. Hydrolysis experiments, which were followed assessing the formation of total, soluble and insoluble reducing sugars (RS), showed that up to 30min of hydrolysis predominantly insoluble reducing sugars were formed, while after this initial hydrolysis stage soluble reducing sugar formation increased significantly, making it thus possible to measure separately EG and ExG activity. FPA activities obtained from the reaction products at different reaction times suggest that EG-activity (FPA(insol)) should be measured between 10 and 20min of hydrolysis. The proposed procedure allows to evaluate the EG and ExG activity contribution to total cellulase activity and to calculate the endo/exo activity ratio of any cellulase preparation.     

Cellulase activity and localization during induced abscission of Coleus blumei

Treatment with dimethipin (2,3-dihydro-5,6-dimethyl-1,4-dithiin 1,1,4,4 tetroxide) inhibited the increase in cellulase activity and decrease in breakstrength associated with the normal course of abscission in Coleus. Application of the surfactant UBI-1126 (Emery OAL 20 in isopropyl alcohol) increased cellulase activity and accelerated the process of abscission in Coleus expiants within 24 h of application. Cellulase activity was localized histochemically at the electron microscopic level in surfactant-treated tissue. The enzyme activity was localized primarily in the cell wall, middle lamella, and paramural bodies of abscission zone cells.     

Tissue and Cell Culture of 'Passe Crassane' Pears: Amylase Pattern of Cultured Tissues Compared with Whole Fruit

Calluses and cell suspension cultures were initiated from young and fully developed fruits and from stem and petiole tissues of ‘Passe Crassane’ pears. On a dry weight basis, proliferation is significantly higher in tissues from full-grown fruits and petioles. Cullus tissues grown in vitro have a protein content up to 25 times higher than the initial quiescent organ. Three amylase fractions (2 β amylases E, and E₂: 1 α amylase E₃) were isolated by gel filtration on Sephadcx G-100 and isoenzymes revealed in these fractions after polyacrylamide gel electrophoresis. Comparison of amylase activity in actively growing cells and in auxin-starved cell suspensions shows that β-amylase activity is mainly a function of cell growth, while α-amylase activity is more related to the age of the cells. The presence of two isoenzymes in the β-amylase E₂ fraction was found to be highly characteristic of young fruits. The same polymorphism was found in all the strain of tissues thus indicating that tissues grown in vitro retain or revert to a juvenile biochemistry. The growth hormone. 2,4-dichlorophenoxyacetic acid, used to support cell division, is demonstrated to be responsible for the reversion of the β-amylase polymorphism from a mature to a juvenile phenotype in mature fruit explants incubated in vitro. The interest of fruit tissue and cell culture for the study of fruit physiology is questioned and discussed.     

Enhancement of polygalacturonase activity during auxin induced para nodulation and endorhizosphere colonization of Azospirillum in rice roots

Effect of different auxins, namely, 2,4-dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA) and indole acetic acid (IAA) and Azospirillum brasilense bioinoculation on the enhancement of polygalacturonase (PG) activity in rice roots during para nodulation and endorhizosphere colonization of Azospirillum was studied under in vitro condition. It was observed that Azospirillum bioinoculation could augment PG activity of rice roots to a lesser extent without any root morphogenesis whereas auxin application together with Azospirillum bioinoculation enhanced PG activity of rice roots to a higher level which resulted in better root morphogenesis (para nodule) and endorhizosphere colonisation of A. brasilense. Among the three auxins tested, 2,4-D, even at lower concentration (0.5 ppm) enhanced the rice root PG activity, root morphogenesis and endorhizosphere colonization of Azospirillum while it was 2.0 ppm with NAA and variable with IAA. It is concluded that there is a positive correlation existing among PG activity, degree of root morphogenesis and endorhizosphere colonization of Azospirillum brasilense in rice roots and the degree of correlation is determined by the chemical composition, concentration and mode of action of the auxin utilised.