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.     

Cellulase involvement in the abscission of peach and pepper leaves is affected by salicylic acid

Phenolic compounds appear to be involved in a number of regulatoryactivities in plants. During the last decade an increasing amountof evidence has brought to light the role played by salicylicacid in a number of physiological processes. Particularly interestingis the inhibitory effect of salicylic acid on ethylene biosynthesis,which might make this natural plant compound a useful tool forcontrolling some of the responses usually promoted by ethylene.Our data show that salicylic acid is actually able to reduceleaf abscission in both peach and pepper plants. Biochemicalanalyses have revealed that the enzyme usually involved in thisphenomenon (cellulase, EC 3.2.1.4 [EC] .) does not increase followingactivation of leaf abscission in plants treated with salicylicacid. In contrast, control plants show a marked increase inthe levels of both enzyme activity and cellulase protein. Flushingplants with exogenous ethylene in the presence of salicylicacid induces an increase in cellulase expression which, however,does not equal the level induced in plants without salicylicacid. Key words: Cellulase, leaf abscission, peach, pepper, salicylic acid     

Characterization of a Non-abscission Mutant in Lupinus angustifolius L.: Physiological Aspects

A single-gene recessive mutant (Abs^-) of Lupinus angustifoliusL. ‘Danja’ that does not abscise any organs wascompared with its parent during continuous exposure of explantsfrom 14 d old seedlings to 10 µl l^-1ethylene. Both endo-(1,4)-ß- D -glucanase (cellulase) and polygalacturonase(PGA) activities increased significantly and progressively inpetiole-stem abscission zones of the parent before the onsetof abscission, and were reflected in a rapid decline in breakstrengthfrom 300 to 70 g within 32 h. In the mutant there was negligibleincrease in hydrolytic enzyme activity, breakstrength declinedslowly (to 180–200 g by 72 h) and there was no abscission.Isoelectric focusing showed two cellulase isoforms (pI 5.0 andpI 8.5) expressed in abscission zones of the parent; these wereexpressed at much lower levels in the mutant. These data areinterpreted to indicate that expression of at least two formsof cellulase activity is enhanced by ethylene in normal petioleabscission zones of lupin. PGA activity also increased in theabscission zone tissue of the parent but to a lesser extentin that of the mutant. We attribute the Abs^-phenotype to mutationof a gene regulating ethylene-responsive expression of abscission-specifichydrolytic enzymes. Copyright 2001 Annals of Botany Company Lupinus angustifolius, abscission, breakstrength, cellulase, ethylene, legume, lupin, mutant, polygalacturonase     

Purification of a cellulase from kidney bean abscission zones

The purification of a cellulase isoenzyme with a pI of 9.5 from kidney bean abscission zones is described. An important step in the purification involved the adsorption of the cellulase isoenzyme onto an affinity column of CF-11 cellulose and the subsequent elution with cellobiose. Native and SDS polyacrylamide gel electrophoresis established that there was only one component in the purified cellulase samples. Antibodies raised against the purified pI 9.5 cellulase precipitated this isoenzyme from crude or purified solutions but did not cross react with pI 4.5 cellulase from 2,4-D-treated abscission zones. The antibody was shown to be monospecific by immunoelectrophoresis and by the fact that it precipitated only a single ¹⁴C-labeled protein from an abscission zone extract heavily labeled with ¹⁴C amino acids.     

Antisense suppression of tomato endo-1,4-β-glucanase Cel2 mRNA accumulation increases the force required to break fruit abscission zones but does not affect fruit softening

Plants of tomato (Lycopersicon esculentum Mill. cv. T5) were transformed with an antisense endo-1,4--glucanase (cellulase, EC 3.2.1.4) Cel2 transgene under the control of the constitutive cauliflower mosaic virus 35S promoter in order to suppress mRNA accumulation of Cel2. In two independent transgenic lines, Cel2 mRNA abundance was reduced by >95% in ripe fruit pericarp and ca. 80% in fruit abscission zones relative to non-transgenic controls. In both transgenic lines the softening of antisense Cel2 fruit pericarp measured using stress-relaxation analysis was indistinguishable from control fruit. No differences in ethylene evolution were observed between fruit of control and antisense Cel2 genotypes. However, in fruit abscission zones the suppression of Cel2 mRNA accumulation caused a significant (P<0.001) increase in the force required to cause breakage of the abscission zone at 4 days post breaker, an increase of 27% in one transgenic line and of 46% in the other transgenic line. Thus the Cel2 gene product contributes to cell wall disassembly occurring in cell separation during fruit abscission, but its role, if any, in softening or textural changes occurring in fruit pericarp during ripening was not revealed by suppression of Cel2 gene expression.     

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.     

Cellulase and polygalacturonase involvement in the abscission of leaf and fruit explants of peach

Ethylene-induced abscission in leaf and fruit explants of peach involves different enzymes. In leaves abscission is accompanied by increased occurrence of cellulase forms differing in isoelectric point (pI 6.5 and 9.5). A polypeptide with a molecular mass of 51 kDa gives in a western blot a strong cross-reaction with an antibody raised against a maturation cellulase from avocado fruit. Cellulase activity is also found in abscising fruit explants but the amount is very low compared to that of the leaf explants. A northern analysis with a cellulase clone from avocado reveals the presence of two hybridizing mRNAs with a size of 2.2 kb and 1.8 kb, respectively. The steady-state level of the 2.2 kb mRNA is significantly increased by treatment with ethylene.Polygalacturonases are not detected in abscising leaves, but are strongly induced by ethylene in fruit explants. Of the three forms found, two are exopolygalacturonases while the third is an endoenzyme. Ethylene activates preferentially the endoenzyme and the basic exoenzyme but depresses the acid exopolygalacturonases. A northern analysis carried out with a cDNA coding for tomato endopolygalacturonase shows hybridization only with one endopolygalacturonase mRNA from in the fruit abscission zone. Treatment with ethylene causes an increase in the steady-state level of this mRNA. The differences in the enzyme patterns observed in fruit and leaf abscission zones and a differential enzyme induction suggest the feasibility to regulate fruit abscission in peach with the aid of antisense RNA genes.     

Exploring the properties of thermostable Clostridium thermocellum cellulase CelE for the purpose of its expression in plants

The main properties (pH and temperature range, stability, substrate specificity) of the modified cellulase CelE (endo--1,4-glucanase) from Clostridium thermocellum have been analyzed with the goal of its expression in plants. The modified enzyme is similar to plant cellulases. Deletions in the N-terminus of the enzyme do not affect its biochemical properties. Based on the present investigation, we conclude that the modified -1,4-glucanase CelEM1, when expressed in plants, will be a good model to study the role of cellulases in plants.     

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.     

Exo- and Endo-Cellular Cellulase and Polygalacturonase in Abscission Zones of Developing Orange Fruits

The activity of cellulase, cellulase-isoenzymes and polygalacturonase (PG) in the shoot/peduncle and calyx abscission zones (AZ-A and AZ-C, respectively) of young and mature Shamouti orange (Citrus sinensis (L.) Osbeck) fruit explants was tested after extraction of total enzymes from either exo- or endo-cellular fractions from fruits treated with ethylene or 2,4-D. Ethylene enhanced and 2,4-D delayed both abscission and the activity of exo- and endo-cellular cellulase and PG. When tested separately in the exo- and endo-cellular fraction, the effects of both growth regulators on the activity of almost all cellulase isoenzymes were similar, irrespective of their location in the tissue. In mature fruits no abscission occurred in AZ-A, and yet the activity of cellulase and PG was regulated by the hormones as in abscising AZs. This was also true for total activity of exo- and endo-cellular cellulase and PG. Similar effects were observed when the activity of cellulase isoenzymes was tested in AZ-A of non-abscising mature fruits. It is suggested that whenever the increase in activity of the hydrolytic enzymes, and especially cellulase, is not followed by abscission, the substrate is either immune or not available to the enzymes.     

Occurrence and Localization of 9.5 Cellulase in Abscising and Nonabscising Tissues

Nitrocellulose tissue prints immunoblotted with 9.5 cellulase antibody were used to demonstrate areas of cellulase localization within Phaseolus vulgaris explants on exposure to ethylene. The 9.5 cellulase was induced in the distal and proximal abscission zone and in the stem. In both abscission zones, the 9.5 cellulase was found in the cortical cells of the separation layer, which develops as a narrow band of cells at the place where fracture occurs. The enzyme was also found associated with the vascular traces of the tissues adjacent to the separation layer extending through the first few millimeters at each side of the separation layer. The two abscission zones differed in the way that cellulase distributed through the separation layer as abscission proceeded. In the distal zone, cellulase appeared first in the cells of the separation layer adjacent to vascular traces and extended toward the periphery. In the proximal zone, 9.5 cellulase accumulated first in the cortical cells that lie in the adaxial side and then extended to the abaxial side. In response to ethylene, 9.5 cellulase was also induced in the vascular traces of the stem and the pulvinus without developing a separation layer. The role of 9.5 cellulase in the vascular traces is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified the same 51-kilodalton protein in both abscising and nonabscising tissues. Therefore, the determinant characteristic of the abscission process is the induction of 9.5 cellulase by cortical cells in the separation layer, and this implies that these cells have a unique mechanism for initiating 9.5 cellulase synthesis.     

Flower abscission in mutant tomato plants

The effect of two mutations of the tomato known as Never ripe (Nr) and ripening inhibitor (rin) on abscission of the flowers was investigated. In the presence of ethylene the rate of abscission of normal and rin explants was similar, while that of Nr explants was delayed. The appearance and subsequent increases in both polygalacturonase (EC 3.2.1.15) and -1-4-glucanase (EC 3.2.1.4) enzyme activities were similar in normal and rin explants, but retarded in Nr explants. Of these two, only polygalacturonase activity was exclusively associated with abscission-zone tissue.Abbreviations PG Polygalacturonase - Nr Never ripe mutation - rin ripening inhibitor mutation     

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.     

Note Floristiche Valdarnesi

Abstract

The involvement of cellulase (endo-b-l,4-glucanase, EC 3.2.1.4) in a number of different cell separation events which occur in higher plants has been well established. Besides their significance for the plant growth and differentiation, these events can be economically important since they also comprise softening of fleshy fruits and abscission of fruits, flowers and leaves. In higher plants cellulase is present in a number of different biochemical isoforms which are encoded by different genes. This finding is in accordance with the wide range of physiological events which require the intervention of cellulase activity, and whose peculiarities and amplitudes can be quite various.