Further comparative studies of pectin esterase in relation to leaf and flower abscission

The possible role of pectin esterase in abscission of bean and coleus leaves was re-examined using improved methods for its extraction. Its possible role in abscission of flowers of tobacco and leaves of soybean. cotton, mulberry, magnolia, and euonymus was also examined. In seven of the eight species, no detectable changes in abscission zone pectin esterase activities immediately preceded abscission. Therefore, pectin esterase probably plays no causal role in abscission in these species.     

The flowering response of coleus in relation to photoperiod and the circadian rhythm of leaf movement

The flowering response of Coleus frederici and Coleus blumei x C. frederici is dependent on the photoperiod; both plants have a critical day length of about 12 hr. The inductive phase, defined as the period when light signals inhibit floral development, started 10 hr after the onset of darkness under 4 and 8-hr photoperiods, and 8 hr after the onset of darkness under a 12-hr photoperiod. However, a fixed temporal relationship between the inductive phase and the minimum leaf position was observed for Coleus frederici. The inductive phase always started 5 hr after the minimum leaf position. This evidence supports the theory that a circadian clock participates in the time measurement process of photoperiodic floral induction.     

Activity of pectin esterase and cellulase in the abscission zone of citrus leaf explants

The activity of pectin esterase and cellulase in abscission of citrus explants was studied. No relation was established between pectin esterase and abscission, while cellulase activity was markedly increased before abscission and for a certain period after excision. IAA and cycloheximide delay abscission and cellulase activity, while ethylene and, to a lesser extent, GA₃ accelerate them. Application of cycloheximide during the lag period and before cellulase activity can be measured, inhibits to a certain extent the formation of cellulase. An escape from the inhibitory effect of cycloheximide is detected when inhibitor is supplied at the end of the lag period.     

Effect of ethylene on auxin transport and metabolism in midrib sections in relation to leaf abscission of woody plants

Abstract The relationship between ethylene-induced leaf abscission and ethylene-induced inhibition of auxin transport in midrib sections of the leaf blade of Citrus sinensis L. Osbeck, Populus deltoides Bart, and Eucalyptus camaldulensis Dehn. was studied. These species differed greatly in their abscission response to ethylene. The kinetic trend of abscission resembled that of the inhibition of auxin transport in all three species. It is suggested that one of the main actions of ethylene in the leaf blade is to inhibit auxin transport in the veinal tissues, thus reducing the amount of auxin transported from the leaf blade to the abscission zone. Ethylene inhibited transport of both IAA (indole-3-acetic acid) and NAA (α-naphthaleneacetic acid) in the midrib sections. However, while ethylene enhanced the conjugation of IAA with aspartic acid and glucose in the apical (absorbing) segment of the midrib sections, it had little effect on the conjugation of NAA. The data indicate that auxin destruction through conjugation does not play a major role in the inhibition of auxin transport by ethylene.     

Methyl jasmonate and bean leaf abscission

The effect of rac-methyl jasmonate, both in solution and as a vapour, on the separation of pulvinar and petiolar tissues in explants containing the distal abscission zone of primary leaves of Phaseolus vulgaris var. Contender was investigated. The effects of rac-methyl jasmonate were compared to those of (±)-abscisic acid, -naphthalene acetic acid, ethylene and 2-chloroethylphosphonic acid. Abscission times were determined in explants prepared from 14-day-old control plants and in explants prepared from plants that had been pretreated for 24h with the ethylene-action inhibitor, silver thiosulphate. While silver-pretreatment, or treatment with -naphthalene acetic acid delayed abscission, treatment with ethylene or 2-chloroethylphosphonic acid accelerated tissue separation. However, (±)-abscisic acid delayed abscission under these conditions. In all instances, treatment with rac-methyl jasmonate had no apparent effect on abscission. The loss of chlorophyll from bean leaf discs incubated in the dark was enhanced by treatment with 2-chloroethylphosphonic acid or (±)-abscisic acid and was retarded in discs incubated in benzyl adenine. While incubation in -naphthalene acetic acid was without effect, incubation in solution of rac-methyl jasmonate also retarded chlorophyll loss when compared to water controls.     

Reactive oxygen species in leaf abscission signaling

Reactive oxygen species (ROS) are produced in response to many environmental stresses, such as UV, chilling, salt and pathogen attack. These stresses also accompany leaf abscission in some plants, however, the relationship between these stresses and abscission is poorly understood. In our recent report, we developed an in vitro abscission system that reproduces stress-induced pepper leaf abscission in planta. Using this system, we demonstrated that continuous production of hydrogen peroxide (H₂O₂) is involved in leaf abscission signaling. Continuous H₂O₂ production is required to induce expression of the cell wall-degrading enzyme, cellulase and functions downstream of ethylene in abscission signaling. Furthermore, enhanced production of H₂O₂ occurs at the execution phase of abscission, suggesting that H₂O₂ also plays a role in the cell-wall degradation process. These data suggest that H₂O₂ has several roles in leaf abscission signaling. Here, we propose a model for these roles.Key words: leaf abscission, reactive oxygen species, H₂O₂, in vitro, ethylene, auxin, pepper, NADPH oxidase     

Role of polygalacturonase in bean leaf abscission

The role of polygalacturonase in leaf abscission was studied in explants of Phaseolus vulgaris L. cv. Red Kidney. Bean polygalacturonase was partially characterized and comparisons were made between the bean enzyme and previously reported higher plant polygalacturonases. Polygalacturonase isolated from bean leaf abscission zones has a pH optimum between 4.5 and 5.0 and hydrolyzed polygalacturonides in an exo-fashion. Activity was found to be higher with a deesterified substrate than with an esterified pectin. No correlation between polygalacturonase activity and abscission was observed. Activity remained virtually constant over the course of abscission in explants aged either in air or in ethylene. The enzyme was primarily localized in the abscission zone, however, indicating a possible involvement in the abscission process. A theoretical model which could explain the relationship between polygalacturonase and bean leaf abscission is discussed.     

Immobilized enzymes: Pectin esterase covalently coupled to dorous glass particles

Pectin esterase (E.C. 3.1.1.11) was covalently immobilized to porous glass particles by reaction of the native protein with pendant, benzoyl azide groups of the carrier. Enzyme loading on the carrier was 0.5 unit per ml as measured by pH stat, assay. Decreasing the size of the immobilized enzyme particles by grinding produced a 12-fold increase in activity suggesting severe internal mass transport restrictions on turnover kinetics, Gross fractionation of the citrus pectin substrate into high and low molecular weight categories and their subsequent use in kinetic characterization shows no effect of molecular weight upon the kinetic behavior of the native enzyme. In contrast the immobilized enzyme displayed a 5-fold increase in the apparent. K_m for the high molecular weight fraction relative to that of the low molecular weight fraction. A striking difference exists in the low pH profile of immobilized pectin esterase relative to the native enzyme. Carrier matrix interactions with the polyelectrolyte substrate are invoked to explain this difference. The thermal stability of the immobilized enzyme is relatively low and displays a half-life of approximately 2 weeks at 25°C.     

Deposition of callose in relation to abscission of citrus leaves

When leaves of Citrus sinensis (L.) Osbeck cv. Shamouti senesce, they become more susceptible to abscission and the proximal 2 mm of their lamina-petiole abscission zones exhibit callose deposition. The degree of senescence, assayed with the DAR-WIN image processor (Telewski et al. 1983), was positively correlated in a linear fashion with callose deposition. Explant of non-senescing leaves were observed. Excision of the leaf at the stem-petiole junction induced callose deposition throughout the petiole, but not in the lamina. Callose deposition began immediately upon excision and reached a maximum at 3 h. It then decreased slightly and remained at the same level for up to 5 days. Exogenous compounds that decrease callose deposition, e.g. laminarase and 2-deoxy-D-glucose, inhibited the rate of abscission of explants. Compounds that promote callose deposition, e.g. uridine diphosphoglucose and mannose, increased the rate of abscission of explants. Exogenous callose, e.g. laminarin, increased the rate of abscission. It is not known how callose might be causally involved in promoting abscission.     

Pectin esterase, polygalacturonase and gel formation in peach pectin fractions

Peaches (Prunus persica cv. Hermoza) were stored at 0C in regular air (RA) or in controlled atmosphere (CA 10% CO2, 3% O2) for 4 weeks and then ripened for 4 days at 20 degrees C. Woolliness developed in the regular air stored fruit while the controlled atmosphere stored fruit ripened normally. In the woolly fruit symptoms of the disorder were greater in the inner mesocarp than in the outer. Polygalacturonase (PG) and pectin esterase (PE) activities differed in the outer and inner mesocarp of the affected fruit. PG activity was low and PE activity was high in the inner mesocarp of the woolly fruit during ripening relative to the outer mesocarp, while in the healthy fruit, activities were similar in both areas. Cell wall fractions of water-soluble, CDTA-soluble and carbonate-soluble pectins were prepared from freshly harvested peaches and incubated with PE and PG from ripe peaches at different ratios. Only the CDTA-soluble fraction formed a gel with peach enzymes, and the rate of gelation increased with increasing amounts of PE relative to PG. Both water-soluble and CDTA-soluble pectin fractions formed gels with commercial PE (extracted from orange peel). The PE extracted from peaches was stable when stored at 0 degrees C for 9 days, while PG activity was stable only for 1 day. We suggest that PE, acting on pectins in the cell wall in vivo may cause gel formation and that the CDTA-soluble polymers have the capacity to bind apoplastic water and create the dry appearance observed in woolly fruit.     

Selective oviposition by a leaf miner in response to temporal variation in abscission

Summary Responses to humidity of net photosynthesis and leaf conductance of single attached leaves were examined in populations of herbs from wet soil sites in Beltsville, Maryland and Davis, California, USA. Plants were grown in controlled environments under three conditions which differed in the magnitude of the day-night temperature difference and in daytime air saturation deficit. No population differences in response were found in Abutilon theophrasti. In Amaranthus hybridus stomatal conductance and net photosynthesis were more reduced by increasing leaf to air water vapor pressure difference (VPD) in the population from Beltsville, but only for the growth condition with a constant 25°C temperature. In Chenopodium album, stomatal conductance was more sensitive to VPD in the population from Davis, but only for the growth condition with 28/22°C day/night temperatures. Population differences in the sensitivity to VPD of leaf conductance were associated with differences in leaf area to root weight ratio. The relative reduction of net photosynthesis as VPD increased was greater than, equal to, or less than the relative decrease in substomatal carbon dioxide partial pressure. The pattern depended on species, and on growth condition. From these results one can not conclude that environmental humidity has been a strong selective force in determining sensitivity to humidity of stomatal conductance.     

Effect of exposure to subfreezing temperatures on ethylene evolution and leaf abscission in citrus

Citrus leaves exposed to subfreezing temperatures evolved ethylene at rates between 0.1 and 38.3 microliters per kilogram fresh weight per hour whereas untreated leaves evolved between 0.01 and 0.50 microliter per kilogram fresh weight per hour. Leaves not injured by freezing temperatures did not abscise, and ethylene evolution was near normal after 2 days. Freeze-injured leaves continued evolving high ethylene levels 4 or 5 days subsequent to freeze injury, and many of the freeze-killed leaves abscised. Supportive evidence suggested freeze-induced ethylene was involved in freeze-induced leaf abscission; whereas freeze-inhibited abscission was not due to a lack of ethylene but injury to other metabolic systems necessary for abscission.     

Interactions of indoleacetic Acid and gibberellic Acid in leaf abscission control

Debladed midribs of citrus leaves showed the typical delay of abscission in response to indoleacetic acid (IAA), and the typical acceleration of abscission in response to gibberellic acid (GA). Interaction experiments with these 2 hormones indicated that the balance of the 2 hormones may be more important in regulating abscission than the quantity of either. The often reported acceleration of abscission with low quantities of IAA did not seem to exist in citrus. IAA did accelerate abscission in this tissue when its application was delayed for at least 24 hours after deblading, which suggests the 2-stage effect is also present in citrus.

When abscission was first delayed with IAA and then allowed to continue, the rate of abscission proceeded at a slower rate than was typical for this tissue. This slower rate was also typical of the effect observed when GA overcame the abscission retarding effect of IAA. The phenylurethane, Barban, blocked the GA acceleration of abscission, but it did not affect the rate of abscission of control or IAA treated midribs.

     

A polygalacturonase from citrus leaf explants: role in abscission

The relationship between polygalacturonase activity and abscission of citrus leaf explants was studied. Determination of polygalacturonase activity in citrus tissues requires concentration of the enzyme, use of a proper assay method, and inhibition of an oxidase present in the extracts which oxidizes the reaction products of the polygalacturonase. The polygalacturonase from citrus leaf explants is an exopolygalacturonase and appears to be a soluble enzyme.     

The time course of xylem differentiation and its relation to deoxyribonucleic Acid synthesis in cultured coleus stem segments

The relationship between DNA synthesis and wound xylem differentiation was investigated in cultured stem segments of Coleus blumei. The addition of 50 micrograms of indoleacetic acid per liter to the culture medium resulted in a 400 to 500% increase in the number of wound vessel members formed in 7 days. However, the time course of wound vessel member formation was similar in segments cultured in the presence and absence of auxin. In either case, no wound vessel members appeared before the 3rd day of culture, while the majority of wound vessel members appeared on the 4th and 5th days of culture. ³H-Thymidine incorporation into DNA was used to measure changes in the DNA synthetic activity of the tissues during the culture period. Comparatively little ³H-thymidine incorporation occurred during the 1st day of culture. Maximum ³H-thymidine incorporation was observed on the 2nd day of culture, 2 days before the peak period of xylem differentiation. The rate of incorporation of ³H-thymidine into DNA decreased with increasing time in culture after the 2nd day. Auxin at 50 micrograms per liter had no effect on the time course of ³H-thymidine incorporation, although somewhat more ³H-thymidine was incorporated into DNA throughout the culture period in the presence of auxin. The magnitude of this effect was small when compared to the effect of auxin on xylem differentiation. The antimetabolite 5-fluorodeoxyuridine was shown to block DNA synthesis in the cultured stem segments. When the tissues were isolated on media containing 10⁻⁶m 5-fluorodeoxyuridine, wound vessel member differentiation was inhibited by approximately 80%, in both the presence and absence of auxin. Thymidine at 10⁻⁵m completely overcame the 5-fluorodeoxyuridine inhibition of wound vessel member formation. 5-Fluorodeoxyuridine was effective in blocking xylogenesis only when this substance was supplied to the tissues during the early part of the culture period. 5-Fluorodeoxyuridine had no effect on xylem differentiation when it was applied after the 3rd day of culture.     

Polygalacturonase expression during leaf abscission of normal and transgenic tomato plants

Polygalacturonase (PG, EC 3.2.1.15), an enzyme commonly found in ripening fruit, has also been shown to be associated with abscission. A zone-specific rise in PG activity accompanies the abscission of both leaves and flowers of tomato (Lycopersicon esculentum Mill.) plants. Studies of transgenic plants expressing an antisense RNA for fruit PG indicate that although the enzyme activity in transgenic fruit is < 1 % of that in untransformed fruit, the PG activity in the leaf abscission zone increases during separation to a similar value to that in untransformed plants. The timing and rate of leaf abscission in transgenic plants are unaffected by the introduction of the antisense gene. A polyclonal antibody raised against tomato fruit PG does not recognise the leaf abscission protein. Furthermore a complementary DNA (cDNA) clone (pTOM6), which has been demonstrated to code for fruit PG, does not hybridise to mRNA isolated from the abscission-zone region of tomato leaves. These results indicate that the PG protein in abscission zones of tomato is different from that in the fruit, and that the gene coding for this protein may also be different.Abbreviation PG polygalacturonase The authors of this paper are grateful to David Jackson of the John Innes Institute, Norwich, UK for his assistance with the in-situ hybridisation work. This research was supported by an Agricultural and Food Research Council Post-Doctoral award to J.E.T., and by a grant to D.G. from the Science and Engineering Research Council Biotechnology Directorate in association with ICI seeds. The work was carried out under Ministry of Agriculture, Food and Fisheries licences.     

Retardation of abscission of citrus leaf and fruitlet explants by brassinolide

Brassinolide (BR), a novel plant growth-regulating steroidal lactone, markedly retarded the abscission of leaf explants of Calamondin (Citrus madurensis Lour.), when dissolved in water and fed through the petiole. BR was effective at concentrations as low as 0.021 M, and showed a stronger effect than IAA which also retarded abscission. Trifluoperazine (TFP), an inhibitor of the calmodulin-calcium complex, accelerated abscission, and this acceleration could be counteracted by a simultaneous addition of IAA or BR, the effect of IAA being stronger. BR in lanolin applied to the cut surface of the leaf blade of the explant showed a weaker abscission-retarding effect than that applied in water via the petiole. BR and IAA also markedly retarded the abscission of fruitlet explants of Calamondin.