Jasmonates promote abscission in bean petiole expiants: Its relationship to the metabolism of cell wall polysaccharides and cellulase activity

Jasmonic acid (JA) and its methyl ester (JA-Me) promoted the abscission of bean petiole expiants in the dark and light, and the activity of these compounds was almost same. JA and JA-Me did not enhance ethylene production in bean petiole expiants in the light, indicating that the abscission-promoting effects of these compounds are not the result of ethylene. Cells in the petiole adjacent to the abscission zone expanded during abscission but not in the pulvinus, and JA-Me promoted cell expansion in the petiole and the pulvinus. JA-Me had no effect on the total amounts of pectic and hemicellulosic polysaccharides in 2-mm segments of the abscission region, which included 1 mm of pulvinus and 1 mm of petiole from the abscission zone. On the other hand, the total amounts of cellulosic polysaccharides in this region were reduced significantly by the addition of JA-Me in the light. JA-Me had no effect on the neutral sugar composition of hemicellulosic polysaccharides during abscission. The decrease in the endogenous levels of UDP-sugars in the petiole adjacent to the abscission zone was accelerated during abscission by the addition of JA-Me in the light. Cellulase activities of pulvinus and petiole in 10-day-old seedlings were enhanced by the addition of JA. These results suggest that the promoting effect of JA or JA-Me on the abscission of bean petiole explants is due to the change of sugar metabolism in the abscission zone, in which the increase in cellulase activity involves the degradation of cell wall polysaccharides. Jasmonic acid (JA) and its methyl ester (JA-Me) are considered to be putative plant hormones for a number of reasons, including their wide occurrence in the plant kingdom, biologic, activities in multiple aspects at low concentrations, and their interaction with other plant hormones (for reviews see Parthier 1991, Hamberg and Gardner 1992, Sembdner and Parthier 1993, Ueda et al. 1994a). We have already reported that JA and JA-Me and C₁₈-unsaturated fatty acids, which are considered to be the substrates of the biosynthesis of jasmonates, are powerful senescence-promoting substances (Ueda et al. 1982b, 1991a). Senescence symptoms induced by these compounds are identical to those of natural senescence. Recently we have also found that JA inhibited indole-3-acetic acid (IAA)-induced elongation of oat (Avena sativa L. cv. Victory) coleoptile segments by inhibiting the synthesis of cell wall polysaccharides (Ueda et al. 1994b, 1995). These facts led us to study the mode of actions of JA and JA-Me on promoting abscission, which is considered the last dramatic phenomenon of senescence. In this paper we report that JA and JA-Me promote abscission in bean (Phaseolus vulgaris L. cv. Masterpiece) petiole expiants and that the changes in the metabolism of cell wall polysaccharides in the petiole and the pulvinus adjacent to the abscission zone are involved in the promotive effects of these compounds.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - DCB 2,6-dichlorobenzonitrile - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - JA jasmonic acid - JA-Me methyl jasmonate - MES 2-(N-morpholino)ethane-sulfonic acid, monohydrate - TCA trichloroacetic acid - Tris 2-amino-2-hydroxymethy-1,3-propanediole     

Inhibition of the synthesis of cell wall polysaccharides in oat coleoptile segments by jasmonic acid: Relevance to its growth inhibition

The inhibitory mode of action of jasmonic acid (JA) on the growth of etiolated oat (Avena sativa L. cv. Victory) coleoptile segments was studied in relation to the synthesis of cell wall polysaccharides using [¹⁴C]glucose. Exogenously applied JA significantly inhibited indoleacetic acid (IAA)-induced elongation of oat coleoptile segments and prevented the increase of the total amounts of cell wall polysaccharides in both the noncellulosic and cellulosic fractions during coleoptile growth. JA had no effect on neutral sugar compositions of hemicellulosic polysaccharides but substantially inhibited the IAA-stimulated incorporation of [¹⁴C]glucose into noncellulosic and cellulosic polysaccharides. JA-induced inhibition of growth was completely prevented by pretreating segments with 30 mm sucrose for 4 h before the addition of IAA. The endogenous levels of UDP-sugars, which are key intermediates for the synthesis of cell wall polysaccharides, were not reduced significantly by JA. Although these observations suggest that the inhibitory mode of action of JA associated with the growth of oat coleoptile segments is relevant to sugar metabolism during cell wall polysaccharide synthesis, the precise site of inhibition remains to be investigated.Abbreviations JA jasmonic acid - ABA abscisic acid - IAA indoleacetic acid - T ₀ minimum stress relaxation time - TFA trifluoroacetic acid - TCA trichloroacetic acid - HPLC high-performance liquid chromatography - EtOAc ethyl acetate - TLC thin-layer chromatography - JA-Me methyl jasmonate - GLC-SIM gas-liquid chromatography-selected ion monitoring     

Thigmomorphogenesis: XXIII. Promotion of foliar senescence by mechanical perturbation of Avena sativa and four other species

Mechanical perturbation (MP, gentle tubbing) promoted the senescence of detached oat ( Avena sativa L. cv. Victory) leaf segments in the dark. The promotion of senescence increased with increase in the number of rubbings and could be seen after 24 h of dark incubation; the maximum effect was reached on day 3. The effect (% of control) of MP on the loss of protein was greater than the effect on chlorophyll (Chl) loss on day 1. However, on day 3 the effect of MP on the loss of Chl became greater than the effect on the loss of protein. Ethephon and 1-aminocyclopropane-1-carboxylic acid (ACC) marginally promoted the loss of Chl by both control and rubbed oat leaf segments, and the effect was additive with MP. Chloramphenicol (CAP), spermine, aminoethoxyvinylglycine (AVG) and Ca²⁺ marginally delayed the loss of Chl and protein in both control and rubbed segments. Kinetin greatly retarded the senescence of all segments. Even in the presence of these substances, the amounts of Chl and protein in the rubbed segments were always less than in their respective controls, thus retaining the effect of the MP. However, abscisic acid (ABA) and cycloheximide (CHI) caused the rubbed oat leaf segments to retain more Chl and protein than their respective control segments. The effect of CHI was actually enhanced by MP. Rubbing promoted the senescence of attached leaves of oats ( Avena sativa L. cv. Victory), maize ( Zea mays L. cv. Early Belle) and pumpkin ( Cucurbita pepo L. cv. Jack-o-lantern) cotyledons in the dark. Rubbing promoted the senescence of oat leaf segments even in light, although to a lesser extent compared to the effect in the dark. The senescence of leaves of pumpkin and cocklebur ( Xanthium strumarium Wallr. var. Pennsylvanicum ) in situ was also enhanced by MP.     

Light-induced h secretion and the relation to senescence of oat leaves

When abraded oat (Avena sativa L. cv Victory) leaf segments are floated on KCl solution, white light causes acidification of the solution external to leaf tissue. The presence of mannitol amplifies the light-induced proton secretion. Mature leaves as well as young ones acidify the medium in light, while senescing leaves (after 3 to 4 days incubated in water in the dark) lose the ability to produce this response to light. The decrease in H⁺ secretion is already measureable after as little as 30 minutes in darkness, while the increase in proteolysis rate was detected only after 6 hours in dark. The decrease in capacity to secrete protons is one of the symptoms of leaf senescence. Moreover, fusicoccin mimics light in stimulating H⁺ pumping and delaying the senescence in the dark. On the other hand vanadate, an apparent inhibitor of plasma membrane H⁺ ATPase, blocks the acidification and promotes the chlorophyll and protein degradation in leaf segments during the 2-day period of incubation. These results, which show a parallel between cessation of H⁺ secretion and acceleration of senescence, may suggest a regulatory role for H⁺ secretion in leaf senescence.     

Modification of chemical properties of cell wall polysaccharides in the inner tissues by white light in relation to the decrease in tissue tension in Pisum sativum epicotyls

When white light irradiation inhibits shoot growth in derooted pea ( Pisum sativum L. cv. Alaska) cuttings, it decreases tissue tension, a driving force for shoot growth, by making the cell wall of the inner tissues mechanically rigid. To elucidate the mechanism by which light affects the mechanical properties of the cell wall in the inner tissues, its effect on the chemical properties of the cell walls was studied by analyzing qualitatively and quantitatively cell wall polysaccharides in the epdidermis and inner tissue of pea epicotyls grown in both dark and light. The amount of polysaccharides per subhook in the cell walls of both tissues increased during a 4-h dark incubation. Light suppressed the increase in hemicellulosic (HC)-II and cellulosic polysaccharides in the inner tissues, while it did not affect the increase in other wall fractions in either the epidermal or subepidermal tissues. No light effect was observed on the neutral sugar compositions of pectin, HC-I or HC-II fractions in either of the tissues. Light increased the mass-average molecular mass of xyloglucan and rhamnoarabinogalactan in HC-II fractions in the inner tissues, while such an effect was not observed in the epidermis. These facts suggest that the light-induced decrease in the tissue tension in pea epicotyls is caused by an increase in the molecular size of xyloglucan, rhamnoarabinogalactan in the HC-II fraction and/or the suppression of the synthesis of HC-II and cellulosic polysaccharides in the inner tissues.     

Degradation of ribulose-1,5-bisphosphate carboxylase and chlorophyll in senescing barley leaf segments triggered by jasmonic acid methylester, and counteraction by cytokinin

Barley ( Hordeum vulgare L. cv. Salome) primary leaf segments responded to the application of a putative plant growth regulator, ± jasmonic acid methylester (JA-Me). with accelerated senescence, as indicated by the loss of chlorophyll and the rapid decrease in activity and immunoreactive protein content of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase, EC 4.1.1.39). The senescence-promoting action of JA-Me differed in light and in darkness; e.g. the initial rates of chlorophyll and RuBP carboxylase breakdown were markedly higher in light than in darkness in the presence of 4.10⁻⁵ M JA-Me. Cytokinin (benzyladenine, 4.10⁻⁵ M ) stopped the loss of chlorophyll and RuBP carboxylase during senescence; however, the rapid drop induced by JA-Me in the early phase of leaf segment senescence could not be prevented by concomitant or previous addition of BA. On the other hand, BA added 24 h after JA-Me application resulted in a recovery of chlorophyll and RuBP carboxylase at the later stages, indicating a possible rapid inactivation of JA-Me in the tissues. The activities of a number of other chloroplastic and cytosolic enzymes were not significantly altered in JA-Me-treated leaf segments compared with controls floated on water. Time-dependent chlorophyll decrease in isolated chloroplasts did not change upon JA-Me addition to the isolated organelles. It is suggested that JA-Me acts on chloroplast senescence by promoting cytoplasic events which eventually bring about the degradation of chloroplast constituents.     

Differential Effect of Auxin on Molecular Weight Distributions of Xyloglucans in Cell Walls of Outer and Inner Tissues from Segments of Dark Grown Squash (Cucurbita maxima Duch.) Hypocotyls

Effects of indole-3-acetic acid (IAA) on the mechanical properties of cell walls and structures of cell wall polysaccharides in outer and inner tissues of segments of dark grown squash (Cucurbita maxima Duch.) hypocotyls were investigated. IAA induced the elongation of unpeeled, intact segments, but had no effect on the elongation of peeled segments. IAA induced the cell wall loosening in outer tissues as studied by the stress-relaxation analysis but not in inner tissues. IAA-induced changes in the net sugar content of cell wall fractions in outer and inner tissues were very small. Extracted hemicellulosic xyloglucans derived from outer tissues had a molecular weight about two times as large as in inner tissues, and the molecular weight of xyloglucans in both outer and inner tissues decreased during incubation. IAA substantially accelerated the depolymerization of xyloglucans in outer tissues, while it prevented that in inner tissues. These results suggest that IAA-induced growth in intact segments is due to the cell wall loosening in outer tissues, and that IAA-accelerated depolymerization of hemicellulosic xyloglucans in outer tissues is involved in the cell wall loosening processes.     

Interaction between Senescence and Wounding in Oat Leaves

A study was made of the influence of wounding on the senescence of standard oat leaf segments in the dark. Wounding was by either subdividing the 3 centimeter long segments into 5 millimeter subsegments, gently scraping the adaxial surface of the segments with a sharp blade, making transverse linear cuts, or by making many small holes with a needle. Wounding considerably delayed the loss of both chlorophyll and protein in the dark and the amount of inhibition was roughly proportional to the intensity of wounding. With surface wounding, the inhibition of senescence was detectable from the first day of dark incubation; other methods caused moderate promotion of senescence for the first 2 days but decreased the loss of chlorophyll and protein thereafter. A number of senescence-modifying substances acted similarly on both unwounded and wounded segments, but the amount of chlorophyll and protein in the wounded segments was always more than in the respective controls. Cytokinins, however, provided an exception, since their effect was actually decreased by wounding. The proteases operating at pH 4.1 and 6.6 were both clearly less active in the wounded leaves than in controls. The possible mechanism of this inhibitory effect of wounding on senescence is discussed.     

Regulation of ethylene biosynthesis during senescence of oat leaf segments

The evolution of endogenous ethylene, the conversion of 1-aminocylopropane-1-car-boxylic acid (ACC) to ethylene and the amounts of ACC (free and conjugated) have been followed during the senescence of oat ( Avena sativa L. cv. Victory) leaf segments. During the first three days of incubation of leaf segments in darkness, endogenous ethylene evolution and ACC-dependent ethylene production displayed a close relationship, both showing an increase followed by a decrease to the basal rate. However, unlike ethylene production, the level of ACC increased during the five days of incubation in the dark without any decline. It is concluded that ACC synthesis does not limit ethylene production, at least in the last stages of leaf senescence when ethylene production markedly decreased. The level of conjugated ACC increased and reached a plateau already at the first day of incubation. Yet, at the progressive stages of senescence, when the level af ACC gradually increased, no further conjugation of ACC could be detected. Thus, conjugation of ACC cannot account for ethylene drop at the last stages of oat leaf senescence.     

Quantitative and qualitative changes in cell wall polysaccharides in relation to growth and cell wall loosening in Lactuca sativa hypocotyls

The correlation between hypocotyl elongation, cell wall loosening and changes in cell wall polysaccharides was studied using intact lettuce seedlings grown in the dark or in light together with gibberellic acid (GA) and/or 5-fluorodeoxyuridine (FUDR). The following results were obtained:
1) The production of pectic, hemicellulosic and cellulosic polysaccharides look place in parallel with hypocotyl elongation, which was substantially affected by different growth conditions.
2) The mole percentage sugar composition of pectic and hemicellulosic polysaccharides changed in response to dark, light, GA, or FUDR treatments.
3) The amounts of xylose and glucose in hemicellulosic polysaccharides and those of galactosc, rhumnose and uronic acid in pectic polysaccharides increased in parallel with hypocotyl elongation.
4) Statistical analysis of the quantitative relationship between sugars composing polysaccharides revealed that the uronic acid content changed in parallel with those of rhamnose and galactose in pectic polysaccharides, and the content of xylose varied in parallel with those of fucose and glucose.
5) The content of hemicellulosic polysaccharides was correlated with cell wall loosening represented by a decrease in the minimum stress-relaxation time. Changes in the stress-relaxation time value were correlated with those in the content of araltinose and galactose in hemicellulosic polysaccharides.
Based on these results, the relationship between hypocotyl elongation, changes in cell wall polysaccharides, and cell wall loosening is discussed with respect to the effect of GA and FUDR on hypocotyl elongation.     

Leaf senescence and lipid peroxidation: Effects of some phytohormones, and scavengers of free radicals and singlet oxygen

During in vitro senescence (chlorophyll loss) of oat ( Avena sativa L. cv. Victory) leaf segments and of leaf discs of Rumex obtusifolius L, the activity of catalase decreases and lipid peroxidation increases. The activity of superoxide dismutase (SOD) decreases in Rumex leaf discs but changes little in oat leaf segments. Kinetin treatment of oat leaf segments, and GA₃ treatment of Rumex leaf discs, inhibit decline in the enzyme activities and increase in the level of lipid peroxidation and strongly inhibit senescence. In either leaf tissue a treatment with ethanol or vitamin E (scavengers of free radicals) or with diphenylisobenzofuran (scavenger of singlet oxygen) results in a strong inhibition of lipid peroxidation and senescence, but does not affect much the decline in the SOD and catalase activities. It is concluded that, i) senscence-associated lipid peroxidation is induced by free radicals and singlet oxygen; and, ii) kinetin and GA₃ inhibit senescence mainly by a modulation of lipid peroxidation through maintaining high levels of such cellular scavengers as SOD and catalase.     

Senescence in Detached Oat Leaves I. Changes in Free Amino Acid Levels

Changes in the levels of free amino acids have been measuredduring light and dark senescence of oat leaf segments. Leaveswere aged either on water, 5 ppm kinetin or 30 ppm abscisicacid. The patterns with which levels of individual amino acidschange differ a great deal in leaves senescing either in darkor light, signifying that different mechanisms may regulateoat leaf senescence in light and dark. Levels of serine andmost of the other amino acids that increase substantially duringdark senescence of oat leaves change parallel to mitochondrialrespiration. Kinetin depresses the rise in amino acids justas it does with respiration in the dark. The synthesis of serineproteases does not seem to be limited by the availability ofendogenous serine. The levels of glutamine increase dramaticallyin leaves kept in light (ca. 2,200% of initial value within7 days) but only a little in the dark, which may reflect a possiblerole of photorespiration during the senescence of oat leavesin the light. Abscisic acid enhances the release of amino acidsmore strongly in the light than dark. The senescence promotingeffect of abscisic acid in the light seems to bring about changesin amino acid levels similar to those that appear in leavessenescing on water in the dark. ¹ Present address: C.F. Kettering Research Laboratory, 150 EastSouth College Street, Yellow Springs, Ohio 45387, U.S.A. (Received June 24, 1981; Accepted October 30, 1981)     

Senescence‐induced loss in photosynthesis enhances cell wall β‐glucosidase activity

A link between senescence‐induced decline in photosynthesis and activity of β‐glucosidase is examined in the leaves of Arabidopsis. The enzyme is purified and characterized. The molecular weight of the enzyme is 58 kDa. It shows maximum activity at pH 5.5 and at temperature of 50°C. Photosynthetic measurements and activity of the enzyme are conducted at different developmental stages including senescence of leaves. Senescence causes a significant loss in total chlorophyll, stomatal conductance, rate of evaporation and in the ability of the leaves for carbon dioxide fixation. The process also brings about a decline in oxygen evolution, quantum yield of photosystem II (PS II) and quantum efficiency of PS II photochemistry of thylakoid membrane. The loss in photosynthesis is accompanied by a significant increase in the activity of the cell wall‐bound β‐glucosidase that breaks down polysaccharides to soluble sugars. The loss in photosynthesis as a signal for the enhancement in the activity of the enzyme is confirmed from the observation that incubation of excised mature leaves in continuous dark or in light with a photosynthesis inhibitor 3‐(3,4‐dichlorophenyl)‐1, 1‐dimethylurea (DCMU) that leads to sugar starvation enhances the activity of the enzyme. The work suggests that in the background of photosynthetic decline, the polysaccharides bound to cell wall that remains intact even during late phase of senescence may be the last target of senescing leaves for a possible source of sugar for remobilization and completion of the energy‐dependent senescence program.     

Influence of Different Cytokinins on the Transpiration and Senescence of Excised Oat Leaves

In order to investigate the possibility that cytokinins control transpiration indirectly through affecting leaf senescence, a direct comparison was made of the effect of different cytokinins on transpiration and senescence of oat leaves (Avena sativa L. cv. Forward). Senescence was assessed by measuring chlorophyll loss. The synthetic cytokinins N⁶ benzyladenine (BA) and kinetin delayed senescence and increased transpiration of oat leaves to a greater extent than did the naturally occurring compounds zeatin, N^b-Δ² isopentenyladenine (i⁶ Ade) and 6-ø-hydroxybenzyladenosine (hyd-BA riboside). During the early stages of the transpiration experiment zeatin showed similar or greater activity than BA. This period was longest when freshly excised leaves were used, was reduced when leaves were used after incubation in distilled water in the dark for 20 h and was eliminated by incubation in cytokinin solution in the dark. After this period the activity of zeatin declined relative to BA. The effect of cytokinins in increasing transpiration occurred only in the light; no effect was observed in the dark. BA showed higher activity than zeatin in senescence tests but both cytokinins were less effective as the tests progressed, this decrease in activity being more rapid when older leaves were used. The results are discussed in relation to the mechanisms by which endogenous cytokinins might control sensecence and transpiration in oat leaves and to the value of the oat leaf senscence and transpiration bioassays as tests for cytokinin activity of plant extracts.     

Effects of growth regulators and light on chloroplasts NAD(P)H dehydrogenase activities of senescent barley leaves

The activities NADH and NADPH dehydrogenases were measured with ferricyanide as electron-acceptor (NADH-FeCN-ox and NADPH-FeCN-ox, respectively) in mitochondria-free chloroplasts of barley leaf segments after receiving various treatments affecting senescence. NADPH-FeCN-ox declined during senescence in the dark, in a way similar to chlorophyll and Hill reaction, and increased when leaf segments were incubated at light. These results suggest that NADPH-FeCN-ox is related to some photosynthetic electron transporter activity (probably ferredoxin-NADP⁺ oxidoreductase). In contrast, NADH-FeCN-ox is notably stable during senescence in the dark and at light. This activity increased during incubation with kinetin or methyl-jasmonate (Me-JA) but decreased when leaf segments were treated with abscisic acid (ABA). The effects of the inhibitors of protein synthesis cycloheximide and chloramphenicol suggest that the changes of NAD(P)H dehydrogenase activities may depend on protein synthesis in chloroplasts. In senescent leaf, chloroplast NADH dehydrogenase might be a way to dissipate NADH produced in the degradation of excess carbon which is released from the degradation of amino acids.Abbreviations ABA abscisic acid - DCPIP 2,6-dichlorophenol-indo-phenol - DOC deoxycholate - Me-JA methyl jasmonate - NADH-FeCN-ox NADH ferricyanide oxidoreductase - NADPH-FeCN-ox NADPH ferricyanide oxidoreductase     

Jasmonic Acid Inhibits the IAA-Induced Elongation of Oat Coleoptile Segments: a Possible Mechanism Involving the Metabolism of Cell Wall Polysaccharides

The effects of jasmonic acid (JA) on the IAA-induced elongationof segments of etiolated oat (Avena sativa L. cv. Victory) coleoptileswere studied. Exogenously applied JA substantially inhibitedIAA-induced elongation of oat coleoptile segments. The inhibitionof the growth of oat coleoptile segments due to JA appeared2 h after the application of JA with IAA. JA did not affectthe consumption of oxygen by the segments, the osmolarity ofthe cell sap or the IAA-induced loosening of cell walls, whichwas recognized as a decrease in the minimum stress-relaxationtime (T₀). JA was extremely effective in preventing increasesin the amount of the cell wall polysaccharides in both the non-cellulosicfraction and the cellulosic fraction during coleoptile growthin the presence and in the absence of IAA. Inhibition of thegrowth of oat coleoptile segments induced by JA was partiallyreversed by the simultaneous addition of sucrose to the testsolution. From these results, it appears that JA inhibits IAA-inducedelongation of oat coleoptile segments by interfering with someaspects of sugar metabolism that are related to the degradationand/or the synthesis of cell wall polysaccharides. (Received March 15, 1994; Accepted August 2, 1994)     

Jasmonates are essential factors inducing gummosis in tulips: mode of action of jasmonates focusing on sugar metabolism

The purpose of this study was to know the mechanism of jasmonates to induce gummosis in tulip (Tulipa gesneriana L. cv. Apeldoorn) shoots, especially on the focus of sugar metabolism. Gummosis in the first internode of tulip plants was induced by the application of methyl jasmonate (JA-Me, 1% w/w in lanolin) and jasmonic acid (JA, 1% w/w in lanolin) 5 days after application and strongly stimulated by the simultaneous application of ethylene-releasing compound, ethephon (2-chloroethylphosphonic acid, 1% w/w in lanolin), although ethephon alone had little effect. JA-Me stimulated ethylene production of the first internodes of tulips, ethylene production increasing up to more than 5 times at day 1 and day 3 after the application. On the other hand, application of ethephon did not increase endogenous levels of jasmonates in tulip stems. Analysis of composition of tulip gums revealed that they were consisted of glucuronoarabinoxylan with an average molecular weight of ca. 700 kDa. JA-Me strongly decreased the total amount of soluble sugars in tulip stems even in 1 day after application, being ca. 50% of initial values 5 days after application, but ethephon did not. However, both JA-Me and ethephon had almost no effect on the neutral sugar compositions of soluble sugars mainly consisting of glucose, mannose and xylose in ratio of 20:2:1 and traces of arabinose. Both JA-Me and ethephon applied exogenously stimulated senescence of tulip shoots shown by the loss of chlorophyll. These results strongly suggest that the essential factor of gummosis in tulips is jasmonates affecting the sugar metabolism in tulip shoots. The mode of action of jasmonates to induce gummosis of tulip shoots is discussed in relation to ethylene production, sugar metabolism and senescence.     

Proteolysis of endogenous substrates in senescing oat leaves : I. Specific degradation of ribulose bisphosphate carboxylase

Proteolysis of ribulose bisphosphate carboxylase (RuBPCase) during senescence was monitored using oat leaf segments (Avena sativa cv Victory), kept in the dark. We here report the development of a novel approach for measuring protein degradation of endogenous substrates both in situ and in vitro in crude extracts using specific antibodies against highly purified polypeptides. The proteolytic products were separated on sodium dodecyl sulfate-gels. They were then electrotransferred onto nitrocellulose paper and identified with specific antibodies to both the large and small subunits of RuBPCase. We could show differences in pH optima between two proteases degrading the subunits of RuBPCase. While both subunits were best hydrolyzed in acid and basic pH, they degraded differently at neutral pH. Furthermore, the large subunit displayed a different pattern of degradative products at the different pH levels. Older leaf segments, which were incubated in darkness, underwent enhanced proteolysis, as compared with young ones. These results show the advantages of the assay in demonstrating: (a) in situ proteolysis of specific substrates in crude extracts without further purification; (b) in vitro differential proteolysis of endogenous substrates during senescence.     

Effects of methyl jasmonate (MeJA) on the dark-induced senescence in oat (Avena sativa L.) leaf segments

To investigate the relationship between methyl jasmonate (MeJA) and ethylene in leaf senescence, we studied the effects of MeJA on ethylene production and ethylene biosynthetic enzyme activities in oat(Avena sativa L.) leaf segments incubated in darkness. MeJA promoted dark-induced senescence judged from the contents of chlorophyll and protein, and increased ethylene production 6 times of the control. MeJA also increased the activities of ethylene biosynthetic enzymes, 1-aminocyclopropane carboxylic acid (ACC) synthase and ACC oxidase as compared to control. In MeJA-treated leaf segments, ACC synthase activity reached its maximum level at 24 h of incubation and ACC oxidase activity peaked at 6 h of incubation. Aminoethoxyvinylglycine (AVG) and Co²⁺, inhibitors of ACC synthase and ACC oxidase respectively, reduced MeJA-induced ethylene production. They also delayed leaf senescence that was promoted by the treatment of MeJA. From these results, we can suggest that MeJA increased the activities of ACC synthase and ACC oxidase, these increased activities lead to increase in ethylene production and this increased ethylene production might promote dark-induced leaf senescence.     

Metabolism of Oat Leaves during Senescence: V. Senescence in Light

A comparison has been made of the progress of senescence in the first leaf of 7-day-old oat plants (Avena sativa cv. Victory) in darkness and in white light. Light delays the senescence, and intensities not over 100 to 200 ft-c (1000-2000 lux) suffice for the maximum effect. In such intensities, chlorophyll loss and amino acid liberation still go on in detached leaves at one-third to one-half the rate observed in darkness; however, when the leaves are attached to the plant, the loss of chlorophyll in 5 days is barely detectable. Transfer of the leaves from 1 or 2 days in the low intensity light to darkness, or vice versa, shows no carryover of the effects of the preceding exposure, so that such treatment affords no evidence for the photoproduction of a stable substance, such as cytokinin, inhibiting senescence. Light causes a large increase in invertaselabile sugar and a smaller increase in glucose, and application of 100 to 300 mm glucose or sucrose in the dark maintains the chlorophyll, at least partially. Correspondingly, short exposure to high light intensity, which increased the sugar content, had a moderate effect in maintaining the chlorophyll. However, 3-(3,4-dichlorphenyl)-1,1-dimethylurea (DCMU) completely prevents the increases in sugars and yet does not prevent the effect of light on senescence, whether determined by chlorophyll loss or by protein hydrolysis. Light causes a 300% increase in the respiration of detached oat leaves, and kinetin lowers that only partly, but unlike the increased respiration associated with senescence in the dark, the increase in the light is fully sensitive to dinitrophenol, and therefore cannot be ascribed to respiratory uncoupling. The increased respiration in light is prevented by DCMU, parallel with the prevention of sugar formation. It is therefore ascribed to the accumulation of soluble sugars, acting as respirable substrate. Also, l-serine does not antagonize the light effect. For all of these reasons, it is concluded that the action of light is not mediated by photosynthetic sugar formation, nor by photoproduction of a cytokinin. Instead, we propose that light exerts its effect by photoproduction of ATP. The action of sugars is ascribed to the same mechanism but by way of respiratory ATP. This hypothesis unifies most of the observed phenomena of the senescence process in oat leaves, and helps to explain some of the divergent findings of earlier workers.