Ethylene as a regulator of senescence in tobacco leaf discs

The regulatory role of ethylene in leaf senescence was studied with excised tobacco leaf discs which were allowed to senesce in darkness. Exogenous ethylene, applied during the first 24 hours of senescence, enhanced chlorophyll loss without accelerating the climacteric-like pattern of rise in both ethylene and CO₂, which occurred in the advanced stage of leaf senescence. Rates of both ethylene and CO₂ evolution increased in the ethylene-treated leaf discs, especially during the first 3 days of senescence. The rhizobitoxine analog, aminoethoxy vinyl glycine, markedly inhibited ethylene production and reduced respiration and chlorophyll loss. Pretreatment of leaf discs with Ag⁺ or enrichment of the atmosphere with 5 to 10% CO₂ reduced chlorophyll loss, reduced rate of respiration, and delayed the climacteric-like rise in both ethylene and respiration. Ag⁺ was much more effective than CO₂ in retarding leaf senescence. Despite their senescence-retarding effect, Ag⁺ and CO₂, which are known to block ethylene action, stimulated ethylene production by the leaf discs during the first 3 days of the senescing period; Ag⁺ was more effective than CO₂. The results suggest that although ethylene production decreases prior to the climacteric-like rise during the later stages of senescence, endogenous ethylene plays a considerable role throughout the senescence process, presumably by interacting with other hormones participating in leaf senescence.     

Relationship between Leaf Water Status and Endogenous Ethylene in Detached Leaves

The pattern of changes in the internal concentration of ethylene in response to water stress was investigated in species with leaves that do abscise and leaves that do not abscise. When leaves which abscise were detached and exposed to dry air for up to 6 hours, a continuous increase of internal ethylene was observed. In water-stressed leaves which do not abscise only a transient rise in ethylene occurred. The peak, which was attained after 30 to 120 minutes, depending on the species studied, was followed by a sharp decline to the initial level. The principal site of ethylene production in response to a short period of water stress was in the blades rather than the petioles in both types of leaves. The internal ethylene level in leaves was reduced by pretreatment with the ethoxy analog of rhizobitoxine (an inhibitor of ethylene biosynthesis) or by maintaining the leaves under subatmospheric pressure. The results obtained by these methods showed that ethylene was not involved in the mechanism of stomatal movement in either turgid or in stressed leaves. Also, the increase in leaf abscisic acid content and the depletion of gibberellins induced by water stress were not related to the internal concentration of ethylene in the detached leaf. The different patterns of drought-induced ethylene production observed in the blades of leaves which exhibit abscission compared with those which do not exhibit abscission may indicate the involvement of ethylene in a primary event in the process of leaf abscission induced by water stress.     

Effect of wounding on ethylene biosynthesis and senescence of detached spinach leaves

Parameters of senescence and ethylene biosynthesis pathway were screened simultaneously in detached spinach leaves and leaf discs. Senescence was enhanced by application of 1-aminocyclopropane-1-carboxylic acid (ACC) and was retarded by amino-ethoxyvinylglycine (AVG). Evidence is presented showing that the bursts of both wound- and climacteric-like ethylene promoted senescence of detached leaves and leaf discs. This ethylene-enhanced leaf senescence was dependent on: (a) ethylene production rates in the tissue; (b) the degree of wounding. Wounding resulted in elevated levels of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), which declined in advanced stages of senescence. The results suggest that wounding might be regarded as one of the primary events in the induction of the senescence syndrome in detached leaves and leaf discs, while ethylene is implicated as a regulator of the rate of the process.     

Use of lysophosphatidylethanolamine, a natural lipid, to retard tomato leaf and fruit senescence

We studied the influence of lysophosphatidylethanolamine (LPE) on the pattern and rate of ethylene production and respiration of tomato ( Lycopersicon esculentum cv. H7155) leaflets and fruit. Leaflets that had been senescing on the plant showed a climacteric-like rise in ethylene production but not in respiration rate which decreased continuously with leaf age. Detached leaflets had a climacteric-like pattern in respiration whether they were incubated in complete darkness or in light. Detached leaflets incubated in the dark had higher rates of ethylene production and CO₂ evolution than did light-incubated leaves. There was no change in the pattern of ethylene production or CO₂ evolution as a result of LPE treatment. However, LPE-treated attached and detached leaflets had consistently lower rates of CO₂ evolution. The reduction in CO₂ evolution by LPE was most pronounced at the climacteric-like peak of the detached leaves. LPE-treated leaflets had a higher chlorophyll content and fresh weight and lower electrolyte leakage than the control. LPE-treated fruits had lower rates of ethylene and CO₂ production than did the control. LPE-treated fruits also had higher pericarp firmness and lower electrolyte leakage than the control. The results of the present study provide evidence that LPE is able to retard senescence of attached leaves and detached leaves and tomato fruits. Several recent studies suggest that lysolipids can act in a specific manner as metabolic regulators. Our results suggest a specific role of lysolipid LPE in aging and senescence     

Production and action of ethylene in senescing leaf discs: effect of indoleacetic Acid, kinetin, silver ion, and carbon dioxide

Supraoptimal concentrations of indoleacetic acid (IAA) stimulated ethylene production, which in turn appeared to oppose the senescence-retarding effect of IAA in tobacco leaf discs. Kinetin acted synergistically with IAA in stimulating ethylene production, but it inhibited senescence. Silver ion and CO(2), which are believed to block ethylene binding to its receptor sites, delayed senescence in terms of chlorophyll loss and stimulated ethylene production. Both effects of Ag(+) were considerably greater than those of CO(2). IAA, kinetin, CO(2), and Ag(+), combined, acted to increase ethylene production further. Although this combination increased ethylene production about 160-fold over that of the control, it inhibited senescence. Treatment with 25 mul/l of ethylene in the presence of IAA enhanced chlorophyll loss in leaf discs and inhibited by about 90% the conversion of l-[3,4-(14)C] methionine to (14)C(2)H(4) suggesting autoinhibition of ethylene production.The results suggest that ethylene biosynthesis in leaves is controlled by hormones, especially auxin, and possibly the rate of ethylene production depends, via a feedback control system, on the rates of ethylene binding at its receptor sites.     

Intact Leaves Exhibit a Climacteric-Like Rise in Ethylene Production before Abscission

The rate of ethylene production by intact, attached leaves of cotton plants (Gossypium hirsutum L.) during aging and senescence was studied using a continuous flow system that allowed air around enclosed leaves to be scrubbed to collect and assay ethylene. Senescence of lower leaves began around 150 d after planting in a controlled environment room. A progressive decline in the ethylene production rate was observed when comparing the 3rd, 6th, and 10th leaves from the base with each other. Ethylene production rates of individual leaves also declined over a 50-d period. However, as leaves began to appear chlorotic, a peak of ethylene production occurred that lasted for about 4 d followed by abscission. This peak involved a 3-fold or greater increase in the rate of ethylene production. The data indicate that intact leaves experience a climacteric-like surge in ethylene production after visible symptoms of senescence appear. This “ethylene climacteric” is apparently the signal that initiates hydrolysis of cell walls in the abscission zone.     

Abscission: potentiating action of auxin transport inhibitors

Reduction in petiolar auxin transport has been proposed as one of the functional actions of endogenous or exogenous ethylene as it regulates intact leaf abscission. If this hypothesis is correct, auxin-transport inhibitors should hasten the rate or amount of abscission achieved with a given level of ethylene. Evidence presented here indicates that the hypothesis is correct. Three auxin transport inhibitors promoted ethylene-induced intact leaf abscission when applied to specific petioles or the entire cotton plant (Gossypium hirsutum L., cv. Stoneville 213). In addition, the transport inhibitors caused rapid abscission of leaves which usually do not abscise under the conditions employed. No stimulation of abscission occurred during the initial 3 to 5 days after plants were treated with transport inhibitors unless such treatments were coupled with exogenous ethylene or that derived from 2-chloroethylphosphonic acid. However, vegetative cotton plants did abscise some of their youngest true leaves during the 2nd and 3rd weeks of exposure to transport inhibitor alone. Taken as a whole, the results indicate that reducing the auxin supply to the abscission zone materially increases sensitivity to ethylene, a condition which favors a role of endogenous ethylene in abscission regulation. Such a role of ethylene indicates the importance of auxin-ethylene interactions in the over-all hormone balance of plants and specific tissues.     

Leaf Age and Ethylene-induced Abscission

Ethylene has been generally credited with promoting the abscission of the oldest leaves on a plant first. Vegetative cotton (Gossypium hirsutum L.) seedlings are an exception to this generalization. Under some conditions the younger, apical, unexpanded, or partially expanded leaves abscise before the less young, basal leaves or cotyledons. The degree or extent of apical leaf abscission increases with ethylene concentration and with plant age from 2 to 5 weeks. The response is promoted by auxin transport inhibitors. Usually the leaves which abscise first are those which have just unfolded and ones apical to the opened but unexpanded leaves. With plants with eight or nine leaves and macroscopic leaf buds, after the initial loss of unexpanded leaves, abscission tends to progress downward from the youngest remaining leaves and upward from the oldest leaves. The findings indicate that some characteristic(s) of apical leaves increases their sensitivity to ethylene. The characteristic may be differences in the abscission process between expanded and unexpanded leaves or differences in the hormone complement of the different leaves. Work is under way to modify this young leaf abscission response in an effort to determine its cause.     

Ethylene production and respiration in aging leaf segments and in disks of fruit tissue of normal and mutant tomatoes

Leaf segments of tomato plants (Lycopersicon esculentum Mill.) of a normal strain and of two nonripening mutants rin and nor were aged in darkness. Respiration in leaf segments of all strains followed a climacteric-like pattern which was accompanied by a similar pattern of ethylene production. l-Methionine-U-(14)C vacuum-infiltrated into leaf segments at the beginning of the climacteric-like rise in respiration was metabolized to ethylene and CO(2) during the subsequent 48 hours to about the same extent in all strains. Pericarp disks of immature fruits of all strains also metabolized l-methionine-U-(14)C to ethylene and CO(2) to about the same extent during the first 48 hours following cutting and vacuum infiltration. Conversion of methionine to ethylene in disks was much more efficient than in aging leaf segments. The apparent capacity for increased production of ethylene in aging leaf segments and in response to wounding in pericap disks of rin and nor is contrasted with the absence of a respiratory climacteric and an associated large increase in ethylene production during natural aging of intact fruits of these two strains.     

Deferral of senescence and abscission by chemical inhibition of ethylene synthesis and action in bean explants

Three compounds known to inhibit ethylene synthesis and/or action were compared for their ability to delay senescence and abscission of bean explants (Phaseolus vulgaris L. cv Contender). Aminoethoxyvinyl-glycine (AVG), AgNO₃, and sodium benzoate were infiltrated into the petiole explants. Their effect on abscission was monitored by measuring the force required to break the abscission zone, and their effect on senescence was followed by measuring chlorophyll and soluble protein in the distal (pulvinus) sections. AVG at concentrations between 1 and 100 micromolar inhibited ethylene synthesis by about 80 to 90% compared to the control during sampling periods of 24 and 48 hours after treatment. This compound also delayed the development of abscission and senescence. Treatment with AgNO₃ at concentrations between 1 and 100 micromolar progressively reduced ethylene production, but to a lesser extent than AVG. The effects of AgNO₃ on senescence and abscission were quite similar to those of AVG. Sodium benzoate at 50 micromolar to 5 millimolar did not inhibit ethylene synthesis during the first 24 hours, but appreciably inhibited ethylene synthesis 48 hours after treatment. It also delayed the development of abscission and senescence. The effects of AVG, Ag⁺, and sodium benzoate suggest that ethylene could play a major role in both the senescence induction phase and the separation phase in bean explants.     

Abscission as a mobilization phenomenon

The possibility that leaf abscission might involve a mobilization phenomenon leading to a localized cellular senescence was examined by following dry weight changes, chlorophyll content, and protein levels in proximal and distal tissue of excised pieces of bean petioles from Phaseolus vulgaris L. var. Red Kidney. Nucleic acid levels were determined as were phosphate pool sizes in conjunction with ³²P labeling patterns. Each of these parameters indicated a mobilization into the tissues proximal to the zone of cellular separation at the expense of the distal tissues, suggesting that mobilization and the consequent development of cellular senescence provide a component in the process of bean leaf abscission.     

Sequence of morphological and physiological events during natural ageing and senescence of a castor bean leaf: sieve tube occlusion and carbohydrate back-up precede chlorophyll degradation

The development of castor bean ( Ricinus communis L. var. sanguineus) leaves from bud break to abscission was studied to determine whether senescence of phloem precedes or follows chlorophyll degradation in the course of natural ageing of leaves. The castor bean leaf blade took 20 days for full expansion and its average life span was 60 days. From the day of full expansion on it suffered a substantial loss in N, a small loss in C, K and P and a gain in Ca, Mg and S. The content of soluble sugars increased with time, paralleled by a decrease of photosynthetic activity. Starch accumulated shortly before chlorophyll breakdown. The amino acid level in the leaves decreased steadily together with nitrate reductase and glutamine synthetase activity. Reactive oxygen species increased and oxidation-protecting compounds decreased during the life span of the leaves. Shortly after full leaf expansion an increasing number of sieve plates showed strong callose depositions when visualized by aniline blue method. At day 40 only half of the sieve tubes appeared functional. Chlorophyll breakdown followed these processes with a time lag of approximately 10 days. The sieve tube sap of ageing leaves had the same sucrose concentrations as young leaves, whereas amino acid concentrations decreased. High levels of reduced ascorbic acid and glutathione together with increasing levels of glutaredoxin indicated oxidative strain during senescence. We speculate that the gradual increase of reactive oxygen species during ageing together with the import of calcium ions lead to the stimulation of callose synthesis in plasmodesmata and sieve plates with the consequence of inhibition of phloem transport leading to carbohydrate back-up in the leaf blade. The latter may finally induce chlorophyll breakdown and, at the end, leaf abscission at the petiole base. Thus phloem blockage would precede and may be causal for chlorophyll degradation in leaf senescence.     

Interrelationship between ethylene and growth regulators in the senescence of lettuce leaf discs

The interrelationship between ethylene and growth regulators in the senescence of romaine lettuce (Lactuca sativa L.) leaves was studied. Gibberellic acid (GA₃), kinetin, and 3-indoleacetic acid (IAA) retarded chlorophyll loss from leaf discs which were floated on hormone solutions. Abscisic acid (ABA) and ethephon enhanced chlorophyll loss and antagonized the senescence-retarding effect of GA₃ and kinetin. A high concentration of IAA (10^–4 M) caused accelerated chlorophyll loss, whereas a similar concentration of kinetin neither retarded nor promoted chlorophyll loss. The ineffectiveness of IAA and kinetin at their supraoptimal concentrations in retarding leaf senescence was related to increased production of ethylene induced in the treated leaf discs. GA₃ was the most effective in retarding chlorophyll loss and did not stimulate ethylene production at all. The senescence-enhancing effect of ABA was not mediated by ethylene. However, the moderately increased production of ethylene, induced by relatively high concentrations of ABA, could act synergistically with the latter to accelerate chlorophyll loss. It is proposed that the effectiveness of exogenously applied hormones, both in enhancing and retarding senescence, is greatly affected by the endogenous ethylene concentration of the treated plant tissue.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 2571-E, 1988 series.     

Ethylene perception inhibitor 1-MCP decreases oxidative damage of leaves through enhanced antioxidant defense mechanisms in soybean plants grown under high temperature stress

Ethylene is a stress hormone involved in early senescence and abscission of vegetative and reproductive organs under stress conditions. Ethylene perception inhibitors can minimize the impact of ethylene-mediated stress. The effects of high temperature (HT) stress during flowering on ethylene production rate in leaf, flower and pod and the effects of ethylene inhibitor on ethylene production rate, oxidative damage and physiology of soybean are not understood. We hypothesize that HT stress induces ethylene production, which causes premature leaf senescence and flower and pod abscission, and that application of the ethylene perception inhibitor 1-Methyl cyclopropene (1-MCP) can minimize HT stress induced ethylene response in soybean. The objectives of this study were to (1) determine whether ethylene is produced in HT stress; (2) quantify the effects of HT stress and 1-MCP application on oxidative injury; and (3) evaluate the efficacy of 1-MCP at minimizing HT-stress-induced leaf senescence and flower abscission. Soybean plants were exposed to HT (38/28 °C) or optimum temperature (OT; 28/18 °C) for 14 d at flowering stage (R2). Plants at each temperature were treated with 1-MCP (1 μg L⁻¹) gas for 5 h or left untreated (control). High temperature stress increased rate of ethylene production in leaves, flowers and pods, production of reactive oxygen species (ROS), membrane damage, and total soluble carbohydrate content in leaves and decreased photosynthetic rate, sucrose content, F_v/F_m ratio and antioxidant enzyme activities compared with OT. Foliar spray of 1-MCP decreased rate of ethylene production and ROS and leaf senescence traits but enhanced antioxidant enzyme activities (e.g. superoxide dismutase and catalase). In conclusion, HT stress increased ethylene production rates, caused oxidative damage, decreased antioxidant enzyme activity, caused premature leaf senescence, increased flower abscission and decreased pod set percentage. Application of 1-MCP lowered ethylene and ROS production, enhanced antioxidant enzyme activity, increased membrane stability, delayed leaf senescence, decreased flower abscission and increased pod set percentage. The beneficial effects of 1-MCP were greater under HT stress compared to OT in terms of decreased ethylene production, decreased ROS production, increased antioxidant protection, decreased flower abscission and increased pod set percentage.     

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.     

Auxin-induced ethylene production as related to auxin metabolism in leaf discs of tobacco and sugar beet

Exogenously supplied indole-3-acetic acid (IAA) stimulated ethylene production in tobacco (Nicotiana glauca) leaf discs but not in those of sugar beet (Beta vulgaris L.). The stimulatory effect of IAA in tobacco was relatively small during the first 24 hours of incubation but became greater during the next 24 hours. It was found that leaf discs of these two species metabolized [1-¹⁴C]IAA quite differently. The rate of decarboxylation in sugar beet discs was much higher than in tobacco. The latter contained much less free IAA but a markedly higher level of IAA conjugates. The major conjugate in the sugar beet extracts was indole-3-acetylaspartic acid, whereas tobacco extracts contained mainly three polar IAA conjugates which were not found in the sugar beet extracts. The accumulation of the unidentified conjugates corresponded with the rise of ethylene production in the tobacco leaf discs. Reapplication of all the extracted IAA conjugates resulted in a great stimulation of ethylene production by tobacco leaf discs which was accompanied by decarboxylation of the IAA conjugates. The results suggest that in tobacco IAA-treated leaf discs the IAA conjugates could stimulate ethylene production by a slow release of free IAA. The inability of the exogenously supplied IAA to stimulate ethylene production in the sugar beet leaf discs was not due to a deficiency of free IAA within the tissue but rather to the lack of responsiveness of this tissue to IAA, probably because of an autoinhibitory mechanism existing in the sugar beet leaf discs.     

Mechanical wounding and abscission in citrus

Fruit detachment force (FDF), ethylene evolution, fruit and leaf drop were determined in Citrus sinensis for periods up to 96 h after mechanical wounding. Injury by removing a thin section of mature fruit flavedo reduced FDF, increased ethylene evolution and promoted abscission. Injuring flavedo 1 cm below the calyx was more effective at reducing FDF than injuring flavedo at the equator or the blossom‐end of mature fruit. Injuring the calyx or peduncle of mature fruit, or injuring three leaves closest to the mature fruit did not reduce FDF. Immature fruitlets either did not abscise or underwent low rates of abscission in response to mechanical wounding, depending on age. Inhibiting ethylene binding in wounded mature fruit with 1‐methylcyclopropene (1‐MCP) increased ethylene evolution compared with wounded fruit alone, but the reduction in FDF was similar. When an ethylene biosynthesis inhibitor (aminoethoxyvinylglycine, AVG) was used, reduction in FDF of wounded mature fruit exposed to AVG was similar to that of wounded fruit alone but ethylene production was markedly reduced. Wounding mature leaf blades in the presence or absence of 1‐MCP resulted in elevated but equal ethylene evolution up to 48 h after wounding, however, no leaf drop occurred. Thereafter, ethylene evolution was higher in 1‐MCP‐treated wounded leaves. Removing up to 77% of the total mature leaf area did not cause leaf drop, nor did wounding tissue across the laminar or petiolar abscission zones. Leaflets of 5 mm length reached nearly 100% abscission after mechanical wounding, whereas wounding leaves 20 mm length resulted in 15% abscission. The data suggest that mechanical wounding of flavedo results in mature fruit abscission, and ethylene binding may not be mandatory to initiate abscission in citrus fruit. The differential response of fruit and leaves at different ages to wounding may be related to potential contribution to carbohydrate accumulation, and production and sensitivity of tissues to an abscission signal(s).     

Role of ethylene in the senescence of detached rice leaves

The role of ethylene in the senescence of detached rice leaves in relation to their changes in 1-aminocyclopropane-1-carboxylic acid (ACC) content and ethylene production was studied. In freshly excised rice leaf segments, ACC level and ethylene production rates were very low. Following incubation, the rates of ethylene production increased and reached a maximum in 12 h, and subsequently declined. The rise of ethylene production was associated with a 20- to 30-fold increase in ACC level.

Ethylene seems to be involved in the regulation of the senescence of detached rice leaves. This conclusion was based on the observations that (a) maximum ethylene production preceded chlorophyll degradation, (b) ACC application promoted chlorophyll degradation, (c) inhibitors of ethylene production and ethylene action retarded chlorophyll degradation, and (d) various treatments such as light, cycloheximide, α,α-dipyridyl, Ni²⁺, and cold temperature, which retarded chlorophyll degradation, also inhibited ethylene production.

Abscisic acid promoted senescence but significantly decreased ethylene production, whereas benzyladenine retarded senescence but promoted ethylene production. This is interpreted to indicate that abscisic acid treatment increased the tissue sensitivity to ethylene, whereas benzyladenine treatment decreased it.

     

Exogenous Polyamines Stimulate Ethylene Synthesis by Soybean Leaf Tissues

Exogenous supply of spermine (Spm) markedly stimulated ethyleneevolution from intact soybean leaves of leaf discs, stronglyincreased the level of free 1-aminocyclopropane-1-carboxylicacid (ACC), and slightly stimulated ethylene forming-enzyme(EFE) activity Spm treatment also resulted in leaf epinastyand accelerated leaf senescence Ethylene stimulation was depressed,but not abolished, by light, and was suppressed by inhibitorsof ACC synthase and EFE activity Spermidine had a less pronouncedstimulatory effect on ethylene production whereas the diaminesputrescine and diaminopropane were without effect These resultscontrast with other reports indicating that di- and polyaminesinhibit ethylene biosynthesis in plants, and extend our previousresults on detached tobacco leaves exogenously treated withpolyamines Glycine max, ethylene, polyamines