Differential induction of three pathogenesis-related genes, PR10, PR1b and PR5 by the ethylene generator ethephon under light and dark in rice (Oryza sativa L.) seedlings

Ethylene has been shown to be involved in triggering pathogenesis-related (PR) gene expression mainly in dicotyledonous species; however, less attention has been devoted identifying and characterizing PR genes in rice (Oryza sativa L.), a monocot and a model of cereal crop genera. Here, we demonstrate that ethylene induces at least three important rice PR genes, the PR10, PR1 basic (PR1b), and PR5 genes in rice (cv. Nipponbare) seedling leaf, upon treatment with the ethylene generator, ethephon (ET), in a light-, time- and dose-dependent manner. Induction of these PR genes was partially blocked by cycloheximide (CHX), a eukaryotic cytoplasmic protein synthesis inhibitor, which indicates an involvement of cytoplasmic de novo protein synthesis in their induction. These results clearly indicate a dynamic role for ethylene in PR genes induction in rice.     

Induction of tomato stress protein mRNAs by ethephon, 2,6-dichloroisonicotinic acid and salicylate

To study the possible involvement of plant hormones in the synthesis of stress proteins in tomato upon inoculation with Cladosporium fulvum, we investigated the induction of mRNAs encoding PR proteins and ethylene biosynthesis enzymes by ethephon, 2,6-dichloroisonicotinic acid (INA) and salicylic acid (SA) by northern blot analysis. Ethephon slightly induced some but not all mRNAs encoding intra- and extracellular PR proteins. INA induced all PR protein mRNAs analysed, except for intracellular chitinase and extracellular PR-4. SA induced all PR protein mRNAs analyzed, except for intracellular chitinase and osmotin. None of the inducers affected the expression of ACC synthase mRNA, whereas all three induced ethylene-forming enzyme (EFE) mRNA.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - HR hypersensitive response - INA 2,6-dichloroisonicotinic acid - PR pathogenesis-related - SA salicylic acid - SAR systemic acquired resistance     

Pathogenesis-related proteins and polyamines in a developmental mutant of tomato, epinastic

The polyamine level and the accumulation of pathogenesis-related (PR) proteins were studied in the ethylene overproducing Epinastic (Epi) tomato (Lycopersicon esculentum Mill.) mutant, as compared with its parent, cv VFN8. Neither a decreased putrescine level nor an enhanced production of PR proteins were detected in Epi, contrary to what could be expected from our previous studies (JM Bellés, J Carbonell, V Conejero [1991] Plant Physiol 96: 1053-1059). However, treatment with the ethylene-releasing compound 2-chloroethylphosphonic acid (ethephon) or silver nitrate at high doses induced a decrease in putrescine content and an enhancing of the synthesis of PR proteins in Epi as ascertained by immunoblot analysis using antisera raised against Rutgers tomato PR proteins.     

Concurrent synthesis and degradation of alcohol dehydrogenase in elicitor-treated and wounded potato tubers

The accumulation of alcohol dehydrogenase (ADH) in arachidonic acid-elicited potato (Solanum tuberosum L.) tuber discs was studied. In accordance with our previous report of the accumulation of Adh mRNA beginning 2 hours after elicitor treatment (DP Matton, CP Constabel, N Brisson [1990] Plant Mol Biol 14: 775-783), immunoprecipitation of ADH from in vivo labeled discs indicated that ADH synthesis occurred as early as 12 hours after treatment. However, levels of ADH activity and protein, as shown by enzyme assay and immunoblot, did not rise in parallel but decreased during the first 24 hours of treatment. After 24 hours, ADH activity and protein began to increase, reaching a several-fold increase at 96 hours after elicitation. Water-treated control discs showed a similar though delayed and less pronounced pattern. These results imply a turnover of ADH following elicitor treatment of potato tuber discs. As shown by nondenaturing gel electrophoresis, the synthesis and degradation involved the same ADH isozyme.     

Whole Plant and Leaf Steady State Gas Exchange during Ethylene Exposure in Xanthium strumarium L

The effects of ethylene evolved from ethephon on leaf and whole plant photosynthesis in Xanthium strumarium L. were examined. Ethylene-induced epinasty reduced light interception by the leaves of ethephon treated plants by up to 60%. Gas exchange values of individual, attached leaves under identical assay conditions were not inhibited even after 36 hours of ethylene exposure, although treated leaves required a longer induction period to achieve steady state photosynthesis. The speed of translocation of recently fixed ¹¹C-assimilate movement was not seriously impaired following ethephon treatment; however, a greater proportion of the assimilate was partitioned downward toward the roots. Within 24 hours of ethephon treatment, the whole plant net carbon exchange rate expressed on a per plant basis or a leaf area basis had dropped by 35%. The apparent inhibition of net carbon exchange rate was reversed by physically repositioning the leaves with respect to the light source. Ethylene exposure also inhibited expansion of young leaves which was partially reversed when the leaves were repositioned. The data indicated that ethylene indirectly affected net C gain and plant growth through modification of light interception and altered sink demand without directly inhibiting leaf photosynthesis.     

Light-dependent kinetics of 2-carboxyarabinitol 1-phosphate metabolism and ribulose-1,5-bisphosphate carboxylase activity in vivo

The light-dependent kinetics of the apparent in vivo synthesis and degradation of 2-carboxyarabinitol 1-phosphate (CA1P) were studied in three species of higher plants which differ in the extent to which this compound is involved in the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase (Rubisco) activity. Detailed studies with Phaseolus vulgaris indicate that both the degradation and synthesis of this compound are light-stimulated, although light is absolutely required only for CA1P degradation. We hypothesize that the steady state level of CAIP at any particular photon flux density (PFD) represents a pseudo-steady state balance between ongoing synthesis and degradation of this compound. The rate of CA1P synthesis in P. vulgaris and the resultant reduction in the total catalytic constant of Rubisco were maximal at 200 micromoles quanta per square meter per second following a step decrease from a saturating PFD, and substantially faster than the rate of synthesis in the dark. Under these conditions an amount of CA1P equivalent to approximately 25% of the Rubisco catalytic site content was synthesized in less than 1 minute. The rate of synthesis was reduced at higher or lower PFDs. In Beta vulgaris, the rate of CA1P synthesis at 200 micromoles quanta per square meter per second was substantially slower than in P. vulgaris. In Spinacea oleracea, an apparent noncatalytic tight-binding of RuBP to deactivated sites on the enzyme was found to occur following a step decrease in PFD. When dark acclimated leaves of P. vulgaris were exposed to a step increase in PFD, the initial rate of CA1P degradation was also found to be dependent on PFD up to a maximum of approximately 300 to 400 micromoles quanta per square meter per second. The rate of degradation of this compound was similar in B. vulgaris. In S. oleracea, a step increase in PFD resulted in noncatalytic RuBP binding to Rubisco followed by an apparent release of RuBP and activation of the enzyme. The in vivo rate of change of Rubisco activity in response to an increase or decrease in PFD was similar between species despite the differences between species in the mechanisms used for the regulation of this enzyme's activity.     

Effects of ethephon and norbornadiene on sterol and glycoalkaloid biosynthesis in potato tuber discs

The accumulation of glycoalkaloids that normally takes place in aerobically incubated potato ( Solanum tuberosum L.) tuber discs has been found to be inhibited by the ethylene-releasing substance ethephon. Using ethephon and the ethylene action inhibitor norborna-2,5-diene, the effect of ethylene on the synthesis of sterols and glycoalkaloids, which partly share their biosynthetic pathway, was investigated.
Control discs showed incorporation of (2-¹⁴C)mevalonic acid into free sterols, steryl esters, steryl glycosides and acylated steryl glycosides at 24 h, thereafter the radioactivity decreased in free sterols and steryl esters concomitant with the appearance of radioactivity in glycoalkaloids. Discs with ethephon additions contained more radioactivity in all sterol classes at all time-points, but no glycoalkaloids were formed.
The enzyme S-adenosyl- l -methionine:sterol C24 methyltransferase (SMT, EC 2. 1. 1. 41), located at one presumed branching point in the sterol and glycoalkaloid pathway, was characterized and found to exhibit similar characteristics as in other plants, but a lower specific activity. The activity of SMT increased in ageing tuber discs and this increase was further stimulated by ethephon, but inhibited by norborna-2,5-diene. The activity of the glycoalkaloid-specific enzyme UDP-glucose:solanidine glucosyltransferase (EC 2. 4. 1) also increased after slicing, but here ethephon additions counteracted the induction. The activity of the sterol-specific UDP-glucose:sterol glucosyltransferase (EC 2. 4. 1) was unaffected by either tuber slicing or ethephon additions.
The results indicate that ethylene stimulates sterol synthesis in wounded potato discs, and that the wound-induction of SMT is regulated by ethylene.     

Mode of action of abscisic Acid in barley aleurone layers : induction of new proteins by abscisic Acid

As part of a continuing effort to elucidate the mode of action of abscisic acid (ABA) in barley (Hordeum vulgare L. cv Himalaya) aleurone layers, we have investigated the induction of several polypeptides by ABA in this tissue. There were nine ABA-induced polypeptides as observed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and considerably more (at least 16 spots) on a two-dimensional gel. These proteins started to show enhanced synthesis 2 to 4 hours after ABA treatment, and their synthesis continued for at least 48 hours. In vitro translation using total RNA isolated from ABA-treated aleurone layers indicated that translatable mRNA levels of these proteins essentially paralleled the levels of in vivo synthesized proteins. The most abundant of the ABA-induced proteins was a 29 kilodalton polypeptide which was also synthesized in tissue incubated without ABA. In vivo synthesis of this protein declined as ABA concentration was decreased, with 1 nanomolar ABA approaching control level. Cell fractionation experiments located the 29 kilodalton major ABA-induced protein in 1,000g and 13,000g pellets; most other induced proteins were in the 80,000g supernatant. The 29 kilodalton protein appeared to be sensitive to degradation by sulfhydryl type proteases. As expected, the induction of these proteins by ABA was suppressed by gibberellic acid. Phaseic acid, the first stable metabolite of ABA, suppressed the gibberellic acid-enhanced α-amylase synthesis but was unable to induce the ABA-induced proteins. None of the ABA-induced proteins were secreted into the incubation medium. A 36 kilodalton ABA-induced protein showed cross-reactivity with antibody against a barley lectin specific for glucosamine, galactosamine, and mannosamine.     

SDS-dependent proteases induced by ABA and its relation to Rubisco and Rubisco activase contents in rice leaves

Protease activities and its relation to the contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase were investigated in detached leaves of rice (Oryza sativa L.) floated on the solutions containing abscisic acid (ABA) or benzyladenine (BA). Rubisco and Rubisco activase contents were decreased during the time course and the decreases were enhanced by ABA and suppressed by BA. The decrease in Rubisco activase was faster than that in Rubisco. SDS-dependent protease activities at 50–70 kDa (rice SDS-dependent protease: RSP) analyzed by the gelatin containing PAGE were significantly enhanced by ABA. RSPs were also increased in attached leaves during senescence. RSPs had the pH optimum of 5.5, suggesting that RSPs are vacuolar protease. Both decrease in Rubisco and Rubisco activase contents and increase in RSPs activities were suppressed by cycloheximide. These findings indicate that the activities of RSPs are well correlated with the decrease in these protein contents. Immunoblotting analysis showed that Rubisco in the leaf extracts was completely degraded by 5 h at pH 5.5 with SDS where it was optimal condition for RSPs. However, the degradation of Rubisco did not proceed at pH 7.5 without SDS where it is near physiological condition for stromal proteins. Rubisco activase was degraded at similar rate under both conditions. These results suggest that RSPs can functions in a senescence related degradation system of chloroplast protein in rice leaves. Rubisco activase would be more susceptible to proteolysis than Rubisco under physiological condition and this could affect the contents of these proteins in leaves.     

Involvement of ethylene in chlorophyll degradation in peel of citrus fruits

The effect of ethylene on chlorophyll degradation in the peel of Robinson tangerine (X Citrus reticulata Blanco) and calamondin (X Citrofortunellamitis [Blanco] Ingram and Moore) fruits was studied. The chlorophyll degrading system in the peel of these two citrus species was not self-sustaining but required ethylene to function. Chlorophyll degradation ceased immediately when fruit were removed from ethylene and held in ethylene-free air at 0.2 atmospheric pressure. However, at atmospheric pressure, chlorophyll degradation continued for 24 hours in the absence of exogenous ethylene. Although chlorophyllase levels were negatively correlated with chlorophyll content in the peel (r = −0.981; P < 0.01), the level of chlorophyllase activity did not change when fruit were removed from ethylene, even though chlorophyll degradation had stopped. From these observations, it was concluded that ethylene is necessary for chlorophyll degradation in the two species of citrus studied, but its primary role is not solely for the induction of chlorophyllase activity.     

Involvement of wound and climacteric ethylene in ripening avocado discs

Avocado (Persea americana Mill. cv Hass) discs (3 mm thick) ripened in approximately 72 hours when maintained in a flow of moist air and resembled ripe fruit in texture and taste. Ethylene evolution by discs of early and midseason fruit was characterized by two distinct components, viz. wound ethylene, peaking at approximately 18 hours, and climacteric ethylene, rising to a peak at approximately 72 hours. A commensurate respiratory stimulation accompanied each ethylene peak. Aminoethoxyvinyl glycine (AVG) given consecutively, at once and at 24 hours following disc preparation, prevented wound and climacteric respiration peaks, virtually all ethylene production, and ripening. When AVG was administered for the first 24 hours only, respiratory stimulation and softening (ripening) were retarded by at least a day. When AVG was added solely after the first 24 hours, ripening proceeded as in untreated discs, although climacteric ethylene and respiration were diminished. Propylene given together with AVG led to ripening under all circumstances. 2,5-Norbornadiene given continuously stimulated wound ethylene production, and it inhibited climacteric ethylene evolution, the augmentation of ethylene-forming enzyme activity normally associated with climacteric ethylene, and ripening. 2,5-Norbornadiene given at 24 hours fully inhibited ripening. When intact fruit were pulsed with ethylene for 24 hours before discs were prepared therefrom, the respiration rate, ethylene-forming enzyme activity buildup, and rate of ethylene production were all subsequently enhanced. The evidence suggests that ethylene is involved in all phases of disc ripening. In this view, wound ethylene in discs accelerates events that normally take place over an extended period throughout the lag phase in intact fruit, and climacteric ethylene serves the same ripening function in discs and intact fruit alike.     

Ethylene-induced Phenylalanine Ammonia-Lyase Activity in Carrot Roots

Ethylene enhanced the activity of phenylalanine ammonialyase in carrot (Daucus carota L., var. “Nauty”) root tissue. Slight increase in enzyme activity was exhibited by root discs incubated in ethylene-free air. It was probably due to the ethylene formed within the sliced tissue. Addition of ethylene to the air stream increased phenylalanine ammonia-lyase activity and the total protein content of the discs until maximum activity was reached after 36 to 48 hours of incubation. The continuous presence of ethylene was required to maintain high level of activity. Ethylene, at a concentration of 10 microliter per liter induced higher activity than at lower or higher concentrations. CO₂ partially inhibited the ethylene-induced activity. Cycloheximide or actinomycin D effectively inhibited the ethylene-induced activity in discs that had not previously been exposed to ethylene. The results appear to support the hypothesis that the mode of action of ethylene may involve both de novo synthesis of the enzyme protein and protection or regulation of activity of the induced enzyme.     

Uptake and Fate of Ethephon ([2-Chloroethyl]phosphonic Acid) in Dormant Weed Seeds

Although ethephon ([2-chloroethyl]phosphonic acid) is often used as a form of liquid ethylene in studies of seed germination, it is not known if ethylene evolved from ethephon in the seed is sufficient to elicit the desired response and/or if ethephon has a regulatory action that alone accounts for the response. For these reasons we studied the uptake and fate of [1,2-¹⁴C]ethephon in dormant seeds of Avena fatua, Sinapis arvensis, Thlaspi arvense, and Chenopodium album. The radioactivity within the seeds was separated into a labile carbon-labeled ethephon/ethylene fraction (64-87%) and, following extraction in methanol-chloroform-water (12:5:3), into fractions associated with insoluble (12-29%) and soluble (3-8%) seed constituents. The radioactivity associated with seed constituents was reduced 5 to 75% by hot alkaline hydrolysis (2.5 n KOH, 70° C for 1 hour). Although a small portion of the ethephon (or metabolite of ethephon/ethylene) taken up by the seeds is tightly bound to the tissues, our results indicate that, at the appropriate external concentrations of ethephon, the amount of ethylene evolved from ethephon within the seeds is sufficient to produce the desired ethylene mediated responses. However, factors affecting the decomposition of ethephon must be considered in the decision as to whether to use ethephon as a liquid supply of ethylene.     

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.     

Identification and kinetics of accumulation of proteins induced by ethylene in bean abscission zones

A two-dimensional gel electrophoresis system that combines a cationic polyacrylamide gel electrophoresis at pH near neutrality with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the spectrum of basic polypeptides that accumulate in bean (Phaseolus vulgaris) abscission zones after treatment with ethylene. Results showed that, as abscission progressed, at least seven basic proteins accumulated in the abscission zone prior to the accumulation of 9.5 cellulase. Six of the seven proteins correspond to pathogenesis-related (PR) proteins. Among them, two isoforms of β-1,3-glucanase and multiple isoforms of chitinase were identified. A 22 kilodalton polypeptide that accumulated to high levels was identified as a thaumatin-like protein by analysis of its N-terminal sequence (up to 20 amino acids) and its serological relationship with heterologous thaumatin antibodies. A 15 kilodalton polypeptide serologically related to PR P1 (p14) from tomato was identified as bean PR P1 (p14)-like protein. The kinetics of accumulation of glucanases, chitinases, thaumatin-like and PR P1 (p14)-like proteins during ethylene treatment were similar and they showed that PR proteins accumulated in abscission zones prior to the increase in 9.5 cellulase. Addition of indoleacetic acid, a potent inhibitor of abscission, reduced the accumulation of these proteins to a similar extent (60%). The synchronized accumulation of this set of PR proteins, early in the abscission process, may play a role in induced resistance to possible fungal attack after a plant part is shed. The seventh protein does not correspond to any previously characterized PR protein. This new 45 kilodalton polypeptide accumulated in abscission zones on exposure to ethylene concomitantly with the increase in 9.5 cellulase. Its N-terminal sequence (up to 15 amino acids) showed some homology with the amino terminal sequence of chitinase. Polyclonal antibodies against chitinase recognized the 45 kilodalton polypeptide, but polyclonal antibodies against the 45 kilodalton protein recognized chitinase weakly. When abscission was inhibited by addition of indoleacetic acid, the accumulation of the 45 kilodalton protein was strongly inhibited (80%). This result suggests that the 45 kilodalton polypeptide may play a more direct role in abscission.     

Polyamines in plants infected by citrus exocortis viroid or treated with silver ions and ethephon

The levels of polyamines in leaves of Gynura aurantiaca DC and tomato, Lycopersicon esculentum Mill. cv Rutgers, infected with citrus exocortis viroid (CEVd) or treated with silver nitrate or ethephon (2-chloroethylphosphonic acid) were measured by HPLC in relation to development of symptoms. Previously it had been demonstrated that treatment of G. aurantiaca plants with silver nitrate or ethephon closely mimicked the effects of viroid infection in the plants. In the studies reported here, a marked decrease in putrescine level was observed in plants infected by CEVd or treated with silver ions or ethephon. There was no significant change in either spermidine or spermine levels. Treatment of G. aurantiaca plants with specific inhibitors of ethylene biosynthesis (aminoethoxyvinylglycine, Co²⁺) or ethylene action (norbornadiene) prevented the decrease of putrescine associated with silver nitrate treatment and had no effect on spermidine or spermine levels. The development of viroid-like symptoms, the production of associated pathogenesis-related proteins, and the rise in protease activity induced by silver nitrate, were all suppressed by exogenous application of putrescine. The decreased level of putrescine as an ethylene-mediated step in the transduction of the viroid and silver or ethephon signaling is discussed.     

Effects of exogenous ethylene on ethylene production in citrus leaf tissue

Exogenous ethylene stimulated ethylene production in intact citrus (Citrus sinensis L. Osbeck cv. “Washington Navel”) leaves and leaf discs following a 24-hour exposure. Studies with leaf discs showed that ethylene production decreased when ethylene was removed by aeration. The extent of stimulation was dependent upon the concentration of exogenous ethylene (1-10 microliters per liter). Silver ion blocked the autocatalytic effect of ethylene at concentrations of 0.5 millimolar and lower, but increased ethylene production at higher concentrations. The stimulating effect of ethylene resulted from the enhancement of both 1-aminocyclopropane-1-carboxylic acid (ACC) formation and the conversion of ACC to ethylene. Whereas autocatalysis was evident following 24 hours incubation, autoinhibition of wound- and mannitol-induced ethylene production was observed during the first 24-hour incubation. Ethylene treatment during this period resulted in a marked decrease in ACC levels and ethylene production rates. Furthermore, in leaf discs treated for 24 hours with ethylene, ethylene production rates increased greatly during the first 2 hours after removal of exogenous ethylene by aeration. This increase was eliminated if the discs were transferred to propylene instead of air, indicating that the autocatalytic effect of ethylene is counteracted by its autoinhibitory effect. It is suggested that autocatalysis involves increased synthesis of ACC synthase and the enzyme responsible for the conversion of ACC to ethylene, whereas autoinhibition involves suppression of the activity of these two enzymes.     

Ethylene-forming Systems in Etiolated Pea Seedling and Apple Tissue

Auxin-induced ethylene formation in etiolated pea (Pisum sativum L. var. Alaska) stem segments was inhibited by inhibitors of RNA and protein synthesis. Kinetics of the inhibitions is described for actinomycin D, cordycepin, α-amanitin, and cycloheximide. α-Amanitin was the most potent and fast-acting inhibitor, when added before induction or 6 hours after induction of the ethylene-forming system. The ethylene-forming system of postclimacteric apple (Malus sylvestris L.) tissue, which is already massively induced, was not further stimulated by auxin. Ethylene production in apples was inhibited least by α-amanitin and most by actinomycin D. The relative responses of the ethylene system in apples to RNA inhibitors were different from the ethylene system of pea stems. However, the protein synthesis inhibitor, cycloheximide, appeared to act equally in both tissue systems. The effect of cycloheximide on ethylene production in postclimacteric apple tissue, already producing large quantities of ethylene, suggests a dynamic regulating system for the synthesis and degradation of the ethylene-forming system.