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.     

Induction of ethylene synthesis and lipid peroxidation in damaged or TMV-infected tobacco leaf tissues by light

The effect of light on ethylene and ethane production in damaged leaf tissues was investigated. When whole leaves of tobacco cv. Samsun NN were damaged with liquid nitrogen, the ethylene formation was the highest, if 100?% of leaves were injured and were kept in the light, the lowest when leaves after 100?% injury were kept in darkness. Ethane production (lipid peroxidation) could be detected only in damaged, but not in control leaves, and was much higher in light than in darkness. In addition, there was a strong degradation of chlorophyll of damaged leaves kept in light. In light aminoethoxy-vinylglycine (AVG) inhibited ethylene formation in control, non-damaged whole leaves effectively, but in leaves with 100?% damage the inhibitory effect was much weaker and similar to the effect of propyl gallate (PG), a free radical scavenger. Both AVG and PG treatments decreased ethylene formation by control leaf discs and discs with 100?% damage. Ethane production was significantly inhibited by PG and slightly by AVG in the case of 100?% damage. Tiron, another free radical scavenger gave similar results on leaf discs as PG did. Paraquat (methylviologen, Pq), as a photosynthesis inhibiting and reactive oxygen species (ROS) producing herbicide produced a large amount of ethylene and ethane in light but very small amount in darkness. In accordance, tobacco mosaic virus (TMV) infection on the necrotic host resulted in significantly larger amount of ethylene and ethane formation in light than in darkness. We conclude that ethylene and ethane production of damaged plant tissues is strongly induced by light and ROS that are involved in this induction.     

Effect of regurgitant from Leptinotarsa decemlineata on wound responses in Solanum tuberosum and Phaseolus vulgaris

The effect of regurgitant from Leptinotarsa decemlineata Say larvae on wound-induced responses was studied using two plant species, Solanum tuberosum L. and Phaseolus vulgaris L. Wounding of one leaf of intact S. tuberosum plants differentially affected ethylene production and activities of peroxidase and polyphenol oxidase. Only polyphenol oxidase activity was stimulated by wounding in both wounded and systemic leaves. Peroxidase activity was not affected by wounding. Wounding caused only a transient increase of ethylene production from wounded leaves. The application of regurgitant to wound surfaces stimulated ethylene production as well as activities of peroxidase and polyphenol oxidase in both wounded and systemic leaves. Wounding significantly enhanced ethylene production and polyphenol oxidase activity in wounded and systemic leaves of P. vulgaris . The application of regurgitant caused an amplification of ethylene production, peroxidase activity, and polyphenol oxidase activity, in both wounded and systemic leaves of bean plants. Several substances were tested for their role as possible endogenous signals in P. vulgaris . Hydrogen peroxide and methyl jasmonate appeared as potential local and systemic signals of ethylene formation in wounded bean plants. Local ethylene production in leaf discs was differentially affected by the regurgitant application in potato versus bean plants. While all tested concentrations of regurgitant caused stimulation of ethylene formation from potato leaf discs, ethylene production was completely inhibited by increasing concentrations of the regurgitant in bean leaf discs. Our data present evidence that ethylene may play an important role in the interaction between plants and herbivores at the level of recognition of a particular herbivore leading to specific induction of signalling cascades.     

Patterns of ehtylene production in senescing leaves

Changes in the patterns of ethylene production, chlorophyll content, and respiration were studied in relation to the senescence of intact leaves and leaf discs. The primary leaves of pinto bean, which abscise readily during natural senescence, and tobacco and sugar beet leaves, which do not abscise, were used. A decrease in the rate of ethylene production and respiration, during the slow phase of chlorophyll degradation, was observed in leaf-blade discs cut from mature leaves and aged in the dark. During rapid chlorophyll loss both ethylene production and respiration increased and then decreased. These climacteric-like patterns were shown by leaf discs of all three species. Discs taken from leaves that had been senescing on the plant also showed a climacteric-like rise in ethylene production but not in respiration, which decreased continuously with leaf age. Climacteric-like patterns in the rise of ethylene and respiration for leaf discs were also shown by the petioles of both bean and tobacco leaves. This indicates that the rise of ethylene and respiration is characteristic of the general process of senescence in leaves and is not restricted to the abscission process. In contrast to the ethylene-forming systems in climacteric fruits and many flowers, the one in leaves declines sharply in the early stages of senescence. The subsequent rise of ethylene production appears to be associated with the rapid phase of chlorophyll breakdown, and may indicate the final stage of the senescence process during which ethylene could be actively involved in inducing leaf abscission.     

Effect of Ethylene on Sucrose Uptake in Root Discs of Sugar Beet

Exogenously-added ethylene stimulated active sucrose uptakein root discs of sugar beet (Beta vulgaris L.) in a log dose-linearresponse manner. The ethylene precursor, 1-aminocyclopropane-1-carboxylicacid (ACC) stimulated both endogenous ethylene production andsucrose uptake. Conversely, an inhibitor of ACC synthesis, aminoethoxyvinylglycine(AVG) inhibited both endogenous ethylene production and sucroseuptake. Exogenously-added ethylene can overcome the AVG effecton sucrose uptake. Root tissue from freshly-harvested sugarbeet plants contain gas-phase ethylene levels slightly belowthat required to stimulate active sucrose uptake. No differenceswere found in gas-phase ethylene levels in the root tissue ofsugar beet cultivars having different concentrations of sucrose.The root tissue has an inherent capacity to synthesize ACC andethylene at high rates. Like ethylene, propylene can stimulate active sucrose uptakein beet root discs, but it is not detected in the gas phaseof the tissue. Acetylene, propane, and ethane had no effecton sucrose uptake. Exogenously-added IAA and ABA each make ethylenesensitivetissue insensitive to ethylene stimulation of sucrose uptake.Other plant hormones have no apparent effect on the ethyleneresponse. The role that ethylene may play on sucrose uptakein root tissue of sugar beet is discussed. (Received February 12, 1986; Accepted April 22, 1986)     

In vivo fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase by reactive oxygen species in an intact leaf of cucumber under chilling-light conditions

Previous studies have demonstrated that the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco) is site-specifically cleaved by a hydroxyl radical (*OH) generated in the illuminated chloroplast lysates or by an artificial *OH-generating system. However, it is not known whether such cleavage of the LSU by reactive oxygen species (ROS) actually occurs in an intact leaf. When leaf discs of chilling-sensitive cucumber (Cucumis sativus L.) were illuminated at 4 degrees C, five major fragments of the LSU were observed. This fragmentation was completely inhibited by ROS scavengers, such as n-propyl gallate (for *OH) and 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron) (for superoxide). FeSO4 stimulated this fragmentation, whereas an iron-specific chelator, deferoxamine, suppressed it. Furthermore, such fragments were identical to those generated from the purified Rubisco by an *OH-generating system in vitro on two-dimensional PAGE. These results indicate that the direct fragmentation of the LSU by reactive oxygen species also occurs in an intact leaf.     

Light-dependence of paraquat-initiated membrane deterioration in bean plants. Evidence for the involvement of superoxide

Treatment of primary bean leaves with 10 mg/I paraquat induces the formation of gel phase lipid in microsomal and chloroplast membranes and enhances the activity of superoxide dismutase, but only if the leaves are exposed to light. These light-dependent changes in membrane lipid phase properties show a close temporal correlation with enhanced O₂⁷ production by illuminated chloroplasts and the onset of lipid peroxidation. Malondialdehyde and ethane, which are both formed during lipid peroxidation, are produced in large amounts by paraquat-treated leaves exposed to light, but not by those maintained in the dark. Electron spin resonance measurements indicate that production of O₂⁷ by illuminated chloroplasts is more than 2-fold greater in the presence of paraquat than in its absence. The identity of the radical formed by illuminated chloroplasts in the presence of paraquat was confirmed by using the diagnostic spin trap 5,5'-dimethyl-l-pyrroline-l-oxide and by establishing that its formation is sensitive to superoxide dismutase. The observations collectively indicate that paraquat-mediated membrane deterioration is light-dependent and attributable to enhanced O₂⁷-production.     

Starch Synthesis from Exogenous Sugars in Tobacco Leaf Discs

Sets of discs were taken from leaves of destarched tobacco plants(Nicotiana tabacum L. cv. xanthii) and floated on solutionsof sucrose or glucose in the dark. Abundant starch was formedin the youngest leaves but there was a marked decline with leafage.By contrast, when replicate sets of discs were floated on waterand illuminated, photosynthetic starch formation was similarin the differently aged leaves. Uptake of sugar, measured bydry weight increases and incorporation of [¹⁴C]sucrose, wasnot dependent on leaf age. The possibility that physiologicalchanges, relating to ageing and import/export status of theleaf, regulate the metabolism of sugar to starch was examined.Increasing retention of sugar in the minor veins is likely tobe a major factor. Invertase activities were measured and foundto be similar in the differently aged leaves. Respiration ratesdeclined with increasing leaf age. Speculations concerning changesin selective permeability of the chloroplast membrane are alsodiscussed.     

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.     

Inactivation in vivo of basic peroxidase and increased content of H2O2 in grapevine leaves post treatment with DTT and paraquat

Substantial differences in the in vivo effect of paraquat (Pq) and DTT on basic peroxidase (GBPx) activity and on H2O2 levels were found in grapevine leaves cv. Sultana. GBPx activity decreased and H2O2 levels increased in illuminated Pq treated leaf-discs. Inactivation of GBPx and accumulation of H2O2 depended on the duration and intensity of the illumination to which discs were exposed. Since GBPx was inactivated directly by H2O2 and not by Pq in leaf extracts, and since GBPx are cytosolic isoenzymes and H2O2 is a stable molecule that can easily permeate chloroplast membranes, we concluded that Pq inactivation of GBPx in vivo is mediated by H2O2. In contrast to the effect induced by Pq, DTT directly inactivated GBPx in leaf extracts. In leaf-discs, however, it reduced GBPx activity in the absence of light, although the levels of H2O2 increased only after exposure of the discs to high irradiance, suggesting that under excess of light, a significant fraction of the photosynthetically produced electrons are dissipated through the water-water cycle and H2O2 accumulates as a consequence of GBPx inactivation.     

The Production of Ethylene by Whole Apple Fruits and by Tissue 1

It is shown that the production of ethylene by whole apples,discs of peel prepared from them, and extracts prepared fromthe discs and supplied with linolenic acid, increases simultaneouslyduring the development of the respiration climacteric in apples.As the climacteric peak is reached, the ability of the extractsto produce ethylene declines and this is associated with a rapidloss of ethylene-producing activity on short term (up to 24h) ageing of the peel discs from which the extracts were obtained. It is suggested that the gmn.ll ethylene production by extracts(with linolenic acid) from pre-climacteric fruits, which arenot themselves evolving sufficient ethylene for its detectionin the ambient atmosphere, may be explained on the grounds thateven before the climacterio the fruit is producing small amountsof ethylene within the tissue; the tissue, as the climactericphase is approached, becomes more sensitive to ethylene andautocatalysed production of the gas then stimulates the fruitinto the respiration climacteric.     

Enhancement by ethylene of cellulysin-induced ethylene production by tobacco leaf discs

Cellulysin-induced ethylene production in tobacco (Nicotiana tabacum L.) leaf discs was enhanced several-fold by prior exposure of the leaf tissue to ethylene. This enhancement in the response of the tissue to Cellulysin increased rapidly during 4 and 8 hours of pretreatment with ethylene and resulted from greater conversion of methionine to ethylene. On treatment with Cellulysin, the content of 1-aminocyclopropane-1-carboxylic acid (ACC) in leaf discs not pretreated with ethylene markedly increased while that of the ethylene-pretreated tissue was only slightly higher than in the tissue incubated in the absence of Cellulysin. Ethylene-treated tissue, however, converted ACC to ethylene at a faster rate than air controls. These data indicate that ethylene stimulates Cellulysin-induced ethylene production by stimulating the conversion of ACC to ethylene. Data are also presented on a possible relation of this phenomenon to ethylene produced by the tobacco leaf upon interaction with its pathogen, Alternaria alternata.     

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     

The Effects of Aqueous Extracts of Red Beet Root on Salt Accumulation and Respiration of Discs of Red Beet Root

An account is given of the preparation of aqueous extracts ofred beet root which are shown to stimulate potassium uptakein beet discs washed for a short period, but inhibit potassiumuptake in discs washed for four days or more. Analysis of extractsshowed them to contain organic anions (especially citrate andmalate) which affect both the metabolic phase of potassium uptakeand respiration of the tissue. The effects of extracts and organicacids on uptake of manganese by beet discs is described andcompared with effects on potassium absorption. The results arediscussed with respect to current theories of salt accumulationand in relation to the hypothesis relating an inhibitor of saltaccumulation to the lag phase of ion uptake by beet discs.     

Acetylcholine and Red-light Influence on Ethylene Evolution from Soyabean Leaf Tissues

Acetylcholine (Ach) and red light inhibited ethylene evolutionfrom excised green soyabean (Glycine max (L.) Merr.) leaf discs.Neostigmine, an Ach-esterase inhibitor, mimicked this effectwhile atropine, an Ach antagonist, increased ethylene evolution.Acetylcholine and red light partially overcame the atropine-inducedresponse. The results were interpreted as an influence of acetylcholineupon the ethylene formation process. Glycine max, ethylene inhibition, leaf discs     

Wound ethylene and 1-aminocyclopropane-1-carboxylate synthase in ripening tomato fruit

Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ACC-synthase activity were compared in intact and wounded tomato fruits (Lycopersicon esculentum Mill.) at different ripening stages. Freshly cut and wounded pericarp discs produced relatively little ethylene and had low levels of ACC and of ACC-synthase activity. The rate of ethylene synthesis, the level of ACC and the activity of ACC synthase all increased manyfold within 2 h after wounding. The rate of wound-ethylene formation and the activity of wound-induced ACC synthase were positively correlated with the rate of ethylene production in the intact fruit. When pericarp discs were incubated overnight, wound ethylene synthesis subsided, but the activity of ACC synthase remained high, and ACC accumulated, especially in discs from ripe fruits. In freshly harvested tomato fruits, the level of ACC and the activity of ACC synthase were higher in the inside parts of the fruit than in the pericarp. When wounded pericarp tissue of green tomato fruits was treated with cycloheximide, the activity of ACC synthase declined with an apparent half life of 30–40 in. The activity of ACC synthase in cycloheximide-treated, wounded pericarp of ripening tomatoes declined more slowly.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

Quantitative relationships between osmotic potential amd epicotyl growth in Vigna radiata as affected by osmotic stress and cotyledon excision

Ethylene biosynthesis in leaf discs of tobacco ( Nicotiana tabacum L. cv. Xanthi), as measured by the conversion of L-[3,4-¹⁴C]-methionine to ¹⁴C₂H₄, was markedly inhibited by exogenous ethylene. This inhibition was accompanied by a decrease in total (free + conjugated) content of 1-aminocyclopropane-1-carboxylic acid (ACC), most of which appeared in its conjugated inactive form. The autoinhibitory effect of ethylene was reversible and could be relieved by Ag⁺. The Ag⁺-treated leaf discs, with or without ethylene, contained only free ACC at an increased level. The results suggest that in tobacco leaves, the autoinhibition of ethylene production resulted from reduction in the availability of free ACC, through both suppression of ACC formation and increased ACC conjugation.     

Response of Phloem Loading and Export to Rapid Changes in Sink Demand

Sink demand was abruptly changed for an illuminated sugar beet source leaf by shading the six to ten other source leaves. Export of recently assimilated, labeled material underwent a transient increase and then returned to a steady rate approximately equal to the pretreatment rate. Uncovering the darkened leaves caused a transient decrease in export of ¹⁴C; following recovery there was a gradual decline. It remains to be established whether export of unlabeled reserves occurs in response to increased sink demand. The possibility that phloem loading increases in response to decreased sieve tube turgor was tested. Phloem loading of exogenous ¹⁴C-sucrose increased when turgor in leaf cells was decreased by floating leaf discs on solutions with up to 1 M mannitol osmoticum. However, the increase appeared to be the result of plasmolysis of mesophyll cells possibly resulting from easier access to minor veins via the free space. Phloem loading in leaf discs continued undiminished even though sieve tube-companion cell sucrose concentration exceeded a calculated value of 1 M. Regulation of export to meet sink demand by a direct response of phloem loading to a turgor or concentration set point does not appear to occur. Phloem loading may be promoted by the influx of water which drives mass flow, increasing phloem loading in response to increased velocity of transport.     

Synergistic interactions between volicitin,jasmonic acid and ethylene mediate insect-induced volatile emission in Zea mays

Plants display differential responses following mechanical damage and insect herbivory. Both caterpillar attack and the application of caterpillar oral secretions (OS) to wounded leaves stimulates volatile emission above mechanical damage alone. Volicitin ( N- 17-hydroxylinolenoyl- l -glutamine), present in beet armyworm (BAW, Spodoptera exigua ) OS, is a powerful elicitor of volatiles in excised maize seedlings ( Zea mays cv. Delprim). We consider some of the mechanistic differences between wounding and insect herbivory in maize by examining the activity of volicitin, changes in jasmonic acid (JA) levels, and volatile emission from both intact plant and excised leaf bioassays. Compared to mechanical damage alone, volicitin stimulated increases in both JA levels and sesquiterpene volatiles when applied to intact plants. In a bioassay comparison, excised leaves were more sensitive and produced far greater volatile responses than intact plants following applications of both volicitin and JA. In the excised leaf bioassay, volicitin applications (10–500 pmol) to wounded leaves resulted in dose dependent JA increases and a direct positive relationship between JA and sesquiterpene volatile emission. Interestingly, volicitin-induced JA levels did not differ between intact and excised bioassays, suggesting a possible interaction of JA with other regulatory signals in excised plants. In addition to JA, insect herbivory is known to stimulate the production of ethylene. Significant increases in ethylene were induced only by BAW herbivory and not by either wounding or volicitin treatments. Using intact plant bioassays, ethylene (at 1 µl l⁻¹ or less) greatly promoted volatile emission induced by volicitin and JA but not mechanical damage alone. For intact plants, wounding, elicitor-induced JA and insect-induced ethylene appear to be important interacting components in the stimulation of insect-induced volatile emission.     

Absence of red-light enhancement of phototropism in pea seedlings at limiting irradiances of blue light

The effect of different light qualities (blue, green, white, red and far-red) on ethylene production in leaf discs and flower petal discs of Begonia × hiemalis cv. Schwabenland Red was studied. All the light qualities, except far-red, reduced the ACC-conversion to ethylene in leaf discs by about 70% at a photosynthetic photon flux density (PPFD) of 20 mol m^–2s^–1.Blue and green light were less inhibitory than white and red light at lower PPFD. In all treatments far-red light at 0.5 mol m^–2s^–1 of photon flux density (PFD) stimulated the ACC-conversion to ethylene in leaf discs by about 60–90% compared to the dark-incubated control. White and red light strongly inhibited the -naphthalene-acetic acid (NAA) stimulated ethylene synthesis in leaf discs. The results may suggest that the ethylene production is controlled by phytochrome in the leaves but not in the petals. Lack of coaction of any light quality with silver ions on ethylene production in leaf and petal discs was also observed.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene forming enzyme - NAA -naphthalene-acetic acid - PFD photon flux density - PPFD photosynthetic photon flux density - RH relative air humidity - SAM S-adenosylmethionine - STS silver thiosulphate