Regulation of Ethylene Biosynthesis in Virus-Infected Tobacco Leaves : II. TIME COURSE OF LEVELS OF INTERMEDIATES AND IN VIVO CONVERSION RATES

Ethylene production was stimulated severalfold during the hypersensitive reaction of Samsun NN tobacco to tobacco mosaic virus (TMV). Exogenous methionine or S-adenosylmethionine (SAM) did not increase ethylene evolution from healthy or TMV-infected leaf discs, although both precursors were directly available for ethylene production. This indicates that ethylene production is not controlled at the level of methionine concentration or availability, nor at the level of SAM production or concentration. In contrast, 1-aminocyclopropane-1-carboxylic acid (ACC) stimulated ethylene production considerably. Thus, ethylene production is primarily limited at the level of ACC production.     

Differential expression of antisense in regenerated tobacco plants transformed with an antisense version of a tomato ACC oxidase gene

Ethylene production was measured during vegetative and reproductive development in normal tobacco plants and in transgenic tobacco plants carrying antisense genes for tomato ACC oxidase driven by the 35S CaMV promoter (Hamilton et al., 1990). When expressed in three independently derived transgenic plants, the antisense ethylene gene failed to affect ethylene production in young/mature leaves or in stems but it did inhibit ethylene production in roots by 37–58%. Ethylene production in developing flowers (i.e. from small unopened flower buds up until open flowers at anthesis) was not affected in transgenic plants but ethylene production in fruits was inhibited by 35%. The most dramatic effect on ethylene production in transgenic plants was seen immediately after wounding leaf tissue, in which case the antisense gene inhibited wound ethylene production by 72%. Thus, the antisense gene composed of a 35S CaMV promoter driving a heterologous ACC oxidase sequence had differential effects on ethylene production in tobacco plants.     

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.     

The modulation of the conversion of l-aminocyclopropane-l-carboxylic acid to ethylene by light

Endogenous ethylene production of tobacco leaves was similar in light and in darkness. However, the rate of conversion of exogenously applied l-aminocyclopropane-l-carboxylic acid (ACC) to ethylene was reversibly inhibited by light. Virus-stimulated ethylene production, during the hypersensitive reaction of tobacco leaves to tobacco mosaic virus, was likewise inhibited by light. Under such circumstances ethylene production is limited at the level of the conversion of ACC to ethylene. Inhibition of the increase in ACC-stimulated ethylene production by cycloheximide and 2-(4-methyl-2,6-dinitroanilino)-N-methyl-propionamide after shifting leaf discs from light to darkness indicated that de novo protein synthsis was involved. Regulation of ACC-dependent ethylene production by reversible oxidation/reduction of essential SH groups, as suggested by Gepstein and Thimann (1980, Planta 149, 196–199) could be excluded. Instead, regulation of the ACC-converting enzyme at the level of both synthesis/degradation and activation/inactivation is suggested. Phytochrome was not involved in light inhibition, but low intensities of either red or blue light decreased the rate of ACC conversion. Dichlorophenyldimethylurea counteracted the inhibitory effect of light, indicating that (part of) the photosynthetic system is involved in the light inhibition. The ethylene production of Pharbitis cotyledons grown in darkness or light, either in the presence of absence of the inhibitor of carotenoid synthesis, SAN 9789 (norflurazon), supported this view.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - DCMU dichlorophenyldimethylurea - MDMP 2-(4-methyl-2,6-dinitroanilino)-N-methyl-propionamide - SAM S-adenosylmethionine - SH groups sulfhydryl groups - TCA trichloroacetic acid - TMV tobacco mosaic virus     

Induction of pathogenesis-related proteins by spermidine exogenously supplied to detached tobacco leaves

Continuous treatment with spermidine or 1-aminocyclopropane-1-carboxylic acid stimulated ethylene production and ethylene-forming enzyme activity and accelerated chlorophyll breakdown in detached tobacco leaves. The treatments also induced the production of eleven major acidic pathogenesis-related proteins, which were also produced during the hypersensitive reaction to tobacco necrosis virus. A delay between the onset of the stimulated ethylene increase and the detection of PR-proteins was found; ethylene production was stimulated after a few hours of treatment, whereas one, three and all the eleven PR-proteins were detected by polyacrylamide gel electrophoresis of fluid extracts after 2, 4 and 6 days of treatments, respectively. The possible causal relationship between stimulation of ethylene production and PR-protein accumulation is discussed.     

在转基因烟草中表达番茄反义ACC合酶基因及其对芽再生的影响

利用从番茄(Lycopersicum esculentum Mill.)果实中分离到的ACC合酶cDNA,反向置于CaMV 35S启动子的控制之下,并转入烟草(Nicotiana tabacum L.)。PCR扩增证明此反义基因已整合到烟草的基因组上。Northern杂交及逆转录PCR分析表明,这种异源反义基因能在转基因烟草组织中表达,并抑制了烟草内源乙烯的合成,对乙烯合成的抑制在芽再生过程中更为明显,同时这也导致了转基因烟草在组织培养过程中芽再生能力的增强。这些结果从基因水平证明,乙烯在芽形成过程中具有重要的调控功能。     

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.     

The Elicitation of Ethylene Biosynthesis by a Trichoderma Xylanase Is Not Related to the Cell Wall Degradation Activity of the Enzyme

A [beta]-1,4-endoxylanase (EIX) isolated from Trichoderma viride elicits plant defense responses in certain tobacco (Nicotiana tabacum L.) cultivars in addition to its xylan degradation activity. It was not clear whether elicitation occurs by cell wall fragments released by the enzymic activity or by the xylanase protein interacting directly with the plant cells. We used protoplasts isolated from tobacco leaves to test whether the cell wall is required for the stimulation of ethylene biosynthesis by EIX. Protoplasts of tobacco (cv Xanthi) responded to treatment with the EIX, as indicated by an increased production of ethylene and the loss of protoplast viability. Protoplasts prepared from ethylene-pretreated leaves produced more ethylene and had higher rates of cell death in response to EIX than protoplasts prepared from nonethylene-treated leaves. Protoplasts of an EIX-insensitive cultivar of tobacco (Hicks) were insensitive to high concentrations of EIX. The addition of a crude cell wall preparation to protoplasts during incubation with EIX did not enhance the induction of ethylene biosynthesis by nonsaturating as well as saturating concentrations of EIX. These data indicate that the xylanase activity of EIX is unrelated to the elicitation of ethylene biosynthesis through the production of some cell wall fragment, since the protein per se appears capable of eliciting ethylene biosynthesis in protoplasts.     

Effect of glyphosate on ethylene production in tobacco callus

Glyphosate (N-phosphonomethylglycine) caused a significant decrease or a slight increase in ethylene production in tobacco callus (Nicotiana tabacum L.) depending on the concentration of indole-3-acetic acid (IAA) present in the medium. IAA stimulated ethylene production, but a pretreatment with glyphosate greatly reduced the IAA-induced ethylene production. Inasmuch as glyphosate treatment promoted the metabolism of IAA, the decrease in ethylene production induced by glyphosate is attributed to the rapid loss of free IAA in the treated tissue.     

The role of ethylene during the infection of Nicotiana tabacum by Colletotrichum destructivum

Two periods of increased ethylene production were observed after inoculation of Nicotiana tabacum by Colletotrichum destructivum. This pathogen exhibits an intracellular hemibiotrophic infection process, with a biotrophic phase followed by a necrotrophic phase. Ethylene production first increased during the biotrophic phase with a peak at 24 h before the necrotrophic phase. A second increase in ethylene occurred late in the necrotrophic phase when the lesions were expanding. Two different 1-aminocyclopropane-1-carboxylic acid synthase genes showed increased expression after the first ethylene peak with a maximum at 24 h before the second ethylene increase. Expression of an 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) gene increased during the first ethylene peak and then declined at the beginning of the second ethylene increase. A second ACO gene showed relatively little change in expression during infection with slightly higher expression at 24 h before the second ethylene increase, and a third ACO gene showed a progressive decline in expression with a major decrease occurring before the second ethylene increase. Inoculation of ethylene-insensitive tobacco with C. destructivum revealed that it was more susceptible than the wild type. The changes in ethylene production and associated gene expression as well as the increased disease susceptibility of ethylene-insensitive tobacco indicate that ethylene plays a role in this interaction, perhaps as a signalling molecule to trigger defense mechanisms.     

氧和二氧化碳对莱阳梨果肉切片乙烯生物合成的调节作用

跃变期的莱阳梨果肉切片保温12h期间,降低空气中O_2浓度使乙烯生成减少,ACC含量相应增加,解除处理后,除0％O_2处理外都能恢复相应的乙烯生成速率。CO_2对乙烯生成有促进和抑制双重作用,处理初期表现出促进,O_2浓度低时更显著,随保温时间延长CO_2表现出抑制作用并继续增强。CO_2浓度增高,乙烯生成的抑制增强,ACC含量变化与乙烯减少之间没有很好的相应关系,解除CO_2处理后乙烯生成速率不能恢复。     

THE ROLE OF ETHYLENE IN COROLLA UNFOLDING IN IPOMOEA NIL (CONVOLVULACEAE)

The roles of ethylene in corolla growth and senescence have been extensively studied; light, temperature, and abscisic acid (ABA) have already been implicated in rapid corolla opening (unfolding) in morning glory. In the present study, a possible interaction between ABA and ethylene production was examined. While applied ABA promoted corolla unfolding, it also promoted ethylene production. Furthermore, the effect of ABA could be eliminated by the ethylene biosynthesis inhibitors, aminoethoxyvinyl glycine (AVG) and cobalt ions. Simultaneously applied aminocyclopropane carboxylic acid (ACC) augmented the ABA response, but ACC applied alone promoted corolla unfolding as effectively as ABA alone. Measurements of ethylene production during eight successive stages of flower opening showed that the ethylene burst widely reported to occur in opened flowers prior to corolla senescence actually begins before the corolla unfolds. Ethylene production seems to be a later part of the sequence of biochemical events that leads to both corolla unfolding and synchronized inrolling and senescence.     

Biochemical Pathway of Stress-induced Ethylene

Ethylene production from beam and tobacco leaves increased rapidly following the application of toxic compounds such as CuSO₄, Endothal, and ozone. Treatments which increased ethylene evolution also increased the conversion of U-¹⁴C-methionine into ethylene. Cycloheximide inhibited the production of chemical stress-induced ethylene. These results suggest that ethylene is produced by the same biochemical pathway forming basal ethylene, auxin-induced ethylene, or that produced during the ripening of climacteric fruit.     

Ethylene response to pollen tube growth in Nicotiana tabacum flowers

In flowers of Nicotiana tabacum L., pollination induces a transient increase in ethylene production by the pistil. The characteristic dynamics of the increase in ethylene correspond to the main steps of the pollen-tube journey into the pistil: penetration into the stigma, growth through the style, entry into the ovary and fertilization. Ethylene is synthesized de novo in the pistil, and its production is reduced in the dark. Ethylene production was monitored in tobacco flowers after pollination with incongruous pollen from three different Nicotiana species, N. rustica, N. repanda and N. trigonophylla, and with pollen from Petunia hybrida. Pollen from all of these different sources can germinate on the stigma surface but each pollen type shows a different behavior and efficiency in penetrating the pistil tissues. Thus, these different crosses provided a model with which to study the response of the pistil to pollination and fertilization. Ethylene evolution upon pollination in tobacco differed in each cross, suggesting that ethylene is correlated with the response to pollen tube growth in the tobacco flower.     

Expression of the ACC synthase and ACC oxidase coding genes after self-pollination and incongruous pollination of tobacco pistils

In tobacco, as in other species, ethylene is produced in response to pollination. Although tobacco is a self-compatible species, it displays unilateral incongruity with other Nicotianaplants. Incongruous pollination also results in ethylene production, but this production differs depending on the pollen used and is related to the extent to which pollen tubes grow in the tobacco style. In the investigation reported here we followed the expression of the ACC synthase- and ACC oxidase-coding genes upon pollination of tobacco pistils and compared self-pollination with incongruous pollination. The pattern of expression of these genes also correlated with pollen-tube growth, although wounding alone cannot explain the results obtained. We also examined the expression of these genes upon pollination of immature tobacco pistils, in which different pollen tubes grew indistinctly inside the tobacco style and reached the ovary at the same rate. In this situation no significant differences in gene expression could be observed between the different pollinations. Ethephon, a substance that produces ethylene, could, in some cases, minimize the arrest of incongruous pollen tubes inside the style.     

Carbohydrates stimulate ethylene production in tobacco leaf discs : I. Interaction with auxin and the relation to auxin metabolism

Various naturally occurring carbohydrates, applied at a concentration range of 1 to 100 mm, stimulated ethylene production for several days in indoleacetic acid (IAA)-treated or untreated tobacco (Nicotiana tabacum L. cv `Xanthi') leaf discs. The lag period for this sugar-stimulated ethylene production was 8 to 12 hours after excision in the untreated leaf discs, but less than 2 hours in the IAA-treated ones. Among the tested carbohydrates, 12 were found to increase synergistically ethylene production, with d-galactose, sucrose, and lactose being the most active; mannitol and l-glucose had no effect. The extent and duration of the increased ethylene production was dependent upon the type of sugar applied, the tissue's age, and the existence of both exogenous IAA and sugar in the medium. Sucrose appeared to elicit a continuous IAA effect for 48 hours, as expressed by increased ethylene production, even when IAA was removed from the medium after a 4-hour pulse. Sucrose stimulated both the uptake and decarboxylation of [1-¹⁴C]IAA, as well as the hydrolysis of the esteric and amide IAA conjugates formed in the tissue after application of free IAA. This gradual hydrolysis was accompanied by a further accumulation of a third IAA metabolite. Moreover, synthetic indole-3-acetyl-l-alanine increased ethylene production mainly with sucrose, and this effect was accompanied by its increased decarboxylation and turnover pattern suggesting that release of free IAA was involved. An esteric IAA conjugate, tentatively identified by GC retention time was found to be the major component (84%) of the naturally occurring IAA conjugates in tobacco leaves. Accordingly the sucrose-stimulated ethylene production in tobacco leaves can be ascribed mainly to the sucrose-stimulated hydrolysis of the esteric IAA conjugate.     

THE ROLES OF PLANT HORMONES IN THE GROWTH OF THE COROLLA OF GAILLARDIA GRANDIFLORA (ASTERACEAE) RAY FLOWERS

Corolla elongation and the roles of plant hormones in this process in Gaillardia grandiflora Van Houtte ray flowers were examined. The sterile ray flowers elongated during a 2-day period, and corolla growth was accompanied by fresh and dry weight increases and epidermal cell elongation (greatest near the base of the corolla) but not by cell division. Corollas excised from young ray flowers were measured during treatment in vitro with solutions of plant growth regulators. They elongated in response to gibberellins and fusicoccin but did not respond to auxins, cytokinins, abscisic acid, ethylene, or inhibitors of ethylene biosynthesis. Sequential and simultaneous hormone applications indicated no additive or synergistic effects between hormones, but auxin did reduce gibberellin-promoted growth. Analyses of endogenous auxins showed no significant variation, and ethylene production decreased prior to elongation, while a 20-fold increase in endogenous gibberellin activity was observed just prior to rapid corolla elongation. It appears that corolla growth in Gaillardia is accomplished by an increase in gibberellin activity alone, that multiple hormone interactions are not important in the control of corolla growth, and that part of the mode of action of gibberellin is acid-induced growth.     

Synergistic biosynthesis of biphasic ethylene and reactive oxygen species in response to hemibiotrophic Phytophthora parasitica in tobacco plants

We observed the biphasic production of ethylene and reactive oxygen species (ROS) in susceptible tobacco (Nicotiana tabacum 'Wisconsin 38') plants after shoot inoculation with Phytophthora parasitica var nicotianae. The initial transient increase in ROS and ethylene at 1 and 3 h (phase I), respectively, was followed by a second massive increase at 48 and 72 h (phase II), respectively, after pathogen inoculation. This biphasic pattern of ROS production significantly differed from the hypersensitive response exhibited by cryptogein-treated wild-type tobacco plants. The biphasic increase in ROS production was mediated by both NADPH oxidase isoforms, respiratory burst oxidase homolog (Rboh) D and RbohF. Conversely, different 1-aminocyclopropane-1-carboxylic acid synthase members were involved in specific phases of ethylene production: NtACS4 in the first phase and NtACS1 in the second phase. Biphasic production of ROS was inhibited in transgenic antisense plant lines expressing 1-aminocyclopropane-1-carboxylic acid synthase/oxidase or ethylene-insensitive3 as well as in transgenic plants impaired in ROS production. All tested transgenic plants were more tolerant against P. parasitica var nicotianae infection as determined based on trypan blue staining and pathogen proliferation. Further, silencing of NtACS4 blocked the second massive increase in ROS production as well as pathogen progression. Pathogen tolerance was due to the inhibition of ROS and ethylene production, which further resulted in lower activation of ROS-detoxifying enzymes. Accordingly, the synergistic inhibition of the second phase of ROS and ethylene production had protective effects against pathogen-induced cell damage. We conclude that the levels of ethylene and ROS correlate with compatible P. parasitica proliferation in susceptible plants.     

Light inhibition of the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in leaves is mediated through carbon dioxide

The mechanism of light-inhibited ethylene production in excised rice (Oryza sativa L.) and tobacco (Nicotiana tabacum L.) leaves was examined. In segments of rice leaves light substantially inhibited the endogenous ethylene production, but when CO₂ was added into the incubation flask, the rate of endogenous ethylene production in the light increased markedly, to a level which was even higher than that produced in the dark. Carbon dioxide, however, had no appreciable effect of leaf segments incubated in the dark. The endogenous level of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, was not significantly affected by lightdark or CO₂ treatment, indicating that dark treatment or CO₂exerted its effect by promoting the conversion of ACC to ethylene. This conclusion was supported by the observations that the rate of conversion of exogenously applied ACC to ethylene was similarly inhibited by light, and this inhibition was relieved in the presence of CO₂. Similar results were obtained with tobacco leaf discs. The concentrations of CO₂ giving half-maximal activity was about 0.06%, which was only slightly above the ambient level of 0.03%. The modulation of ACC conversion to ethylene by CO₂ or light in detached leaves of both rice and tobacco was rapid and fully reversible, indicating that CO₂ regulates the activity, but not the synthesis, of the enzyme converting ACC to ethylene. Our results indicate that light inhibition of ethylene production in detached leaves is mediated through the internal level of CO₂, which directly modulates the activity of the enzyme converting ACC to ethylene.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid Recipient of a Republic of China National Science Council Fellowship