Carbohydrates Stimulate Ethylene Production in Tobacco Leaf Discs : III. Stimulation of Enzymic Hydrolysis of Indole-3-Acetyl-l-Alanine

The sucrose-stimulated in vivo hydrolysis of indole-3-acetyl-l-alanine (IAAIa) in tobacco (Nicotiana tabacum L.) leaf discs was confirmed by in vitro analysis of an IAAIa-hydrolyzing enzyme isolated from the same tissue. The enzymic activity could be stimulated by either aging of the tissue or by application of external IAA or sucrose. A combination of the above three treatments yielded maximal activity.     

Stimulation of Ethylene Production by Exogenous Spermidine in Detached Tobacco Leaves in the Light

Exogenous supply of Spd and Spin stimulated ethylone production in detached tobacco leaves kept in the light. Stimulation, that was first detected after 9 but not 6 h of treatment, linearly increased with concentration and was maintained for several h after returning treated leaves to deionized water. Stimulation of ethylene production was prevented by AVG and Co²⁺ and was accompanied by increased activity of ACC synthase and ethylene-forming enzyme. Put, and other diamines, did not give any stimulatory effect. Stimulation was not accompanied by apparent damage of plasmalomina nor was it reversed by Ca⁺ or Put, suggesting that the cationic properties of polyamines are probably not involved. Stimulation might be due to endogenous polyamine accumulation high enough to inhibit the activity of S-adenosylmethionine decarboxylase, so that all S-adenosyhnethionine might be committed to the ethylene pathway. The stimulatory effect of poly a nines acquires particular interest considering that they have so fur been doscribod to inhibit ethylone production in darkened plant tissues. This finding suggests that polyamines may play a regulatory role in plant development by modulating ethylene bio-synthesis under the control of light.     

Stimulation of Net Photosynthesis in Cucumber Leaf Discs: Effect of K-Glyoxylate and KCL

Incubation of leaf discs of Cucumis sativus in 15 mol m^–3K-glyoxylate (pH 4.6) doubled the rate of net photosynthesisat limiting CO₂ or HCO^–₃ compared with discs floated ondistilled water. When both control and treated discs were incubatedin Mes-TMAOH buffer at pH 5.0, K-glyoxylate still increasednet photosynthesis by as much as 70%. The tetramethylammoniumsalt (TMA-glyoxylate) was without effect but KCl enhanced netphotosynthesis. Both KCl and K-glyoxylate increased stomatalaperture at pH 5.0. At pH 7.5 (Mops-TMAOH), neither stomatalaperture or photosynthesis was altered by K-glyoxylate, KClor TMA-glyoxylate. None of these salts was found to stimulatephotosynthesis in isolated cucumber mesophyll cells over a rangeof pH values although the cells incorporated as much ¹⁴C-glyoxylateas did leaf discs. The data suggest that enhanced photosynthesisin leaf discs is not due directly to a stimulation of mesophyllcell photosynthesis but rather is a consequence of increasedCO₂ availability and decreased stomatal resistance at low pHin the presence of potassium. Key words: Cucumber, Photosynthesis, Potassium glyoxylate     

Stimulation by Ethylene of Chlorophyll Biosynthesis in Dark-grown Cucumber Cotyledons

Five-day-old etiolated cucumber (Cucumis sativus L. var. Alpha Green) cotyledons produced more chlorophyll over a 4-hour illumination period after a prolonged exposure (12 to 72 hours) in the dark to ethylene concentrations ranging from 0.1 to 10 μl/l. Intact seedlings and excised cotyledons responded in the same way to this treatment. This effect does not involve a shortening of the lag phase of chlorophyll accumulation. Exposure of cotyledons to ethylene during the illumination period did not produce the same stimulatory effect on chlorophyll synthesis and, under certain conditions, chlorophyll synthesis was slightly inhibited by exposure to ethylene in the light.     

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.     

Pathway of Salicylic Acid Biosynthesis in Healthy and Virus-Inoculated Tobacco

Salicylic acid (SA) is a likely endogenous regulator of localized and systemic disease resistance in plants. During the hypersensitive response of Nicotiana tabacum L. cv Xanthi-nc to tobacco mosaic virus (TMV), SA levels rise dramatically. We studied SA biosynthesis in healthy and TMV-inoculated tobacco by monitoring the levels of SA and its likely precursors in extracts of leaves and cell suspensions. In TMV-inoculated leaves, stimulation of SA accumulation is accompanied by a corresponding increase in the levels of benzoic acid. 14C-Tracer studies with cell suspensions and mock-or TMV-inoculated leaves indicate that the label moves from trans-cinnamic acid to SA via benzoic acid. In healthy and TMV-inoculated tobacco leaves, benzoic acid induced SA accumulation. o-Coumaric acid, which was previously reported as a possible precursor of SA in other species, did not increase SA levels in tobacco. In healthy tobacco tissue, the specific activity of newly formed SA was equal to that of the supplied [14C]benzoic acid, whereas in TMV-inoculated leaves some isotope dilution was observed, presumably because of the increase in the pool of endogenous benzoic acid. We observed accumulation of pathogen-esis-related-1 proteins and increased resistance to TMV in benzoic acid- but not in o-coumaric acid-treated tobacco leaves. This is consistent with benzoic acid being the immediate precursor of SA. We conclude that in healthy and virus-inoculated tobacco, SA is formed from cinnamic acid via benzoic acid.     

乙烯生物合成途径及其相关基因工程的研究进展(综述)

在对植物激素乙烯生理功能作简要回顾的基础上着重对乙烯的生物合成途径中的关键酶,包括腺苷蛋氨酸合成酶、ACC合成酶及ACC氧化酶的性质和基因的研究进展作了综述,同时展现出了与调控内源乙烯生物合成有关的基因工程的整体轮廓。     

Metabolic Regulation in the Senescing Tobacco Leaf: II. Changes in Glycolytic Metabolite Levels in the Detached Leaf

Yellowing of detached mature tobacco leaves standing in water in the dark was accompanied by a strong “climacteric rise” in respiration rate. During this period the ATP level and energy charge of the adenylate system also rose. The levels of glycolytic intermediates between glucose 1-phosphate and triose phosphates rose, those between 3-phosphoglycerate and phosphoenolpyruvate fell, and pyruvate rose. On the assumption of a drop in NAD/NADH ratio, as found by other workers in wheat leaves, the reverse crossover between triose phosphates and 3-phospholglycerate was attributed to inhibition of glyceraldehyde 3-phosphate dehydrogenase. The forward crossover between phosphoenolpyruvate and pyruvate was taken to indicate activation of pyruvate kinase, possibly by fructose diphosphate. Secondary large rises in pyruvate and fructose diphosphate occurred well after the climacteric peak had been passed. No evidence was found for participation of phosphofructokinase in metabolic control in the yellowing leaf. Possible limitations to the use of the crossover theorem in the present situation, such as changes in compartmentation and in flux through branch points, are emphasized.     

Ureide Catabolism of Soybeans : II. Pathway of Catabolism in Intact Leaf Tissue

Allantoin catabolism studies have been extended to intact leaf tissue of soybean (Glycine max L. Merr.). Phenyl phosphordiamidate, one of the most potent urease inhibitors known, does not inhibit ¹⁴CO₂ release from [2,7-¹⁴C]allantoin (urea labeled), but inhibits urea dependent CO₂ release ≥99.9% under similar conditions. Furthermore, ¹⁴CO₂ and [¹⁴C] allantoate are the only detectable products of [2,7-¹⁴C]allantoin catabolism. Neither urea nor any other product were detected by analysis on HPLC organic acid or organic base columns although urea and all commercially available metabolites that have been implicated in allantoin and glyoxylate metabolism can be resolved by a combination of these two columns. In contrast, when allantoin was labeled in the two central, nonureido carbons ([4,5-¹⁴C]allantoin), its catabolism to [¹⁴C]allantoate, ¹⁴CO₂, [¹⁴C]glyoxylate, [¹⁴C]glycine, and [¹⁴C]serine in leaf discs could be detected. These data are fully consistent with the metabolism of allantoate by two amidohydrolase reactions (neither of which is urease) that occur at similar rates to release glyoxylate, which in turn is metabolized via the photorespiratory pathway. This is the first evidence that allantoate is metabolized without urease action to NH₄⁺ and CO₂ and that carbons 4 and 5 enter the photorespiratory pathway.     

Autoinhibition of Ethylene Production in Citrus Peel Discs : SUPPRESSION OF 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHESIS

Wound ethylene formation induced in flavede tissue of citrus fruit (Citrus paradisi MacFad. cv. Ruby Red) by slicing was almost completely inhibited by exogenous ethylene. The inhibition lasted for at least 6 hours after removal of exogenous ethylene and was then gradually relieved. The extent of inhibition was dependent upon the concentration of ethylene (1 to 10 microliters/liter) and the duration of treatment. The increase in wound ethylene production in control discs was paralleled by an increase in 1-aminocyclopropane-1-carboxylic acid (AAC) content, whereas in ethylene-treated discs there was little increase in ACC content. Application of ACC completely restored ethylene production in ethylene-pretreated discs, indicating that the conversion of ACC to ethylene is not impaired by the presence of ethylene. Thus, autoinhibition of ethylene synthesis was exerted by reducing the availability of ACC. Ethylene treatment resulted in a decrease in extractable ACC synthase activity, but this decrease was too small to account for the marked inhibition of ACC formation. The data indicate that autoinhibition of ethylene production in citrus flavede discs results from suppression of ACC formation through repression of the synthesis of ACC synthase and inhibition of its activity.     

Ethylene Biosynthesis and Cadmium Toxicity in Leaf Tissue of Beans (Phaseolus vulgaris L.)

Stress ethylene production in bean (Phaseolus vulgaris L., cv. Taylor's Horticultural) leaf tissue was stimulated by Cd²⁺ at concentrations above 1 micromolar. Cd²⁺-induced ethylene biosynthesis was dependent upon synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase. Activity of ACC synthase and ethylene production rate peaked at 8 h of treatment. The subsequent decline in enzyme activity was most likely due to inactivation of the enzyme by Cd²⁺, which inhibited ACC synthase activity in vitro at concentrations as low as 0.1 micromolar. Decrease in ethylene production rate was accompanied by leakage of solutes and increasing inhibition of ACC-dependent ethylene production. Ca²⁺, present during a 2-hour preincubation, reduced the effect of Cd²⁺ on leakage and ACC conversion. This suggests that Cd²⁺ exerts its toxicity through membrane damage and inactivation of enzymes. The possibility of an indirect stimulation of ethylene biosynthesis through a wound signal from injured cells is discussed.     

Ethylene Stimulation During the Hypersensitive Reaction of Soybean to Tobacco Necrosis Virus under Different Photoperiods

Abstract The production of stress ethylene was increased in soybean leaves hypersensitively responding to tobacco necrosis virus, independently of photoperiod. However, only little increase occurred under continuous darkness, whereas most occurred under continuous darkness, whereas most occurred under continuous light. Ethylene stimulation paralleled accumulation of 1-aminocyclopropane-1-carboxylic (ACC) and its conversion to ethylene. Continuous darkness substantially inhibited viral antigen accumulation but not lesion area in comparison to continuous light. Ethylene release, viral lesion area and antigen accumulation were substantially increased when darkened leaf tissues were fed with glucose, this suggesting that dark inhibition was due to energy and/or, metabolic depletion. Co²⁺ and aminoethoxyvinylglycine, which completely inhibited stress ethylene, and ACC, which conspicuously increased it, had no effect on both viral lesion and antigen accumulation.
These results indicate that stress ethylene developing during a HR to virus does not affect the localizing mechanism operating during it.     

Regulation of Ethylene Biosynthesis in Virus-Infected Tobacco Leaves : I. DETERMINATION OF THE ROLE OF METHIONINE AS THE PRECURSOR OF ETHYLENE

The hypersensitive reaction of Samsun NN tobacco leaves to tobacco mosaic virus (TMV) was accompanied by a large increase in ethylene production, just before necrotic local lesions became visible. Normal and virus-induced ethylene production were both largely inhibited by 0.1 millimolar aminoethoxyvinylglycine indicating that methionine is a main ethylene precursor.     

Identification of Genetically Induced Lesions and the Sites of Action of Inhibitors Affecting Photorespiration by Simple Tests on Leaf Discs

Turner, J. C. and Hall, N. P. 1988. Identification of geneticallyinduced lesions and sites of action of inhibitors affectingphotorespiration by simple tests on leaf discs.—J. exp,Bot. 39: 345-351. Six photorespiratory mutants of barley deficient in catalaseand two mutants lacking phospho-glycollate phosphatase wereidentified by a novel screening method using leaf discs. Leaf discs were punched directly into an appropriate bufferedreagent in which the enzymes diffused from the cut edges ofthe discs causing a change in the colour reagents. The reactionswere observed from 15 min onwards depending on which enzymeactivity was being followed. Hundreds of plants can be screenedrapidly for major differences in enzyme activity. The methods depend on the formation of a red product in thereaction of hydrogen peroxide (H₂O₂) with 4-aminoantipyrenein the presence of peroxidase. To detect P-glycollatc phosphataseand glycollate oxidase, the product of the linked reactions,H₂O₂, was measured. For catalase, the disappearance of addedH₂O₂ is followed. By omitting peroxidase from the colour reagentmixture, peroxidase activity in leaf discs can be measured. The method was evaluated by applying it to existing enzyme deficientmutants of barley lacking P-glycollate phosphatase and catalase.Further mutant plants were detected by this method. The techniquecould also be used to screen for inhibitors of the glycollatepathway for use as herbicides. Key words: Phosphoglycollate phosphatase, glycollate oxidase, catalase, peroxidase, hydrogen peroxide     

Auxin Biosynthesis in Pea: Characterization of the Tryptamine Pathway

One pathway leading to the bioactive auxin, indole-3-acetic acid (IAA), is known as the tryptamine pathway, which is suggested to proceed in the sequence: tryptophan (Trp), tryptamine, N-hydroxytryptamine, indole-3-acetaldoxime, indole-3-acetaldehyde (IAAld), IAA. Recently, this pathway has been characterized by the YUCCA genes in Arabidopsis (Arabidopsis thaliana) and their homologs in other species. YUCCA is thought to be responsible for the conversion of tryptamine to N-hydroxytryptamine. Here we complement the genetic findings with a compound-based approach in pea (Pisum sativum), detecting potential precursors by gas chromatography/tandem-mass spectrometry. In addition, we have synthesized deuterated forms of many of the intermediates involved, and have used them to quantify the endogenous compounds, and to investigate their metabolic fates. Trp, tryptamine, IAAld, indole-3-ethanol, and IAA were detected as endogenous constituents, whereas indole-3-acetaldoxime and one of its products, indole-3-acetonitrile, were not detected. Metabolism experiments indicated that the tryptamine pathway to IAA in pea roots proceeds in the sequence: Trp, tryptamine, IAAld, IAA, with indole-3-ethanol as a side-branch product of IAAld. N-hydroxytryptamine was not detected, but we cannot exclude that it is an intermediate between tryptamine and IAAld, nor can we rule out the possibility of a Trp-independent pathway operating in pea roots.Auxin is a key plant growth hormone, involved in processes as diverse as branching, gravitropism, phototropism, and seed development (Davies, 2004). However, the biosynthetic pathways leading to the main auxin in plants, indole-3-acetic acid (IAA), are not well understood. Although there is good evidence that the amino acid Trp is an early precursor (Gibson et al., 1972; Wright et al., 1991; Tsurusaki et al., 1997), several routes from Trp to IAA have been proposed, and for any given species it is not clear which route or routes occur. The possible Trp-dependent pathways in higher plants are the indole-3-pyruvic acid (IPyA) pathway (Stepanova et al., 2008; Tao et al., 2008), the tryptamine (YUCCA) pathway (Zhao et al., 2001), the indole-3-acetaldoxime (IAOx) pathway (Bartel et al., 2001), and the indoleacetamide pathway (Pollmann et al., 2002), on the basis of the first metabolite of Trp (Fig. 1). In addition, a possible Trp-independent pathway has been proposed (Normanly et al., 1993), bypassing Trp completely, further complicating the process of IAA biosynthesis.Open in a separate windowFigure 1.Left: The putative tryptamine (red), Trp-independent (light blue), IPyA (green), indoleacetamide (yellow), and IAOx (dark blue) biosynthetic pathways to IAA in Arabidopsis. The steps shown in gray appear not to occur in peas. Right: The simplified pathway scheme suggested to occur in pea based on the present results and Sugawara et al. (2009). N-hydroxytryptamine was not detected as a metabolite in this study, suggesting that tryptamine might be converted directly to IAAld in pea roots. The Trp-independent, indoleacetamide, and IPyA pathways were not studied.Since 2001, there has been renewed interest in the tryptamine route to IAA, after the discovery and functional analysis of the Arabidopsis (Arabidopsis thaliana) YUCCA gene, reported to encode the enzyme for converting tryptamine to N-hydroxytryptamine (Zhao et al., 2001, 2002). On the basis of the Zhao et al. (2001, 2002) reports, tryptamine pathways have generally been proposed in the sequence: Trp, tryptamine, N-hydroxytryptamine, IAOx, indole-3-acetaldehyde (IAAld), IAA (Fig. 1; Woodward and Bartel, 2005). Recently, however, Sugawara et al. (2009) suggested that IAOx be removed from the tryptamine pathway, and that IAOx-dependent IAA biosynthesis operates only in the Brassicaceae. In Arabidopsis, this pathway can be important, at least in some circumstances, because when a side branch is impaired, as in the sur1 mutant, IAA levels increase dramatically (Sugawara et al., 2009). On the other hand, the IAOx pathway is not the only pathway operating in Arabidopsis, because genetically blocking the step Trp to IAOx does not always reduce IAA content, compared with wild-type plants (Sugawara et al., 2009). This means that in Arabidopsis, the tryptamine and/or IPyA and/or indoleacetamide pathways compensate for the loss of the IAOx pathway. Interestingly, Sugawara et al. (2009) do not include IAAld in their tryptamine pathway, and their model implies instead that N-hydroxytryptamine is directly converted to IAA.Turning to other species, it has been reported that tryptamine is not present in pea (Pisum sativum; Schneider et al., 1972), despite being present in tomato (Solanum lycopersicum; Cooney and Nonhebel, 1991), rice (Oryza sativa; Ishihara et al., 2008), Arabidopsis (Sugawara et al., 2009), and barley (Hordeum vulgare; Schneider et al., 1972). In tomato, although tryptamine is relatively abundant, and early metabolism studies indicated the conversion of tryptamine to IAA via IAAld (Schneider et al., 1972), Cooney and Nonhebel (1991) cast doubt on the role of tryptamine after studying patterns of labeling after incubation of plants with deuterated water. Again, in tobacco (Nicotiana tabacum), Songstad et al. (1990) showed that while tobacco plants overexpressing a Trp decarboxylase accumulated very high levels of tryptamine, IAA levels were unaffected. Although this result has been interpreted as evidence against the involvement of tryptamine (Bartel et al., 2001), another explanation is that excess tryptamine is converted to compounds via a side branch or side branches, although these are not well studied. Finally, the compound N-hydroxytryptamine is relatively unknown, with no reports of its presence in plants to date.It is clear, therefore, that the tryptamine pathway to IAA remains poorly understood. In this article, we further characterize the pathway, using the garden pea as a model species. We report on the presence/absence and levels of the putative endogenous intermediates, as determined by gas chromatography/tandem mass spectrometry (GC/MS/MS), and investigate their metabolic fates using [¹⁴C] and deuterated versions of the compounds. Our evidence indicates that key elements of the tryptamine pathway are operative in pea roots.     

Biosynthesis of limonoids in Citrus: Sites and translocation

Sites of limonoid biosynthesis were located in Citrus limon. The stem was found to be the major site of nomilin biosynthesis from acetate. Epicotyl, hypocotyl and root tissues were also capable of biosynthesizing nomilin from acetate, but leaves, fruits and seeds did not show this capacity under the conditions used. All the tissues tested were capable of biosynthesizing other limonoids starting from nomilin. C. limon was capable of translocating nomilin from the stem to other sites.