Regulation of ethylene biosynthesis in virus-infected tobacco leaves

During the hypersensitive reaction of tobacco (Nicotiana tabacum L.) cv. Samsun NN to tobacco mosaic virus (TMV), the appearance of local lesions was accompanied by a large burst of ethylene. Biosynthesis of both basal and virus-stimulated ethylene production was investigated in vivo by labeling experiments, the use of specific inhibitors, and the determination of the concentration of the probable precursor and intermediates.Determination by labeling of the role of a specific compound as a precursor in a particular biosynthetic pathway may be complicated. By comparing the specific radioactivities of, on the one hand, the endogenous precursor pool, and, on the other hand, the product involved, a quantitative estimate of precursor-product conversion can be obtained. However, this ratio is altered by unequal distribution of the labeled precursor and/or the product formation within an individual plant or animal, within a specific organ or tissue, or even within cells, leading to erroneous conclusions.The main biosynthetic pathway of ethylene in plant tissues has been established as methionine ->S-adenosylmethionine (SAM) ->1-aminocyclopropane-l-carboxylic acid (ACC) ->ethylene. In this research we investigated the role of methionine as ethylene precursor in a pathological situation suchas a hypersensitive reaction. Non-infected and hypersensitively- reacting tobacco leaves were labeled with L-(U- 14C)methionine by petiolar uptake. Under these conditions the labeled methionine was retained mainly in the veins, whereas the virus- induced ethylene production was restricted to the immediate vicinity of the developing lesions in interveinal tissues, preventing the assessment of the importance of methionine as the ethylene precursor (Chapter I). This complication was avoided by labeling leaves by vacuum infiltration. A comparison of the specific radioactivities of the methionine pool and the ethylene produced by non-infected or hypersensitively-reacting leaves, was highly indicative of methionine being the only ethylene precursor in both cases.By using aminoethoxyvinylglycine (AVG), a specific inhibitor of methionine-derived ethylene synthesis, and by determination of endogenous concentrations of ACC, methionine was further demonstrated to be the only precursor of ethylene in both noninfected and TMV-infected Samsun NN tobacco. Even the small amount of ethylene emanated in the presence of AVG was derived from methionine.Endogenous concentrations of both methionine and SAM remained constant up to 4 days after inoculation. Furthermore, exogenously applied methionine or SAM did not increase ethylene production in non-infected leaf discs, although both precursors were directly available for ethylene production. In contrast, ethylene production was increased severalfold upon incubation of leaf discs in solutions of ACC. Thus, ethylene production in tobacco was not regulated at the level of the concentration or availability of either methionine or SAM, but was primarily limited at the level of ACC production (Chapter III).The sharp peak in ethylene production near the time of lesion appearance was preceded by a strong rise in ACC production, peaking 8 h earlier. As a result, ACC accumulated in the tissue. Only after lesions had become macroscopically visible, the capacity of the leaf to convert ACC to ethylene increased severalfold, associated with a sharp decrease in ACC content and a large rise in ethylene evolution. Thus, virus-stimulated ethylene production during a hypersensitive reaction turned out to be regulated at the level of both the production of ACC and its conversion to ethylene (Chapter III).Investigation of genetically different host/virus combinations revealed that an increased ethylene production after virus infection was determined neither by the genetic constitution of the host plant, nor by the properties of the infecting virus, but was related exclusively to the type of symptoms expressed. No rise in ACC production occurred in combinations leading to systemic mosaic symptoms.The rise in ACC production in hypersensitively-reacting combinations depended on both RNA and protein synthesis, suggesting the ACC-synthase to be synthesized de novo. So far efforts to find an ACC- synthase- inducing factor have failed: the increase in ACC production could not be mimicked by local membrane damage, and no ACC-synthasestimulating agent could be isolated from leaves with developing lesions.Light inhibited the conversion of ACC to ethylene via (part of) the photosynthetic system. Inhibiting protein synthesis during the shift from light to darkness abolished the increase in ACC conversion, indicating that the enzyme is synthesized de novo. However, the rapid decrease upon a shift from darkness to light cannot be easily explained and may involve both active degradation and/or inactivation (Chapter V).After primary infection of hypersensitively-responding plants the ACC-converting capacity was increased systemically within the plant. As. no ACC accumulated upon challenge inoculation of systemically-resistant leaves. acquired resistance may be related to the increased capacity to convert ACC to ethylene (Chapter IV).The involvement of virus-stimulated ethylene production in virus localization was further investigated by studying effects of temperature, light conditions, and leaf age on both ethylene production and lesion size (Chapter VI). In non-infected leaves, both endogenous and ACC-stimulated ethylene production increased with increasing temperature up to 35°C, and decreased with increasing leaf age. Light inhibited only the conversion of ACC to ethylene. Temperature, light and leaf age similarly affected the pattern of virus - stimulated ethylene production; enhanced localization of the virus in old leaves was associated with a sharp peak in ethylene production near the time of lesion appearance. In contrast, large lesions developed in continuous light or in young leaves, where virus-induced ethylene production increased only gradually from lesion appearance onwards. Hence, an early burst of ethylene and the virus localizing reaction are closely connected.The expression of the N gene in Samsun NN tobacco, which confers hypersensitivity towards all strains of TMV, does not occur above 28°C. From experiments in which temperature was shifted from 20° to 30°C and back, the N gene was demonstrated to be involved only in the initiation, and not in the realization of the hypersensitive reaction. N -gene activity is required for at least 6 h between 16 and 24 h after inoculation for both stimulation of ethylene production and local lesions to develop

Regulation of ethylene biosynthesis in virus-infected tobacco leaves

During the hypersensitive reaction of tobacco (Nicotiana tabacum L.) cv. Samsun NN to tobacco mosaic virus (TMV), the appearance of local lesions was accompanied by a large burst of ethylene. Biosynthesis of both basal and virus-stimulated ethylene production was investigated in vivo by labeling experiments, the use of specific inhibitors, and the determination of the concentration of the probable precursor and intermediates.Determination by labeling of the role of a specific compound as a precursor in a particular biosynthetic pathway may be complicated. By comparing the specific radioactivities of, on the one hand, the endogenous precursor pool, and, on the other hand, the product involved, a quantitative estimate of precursor-product conversion can be obtained. However, this ratio is altered by unequal distribution of the labeled precursor and/or the product formation within an individual plant or animal, within a specific organ or tissue, or even within cells, leading to erroneous conclusions.The main biosynthetic pathway of ethylene in plant tissues has been established as methionine ->S-adenosylmethionine (SAM) ->1-aminocyclopropane-l-carboxylic acid (ACC) ->ethylene. In this research we investigated the role of methionine as ethylene precursor in a pathological situation suchas a hypersensitive reaction. Non-infected and hypersensitively- reacting tobacco leaves were labeled with L-(U- 14C)methionine by petiolar uptake. Under these conditions the labeled methionine was retained mainly in the veins, whereas the virus- induced ethylene production was restricted to the immediate vicinity of the developing lesions in interveinal tissues, preventing the assessment of the importance of methionine as the ethylene precursor (Chapter I). This complication was avoided by labeling leaves by vacuum infiltration. A comparison of the specific radioactivities of the methionine pool and the ethylene produced by non-infected or hypersensitively-reacting leaves, was highly indicative of methionine being the only ethylene precursor in both cases.By using aminoethoxyvinylglycine (AVG), a specific inhibitor of methionine-derived ethylene synthesis, and by determination of endogenous concentrations of ACC, methionine was further demonstrated to be the only precursor of ethylene in both noninfected and TMV-infected Samsun NN tobacco. Even the small amount of ethylene emanated in the presence of AVG was derived from methionine.Endogenous concentrations of both methionine and SAM remained constant up to 4 days after inoculation. Furthermore, exogenously applied methionine or SAM did not increase ethylene production in non-infected leaf discs, although both precursors were directly available for ethylene production. In contrast, ethylene production was increased severalfold upon incubation of leaf discs in solutions of ACC. Thus, ethylene production in tobacco was not regulated at the level of the concentration or availability of either methionine or SAM, but was primarily limited at the level of ACC production (Chapter III).The sharp peak in ethylene production near the time of lesion appearance was preceded by a strong rise in ACC production, peaking 8 h earlier. As a result, ACC accumulated in the tissue. Only after lesions had become macroscopically visible, the capacity of the leaf to convert ACC to ethylene increased severalfold, associated with a sharp decrease in ACC content and a large rise in ethylene evolution. Thus, virus-stimulated ethylene production during a hypersensitive reaction turned out to be regulated at the level of both the production of ACC and its conversion to ethylene (Chapter III).Investigation of genetically different host/virus combinations revealed that an increased ethylene production after virus infection was determined neither by the genetic constitution of the host plant, nor by the properties of the infecting virus, but was related exclusively to the type of symptoms expressed. No rise in ACC production occurred in combinations leading to systemic mosaic symptoms.The rise in ACC production in hypersensitively-reacting combinations depended on both RNA and protein synthesis, suggesting the ACC-synthase to be synthesized de novo. So far efforts to find an ACC- synthase- inducing factor have failed: the increase in ACC production could not be mimicked by local membrane damage, and no ACC-synthasestimulating agent could be isolated from leaves with developing lesions.Light inhibited the conversion of ACC to ethylene via (part of) the photosynthetic system. Inhibiting protein synthesis during the shift from light to darkness abolished the increase in ACC conversion, indicating that the enzyme is synthesized de novo. However, the rapid decrease upon a shift from darkness to light cannot be easily explained and may involve both active degradation and/or inactivation (Chapter V).After primary infection of hypersensitively-responding plants the ACC-converting capacity was increased systemically within the plant. As. no ACC accumulated upon challenge inoculation of systemically-resistant leaves. acquired resistance may be related to the increased capacity to convert ACC to ethylene (Chapter IV).The involvement of virus-stimulated ethylene production in virus localization was further investigated by studying effects of temperature, light conditions, and leaf age on both ethylene production and lesion size (Chapter VI). In non-infected leaves, both endogenous and ACC-stimulated ethylene production increased with increasing temperature up to 35°C, and decreased with increasing leaf age. Light inhibited only the conversion of ACC to ethylene. Temperature, light and leaf age similarly affected the pattern of virus - stimulated ethylene production; enhanced localization of the virus in old leaves was associated with a sharp peak in ethylene production near the time of lesion appearance. In contrast, large lesions developed in continuous light or in young leaves, where virus-induced ethylene production increased only gradually from lesion appearance onwards. Hence, an early burst of ethylene and the virus localizing reaction are closely connected.The expression of the N gene in Samsun NN tobacco, which confers hypersensitivity towards all strains of TMV, does not occur above 28°C. From experiments in which temperature was shifted from 20° to 30°C and back, the N gene was demonstrated to be involved only in the initiation, and not in the realization of the hypersensitive reaction. N -gene activity is required for at least 6 h between 16 and 24 h after inoculation for both stimulation of ethylene production and local lesions to develop