Evolution of Ethylene Production, ACC and Conjugated ACC Levels Accompanying Symptom Development in Tomato and Gynura aurantiaca DC Leaves Infected with Citrus Exocortis Viroid (CEV)

Ethylene production, ACC and Conjugated ACC levels as related to symptom development in leaves of tomato and Gynura aurantiaca infected with citrus exocortis viroid (CEV) were studied. In both hosts, the infection induced an increase in ethylene production and ACC levels and a strong accumulation of Conjugated ACC, Contrary to what happens with the stimulation of ethylene production and the enhancing of ACC content which maintain a steady level as leaves and their symptoms develop, conjugated ACC accumulation increased with the intensification of symptoms.     

No difference in the regulation pattern of calcium on ethylene biosynthesis between wild-type and never-ripe tomato fruit at mature green stage

This work investigated how calcium regulates the ethylene biosynthesis in the fruits of wild-type tomato (Lycopersicon esculentum L.) and their ethylene receptor never-ripe (Nr) mutants. In Nr tomato, the ethylene perception was blocked. When both materials were treated with calcium, the content of 1-aminocyclopropane-1-carboxylic acid (ACC)/malonyl-ACC and the activity of ACC oxidase (ACO) in tomato fruit discs increased, whereas the production of ethylene, content of malondialdehyde, and membrane permeability decreased. Calcium treatment did not affect the activity of ACC synthase, which is the first committed step in the ethylene biosynthesis pathway. The expression of LeACO1 in mature green fruit was inhibited significantly by calcium treatment in wild-type and Nr tomatoes, but the expression of LeACS2, the key ACC synthase gene in ethylene synthesis during tomato fruit maturing, was not affected. These results revealed that the effect of calcium on ethylene biosynthesis in tomato mature green fruit was independent of ethylene perception. The results also revealed that the targeting step of calcium preventing ethylene production was located at the ACC conversion to ethylene, by means of inhibiting ACC availability for ACO through enhancing cell membrane integrity and by means of preventing LeACO1 gene expression. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 1, pp. 60–67. The text was submitted by the authors in English.     

Suppression by Citrus Exocortis Viroid Infection of the Naturally Occurring Inhibitor of the Conversion of 1-aminocyclopropane-1-carboxylic Acid to Ethylene by Tomato Microsomes

Citrus exocortis viroid (CEVd) infection of tomato cell cultures suppresses the constitutive inhibitor which blocks the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by tomato microsomes. The inhibitor is associated to microsomal membranes and is also found in the water soluble fraction co-isolated from the cells. The inhibitory effect is concentration dependent, heat stable and could be removed from solution by dialysis. Its possible relationship with regulation of the viroid-induced ethylene production is discussed.     

Biosynthesis of ethylene in higher plants: the metabolic site of inhibition by phosphate*

Abstract. Phosphate inhibited endogenous as well as 1-aminocyclopropane-1-carboxylic acid (ACC)-stimulated ethylene synthesis in slices of tomato fruit, segments of carrot root and pea hypocotyls. ACC concentrations of up to 10 mol m^?3 did not overcome this inhibition. Phosphate inhibited the conversion of ¹⁴C ACC to ethylene in tomato fruit and vegetative tissue. Enzymatic conversion of ACC to ethylene by pea seedling homogenate was also inhibited by phosphate with a linear concentration dependency. The formation of ACC from S-adenosylmethionine (SAM) by extracts of pink tomatd fruit was slightly, but not significantly, affected by phosphate. However, the SAM to ACC conversion was greater when extracts from tomato fruit were made in phosphate rather than in HEPES-KOH buffer. Non-enzymatic ethylene synthesis from ACC in a model system was stimulated by phosphate. We suggest that phosphate is an inhibitor of ethylene biosynthesis in higher plants and that one site of its control is the conversion of ACC to ethylene.     

Reduced symptoms of Verticillium wilt in transgenic tomato expressing a bacterial ACC deaminase

Ethylene evolved during compatible or susceptible disease interactions may hasten and/or worsen disease symptom development; if so, the prevention of disease-response ethylene should reduce disease symptoms. We have examined the effects of reduced ethylene synthesis on Verticillium wilt (causal organism, Verticillium dahliae) of tomato by transforming tomato with ACC deaminase, which cleaves ACC, the immediate biosynthetic precursor of ethylene in plants. Three promoters were used to express ACC deaminase in the plant: (i) CaMV 35S (constitutive expression); (ii) rolD (limits expression specifically to the site of Verticillium infection, i.e. the roots); and (iii) prb-1b (limits expression to certain environmental cues, e.g. disease infection). Significant reductions in the symptoms of Verticillium wilt were obtained for rolD- and prb-1b-, but not for 35S-transformants. The pathogen was detected in stem sections of plants with reduced symptoms, suggesting that reduced ethylene synthesis results in increased disease tolerance. The effective control of formerly recalcitrant diseases such as Verticillium wilt may thus be obtained by preventing disease-related ethylene production via the tissue-specific expression of ACC deaminase.     

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.     

The sequence of a viroid from grapevine closely related to severe isolates of citrus exocortis viroid.

The primary structure of a grapevine viroid (GVs) isolated in Spain was determined. The sequence consisted of 369 nucleotide residues forming a circular molecule. GVs presented extensive homology with viroids of the potato spindle tuber viroid (PSTV) group, that was specially high in the case of citrus exocortis viroid (CEV) both with variants found in isolates inducing severe (92% with CEV-A) and mild (89% with CEV-DE26) symptoms on tomato. The secondary structure proposed for GVs showed that the changes in the sequence in relation to CEV-A generated modifications of the secondary structure particularly important in the left terminal (Tl), variable (V) and pathogenesis (P) viroid domains that have been postulated. Nevertheless it was noted in GVs a central core in the P domain that is conserved in the class A sequence variants characteristic of severe isolates, but not in the class B ones found in mild isolates of CEV. These observations indicate that GVs should be considered as a severe isolate of CEV from grapevine (CEV-g), a suggestion that correlates with the biological properties of CEV-g both in tomato and in Gynura aurantiaca. The presence of this central core in the P domain seems to characterize all the variants of CEV inducing severe symptoms in tomato.     

Biosynthesis of Auxin-Induced Ethylene. Effects of Indole-3-Acetic Acid, Benzyladenine and Abscisic Acid on Endogenous Levels of 1-Aminocyclopropane-l-Carboxylic Acid (ACC) and ACC Synthase

Auxin-induced ethylene biosynthesis and its regulatory stepsin etiolated mung bean hypocotyl segments were examined. Theendogenous content of 1-aminocyclopropane- 1-carboxylic acid(ACC), an immediate precursor of ethylene, increased correspondingto the rate of ethylene production. Benzyladenine (BA), whichis a synergistic stimulator of auxin-induced ethylene production,increased the ACC content parallel to the rate of ethylene productionin the presence of IAA, but failed to increase the ACC contentin the absence of IAA while ethylene production was significantlystimulated by BA. Abscisic acid (ABA) inhibited the formationof ACC. The ACC synthase activity in the tissue was increasedby IAA, and the increase was further promoted by the presenceof BA. Cycloheximide severely inhibited the development of auxin-inducedACC synthase. The enzymatic properties of mung bean ACC synthasewere similar to those of the tomato fruit enzyme. Aminoethoxyvinylglycine(AVG) and aminooxyacetic acid, which inhibit the ACC synthasereaction, stimulated the development of ACC synthase. The regulatorymechanisms of the growth regulators are discussed in relationto ACC formation. (Received December 3, 1980; Accepted January 22, 1981)     

Inhibition of ethylene synthesis in tomato plants subjected to anaerobic root stress

Enhanced ethylene production and leaf epinasty are characteristic responses of tomato (Lycopersicon esculentum Mill.) to waterlogging. It has been proposed (Bradford, Yang 1980 Plant Physiol 65: 322-326) that this results from the synthesis of the immediate precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), in the waterlogged roots, its export in the transpiration stream to the shoot, and its rapid conversion to ethylene. Inhibitors of the ethylene biosynthetic pathway are available for further testing of this ACC transport hypothesis: aminooxyacetic acid (AOA) or aminoethoxyvinylglycine (AVG) block the synthesis of ACC, whereas CO²⁺ prevents its conversion to ethylene. AOA and AVG, supplied in the nutrient solution, were found to inhibit the synthesis and export of ACC from anaerobic roots, whereas Co²⁺ had no effect, as predicted from their respective sites of action. Transport of the inhibitors to the shoot was demonstrated by their ability to block wound ethylene synthesis in excised petioles. All three inhibitors reduced petiolar ethylene production and epinasty in anaerobically stressed tomato plants. With AOA and AVG, this was due to the prevention of ACC import from the roots as well as inhibition of ACC synthesis in the petioles. With Co²⁺, conversion of both root- and petiole-synthesized ACC to ethylene was blocked. Collectively, these data support the hypothesis that the export of ACC from low O₂ roots to the shoot is an important factor in the ethylene physiology of waterlogged tomato plants.     

Promotion by Ethylene of the Capability to Convert 1-Aminocyclopropane-1-carboxylic Acid to Ethylene in Preclimacteric Tomato and Cantaloupe Fruits

The intact fruits of preclimacteric tomato (Lycopersicon esculentum Mill) or cantaloupe (Cucumis melo L.) produced very little ethylene and had low capability of converting 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. When these unripe tomato or cantaloupe fruits were treated with ethylene for 16 hours there was no increase in ACC content or in ethylene production rate, but the tissue's capability to convert ACC to ethylene increased markedly. Such an effect was also observed in fruits of tomato mutants rin and nor, which do not undergo ripening and the climacteric increase in ethylene production during the senescence. The development of this ethylene-forming capability induced by ethylene increased with increasing ethylene concentration (from 0.1 to 100 microliters per liter) and duration (1 to 24 hours); when ethylene was removed this capability remained high for sometime (more than 24 hours). Norbornadiene, a competitive inhibitor of ethylene action, effectively eliminated the promotive effect of ethylene in tomato fruit. These data indicate that the development of the capability to convert ACC to ethylene in preclimacteric tomato and cantaloupe fruits are sensitive to ethylene treatment and that when these fruits are exposed to exogenous ethylene, the increase in ethylene-forming enzyme precedes the increase in ACC synthase.     

Regulation of Ethylene Biosynthesis in Avocado Fruit during Ripening

Preclimacteric avocado (Persea americana Mill.) fruits produced very little ethylene and had only a trace amount of l-aminocyclopropane-1-carboxylic acid (ACC) and a very low activity of ACC synthase. In contrast, a significant amount of l-(malonylamino)cyclopropane-1-carboxylic acid (MACC) was detected during the preclimacteric stage. In harvested fruits, both ACC synthase activity and the level of ACC increased markedly during the climacteric rise reaching a peak shortly before the climacteric peak. The level of MACC also increased at the climacteric stage. Cycloheximide and cordycepin inhibited the synthesis of ACC synthase in discs excised from preclimacteric fruits. A low but measurable ethylene forming enzyme (EFE) activity was detected during the preclimacteric stage. During ripening, EFE activity increased only at the beginning of the climacteric rise. ACC synthase and EFE activities and the ACC level declined rapidly after the climacteric peak. Application of ACC to attached or detached fruits resulted in increased ethylene production and ripening of the fruits. Exogenous ethylene stimulated EFE activity in intact fruits prior to the increase in ethylene production. The data suggest that conversion of S-adenosylmethionine to ACC is the major factor limiting ethylene production during the preclimacteric stage. ACC synthase is first synthesized during ripening and this leads to the production of ethylene which in turn induces an additional increase in ACC synthase activity. Only when ethylene reaches a certain level does it induce increased EFE activity.     

Brassinosteroid-induced epinasty in tomato plants

The effects of root treatments of brassinosteroid (BR) on the growth and development of hydroponically grown tomato plants (Lycopersicon esculentum Mill cv Heinz 1350) were evaluated. There was a dramatic increase in petiole bending when the plants were treated with 0.5 to 1.0 micromolar BR. The leaf angle of the treated plants was almost three times that of untreated controls. BR-induced epinasty appeared to be due to stimulation of ethylene production. Excised petioles from BR-treated plants produced more than twice as much ethylene as did untreated controls. As ethylene production increased, the degree of petiole bending also increased, and inhibition of ethylene production by AOA or CoCl₂ also inhibited epinasty. BR-treated plants had increased levels of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaf tissue. ACC appeared to accumulate primarily in the petioles with the greatest amount of ACC accumulating in the youngest petioles. Time course evaluations revealed that BR treatment stimulated ACC production. As ACC accumulated, ethylene increased, resulting in epinasty. Little or no ACC was found in the xylem sap, indicating that there was a signal transported from the roots which stimulated ACC synthesis in the leaf tissue.     

Role of the mitochondria in the conversion of 1-aminocyclopropane 1-carboxylic acid to ethylene in plant tissues

Intact plant mitochondria, isolated from climacteric (Lycopersicon esculentum, Mill., tomato) or non-climacteric (Solanum tuberosum, L., potato) tissues, and purified on Percoll density gradients, were unable to convert 1-aminocyclopropane 1-carboxylic acid (ACC) to ethylene. Energization or sonication did not enhance ethylene production. For both tissues, the low activity of ACC conversion found in crude mitochondrial fractions from both tissues was increased by sonication. After mitochondrial purification, this activity was located on top of the gradient together with the microsomal membrane fraction containing a high lipoxygenase activity. Addition of exogenous lipoxygenase and linoleic acid to isolated tomato or potato mitochondria greatly enhanced ACC conversion (to approx. 300 pmol h⁻¹ mg⁻¹ protein). Direct measurements of ACC uptake by mitochondria indicated that ACC uptake is not dependent on energization.     

Effects of Low O(2) Root Stress on Ethylene Biosynthesis in Tomato Plants (Lycopersicon esculentum Mill cv Heinz 1350)

Low O₂ conditions were obtained by flowing N₂ through the solution in which the tomato plants (Lycopersicon esculentum Mill cv Heinz 1350) were growing. Time course experiments revealed that low O₂ treatments stimulated 1-aminocyclopropane-1-carboxylate (ACC) synthase production in the roots and leaves. After the initiation of low O₂ conditions, ACC synthase activity and ACC content in the roots increased and reached a peak after 12 and 20 hours, respectively. The conversion of ACC to ethylene in the roots was inhibited by low levels of O₂, and ACC was apparently transported to the leaves where it was converted to ethylene. ACC synthase activity in the leaves was also stimulated by low O₂ treatment to the roots, reaching a peak after 24 hours. ACC synthase levels were enhanced by cobalt chloride and aminooxyacetic acid (AOA), although they inhibited ethylene production. Cobalt chloride enhanced ACC synthase only in combination with low O₂ conditions in the roots. Under aeration, AOA stimulated ACC synthase activity in both the roots and leaves. However, in combination with low O₂ conditions, AOA caused a stimulation in ACC synthase activity in the leaves and no effect in the roots.     

Galactose inhibits the conversion of 1-aminocyclopropane-1-carboxylic Acid to ethylene in aged tobacco leaf discs

d-Galactose has been shown to have toxic and growth inhibitory effects in plants. When applied at levels of 50 millimolar to tobacco (Nicotiana tabacum L. cv Xanthi) leaf discs galactose caused a rapid increase in ethylene production during the first 2 days of incubation, followed by a rapid return to the basal level on the third day. This pattern of galactose-stimulated ethylene production was accompanied by increased formation of 1-aminocyclopropane-1-carboxylic acid (ACC), which accumulated without being metabolized to ethylene or to the ACC-conjugate. The inhibitory effect of galactose (50 millimolar) on the conversion of ACC of ethylene was relieved partially by d-glucose or sucrose (50 millimolar), and completely by CO₂ (10%), which were shown to enhance this conversion by themselves. Consequently, application of galactose plus any one of these compounds increased ethylene production and decreased free ACC levels. The data suggest that galactose toxicity may result in both an increased ethylene production as well as in accumulation of free ACC in aged discs. The increased ethylene production rates and ACC levels may, in turn, play a role in the development of symptoms associated with galactose toxicity.     

Stress-induced Ethylene Production in the Ethylene-requiring Tomato Mutant Diageotropica

Ethylene synthesis in vegetative tissues is thought to be controlled by indoleacetic acid (IAA). However, ethylene synthesis in the diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) was much less sensitive to IAA than in the normal variety (VFN8). Yet, mechanical wounding stimulated ethylene production by the mutant. The dgt tomato provides an opportunity to study the regulation of stress ethylene independent of IAA effects. Waterlogging (i.e. anaerobic stress) stimulated production of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), in the roots. The ACC was transported to the shoot where it was converted to ethylene. The dgt mutant efficiently utilized ACC for ethylene synthesis under aerobic conditions. The results confirm that the genetic lesion in dgt is located at a step prior to the formation of ACC. Furthermore, induction of ethylene synthesis by anaerobic or mechanical stresses in this mutant is independent of IAA action.     

Variations in the "cross protection" effect between two strains of citrus exocortis viroid

In a survey conducted to evaluate the biological properties of several field sources that induced a severe exocortis reaction on citron (Citrus medica L.), a viroid isolate which induced mild symptoms on Gynura aurantiaca was detected. This isolate was characterised as a strain of the citrus exocortis viroid (CEV) by size and homology, and was designated as CEV-129. Cross protection assays using CEV-129 as a “protecting” strain against the severe type strain of CEV demonstrated that a mild strain of CEV could provide apparent ‘protection’ against challenge inoculations with the severe strain. The protection effect, however, displayed a variability which ranged from only a brief delay to almost total impairment of symptom expression. The level of protection was dependent upon the length of the interval between the inoculations with the mild and the severe strains. In all cases the effect was temporary since the symptoms and viroid concentration which ultimately prevailed reflected the predominance of the severe strain in the mixed infection. The interpretation of these results and their relationship with previous reports of ‘cross protection’ reactions are discussed along with the consideration of the efficacy and limitations of this term with viroid infection.     

Regulatory Role of Cystathionine-γ-Synthase and de novo Synthesis of Methionine in Ethylene Production during tomato Fruit Ripening

The essential amino acid methionine is a substrate for the synthesis of S-adenosyl-methionine (SAM), that donates its methyl group to numerous methylation reactions, and from which polyamines and ethylene are generated. To study the regulatory role of methionine synthesis in tomato fruit ripening, which requires a sharp increase in ethylene production, we cloned a cDNA encoding cystathionine γ-synthase (CGS) from tomato and analysed its mRNA and protein levels during tomato fruit ripening. CGS mRNA and protein levels peaked at the “turning” stage and declined as the fruit ripened. Notably, the tomato CGS mRNA level in both leaves and fruit was negatively affected by methionine feeding, a regulation that Arabidopsis, but not potato CGS mRNA is subject to. A positive correlation was found between elevated ethylene production and increased CGS mRNA levels during the ethylene burst of the climacteric ripening of tomato fruit. In addition, wounding of pericarp from tomato fruit at the mature green stage stimulated both ethylene production and CGS mRNA level. Application of exogenous methionine to pericarp of mature green fruit increased ethylene evolution, suggesting that soluble methionine may be a rate limiting metabolite for ethylene synthesis. Moreover, treatment of mature green tomato fruit with the ethylene-releasing reagent Ethephon caused an induction of CGS mRNA level, indicating that CGS gene expression is regulated by ethylene. Taken together, these results imply that in addition to recycling of the methionine moieties via the Yang pathway, operating during synthesis of ethylene, de novo synthesis of methionine may be required when high rates of ethylene production are induced.