Characterization of an Ethylene Overproducing Mutant of Tomato (Lycopersicon esculentum Mill. Cultivar VFN8)

Ethylene production rates and tissue ethylene concentrations were determined for the single-gene, Epinastic (Epi) tomato (Lycopersicon esculentum Mill.) mutant, and its parent, cv VFN8. The Epi phenotype was characterized by severe leaf epinasty, thickened stems and petioles, and a compact growth habit. In 4-day-old seedlings, ethylene production was significantly higher in Epi than in VFN8. Ethylene production rates also were higher for excised root, hypocotyl, cotyledon, and shoot tissue of 14-day-old Epi seedlings as compared with VFN8. The greatest difference in the ethylene production rate was observed in excised Epi shoot tissue, which was more than 2.5 times higher than in VFN8. Tissue ethylene concentrations of 19−, 25−, and 31-day-old Epi plants were 8, 172, and 307% higher than for VFN8, corresponding to increasing expression of the Epi phenotypic characteristics with age. The highest ethylene concentrations occurred in the shoot apex of both genotypes. Higher ethylene concentrations in Epi resulted from greater 1-aminocyclopropane-1-carboxylic acid content rather than increased ethylene-forming enzyme activity. The elevated ethylene levels in Epi did not result from increased auxin sensitivity. The sensitivity of root growth to inhibition by ethylene did not differ between VFN8 and Epi. Although elevated levels of ethylene in Epi plants apparently exacerbate its epinastic growth characteristics, other evidence indicates that this may not be the fundamental lesion. This mutant may provide a unique system for investigating the regulation of ethylene biosynthesis and the role of target cell types in plant development.     

Degradation of tobacco pathogenesis-related proteins : evidence for conserved mechanisms of degradation of pathogenesis-related proteins in plants

Tobacco (Nicotiana tabacum L.) leaves were found to contain an extracellular proteinase that endoproteolytically cleaves tobacco pathogenesis-related (PR) proteins. This proteinase was partially purified from tobacco leaves and characterized as an aspartyl proteinase with a pH optimum around pH 3 and a molecular mass of 36,000 to 40,000 daltons. In vitro, the enzyme cleaved purified tobacco and tomato PR proteins into discrete fragments. The characteristics of this proteinase were similar to pepsin and identical to those displayed by a previously described tomato 37-kilodalton aspartyl proteinase active against tomato PR proteins (I Rodrigo, P Vera, V Conejero [1989] Eur J Biochem 184: 663-669), suggesting that these extracellular proteases could play a role in a conserved mechanism for PR protein turnover in plants.     

Polyamines in plants infected by citrus exocortis viroid or treated with silver ions and ethephon

The levels of polyamines in leaves of Gynura aurantiaca DC and tomato, Lycopersicon esculentum Mill. cv Rutgers, infected with citrus exocortis viroid (CEVd) or treated with silver nitrate or ethephon (2-chloroethylphosphonic acid) were measured by HPLC in relation to development of symptoms. Previously it had been demonstrated that treatment of G. aurantiaca plants with silver nitrate or ethephon closely mimicked the effects of viroid infection in the plants. In the studies reported here, a marked decrease in putrescine level was observed in plants infected by CEVd or treated with silver ions or ethephon. There was no significant change in either spermidine or spermine levels. Treatment of G. aurantiaca plants with specific inhibitors of ethylene biosynthesis (aminoethoxyvinylglycine, Co²⁺) or ethylene action (norbornadiene) prevented the decrease of putrescine associated with silver nitrate treatment and had no effect on spermidine or spermine levels. The development of viroid-like symptoms, the production of associated pathogenesis-related proteins, and the rise in protease activity induced by silver nitrate, were all suppressed by exogenous application of putrescine. The decreased level of putrescine as an ethylene-mediated step in the transduction of the viroid and silver or ethephon signaling is discussed.     

Ineffectiveness of ethylene biosynthetic and action inhibitors in phenotypically reverting theEpinastic mutant of Tomato (Lycopersicon esculentum mill.)

Five-day-old, dark-grown seedlings of theEpinastic (Epi) tomato mutant (Lycopersicon esculentum Mill.) and its parent, cultivar VFN8, were used as a system for assessing the role of ethylene in theEpi phenotype. The distinguishing features ofEpi seedlings are an increase in hypocotyl diameter and reduced hypocotyl length. Treatment of VFN8 seedlings with 0.5 l/liter ethylene closely mimicked theEpi phenotype. The rate of ethylene production by 5-day-old, dark-grownEpi seedlings was double that of VFN8 seedlings. Nevertheless, treatment ofEpi seedlings with inhibitors of ethylene biosynthesis (aminoethoxyvinylglycine or Co²⁺) or ethylene action (silver thiosulfate or norbornadiene) failed to normalize theEpi phenotype.Epi seedlings grown in sealed jars containing ethylene and CO₂ adsorbants also expressed the characteristicEpi phenotype. The results indicate that the physiological lesion resulting from theEpi gene mutation is not simply an overproduction of ethylene.     

Purification and serological characterization of three basic 15-kilodalton pathogenesis-related proteins from tomato

In tomato (Lycopersicon esculentum) several acidic and basic apoplastic pathogenesis-related (PR) proteins are induced upon inoculation with virulent or avirulent races of Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif). One of the most predominant and best characterized tomato PR proteins is P14, a basic protein that shows homology to the tobacco (Nicotiana tabacum) PR-1 protein family. To investigate whether, by analogy with these tobacco PR-1 proteins, P14 also belongs to a family of differently charged isomers, the abundantly occurring PR proteins with molecular masses around 15 kilodaltons (kD) were purified from apoplastic fluids isolated from C. fulvum-infected tomato. Three basic proteins migrating similarly to P14 on sodium dodecyl sulfate polyacrylamide gels were purified to homogeneity by gel filtration followed by high resolution liquid chromatography. Two proteins (15.5 kD, isoelectric point [pl] 10.9 and 10.7 appeared to be serologically related to each other and to the tobacco PR-1 proteins. A third protein (15 kD, pl 10.4) was not serologically related to any other tomato PR protein but was found to be related to PR-R from tobacco.     

Short-term salt tolerance mechanisms in differentially salt tolerant tomato species

The physiological changes induced by a daily increase of NaCl level, over a period of 4 d, were studied in leaves of the salt-sensitive cultivated tomato species Lycopersicon esculentum and its wild salt-tolerant relative Lycopersicon pennellii. A higher solute contribution to the osmotic adjustment was observed in NaCl-treated leaves of L. pennellii than in those of L. esculentum. This response together with the higher accumulation of inorganic solutes in the wild species and of organic solutes in the cultivated species verified the different salt tolerance mechanisms operating in the two species in the short-term. With regard to the changes induced by salt stress on the free polyamine levels, the putrescine and spermine levels increased with salinity, whereas the spermine levels decreased in both tomato species; nevertheless, the main difference between the two species lays in an earlier and greater accumulation of putrescine induced by salinity in L. pennellii than in L. esculentum. The changes in putrescine levels were associated to changes in amino acids related to its synthesis, and the changes were different in both species. In L. esculentum, the high concentrations of some intermediate compounds (glutamate and arginine) were related to the low accumulation rate of both proline and putrescine. In contrast, in L. pennellii, important reductions in glutamate and arginine levels were found at the end of the salinization period. Moreover, in this last situation, a decline in the putrescine level ran parallel to a high proline accumulation, which suggests that the higher the stress level, the higher the deviation of glutamate to proline occurring in the salt tolerant species. It could be concluded that an early accumulation of the diamine putrescine seems to be associated with salt tolerance in the short-term.     

Auxin and Ethylene Regulation of Petiole Epinasty in Two Developmental Mutants of Tomato, diageotropica and Epinastic

The epinastic growth responses of petioles to auxin and ethylene were quantified in two developmental mutants of tomato (Lycopersicon esculentum Mill.). In the wild type parent line, cultivar VFN8, the epinastic response of excised petiole sections was approximately log-linear between 0.1 and 100 micromolar indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations, with a greater response to 2,4-D at any concentration. When ethylene synthesis was inhibited by aminoethoxyvinylglycine (AVG), epinasty was no longer induced by auxin, but could be restored by the addition of ethylene gas. In the auxin-insensitive mutant, diageotropica (dgt), no epinastic response to IAA was observed at IAA concentrations that effectively induced epinasty in VFN8. In the absence of added IAA, epinastic growth of dgt petioles in 1.3 microliters per liter exogenous ethylene gas was more than double that of VFN8 petioles. IAA had little additional effect in dgt, but promoted epinasty in VFN8. These results confirm that tomato petiole cells respond directly to ethylene and make it unlikely that the differential growth responsible for epinasty results from lateral auxin redistribution. The second mutant, Epinastic (Epi), exhibits constitutively epinasty, cortical swelling, and root branching symptomatic of possible alternation in auxin or ethylene regulation of growth. Only minor quantitative differences were observed between the epinastic responses to auxin and ethylene of VFN8 and Epi. However, in contrast to VFN8, when ethylene synthesis or action was inhibited in Epi, auxin still induced 40 to 50% of the epinastic response observed in the absence of inhibitors. This indicates that the target cells for epinastic growth in Epi are qualitatively different from those of VFN8, having gained the ability to grow differentially in response to auxin alone. The dgt and Epi mutants provide useful systems in which to study the genetic determination of target cell specificity for hormone action.     

Effect of Ethephon on Protein Degradation and the Accumulation of ;Pathogenesis-Related' (PR) Proteins in Tomato Leaf Discs

The effect of ethephon (2-chloroetylphosphonic acid) on the degradation of proteins and on the induction of Lycopersicon esculentum pathogenesis-related (PR) proteins was studied in tomato leaf discs. The rate of ribulose, -1,5-bisphosphate carboxylase/oxygenase (Rubisco) degradation was maximal in discs after 48 hours of incubation with 1 millimolar ethephon, leading to complete disappearance of Rubisco after 96 hours. This effect was correlated with an increase in PR protein synthesis and the induction of the previously reported alkaline proteolytic enzyme PR-P69 (P Vera, V Conejero [1988] Plant Physiol 87: 58-63). In vivo pulse-chase experiments demonstrated that ethephon not only affected Rubisco content but that of many other ³⁵S-labeled proteins as well, indicating that ethylene activates a general and nonspecific mechanism of protein degradation. This effect was partially inhibited in vivo by the action of pCMB, a selective inhibitor of cysteine-proteinases such as P69. These data reinforce the hypothesis that P69 and perhaps other PR proteins are involved in the mechanism of accelerated protein degradation activated by ethylene.     

Polyamine content of long-keeping alcobaca tomato fruit

Fruit of tomato landrace Alcobaca, containing the recessive allele alc, ripen more slowly, with a reduced level of ethylene production, and have prolonged keeping qualities. The levels of polyamines in pericarp tissues of alc and `wild type' Alc (cv Rutgers and Alcobaca-red) fruit were measured by HPLC in relation to ripening. Putrescine was the predominant polyamine with a lower content of spermidine, while spermine was just detectable. The level of putrescine was high at the immature green stage and declined in the mature green stage. In Alc fruit the decline persisted but in alc fruit the putrescine level increased during ripening to a level similar to that present at the immature green stage. There was no pronounced change or difference in spermidine levels. The enhanced polyamine level in alc fruit may account for their ripening and storage characteristics.     

Quantification of Indole-3-Acetic Acid in Dark-Grown Seedlings of the Diageotropica and Epinastic Mutants of Tomato (Lycopersicon esculentum Mill.)

Endogenous indoleacetic acid (IAA) levels were examined in 7-day-old, dark-grown tomato seedlings (Lycopersicon esculentum Mill. cv VFN8), and in two single-gene mutants, Epinastic and diageotropica. Gas chromatography-mass spectrometry was employed to quantify IAA using ¹³C₆-[benzene ring]indoleacetic acid as internal standard. IAA concentrations ranged from 89 to 134 nanograms per gram dry weight and were not significantly different for the three genotypes. Ethylene over-production by dark-grown Epi seedlings is not likely to result from increased IAA. Assuming similar recovery percentages for each genotype, indole-3-ethanol, a purported storage form of IAA, was identified by GC-MS and found to be more prevalent in the parent tomato, VFN8, with only trace amounts observed in Epi. No IEt was detected by high performance liquid chromatography/fluorescence in dgt (detection limit >100 picograms).     

Role of putrescine in ovary and embryo development in fruit bat Cynopterus sphinx during embryonic diapause

The aim of this study was to evaluate the effect of putrescine on ovarian activity and the rate of embryonic development in Cynopterus sphinx during delayed development. The result showed the presence of a rate‐limiting enzyme, ornithine decarboxylase‐1, in both ovary and utero‐embryonic unit of C. sphinx suggests a synthesis of putrescine in these sites. The corpus luteum showed increased, whereas utero‐embryonic unit showed decreased production of putrescine during delayed development as compared with the normal development. The bat treated in vivo with putrescine during delayed development showed increase in progesterone and estradiol synthesis, correlated with increased expression of luteinizing hormone receptor, steroidogenic acute receptor protein, and 3β‐hydroxysteroid dehydrogenase through extracellular signal‐regulated kinase (ERK1/2)‐mediated pathway in the ovary; but showed increase in the weight and expression of progesterone receptor (PR), B‐cell lymphoma 2, proliferating cell nucleus antigen, and vascular endothelial growth factor proteins in utero‐embryonic unit. The in vitro treatment of putrescine showed stimulatory whereas treatment with an inhibitor of putrescine, 2‐difluoromethylornithine caused an inhibitory effect on ovarian progesterone synthesis and cell proliferation, and cell survival in the utero‐embryonic unit. In conclusion, the putrescine showed two separate roles during embryonic diapause, high concentration of putrescine in the ovary may support corpus luteum and basal synthesis of progesterone, whereas a low level of putrescine causes retarded embryonic development by inhibiting cell proliferation in the utero‐embryonic unit. The bat treated with putrescine either directly promotes cell proliferation, cell survival, and angiogenic activities or acts indirectly increasing PR on utero‐embryonic unit thereby activating development in delayed embryo in C. sphinx.     

Pathogenesis and stress related,as well as metabolic proteins are regulated in tomato stems infected with Ralstonia solanacearum

A comparative proteome analysis was initiated to systematically investigate the physiological response of tomato (Solanum lycopersicum) to infection with Ralstonia solanacearum, causal agent of bacterial wilt. Plants of the susceptible tomato recombinant inbred line NHG3 and the resistant NHG13 were either infected or not infected with R. solanacearum and subsequently used for proteome analysis. Two-dimensional isoelectric focussing/sodium dodecyl-sulphate polyacrylamide gel electrophoresis (2-D IEF/SDS-PAGE) allowed the separation of about 650–690 protein spots per analysis. Twelve proteins were of differential abundance in susceptible plants in response to bacterial infection, while no differences were observed in the resistant genotype. LC-MS/MS analysis of these spots revealed 12 proteins, six of which were annotated as plant and six as bacterial proteins. Among the plant proteins, two represent pathogenesis related (PR) proteins, one stress response protein, one enzyme of carbohydrate and energy metabolism, and one hypothetical protein. A constitutive difference between resistant and susceptible lines was not found.     

Polyamine metabolism in ripening tomato fruit : I. Identification of metabolites of putrescine and spermidine

The metabolism of [1,4-¹⁴C]putrescine and [terminal methylene-³H]spermidine was studied in the fruit pericarp (breaker stage) discs of tomato (Lycopersicon esculentum Mill.) cv Rutgers, and the metabolites identified by high performance liquid chromatography and gas chromatography-mass spectrometry. The metabolism of both putrescine and spermidine was relatively slow; in 24 hours about 25% of each amine was metabolized. The ¹⁴C label from putrescine was incorporated into spermidine, γ-aminobutyric acid (GABA), glutamic acid, and a polar fraction eluting with sugars and organic acids. In the presence of gabaculine, a specific inhibitor of GABA:pyruvate transaminase, the label going into glutamic acid, sugars and organic acids decreased by 80% while that in GABA increased about twofold, indicating that the transamination reaction is probably a major fate of GABA produced from putrescine in vivo. [³H]Spermidine was catabolized into putrescine and β-alanine. The conversion of putrescine into GABA, and that of spermidine into putrescine, suggests the presence of polyamine oxidizing enzymes in tomato pericarp tissues. The possible pathways of putrescine and spermidine metabolism are discussed.     

Identification of Several Pathogenesis-Related Proteins in Tomato Leaves Inoculated with Cladosporium fulvum (syn. Fulvia fulva) as 1,3-beta-Glucanases and Chitinases

Inoculation of tomato (Lycopersicon esculentum) leaves with Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif) results in a marked accumulation of several pathogenesis-related (PR) proteins in the apoplast. Two predominant PR proteins were purified from apoplastic fluid by ion exchange chromatography followed by chromatofocusing. One protein (molecular mass [M_r] 35 kilodaltons [kD], isoelectric point [pI] ~6.4) showed 1,3-β-glucanase activity, while the other one (M_r26 kD, pI ~6.1) showed chitinase activity. Identification of the products that were released upon incubation of the purified enzymes with laminarin or regenerated chitin revealed that both enzymes showed endo-activity. Using antisera raised against these purified enzymes from tomato and against chitinases and 1,3-β-glucanases isolated from other plant species, one additional 1,3-β-glucanase (M_r33 kD) and three additional chitinases (M_r 27, 30, and 32 kD) could be detected in apoplastic fluids or homogenates of tomato leaves inoculated with C. fulvum. Upon inoculation with C. fulvum, chitinase and 1,3-β-glucanase activity in apoplastic fluids increased more rapidly in incompatible interactions than in compatible ones. The role of these hydrolytic enzymes, potentially capable of degrading hyphal walls of C. fulvum, is discussed in relation to active plant defense.     

Polyamine Metabolism in Ripening Tomato Fruit : II. Polyamine Metabolism and Synthesis in Relation to Enhanced Putrescine Content and Storage Life of a/c Tomato Fruit

The fruit of the Alcobaca landrace of tomato (Lycopersicon esculentum Mill.) have prolonged keeping qualities (determined by the allele a/c) and contain three times as much putrescine as the standard Rutgers variety (A/c) at the ripe stage (ARG Dibble, PJ Davies, MA Mutschler [1988] Plant Physiol 86: 338-340). Polyamine metabolism and biosynthesis were compared in fruit from Rutgers and Rutgers-a/c—a near isogenic line possessing the allele a/c, at four different stages of ripening. The levels of soluble polyamine conjugates as well as wall bound polyamines in the pericarp tissue and jelly were very low or nondetectable in both genotypes. The increase in putrescine content in a/c pericarp is not related to normal ripening as it occurred with time and whether or not the fruit ripened. Pericarp discs of both normal and a/c fruit showed a decrease in the metabolism of [1,4-¹⁴C]putrescine and [terminal labeled-³H]spermidine with ripening, but there were no significant differences between the two genotypes. The activity of ornithine decarboxylase was similar in the fruit pericarp of the two lines. Arginine decarboxylase activity decreased during ripening in Rutgers but decreased and rose again in Rutgers-a/c fruit, and as a result it was significantly higher in a/c fruit than in the normal fruit at the ripe stage. The elevated putrescine levels in a/c fruit appear, therefore, to be due to an increase in the activity of arginine decarboxylase.     

The possible involvement of polyamines in the development of tomato fruits in vitro

The apparent involvement of ornithine decarboxylase (ODC) and putrescine in the early stages of fruit growth in tomato (Lycopersicon esculentum Mill.) has been previously described. Further evidence presented here supports the direct involvement of ODC and putrescine in the cell division process in tomato fruits. In tomato fruits grown in vitro, in which basic growth processes are inhibited, the activity of ODC and arginine decarboxylase (ADC) and the level of free polyamines were reduced. While ODC and ADC activity was correlated with the period of cell division in the tomato fruit, the free polyamine content was correlated with the DNA content, cell size, and fruit fresh weight. The addition of exogenous putrescine, however, did not restore the basic growth processes in the fruits grown in vitro.     

Snow-Mold-Induced Apoplastic Proteins in Winter Rye Leaves Lack Antifreeze Activity

During cold acclimation, winter rye (Secale cereale L.) plants secrete antifreeze proteins that are similar to pathogenesis-related (PR) proteins. In this experiment, the secretion of PR proteins was induced at warm temperatures by infection with pink snow mold (Microdochium nivale), a pathogen of overwintering cereals. A comparison of cold-induced and pathogen-induced proteins showed that PR proteins accumulated in the leaf apoplast to a greater level in response to cold. The PR proteins induced by cold and by snow mold were similar when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting. Both groups of PR proteins contained glucanase-like, chitinase-like, and thaumatin-like proteins, and both groups exhibited similar levels of glucanase and chitinase activities. However, only the PR proteins induced by cold exhibited antifreeze activity. Our findings suggest that the cold-induced PR proteins may be isoforms that function as antifreeze proteins to modify the growth of ice during freezing while also providing resistance to the growth of low-temperature pathogens in advance of infection. Both functions of the cold-induced PR proteins may improve the survival of overwintering cereals.     

Induction of defense responses in tomato against Pseudomonas syringae pv. tomato by regulating the stress ethylene level with Methylobacterium oryzae CBMB20 containing 1-aminocyclopropane-1-carboxylate deaminase

This study aimed to examine the induction of defense responses in tomato elicited by Methylobacterium oryzae CBMB20 as a consequence of reduced stress ethylene level possibly through its ACC deaminase activity. Significantly increased activities of pathogenesis-related (PR) proteins and defense enzymes such as β-1,3-glucanase, phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase were noted in M. oryzae CBMB20 pretreated and challenged with Pseudomonas syringae pv. tomato (Pst) compared to either control or M. oryzae-treated tomato plants in both growth chamber and greenhouse conditions. Increased PR proteins and defense enzyme activities were correlated with the reduction of stress ethylene level. M. oryzae CBMB20 reduced the stress ethylene level about 27% and 55% when challenged with Pst, in growth chamber and greenhouse on day 7 respectively and the effect was comparable to that of the chemical ethylene biosynthesis inhibitor AVG, L-α-(2-aminoethoxyvinyl)-glycine hydrochloride. As a consequence of reduced stress ethylene level and its effect on defense response in crop plants, the disease severity was reduced 26% in M. oryzae CBMB20-treated plants challenged with pathogen. Therefore, inoculation of M. oryzae CBMB20 would induce the defense enzymes and contribute to the enhanced resistance of tomato plants against the pathogen Pst.     

Utilization of putrescine in tobacco cell lines resistant to inhibitors of polyamine synthesis

Three tobacco cell lines have been analyzed which are resistant to lethal inhibitors of either putrescine production or conversion of putrescine into polyamines. Free and conjugated putrescine pools, the enzymic activities (arginine, ornithine, and S-adenosylmethionine decarboxylases), and the growth characteristics during acidic stress were measured in suspension cultures of each cell line. One cell line, resistant to difluoromethylornithine (Dfr1) had a very low level of ornithine decarboxylase activity which was half insensitive to the inhibitor in vitro. Intracellular free putrescine in Dfr1 was elevated 10-fold which was apparently due to a 20-fold increase in the arginine decarboxylase activity. The increased free putrescine titer was not reflected in an increased level of spermidine, spermine, or putrescine conjugation. Dfr1 cultures survived acidic stress at molarities which were lethal to wild type cultures. Two other mutants, resistant to methylglyoxal bis(guanylhydrazone) (Mgr3, Mgr12), had near normal levels of the three decarboxylases and normal titers of free putrescine, spermidine, and spermine. Both mutants however had elevated levels of conjugated putrescine. Mgr12 had an increased sensitivity to acidic medium. These results suggest that increased levels of free putrescine production may enhance the ability of tobacco cells to survive acid stress. This was supported by the observation that cytotoxic effects of inhibiting arginine decarboxylase in wild type cell lines were dependent on the acidity of the medium.