Bean abscission cellulase : characterization of a cDNA clone and regulation of gene expression by ethylene and auxin

The physiology and anatomy of abscission has been studied in considerable detail; however, information on the regulation of gene expression in abscission has been limited because of a lack of probes for specific genes. We have identified and sequenced a 595 nucleotide bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase cDNA clone (pBACl). The bean cellulase cDNA has extensive nucleic and amino acid sequence identity with the avocado cellulase cDNA pAV363. The 2.0 kilobase bean mRNA complementary to pBACl codes for a polypeptide of approximately 51 kilodalton (shown by hybrid-selection followed by in vitro translation). Bean cellulase antiserum is shown to immunoprecipitate a 51 kilodalton polypeptide from the in vitro translation products of abscission zone poly(A)⁺ RNA. Ethylene initiates bean leaf abscission and tissue-specific expression of cellulase mRNA. If ethylene treatment of bean explants was discontinued after 31 h and then 2,5-norbornadiene given to inhibit responses resulting from endogenously synthesized ethylene, polysomal cellulase mRNA hybridizing to pBACl decreased. Thus, ethylene is required not only to initiate abscission and cellulase gene expression but also to maintain continued accumulation of cellulase mRNA. Explants treated with auxin 4 hours prior to a 48 hour treatment with ethylene showed no substantial accumulation of RNA hybridizing to pBACl or expression of cellulase activity.     

Wound ethylene and 1-aminocyclopropane-1-carboxylate synthase in ripening tomato fruit

Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ACC-synthase activity were compared in intact and wounded tomato fruits (Lycopersicon esculentum Mill.) at different ripening stages. Freshly cut and wounded pericarp discs produced relatively little ethylene and had low levels of ACC and of ACC-synthase activity. The rate of ethylene synthesis, the level of ACC and the activity of ACC synthase all increased manyfold within 2 h after wounding. The rate of wound-ethylene formation and the activity of wound-induced ACC synthase were positively correlated with the rate of ethylene production in the intact fruit. When pericarp discs were incubated overnight, wound ethylene synthesis subsided, but the activity of ACC synthase remained high, and ACC accumulated, especially in discs from ripe fruits. In freshly harvested tomato fruits, the level of ACC and the activity of ACC synthase were higher in the inside parts of the fruit than in the pericarp. When wounded pericarp tissue of green tomato fruits was treated with cycloheximide, the activity of ACC synthase declined with an apparent half life of 30–40 in. The activity of ACC synthase in cycloheximide-treated, wounded pericarp of ripening tomatoes declined more slowly.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

Proteinase inhibitors I and II in fruit of wild tomato species: Transient components of a mechanism for defense and seed dispersal

The juice of unripe fruit from a wild species of tomato, Lycopersicon peruvianum (L.) Mill., LA 107, contains over 50% of its soluble proteins as the sum of two proteinase inhibitors. These are the highest levels of proteinase inhibitors and highest percentage of soluble proteins as proteinase inhibitors of any plant or animal tissue found to date. Fruit of the modern tomato, L. esculentum Mill., contains only negligible quantities of the two inhibitors. The two proteinase inhibitors in the fruit of L. peruvianum are members of the Inhibitor I and II families previously found in potato tubers and in leaves of wounded potato and tomato plants. The levels of the two inhibitors in the unripe fruit decrease significantly during ripening. Unripe fruit from other wild Lycopersicon species such as L. parviflorum Rick, Kesicki, Fobes et Holle, L. hirsutum Humb. et Bonpe., L. pimpinellifolium Mill., and other lines of L. peruvianum contain moderate levels of the inhibitors that also decrease during ripening. Another wild tomato species, L. pennellii Corr., is similar to L. esculentum in not containing the two proteinase inhibitors in either unripe or ripe fruit. The transient levels of the inhibitors in fruit of wild species indicate that they are present in unripe fruit as defensive chemicals against insects, birds or small mammals and their disappearance during ripening may render them edible to facilitate seed dispersal. High levels of mRNAs coding for Inhibitors I and II in unripe fruit of L. peruvianum, LA 107, indicate that strong promoters may regulate the developmentally expressed proteinase-inhibitor genes in tomato fruit that may have a substantial potential for use in genetic-engineering experiments to enhance the production of large quantities of proteinase inhibitors or other proteins in field tomatoes.Abbreviations poly(A)⁺ mRNA polyadenylated mRNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide electrophoresis Project 1791, College of Agriculture and Home Economics Research Center, Washington, State University     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Cloning of a Complementary DNA that Encodes an Acidic Chitinase which is Induced by Ethylene and Expression of the Corresponding Gene

A complementary DNA encoding an ethylene-inducible acidic chitinaseof azuki bean (Vigna angularis) was isolated, and its completenucleotide sequence was determined. The nucleotide and deducedamino-acid sequence were very similar to those of an acidicchitinase from cucumber leaves that had been infected with tobacconecrosis virus. The mRNA for the acidic chitinase was not detectedin leaves of azuki bean that had not been treated with ethylene,but it appeared 3 h after initiation of treatment with ethyleneand its level gradually increased over a period of 19 h. ThemRNA also accumulated in response to salicylate or wounding.The expression of the gene in response to wounding was suppressedby 2,5-norbornadiene, but that in response to salicylate wasnot affected by this inhibitor. (Received May 19, 1992; Accepted November 2, 1992)     

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.     

Properties and Partial Purification of 1-Aminocyclopropane-1-carboxylate Synthase

We studied the regulation of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in tomato (Lycopersicon esculentum Mill.) fruit tissue and attempted the purification of this enzyme. The increase of ACC synthase activity in wounded tomato pericarp was inhibited by cordycepin and cycloheximide. Density labeling studies showed a 0.75% increase in the buoyant density of ACC synthase isolated from tomato pericarp tissue that had been incubated on ²H₂O as compared to ACC synthase from H₂O-treated tissue. These data are consistent with the hypothesis that ACC synthase is synthesized de novo following wounding of tomato pericarp tissue. SDS-gel electrophoresis and fluorography showed that the pattern of incorporation of l-[³⁵S]methionine into protein changed with time after wounding of the tissue. Radioactive protein bands that were not detected 1 hour after wounding, became apparent 2 to 3 hours after wounding.     

Inhibition of Biosynthesis of Polyisoprenol Sugars in Chick Embryo Microsomes by Tunicamycin

Changes in the phytoalexin content in unripe fruit of banana, Musa acuminata, were analyzed after various treatments. The results show that level of hydroxyanigorufone started to increase 1-2 day after either wounding or inoculation with conidia of Colletotrichum musae. Inoculation followed by wounding induced the formation of many other phenylphenalenones. The accumulation of hydroxyanigorufone decreased, after its transient maximum, on ripening by exposure of the wounded fruit to ethylene. The level of production of hydroxyanigorufone in ripe fruit treated by wounding and/or by inoculation was much lower than that in unripe fruit. 2-Aminooxyacetic acid, an inhibitor of phenylalanine ammonia-lyase (PAL), inhibited the accumulation of hydroxyanigorufone in wounded fruit, and the PAL activity increased after wounding and ethylene treatment, respectively. Feeding experiments with [1-¹³C] and [2-¹³C]cinnamic acids, and [2-¹³C]malonate show that two molecules of cinnamic acid and one of malonate were incorporated into each molecule of hydroxyanigorufone. The phytoalexins isolated from fruit to which deuterated hydroxyanigorufone and irenolone had been administered revealed that 2-(4′-hydroxyphenyl)-1,8-naphthalic anhydride was biosynthesized from hydroxyanigorufone rather than from irenolone.     

Characterization and Induction of the Activity of 1-Aminocyclopropane-1-Carboxylate Oxidase in the Wounded Mesocarp Tissue of Cucurbita maxima

1-Aminocyclopropane-1-carboxylate (ACC) oxidase (ethylene-formingenzyme) was isolated from wounded mesocarp tissue of Cucurbitamaxima (winter squash) fruit, and its enzymatic properties wereinvestigated. The enzyme required Fe²⁺ and ascorbate for itsactivity as well as ACC and O₂ as substrates. The in vitro enzymeactivity was enhanced by CO₂. The apparent K_m value for ACCwas 175 µM under atmospheric conditions. The enzyme activitywas inhibited by sulfhydryl inhibitors and divalent cationssuch as Co²⁺, Cu²⁺, and Zn²⁺. ACC oxidase activity was induced at a rapid rate by woundingin parallel with an increase in the rate of ethylene production.The exposure of excised discs of mesocarp to 2,5-norbornadiene(NBD),an inhibitor of ethylene action, strongly suppressed inductionof the enzyme, and the application of ethylene significantlyaccelerated the induction of the activity of ACC oxidase inthe wounded mesocarp tissue. These results suggests that endogenousethylene produced in response to wounding may function in promotingthe induction of ACC oxidase. (Received January 13, 1993; Accepted April 15, 1993)     

A Complex Array of Proteins Related to the Multimeric Leucine Aminopeptidase of Tomato

Leucine aminopeptidase (LAP) mRNAs are induced in response to mechanical wounding, pathogen infection, and insect infestation (V. Pautot, F.M. Holzer, B. Reisch, L.L. Walling [1993] Proc Natl Acad Sci USA 90: 9906-9910). Polyclonal antibodies to a glutathione S-transferase-LAP fusion protein and affinity-purified antibodies recognizing LAP antigenic determinants detected four classes of polypeptides in tomato (Lycopersicon esculentum) leaves. All four classes had multiple polypeptides in two-dimensional polyacrylamide gel electrophoresis immunoblots. Although antigenically related to the wound-induced tomato LAP proteins, the 77- and 66-kD LAP-like proteins accumulated in both healthy and wounded leaves. Two classes of 55-kD polypeptides with distinctive isoelectric points were designated as plant LAPs; only the acidic LAP proteins accumulated to high levels after mechanical wounding or Pseudomonas syringae pv tomato infection of tomato leaves. The temporal accumulation of LAP mRNAs was correlated with the increase in acidic LAP protein subunits. A slow-migrating LAP activity was detected using a native gel assay after wounding. The molecular mass of the native wound-induced LAP enzyme was 353 kD. The 55-kD acidic LAP proteins were associated with induced LAP activity, whereas the neutral LAPs and the LAP-like proteins were not associated with this exopeptidase. A second, fast-migrating aminopeptidase was detected in both healthy and wounded tomato leaves. Cell fractionation experiments revealed that wound-induced LAP is a soluble enzyme.     

Molecular details of tomato extensin and glycine-rich protein gene expression

In a recent publication (Plant Molecular Biology 16: 547–565 (1991)) Showalter et al. described the isolation and initial characterization of fifteen extensin and extensin-like tomato cDNAs. These cDNAs were determined to fall into five distinct classes; class I and II clones encoded extensins, class III and V clones encoded glycine-rich proteins (GRPs), and class IV clones encoded a portion of a GRP sequence on one DNA strand and a portion of an extensin sequence on the other DNA strand. In this publication, a more detailed analysis of the expression of these cDNA classes was performed with respect to wounding in various tomato organs, development, kinetics and systemic extent of the wound response, ethylene treatment, abscisic acid (ABA) treatment, and drought stress by using RNA gel blot hybridizations. In general, extensin gene expression was readily detected in stems and roots, but not in leaves. With both class I and II extensin cDNA probes, wound-induced accumulation of mRNA in stems was first detected between 4 and 8 h after wounding with maximal accumulation occurring after 12 h. Moreover, these extensin wound responses were detected locally at the wound site but not systemically. Expression of the class III GRP was largely limited to wounded stem tissue. Initial detection and maximal accumulation of the class III GRP mRNA was similar to the extensins mRNAs; however, this GRP wound response occurred both locally and systemically. Additionally, abscisic acid treatment and drought stress resulted in the marked accumulation of the class III GRP mRNA in tomato stems, but did not alter the expression of the other cDNA classes. In contrast, expression of the class V GRP occurred in stems and roots and to a lesser extent in leaves and decreased in response to wounding over a 24 h time period. The class V GRP wound response was further characterized by an early, transient accumulation of mRNA occurring 2–4 h after wounding in stems and by its local nature.     

Expression of allene oxide synthase determines defense gene activation in tomato

Allene oxide synthase (AOS; hydroperoxide dehydratase; EC 4.2.1.92) catalyzes the first step in the biosynthesis of jasmonic acid from lipoxygenase-derived hydroperoxides of free fatty acids. Using the AOS cDNA from tomato (Lycopersicon esculentum), in which the role of jasmonic acid in wound-induced defense gene activation has been best described, we examined the kinetics of AOS induction in response to wounding and elicitors, in parallel with that of the wound-inducible PIN II (proteinase inhibitor II) gene. AOS was induced in leaves by wounding, systemin, 12-oxophytodienoic acid, and methyl jasmonate. The levels of AOS mRNA started declining by 4 h after induction, whereas the levels of PIN II mRNA continued to increase up to 20 h after induction. Salicylic acid inhibited AOS and PIN II expression, and the addition of 12-oxophytodienoic acid or methyl jasmonate did not prevent the inhibition of PIN II expression in the presence of salicylic acid. Ethylene induced the expression of AOS, but the presence of ethylene alone did not produce an optimal induction of PIN II. The addition of silver thiosulfate, an ethylene action inhibitor, prevented the wound-induced expression of both AOS and PIN II. Products of hydroperoxide lyase affected neither AOS nor PIN II, but induced expression of prosystemin. Based on these results, we propose an updated model for defense gene activation in tomato.     

Changes in Gene Expression during Tomato Fruit Ripening

Total proteins from pericarp tissue of different chronological ages from normally ripening tomato (Lycopersicon esculentum Mill. cv Rutgers) fruits and from fruits of the isogenic ripening-impaired mutants rin, nor, and Nr were extracted and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Analysis of the stained bands revealed increases in 5 polypeptides (94, 44, 34, 20, and 12 kilodaltons), decreases in 12 polypeptides (106, 98, 88, 76, 64, 52, 48, 45, 36, 28, 25, and 15 kilodaltons), and fluctuations in 5 polypeptides (85, 60, 26, 21, and 16 kilodaltons) as normal ripening proceeded. Several polypeptides present in ripening normal pericarp exhibited very low or undetectable levels in developing mutant pericarp. Total RNAs extracted from various stages of Rutgers pericarp and from 60 to 65 days old rin, nor, and Nr pericarp were fractionated into poly(A)⁺ and poly(A)⁻ RNAs. Peak levels of total RNA, poly(A)⁺ RNA, and poly(A)⁺ RNA as percent of total RNA occurred between the mature green to breaker stages of normal pericarp. In vitro translation of poly(A)⁺ RNAs from normal pericarp in rabbit reticulocyte lysates revealed increases in mRNAs for 9 polypeptides (116, 89, 70, 42, 38, 33, 31, 29, and 26 kilodaltons), decreases in mRNAs for 2 polypeptides (41 and 35 kilodaltons), and fluctuations in mRNAs for 5 polypeptides (156, 53, 39, 30, and 14 kilodaltons) during normal ripening. Analysis of two-dimensional separation of in vitro translated polypeptides from poly(A)⁺ RNAs isolated from different developmental stages revealed even more extensive changes in mRNA populations during ripening. In addition, a polygalacturonase precursor (54 kilodaltons) was immunoprecipitated from breaker, turning, red ripe, and 65 days old Nr in vitro translation products.     

Disruption of the Polar Auxin Transport System in Cotton Seedlings following Treatment with the Defoliant Thidiazuron

The effect of the defoliant thidiazuron (TDZ) on basipetal auxin transport in petiole segments isolated from cotton (Gossypium hirsutum L. cv LG102) seedlings was examined using the donor/receiver agar block technique. Treatment of intact seedlings with TDZ at concentrations of 1 micromolar or greater resulted in a dose-dependent inhibition of ¹⁴C-IAA transport in petiole segments isolated 1 or 2 days after treatment. Using 100 micromolar TDZ, the inhibition was detectable 19 hours after treatment and was complete by 27 hours. Both leaves and petiole segments exhibited a marked increase in ethylene production following treatment with TDZ at concentrations of 0.1 micromolar or greater. The involvement of ethylene in this TDZ response was evaluated by examining the effects of two inhibitors of ethylene action: silver thiosulfate, 2,5-norbornadiene. One day after treatment, both inhibitors effectively antagonized the TDZ-induced inhibition of auxin transport. Two days after TDZ treatment both inhibitors were ineffective. The decrease in IAA transport in TDZ treated tissues was associated with increased metabolism of IAA. The transport of ¹⁴C-2,4-dichlorophenoxyacetic acid was also inhibited by TDZ treatment. This inhibition was not accompanied by increased metabolism. Incorporation of TDZ into the receiver blocks had no effect on auxin transport. The ability of the phytotropin N-1-naphthylphthalamic acid to stimulate IAA uptake from a bathing medium was reduced in TDZ-treated tissues. This reduction is thought to reflect a decline in the auxin efflux system following TDZ treatment.     

DNA fragmentation is regulated by ethylene during carpel senescence in Pisum sativum

Treatment of the pea carpel with the ethylene action inhibitors, silver thiosulphate and 2,5-norbornadiene, retarded its senescence and extended the growth time in which the unpollinated carpel was able to respond to gibberellins. Cells of the senescent carpel have characteristics of apoptotic cells, such as nuclei condensation and internucleosomal DNA fragmentation. Both DNA laddering and condensed nuclei can be prevented by the use of the ethylene action inhibitors, while ethylene treatment accelerates DNA fragmentation. It is postulated that ethylene produced by the flower after anthesis determines the fate of the ovary/ovule; in other words, if the ovary receives no additional stimulus (pollination or gibberellin treatment), it undergoes a senescence process, with some cells showing characteristics similar to those of programmed cell death already identified in animal systems.