Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria

Biotic stress like pathogenic infection increases ethylene biosynthesis in plants and ethylene inhibitors are known to alleviate the severity of plant disease incidence. This study aimed to reduce the bacterial spot disease incidence in tomato plants caused by Xanthomonas campestris pv. vesicatoria (XCV) by modulating stress ethylene with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Methylobacterium strains. Under greenhouse condition, Methylobacterium strains inoculated and pathogen challenged tomato plants had low ethylene emission compared to pathogen infected ones. ACC accumulation and ACC oxidase (ACO) activity with ACO related gene expression increased in XCV infected tomato plants over Methylobacterium strains inoculated plants. Among the Methylobacterium spp., CBMB12 resulted lowest ACO related gene expression (1.46 Normalized Fold Expression), whereas CBMB20 had high gene expression (3.42 Normalized Fold Expression) in pathogen challenged tomato. But a significant increase in ACO gene expression (7.09 Normalized Fold Expression) was observed in the bacterial pathogen infected plants. In contrast, Methylobacterium strains enhanced β-1,3-glucanase and phenylalanine ammonia-lyase (PAL) enzyme activities in pathogen challenged tomato plants. The respective increase in β-1,3-glucanase related gene expressions due to CBMB12, CBMB15, and CBMB20 strains were 66.3, 25.5 and 10.4% higher over pathogen infected plants. Similarly, PAL gene expression was high with 0.67 and 0.30 Normalized Fold Expression, in pathogen challenged tomato plants inoculated with CBMB12 and CBMB15 strains. The results suggest that ethylene is a crucial factor in bacterial spot disease incidence and that methylobacteria with ACC deaminase activity can reduce the disease severity with ultimate pathogenesis-related protein increase in tomato.     

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.     

ACC deaminase from plant growth-promoting bacteria affects crown gall development

In addition to the well-known roles of indoleacetic acid and cytokinin in crown gall formation, the plant hormone ethylene also plays an important role in this process. Many plant growth-promoting bacteria (PGPB) encode the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which can degrade ACC, the immediate precursor of ethylene in plants, to alpha-ketobutyrate and ammonia and thereby lower plant ethylene levels. To study the effect of ACC deaminase on crown gall development, an ACC deaminase gene from the PGPB Pseudomonas putida UW4 was introduced into Agrobacterium tumefaciens C58, so that the effect of ACC deaminase activity on tumour formation in tomato and castor bean plants could be assessed. Plants were also coinoculated with A. tumefaciens C58 and P. putida UW4 or P. putida UW4-acdS- (an ACC deaminase minus mutant strain). In both types of experiments, it was observed that the presence of ACC deaminase generally inhibited tumour development on both tomato and castor bean plants.     

Plant encoded 1-aminocyclopropane-1-carboxylic acid deaminase activity implicated in different aspects of plant development

Proper plant development is dependent on the coordination and tight control of a wide variety of different signals. In the study of the plant hormone ethylene, control of the immediate biosynthetic precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is of interest as the level of ethylene can either help or hinder plant growth during times of stress. It is known that ACC can be reversibly removed from the biosynthesis pathway through conjugation into other compounds. We recently reported that plants can also irreversibly remove ACC from ethylene production through the activity of a plant encoded ACC deaminase. Heretofore only found in bacteria, we showed that there was ACC deaminase activity in both Arabidopsis and in developing wood of poplar. Here we extend this original work and show that there is also ACC deaminase activity in tomato plants, and that this activity is regulated during tomato fruit development. Further, using an antisense construct of AtACD1 in Arabidopsis, we investigate the role of ACC deamination during salt stress. Together these studies shed light on a new level of control during ethylene production in a wide variety of plant species and during different plant developmental stages.Key words: tomato fruit ripening, wood development, stress response, hormone, antisense, synthesisHormones are a class of signaling molecules produced and sensed at very low levels; therefore control of their biosynthesis is crucial for proper plant development. The plant hormone ethylene has been studied for over a century and can positively impact plant development, such as in the initiation of fruit ripening, but ethylene accumulation can also induce widespread damage during stress responses.¹ Ethylene is produced in two steps from the S-adenosylmethionine (SAM) that is derived from the Yang cycle.² In the first committed step, SAM is converted into 1-aminocyclopropane-1-carboxcylic acid (ACC) via the action of ACC SYNTHASEs (ACSs).³ ACC is then converted into ethylene by ACC OXIDASEs (ACOs), a particular adaptation of flowering plants.⁴ Once ACC is produced, there are few proven pathways that can divert it from conversion into ethylene. ACC can be conjugated into malonyl-1-aminocyclopropane- 1-carboxylic acid (MACC) through the activity of ACC malonyl transferase⁵ or to 1-(γ-L-glutamyl-amino) cyclopropane-1-carboxylic acid (GACC) via γ-glutamyltranspeptidase.⁶ In bacteria, another pathway exists that can break down ACC obtained from plants through an irreversible deamination process.⁷ Through heterologous expression of bacterial ACC DEAMINASEs (ACDs) in plants it has been possible to engineer plants that have reduced production of ethylene by affecting the native pools of ACC.⁸ Until recently no ACC deaminase pathway has ever been proven in plants, although a number of different plant genomes encode genes which bear sequence homology to bacterial ACDs. Should these genes code for active ACDs, this would provide an additional level of control for ethylene production beyond the activity of ACSs and ACOs. Recently we reported that Arabidopsis and Populus have inherent ACC deaminase activity, and we showed that this activity in Arabidopsis is due, in part, to the product of ACC DEAMINASE1 (AtACD1) (At1g48420).⁹ This discovery raises many questions concerning the role of ACC deaminases during ethylene mediated processes in a number of different plant models. We report here some of our preliminary findings in the areas of tomato fruit ripening and salt stress in Arabidopsis.As precise control of ethylene levels is essential during climacteric fruit development, in parallel with our reported studies we also studied ACC deaminase activity in developing tomato fruit. Ethylene production during ripening in tomato is controlled by ethylene receptor turnover¹⁰ and conjugation of ACC by MACC and GACC.^6,11,12 We found that tomatoes also have inherent ACD activity, and that this activity varies over ripening of the fruit (Fig. 1; solid line). During the immature green stage in tomato development ACC deaminase activity was low. This activity increased significantly during the ‘late breaker’ stage, just prior to the orange/red stage of development, and then decreased during later stages of tomato ripening. Also shown in this figure are the predicted levels of ethylene during fruit development. It is interesting to note that the highest amount of ACC deaminase activity coincides with the drop in ethylene levels soon after the breaker stage (Fig. 1; dashed line; based on Brady¹³). Our data would suggest that, in addition to ethylene receptor turnover and GACC and MACC activity, ACC deaminase activity may also help control ethylene levels. It has already been shown that constitutive expression of a bacterial ACC deaminase in tomato can delay the rate of tomato fruit ripening by reduction of ethylene production.⁸ Although ACD activity is evident during ripening in tomato, the gene responsible has not been identified. Recently a tomato gene with sequence similarity to bacterial ACC deaminases was tested for ACD activity. It was found that, despite the close sequence similarity, this gene (accession number {"type":"entrez-nucleotide","attrs":{"text":"EU639448","term_id":"186920268","term_text":"EU639448"}}EU639448) did not have ACD activity.¹⁴ Therefore, additional work must be done to isolate the gene responsible for the ACD activity we demonstrate in tomato fruit.Open in a separate windowFigure 1Tomato fruits exhibit AC deaminase activity during ripening. A plot of ACC Deaminase activity (Solid Line) with known levels of ethylene production during ripening (Dashed Line; Brady¹³) superimposed over pictures of the corresponding stage of tomato development. *Indicates significant increase in activity (†nmol mg⁻¹ hr⁻¹). AC deaminase activity analysis was performed on total tomato fruit protein as per Penrose and Glick (2003).²¹Our discovery of a plant encoded ACC deaminase in Arabidopsis allows us, for the first time, to downregulate ACC deaminase activity and investigate how this affects plant development. Previously, we showed that downregulation of AtACD1 using antisense resulted in up to a 30% reduction in ACD activity and up to a 2.5-fold increase in the evolution of ethylene.⁹ We showed that this difference in ACD activity was sufficient to alter hypocotyl elongation during Arabidopsis germination on different concentrations of ACC. It was unknown, however, if this difference was sufficient to affect other areas of development, such as stress response, in Arabidopsis. The expression of bacterial ACC deaminases in plants are known to increase plant resistance to a number of stressors due to decreased ethylene evolution.^15–18 Based on microarray data, it is known that AtACD1 expression is upregulated 150% during salt stress¹⁹ and functionally it has been demonstrated that ACC production is increased in salt stressed roots²⁰ and overexpression of bacterial ACDs in canola increases salt tolerance.¹⁸ It was unknown, however, if a reduction in native ACD activity would result in reduced vigour of plants grown on increasing concentrations of sodium chloride. We observed that there was no significant difference in rosette size, leaf production or percent dry weight between wildtype and three independent Arabidopsis lines expressing the AtACD1 antisense construct when grown on MS media without salt (Fig. 2A–C). As the concentration of salt increased in the growth media it was found that the antisense lines also did not differ from wildtype in their growth. The lack of a definitive phenotype under salt stress may mean that the level of reduced ACD activity achieved in the AtACD1 antisense lines was not sufficient to quantifiably affect the development of Arabidopsis. Additionally, as ethylene is not the only factor that affects a plant’s survival during times of salt stress, it is also possible that the plants were able to compensate for increased ethylene production in the AtACD1 antisense lines to promote normal plant development. This finding highlights the complex nature of the different signals involved in a plant’s response to salt stress and the need for a better understanding of the role of plant ACDs and how the plant may compensate for altered ACD activity.Open in a separate windowFigure 2Growth and development of Arabidopsis wildtype and three Antisense AtACD1 lines on increasing concentrations of salt. Stratified wildtype Arabidopsis (Col-0) and three independent transgenic lines expressing an antisense construct of AtACD1 (A1, A2, A3) were sown on 0 mM NaCl (Dark Grey Bars), 100 mM NaCl (White Bars), 125 mM NaCl (Black Bars) and 150 mM NaCl (Light Grey Bars) and allowed to germinate and grow for 2 weeks under long-day conditions (16 h light/8 h dark) at a light intensity of 130 to 190 µE m-2s⁻¹ at the rosette level at 21°C in Econair AC -60 growth chambers. Plants were analyzed for rosette diameter (A), leaf production (B) and percent dry weight (C). Error bars are ± SE.In the known framework of ethylene synthesis our work has shown that plants do have the ability to reduce ethylene synthesis by irreversibly deaminating ACC through the action of a native ACC deaminase. Further to our first study, we show here that there is inherent ACC deaminase activity in tomatoes and that this activity varies during tomato ripening in a manner consistent with a factor that is involved in the regulation of ethylene levels. We also show here that transgenic Arabidopsis lines with a mild reduction in ACD1 activity do not have an obvious affect on mediation of salt stress. This finding, however, does not preclude a role for ACD1 in mediating other aspects of plant development or in affecting plant development during other types of plant stress (i.e., drought). Therefore, there still remain many questions to answer concerning the role of plant encoded ACC deaminases and many exciting avenues of ethylene regulation to pursue. The identification and exploitation of tomato, poplar and other plant ACC deaminases could be used to alter fruit ripening, wood production and stress tolerance—all aspects of plant development that are economically and scientifically important.     

Promotion of tomato (Lycopersicon esculentum Mill.) plant growth by rhizosphere competent 1-aminocyclopropane-1-carboxylic acid deaminase-producing streptomycete actinomycetes

The ability of streptomycete actinomycetes to promote growth of tomato through the production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase was evaluated under gnotobiotic and greenhouse conditions. To achieve this, 64 isolates of Streptomyces spp. obtained from a tomato rhizosphere in the United Arab Emirates were initially selected for their ability to produce ACC deaminase as well as indole-3-acetic acid (IAA) and subsequently for their rhizosphere competence as root colonizers. Of the two selected ACC deaminase-producing isolates showing exceptional rhizosphere competence, S. filipinensis no. 15 produced both ACC deaminase and IAA, whilst S. atrovirens no. 26 did not produce IAA. Under greenhouse conditions, the application of S. filipinensis no. 15 or S. atrovirens no. 26 resulted in the reduction of the endogenous levels of ACC, the immediate precursor of ethylene, in both roots and shoots and increased plant growth. Plant growth promotion was most pronounced in the presence of S. filipinensis no. 15 compared to S. atrovirens no. 26. This relative superiority in performance shows the advantage conferred to S. filipinensis no. 15 due to its ability to produce both IAA and ACC deaminase. In comparison, an ACC deaminase-producing isolate of S. albovinaceus no. 41 which was neither rhizosphere-competent nor capable of producing IAA, failed to promote plant growth compared to S. filipinensis no. 15 or S. atrovirens no. 26 although the growth promotion obtained by S. albovinaceus no. 41 was significant compared to control. The application of S. globosus no. 8, which was not rhizosphere-competent and did not produce detectable levels of ACC deaminase or IAA did not promote plant growth. These results indicate the importance of rhizosphere competence. In conclusion I report the production of ACC deaminase by streptomycete actinomycetes and its ability to enhance plant growth through reduction in the in planta levels of endogenous ACC and the consequent lowering of endogenous ethylene levels in plant tissues.     

Does light inhibit ethylene production in leaves?

The effect of light on the rate of ethylene production was monitored using two different techniques—leaf segments incubated in closed flasks versus intact plants in a flow-through open system. Three different plants were used, viz sunflower (Helianthus annuus), tomato (Lycopersicon esculentum), and soybean (Glycine max). Experiments were conducted both in the presence and absence of 1-aminocyclopropane-1-carboxylic acid (ACC).

The results obtained indicate that, in all three species studied, light strongly inhibits ethylene production when cut leaf segments are incubated in the presence of ACC in closed flasks. When ethylene measurements are made with ACC-sprayed intact plants using a continuous flow system, the effect of light on ethylene production is only marginal. In leaf segments of sunflower and soybean incubated only in distilled H₂O in closed flasks, light promotes ethylene production. In tomato, there is no difference between the rate of ethylene production between light and darkness under such conditions. When measurements are made with intact plants in a continuous flow system, the rate of ethylene production is almost identical in light and darkness, in the three plants studied.

It is concluded that the effect of light on cut leaf segments incubated in the presence of ACC in closed flasks can be attributed to the techniques used for these measurements. Light has little effect on ethylene production by intact plants in an open system.

     

Rhizobium leguminosarum Biovar viciae 1-Aminocyclopropane-1-Carboxylate Deaminase Promotes Nodulation of Pea Plants

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.     

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.     

Cloning and Characterization of a Plasmid Encoded ACC Deaminase from an Indigenous Pseudomonas fluorescens FY32

In addition to the characterized mechanisms responsible for many direct effects of plant growth promoting bacteria (PGPB) on plants, it has been suggested that a number of PGPB contain the enzyme ACC deaminase that catalyzes degradation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, into α-ketobutyrate and ammonia. As part of an effort to obtain an ACC deaminase encoding gene from a collection of soil samples, only one bacterial isolate, Pseudomonas fluorescens FY32 was capable of growing on ACC as a sole source of nitrogen. The ACC deaminase gene was amplified from the above isolate by polymerase chain reaction (PCR) giving an expected DNA fragment, 1017 bp. Sequence analysis of the fragment showed that it was highly homologous (94% and 98% identities at nucleotide and amino acid levels, respectively) to the previously characterized acdS gene from Pseudomonas sp. 6G5. Furthermore, fusion of the ACC deaminase ORF with lacZ gene resulted in the expression of active enzyme in Escherichia coli. In addition, further analyses revealed that the acdS gene was plasmid-encoded so that a large plasmid (pFY32) with almost 50 kb in size was identified from this bacterium. Furthermore, transfer of pFY32 into E. coli DH5α proved its ACC deaminase activity. This result was in accordance with previous reports suggesting horizontal transfer of the acdS gene. However, it needs more investigation to identify whether this pFY32 plasmid has undergone lateral gene transfer during the evolutionary process.     

Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants.

Synthesis of the phytohormone ethylene is believed to be essential for many plant developmental processes. The control of ripening in climacteric fruits and vegetables is among the best characterized of these processes. One approach to reduce ethylene synthesis in plants is metabolism of its immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). Soil bacteria containing an enzyme, ACC deaminase, were identified by their ability to grow on ACC as a sole nitrogen source. The gene encoding ACC deaminase was cloned and introduced into tomato plants. Reduction in ethylene synthesis in transgenic plants did not cause any apparent vegetative phenotypic abnormalities. However, fruits from these plants exhibited significant delays in ripening, and the mature fruits remained firm for at least 6 weeks longer than the nontransgenic control fruit. These results indicated that ACC deaminase is useful for examining the role of ethylene in many developmental and stress-related processes in plants as well as for extending the shelf life of fruits and vegetables whose ripening is mediated by ethylene.     

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.     

Expression of an Exogenous 1-Aminocyclopropane-1-Carboxylate Deaminase Gene in Sinorhizobium meliloti Increases Its Ability To Nodulate Alfalfa

1-Aminocyclopropane-1-carboxylate (ACC) deaminase has been found in various plant growth-promoting rhizobacteria, including rhizobia. This enzyme degrades ACC, the immediate precursor of ethylene, and thus decreases the biosynthesis of ethylene in higher plants. The ACC deaminase of Rhizobium leguminosarum bv. viciae 128C53K was previously reported to be able to enhance nodulation of peas. The ACC deaminase structural gene (acdS) and its upstream regulatory gene, a leucine-responsive regulatory protein (LRP)-like gene (lrpL), from R. leguminosarum bv. viciae 128C53K were introduced into Sinorhizobium meliloti, which does not produce this enzyme, in two different ways: through a plasmid vector and by in situ transposon replacement. The resulting ACC deaminase-producing S. meliloti strains showed 35 to 40% greater efficiency in nodulating Medicago sativa (alfalfa), likely by reducing ethylene production in the host plants. Furthermore, the ACC deaminase-producing S. meliloti strain was more competitive in nodulation than the wild-type strain. We postulate that the increased competitiveness might be related to utilization of ACC as a nutrient within the infection threads.     

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.     

Xylem Transport of 1-Aminocyclopropane-1-carboxylic Acid, an Ethylene Precursor, in Waterlogged Tomato Plants

Waterlogging is known to cause an increase in ethylene synthesis in the shoot which results in petiole epinasty. Evidence has suggested that a signal is synthesized in the anaerobic roots and transported to the shoot where it stimulates ethylene synthesis. Experimental data are presented showing that 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, serves as the signal. Xylem sap was collected from detopped tomato plants (Lycopersicon esculentum Mill. cv. VFN8). ACC in the sap was quantitated by a sensitive and specific assay, and its tentative chemical identity verified by paper chromatography. ACC levels in both roots and xylem sap increased markedly in response to waterlogging or root anaerobiosis. The appearance of ACC in the xylem sap of flooded plants preceded both the increase in ethylene production and epinastic growth, which were closely correlated. Plants flooded and then drained showed a rapid, simultaneous drop in ACC flux and ethylene synthesis rate. ACC supplied through the cut stem of tomato shoots at concentrations comparable to those found in xylem sap caused epinasty and increased ethylene production. These data indicate that ACC is synthesized in the anaerobic root and transported to the shoot where it is readily converted to ethylene.     

The interconversion of ACC deaminase and <Emphasis Type="SmallCaps">d</Emphasis>-cysteine desulfhydrase by directed mutagenesis

Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. These enzymes cleave ACC, the immediate precursor of ethylene in plants, into ammonia and alpha-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. In the work reported here, it was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather it utilizes D: -cysteine as a substrate, and in fact encodes a D: -cysteine desulfhydrase. Kinetic characterization of the tomato enzyme indicates that it is similar to other, previously characterized, D: -cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering only two amino acid residues within the predicted active site served to change the enzyme from D: -cysteine desulfhydrase to ACC deaminase. Conversely, by altering two amino acid residues at the same positions within the active site of ACC deaminase from Pseudomonas putida UW4 the enzyme was converted into D: -cysteine desulfhydrase. Therefore, it is possible that a change in these two residues may have occurred in an ancestral protein to result in two different enzymatic activities.     

Detection and Characterization of ACC Deaminase in Plant Growth Promoting Rhizobacteria

The enzyme 1-aminocyclopropane-1-carboxylate deaminase converts ACC, the precursor of the plant hormone ethylene to α-ketobutyrate and ammonium. The enzyme has been identified in few soil bacteria, and is proposed to play a key role in plant growth promotion. In this study, the isolates of plant growth promoting rhizobacteria were screened for ACC deaminase activity based on their ability to grow on ACC as a sole nitrogen source. The selected isolates showed the presence of other plant growth promoting characteristics such as IAA production, phosphate solubilization and siderophore production. The role of ACC deaminase in lowering ethylene production under cadmium stress condition was also studied by measuring in vitro ethylene evolution by wheat seedlings treated with ACC deaminase positive isolates. Nucleic acid hybridization confirmed the presence of ACC deaminase gene (acdS) in the bacterial isolates.