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Summary• Decreased soil water availability can stimulate production of the plant hormone ethylene and inhibit plant growth. Strategies aimed at decreasing stress ethylene evolution might attenuate its negative effects.• An environmentally benign (nonchemical) method of modifying crop ethylene relations -soil inoculation with a natural root-associated bacterium Variovorax paradoxus 5C-2 (containing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase that degrades the ethylene precursor ACC), was assessed with pea (Pisum sativum) plants grown in drying soil.• Inoculation with V. paradoxus 5C-2, but not with a transposome mutant with massively decreased ACC deaminase activity, improved growth, yield and water-use efficiency of droughted peas. Systemic effects of V. paradoxus 5C-2 included an amplified soil drying-induced increase of xylem abscisic acid (ABA) concentration, but an attenuated soil drying-induced increase of xylem ACC concentration. A local bacterial effect was increased nodulation by symbiotic nitrogen-fixing bacteria, which prevented a drought-induced decrease in nodulation and seed nitrogen content.• Successfully deploying a single bacterial gene in the rhizosphere increased yield and nutritive value of plants grown in drying soil, via both local and systemic hormone signalling. Such bacteria may provide an easily realized, economic means of sustaining crop yields and using irrigation water more efficiently in dryland agriculture.

PCR was used to rapidly identify and isolate 1-aminocyclopropane-1-carboxylate (ACC) deaminase genes from bacteria. The Shimodaira-Hasegawa test was used to assess whether phylogenetically anomalous gene placements suggestive of horizontal gene transfer (HGT) were significantly favored over vertical transmission. The best maximum likelihood (ML) ACC deaminase tree was significantly more likely than four alternative ML trees, suggesting HGT.Plant growth-promoting bacteria include a diverse group of free-living soil bacteria that can stimulate the growth of plants by different direct or indirect mechanisms (5). Relatively recently, it was discovered that many plant growth-promoting bacteria contain the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase and that this enzyme can cleave the ethylene precursor ACC to ␣-ketobutyrate and ammonia and thereby lower the level of ethylene in developing or stressed plants (5,6,7,9,11,13). The gene encoding this enzyme has been isolated from a few strains of Pseudomonas spp., Rhizobium leguminosarum of the yeast Hansenula saturnus, and the fungus Penicillium citrinum. The crystal structure has been determined for the yeast (15) and bacterial (12) ACC deaminase enzymes; the biochemical and thermodynamic properties of the ACC deaminase from Pseudomonas putida UW4 have been measured (10). Here, using PCR with degenerate primers, we have successfully isolated ACC deaminase genes from a range of both gram-positive and gram-negative bacterial species. Using the biochemical assay procedure, it was ascertained that all of these putative genes encoded functional ACC deaminase. Furthermore, we propose that ACC deaminase genes did not evolve vertically but instead have undergone horizontal gene transfer (HGT).The bacterial strains used in this work are listed in Table 1. The bacteria, except for rhizobial strains, were routinely maintained on Bacto Pseudomonas F as described previously (1, 2, 3).For genus and species identification, a colony PCR was performed with live cells cultured on solid Bacto Pseudomonas F or M79 medium as described previously (17). Species assignment was confirmed by submitting the 16S rRNA sequences to the Ribosomal Database Project II (http://rdp.cme.msu.edu) and comparing them with their nearest phylogenetic relatives.Degenerate primers DegACC5Ј (5Ј-GGBGGVAAYAARM YVMGSAAGCTYGA) and DegACC3Ј (5Ј-TTDCCHKYRT ANACBGGRTC) were designed based upon stretches of conserved base pairs towards the N terminus of the protein and around the putative pyridoxal phosphate cofactor binding domain of the protein (20), whereas for 3Ј primer design, a conserved region close to the carboxyl terminus of the protein was utilized. This allows for the amplification of a fragment of approximately 750 bp. Thus, by using this PCR method, bacterial colonies can be quickly screened for the presence of the ACC deaminase gene. Nucleotide sequences were aligned using MUSCLE v3.52 (4, 16) and refined by eye. Phylogenetic analyses were performed using PAUP* v4.10b via an automated script (...

The role of bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity in the interaction between tomato (Lycopersicon esculentum=Solanum lycopersicum) and Pseudomonas brassicacearum was studied in different strains. The phytopathogenic strain 520-1 possesses ACC deaminase activity, an important trait of plant growth-promoting rhizobacteria (PGPR) that stimulates root growth. The ACC-utilizing PGPR strain Am3 increased in vitro root elongation and root biomass of soil-grown tomato cv. Ailsa Craig at low bacterial concentrations (10(6) cells ml-1 in vitro and 10(6) cells g-1 soil) but had negative effects on in vitro root elongation at higher bacterial concentrations. A mutant strain of Am3 (designated T8-1) that was engineered to be ACC deaminase deficient failed to promote tomato root growth in vitro and in soil. Although strains T8-1 and 520-1 inhibited root growth in vitro at higher bacterial concentrations (>10(6) cells ml-1), they did not cause disease symptoms in vitro after seed inoculation, or in soil supplemented with bacteria. All the P. brassicacearum strains studied caused pith necrosis when stems or fruits were inoculated with a bacterial suspension, as did the causal organism of this disease (P. corrugata 176), but the non-pathogenic strain Pseudomonas sp. Dp2 did not. Strains Am3 and T8-1 were marked with antibiotic resistance and fluorescence to show that bacteria introduced to the nutrient solution or on seeds in vitro, or in soil were capable of colonizing the root surface, but were not detected inside root tissues. Both strains showed similar colonization ability either on root surfaces or in wounded stems. The results suggest that bacterial ACC deaminase of P. brassicacearum Am3 can promote growth in tomato by masking the phytopathogenic properties of this bacterium.

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The technique of RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) was used to study changes in gene expression over time in canola roots treated with the 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing plant-growth-promoting bacterium Enterobacter cloacae UW4 and to compare the changes with those in a mutant of E. cloacae UW4 in which the ACC deaminase structural gene acdS was replaced by homologous recombination with acdS with an intentional knockout containing a tetracycline resistance gene. Genes that were either up- or down-regulated over a three-day period in canola plants treated with wild-type or mutant bacteria were isolated, cloned, and sequenced; all appeared to have high homology with Arabidopsis thaliana genes. The upregulated genes included a cell division cycle protein 48 homolog and a eukaryotic translation initiation factor 3 subunit 7 gene homolog. The downregulated genes included one encoding a glycine-rich RNA binding protein with a function in RNA processing or binding during ethylene-induced stress, which is expressed only in roots, and another gene thought to be involved in a defense signaling pathway. All RAP-PCR results were verified using Northern blotting. These data, indicate that roots isolated from canola seeds treated with the ACC deaminase-producing E. cloacae UW4 upregulate genes involved in cell division and proliferation but down-regulate stress genes. This data is in agreement with a model in which ACC deaminase-containing plant-growth-promoting bacteria reduce plant stress and induce root elongation and proliferation in plants, largely by lowering ethylene levels.

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