Abstract
1-Aminocyclopropane-1-carboxylate (ACC) deaminase-mediated reduction of ethylene generation in plants under abiotic stresses is a key mechanism by which bacteria can promote plant growth. Misidentification of ACC deaminase and the ACC deaminase structure gene (acdS) can lead to overestimation of the number of bacteria containing ACC deaminase and their function in ecosystems. Previous non-specific amplification of acdS homologs has led to an overestimation of the horizontal transfer of acdS genes. Here, we designed consensus-degenerate hybrid oligonucleotide primers (acdSf3, acdSr3 and acdSr4) based on differentiating the key residues in ACC deaminases from those of homologs for specific amplification of partial acdS genes. PCR amplification, sequencing and phylogenetic analysis identified acdS genes from a wide range of proteobacteria and actinobacteria. PCR amplification and a genomic search did not find the acdS gene in bacteria belonging to Pseudomonas stutzeri or in the genera Enterobacter, Klebsiella or Bacillus. We showed that differentiating the acdS gene and ACC deaminase from their homologs was crucial for the molecular identification of bacteria containing ACC deaminase and for understanding the evolution of the acdS gene. We provide an effective method for screening and identifying bacteria containing ACC deaminase.
Highlights
1-Aminocyclopropane-1-carboxylate (ACC) is the immediate biosynthetic precursor to the plant hormone ethylene that regulates plant development and responses to environmental stress
ACC deaminase (ACCD)-producing bacteria associated with plants can act as a sink for plant ACC by taking up and cleaving ACC released from the plant cells and can reduce ethylene generation in plants; in this way they can reduce the extent of growth inhibition caused by high-level ethylene, under abiotic stresses (Glick, Penrose and Li 1998)
The bacteria consisted of 44 known ACCDproducing isolates (18 Burkholderia, 10 Herbaspirillum and 16 Pseudomonas) (Li et al 2011), the nitrogen-fixing Pseudomonas stutzeri strain A1501 (Yan et al 2008), Bradyrhizobium sp. strain DGR24 isolated from a nodule of soybean, Bradyrhizobium sp. strain LR51 isolated from a nodule of mung bean and 327 other isolates obtained by culturing material from sugarcane roots and rhizosphere soils, soybean nodules, mung bean nodules and black locust nodules on a nitrogen-deficient DF medium (Li et al 2011) and a modified Ashby medium (Lin et al 2012)
Summary
1-Aminocyclopropane-1-carboxylate (ACC) is the immediate biosynthetic precursor to the plant hormone ethylene that regulates plant development and responses to environmental stress (reviewed by Bleecker and Kende 2000). Amplification of the acdS gene by PCR with degenerate primers has been widely used for molecular identification of ACCD-producing bacteria (Shah et al 1998; Ma et al 2003; Hontzeas et al 2005; Blaha et al 2006; Caballero-Mellado et al 2007; Govindasamy et al 2008; Onofre-Lemus et al 2009; Nikolic, Schwab and Sessitsch 2011). These degenerate primers are either non-specific or only effective for amplification of acdS genes from a narrow range of bacteria
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