Effect of mustard rhizobacteria on wheat growth promotion under cadmium stress: characterization of acdS gene coding ACC deaminase

Abstract

Ten mustard rhizobacterial isolates that utilize 1-aminocyclopropane-1-carboxylate (ACC) as sole nitrogen source were screened for plant growth-promoting traits. These isolates enhanced root elongation significantly and minimized ethylene synthesis in wheat seedlings under induced cadmium stress condition. The presence of acdS gene coding for ACC deaminase was tested through gene-specific PCR amplification and further confirmed by nucleic acid hybridization. acdS gene from isolates Ps 2-3 and Ps 7-12 selected on the basis of wheat growth promotion was cloned and sequenced. Sequence analysis showed an open reading frame of 1017 bp encoding a complete polypeptide with an identity of 86.4 % between each other and sharing 84–95 % similarity with the earlier reported acdS genes. The deduced protein sequences were highly conserved and shared a 95.2 % homology at the amino acid level. Eight and thirteen new amino acid residues were found in the protein sequence of Ps 2-3 and Ps 7-12 with variation in numbers of different peptidase acting sites and protein motifs. Based on 16S rDNA sequencing, the isolates Ps 2-3 and Ps 7-12 were identified as Pseudomonas sp. and Pseudomonas fluorescens, respectively.

Introduction

The growth of plants in the natural environment is influenced by large numbers of biotic and abiotic stresses. Synthesis of high levels of ethylene under different stress conditions triggers onset of plant defense mechanisms followed by exaggerate symptoms leading to senescence in crop plants (Abeles et al. 1992; Pierik et al. 2006). Although a burst of ethylene is necessary to break seed dormancy in many plants, sustained higher levels of ethylene synthesis effectively inhibit root elongation and consequent plant growth. Interestingly, certain plant growth-promoting rhizobacteria (PGPR) are known for their ability to lower the ethylene levels through enzymatic control exerted by 1-aminocyclopropane-1-carboxylate (ACC) deaminase (EC 4.1.99.4). This enzyme hydrolyses ACC, the immediate precursor of ethylene into α-ketobutyrate and ammonia (Honma and Shimomura 1978; Sheehy et al. 1991), thereby reduces the endogenous ethylene level to enhance root elongation in plants (Hall et al. 1996; Glick et al. 1998). In addition, plants treated with ACC deaminase-containing PGPR are highly resistant to the deleterious effects of stress ethylene synthesized as a consequence of various biotic and abiotic stresses (Wang et al. 2000; Grichko and Glick 2001; Mayak et al. 2004; Saravanakumar and Samiyappan 2007).

The ACC deaminase has been isolated from few Pseudomonas species, yeast Hansenula saturnus and the fungus Penicillium citrinum (Sheehy et al. 1991; Jia et al. 1999; Yao et al. 2000). According to Jia et al. (1999), ACC deaminase of Penicillium citrinum was quite distinct from all other known ACC deaminases at the biochemical level. Several partial acdS gene fragments have been cloned from different bacterial species gave supportive evidence to the existence of different ACC deaminase genes in bacteria isolated from various ecosystems (Belimov et al. 2001; Hontzeas et al. 2005; Blaha et al. 2006; Govindasamy et al. 2008a).

There have been reports on soybean–bacterial endophytes-promoted plant growth through the reduction of biotic stress caused by fungal and bacterial pathogens (Senthilkumar et al. 2009; Mageshwaran et al. 2011). Similarly, rhizobacteria from different crops for their plant growth-promoting traits including the detection of ACC deaminase activity have also been reported (Govindasamy et al. 2008a, b; 2009). Here, we report the ACC deaminase-containing rhizobacteria isolated from Indian mustard, which affects wheat growth by lowering stress ethylene synthesis in the presence of cadmium (Cd)-induced stress and also the initial characterization of their acdS genes encoding the enzyme.

Materials and methods

Bacterial isolates, media and culture conditions

A total of 128 bacterial isolates purified from rhizosphere soils supporting eight varieties of Indian mustard. Pseudomonas fluorescens 913, having ACC deaminase activity, was a kind gift of Prof. B N Johri, GB Pant University of Agriculture and Technology, Pantnagar, India. The isolates were grown at 28 ± 2 °C in tryptic soy broth (TSB) or Luria Bertani (LB) medium. DF minimal medium was used for screening bacterial isolates for ACC deaminase activity (Penrose and Glick 2003). Escherichia coli DH5α and its transformants carrying plasmids were grown at 37 °C in LB medium with appropriate antibiotics.

Screening for ACC deaminase activity

The bacterial isolates were screened for potential utilization of ACC as described previously by Govindasamy et al. (2009). The ACC positive isolates were further screened for their ability to produce indole-3-acetic acid (IAA) (Bric et al. 1991), siderophore (Schwyn and Neilands 1987) and HCN (Bakker and Schippers 1987) and to solubilize mineral phosphate (Pikovskya 1948). The quantitative estimation of IAA was performed by the spectrophotometric method using Salkowski reagent (Patten and Glick 2002). For the ACC deaminase enzyme assay, the bacterial isolates were initially induced for ACC deaminase activity by growing them in DF minimal medium supplemented with 3 mmol/L ACC (Penrose and Glick 2003). The enzyme activity of the bacterial cell extracts was further determined by measuring the amount of α-ketobutyrate production (Honma and Shimomura 1978).

Root elongation and growth promotion of wheat seedlings

The effect of bacterial isolates on root elongation and plant growth of wheat cv. HD 2687 seedlings on soft agar under gnotobiotic conditions was assessed. Surface-sterilized seeds were imbibed in bacterial suspension (A600 nm = 1.0) along with 0.1 % (w/v) Carboxy Methyl Cellulose (CMC) for 4 h and air-dried. The bacterized seeds were placed on soft agar plates (six seeds per plate) and initially incubated at 20 °C in the dark for 12 h, followed by intermittent light in a growth chamber. Root length, shoot length and fresh weight of the seedlings were recorded on 8 days incubation. The dry weight of seedlings was also recorded by keeping them in hot air oven at 60 °C for 7 days for complete desiccation.

Ethylene production assay in wheat seedlings under induced Cd stress conditions

Wheat cv. HD 2687 seeds were treated with bacterial suspension (A600 nm = 1.0) and six seeds were placed in each 25-mL Erlenmeyer flask containing a piece of filter paper with the addition of 2 mL of sterile deionized water. Each treatment was replicated five times. subsequent to incubation for 8 days in a growth chamber at 20 °C with a 12-h light/dark cycle of photoperiod, the excess liquid was drained and 2 mL of 150-ppm (w/v) of cadmium chloride (CdCl₂) solution was added to induce stress. The flasks were maintained under airtight condition using subaseal. After 36 h of incubation, the gas samples in the headspace were analyzed by gas chromatography (Shimadzu, Tokyo, Japan) equipped with a hydrogen flame ionization detector and an AT-1 column.

Isolation and hybridization of ACC deaminase genes

Genomic DNA of the bacterial isolates was extracted described by Masterson et al. (1985). PCR was performed in a MJ research thermal cycler PTC-200 using the degenerate primers 5’- ATG AAY CTG MAN CGN TTY SMN CGB TAY CC -3’ and 5’-TYA GCC RTY NCG RAA NAD RAA RCT RTA NSC -3’ to amplify complete ACC deaminase structural gene (acdS). Reaction mixture of 100 μL volume was prepared by mixing genomic DNA with 10× polymerase buffer, 1.5 mmol/L MgCl₂, 0.5 μmol/L of each primer, 200 μmol/L dNTPs and 1 U of Taq DNA polymerase followed by 30 cycles of amplification.

DNA dot blot and Southern hybridization were performed using the techniques described by Sambrook and Russell (2001). An ∼800-bp length of acdS from P. fluorescens strain 913 was amplified (Blaha et al. 2006), radio-labeled with α-³²P dCTP (BARC, India) and used as a hybridization probe (Govindasamy et al. 2009).

Cloning, sequencing and phylogenetic analysis

PCR product of two isolates Ps 2-3 and Ps 7-12 was gel-purified, ligated and transformed into E. coli DH5α following standard protocols (Sambrook and Russell 2001). Sequencing was carried out using modified Tag-FS enzyme with fluorescently labeled dideoxy-terminator chemistry. Sequences were subjected to BLAST analysis (Altschul et al. 1997) and their deduced protein sequences were analyzed using software GeneRunner v.3.05 (Hastings software) and BioEdit version 7.0 (Ibis Biosciences, Carlsbad, CA, USA).

Phylogenetic analysis of acdS and deduced AcdS sequences were conducted using software Treecon v.1.3b (Yves van de Peer, University of Antwerp). Distances between sequence pairs, the deduced phylogenetic tree, and bootstrap values were all computed using the global gap removal option. Phylogenetic tree for acdS sequences was generated by the neighbor-joining method (NJ), using both the Kimura 2 Parameter (K2P) and Jukes-Cantor (JC; with gamma parameter), and maximum parsimony method (MP). For deduced AcdS sequences, a tree was generated by the NJ method using both the Poisson corrections and observed divergence and by the MP method. Nodal robustness of the trees was assessed using 1000 bootstrap replicates.

Identification of bacterial isolates by 16S rDNA sequencing

PCR amplification of 16S rDNA from isolates Ps 2-3 and Ps 7-12 was carried out with universal primers PA and PH as per Blaha et al. (2006). The cloning and sequencing of 16S rDNA was similar to that explained for ACC deaminase gene. The sequences of 16S rDNA obtained were subjected to BLAST analysis.

Statistical analysis and nucleotide sequences accession numbers

The data on root elongation, plant growth measurements and ethylene assay were analyzed by analysis of variance (ANOVA) and the treatment means were compared by Duncan’s multiple-range test (DMRT). All hypotheses were tested at the 95 % confidence interval (α = 0.05). The full length sequences of 16S rDNA and acdS gene of these two isolates were submitted to GenBank of NCBI with accession numbers EU520398, EU520401, EU854429 and EU854430, respectively.

Results

Screening rhizobacteria for ACC deaminase and other PGP traits

Ten of 128 bacterial isolates from mustard rhizosphere grew well in DF minimal medium containing ACC as the sole N source. Rhizobacteria strain A48 exhibited the lowest ACC deaminase activity (2.76 μmol/h α-ketobutyrate/mg protein) while isolates Ps 2-3 and Ps 7-12 showed high level of enzyme activity, viz., 5.15 and 4.98 μmol/h α-ketobutyrate/mg protein, respectively (Table 1).

Table 1 ACC deaminase enzyme assay, IAA production assay and other PGP traits of the selected mustard rhizobacterial isolates

Rhizobacterial isolates positive for ACC deaminase activity were also positive for IAA production (concentrations ranged from 8.32 to 46.80 μg/mL, the maximum being produced by isolate Ps 2-3 and Ps 7–12) and P-solubilization. Six of the 10 isolates (Ps 2-3, Ps 2-12, Ps 3-7, Ps 4-17, Ps 7-9, Ps 7-12) produced siderophore and seven isolates were found positive in the plate assay for HCN production by change in the color of picric acid-treated filter paper into brownish red (Table 1).

Effect of acdS⁺ isolates on root elongation, plant biomass and ethylene production

The wheat seedlings treated with rhizobacterial isolates Ps 2-3, Ps 7-12 and Fb600 had an increased root length of 20.70, 19.48 and 19.42 cm, respectively, which was on a par with Pf 913 (Table 2; Fig. 1). Shoot length was insignificantly different among treatments. However, bacterization with Ps 2-3, Ps 7-9, Ps 7-12 and Fb600 resulted in significantly higher shoot growth, fresh and dry weights when compared with the controls (Table 2). Under Cd stress, these treatments showed 0.39, 0.40 and 0.51 pmol of ethylene production per gram fresh weight, respectively. Stress ethylene production in the wheat seedlings was reduced to the tune of ∼72 % when treated with Ps 2-3 and Ps 7-12 (Table 2).

Table 2 Effect of ACC deaminase containing mustard rhizobacterial isolates on plant growth and in vitro plant ethylene production in wheat (cv. HD 2687) seedlings under gnotobiotic conditions

Fig. 1

Effect of ACC deaminase containing mustard rhizobacterial isolates on root elongation of wheat (cv. HD 2687) seedlings under gnotobiotic conditions

Isolation, sequencing and characterization of ACC deaminase gene

PCR yielded an amplicon of ∼1000 bp size in all the bacterial isolates except A48. However, positive signal was obtained in A48 in dot blot and Southern hybridization (Fig. 2). In BLASTN analysis, sequences of Ps 2-3 and Ps 7-12 showed 87 and 89 % identity, respectively, with transcriptional regulator (acdR) gene and ACC deaminase (acdS) gene of Pseudomonas fluorescens strain 2P24. The acdS gene of Ps 2-3 and Ps 7-12 contained an open reading frame of 1017 bp nucleotides encoding a protein of 338 amino acids length. The G + C content was 59.6 and 61.3 % for Ps 2-3 and Ps 7-12, respectively, and 86.4 % identical to each other. About 14 % of the ORF (137 nucleotides) were dissimilar especially in the wobble position constituting of 111 codons (Fig. 3). Multiple alignments of nucleotide sequences of Ps 2-3 and Ps 7-12 with sequences of complete ACC deaminase genes available in the GenBank databases showed sequence homology with Pseudomonas putida strain UW4 (PpUW4) as ∼84 and ∼85 %, respectively. Sequence identity of Ps 2-3 and Ps 7-12 was 74.5 and 76.2 %, respectively, when compared to ACC deaminase gene sequence of Pseudomonas sp. strain ACP (PspACP). However, the deduced amino acid sequences of Ps 2-3 and Ps 7-12 showed 96 and 95 % similarity respectively, with the ACC deaminase protein of P. putida UW4.

Fig. 2

Nucleic acid dot blot hybridization showing the presence of acdS gene in mustard rhizobacterial isolates. 1 Ps 1-12; 2 Ps 2-3; 3 Ps 2-12; 4 Ps 3-7; 5 Ps 4-17; 6 Ps 5-1; 7 Ps 7-12; 8 Ps 7-9; 9 A48; 10 Fb600; 11 DH5α; 12 Pf 913

Fig. 3

Alignment of the nucleotide sequences of acdS genes of mustard rhizobacteria Ps 2-3 and Ps 7-12. Identical nucleotides are shown as dots

A high level of conservation (95.2 %) was observed in Ps 2-3 and Ps 7-12 and only 16 amino acids of 338 were found to be different between them. Additionally, 8 and 13 new amino acids were observed in the AcdS protein sequence of Ps 2-3 (positions K⁴, A⁷, Q³³, E¹²⁵, I²²², D²⁷³, F³⁰³ and D³⁰⁴) and Ps 7-12 (positions H⁴, E⁷, Q³³, E¹²⁵, D¹⁴⁶, S¹⁵⁰, E¹⁸¹, Q¹⁸⁵, T¹⁸⁹, Q²¹⁸, I²²², K²³⁰ and D³⁰⁴), respectively (Fig. 4). Ten different types of peptidase sites were present in the deduced protein sequences of both isolates. Hydroxylamine (NH₂OH) acting site was absent in Ps 2-3 compared to Ps 7-12 AcdS protein. However, an additional number of peptidase acting sites for S. aureus V₈ protease (Endoproteinase Glu-C) and Chymotrypsin in Ps 7-12, and Carboxypeptidase B site and Modified trypsin (Trypsin) site in Ps 2-3 AcdS proteins, were observed (Table 3). Three types of motifs with varying patterns, viz., protein kinase phosphorylation site, N-glycosylation site and N-myristoylation site, were present. An additional N-myristoylation motif site was observed in the deduced protein sequence of Ps 7-12 (Table 4).

Fig. 4

Alignment of the deduced amino acid sequences from acdS genes of mustard rhizobacteria Ps 2-3 and Ps 7-12 with Pseudomonas sp. ACP and P. putida UW4. The identical amino acid sequences are showed as dots. The dissimilar amino acids are showed in boxes. The new amino acids are indicated as stars

Table 3 Number of different types of peptidase acting sites present in the deduced amino acid sequences from acdS gene of mustard rhizobacteria Ps 2-3 and Ps 7-13

Table 4 Number and pattern of different types of motifs present in the deduced amino acid sequences from acdS gene of mustard rhizobacteria Ps 2-3 and Ps 7-13

Phylogenetic analysis of acdS and deduced AcdS sequences

A distinct polymorphism was observed between our isolates and others existing in the database. Two distinct clades were observed which were different from Pseudomonas sp. ACP, Rhizobium leguminosarum 128C53K and Pseudomonas plecoglossicida AM10. acdS sequence of isolate Ps 7-12 was more similar to strains Ps 2P24, Pf 17, CH-CRS-8 and PSNL (Fig. 5), but the deduced amino acid sequences of Ps 2-3 showed better homology to other P. fluorescens AcdS protein sequences (Fig. 6).

Fig. 5

Phylogenetic relationship of mustard rhizobacteria Ps 2-3 and Ps 7-12 based on nucleotide sequence of complete acdS genes. The numbers at the nodes indicate the level of bootstrap support, based on neighbour-joining analysis of 1000 replicated datasets

Fig. 6

Phylogenetic relationship of mustard rhizobacteria Ps 2-3 and Ps 7-12 based on complete amino acid sequences deduced from acdS gene sequences. The numbers at the nodes indicate the level of bootstrap support, based on neighbour-joining analysis of 1000 replicated datasets

Identification of rhizobacterial isolates by 16S rDNA sequencing

Isolates Ps 2-3 and Ps 7-12 were closely related to Pseudomonas sp. and Pseudomonas fluorescens, respectively, based on BLASTN analysis of their full length 16S rDNA sequences.

Discussion

The cyclopropanoid amino acid, 1-aminocyclopropane-1-carboxylic acid (ACC), is known to participate only in the ethylene biosynthesis pathway; it is likely that the only biochemical consequence to the plant of ACC degradation is inhibition of ethylene synthesis (Abeles et al. 1992). The ability to hydrolyze ACC with ACC deaminase and utilize the reaction products as nutrients is considered to be phyto-beneficial trait found in some plant growth-promoting rhizobacteria (Glick 1995; Glick et al. 1998). In the present study, we have identified ten ACC deaminase producing rhizobacteria out of a large number of rhizobacteria screened, which were isolated from eight varieties of Indian mustard. Their ability to utilize ACC as the sole nitrogen source, suggesting production of ACC deaminase enzyme, was confirmed by in vitro estimation of α-ketobutyrate. ACC deaminase enzyme activity of these bacterial isolates significantly promoted root elongation in the treated seedlings as compared to unbacterized and E. coli-treated control seedlings. On the other hand, the ability of these bacterial isolates to utilize ACC was associated with other properties, such as production of IAA, phosphate solubilization, siderophore and HCN formation, and could probably also affect their interaction with plants (Glick et al. 1999; Dey et al. 2004).

Differential AcdS enzyme activity of the bacterial isolates significantly correlated to their potential to lower stress ethylene synthesis in the inoculated wheat seedlings. Although the endogenous level of ACC and its related compounds was not quantified, the efficiency of these isolates to reduce ethylene level was found to vary from 32 to 72 % as compared to the unbacterized and E. coli-treated controls in which ethylene production was maximum under cadmium-induced stress. These results were consistent with the proposed model that AcdS⁺ bacteria are closely associated with plant roots and act as a sink for a significant amount of ACC present in the root exudates (Glick et al. 1998; Penrose et al. 2001). Reduction in ACC concentration within the plant resulted in reduced amounts of ethylene synthesis, and thus decreased the extent of ethylene inhibition on root elongation and improved the plant survival under stress (Penrose and Glick 2001; Pierik et al. 2006). Even though IAA production was shown by all AcdS⁺ isolates, and bacterial IAA at low levels is known to have a positive effect on plant root growth (Patten and Glick 2002), the result obtained from the Cd-treated seedlings with low-stress ethylene synthesis clearly implicated the role of ACC deaminase enzyme of the isolates on root elongation and plant growth promotion under abiotic stress conditions. These results are in accordance with others (Grichko and Glick 2001; Mayak et al. 2004; Saravanakumar and Samiyappan 2007).

Characterization of the acdS gene in Ps 2-3 and Ps 7-12 isolates (selected for higher in vitro enzymatic activity and better reduction of stress ethylene in plants) showed that it encoded a complete protein of 338 amino acids in length. As most of the nucleotide dissimilarities were observed in the wobble position, the deduced amino acid sequences showed better homology than nucleotide sequences. Isolate Ps 7-12 branched closer with other strains Pf 2P24, Pf 17, CH-CRS-8 and PSNL based on nucleotide sequences; however, Ps 2-3 shared more affinity with them based on amino acid sequences. Variation in numbers of different peptidase acting/cleaving sites and the presence of different types of motif sites were observed between the two AcdS protein sequences with an additional N-myristoylation site in Ps 7-12. This motif site plays an important role in plant responses to environmental stress by membrane targeting and signal transduction during plant–rhizobacterial interactions. Deduced amino acid sequence analysis indicated the conservation of key residues, for example, K⁵¹ which acts as a PLP binding site, S⁷⁸ which act as nucleophile in the enzymatic attack, C¹⁶²/A¹⁶² which is important for enzymatic activity of protein, Y²⁹⁴ which is involved in substrate/product transportation, and E²⁹⁵ which stabilizes the PLP binding as required for the protein confirmation and enzymatic activity in P. fluorescens ACP and H. saturnus (Sheehy et al. 1991; Yao et al. 2000). These conserved key residues were also observed in the AcdS sequence of isolates Ps 2-3 and Ps 7-12. In addition, there were 8 (K⁴, A⁷, Q³³, E¹²⁵, I²²², D²⁷³, F³⁰³ and D³⁰⁴) and 13 (H⁴, E⁷, Q³³, E¹²⁵, D¹⁴⁶, S¹⁵⁰, E¹⁸¹, Q¹⁸⁵, T¹⁸⁹, Q²¹⁸, I²²², K²³⁰ and D³⁰⁴) new amino acid residues observed in Ps 2-3 and Ps 7-12, respectively, as compared to earlier reports. These results are consistent with the possibility that there were a limited number of pre-existing gene containing a few motifs for ACC deaminase genes (Hontzeas et al. 2005; Blaha et al. 2006) that could be mutated (without large numbers of changes) to encode ACC deaminase protein and could be evolved through multiple horizontal gene transfer.

The present study demonstrated the growth-promoting ability of mustard rhizobacteria containing ACC deaminase in wheat seedlings through modulation of stress ethylene synthesis. Nucleotide sequence variations were observed in acdS of Pseudomonas sp. Ps 2-3 and P. fluorescens Ps 7-12, but at the same time key amino acid residues of its AcdS protein were highly conserved. However, overexpression of these cloned genes and the potentiality of these characterized genes in the rhizobacterial isolates Ps 2-3 and Ps 2-17 for growth promotion of wheat should be validated under field experiments with appropriate abiotic stress conditions.