Journal of Plant Physiology 174 (2015) 131–136
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Short Communications
Cloning of nitric oxide associated 1 (NOA1) transcript from oil palm (Elaeis guineensis) and its expression during Ganoderma infection Yee-Min Kwan a , Sariah Meon a,b , Chai-Ling Ho a,c , Mui-Yun Wong a,b,∗ a
Laboratory of Plantation Crops, Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia b c
a r t i c l e
i n f o
Article history: Received 5 August 2014 Received in revised form 9 October 2014 Accepted 9 October 2014 Available online 18 October 2014 Keywords: Circular permutated GTPase Ganoderma Nitric oxide NOA1 Oil palm
s u m m a r y Nitric oxide associated 1 (NOA1) protein is implicated in plant disease resistance and nitric oxide (NO) biosynthesis. A full-length cDNA encoding of NOA1 protein from oil palm (Elaeis guineensis) was isolated and designated as EgNOA1. Sequence analysis suggested that EgNOA1 was a circular permutated GTPase with high similarity to the bacterial YqeH protein of the YawG/YlqF family. The gene expression of EgNOA1 and NO production in oil palm root tissues treated with Ganoderma boninense, the causal agent of basal stem rot (BSR) disease were profiled to investigate the involvement of EgNOA1 during fungal infection and association with NO biosynthesis. Real-time PCR (qPCR) analysis revealed that the transcript abundance of EgNOA1 in root tissues was increased by G. boninense treatment. NO burst in Ganoderma-treated root tissue was detected using Griess reagent, in advance of the up-regulation of the EgNOA1 transcript. This indicates that NO production was independent of EgNOA1. However, the induced expression of EgNOA1 in Ganoderma-treated root tissues implies that it might be involved in plant defense responses against pathogen infection. © 2014 Elsevier GmbH. All rights reserved.
Introduction Nitric oxide (NO) is an ubiquitous biological messenger involved in a broad spectrum of plant physiological processes such as seed germination, pollen tube growth, stomatal movement, leaf maturation and senescence, floral transition, root organogenesis, stress tolerance and disease resistance (Ferrarini et al., 2008; FrÖhlich and Durner, 2011). The rapid burst of NO as elicited by avirulent
Abbreviations: BSR, basal stem rot; CPG, circularly permutated GTPase; CTD, Cterminal domain; DAI, day after inoculation; Cq , quantification cycle; EST, expressed sequence tag; FFB, fresh fruit bunch; GTP, guanosine triphosphate GTPases; HAS, hydrophobic amino acid substituted for catalytic glutamine residue; HR, hypersensitive response; JA, jasmonic acid; NAD, NADH dehydrogenase subunit 5-like; Ni-NOR, nitrite-NO reductase; NO, nitric oxide; NOA1, nitric oxide associated 1; NR, nitrate reductase; NTC, non-template control; ORF, open reading frame; PAMP, pathogen associated molecule patterns; PRRs, PAMP recognition receptors; PTI, PAMP-triggered immunity; RACE, rapid amplification of cDNA ends; ROS, reactive oxygen species; RWB, rubber wood block; SA, salicylic acid; TRAFAC, after translation factors; UTR, untranslated region; ZBD, zinc-binding domain. ∗ Corresponding author at: Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia. Tel.: +60 389474852; fax: +60 389381014. E-mail address: [email protected] (M.-Y. Wong). http://dx.doi.org/10.1016/j.jplph.2014.10.003 0176-1617/© 2014 Elsevier GmbH. All rights reserved.
pathogen in mouse-ear cress/Pseudomonas syringae pv. tomato and oat/Puccinia coronata pv. avenea is one of the hallmark events that mediates defense responses (Tada et al., 2004). The NO mediated plant defense responses include hypersensitive response (HR), activation of salicylic acid (SA) and jasmonic acid (JA) signaling pathway, accumulation of antimicrobial compounds, cell wall lignification, modulation of defense related gene and posttranslational protein modification (Ferrarini et al., 2008; Misra et al., 2010). NO biosynthesis in plants is divided into oxidative and reductive pathways involving various enzymes and non-enzymatic mechanisms (FrÖhlich and Durner, 2011; Thakur and Sohal, 2013). Nitric oxide synthases (NOSs) (EC 1.14.13.39) are NO generators in the mammalian system, involving the oxidation of l-arginine to lcitrulline and NO. NOS homologs are widely distributed in animals, fungi, green algae and bacteria, but are absent in plants. Although NOS activity has been detected in plants but a novel plant NOS has not yet been identified. The Arabidopsis thaliana NO synthase 1 (AtNOS1) was initially reported as a putative plant NOS, but was later renamed A. thaliana NO associated 1 (AtNOA1), as it was soon indicated as a circular permuted GTPase (cpGTPase) of the YawG/YlqF family (Zemojtel et al., 2006; Moreau et al., 2008). The defective arginine-dependent NO synthesis activity in recombinant
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AtNOS1 protein and the contradictory NO accumulation responses in NOA1-silenced mutants have affirmed that AtNOS1 is not an authentic NOS per se (Van Ree et al., 2011). The involvement of NOA1 in NO biosynthesis is currently identified as sucrose dependent and nitrate reductase (NR)-related (Gas et al., 2009; Van Ree et al., 2011). The indirect regulatory effect of NOA1 in NO production was demonstrated with impaired NO accumulation in NOA1-silenced A. thaliana and Nicotiana benthamiana (Guo et al., 2003; Asai and Yoshioka, 2009). Apart from NO biosynthesis, NOA1 is implicated in plastid function, ribosome biogenesis, and enhanced tolerance to both abiotic and biotic stresses (Flores-Perez et al., 2008; Qiao et al., 2009; Liu et al., 2010). Increased susceptibility to pathogens in NOA1 knockout A. thaliana and N. benthamiana had demonstrated the role of NOA1 in disease resistance (Zeidler et al., 2004; Asai and Yoshioka, 2009). The NOA1 mediated defense mechanism was observed be to associated with the induced accumulation of oleic acid, jasmonates and carbon-based secondary metabolites, expression of salicylic acid (SA)-defense responsive gene and elevated NO production (Kato et al., 2008; Wünsche et al., 2011; Mandal et al., 2012). The African oil palm (Elaeis guineensis) is commercially cultivated for vegetable oil, biofuel and oleochemicals. The outbreak of the destructive basal stem rot (BSR) disease in the oil palm nursery and plantations, caused by the white rot fungi, Ganoderma species is a major challenge to oil palm cultivation (Paterson et al., 2009). BSR disease has resulted in severe economic loss due to reduction in fresh fruit bunch (FFB) and collapse of standing palms (Chung, 2011). Ganoderma is spread by airborne basidiospores, insects assisted spore transfer and mycelial root contact (Sanderson, 2005; Paterson, 2007). The current disease management strategies include soil mounding, mechanical removal, clean clearing, legume cover crops, fungicide application, biological control, fertilizer and biofertilizer input management to prolong the lifespan of infected palms, but these are not effective in disease eradication (Chung, 2011). The low effectiveness of these approaches is caused by the resistant pseudosclerotia structure of the Ganoderma fungi and the lack of early disease symptoms (Rees et al., 2009). The different susceptibility to Ganoderma infection exhibited by planting materials from different geographical origins such as Cameroon, Nigeria and Zaire has unraveled the prospect of oil palm genetic improvement programs (Durand-Gasselin et al., 2005). Breeding of resistant or tolerant planting materials is effective in disease eradication or delay of disease development. This reduces Ganoderma inoculum for subsequent replantings. Chitinase, glucanase, isoflavone reductase, metallothioneins, metallothionein-like protein, early methionine-labeled polypeptides and stearoyl-acyl carrier protein desaturase (SAD) have been reported to express differentially after Ganoderma infection (Yeoh et al., 2012; Tan et al., 2013; Tee et al., 2013). Nevertheless, the identification of defense associated genes against Ganoderma is ongoing. These genes are important in providing insight into the molecular events during plant–pathogen interaction and for the development of expressed markers for large scale screening of resistant or tolerant planting materials. In the present study, a transcript encoding NOA1 protein from oil palm, designated as EgNOA1, was isolated and characterized. Oil palm seedlings were artificially inoculated with Ganoderma boninense to investigate the expression profile of EgNOA1 in root tissue and its potential as expressed markers for early disease detection and/or fungal resistance. In addition, the association between EgNOA1 gene expression and NO biosynthesis was also investigated.
Materials and methods Isolation of full length cDNA sequence Total RNA was extracted from three month old oil palm (Elaeis guineensis Jacq., Dura × Pisifera, GH500 series) root tissue by adopting the protocol outlined in Chan et al. (2007). RNA samples were treated with DNase I (Fermentas, Lithuania) according to manufacturer’s instructions to eliminate traces of genomic DNA contamination. The integrity of RNA samples was examined using agarose gel electrophoresis and purity was assessed using the NanoDrop ND-1000 UV–Vis Spectrophotometer (NanoDrop Technologies, USA) at absorbance ratio of A260/280 and A260/230. Gene specific primers were designed according to two expressed sequence tags (ESTs) (i.e. EL694732 and 17871) retrieved from the oil palm EST library (Ho et al., 2007) that matched (E-value < 10−5 ) the NOA1 protein in the GenBank non-redundant protein database. Gene specific primers were designed using Primer3 Input software (version 0.4.0) (www.bioinfopop.ufv.br/sistema/primer3). The primers designed for 5 - and 3 -rapid amplification of cDNA ends (RACE) PCR were: 17871-R1 (5 -TTC CTG GAA CAA CCC TTG GTC-3 ) and EL694732-F1 (5 -TTC CTG GAA CAA CCC TTG GTC3 ), respectively. The primers used for nested 5 - and 3 -RACE PCR were: 17871-R2 (5 -TCC TGG AAC AAC CCT TGG TCC CAT TG-3 ) and EL694732-F2 (5 -TCC TGG AAC AAC CCT TGG TCC CAT TG-3 ), respectively. Template for RACE-PCR was synthesized from 10 g of total RNA using ExactSTART Eukaryotic mRNA 5 - and 3 -RACE kit (Epicentre, USA) according to manufacturer’s instructions. RACEPCR products were cloned into the pDrive vector (Qiagen, German) and sequenced using T7 and SP6 primers. The resulting full-length cDNA sequence was designated as EgNOA1. Bioinformatics analysis Sequence similarity and catalytic domain of EgNOA1 was analyzed using Basic Local Alignment Search Tool (BLASTx) and conserved domain database (www.ncbi.nlm.nih.gov/Structure/ cdd/wrpsb.cgi) at the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). Molecular weight and isoelectric point of the EgNOA1 protein was calculated using ExPASy ProtParam program (www.expasy.ch/tools/protparam.htm). Subcellular location of EgNOA1 was predicted using WoLFPSORT (www.wolfpsort.org). Sequence alignment of different plant NOA homologs was performed using the ClustalW method (www.ebi.ac.uk/clustalw). The phylogenetic tree was constructed using neighbour joining method implemented in Molecular Evolutionary Genetics Analysis (MEGA) version 5.1 program (Tamura et al., 2011). Plant materials and treatments A total of 70 4-month-old oil palm seedlings (E. guineensis Jacq., Dura × Pisifera, GH500 series) was purchased from Sime Darby Seeds & Agricultural Services Sdn. Bhd. (Banting, Malaysia). The seedlings were equally divided for two treatments, either treated with one-month-old Ganoderma boninense PER71 colonized rubber wood blocks (RWB) or sterilized RWBs (served as control). Inoculation was carried out through direct sitting technique by adopting the protocol outlined by Zaiton (2006). Five seedlings were harvested from both treatments at 3, 7, 14, 21, 28, 56 and 96 days after inoculation (DAI). The roots were washed and examined for visible symptoms of G. boninense infection, including mycelial colonization on root surfaces and necrotic lesions. Samples of roots were flash frozen in liquid nitrogen and stored at −80 ◦ C until required.
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Fig. 1. Multiple sequence aligment of EgNOA1 with other plant NOA1 homologs. Plant NOA1 proteins have three discrete conserved domains – zinc binding domain (ZBD), circularly permuted GTPase (CPG) domain arranged in G4-G5-G1-G2-G3 order and the C-terminal domain (CTD). The residues that denote the ZBD are indicated within the arrows and the four conserved cysteine residues are indicated in the box. The CTD region is indicated within the arrows. The motif residues of the CPG domain are indicated in the box and the conserved residues are shaded in grey. Gaps are represented by dashes. Numbering of the amino acid is indicated on the right. Protein identification in the alignment – AtNOA1: A. thaliana NOA1 (NP190329), MtNOA1: Medicago truncatula NOA1 (ADK47527), OsNOA1: O. sativa NOA1 (NP001045614), RcNOA1: R. communis NOA1 (XP002510962) and SbNOA1: S. bicolor NOA1 (XP002451348).
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RNA extraction and reverse transcription RNA extraction was performed on root samples pooled from five biological replicates harvested from each treatment at respective sampling intervals. Total RNA was isolated from root samples using Total RNA Purification Kit (Norgen Biotek Corporation, Canada) according to instructions from the manufacturer. Oncolumn DNase I (Fermentas, Lithuania) digestion was carried out to eliminate traces of genomic DNA. First strand cDNA was synthesized from 1 g of total RNA using Maxima Reverse Transcriptase (Fermentas, Lithuania) with combination of oligo-dT and random primer.
Real-time PCR (qPCR) The transcript abundance of EgNOA1 in root samples was profiled using qPCR in the BioRad CFX96 real time PCR system (BioRad, USA). Reaction mixture of 25 L total volume contained 1× Brilliant III Ultra-Fast SYBR Green Master Mix (Agilent Technologies, USA), 100 ng first strand cDNA and 0.2 M forward and reverse primers (Supplementary Table S1, Ooi et al. 2012). The qPCR reaction was performed under the following temperature regime: initial denaturation at 95 ◦ C for 3 min; 40 cycles of 95 ◦ C for 5 s and 55 ◦ C for 10 s. Three technical triplicates were performed from the same RNA preparation for each treatment at corresponding sampling intervals and a non-template control (NTC) was included. Melt curve analysis was performed at 65–95 ◦ C with temperature increment of 0.5 ◦ C for every 10 s. The relative transcript abundance of EgNOA1 was normalized using manganese superoxide dismutase (MSD), NADH dehydrogenase subunit 5-like (NAD) and ubiquitin according to the Livak 2−Cq method (Vandesompele et al., 2002). Reference genes for data normalization were selected using NormFinder (Andersen et al., 2004) and BestKeeper (Pfaffl et al., 2004) algorithms based on gene expression stability. The relative expression fold change of EgNOA1 in the G. boninense-treated sample was obtained by comparison with its transcript abundance in control samples (calibrator) at corresponding sampling intervals. The significant difference in gene expression was fixed at ≥2 expression fold change based on the correlation (0.80) between microarray
and qPCR results when ≥1.4 expression fold change was detected (Morey et al., 2006; Tan et al., 2013). Supplementary Table S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph. 2014.10.003. Griess assay NO concentration in 2 g root samples pooled from equal weight of five biological replicates harvested from each treatment at respective sampling intervals was quantified using Griess assay according to the protocol outlined by Yucel et al. (2012). The formation of red violet azo compound following nitrite reaction with Griess reagent (Promega, USA) was measured using a microplate reader (Rayto RT-2100C) at 540 nm. Samples were tested in triplicate and data was analyzed using T-test at p = 0.01 level. Results and discussion Sequence analysis of EgNOA1 The cDNA sequence contains a 5 -untranslated region (UTR) of 47 bp upstream of the predicted start codon (ATG) and the longest 3 -UTR of 219 bp downstream of the translation stop codon (TGA). EgNOA1 (GenBank accession number KF601427) has an open reading frame (ORF) of 1674 bp that encodes a polypeptide of 558 amino acids with a predicted molecular weight of 61.16 kDa and isoelectric point of 9.42. The EgNOA1 protein was predicted to be targeted to chloroplast and mitochondria by WoLFPSORT. The closest homologs of EgNOA1 were amino acid sequence of NOA1 from rice (Oryza sativa NP001045614) (81%), castor bean (Ricinus communis XP002510962) (81%) and mouse-ear cress (Arabidopsis thaliana NP190329) (81%). Besides, the EgNOA1 shares a rather similar sequence identity with the YqeH protein from Bacillus subtilis (BAA12444) (31%). Multiple sequence alignment of different plant NOA1 homologs revealed a high level of sequence homology (Fig. 1). Phylogenetic analysis indicated a sequence conservation pattern among the NOA1 proteins from monocots/dicots and the family (Fig. 2). NOA1 proteins from dicotyledonous and monocotyledonous plants were grouped into separate clades.
Fig. 2. Phylogenetic analysis of EgNOA1 and NOA1 proteins from plants, bacteria and human. The phylogenetic tree was generated using MEGA 5.1 based on neighbour joining method. Bootstrap values obtained from 1000 replications are indicated as precentage at tree nodes. The branch length is drawn to scale and in the same unit as evolutionary distance, number of amino acid sunstituition per site. The scale bar represents 20% of amino acid differences between protein sequences. Protein identification in the phylogenetic tree – AtNOA1: A. thaliana NOA1 (NP190329), BjNOA1: B. juncea NOA1 (ACX61572), MtNOA1: M. truncatula NOA1 (ADK47527), NbNOA1: N. benthamiana NOA1 (BAF93184), OsNOA1: O. sativa NOA1 (NP001045614), PsNOA1: P. sitchensis NOA1 (ABR16951), RcNOA1: R. communis NOA1 (XP002510962), SbNOA1: S. bicolor NOA1 (XP002451348), StNOA1: S. tuberosum NOA1 (ADD63989), VvNOA1: V. vinifera NOA1 (XP002268388), ZmNOA1: Z. mays NOA1 (NP001168044), Bacteria YqeH: B. subtilis YqeH (BAA12444) and Human NOA1: H. sapiens NOA1 (NP115689).
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The EgNOA1 has all three active domains reported on AtNOA1 (Moreau et al., 2008; Sudhamsu et al., 2008), arranged in the order: N-terminal zinc-binding domain (ZBD), circularly permuted GTPase (CPG) domain and C-terminal domain (CTD). The CPG domain of EgNOA1 containing five motif residues, arranged in the permutated order were G4 (TKID) (220–223 residues), G5 (SSK) (256–258 residues), G1 (GSANVGKS) (283–290 residues), G2 (T) (324 residues) and G3 (DTPG) (341–344 residues). EgNOA1 is a HAS (hydrophobic amino acid substituted for catalytic glutamine residue) GTPase with a hydrophobic residue, valine (residue 345) in the G3 motif, replacing the glutamine or histamine residue found in the small GTPases (Anand et al., 2006). HAS GTPases have been reported with enhanced GTP hydrolysis efficiency due to valine mediated conformational alteration of the nucleotide binding pocket (Mishra et al., 2005). EgNOA1 is classified under the YqeH cpGTPase of the YlqF related GTPase superfamily which belongs to the TRAFAC (after translation factors) class (Leipe et al., 2002). The YqeH protein from B. subtilis was reported to restore stunted growth and greening defects in A. thaliana Atnoa1/rif1 mutant (Flores-Perez et al., 2008; Sudhamsu et al., 2008). The YqeH protein is implicated in GTPase activity, cellular growth, RNA binding, ribosome biogenesis and assembly in bacterial cells (Loh et al., 2007; Uicker et al., 2007). The predicted RNA/ribosome binding activities of EgNOA1 protein are supported by ZBD, implicated in ligand, protein and nucleic acid binding whereas the CTD is reported in RNA binding (Sudhamsu et al., 2008). Expression profile of EgNOA1 and NO burst upon Ganoderma infection The expression profile of EgNOA1 and NO concentration in Ganoderma-infected root tissues were analyzed to examine the involvement of EgNOA1 during fungal infection and its association with NO biosynthesis. The transcript abundance of EgNOA1 in Ganoderma-infected root tissue at 3, 7, 14, 21, 28, 56 and 96 DAI compared to the controls at the respective sampling intervals was profiled using qPCR. The increase in expression level of two-fold or above was significant, suggesting an increase in the total activity of NOA1 protein in Ganoderma-infected root tissue (Yeoh et al., 2012). NOA1 is implicated in plant defense as plant resistance towards pathogens was compromised by the silence of NOA1 (Zeidler et al., 2004; Asai and Yoshioka, 2009). Although the NOA1 mediated defense mechanism remains unclear, increased expression of NOA1 has been reported in A. thaliana, C. sativa and N. benthamiana upon
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Fig. 3. Relative abundance of EgNOA1 transcripts in oil palm root tissue in response to Ganoderma-treatment at respective sampling intervals. The transcript abundance of EgNOA1 is represented in fold change (number above each bar) compared to that of the control at the respective sampling intervals after being normalized with three endogenous controls. The bars are means ± standard error calculated from the results of three technical replicates. The asterisk (*) indicates significant up-regulation of EgNOA1 in Ganoderma-treated root tissues at 96 DAI.
pathogen infection (Guo et al., 2003; Kato et al., 2008; Mansour, 2011). In agreement with this, the transcript abundance of EgNOA1 was up-regulated (2.9-fold) at 96 DAI in Ganoderma-infected root tissues compared to that of the control (Fig. 3). In this study, EgNOA1 was demonstrated to be constitutively expressed in root tissues, and the expression level gradually increased with the colonization of G. boninense. The induced expression of EgNOA1 is an indication of the oil palm defense system activation against invading pathogenic G. boninense. Recent studies have reported that NOA1 mediated induction of JA and salicylic acid defense responses enhanced plant tolerance to biotic stress (Wünsche et al., 2011; Mandal et al., 2012). The concentration of NO in root tissue was determined from the sum of nitrate and nitrite as NO is rapidly oxidized to nitrite and/or nitrate by oxygen in biological systems (Yucel et al., 2012). NO burst is known as one of the earliest events of plant defense response against pathogen attack (Torres and Jones, 2006; Hong et al., 2008). At 14 DAI, NO concentration in Ganoderma-treated root tissues was higher (30.49 ± 0.94 M) compared to that of the control (22.18 ± 0.81 M) at P < 0.01 (Fig. 4). There were no significant differences (P > 0.01) in NO concentration in Ganoderma-infected root tissues compared to the control at 3–7 DAI and at 28–96 DAI.
35 30.49
Nitrite concentration (µM)
30 25
Control
Ganoderma-treated
27.06 24.52 22.60
22.18
24.12
18.25
20
17.42
16.89
15
12.01
10.86 11.05
10.91 10.82
56
96
10 5 0
3
7
14
21
28
Day after inoculation Fig. 4. NO concentration in Ganoderma-treated and control oil palm root tissues at respective sampling intervals. The bars represent means ± standard error (n = 3). The asterisk (*) indicates significant up-regulation of NO concentration in Ganoderma-treated root tissue compared to that of the control at P < 0.01 as determined by T-test.
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However, a fluctuation pattern in NO production was observed among 3–56 DAI. Higher NO concentrations were detected in root tissues at three DAI and 28 DAI is which is attributed to environmental stress such as transplanting shock (3 DAI) and drought stress (28 DAI) (Hao and Zhang, 2009). NO burst was induced by the infection of G. boninense leading to the initiation of oil palm defense mechanism. This was reflected by the appearance of lesions on Ganoderma-infected roots which were first observed at 56 DAI. NO burst was induced upon plant cell recognition of elicitors (e.g. ligninolytic enzyme such as peroxidases and laccases) released by G. boninense to penetrate the plant cell wall (Paterson et al., 2009). The elicitors are recognized by pathogen associated molecule patterns (PAMP) recognition receptors (PRRs) followed by the activation of PAMP-triggered immunity (PTI) (Kawano et al., 2010). However, the source of NO in the Ganoderma-infected root tissues remains elusive given that plant NO biosynthesis has been identified from nitrate reductase (NR), nitrite-NO reductase (NiNOR) and polyamine oxidases (FrÖhlich and Durner, 2011; Pauly et al., 2011). NO burst had occurred in advance to the up-regulation of EgNOA1. The results did not show EgNOA1 dependent NO production in Ganoderma-infected root tissues, but there was differential EgNOA1 overexpression and NO production. It has been known that NOA1-knock out mutants were impaired in NO accumulation during pathogen invasion, but the mechanism remains unclear (Guo et al., 2003). Recent reports have suggested that the NO impairment is an indirect effect of interrupted chloroplast metabolism associated with the co-localization of NOA1 protein in the chloroplast and mitochondria (Moreau et al., 2008; Gas et al., 2009). Conclusions In conclusion, a full-length cDNA encoding nitric oxide associated 1 (EgNOA1) protein from oil palm was isolated. The transcript abundance of EgNOA1 was up-regulated in Ganoderma-treated oil palm root tissues at a later stage and did not coincide with NO burst. Functional characterization of EgNOA1 protein is useful in elucidating the role of EgNOA1 in plant defense. Acknowledgements The authors would like to acknowledge the Ministry of Science, Technology and Innovation, Malaysia for funding this research via ScienceFund (Project number 05-01-04-SF0950). Y.-M. Kwan was supported by scholarship from the Ministry of Higher Education, Malaysia. We would also like to thank the GanoDROP Laboratory of Malaysian Palm Oil Board (MPOB) for providing Ganoderma boninense PER71 culture. References Anand B, Verma SM, Prakash B. Nucleic Acids Res 2006;34:2196–205. Andersen C, Jensen J, Ørntoft T. Cancer Res 2004;64:5245–50.
Asai S, Yoshioka H. Mol Plant Microbe Interact 2009;22:619–29. Chan KL, Ho CL, Namasivayam P, Napis S. Nat Protoc 2007;184. Chung GF. The Planter 2011;87:325–39. Durand-Gasselin T, Asmady H, Flori A, Jacquemard JC, Hayun Z, Breton F, et al. Mycopathologia 2005;159, 93–00. Ferrarini A, de Stefano M, Baudouin E, Pucciariello C, Polverari A, Puppo A, et al. Mol Plant Microbe Interact 2008;21:781–90. Flores-Perez U, Sauret-Gueto S, Gas E, Jarvis P, Rodriguez-Concepcion M. Plant Cell 2008;20:1303–15. FrÖhlich A, Durner J. Plant Sci 2011;181:401–4. Gas E, Flores-Perez U, Sauret-Gueto S, Rodriguez-Concepcion M. Plant Cell 2009;21:18–23. Guo F, Okamoto M, Crawford NM. Science 2003;302:100–3. Hao G, Zhang J. Hayat S, Mori M, Pichtel J, Ahmad A, editors. Nitric oxide in plant physiology. Germany: Wiley-VCH; 2009. p. 115–38. Ho CL, Kwan YY, Choi MC, Tee SS, Ng WH, Lim KA, et al. BMC Genomics 2007;8:381–92. Hong JK, Yun BW, Kang JG, Raja MU, Kwon E, Sorhagen K, et al. J Exp Bot 2008;59:147–54. Kato H, Asai S, Yamamoto-Katou A, Yoshioka H, Doke N, Kawakita K. J Gen Plant Pathol 2008;74:15–23. Kawano Y, Chen L, Shimamoto K. Rice 2010;3:112–21. Leipe DD, Wolf YI, Koonin EV, Aravind L. J Mol Microbiol 2002;317:41–72. Liu H, Lau E, Lam MPY, Chu H, Li S, Huang G, et al. New Phytol 2010;187, 83–05. Loh PC, Morimoto T, Matsuo Y, Oshima T, Ogasawara N. Genes Genet Syst 2007;82:281–9. Mandal MK, Chandra-Shekara AC, Jeong RD, Yu K, Zhu S, Chandra B, et al. Plant Cell 2012;24:1654–74. Mansour S. Pathotypes of Pseudoperonospora cubensis (Berk. Et Curt.) Rostow, causal agent of downy mildew and induced defense responses in the host. Serdang: Universiti Putra Malaysia; 2011. Mishra R, Gara SK, Mishra S, Prakash B. Proteins 2005;59:332–8. Misra AN, Misra M, Singh R. J Med Plants Res 2010;4:2729–39. Moreau M, Lee GI, Wang Y, Crane BR, Klessig DF. J Biol Chem 2008;283:32957–67. Morey JS, Ryan JC, Van Dolah FM. Biol Proced Online 2006;8:175–93. Ooi S, Choo C, Ishak Z, Abdullah MO. Plant Cell Tiss Org 2012;110:201–12. Paterson RRM. Crop Prot 2007;26:1369–76. Paterson RRM, Sariah M, Lima N. J Phytopathol 2009;157:649–56. Pauly N, Ferrari C, Andrio E, Marino D, Piardi S, Brouquisse R, et al. J Exp Bot 2011;62:939–48. Pfaffl M, Tichopad A, Prgomet C, Neuvians T. Biotechnol Lett 2004;26:509–15. Qiao W, Xiao S, Yu L, Fan L. Environ Exp Bot 2009;65:90–8. Rees RW, Flood J, Hasan Y, Potter U, Cooper RM. Plant Pathol 2009;58:982–9. Sanderson FR. Mycopathologia 2005;159:139–41. Sudhamsu J, Lee GI, Klessig DF, Crane BR. J Biol Chem 2008;283:32968–76. Tada Y, Mori T, Shinogi T, Yao N, Takahashi S, Betsuyaku S. Mol Plant Microbe Interact 2004;17:245–53. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. Mol Biol Evol 2011;28:2731–9. Tan YC, Yeoh KA, Wong MY, Ho CL. J Plant Physiol 2013;170:1455–60. Tee SS, Tan YC, Abdullah F, Ong-Abdullah M, Ho CL. Tree Genet Genomes 2013;9:377–86. Thakur B, Sohal BS. ISRN Biochem 2013;2013:1–10. Torres MA, Jones JDG, Dangl JL. Plant Physiol 2006;141:373–8. Uicker WC, Schaefer L, Koenigsknecht M, Britton RA. J Bacteriol 2007;189:2926–9. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Genome Biol 2002;3:1–11 [research0034]. Van Ree K, Gehl B, Chehab EW, Tsai YC, Braam J. Plant J 2011;68:225–33. Wünsche H, Baldwin IT, Wu J. J Integr Plant Biol 2011;53:619–31. Yeoh KA, Othman A, Meon S, Abdullah F, Ho CL. J Plant Physiol 2012;169:1565–70. Yucel AA, Gulen S, Dincer S, Yucel AE, Yetkin GI. J Exp Integr Med 2012;2: 167–71. Zaiton S. Bacterial endophytes of oil palm (Elaeis Guineensis) and antagonistic activity against Ganoderma boninense). Serdang: Universiti Putra Malaysia; 2006. Zeidler D, Zahringer U, Gerber I, Dubery I, Hartung T, Bors W, et al. Proc Natl Acad Sci U S A 2004;101:15811–6. Zemojtel T, Fröhlich A, Palmieri MC, Kolanczyk M, Mikula I, Wyrwicz LS, et al. Trends Plant Sci 2006;11:524–5.
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Short Communications
Cloning of nitric oxide associated 1 (NOA1) transcript from oil palm (Elaeis guineensis) and its expression during Ganoderma infection Yee-Min Kwan a , Sariah Meon a,b , Chai-Ling Ho a,c , Mui-Yun Wong a,b,∗ a
Laboratory of Plantation Crops, Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia b c
a r t i c l e
i n f o
Article history: Received 5 August 2014 Received in revised form 9 October 2014 Accepted 9 October 2014 Available online 18 October 2014 Keywords: Circular permutated GTPase Ganoderma Nitric oxide NOA1 Oil palm
s u m m a r y Nitric oxide associated 1 (NOA1) protein is implicated in plant disease resistance and nitric oxide (NO) biosynthesis. A full-length cDNA encoding of NOA1 protein from oil palm (Elaeis guineensis) was isolated and designated as EgNOA1. Sequence analysis suggested that EgNOA1 was a circular permutated GTPase with high similarity to the bacterial YqeH protein of the YawG/YlqF family. The gene expression of EgNOA1 and NO production in oil palm root tissues treated with Ganoderma boninense, the causal agent of basal stem rot (BSR) disease were profiled to investigate the involvement of EgNOA1 during fungal infection and association with NO biosynthesis. Real-time PCR (qPCR) analysis revealed that the transcript abundance of EgNOA1 in root tissues was increased by G. boninense treatment. NO burst in Ganoderma-treated root tissue was detected using Griess reagent, in advance of the up-regulation of the EgNOA1 transcript. This indicates that NO production was independent of EgNOA1. However, the induced expression of EgNOA1 in Ganoderma-treated root tissues implies that it might be involved in plant defense responses against pathogen infection. © 2014 Elsevier GmbH. All rights reserved.
Introduction Nitric oxide (NO) is an ubiquitous biological messenger involved in a broad spectrum of plant physiological processes such as seed germination, pollen tube growth, stomatal movement, leaf maturation and senescence, floral transition, root organogenesis, stress tolerance and disease resistance (Ferrarini et al., 2008; FrÖhlich and Durner, 2011). The rapid burst of NO as elicited by avirulent
Abbreviations: BSR, basal stem rot; CPG, circularly permutated GTPase; CTD, Cterminal domain; DAI, day after inoculation; Cq , quantification cycle; EST, expressed sequence tag; FFB, fresh fruit bunch; GTP, guanosine triphosphate GTPases; HAS, hydrophobic amino acid substituted for catalytic glutamine residue; HR, hypersensitive response; JA, jasmonic acid; NAD, NADH dehydrogenase subunit 5-like; Ni-NOR, nitrite-NO reductase; NO, nitric oxide; NOA1, nitric oxide associated 1; NR, nitrate reductase; NTC, non-template control; ORF, open reading frame; PAMP, pathogen associated molecule patterns; PRRs, PAMP recognition receptors; PTI, PAMP-triggered immunity; RACE, rapid amplification of cDNA ends; ROS, reactive oxygen species; RWB, rubber wood block; SA, salicylic acid; TRAFAC, after translation factors; UTR, untranslated region; ZBD, zinc-binding domain. ∗ Corresponding author at: Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia. Tel.: +60 389474852; fax: +60 389381014. E-mail address: [email protected] (M.-Y. Wong). http://dx.doi.org/10.1016/j.jplph.2014.10.003 0176-1617/© 2014 Elsevier GmbH. All rights reserved.
pathogen in mouse-ear cress/Pseudomonas syringae pv. tomato and oat/Puccinia coronata pv. avenea is one of the hallmark events that mediates defense responses (Tada et al., 2004). The NO mediated plant defense responses include hypersensitive response (HR), activation of salicylic acid (SA) and jasmonic acid (JA) signaling pathway, accumulation of antimicrobial compounds, cell wall lignification, modulation of defense related gene and posttranslational protein modification (Ferrarini et al., 2008; Misra et al., 2010). NO biosynthesis in plants is divided into oxidative and reductive pathways involving various enzymes and non-enzymatic mechanisms (FrÖhlich and Durner, 2011; Thakur and Sohal, 2013). Nitric oxide synthases (NOSs) (EC 1.14.13.39) are NO generators in the mammalian system, involving the oxidation of l-arginine to lcitrulline and NO. NOS homologs are widely distributed in animals, fungi, green algae and bacteria, but are absent in plants. Although NOS activity has been detected in plants but a novel plant NOS has not yet been identified. The Arabidopsis thaliana NO synthase 1 (AtNOS1) was initially reported as a putative plant NOS, but was later renamed A. thaliana NO associated 1 (AtNOA1), as it was soon indicated as a circular permuted GTPase (cpGTPase) of the YawG/YlqF family (Zemojtel et al., 2006; Moreau et al., 2008). The defective arginine-dependent NO synthesis activity in recombinant
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AtNOS1 protein and the contradictory NO accumulation responses in NOA1-silenced mutants have affirmed that AtNOS1 is not an authentic NOS per se (Van Ree et al., 2011). The involvement of NOA1 in NO biosynthesis is currently identified as sucrose dependent and nitrate reductase (NR)-related (Gas et al., 2009; Van Ree et al., 2011). The indirect regulatory effect of NOA1 in NO production was demonstrated with impaired NO accumulation in NOA1-silenced A. thaliana and Nicotiana benthamiana (Guo et al., 2003; Asai and Yoshioka, 2009). Apart from NO biosynthesis, NOA1 is implicated in plastid function, ribosome biogenesis, and enhanced tolerance to both abiotic and biotic stresses (Flores-Perez et al., 2008; Qiao et al., 2009; Liu et al., 2010). Increased susceptibility to pathogens in NOA1 knockout A. thaliana and N. benthamiana had demonstrated the role of NOA1 in disease resistance (Zeidler et al., 2004; Asai and Yoshioka, 2009). The NOA1 mediated defense mechanism was observed be to associated with the induced accumulation of oleic acid, jasmonates and carbon-based secondary metabolites, expression of salicylic acid (SA)-defense responsive gene and elevated NO production (Kato et al., 2008; Wünsche et al., 2011; Mandal et al., 2012). The African oil palm (Elaeis guineensis) is commercially cultivated for vegetable oil, biofuel and oleochemicals. The outbreak of the destructive basal stem rot (BSR) disease in the oil palm nursery and plantations, caused by the white rot fungi, Ganoderma species is a major challenge to oil palm cultivation (Paterson et al., 2009). BSR disease has resulted in severe economic loss due to reduction in fresh fruit bunch (FFB) and collapse of standing palms (Chung, 2011). Ganoderma is spread by airborne basidiospores, insects assisted spore transfer and mycelial root contact (Sanderson, 2005; Paterson, 2007). The current disease management strategies include soil mounding, mechanical removal, clean clearing, legume cover crops, fungicide application, biological control, fertilizer and biofertilizer input management to prolong the lifespan of infected palms, but these are not effective in disease eradication (Chung, 2011). The low effectiveness of these approaches is caused by the resistant pseudosclerotia structure of the Ganoderma fungi and the lack of early disease symptoms (Rees et al., 2009). The different susceptibility to Ganoderma infection exhibited by planting materials from different geographical origins such as Cameroon, Nigeria and Zaire has unraveled the prospect of oil palm genetic improvement programs (Durand-Gasselin et al., 2005). Breeding of resistant or tolerant planting materials is effective in disease eradication or delay of disease development. This reduces Ganoderma inoculum for subsequent replantings. Chitinase, glucanase, isoflavone reductase, metallothioneins, metallothionein-like protein, early methionine-labeled polypeptides and stearoyl-acyl carrier protein desaturase (SAD) have been reported to express differentially after Ganoderma infection (Yeoh et al., 2012; Tan et al., 2013; Tee et al., 2013). Nevertheless, the identification of defense associated genes against Ganoderma is ongoing. These genes are important in providing insight into the molecular events during plant–pathogen interaction and for the development of expressed markers for large scale screening of resistant or tolerant planting materials. In the present study, a transcript encoding NOA1 protein from oil palm, designated as EgNOA1, was isolated and characterized. Oil palm seedlings were artificially inoculated with Ganoderma boninense to investigate the expression profile of EgNOA1 in root tissue and its potential as expressed markers for early disease detection and/or fungal resistance. In addition, the association between EgNOA1 gene expression and NO biosynthesis was also investigated.
Materials and methods Isolation of full length cDNA sequence Total RNA was extracted from three month old oil palm (Elaeis guineensis Jacq., Dura × Pisifera, GH500 series) root tissue by adopting the protocol outlined in Chan et al. (2007). RNA samples were treated with DNase I (Fermentas, Lithuania) according to manufacturer’s instructions to eliminate traces of genomic DNA contamination. The integrity of RNA samples was examined using agarose gel electrophoresis and purity was assessed using the NanoDrop ND-1000 UV–Vis Spectrophotometer (NanoDrop Technologies, USA) at absorbance ratio of A260/280 and A260/230. Gene specific primers were designed according to two expressed sequence tags (ESTs) (i.e. EL694732 and 17871) retrieved from the oil palm EST library (Ho et al., 2007) that matched (E-value < 10−5 ) the NOA1 protein in the GenBank non-redundant protein database. Gene specific primers were designed using Primer3 Input software (version 0.4.0) (www.bioinfopop.ufv.br/sistema/primer3). The primers designed for 5 - and 3 -rapid amplification of cDNA ends (RACE) PCR were: 17871-R1 (5 -TTC CTG GAA CAA CCC TTG GTC-3 ) and EL694732-F1 (5 -TTC CTG GAA CAA CCC TTG GTC3 ), respectively. The primers used for nested 5 - and 3 -RACE PCR were: 17871-R2 (5 -TCC TGG AAC AAC CCT TGG TCC CAT TG-3 ) and EL694732-F2 (5 -TCC TGG AAC AAC CCT TGG TCC CAT TG-3 ), respectively. Template for RACE-PCR was synthesized from 10 g of total RNA using ExactSTART Eukaryotic mRNA 5 - and 3 -RACE kit (Epicentre, USA) according to manufacturer’s instructions. RACEPCR products were cloned into the pDrive vector (Qiagen, German) and sequenced using T7 and SP6 primers. The resulting full-length cDNA sequence was designated as EgNOA1. Bioinformatics analysis Sequence similarity and catalytic domain of EgNOA1 was analyzed using Basic Local Alignment Search Tool (BLASTx) and conserved domain database (www.ncbi.nlm.nih.gov/Structure/ cdd/wrpsb.cgi) at the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). Molecular weight and isoelectric point of the EgNOA1 protein was calculated using ExPASy ProtParam program (www.expasy.ch/tools/protparam.htm). Subcellular location of EgNOA1 was predicted using WoLFPSORT (www.wolfpsort.org). Sequence alignment of different plant NOA homologs was performed using the ClustalW method (www.ebi.ac.uk/clustalw). The phylogenetic tree was constructed using neighbour joining method implemented in Molecular Evolutionary Genetics Analysis (MEGA) version 5.1 program (Tamura et al., 2011). Plant materials and treatments A total of 70 4-month-old oil palm seedlings (E. guineensis Jacq., Dura × Pisifera, GH500 series) was purchased from Sime Darby Seeds & Agricultural Services Sdn. Bhd. (Banting, Malaysia). The seedlings were equally divided for two treatments, either treated with one-month-old Ganoderma boninense PER71 colonized rubber wood blocks (RWB) or sterilized RWBs (served as control). Inoculation was carried out through direct sitting technique by adopting the protocol outlined by Zaiton (2006). Five seedlings were harvested from both treatments at 3, 7, 14, 21, 28, 56 and 96 days after inoculation (DAI). The roots were washed and examined for visible symptoms of G. boninense infection, including mycelial colonization on root surfaces and necrotic lesions. Samples of roots were flash frozen in liquid nitrogen and stored at −80 ◦ C until required.
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Fig. 1. Multiple sequence aligment of EgNOA1 with other plant NOA1 homologs. Plant NOA1 proteins have three discrete conserved domains – zinc binding domain (ZBD), circularly permuted GTPase (CPG) domain arranged in G4-G5-G1-G2-G3 order and the C-terminal domain (CTD). The residues that denote the ZBD are indicated within the arrows and the four conserved cysteine residues are indicated in the box. The CTD region is indicated within the arrows. The motif residues of the CPG domain are indicated in the box and the conserved residues are shaded in grey. Gaps are represented by dashes. Numbering of the amino acid is indicated on the right. Protein identification in the alignment – AtNOA1: A. thaliana NOA1 (NP190329), MtNOA1: Medicago truncatula NOA1 (ADK47527), OsNOA1: O. sativa NOA1 (NP001045614), RcNOA1: R. communis NOA1 (XP002510962) and SbNOA1: S. bicolor NOA1 (XP002451348).
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RNA extraction and reverse transcription RNA extraction was performed on root samples pooled from five biological replicates harvested from each treatment at respective sampling intervals. Total RNA was isolated from root samples using Total RNA Purification Kit (Norgen Biotek Corporation, Canada) according to instructions from the manufacturer. Oncolumn DNase I (Fermentas, Lithuania) digestion was carried out to eliminate traces of genomic DNA. First strand cDNA was synthesized from 1 g of total RNA using Maxima Reverse Transcriptase (Fermentas, Lithuania) with combination of oligo-dT and random primer.
Real-time PCR (qPCR) The transcript abundance of EgNOA1 in root samples was profiled using qPCR in the BioRad CFX96 real time PCR system (BioRad, USA). Reaction mixture of 25 L total volume contained 1× Brilliant III Ultra-Fast SYBR Green Master Mix (Agilent Technologies, USA), 100 ng first strand cDNA and 0.2 M forward and reverse primers (Supplementary Table S1, Ooi et al. 2012). The qPCR reaction was performed under the following temperature regime: initial denaturation at 95 ◦ C for 3 min; 40 cycles of 95 ◦ C for 5 s and 55 ◦ C for 10 s. Three technical triplicates were performed from the same RNA preparation for each treatment at corresponding sampling intervals and a non-template control (NTC) was included. Melt curve analysis was performed at 65–95 ◦ C with temperature increment of 0.5 ◦ C for every 10 s. The relative transcript abundance of EgNOA1 was normalized using manganese superoxide dismutase (MSD), NADH dehydrogenase subunit 5-like (NAD) and ubiquitin according to the Livak 2−Cq method (Vandesompele et al., 2002). Reference genes for data normalization were selected using NormFinder (Andersen et al., 2004) and BestKeeper (Pfaffl et al., 2004) algorithms based on gene expression stability. The relative expression fold change of EgNOA1 in the G. boninense-treated sample was obtained by comparison with its transcript abundance in control samples (calibrator) at corresponding sampling intervals. The significant difference in gene expression was fixed at ≥2 expression fold change based on the correlation (0.80) between microarray
and qPCR results when ≥1.4 expression fold change was detected (Morey et al., 2006; Tan et al., 2013). Supplementary Table S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph. 2014.10.003. Griess assay NO concentration in 2 g root samples pooled from equal weight of five biological replicates harvested from each treatment at respective sampling intervals was quantified using Griess assay according to the protocol outlined by Yucel et al. (2012). The formation of red violet azo compound following nitrite reaction with Griess reagent (Promega, USA) was measured using a microplate reader (Rayto RT-2100C) at 540 nm. Samples were tested in triplicate and data was analyzed using T-test at p = 0.01 level. Results and discussion Sequence analysis of EgNOA1 The cDNA sequence contains a 5 -untranslated region (UTR) of 47 bp upstream of the predicted start codon (ATG) and the longest 3 -UTR of 219 bp downstream of the translation stop codon (TGA). EgNOA1 (GenBank accession number KF601427) has an open reading frame (ORF) of 1674 bp that encodes a polypeptide of 558 amino acids with a predicted molecular weight of 61.16 kDa and isoelectric point of 9.42. The EgNOA1 protein was predicted to be targeted to chloroplast and mitochondria by WoLFPSORT. The closest homologs of EgNOA1 were amino acid sequence of NOA1 from rice (Oryza sativa NP001045614) (81%), castor bean (Ricinus communis XP002510962) (81%) and mouse-ear cress (Arabidopsis thaliana NP190329) (81%). Besides, the EgNOA1 shares a rather similar sequence identity with the YqeH protein from Bacillus subtilis (BAA12444) (31%). Multiple sequence alignment of different plant NOA1 homologs revealed a high level of sequence homology (Fig. 1). Phylogenetic analysis indicated a sequence conservation pattern among the NOA1 proteins from monocots/dicots and the family (Fig. 2). NOA1 proteins from dicotyledonous and monocotyledonous plants were grouped into separate clades.
Fig. 2. Phylogenetic analysis of EgNOA1 and NOA1 proteins from plants, bacteria and human. The phylogenetic tree was generated using MEGA 5.1 based on neighbour joining method. Bootstrap values obtained from 1000 replications are indicated as precentage at tree nodes. The branch length is drawn to scale and in the same unit as evolutionary distance, number of amino acid sunstituition per site. The scale bar represents 20% of amino acid differences between protein sequences. Protein identification in the phylogenetic tree – AtNOA1: A. thaliana NOA1 (NP190329), BjNOA1: B. juncea NOA1 (ACX61572), MtNOA1: M. truncatula NOA1 (ADK47527), NbNOA1: N. benthamiana NOA1 (BAF93184), OsNOA1: O. sativa NOA1 (NP001045614), PsNOA1: P. sitchensis NOA1 (ABR16951), RcNOA1: R. communis NOA1 (XP002510962), SbNOA1: S. bicolor NOA1 (XP002451348), StNOA1: S. tuberosum NOA1 (ADD63989), VvNOA1: V. vinifera NOA1 (XP002268388), ZmNOA1: Z. mays NOA1 (NP001168044), Bacteria YqeH: B. subtilis YqeH (BAA12444) and Human NOA1: H. sapiens NOA1 (NP115689).
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The EgNOA1 has all three active domains reported on AtNOA1 (Moreau et al., 2008; Sudhamsu et al., 2008), arranged in the order: N-terminal zinc-binding domain (ZBD), circularly permuted GTPase (CPG) domain and C-terminal domain (CTD). The CPG domain of EgNOA1 containing five motif residues, arranged in the permutated order were G4 (TKID) (220–223 residues), G5 (SSK) (256–258 residues), G1 (GSANVGKS) (283–290 residues), G2 (T) (324 residues) and G3 (DTPG) (341–344 residues). EgNOA1 is a HAS (hydrophobic amino acid substituted for catalytic glutamine residue) GTPase with a hydrophobic residue, valine (residue 345) in the G3 motif, replacing the glutamine or histamine residue found in the small GTPases (Anand et al., 2006). HAS GTPases have been reported with enhanced GTP hydrolysis efficiency due to valine mediated conformational alteration of the nucleotide binding pocket (Mishra et al., 2005). EgNOA1 is classified under the YqeH cpGTPase of the YlqF related GTPase superfamily which belongs to the TRAFAC (after translation factors) class (Leipe et al., 2002). The YqeH protein from B. subtilis was reported to restore stunted growth and greening defects in A. thaliana Atnoa1/rif1 mutant (Flores-Perez et al., 2008; Sudhamsu et al., 2008). The YqeH protein is implicated in GTPase activity, cellular growth, RNA binding, ribosome biogenesis and assembly in bacterial cells (Loh et al., 2007; Uicker et al., 2007). The predicted RNA/ribosome binding activities of EgNOA1 protein are supported by ZBD, implicated in ligand, protein and nucleic acid binding whereas the CTD is reported in RNA binding (Sudhamsu et al., 2008). Expression profile of EgNOA1 and NO burst upon Ganoderma infection The expression profile of EgNOA1 and NO concentration in Ganoderma-infected root tissues were analyzed to examine the involvement of EgNOA1 during fungal infection and its association with NO biosynthesis. The transcript abundance of EgNOA1 in Ganoderma-infected root tissue at 3, 7, 14, 21, 28, 56 and 96 DAI compared to the controls at the respective sampling intervals was profiled using qPCR. The increase in expression level of two-fold or above was significant, suggesting an increase in the total activity of NOA1 protein in Ganoderma-infected root tissue (Yeoh et al., 2012). NOA1 is implicated in plant defense as plant resistance towards pathogens was compromised by the silence of NOA1 (Zeidler et al., 2004; Asai and Yoshioka, 2009). Although the NOA1 mediated defense mechanism remains unclear, increased expression of NOA1 has been reported in A. thaliana, C. sativa and N. benthamiana upon
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Fig. 3. Relative abundance of EgNOA1 transcripts in oil palm root tissue in response to Ganoderma-treatment at respective sampling intervals. The transcript abundance of EgNOA1 is represented in fold change (number above each bar) compared to that of the control at the respective sampling intervals after being normalized with three endogenous controls. The bars are means ± standard error calculated from the results of three technical replicates. The asterisk (*) indicates significant up-regulation of EgNOA1 in Ganoderma-treated root tissues at 96 DAI.
pathogen infection (Guo et al., 2003; Kato et al., 2008; Mansour, 2011). In agreement with this, the transcript abundance of EgNOA1 was up-regulated (2.9-fold) at 96 DAI in Ganoderma-infected root tissues compared to that of the control (Fig. 3). In this study, EgNOA1 was demonstrated to be constitutively expressed in root tissues, and the expression level gradually increased with the colonization of G. boninense. The induced expression of EgNOA1 is an indication of the oil palm defense system activation against invading pathogenic G. boninense. Recent studies have reported that NOA1 mediated induction of JA and salicylic acid defense responses enhanced plant tolerance to biotic stress (Wünsche et al., 2011; Mandal et al., 2012). The concentration of NO in root tissue was determined from the sum of nitrate and nitrite as NO is rapidly oxidized to nitrite and/or nitrate by oxygen in biological systems (Yucel et al., 2012). NO burst is known as one of the earliest events of plant defense response against pathogen attack (Torres and Jones, 2006; Hong et al., 2008). At 14 DAI, NO concentration in Ganoderma-treated root tissues was higher (30.49 ± 0.94 M) compared to that of the control (22.18 ± 0.81 M) at P < 0.01 (Fig. 4). There were no significant differences (P > 0.01) in NO concentration in Ganoderma-infected root tissues compared to the control at 3–7 DAI and at 28–96 DAI.
35 30.49
Nitrite concentration (µM)
30 25
Control
Ganoderma-treated
27.06 24.52 22.60
22.18
24.12
18.25
20
17.42
16.89
15
12.01
10.86 11.05
10.91 10.82
56
96
10 5 0
3
7
14
21
28
Day after inoculation Fig. 4. NO concentration in Ganoderma-treated and control oil palm root tissues at respective sampling intervals. The bars represent means ± standard error (n = 3). The asterisk (*) indicates significant up-regulation of NO concentration in Ganoderma-treated root tissue compared to that of the control at P < 0.01 as determined by T-test.
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However, a fluctuation pattern in NO production was observed among 3–56 DAI. Higher NO concentrations were detected in root tissues at three DAI and 28 DAI is which is attributed to environmental stress such as transplanting shock (3 DAI) and drought stress (28 DAI) (Hao and Zhang, 2009). NO burst was induced by the infection of G. boninense leading to the initiation of oil palm defense mechanism. This was reflected by the appearance of lesions on Ganoderma-infected roots which were first observed at 56 DAI. NO burst was induced upon plant cell recognition of elicitors (e.g. ligninolytic enzyme such as peroxidases and laccases) released by G. boninense to penetrate the plant cell wall (Paterson et al., 2009). The elicitors are recognized by pathogen associated molecule patterns (PAMP) recognition receptors (PRRs) followed by the activation of PAMP-triggered immunity (PTI) (Kawano et al., 2010). However, the source of NO in the Ganoderma-infected root tissues remains elusive given that plant NO biosynthesis has been identified from nitrate reductase (NR), nitrite-NO reductase (NiNOR) and polyamine oxidases (FrÖhlich and Durner, 2011; Pauly et al., 2011). NO burst had occurred in advance to the up-regulation of EgNOA1. The results did not show EgNOA1 dependent NO production in Ganoderma-infected root tissues, but there was differential EgNOA1 overexpression and NO production. It has been known that NOA1-knock out mutants were impaired in NO accumulation during pathogen invasion, but the mechanism remains unclear (Guo et al., 2003). Recent reports have suggested that the NO impairment is an indirect effect of interrupted chloroplast metabolism associated with the co-localization of NOA1 protein in the chloroplast and mitochondria (Moreau et al., 2008; Gas et al., 2009). Conclusions In conclusion, a full-length cDNA encoding nitric oxide associated 1 (EgNOA1) protein from oil palm was isolated. The transcript abundance of EgNOA1 was up-regulated in Ganoderma-treated oil palm root tissues at a later stage and did not coincide with NO burst. Functional characterization of EgNOA1 protein is useful in elucidating the role of EgNOA1 in plant defense. Acknowledgements The authors would like to acknowledge the Ministry of Science, Technology and Innovation, Malaysia for funding this research via ScienceFund (Project number 05-01-04-SF0950). Y.-M. Kwan was supported by scholarship from the Ministry of Higher Education, Malaysia. We would also like to thank the GanoDROP Laboratory of Malaysian Palm Oil Board (MPOB) for providing Ganoderma boninense PER71 culture. References Anand B, Verma SM, Prakash B. Nucleic Acids Res 2006;34:2196–205. Andersen C, Jensen J, Ørntoft T. Cancer Res 2004;64:5245–50.
Asai S, Yoshioka H. Mol Plant Microbe Interact 2009;22:619–29. Chan KL, Ho CL, Namasivayam P, Napis S. Nat Protoc 2007;184. Chung GF. The Planter 2011;87:325–39. Durand-Gasselin T, Asmady H, Flori A, Jacquemard JC, Hayun Z, Breton F, et al. Mycopathologia 2005;159, 93–00. Ferrarini A, de Stefano M, Baudouin E, Pucciariello C, Polverari A, Puppo A, et al. Mol Plant Microbe Interact 2008;21:781–90. Flores-Perez U, Sauret-Gueto S, Gas E, Jarvis P, Rodriguez-Concepcion M. Plant Cell 2008;20:1303–15. FrÖhlich A, Durner J. Plant Sci 2011;181:401–4. Gas E, Flores-Perez U, Sauret-Gueto S, Rodriguez-Concepcion M. Plant Cell 2009;21:18–23. Guo F, Okamoto M, Crawford NM. Science 2003;302:100–3. Hao G, Zhang J. Hayat S, Mori M, Pichtel J, Ahmad A, editors. Nitric oxide in plant physiology. Germany: Wiley-VCH; 2009. p. 115–38. Ho CL, Kwan YY, Choi MC, Tee SS, Ng WH, Lim KA, et al. BMC Genomics 2007;8:381–92. Hong JK, Yun BW, Kang JG, Raja MU, Kwon E, Sorhagen K, et al. J Exp Bot 2008;59:147–54. Kato H, Asai S, Yamamoto-Katou A, Yoshioka H, Doke N, Kawakita K. J Gen Plant Pathol 2008;74:15–23. Kawano Y, Chen L, Shimamoto K. Rice 2010;3:112–21. Leipe DD, Wolf YI, Koonin EV, Aravind L. J Mol Microbiol 2002;317:41–72. Liu H, Lau E, Lam MPY, Chu H, Li S, Huang G, et al. New Phytol 2010;187, 83–05. Loh PC, Morimoto T, Matsuo Y, Oshima T, Ogasawara N. Genes Genet Syst 2007;82:281–9. Mandal MK, Chandra-Shekara AC, Jeong RD, Yu K, Zhu S, Chandra B, et al. Plant Cell 2012;24:1654–74. Mansour S. Pathotypes of Pseudoperonospora cubensis (Berk. Et Curt.) Rostow, causal agent of downy mildew and induced defense responses in the host. Serdang: Universiti Putra Malaysia; 2011. Mishra R, Gara SK, Mishra S, Prakash B. Proteins 2005;59:332–8. Misra AN, Misra M, Singh R. J Med Plants Res 2010;4:2729–39. Moreau M, Lee GI, Wang Y, Crane BR, Klessig DF. J Biol Chem 2008;283:32957–67. Morey JS, Ryan JC, Van Dolah FM. Biol Proced Online 2006;8:175–93. Ooi S, Choo C, Ishak Z, Abdullah MO. Plant Cell Tiss Org 2012;110:201–12. Paterson RRM. Crop Prot 2007;26:1369–76. Paterson RRM, Sariah M, Lima N. J Phytopathol 2009;157:649–56. Pauly N, Ferrari C, Andrio E, Marino D, Piardi S, Brouquisse R, et al. J Exp Bot 2011;62:939–48. Pfaffl M, Tichopad A, Prgomet C, Neuvians T. Biotechnol Lett 2004;26:509–15. Qiao W, Xiao S, Yu L, Fan L. Environ Exp Bot 2009;65:90–8. Rees RW, Flood J, Hasan Y, Potter U, Cooper RM. Plant Pathol 2009;58:982–9. Sanderson FR. Mycopathologia 2005;159:139–41. Sudhamsu J, Lee GI, Klessig DF, Crane BR. J Biol Chem 2008;283:32968–76. Tada Y, Mori T, Shinogi T, Yao N, Takahashi S, Betsuyaku S. Mol Plant Microbe Interact 2004;17:245–53. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. Mol Biol Evol 2011;28:2731–9. Tan YC, Yeoh KA, Wong MY, Ho CL. J Plant Physiol 2013;170:1455–60. Tee SS, Tan YC, Abdullah F, Ong-Abdullah M, Ho CL. Tree Genet Genomes 2013;9:377–86. Thakur B, Sohal BS. ISRN Biochem 2013;2013:1–10. Torres MA, Jones JDG, Dangl JL. Plant Physiol 2006;141:373–8. Uicker WC, Schaefer L, Koenigsknecht M, Britton RA. J Bacteriol 2007;189:2926–9. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Genome Biol 2002;3:1–11 [research0034]. Van Ree K, Gehl B, Chehab EW, Tsai YC, Braam J. Plant J 2011;68:225–33. Wünsche H, Baldwin IT, Wu J. J Integr Plant Biol 2011;53:619–31. Yeoh KA, Othman A, Meon S, Abdullah F, Ho CL. J Plant Physiol 2012;169:1565–70. Yucel AA, Gulen S, Dincer S, Yucel AE, Yetkin GI. J Exp Integr Med 2012;2: 167–71. Zaiton S. Bacterial endophytes of oil palm (Elaeis Guineensis) and antagonistic activity against Ganoderma boninense). Serdang: Universiti Putra Malaysia; 2006. Zeidler D, Zahringer U, Gerber I, Dubery I, Hartung T, Bors W, et al. Proc Natl Acad Sci U S A 2004;101:15811–6. Zemojtel T, Fröhlich A, Palmieri MC, Kolanczyk M, Mikula I, Wyrwicz LS, et al. Trends Plant Sci 2006;11:524–5.
