Journal of Plant Physiology 174 (2015) 131–136

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Journal of Plant Physiology

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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
b
Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia
c
Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang,
Selangor Darul Ehsan, Malaysia

a r t i c l e i n f o s u m m a r y

Article history: Nitric oxide associated 1 (NOA1) protein is implicated in plant disease resistance and nitric oxide (NO)
Received 5 August 2014 biosynthesis. A full-length cDNA encoding of NOA1 protein from oil palm (Elaeis guineensis) was isolated
Received in revised form 9 October 2014 and designated as EgNOA1. Sequence analysis suggested that EgNOA1 was a circular permutated GTPase
Accepted 9 October 2014
with high similarity to the bacterial YqeH protein of the YawG/YlqF family. The gene expression of EgNOA1
Available online 18 October 2014
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
Keywords:
association with NO biosynthesis. Real-time PCR (qPCR) analysis revealed that the transcript abundance
Circular permutated GTPase
Ganoderma
of EgNOA1 in root tissues was increased by G. boninense treatment. NO burst in Ganoderma-treated root
Nitric oxide tissue was detected using Griess reagent, in advance of the up-regulation of the EgNOA1 transcript. This
NOA1 indicates that NO production was independent of EgNOA1. However, the induced expression of EgNOA1
Oil palm 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 pathogen in mouse-ear cress/Pseudomonas syringae pv. tomato and

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 matu-
ration 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, C-
terminal 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, hyper-
sensitive 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 transla-
tion 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: muiyun@upm.edu.my (M.-Y. Wong).
oat/Puccinia coronata pv. avenea is one of the hallmark events that
mediates defense responses (Tada et al., 2004). The NO medi-
ated plant defense responses include hypersensitive response
(HR), activation of salicylic acid (SA) and jasmonic acid (JA) sig-
naling pathway, accumulation of antimicrobial compounds, cell
wall lignification, modulation of defense related gene and post-
translational 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 mecha-
nisms (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 l-
citrulline 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

http://dx.doi.org/10.1016/j.jplph.2014.10.003
0176-1617/© 2014 Elsevier GmbH. All rights reserved.
132 Y.-M. Kwan et al. / Journal of Plant Physiology 174 (2015) 131–136
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 depend-
ent and nitrate reductase (NR)-related (Gas et al., 2009; Van Ree
et al., 2011). The indirect regulatory effect of NOA1 in NO pro-
duction 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 func-
tion, 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 knock-
out 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, jas-
monates and carbon-based secondary metabolites, expression of
salicylic acid (SA)-defense responsive gene and elevated NO pro-
duction (Kato et al., 2008; Wünsche et al., 2011; Mandal et al.,
2012).
The African oil palm (Elaeis guineensis) is commercially culti-
vated 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, Gano-
derma 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 basid-
iospores, insects assisted spore transfer and mycelial root contact
(Sanderson, 2005; Paterson, 2007). The current disease manage-
ment strategies include soil mounding, mechanical removal, clean
clearing, legume cover crops, fungicide application, biological con-
trol, fertilizer and biofertilizer input management to prolong the
lifespan of infected palms, but these are not effective in dis-
ease 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 exhib-
ited 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 mate-
rials is effective in disease eradication or delay of disease
development. This reduces Ganoderma inoculum for subsequent
replantings. Chitinase, glucanase, isoflavone reductase, metallo-
thioneins, 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 impor-
tant 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 boni-
nense to investigate the expression profile of EgNOA1 in root tissue
and its potential as expressed markers for early disease detec-
tion and/or fungal resistance. In addition, the association between
EgNOA1 gene expression and NO biosynthesis was also investi-
gated.
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 adopt-
ing 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 Tech-
nologies, 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 soft-
ware (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 GTC-
3 ), 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. RACE-
PCR 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 ana-
lyzed 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 Infor-
mation (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). Sub-
cellular 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 Evo-
lutionary 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). Inocu-
lation was carried out through direct sitting technique by adopting
the protocol outlined by Zaiton (2006). Five seedlings were har-
vested 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 coloniza-
tion on root surfaces and necrotic lesions. Samples of roots were
flash frozen in liquid nitrogen and stored at −80 ◦ C until required.
 Y.-M. Kwan et al. / Journal of Plant Physiology 174 (2015) 131–136 133

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).
134 Y.-M. Kwan et al. / Journal of Plant Physiology 174 (2015) 131–136

RNA extraction and reverse transcription
RNA extraction was performed on root samples pooled from
five biological replicates harvested from each treatment at respec-
tive sampling intervals. Total RNA was isolated from root samples
using Total RNA Purification Kit (Norgen Biotek Corporation,
Canada) according to instructions from the manufacturer. On-
column DNase I (Fermentas, Lithuania) digestion was carried out
to eliminate traces of genomic DNA. First strand cDNA was synthe-
sized 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 pro-
filed 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 dena-
turation 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). Refer-
ence genes for data normalization were selected using NormFinder
(Andersen et al., 2004) and BestKeeper (Pfaffl et al., 2004) algo-
rithms 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 sig-
nificant 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 for-
mation 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 tripli-
cate 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 read-
ing frame (ORF) of 1674 bp that encodes a polypeptide of 558 amino
acids with a predicted molecular weight of 61.16 kDa and isoelec-
tric 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 (Ara-
bidopsis 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 con-
servation 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).
 Y.-M. Kwan et al. / Journal of Plant Physiology 174 (2015) 131–136 135

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 bind-
ing pocket (Mishra et al., 2005).
EgNOA1 is classified under the YqeH cpGTPase of the YlqF related Fig. 3. Relative abundance of EgNOA1 transcripts in oil palm root tissue in response
GTPase superfamily which belongs to the TRAFAC (after translation to Ganoderma-treatment at respective sampling intervals. The transcript abundance
of EgNOA1 is represented in fold change (number above each bar) compared to
factors) class (Leipe et al., 2002). The YqeH protein from B. subtilis
that of the control at the respective sampling intervals after being normalized
was reported to restore stunted growth and greening defects in A. with three endogenous controls. The bars are means ± standard error calculated
thaliana Atnoa1/rif1 mutant (Flores-Perez et al., 2008; Sudhamsu from the results of three technical replicates. The asterisk (*) indicates significant
et al., 2008). The YqeH protein is implicated in GTPase activity, cel- up-regulation of EgNOA1 in Ganoderma-treated root tissues at 96 DAI.
lular growth, RNA binding, ribosome biogenesis and assembly in
bacterial cells (Loh et al., 2007; Uicker et al., 2007). The predicted
pathogen infection (Guo et al., 2003; Kato et al., 2008; Mansour,
RNA/ribosome binding activities of EgNOA1 protein are supported
2011). In agreement with this, the transcript abundance of EgNOA1
by ZBD, implicated in ligand, protein and nucleic acid binding
was up-regulated (2.9-fold) at 96 DAI in Ganoderma-infected root
whereas the CTD is reported in RNA binding (Sudhamsu et al., 2008).
tissues compared to that of the control (Fig. 3). In this study,
EgNOA1 was demonstrated to be constitutively expressed in root
Expression profile of EgNOA1 and NO burst upon Ganoderma tissues, and the expression level gradually increased with the col-
infection onization of G. boninense. The induced expression of EgNOA1 is an
indication of the oil palm defense system activation against invad-
The expression profile of EgNOA1 and NO concentration in ing pathogenic G. boninense. Recent studies have reported that
Ganoderma-infected root tissues were analyzed to examine the NOA1 mediated induction of JA and salicylic acid defense responses
involvement of EgNOA1 during fungal infection and its associa- enhanced plant tolerance to biotic stress (Wünsche et al., 2011;
tion with NO biosynthesis. The transcript abundance of EgNOA1 in Mandal et al., 2012).
Ganoderma-infected root tissue at 3, 7, 14, 21, 28, 56 and 96 DAI The concentration of NO in root tissue was determined from
compared to the controls at the respective sampling intervals was the sum of nitrate and nitrite as NO is rapidly oxidized to nitrite
profiled using qPCR. The increase in expression level of two-fold or and/or nitrate by oxygen in biological systems (Yucel et al., 2012).
above was significant, suggesting an increase in the total activity of NO burst is known as one of the earliest events of plant defense
NOA1 protein in Ganoderma-infected root tissue (Yeoh et al., 2012). response against pathogen attack (Torres and Jones, 2006; Hong
NOA1 is implicated in plant defense as plant resistance towards et al., 2008). At 14 DAI, NO concentration in Ganoderma-treated root
pathogens was compromised by the silence of NOA1 (Zeidler et al., tissues was higher (30.49 ± 0.94 ␮M) compared to that of the con-
2004; Asai and Yoshioka, 2009). Although the NOA1 mediated trol (22.18 ± 0.81 ␮M) at P < 0.01 (Fig. 4). There were no significant
defense mechanism remains unclear, increased expression of NOA1 differences (P > 0.01) in NO concentration in Ganoderma-infected
has been reported in A. thaliana, C. sativa and N. benthamiana upon root tissues compared to the control at 3–7 DAI and at 28–96 DAI.

35
30.49
Control Ganoderma-treated
30 27.06
Nitrite concentration (µM)

24.52
24.12
25 22.18
22.60

18.25
20 17.42 16.89

15 12.01
10.86 11.05 10.91 10.82
10

0
3 7 14 21 28 56 96
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.
136 Y.-M. Kwan et al. / Journal of Plant Physiology 174 (2015) 131–136
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 environ-
mental 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 elic-
itors (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 (Ni-
NOR) 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 produc-
tion 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 dur-
ing 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 associ-
ated 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 associ-
ated 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 boni-
nense PER71 culture.
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