Journal of Experimental Botany, Vol. 64, No. 17, pp. 5297–5307, 2013
doi:10.1093/jxb/ert295 Advance Access publication 16 September, 2013
Research paper
Mining the genome of Arabidopsis thaliana as a basis for the
identification of novel bioactive peptides involved in oxidative
stress tolerance
Barbara De Coninck1,*, Delphine Carron1,*, Patrizia Tavormina1, Lander Willem1, David J. Craik2, Christine Vos1,
Karin Thevissen1, Janick Mathys1,† and Bruno P. A. Cammue1,§
1 Centre for Microbial and Plant Genetics, KU Leuven, 3001 Heverlee, Belgium
Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
† Present address: Bioinformatics Training and Service facility of the VIB, 9052 Ghent, Belgium.
2 * These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: [email protected]
§ Received 30 April 2013; Revised 19 July 2013; Accepted 6 August 2013
Abstract
Although evidence has accumulated on the role of plant peptides in the response to external conditions, the number
of peptide-encoding genes in the genome is still underestimated. Using tiling arrays, we identified 176 unannotated
transcriptionally active regions (TARs) in Arabidopsis thaliana that were induced upon oxidative stress generated by
the herbicide paraquat (PQ). These 176 TARs could be translated into 575 putative oxidative stress-induced peptides
(OSIPs). A high-throughput functional assay was used in the eukaryotic model organism Saccharomyces cerevisiae
allowing us to test for bioactive peptides that increase oxidative stress tolerance. In this way, we identified three
OSIPs that, upon overexpression in yeast, resulted in a significant rise in tolerance to hydrogen peroxide (H2O2). For
one of these peptides, the decapeptide OSIP108, exogenous application to H2O2-treated yeast also resulted in significantly increased survival. OSIP108 is contained within a pseudogene and is induced in A. thaliana leaves by both the
reactive oxygen species-inducer PQ and the necrotrophic fungal pathogen Botrytis cinerea. Moreover, infiltration and
overexpression of OSIP108 in A. thaliana leaves resulted in increased tolerance to treatment with PQ. In conclusion,
the identification and characterization of OSIP108 confirms the validity of our high-throughput approach, based on
tiling array analysis in A. thaliana and functional screening in yeast, to identify bioactive peptides.
Key words: Arabidopsis thaliana, bioactive peptide, hydrogen peroxide, paraquat, Saccharomyces cerevisiae, sORF, tiling array.
Introduction
Bioactive peptides have been identified from both plant and
animal sources. In general, they are considered to be encrypted
as inactive fragments in mature proteins. When released via
enzymatic hydrolysis, they can exert various functional activities such as antimicrobial, antihypertensive, antithrombotic
and antioxidative activity (Kim and Wijesekara, 2010; Sarmadi
and Ismail, 2010). Very recently, Brand et al. (2012) developed
a method to identify putative antimicrobial peptides encrypted
in protein sequences from plants. Here, we have focused on
bioactive peptides involved in oxidative stress tolerance, not
encrypted in mature proteins but encoded by small open reading frames (sORFs) in the genome of Arabidopsis thaliana.
Oxidative stress is generated by the production of toxic
compounds called reactive oxygen species (ROS). These are
Abbreviations: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; MIC, minimal inhibitory concentration; ncRNA, non-coding RNA; OSIP, oxidative stressinduced peptide; PQ, paraquat; qRT-PCR, quantitative reverse transcriptase-PCR; ROS, reactive oxygen species; sORF, small open reading frame; TAR, transcriptionally active region.
© The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For
permissions, please email: [email protected]
5298 | De Coninck et al.
small, highly reactive molecules such as hydrogen peroxide (H2O2), superoxide (O2–) and hydroxyl (OH•) radicals,
containing one or more oxygen atoms. ROS production
is induced in plants following various biotic and abiotic
stress conditions, such as infection with phytopathogens
(Govrin and Levine, 2000; Schouten et al., 2002; Heller and
Tudzynski, 2011; O’Brien et al., 2012) and cold and heat stress
(Lukatkin, 2002; Volkov et al., 2006), respectively. An important feature of the plant’s response to stress comprises the
production of peptides with a signalling role, antimicrobial
activity or antioxidant functions (Sels et al., 2008; Fukuda
and Higashiyama, 2011; Katsir et al., 2011; Marshall et al.,
2011; Matsubayashi, 2011; Dubreuil-Maurizi and Poinssot,
2012), some of which have also been implicated in oxidative stress tolerance, such as the A. thaliana peptides for late
embryogenesis abundant 5 (AtLEA5; Mowla et al., 2006) and
mitochondrial encoded ATP synthase (AtMtATP6; Zhang
et al., 2008) and the unknown peptide encoded by At1g21520
(Luhua et al., 2008). AtLEA5 encodes a ‘late embryogenesis abundant’-like peptide that is expressed constitutively in
roots and reproductive organs. In leaves, AtLEA5 transcripts
are repressed when exposed to light but are abundant in the
dark or under conditions of oxidative stress. Moreover, overexpression of AtLEA5 in A. thaliana confers tolerance to
H2O2 (Mowla et al., 2006). AtMtATP6 is a component of
the mitochondrial F1F0-ATPase, an enzyme involved in oxidative respiration. Overexpression of the corresponding gene
in A. thaliana increases tolerance to paraquat (PQ), an oxidative stress-generating herbicide (Zhang et al., 2008). In recent
years, it has become clear that genes encoding peptides are
strongly underrepresented in the current genome annotation
of several organisms, including A. thaliana. The main reason
for this underrepresentation is the minimum length threshold of 330 nt that is imposed by gene annotation software to
avoid false-positive predictions (Haas et al., 2005).
As an extension of classical microarray analysis performed
on annotated gene-based arrays, tiling array technology
focuses on the entire non-repetitive part of a genome, including intergenic regions and introns, and has often been used
to identify previously undetected transcripts, including noncoding RNA (ncRNA) in A. thaliana (Yamada et al., 2003;
Stolc et al., 2005; Hanada et al., 2007; Matsui et al., 2008).
Using this technology, it was indeed shown that the annotation in A. thaliana is far from complete, as these studies
identified many unannotated transcripts in non-stress and
abiotic (drought, cold, and salt) stress conditions. Evidence is
increasing that sORFs, including those occurring in ncRNA,
can also encode bioactive peptides (Sousa et al., 2001; Narita
et al., 2004; Castellana et al., 2008; Podkowinski et al., 2009;
Hanada et al., 2013). During the past decade, different
approaches have been developed to isolate and identify novel
plant bioactive peptides, including a combination of various
chromatography techniques (Sagar et al., 2012) as well as the
development of bioinformatics tools predicting peptides with
specific bioactivities (Torrent et al., 2012). However, a weak
point in such high-throughput approaches often is the proof
of desired functionality of the predicted peptide sequences
in vivo.
In this study, we used a combination of high-throughput
identification with tiling array technology and functional
screening in yeast to identify sORFs upregulated in A. thaliana
during oxidative stress, and active in governing oxidative stress
tolerance. The tiling array analysis resulted in 176 transcriptionally active regions (TARs), which could be translated into 575
putative oxidative stress-induced peptides (OSIPs). To investigate the potential of these peptides in governing oxidative stress
tolerance, they were overexpressed in yeast and the resulting
transformants were screened for increased oxidative stress tolerance. It has been demonstrated that the lower eukaryote yeast is
a potent model system for studying processes in higher eukaryotic organisms (Botstein et al., 1997). Moreover, the previously
mentioned peptides AtLEA5 and AtMtATP6 were shown to
increase oxidative stress tolerance in yeast upon overexpression
(Mowla et al., 2006; Zhang et al., 2008). Overall, this study presents a strategy for the identification of novel bioactive peptides
and resulted in the discovery of OSIP108, a decapeptide that
increases oxidative stress tolerance in yeast and in planta when
overexpressed and upon exogenous application.
Materials and methods
Plant material, growth conditions, treatments, and yeast strains
Seeds of A. thaliana wild-type ecotype Columbia-0 (Col-0) were
obtained from the European A. thaliana Stock Centre (http://arabidopsis.info/). They were sown on ‘Zaai- en Stekgrond’ (DCM, SintKatelijne-Waver, Belgium) and grown in a growth chamber at 21 °C,
75% humidity and a 12 h day/night cycle with a light intensity of
approximately 120 µmol m–2 s–1.
Rosette leaves from soil-grown 4-week-old A. thaliana plants were
sprayed with PQ (25 µM methyl 98% viologen dichloride hydrate;
Sigma-Aldrich, St Louis, MO, USA) to obtain an equal and proportional distribution. Control plants were mock treated by spraying
with H2O. The treated plants were kept at 100% relative humidity
for 48 h, and all aboveground plant material was then harvested in
liquid nitrogen and stored at –80 °C until further analysis.
Spores from Botrytis cinerea B05-10 were harvested as described
previously (Broekaert et al., 1990). Spores (2 × 107 ml–1) were kept in
25% glycerol at –80 °C until further use. Plants were sprayed evenly
with a spore suspension of 5 × 105 spores ml–1 in ½ potato dextrose
broth (PDB). Control plants were mock treated by spraying with ½
PDB. After inoculation, plants were kept at 100% relative humidity.
The yeast strain used was Saccharomyces cerevisiae strain BY4741
(Euroscarf, Germany), which was cultivated in yeast peptone dextrose (1% yeast extract, 2% peptone, 2% glucose). Yeast strains
transformed with the pYES-DEST52 Gateway® vector (Invitrogen,
Carlsbad, USA) were cultured in SC–ura (Glu) [comprising 0.8 g
l–1 of CSM–ura (complete amino acid supplement mixture minus
uracil, Bio 101 Systems; 6.5 g l–1 of yeast nitrogen base; 20 g l–1 of
glucose). Expression of the OSIPs was induced in SC–ura (Gal).
Isolation of total RNA
RNA was isolated by the guanidinium isothiocyanate method
(TRIzol reagent; Invitrogen) followed by purification using an
RNeasy Mini kit (Qiagen, Valencia, USA). First, ~100 mg of plant
material was crushed in liquid nitrogen, 1 ml of TRIzol reagent was
added, and samples were mixed and incubated for 5 min at room
temperature. Following centrifugation (20 000g, 10 min, 4 °C) the
supernatant was transferred to a fresh tube and 200 µl of chloroform
was added. The thoroughly mixed samples were incubated for 2 min
at room temperature and the upper aqueous phase was transferred
to a fresh tube after centrifugation (20 000g, 15 min, 4 °C). An equal
Oxidative stress-protectant activity of a bioactive plant peptide | 5299
volume of 70% ethanol was added after which the samples were
thoroughly shaken and applied to a Qiagen column. The procedure
was continued according to the manufacturer’s instructions.
Target preparation and tiling array hybridization
Preparation and labelling of mRNA and hybridization on
GeneChip® Arabidopsis Tiling 1.0R Arrays (Affymetrix, Santa
Clara, USA) were performed by the VIB Nucleomics Core Facility
(http://www.nucleomics.be). In summary, RNA was converted to
double-stranded cDNA in a reverse transcription reaction with
a poly(dT) primer. Subsequently, the sample was converted and
amplified to antisense cRNA and labelled with biotin in an in vitro
transcription reaction according to the Affymetrix One Cycle Target
Labeling Kit. cRNA was subsequently random primed to generate ds cDNA according to the Affymetrix WT Double-Stranded
cDNA Synthesis Kit, and then fragmented and labelled with biotin in a terminal labelling reaction according to the Affymetrix WT
Double-Stranded DNA Terminal Labeling Kit. Two independent
biological replicates were used for each condition. Scanning and
image processing were performed by the Nucleomics Core Facility
using Affymetrix equipment and software.
Identification of short unannotated and PQ-induced genomic
regions
Background correction, log2 transformation, and inter-slide normalization were performed with the Affymetrix Tiling Analysis
Software version 1.1, Build 2. As the tiling arrays were based on
the A. thaliana genome sequence of the 2007 version of the TAIR
database, the corresponding TAIR7 genome annotation was used
for analysis of the tiling array data. All steps of our strategy are
illustrated in Supplementary Fig. S1 at JXB online. In a first step,
the average log2 intensities of biological replicates were calculated
and a threshold value of 8.8 was chosen to identify transcriptionally
active probes. This threshold was determined by comparing the log2
intensity distribution of genomic regions with known expression
status (expressed and not expressed). Probes with log2 intensities
below the threshold that were surrounded by transcriptionally active
probes were considered active and vice versa to remove the effect of
probes with poor hybridization properties. Secondly, groups of 4–13
successive transcriptionally active probes were combined into short
TARs. Selection of even smaller regions would lead to a high number of false positives and to the identification of very small genes
that are difficult to amplify in later stages, while all active regions of
14–15 successive probes were already annotated (TAIR7), supporting our choice of size limits. In a third step, TARs were withheld
if the number of transcriptionally active probes was smaller in the
control than in the PQ treatment and if the average log2 intensity of
all probes in the smaller of the two regions was at least 1.4 higher
in the PQ treatment than in the control. Both rules were based on
the tiling array results for genomic regions that are known to be
induced upon PQ treatment. The former rule was added because
of the frequent occurrence of probes having zero intensity in one of
the four hybridizations. In small regions, these zero values can have
a substantial influence on the average log2 intensity leading to falsepositive calls. The threshold for upregulation by PQ was refined by
comparing quantitative reverse transcriptase-PCR (qRT-PCR) and
tiling array results for 30 selected TARs. All upregulations of 1.4 or
more (corresponding to 2.6-fold or more) in the tiling arrays could
be confirmed by qRT-PCR supporting our choice of threshold (data
not shown). Finally, each selected region was compared with the
A. thaliana TAIR7 genome annotation and only regions that were
located in intergenic regions, introns, or pseudogenes were withheld.
Selection of ORFs
Using the TAIR7 genome sequence, the corresponding DNA
sequences of all selected regions were retrieved. On each side of the
active region an extra sequence of 50 bp was selected to minimize
the chance of missing the start or stop codon. Each sequence was
translated in the six possible reading frames and peptide sequences
that were located entirely in the outer 50 bp or were shorter than 10
aa were discarded. The latter decision was made to avoid too many
false positives.
Construction of a mini library in yeast
ORFs were amplified via PCR with specific primers for each
selected ORF (Primer3 software; Rozen and Skaletsky, 2000)
(Supplementary Table S1 at JXB online). For transformation using
Gateway® technology and efficient translation in yeast, the sequence
5′-CACCAAAA-3′ was added to each forward primer. The pool of
full coding sequences of a selection of 138 potential OSIP genes
was cloned into the pENTRTM plasmid (Invitrogen) using the
pENTRTM/D-TOPO® cloning system according to the manufacturer’s instructions and the presence of each ORF in the resulting
pool of Escherichia coli transformants was confirmed by PCR. The
inserts were transferred to the pYES-DEST52 Gateway® vector
behind a galactose-inducible promoter and used for transformation
to S. cerevisiae BY4741 using the Gietz protocol (Gietz et al., 1995).
The presence of each ORF in the pool of yeast transformants was
confirmed by PCR.
Oxidative stress assays in yeast
Oxidative stress tolerance of individual yeast transformants was
examined by determination of the minimal inhibitory concentration (MIC) for H2O2, using a two-fold dilution series (0–20 mM) in
SC–ura (Gal) inoculated with 1/200 of an overnight culture (2 × 108
cells ml–1) in SC–ura (Glu). The resistance of yeast transformants
against oxidative stress induced by H2O2 was additionally assayed
by spotting 15 µl of 240 mM H2O2 on SC–ura (Glu) and SC–ura
(Gal) agar, inoculated with 1/200 of an overnight yeast culture
as described previously (Aerts et al., 2006). After 48 h of incubation at 30 °C, the diameters of the halos on the SC–ura (Gal) and
SC–ura (Glu) plates were measured and the relative difference in
halo diameter for each transformant was determined as: relative
difference=(diametergal–diameterglu)/diameterglu.
Survival of wild-type yeast in PBS upon H2O2 treatment in the
presence of 200 µM OSIP in 1% dimethyl sulphoxide (DMSO) was
assessed as described previously (Aerts et al., 2006). Control treatment consisted of 1% DMSO. In short, an overnight culture of
S. cerevisiae BY4741 was washed in PBS and diluted to an optical
density at 600 nm of 0.05. The cells were incubated with 200 µM
of OSIP108 at 30 °C and afterwards treated with different concentrations of H2O2 (1.25, 2.5 and 5 mM). Survival was determined by
plating out the cells 3 h after H2O2 treatment.
Peptide synthesis
OSIP108 and the [C3A]OSIP108 mutant were synthesized using
standard fmoc chemistry on a microwave automatic synthesizer
(Liberty, CEM) as described previously (Skjærbæk et al., 1997).
The mass and purity of the synthetic peptides were confirmed by
mass spectrometry and reversed-phased high-performance liquid
chromatography (data not shown). The peptides had a purity of
≥95%.
In vitro H2O2-scavenging activity assay
Peroxidase activity was tested using an Amplex® Red Hydrogen
Peroxide/Peroxidase activity assay (Invitrogen). To this end, 100 µM
of OSIP108, glutathione and [C3A]OSIP108 were co-incubated
with 0.75 and 1.25 mM H2O2, 0.5 mU ml–1 of horseradish peroxidase, and 50 µM Amplex red reagent. After 30 min of incubation
at room temperature, the fluorescence was measured (560–590 nm)
(Synergy™MX, Biotek).
5300 | De Coninck et al.
qRT-PCR analysis
RNA was isolated from leaves using Plant RNA Isolation
Reagent (Invitrogen), treated with DNAse (Westburg, Leusden,
The Netherlands) and single-strand cDNA was synthesized using
SuperScript III reverse transcriptase (Invitrogen). Primers were
designed using Primer Express 3.0 (Supplementary Table S1). The
qRT-PCR analysis was carried out using the StepOnePlus System
and Power SYBR Green PCR Master Mix (Applied Biosystems,
Carlsbad, USA). The PCR parameters were: initial denaturation
(10 min at 95 °C), 40 cycles of amplification (10 s at 95 °C, 10 s at
58 °C, and 10 s at 72 °C), and a melting curve stage (15 s at 95 °C,
1 min at 60 °C increased to 95 °C with steps of 0.3 °C). The last stage
was used to verify the specificity of each PCR. Transcript levels were
normalized to the respective transcript level of the reference gene
elongation-factor 1α (EF1α; At5g60390). Relative log2 ratios of
treated samples compared with the mock treatment were calculated
based on the ΔΔCt method (Livak and Schmittgen, 2001). All reactions were performed in triplicate and carried out in Fast Optical
96-Well Reaction Plates (MicroAmp).
Peptide infiltration of A. thaliana leaves and subsequent PQ
treatment
One leaf of soil-grown 4-week-old Col-0 plants was syringe infiltrated with 100 µM of OSIP108 or [C3A]OSIP108 dissolved in 1%
DMSO. Control plants were mock treated with 1% DMSO. The
plants were incubated for 24 h in the growth chamber preceding PQ
treatment. Leaves were inoculated with a 5 µl drop of PQ (100 µM).
The lesion diameters caused by the PQ treatment were measured 2
d after the treatment. The experiment was repeated twice with 13
plants per experiment.
Construction of A. thaliana 35S::OSIP108-overexpressing plants
The OSIP108-coding sequence was cloned in the pENTR-DTOPO® vector using primers designed with Primer3 software
(Supplementary Table S1) and a pENTR-D-TOPO® cloning kit
(Invitrogen), according to the manufacturer’s instructions. The
insert was first transferred to the pB2GW7 plasmid and then to
Agrobacterium tumefaciens PMP90. A. thaliana Col-0 plants were
transformed via Agrobacterium tumefaciens floral dip (Clough and
Bent, 1998). Transgenic seedlings (T1) were selected on a sand/perlite mixture (1/3 sand, 2/3 perlite) irrigated with H2O containing the
BASTA herbicide (Bayer NV, Belgium) at a final concentration of
25 µM for the active constituent phosphinotricin. A T3 homozygous line (35S::OSIP108) with a single T-DNA insert was selected
via segregation analysis, and transgene expression was analysed by
qRT-PCR. 35S::OSIP108 and wild-type plants were sprayed evenly
with 40 µM PQ. At 7 d post-treatment, pictures were taken with a
digital camera (Canon Eos 450D). ASSESS 2.0 (Lamari, 2002) was
used to quantify the chlorotic leaf area. Six sets of four plants per
line were selected with the manual selection tool and analysed in the
‘Classical panel’ to perform measurements. The number of pixels of
the total leaf area (chlorotic+green) was measured by selecting suitable wavelengths with settings ‘RGB’ and ‘G’. The number of pixels
of the chlorotic leaf area was measured with settings ‘RGB’and ‘B’.
The percentage of chlorotic leaf area was calculated as: pixels chlorotic leaf area/pixels total leaf area. The experiment was repeated
twice.
Statistical analysis
Experiments were statistically analysed using Statistica® (Release 7;
Statsoft, Tulsa, USA). The data of exogenous application of OSIP108
and its effect on PQ tolerance in planta were analysed by analysis of
variance (ANOVA). The Tukey HSD test was applied for multiple
comparisons of group means. The data of the oxidative stress-protectant effect of the yeasts overexpressing the different OSIPs were
analysed using non-parametric Kruskal–Wallis ANOVA by ranks,
because of the smaller sample size. When the Kruskal–Wallis statistic was significant, comparisons of treatments versus control were
calculated as described by Siegel and Castellan (1988). The effect of
exogenous application of OSIP108 to yeast, the expression analysis
of OSIP108, and the PQ treatment of OSIP-overexpressing plants
were analysed using Student’s t-test.
Results
Identification of OSIP genes in A. thaliana using
tiling arrays
Tiling array analysis (Supplementary Fig. S1) was performed
on mRNA extracted from leaves treated with PQ and H2O
(control), resulting in the identification of 92 844 and 86 272
TARs, respectively (GEO GSE49001). A total of 176 short
unannotated TARs were induced by PQ treatment compared
with the control treatment (Supplementary Table S2 at JXB
online and Fig. S1). The bulk of these TARs were intergenic,
although 70 and 18 OSIP-encoding regions were located in
pseudogenes and introns of annotated genes, respectively.
The DNA sequences of the selected TARs were translated in
all reading frames, and ORFs of at least 30 bp were retained,
resulting in a list of 575 potential OSIPs (Supplementary
Fig. S1 and Table S3 at JXB online), ordered based on the
induction level of the corresponding genes in the tiling array
(Fig. 1A).
Several potential OSIPs increase the oxidative stress
tolerance of yeast when overexpressed
To screen for OSIPs that effectively improve oxidative stress
tolerance, we selected 138 OSIP-encoding ORFs that were
at least 8-fold (log2 ratio=3) induced by PQ (Fig. 1A). These
ORFs were cloned behind a galactose-inducible promoter
and overexpressed in yeast. As PQ induces oxidative stress
in yeast during respiration, which occurs upon cultivation
in glycerol but not in galactose, we screened for yeast transformants with increased tolerance against H2O2 in galactose-containing medium. As positive controls, we included
AtLEA5 and AtMtATP6. The MIC for H2O2 of 500 individual yeast transformants was determined and we identified
four yeast transformants characterized by a 2-fold increase
in H2O2 tolerance (MIC=5 mM H2O2), compared with the
other 496 yeast transformants (MIC=2.5 mM H2O2) (data
not shown). A similar increase in MIC value was observed for
yeast transformants overexpressing AtLEA5 or AtMtATP6.
Upon sequencing of these four H2O2-tolerant yeast transformants, we identified two containing OSIP108, one containing
OSIP152, and one containing OSIP163 (Fig. 1A). The log2
ratios of OSIP108, OSIP163, and OSIP152 upon PQ treatment in the tiling array were 5.42, 5.07, and 4.74, respectively
(Fig. 1B). To validate these expression data in A. thaliana, the
expression levels of the three selected OSIPs were assessed
using qRT-PCR analysis under the same conditions and at
the same time point as used in the tiling array analysis. The
log2 ratios of OSIP108, OSIP163, and OSIP152 were 5.32,
5.68, and 5.01, respectively (Fig. 1B), which are in line with
Oxidative stress-protectant activity of a bioactive plant peptide | 5301
Fig. 1. Expression levels of 575 potential OSIPs of A. thaliana. (A) The OSIPs are ranked in the graph based on their log2 ratio,
derived from the tiling array analysis. In total, 138 ORFs with a log2 ratio >3 (represented by the baseline on the graph) were used for
overexpression in yeast. (B) Expression levels of OSIP108, OSIP152, and OSIP163 were further quantified using qRT-PCR. Log2 ratios
determined via tiling array analysis and qRT-PCR are indicated. Values are means ±SD of three biological replicates.
the normalized log2 ratios from the tiling arrays, thereby confirming the observed induction after treatment with PQ.
Next, we assessed the tolerance to H2O2 of the three
selected yeast transformants and the two positive control
transformants in a halo test by spotting 240 mM H2O2 on to
agar plates inoculated with the specific yeast transformants.
The diameters of the halos on galactose-containing medium
and glucose-containing medium were measured and the relative difference was determined to quantify the effect of the
OSIP on oxidative stress tolerance (Fig. 2). The relative difference for OSIP108-overexpressing yeast (0.078 ± 0.019) was
significantly lower than yeast transformants overexpressing
negative controls (0.350 ± 0.029), which consisted of four
yeast OSIP transformants that previously showed no altered
H2O2 MIC value. These data indicated that OSIP108 can protect yeast cells against the lethal effect of the oxidative stress
agent H2O2. The yeast transformants expressing OSIP152
(0.405 ± 0.041), OSIP163 (0.267 ± 0.067), AtMtATP6
(0.273 ± 0.057), or AtLEA5 (0.400 ± 0.064) did not show significantly smaller halos compared with the negative controls.
These data indicated a significant 4.5-fold reduction in halo
diameter for OSIP108-overexpressing yeast transformants
compared with control transformants, and hence increased
oxidative stress tolerance upon overexpression of OSIP108
in yeast.
Exogenous application of OSIP108 to yeast increases
survival upon H2O2 treatment
Based on the data obtained above, further experiments on
oxidative stress tolerance of yeast and plants focused on
the effect of OSIP108, a decapeptide with the amino acid
sequence MLCVLQGLRE. In the first instance, the tolerance to H2O2-induced oxidative stress was analysed in yeast
co-incubated with the chemically synthesized OSIP108 peptide. Survival of yeast cells was determined upon treatment
with 1.25, 2.5, and 5 mM H2O2, and simultaneous application
of 200 µM OSIP108. OSIP108 significantly increased (15%)
the survival of H2O2-treated yeast compared with the mock
treatment (1% DMSO) (Fig. 3).
OSIP108 has in vitro H2O2 scavenging activity
As it is known that thiol groups, e.g. from the cysteine present in OSIP108, are effective ROS scavengers, we examined
the H2O2 scavenging activity of native OSIP108 and its analogue [C3A]OSIP108, in which cysteine on position three
was replaced by alanine, with an Amplex® Red Hydrogen
Peroxide/Peroxidase Assay kit. This test is based on the formation of a fluorescent product, resorufin, upon reaction
of the Amplex Red reagent with H2O2 in the presence of a
5302 | De Coninck et al.
Fig. 2. Overexpression of OSIP108 results in increased
oxidative stress tolerance in a yeast halo test. The specific yeast
transformants were grown overnight at 30 °C and inoculated
on agar plates. The negative controls consisted of four yeast
transformants that previously showed no altered H2O2 MIC value.
In addition yeast transformants expressing AtMtATP6 or AtLEA5,
peptides shown to increase oxidative stress tolerance of yeast, were
included. A drop of 15 µl of 240 mM H2O2 was applied to the centre
of the agar plates. After 48 h of incubation, the diameters of the
halos on the SC–ura (Gal) and SC–ura (Glu) plates were measured
and the relative differences in halo diameter for each transformant
were compared. Each bar represents the mean of triplicate samples
and the figure is a representative of two independent experiments.
Error bars represent SD and significant differences are indicates by
an asterisk (P<0.05 Kruskal–Wallis test).
Fig. 3. Exogenous application of OSIP108 increases survival
of yeast upon H2O2 treatment. Survival of the yeast cells was
determined by plating the cultures 3 h after treatment with 1.2,
2.5, or 5 mM H2O2 upon co-incubation with 200 µM OSIP108
or mock-treated yeast (1% DMSO). Each bar represents the
mean of three samples and this figure is a representative of three
independent experiments. Error bars represent SD and significant
differences are indicates by an asterisk (P<0.05, Student’s t-test).
peroxidase. We tested whether resorufin was less prone to
peroxidation by horseradish peroxidase in the presence of
OSIP108, due to H2O2 scavenging. A significant decrease in
fluorescence was observed upon addition of either 100 µM
OSIP108 or glutathione, an antioxidant tripeptide, compared
Fig. 4. OSIP108 and glutathione effectively scavenge H2O2 in an
in vitro assay. OSIP108 (100 µM), glutathione, and [C3A]OSIP108
were co-incubated with 0.75 or 1.25 mM H2O2, 0.5 mU ml–1 of
horseradish peroxidase, and 50 µM Amplex Red reagent. H2O2
scavenging resulted in decreased formation of the fluorescent
resorufin.
with the mock treatment (1% DMSO) (Fig. 4). These results
showed that the H2O2 scavenging activity of OSIP108 was
similar to that of glutathione. Addition of [C3A]OSIP108 did
not result in significantly decreased fluorescence.
OSIP108 is induced by PQ and B. cinerea in
A. thaliana leaves
The coding sequence of OSIP108 is contained within the
pseudogene At3g61185, which has been suggested to encode a
potential defensin-like peptide within its C-terminal sequence
(Silverstein et al., 2007). The sequence of OSIP108 is, however, not in frame with the defensin-like peptide (Fig. 5).
The upstream region of the At3g61185 gene in A. thaliana
comprises the genes At3g61180 and At3g61182 encoding a
RING/U-box superfamily protein and a low-molecularweight cysteine-rich protein (LCR54), respectively (Fig. 5). In
order to gain more insight into the role of OSIP108 in planta,
a more detailed expression analysis by qRT-PCR of OSIP108
in A. thaliana was performed at different time points after
PQ treatment (Fig. 6). Significant induction of OSIP108 was
detected at 48 h (21-fold), 72 h (7.2-fold), and 96 h (18-fold)
after treatment. As oxidative stress is often linked with biotic
stress, expression of OSIP108 was additionally analysed in
A. thaliana leaves at different time points after inoculation
with the necrotrophic pathogen B. cinerea, reported to induce
ROS in plant tissues upon infection (Govrin and Levine,
2000; Heller and Tudzynski, 2011) (Fig. 6). Again, a comparable significant induction of OSIP108 was detected at 24 h
(11-fold), 48 h (26-fold), and 72 h (20-fold) after B. cinerea
inoculation.
Exogenous application of OSIP108 to A. thaliana
leaves increases PQ tolerance
To determine whether OSIP108 also boosts oxidative
stress tolerance in plants, leaves of A. thaliana were syringe
Oxidative stress-protectant activity of a bioactive plant peptide | 5303
Fig. 5. Schematic overview of the genomic location of OSIP108. The TAR is displayed as a grey bar, the coding sequence of OSIP108
as a black arrow and the putative defensin-like peptide (DEFL) as a dotted arrow. OSIP108 and DEFL are not within the same reading
frame. The arrow indicates the translation direction. Numbering begins at the start codon from the pseudogene At3g61185. Upstream
genes are indicated (not to scale).
the percentage of chlorotic leaf area was three times less in
35S::OSIP108 plants (14.99 ± 4.39%) (Fig. 8). As such, it
could be shown that overexpression of OSIP108 in A. thaliana resulted in a significantly higher tolerance to PQ (P<0.01).
These data confirmed the ability of OSIP108 to increase oxidative stress tolerance not only in yeast but also in plants.
Discussion
Fig. 6. OSIP108 is induced in leaves of A. thaliana upon PQ
treatment and B. cinerea inoculation. Expression analysis via qRTPCR was performed at 24, 48, 72, and 96 h after PQ treatment
(leaves sprayed evenly with 25 µM PQ) and B. cinerea inoculation
(leaves sprayed evenly with a spore suspension of 5 × 105 spores
ml–1 in ½ PDB). Plants were mock treated with water and ½
PDB, respectively. Each bar represents the mean fold change
(treatment/control) (±SEM) of at least three independent replicates.
Significantly induced expression is indicated by an asterisk
(P<0.05, one-sample Student’s t-test).
infiltrated with OSIP108. Subsequently, the plants were
treated with PQ and the diameters of the resulting lesions
were measured (Fig. 7). Two d after PQ application, the
average lesion diameter of plants pre-treated with OSIP108
(2.92 ± 1.93 mm, P<0.01) was significantly lower than the
average lesion diameter of the control plants (5.87 ± 2.79 mm).
Interestingly, the cysteine at position 3 seemed to be crucial
in this oxidative stress-protectant activity, as no difference
in lesion diameter was observed between control and [C3A]
OSIP108 (6.00 ± 2.22 mm) treatment.
Overexpression of OSIP108 increases PQ tolerance in
A. thaliana
In order to prove the bioactivity of OSIP108 in planta, this
peptide was constitutively overexpressed in A. thaliana. The
homozygous T3 overexpression line 35S::OSIP108 (175
364.8 ± 1.65 fold change) and wild-type plants were sprayed
with 40 µM PQ. At 7 d post-application, ASSESS software
was used to determine the percentage of total chlorotic
leaf area. Compared with wild-type plants (47.43 ± 9.08%),
Peptides have been shown to play crucial roles in plant
defence responses and development. Whereas some peptides
have been reported to have a signalling role in the plant and
are often referred to as peptide hormones, others exhibit
direct antimicrobial activity (Sels et al., 2008; Fukuda and
Higashiyama, 2011; Katsir et al., 2011; Marshall et al., 2011;
Matsubayashi, 2011). The latter group generally consists of
pathogenesis-related peptides such as the cysteine-rich plant
defensins, thionins, knottins, hevein-like peptides, snakins, lipid transfer proteins, and cyclotides (Sels et al., 2008;
Hammami et al., 2009; Marshall et al., 2011). Like most
signalling peptides, they contain a signal sequence, resulting
in secretion of the peptide. A first group of secreted peptide
hormones are initially synthesized as pre-propeptides and
then undergo major post-translational modifications and
proteolytic processing, resulting in the formation of a small
peptide (Matsubayashi, 2011). This type of peptide signal
has been implicated in both plant defence and development,
including for instance the 18 aa tomato systemin, which is
processed from a 200 aa precursor (Constabel et al., 1995;
Farrokhi et al., 2008) and CLAVATA3, which is translated as
a 96 aa peptide and is post-translationally processed to a 13
aa peptide (Katsir et al., 2011). A second group of secreted
peptide hormones are cysteine-rich peptides (Marshall et al.,
2011; Matsubayashi, 2011) such as RALF and SCR/SP11.
Some peptide hormones have been reported to be encoded
by sORFs and have no signal sequence. Two examples of
this class are ENOD40 and POLARIS. The ENOD40 gene
is found in several plant species, in which it has a role in new
organ initiation. ENOD40 contains two sORFs, one encoding
ENOD40A with a similar size as OSIP108 and also containing a cysteine residue, and the second encoding a 27 aa peptide (Sousa et al., 2001). The POLARIS gene encodes three
putative peptides of 8, 9, and 36 aa, respectively. It was demonstrated that the 36 aa peptide is implicated in root growth
5304 | De Coninck et al.
Fig. 7. Exogenous application of OSIP108 to A. thaliana leaves
increases their tolerance to PQ. OSIP108 (100 µM) was used
for syringe infiltration of leaves from A. thaliana, followed by
application of a 5 µl drop of PQ (100 µM). The lesion diameters
of plants pre-treated with OSIP108 or [C3A]OSIP108 and mocktreated plants (treated with 1% DMSO only) were measured 2 d
post-application. Each bar represents the mean (±SEM) of at least
13 plants, and this figure is a representative of two independent
experiments. Different letters indicate significant differences
(P<0.01) according to an ANOVA Tukey HSD test.
Fig. 8. 35S::OSIP108 overexpression plants are more tolerant
to PQ treatment than wild-type plants. A 35S::OSIP108
overexpression line and wild-type plants were evenly sprayed
with 40 µM PQ. At 7 d post-application, ASSESS software was
used to determine the percentage of chlorotic leaf area. Each
bar represents the mean (±SEM) of six sets of four plants and
this figure is a representative of two independent experiments.
Significant differences are indicated by an asterisk (P<0.01,
Student’s t-test).
and this peptide is proposed to play a role in auxin, ethylene,
and cytokinin crosstalk (Casson et al., 2002; Liu et al., 2010),
although it cannot be excluded yet that the shorter peptide
forms are also active. However, the discovery of new sORFs
encoding peptides and the elucidation of their bioactivity is
challenging. To this end, we adopted a novel approach combining tiling arrays with a functional characterization assay
in yeast.
Using this genome-wide approach based on tiling array
analysis, we identified 176 previously unannotated TARs in
the A. thaliana genome that were upregulated upon PQ treatment. Translation of these TARs gave rise to 575 sORFs
that potentially encoded OSIPs, with variable length (from
10 to >100 aa) and without sequence homology. In recent
years, tiling arrays on A. thaliana plants subjected to different
treatments have revealed the presence of many unannotated
transcripts, most of them occurring in intergenic regions and
pseudogenes (Thibaud-Nissen et al., 2006; Hanada et al.,
2007; Matsui et al., 2008). Our study indicated that OSIP108
is also located in a pseudogene (At3g61185). However, the
functional significance of most of these transcripts identified
in tiling arrays, often regarded as ncRNA, is largely unknown
(Guttman and Rin, 2012; Jin et al., 2013). It cannot be
ruled out that ncRNAs contain sORFs that encode peptides
(Kageyama et al., 2011), but determination as to whether a
transcript is non-coding is challenging. Here, a high-throughput functional assay was used to identify OSIPs that significantly increased oxidative stress tolerance in the eukaryotic
model yeast S. cerevisiae upon overexpression. The use of
yeast to characterize plant proteins has been proven previously, for example for AtLEA5, AtMtATP6, and the defensin
AhPDF1.1 (Mirouze et al., 2006; Mowla et al., 2006; Zhang
et al., 2008). Via this approach, OSIP108 was identified, and
this bioactive peptide was proven to increase oxidative stress
tolerance in both S. cerevisiae and A. thaliana, when applied
exogenously or overexpressed.
Like glutathione, OSIP108 is also able to scavenge ROS,
as OSIP108 inhibited the formation of resorufin to the same
extent as glutathione. The activity of glutathione results
from the presence of the highly reactive nucleophilic thiol
group in the cysteine residue (Deneke, 2000). Similarly, we
demonstrated that the oxidative stress-protectant activity
of OSIP108 upon infiltration in planta was completely abolished by replacement of the cysteine by alanine. However,
having a cysteine in the peptide sequence was not enough
to induce oxidative stress tolerance, as several of the OSIPs
(Supplementary Table S3) contained a cysteine but did not
exhibit this protectant activity.
We further observed that OSIP108 is induced not only by
PQ but also by the necrotrophic fungal pathogen B. cinerea
in A. thaliana leaves, which is not surprising as ROS are also
reportedly implicated in the defence response against pathogens. Additionally, we compared our results with those of
other tiling array studies in A. thaliana using the AtTAX
browser (Zeller et al., 2009), and the gene encoding OSIP108
seemed to be transcriptionally active under various conditions, including abiotic stress by cold or osmotic stress
treatment.
More research is necessary to characterize the role of
OSIP108 in planta. Unfortunately, so far we have been unable
to detect the OSIP108 peptide in PQ-treated A. thaliana using
both liquid chromatography/mass spectrometry and western
blotting (data not shown). This is, however, also the case for
the previously mentioned peptides POLARIS and ENOD40
for which in planta existence could only be proven in an indirect way. ENOD40 was assumed to be translated based on
translational fusions and in vitro translation (Sousa et al.,
2001; Rohrig et al., 2002; Podkowinski et al., 2009). However,
Oxidative stress-protectant activity of a bioactive plant peptide | 5305
Medicago truncatula ENOD40 could not be detected in roots
using antibodies (Sousa et al., 2001) and soybean ENOD40Aspecific antibodies were not able to detect the peptide in
soybean nodules. Nevertheless, these antibodies recognized
several compounds with high molecular masses, which might
suggest that the peptide forms large aggregates (Rohrig et al.,
2002). The assumption of translation of the POLARIS gene
is based on the observation that the pls mutant phenotype
could be partially rescued by the ORF encoding the 36 aa
peptide (Casson et al., 2002). Very recently, Hanada et al.
(2013) showed that 10% of 473 selected sORFs, identified
in intergenic regions of A. thaliana, resulted in phenotypic
altered morphogenic-related effects when overexpressed in
A. thaliana. Moreover, recent findings in Drosophila (Kondo
et al., 2010), human cells (Slavoff et al., 2013), and plants
(Castellana et al., 2008; Yang et al., 2011) suggest that sORFs
can be translated into peptides. For example, evidence of
translation (Castellana et al., 2008) was found for one of
the sORFs from Hanada et al. (2013), whose overexpression
resulted in an altered phenotype.
In conclusion, the identification and characterization
of OSIP108 confirms the validity of our high-throughput
approach, based on tiling arrays and functional screening in
yeast, to identify bioactive peptides in A. thaliana. Our results
on the protective activity of OSIP108 in A. thaliana against
the ROS-inducing agent PQ indicates its potential to apply
OSIP108 as a molecular trait to generate crops with higher
resistance to oxidative stress conditions (Apel and Hirt,
2004). The protective role of OSIP108 might also be interesting for potential applications outside plant research, as oxidative stress has been implicated in the progression of several
human diseases, including Alzheimer’s disease, Parkinson’s
disease, and amyotrophic lateral sclerosis (Barnham et al.,
2004). The damage caused by ROS to lipids, proteins, and
nucleic acids plays an important role in the pathogenic events
of these neurodegenerative diseases (Jellinger, 2009). Several
peptides have been shown to increase tolerance against oxidative stress in this respect. For example, overexpression of
the peptide DSEP/dermcidin, initially purified from the secretions of oxidatively stressed neural cell lines, increased the tolerance of human neural cells to oxidative stress (Cunningham
et al., 2002). In this context, preliminary data from our group
have shown recently that the observed oxidative stress-protectant activity of OSIP108 in yeast and plants can be further extrapolated to mammalian cellular models for oxidative
stress-related diseases (P. Spincemaille, unpublished results),
indicating the potential of this newly discovered plant peptide
in medical treatments.
Acknowledgements
DC and PT acknowledge the receipt of PhD grants from
‘IWT-Vlaanderen’ (SB-71024 and SB-111657). We also
acknowledge the receipt of a postdoctoral fellowship from
‘FWO-Vlaanderen’ to BDC (FWO/12A7213N), from the
Industrial Research Fund (KU Leuven) to KT and from the
KU Leuven (PDM/12/126) to CV. This work was supported
by ‘FWO-Vlaanderen’ (G.A062.10N and G.0414.09) and
‘Bijzonder Onderzoeksfonds KU Leuven’ (GOA/2008/11).
DJC thanks NHMRC (Australia) for a Professorial
Fellowship (APP1026501). We thank Chia Chia Tan for the
peptide synthesis.
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