ORIGINAL PAPER
Stress-related genes define essential steps in the response
of maize seedlings to smoke-water
Vilmos Soós & Endre Sebestyén & Angéla Juhász &
János Pintér & Marnie E. Light & Johannes Van Staden &
Ervin Balázs
Received: 12 September 2008 /Revised: 3 December 2008 /Accepted: 21 December 2008 / Published online: 13 January 2009
# Springer-Verlag 2009
Abstract Smoke from burning vegetation is widely recog-
nised as a germination cue for seed germination and recent
reports suggest that smoke treatments can improve seedling
vigour also. We investigated the effect of smoke-water on
seedling vigour and changes of the global transcriptome in
the early post-germination phase in maize. Application of
smoke-water improved the germination characteristics and
seedling vigour. The transcriptional response of embryos
and emerging radicles 24 and 48 h after the onset of
smoke treatment was investigated. The microarray study
revealed a number of smoke-responsive genes amongst
which stress- and abscisic acid (ABA)-related genes were
over-represented. The global promoter analysis of the
smoke-responsive genes revealed a tight correlation with
the results obtained from Gene Ontology annotations.
This concerted over-expression shows that smoke treat-
ment induces stress and ABA-related responses in the
early post-germination phase which leads to better
adaptation to environmental stress factors occurring
during germination, eventually resulting in greater seed-
ling vigour.
Keywords Gene expression . Germination .Maize .
Smoke . Zea mays
Introduction
Smoke from burning vegetation is known to be an
important environmental cue (Van Staden et al. 2000) and
numerous species, mainly from South African fynbos
(Brown 1993; Brown et al. 2003), Western Australian
kwongan (Roche et al. 1997; Bell 1999) and Californian
chaparral (Keeley and Fotheringham 1998) have shown an
improved germination response to treatments using aerosol
smoke or smoke-water. Many species from these fire-prone
environments germinate in response to smoke treatments
and several weed species, many from non-fire prone
regions, respond to various smoke treatments (Adkins and
Peters 2001).
Bioactivity-guided fractionation of smoke-water, using
achenes of Lactuca sativa cv. Grand Rapids as a test
system, has led to the isolation of a butenolide compound,
3-methyl-2H-furo[2,3-c]pyran-2-one, from plant-derived
smoke (Van Staden et al. 2004). Likewise, Flematti et al.
(2004) were able to isolate the identical compound from
smoke obtained from burning cellulose. This butenolide-
type compound is active in promoting germination at
Funct Integr Genomics (2009) 9:231–242
DOI 10.1007/s10142-008-0105-8
Electronic supplementary material The online version of this article
(doi:10.1007/s10142-008-0105-8) contains supplementary material,
which is available to authorized users.
V. Soós : E. Sebestyén : A. Juhász : E. Balázs (*)
Department of Applied Genomics,
Agricultural Research Institute of the Hungarian Academy
of Sciences (ARI-HAS),
Brunszvik u. 2,
2462 Martonvásár, Hungary
e-mail: balazs@mail.mgki.hu
J. Pintér
Department of Maize Breeding,
Agricultural Research Institute of the Hungarian Academy
of Sciences,
Brunszvik u. 2,
2462 Martonvásár, Hungary
M. E. Light : J. Van Staden
Research Centre for Plant Growth and Development,
School of Biological and Conservation Sciences,
University of KwaZulu-Natal Pietermaritzburg,
Private Bag X01,
Scottsville 3209, South Africa

concentrations as low as 10−9 M (Flematti et al. 2004; Van
Staden et al. 2004). Thus, the action of smoke in promoting
the germination of seeds of many species is mainly
attributed to the presence of this compound in smoke.
However, it was shown that dual regulatory cues do exist in
the smoke which can have either promoting or inhibitory
effect on germination (Light et al. 2002) suggesting the
presence of compounds with differing activities in smoke.
To date, there is relatively little documented work on the
post-germination effects of smoke (Sparg et al. 2005).
Baxter and Van Staden (1994) reported that the seedlings of
the fire-climax grass Themeda triandra Forssk. from
smoke-treated seeds grew more vigorously without any
morphological abnormalities. A similar effect was observed
for Erica species and species of Asteraceae (Brown 1993).
More recently, Sparg et al. (2005) stated that although
smoke treatment may not necessarily have an effect at
the germination stage, it may play a role at the post-
germination stage and suggested that in previous studies
where many species have not responded to smoke treat-
ments, these species may show some response at their
post-germination stages, i.e. improved seedling vigour.
Therefore, it may be necessary to extend germination
studies to include an assessment of seedling vigour when
smoke is used as a germination treatment. All these
previous studies have been conducted mainly on wild
species, with no evidence to suggest whether this post-
germination effect of smoke can be observed in commercial
crop plants.
Maize (Zea mays L.) is one of the most widely cultivated
crops and is a large component of human and animal diets
in many countries. It is considered to be a crop with the
most biotechnological potential for industrial applications
(McLaren 2005). Different kinds of stresses are the major
constraints for maize production worldwide, since this crop
is largely grown in areas in which unfavourable conditions
(drought, heat and salt stress) are predominant. In these
areas, seedling vigour is an important agronomic trait for
the establishment of seedlings which can help young plants
to overcome such adverse effects that usually result in lower
yields. To date, there are only a few reports discussing the
effect of smoke on the post-germination stage, although the
use of fire and smoke in maize agrotechnology is not a new
practice. For example, in South Africa, some rural farmers
store maize seed lots over a fireplace subjecting the seeds
to smoke and heat (Modi 2002). This storage method
improved the germination rate, final germination in
comparison with untreated seeds and produced significantly
more vigorous seedlings, which were heavier and taller, in
comparison with untreated seeds. Thus, smoke treatments
have the potential to improve not only the percentage
germination but also the seedling vigour of commercially
bred maize seeds (Sparg et al. 2006).
High seedling vigour and survival rate often can be
attributable to the induction of abscisic acid (ABA)-signal
transduction (summarised by Chandler and Robertson
1994), stress-related events in the cell and subsequent
increased stress tolerance (Khajeh-Hosseini et al. 2003;
Soeda et al. 2005). During the germination process, the re-
induction of the seed maturation program (Rajjou et al.
2006) and osmopriming of germinating seeds also results in
the induction of stress-responsive genes (Soeda et al. 2005)
which was found to correlate to seed stress tolerance and
higher vigour. Smoke-water can potentially be used as
germination priming agent to ensure synchronous germina-
tion and optimal seedling establishment especially under
adverse conditions (Sparg et al. 2006). It was hypothesised
that stress-related genes may play a major role in smoke
action and the improvement in seedling vigour elicited by
smoke implies huge shifts in the gene expression pattern.
To achieve a better understanding of the effect of smoke
on germination characteristics, seedling vigour and the
molecular background of smoke action, we analysed gene
expression in the early post-germination phase in an inbred
maize line.
Materials and methods
Plant material
The HMV5405 inbred maize line has been developed in
ARI-HAS (Martonvásár, Hungary). The line originates
from the Iodent family and possesses good general and
specific combining ability; therefore, the HMV5405 is
widely used as parent in maize hybrid production.
However, it has a low germination percentage and low
seedling vigour, which makes the production of basic seed
difficult. Contrary to the efforts that have been made to
overcome these disadvantages, the only solution to improve
seedling vigour is the application of growth stimulants and
germination cues.
Growth conditions, germination and vigour tests
For the germination time course tests, kernels (seeds) of Z.
mays L. MV5405 (130 pieces) in four replicates were
decontaminated in 3% sodium hypochlorite containing
Tween 20 and 70% EtOH (10 min each) and then germi-
nated in an illuminated environmental chamber (25°C,
100 μmol m−2 s−1 light intensity) on tissue paper placed in
Petri dishes. The batches of kernels were treated with 20 ml
water (control), 1:500 or 1:1,000 (v/v) dilution of smoke-
water, 100 μM ABA (Sigma) and 1:500 smoke-water
containing 100 μM ABA. Germinated kernels were scored
every 12 h for 9 days. The standard vigour test developed
232 Funct Integr Genomics (2009) 9:231–242

for determining the viability of the seedlings of commercial
seedlots (‘paper roll test’) was applied in the vigour
experiment. Eight replicates of 25 kernels each were
imbibed in distilled water or smoke-water (1:500 or
1:1,000 dilutions) for 48 h. Thereafter, the kernels were
transferred to moistened filter paper and placed at 5°C for
2 days. The chilling was followed by a 7-day period during
which the kernels were kept at 25°C with a light intensity
of 100 μmol m−2 s−1. This experiment was repeated with
the same temperature and light regimes excluding the
chilling period. Germination percentage, root and shoot
length were measured after 9 days and seedling vigour was
determined using the equation:
V ¼ Σ Ls þ Lrð Þ
GP;
where Ls and Lr are shoot and root length in millimeters,
respectively, and GP is germination percentage (Dhindwal
et al. 1991). Seedling dry mass and kernel water uptake
were determined gravimetrically after drying the samples in
an oven set to 100°C until they reached constant weight.
The smoke-water was prepared from burnt Themeda
triandra Forssk. (Poaceae), according to the method out-
lined in Baxter et al. (1994).
RNA isolation
For RNA isolation from control and smoke-treated (1:500)
kernels, identical conditions were applied as for the
germination time course, tests and samples were harvested
24 and 48 h after treatment. Embryos in the same
developmental stage and size were chosen. The embryos
of kernels with ruptured testa at 24 h and embryos of
kernels with emerged radicle (3–4 mm) at 48 h were
harvested from both control and treated samples. Total
RNA was isolated from the maize kernel embryos using
TRIzol reagent (Invitrogen) and cleaned up with Qiagen
RNeasy Plant Mini Kit (Qiagen) applying a few modifica-
tions. RNA was then treated with RNase-free DNase I
(Promega) according to the manufacturer’s instructions.
The concentration of RNA was determined with a Nano-
drop ND-1000 spectrophotometer (NanoDrop). The RNA
integrity number (RIN) of the samples was determined
using the Agilent BioAnalyzer. Only samples with a RIN≥
8 were considered for further analysis.
Microarray platform, labelling, hybridisation
and image acquisition
In the microarray study, ten individual germinating kernels
from each of four replicates were chosen and the RNA
samples were pooled. Three technical replicates were
applied at each time point for the microarray analysis.
Microarray slides (46 K) were obtained from the University
of Arizona Maize Array Project (http://www.maizearray.
org). The detailed array annotation and composition is
available at the Internet site. All the experimental proce-
dures were carried out according to the manufacturer of the
slides, with a few modifications. In brief, 450 ng total RNA
was amplified and aminoallyl-UTP was incorporated using
101 TargetAmp Kit (Epicentre) and the resulting aaRNA
was labelled with Cy3 and Cy5 (Amersham). The dye-
labelled probes were then cleaned up (Qiagen), mixed with
the corresponding samples, concentrated, re-suspended in
the hybridisation solution and incubated at 42°C overnight
in a hybridisation oven. Finally, slides were washed with
different concentrations of SSC at room temperature.
Scanning was performed using an Amersham Typhoon
Trio+ scanner at 10 μm resolution and default settings. The
detection of signal intensities and the grid adjustment were
accomplished with ArrayVision software version 8.0
(Amersham). The intensity value of each spot and back-
ground region multiplied by its area was used as signal
intensity for further analysis.
Microarray data normalisation and analysis
Raw intensity data were imported into the R 2.6.2 software
(R Development Core Team 2008) after pre-processing it
with custom made Perl scripts. Further analysis was carried
out using the LIMMA (Smyth 2005) package of BIO-
CONDUCTOR (Gentleman et al. 2004). Background
correction was done using the multi-array analysis method
(Irizarry et al. 2003). Normalisation of data within arrays
was done using the ‘loess’ method (Yang et al. 2002). To
normalise the data between arrays, the ‘Aquantile’ method
was used (Yang and Thorne 2003). Besides these, the
relative reliability of each array was estimated and the data
weighted accordingly, based on the method described by
Ritchie et al. (2006). The microarray data for each gene
were fitted to a linear model, and statistics were generated
using the lmFit and eBayes functions (Smyth 2004) of the
LIMMA package. The P values were adjusted for multiple
testing using the Benjamini and Hochberg (1995) method.
Genes with adjusted P values of <0.05 and fold change ≥2
were considered as differentially expressed.
Gene ontology and promoter analysis
The differentially expressed genes were subjected to
further analysis. Based on the available chip annotation,
the genes were assigned into different Gene Ontology
categories (Ashburner et al. 2000), and the significant
over-representation of particular categories in the 24 and
48 h up- and down-regulated gene sets were determined
with a modified version of the GeneMerge software
(Castillo-Davis and Hartl 2003) optimised for speed.
Funct Integr Genomics (2009) 9:231–242 233

The cDNA sequences were searched against the available
maize genome sequence (http://www.maizegenome.org) with
a locally installed version of BLAST (Altschul et al. 1997)
and the promoter sequences up to 1,500 bp were extracted in
the case of a perfect hit. The sequences were analysed with
the TRANSFAC software suite version 12.1 (Matys et al.
2006), and a search was carried out for known transcription
factor binding sites. The frequencies of the binding sites up
to 8 bp length in all available maize promoter sequences and
the 24 and 48 h up- and down-regulated gene promoters
were calculated with the compseq program of the European
Molecular Biology Open Software Suite (Rice et al. 2000).
The over- or under-representation of a given transcription
factor binding site in the promoter regions of the smoke
specific up- or down-regulated genes was calculated as the
ratio of the observed frequency in the studied promoters and
expected frequency in all maize promoters.
Real-time PCR
The mRNA samples (200 ng) extracted from three
independent biological replicates were reverse transcribed
with SuperScript III reverse transcriptase (Invitrogen).
Real-time polymerase chain reaction (PCR) was performed
with an Applied Biosystems 7500 real-time PCR system
using SYBR Green detection chemistry (Applied Bio-
systems) and gene-specific primers. The reactions were
performed in quadruplicate. Confirmation of specific
product amplification was done by Tm analysis using the
dissociation curve option. PCR efficiency (derived from
the log slope of the fluorescence versus cycle number in
the exponential phase of each amplification plot) for all
primer pairs ranged from 95.5% to 98.0%. Maize actin
(AY103587.1) and GAPDH (NM001111944) were also
selected as potential internal controls and their expression
was checked using PCR and microarray data (data not
shown). Based on the preliminary findings, actin was
selected and used in further experiments. The relative ratio
of threshold cycle (Ct) values between the actin and the
specific gene and their standard deviations were calculated
for each sample.
Results
Germination and imbibition characteristics
Germination curves were recorded for control and smoke-
treated (1:500 and 1:1,000 dilutions) maize kernels to
determine the most effective concentration of the smoke-
water. In general, smoke-water improved the germination of
maize (Fig. 1a). Compared with the control, smoke-water at
a concentration of 1:500 resulted in significantly higher
germination with a mean of 64±1.45% whilst the 1:1,000
concentration only had a slight effect. Besides, ruptured
kernels were first observed in smoke-treated kernels (1:500
and 1:1,000 smoke-water treatments). Since the microarray
data (presented later) suggested a possible involvement of
ABA-related pathways in smoke action, ABA (100 μM)
and ABA (100 μM) + smoke-water (1:500) were also
applied to maize kernels. ABA treatment caused a slower
germination rate in the first half of the experiment but later
on, the germination percentage was the same as for
controls. However, smoke-water applied simultaneously
with ABA resulted in significantly lower germination
percentage throughout the course of the experiment.
Fig. 1 Germination characteristics of MV5405 inbred maize kernels.
a Germination time course of control and treated kernels. Treatments
as follows: smoke-water 1:500 dilution (S500) and 1:1,000 dilution
(S1000), ABA at 100 μM (ABA) and 100 μM ABA + 1:500 smoke-
water (ABAS). b Relative water content of germinating maize kernels.
Treatments, as above excluding the 1:1,000 smoke-water treatment
234 Funct Integr Genomics (2009) 9:231–242

The water uptake of kernels treated with smoke-water
(1:500 and 1:1,000 dilutions), ABA (100 μM) and ABA
(100 μM) + smoke-water (1:500) were recorded to outline
the early post-germination phase (or phase III of germina-
tion). Imbibition of the kernels was not affected by any of
the treatments used, as water content was quite similar in all
cases during the course of the experiment (Fig. 1b). The
rapid initial imbibition ended after 24 h, when the coat
started to rupture. Thereafter, the water content reached a
plateau phase, and 48 h onwards, a massive increase in
water uptake was observed which coincided with radicle
elongation and emergence.
Effect of smoke-water on seedling vigour
Seedling vigour tests were carried out under two different
temperature regimes. Applying smoke-water at both con-
centrations resulted in higher vigour compared to the
control during both regimes (data not shown). When the
chilling period was abandoned, the joint application of
smoke and ABA slightly decreased the seedling vigour
(data not shown). The effect of the smoke-water was,
however, more pronounced if a short chilling period was
introduced. The highest seedling vigour was observed in
this experiment after treating the kernels with smoke-water
at 1:500 dilution (Fig. 2a). These seedlings had significantly
longer roots and shoots, more roots and they produced
significantly higher dry mass after 9 days compared to the
control (Fig. 2 b1–b4). If the smoke-water was applied
together with 100 μM ABA, the vigour decreased com-
pared to water or ABA alone. This negative effect on
vigour was mainly attributed to the lower germination
percentage and shorter root length.
Transcriptome profile of smoke response
in the early post-germination phase
Based on the germination characteristics and imbibition
tests, 24 and 48 h were chosen as the time points to collect
samples for the microarray study (i.e. the earliest time
points at the beginning of phase III of germination). The
main objective of choosing these time points was that
smoke-responsive genes would be difficult to ascertain at
later stages following radicle emergence. In order to obtain
homogenic samples in terms of developmental stage,
embryos were collected either at 24 h when the embryos
just ruptured the testa or at 48 h when radicles had reached
a length of 3–4 mm. During this stage, the kernels had just
entered phase III of germination which is characterised by
rapid and pronounced water uptake. Genome-wide detec-
tion of smoke-responsive genes was performed by com-
paring smoke-treated kernels with control samples. The
microarray platform used covers nearly 98% of maize
genes. Thus, the transcriptome profile obtained represents
approximately all the genes potentially involved in smoke
action. According to the stringency levels (adjusted P
value <0.05 and fold change ≥2), a total of 1,842 genes
(721 up- and 1,121 down-regulated) showed differential
Fig. 2 Vigour of control and treated MV5405 inbred maize seedlings.
a The vigour index is indicated below the treatment name. The
seedling dry mass (g/seedling without kernel) and SE are in italics.
b1–b4 Morphology of 9-day-old control and treated seedlings. b1
Water control; b2 100 μM ABA; b3 1:500 dilution smoke-water; b4
100 µM ABA + 1:500 dilution smoke-water
Funct Integr Genomics (2009) 9:231–242 235

expression at 24 h. Likewise, 1,652 genes (887 up- and 765
down-regulated) showed differential expression at 48 h
(see Supplementary Table S1). Tables 1 and 2 show the
20 most strongly up- and down-regulated genes in the 24
and 48 h experiments, respectively. At 24 h after smoke-
water treatment, the transcript abundance of a sulfiredoxin-
like protein (MZ00020514), putative LRR receptor-like
kinase 2 (MZ00000704), ubiquitin-activating enzyme E1 1.
(MZ00041434), DIE2/ALG10 family protein (MZ00044608),
heat shock factor RHSF3 (MZ00025151) and several un-
known genes were amongst the highest up-regulated genes.
Genes that were down-regulated at 24 h included a B-type
cyclin homologue (MZ00044400), a parathymosin-like pro-
Table 1 Smoke-responsive genes in the 24 h experiment
Maize array ID Fold
change
Annotation
MZ00020514 27.9 Sulfiredoxin-like protein
MZ00000704 25.3 Putative LRR receptor-like kinase 2
MZ00041434 20.1 Ubiquitin-activating enzyme E1 1.
MZ00044608 10.8 DIE/ALG protein
MZ00021574 9.4 None
MZ00014903 9.4 Putative deoxyuridine triphosphatase
MZ00025151 9.0 Putative heat shock transcription factor
MZ00042191 8.5 Zea mays NAS2 like protein
MZ00048436 7.9 Transcription factor AP2D23-like
MZ00018324 6.5 None
MZ00014843 6.4 Putative methyl-binding domain protein
MBD111
MZ00018343 6.4 None
MZ00041286 5.9 Putative actin-depolymerising factor
MZ00032953 5.9 Putative transcription factor
E2F/dimerisation partner
MZ00004981 5.8 None
MZ00023856 5.7 Ribosomal protein L7Ae-like
MZ00027074 5.7 Copine III-like
MZ00032624 5.6 Glutathione S-transferase (GST) 39
MZ00023897 5.5 Putative DNA-binding protein GBP16
MZ00041376 5.5 Floral organ regulator 1
MZ00044400 −10.0 Cyclin type B-like
MZ00004143 −8.0 None
MZ00036429 −5.8 Parathymosin-like
MZ00035829 −5.7 None
MZ00033434 −5.7 Putative cellulase
MZ00042427 −5.1 None
MZ00046452 −4.9 Myb-like protein
MZ00032728 −4.5 None
MZ00024244 −4.5 Similar to splicing factor/activator protein
MZ00030105 −4.4 Contains tetratricopeptide repeat
MZ00004850 −4.4 Mini-chromosome maintenance protein
MZ00007375 −4.4 Putative 40S ribosomal protein S15
MZ00047595 −4.3 None
MZ00056286 −4.2 Poly-pyrimidine tract-binding protein-like
MZ00026860 −3.9 Fatty aldehyde dehydrogenase 1
MZ00005353 −3.9 None
MZ00015740 −3.9 None
MZ00032962 −3.8 Putative cyclic nucleotide-binding
transporter 1
MZ00044097 −3.8 Putative holocarboxylase synthetase
MZ00020180 −3.8 None
Top 20 up- and down-regulated genes were selected and displayed
Table 2 Smoke-responsive genes in the 48 h experiment
Maize array ID Fold
change
Annotation
MZ00026223 19.6 Contains leucine zipper
MZ00033282 18.9 Expressed protein
MZ00035162 15.4 None
MZ00055869 11.6 None
MZ00036882 11.3 None
MZ00024571 10.9 Auxin-regulated protein
MZ00019209 8.8 Putative zinc finger protein
MZ00001222 8.3 None
MZ00047597 8.3 None
MZ00055822 8.2 None
MZ00048220 8.1 AP domain containing transcription factor
MZ00001329 8.1 None
MZ00041357 8.0 None
MZ00043490 7.6 Transport protein particle component
Bet3-like protein
MZ00014903 7.5 Putative deoxyuridine triphosphatase
MZ00042879 7.5 Putative 60S ribosomal protein L37a
MZ00022728 7.4 None
MZ00036981 7.2 Transcription factor, MADS-box
MZ00018793 7.1 None
MZ00035697 7.1 Expressed protein
MZ00044747 −12.3 None
MZ00030535 −11.6 None
MZ00016593 −10.3 Putative vicilin
MZ00007309 −10.0 Putative calcium-dependent protein kinase
MZ00022141 −9.8 Receptor protein kinase PERK1-like
protein
MZ00005478 −9.8 Deoxyribodipyrimidine photolyase
MZ00032617 −9.5 Putative Sm protein F
MZ00029468 −9.3 Putative geranylgeranyl diphosphate
synthase
MZ00017582 −9.2 None
MZ00027351 −8.8 None
MZ00044410 −8.5 Putative glycerol 3-phosphate permease
MZ00031823 −7.5 NPK1-related protein kinase-like protein
MZ00005032 −7.2 Putative oxidoreductase, FAD-binding
MZ00019288 −7.2 Putative Rab geranylgeranyl
transferase, a subunit
MZ00013558 −7.1 Protein kinase-like
MZ00043179 −6.7 Putative protein-L-isoaspartate
O-methyltransferase
MZ00034227 −6.7 Putative NEC1
MZ00051069 −6.4 None
MZ00001125 −6.4 Putative peptide transporter
MZ00028057 −6.1 Putative TATA box binding protein-
associated factor
Top 20 up- and down-regulated genes were selected and displayed
236 Funct Integr Genomics (2009) 9:231–242

tein (MZ00036429) and a tetratricopeptide repeat containing
protein (MZ00030105). The transcription profile of 48 h
samples showed very few overlaps with the 24-h data.
Amongst others, leucine zipper (MZ00026223) and zinc finger
(MZ00019209) proteins, AP domain containing transcription
factor (MZ00048220), auxin induced protein (MZ00024571)
and genes with unknown function were the most abundant
transcripts. Conversely, a putative vicilin (MZ00016593), a
putative calcium-dependent protein kinase (MZ00007309), a
putative glycerol 3-phosphate permease (MZ00044410) and
other unknown genes were down-regulated at 48 h.
Validation of microarray data by quantitative real-time
RT-PCR
To confirm the reliability of the microarray results,
differential expression was corroborated for 12 genes using
quantitative real-time reverse transcriptase PCR. The
expression pattern observed in the microarray experiments
was consistent with the genes analysed by real-time PCR
(Fig. 3; Operon oligo identifiers are in brackets). As
suggested by microarray results, the relative transcript
abundance of an unknown gene similar to DIE/ALG
protein (MZ00044608), the putative LRR receptor-like
kinase 2 (MZ00000704), the cytochrome P450 gene
(MZ00022704), sulfiredoxin-like protein (MZ00020514)
and ubiquitin-activating enzyme E1 1. (MZ00041434) were
up-regulated after 24 h of smoke-water treatment. The
expression of the putative zinc finger protein (MZ00019209),
the gene similar to basic leucine zipper family protein
(MZ00026223), was up-regulated after 48 h whilst the
unknown gene (MZ00030105) which contains a tetratricopep-
tide repeat was down-regulated. We tested the transcript
abundance of several known stress or ABA-related genes,
such as a putative MYB transcription factor (MZ00004101),
calcineurin B-like protein 9 (MZ00024064), putative protein
kinase (MZ00047075) and unknown gene (MZ00051069),
which were up- or down-regulated in line with the expression
pattern obtained from the microarray study.
Gene ontology analysis of differentially expressed genes
The genes up-regulated by more or equal to twofold due to
smoke-water treatment (Supplementary Table S1) were
associated with different Gene Ontology terms based on
the Gene Ontology assignments available (Supplementary
Table S3). As the ‘biological function’ functional category
seemed to be the most informative in this case, the other
two (i.e. ‘molecular function’ and ‘cellular localisation’)
were excluded from further analysis. As revealed by the
Gene Ontology annotation, the most pronounced differ-
ences in the gene expression pattern after smoke-water
treatment correspond to translation and categories of
‘embryonic development ending in seed dormancy’, ‘cell
growth’ and ‘seed germination’. A number of Gene
Ontology terms involved in stress and ABA responsiveness
were highly enriched in the smoke-treated gene list. Gene
Ontology terms such as ‘response to cold’, ‘response to
water deprivation’, ‘response to salt stress’ and ‘response to
heat’ were ranked the highest amongst smoke-responsive
genes. The up-regulation of stress-related genes was robust
and extensive amongst the responses identified in this
work. Amongst the annotated genes, the stress-responsive
and hormone-related genes accounted for the significant
proportion of the smoke-responsive genes. At 24 h, genes
involved in stress responses (cold, water deprivation, salt
and osmotic stresses) were up-regulated and this tendency
continued at 48 h, although to a lower extent. After 48 h,
the enrichment of Gene Ontology terms such as ‘electron
Fig. 3 Expression analysis of some selected genes in response to
smoke treatment after 24 and 48 h. Relative transcript abundance
(RQ, relative quantification number) was calculated and normalised
with respect to the actin transcript level. At each time point, the
control was set as the calibrator. Data shown represent the mean
values obtained from four independent amplification reactions (n=4).
The experiment was repeated three times with similar results. Error
bars represent standard deviation. Operon oligo identifiers are shown:
MZ00051069—unknown {Oryza sativa (japonica cultivar group)};
MZ00044608—unknown {O. sativa (japonica cultivar group)}, similar
to DIE/ALG protein; MZ00026223—NA, similar to basic leucine
zipper family protein; MZ00000704—putative LRR receptor-like
kinase 2 {O. sativa (japonica cultivar group)}; MZ00022704—
cytochrome P450; MZ00020514—sulfiredoxin-like protein {O. sativa
(japonica cultivar group)}; MZ00030105—unknown protein {O. sativa
(japonica cultivar group)}, contains tetratricopeptide repeat;
MZ00041434—ubiquitin-activating enzyme E1 1. {Triticum aestivum};
MZ00019209—putative zinc finger protein {O. sativa (japonica cultivar
group)}; MZ00004101—putative MYB transcription factor {O. sativa
(japonica cultivar group)}; MZ00024064—calcineurin B-like protein 9
{O. sativa (japonica cultivar group)}; MZ00047075—putative protein
kinase {O. sativa (japonica cultivar group)}
Funct Integr Genomics (2009) 9:231–242 237

transport’ and ‘ribosome biogenesis and assembly’ were the
most prevalent but categories related to perception of
environmental stimuli and abiotic stress like ‘red and far-
red signaling pathway’, ‘response to ozone’ and ‘response
to hypoxia’ were also over-represented. Surprisingly,
various biotic stress-related terms such as ‘detection of
bacteria’, ‘response to insect’, ‘response to chitin’, ‘defense
response signaling pathway’ and ‘systemic acquired resis-
tance’ were significantly enriched in the up-regulated gene
list. However, the terms ‘response to fungus’ and ‘response
to wounding’ were over-represented in the down-regulated
gene list. In terms of hormone action, most genes showing
up-regulation in both time points were related to ABA and
ABA-mediated stress responses, whilst other hormone-
responsive genes (such as auxin, ethylene, jasmonic acid-
related genes) showed altered expression patterns. After
48 h, genes involved in the auxin-mediated signaling
pathway were more prevalent in the Gene Ontology terms
list. Ethylene and salicylic acid dependent systemic
resistance-related genes were also abundant. However,
salicylic acid-responsive genes were up-regulated after
48 h. As a general rule, fewer Gene Ontology terms were
significantly enriched in the 48 h data, compared to the 24 h
data. This might be attributed to the fact that at 48 h, a large
number of genes and biological processes are in operation,
and the microarray results contained more false positive and
false negative hits, causing the Gene Ontology analysis to
be less sensitive.
Promoter analysis of smoke-responsive genes
Cis-acting regulatory elements are the key points for the
regulation of gene expression and the determination of a
comprehensive set of smoke-related responses in the early
post-germination phase provided an opportunity to search
for elements common to their promoter region. These
known promoter motifs were searched for in 1,500-bp
regions upstream of the predicted start codon of the
smoke-regulated genes. The cis-acting element composition
(Supplementary Table S2) of smoke-responsive genes
reflects similar characteristics and involvement of biologi-
cal processes similar to that observed through the Gene
Ontology annotation. As expected, motifs and binding sites
related to organogenesis, meristem development and house-
keeping functions were the most pronounced in the
promoters of differentially expressed genes of smoke-
treated maize. The Cab140 and the seed specific CANNTG
motif occurred in a large proportion of the promoters
investigated. Different stress motifs, especially biotic stress,
cold- and dehydration-responsive elements (DREs), ABA/
glucose signalling and ABA-related motifs, present the
second largest group of the found motifs. Dehydration-
responsive elements, ABA-responsive elements (ABREs)
and ABRE/MYC recognition sites, Gt-box (pathogenesis
related), Sp8b (at both time points), Sph and Sph-box
motifs (at 48 h) are all over-represented after application of
smoke-water. Similarly to Gene Ontology annotation, the
incidence of stress and ABA-related cis-acting elements
were higher at 24 h than at 48 h, but these motifs were very
well represented at this time point also. At 48 h, WRKY1
was one of the most pronounced whilst at 24 h, ABA-
related motifs were more abundant.
Discussion
Many reports have been published in the past few years
describing the physiological effects of smoke treatments on
seed germination, but only a few have discussed the
possible mode of action (Van Staden et al. 2000). This is
the first report in which the effect of smoke on improving
seedling vigour and the post-germination phase are
assessed with respect to the molecular background of the
phenomena. In agreement with a previous investigation
(Sparg et al. 2006), our results show that smoke can
considerably increase seedling vigour and dry mass in the
MV5405 maize inbred line. A similar improvement of
seedling vigour was observed when smoke was applied to
Erica species and species of Asteraceae (Brown 1993),
South African indigenous medicinal plants (Sparg et al.
2005) and arable weeds (Daws et al. 2007). In addition,
smoke treatment resulted in earlier germination, higher
germination percentage in line with the findings of the vast
majority of the publications discussing the effect of smoke
on seed germination.
Smoke-water treatment considerably affected the tran-
scription profile of young seedlings just entering the early
post-germination stage. The common genes that were up-
regulated by smoke-water have been described previously
as stress-related genes, some of which have been well
characterised in this regard (Zhu 2002; Kirch et al. 2005;
Dreher and Callis 2007). Plant response to smoke in the
early post-germination phase, according to the present
microarray results, may be caused by reactions that are
similar to those occurring during abiotic and biotic stress.
Stress-related genes (cold, heat, drought, salt, wounding)
are up-regulated and the abundance of ABA-stimulated
transcripts were also pronounced. The proposed crosstalk
between stress response pathways and the smoke response
may be mediated by ABA. This finding is supported by the
fact that besides the occurrence of promoter motifs involved
in developmental regulation and stress-related events, the
most frequent motifs were the ABA-related elements such
as ABRE, ABRE/MYC recognition sites, Sph and Sph-box
(at 48 h). Although these motifs can be found widespread in
the promoter region of stress-related genes, this high
238 Funct Integr Genomics (2009) 9:231–242

Table 3 The most significant Gene Ontology terms in the different gene lists
24 h 48 h
Terms in up-regulated genes Terms in down-regulated genes Terms in up-regulated genes Terms in down-regulated genes
Nucleosome assembly 5 Response to salicylic acid
stimulus
18 Negative regulation of gene
expression, epigenetic
2 Mitochondrial fission 4
Translation 25 Response to absence of light 2 Ribosome biogenesis and
assembly
7 Pollen maturation 14
Response to virus 10 Cellular response to water
deprivation
3 Phloem histogenesis 2 Defense response to fungus 18
Nuclear mRNA splicing,
via spliceosome
5 Polysaccharid biosynthetic
process
3 Nucleosome assembly 3 Protein amino acid auto-
phosphorylation
19
RNA-dependent DNA
replication
3 Starch biosynthetic process 3 Nucleotide-excision repair 2 Cell growth 16
Chromosome
organisation and
biogenesis
3 Cell morphogenesis 3 Negative regulation of
transcription by glucose
2 Embryonic development
ending in seed dormancy
25
DNA replication
initiation
3 Trichome morphogenesis 4 Developmental growth 2 Transmembrane receptor
protein tyrosine kinase
signaling pathway
19
Embryonic pattern
specification
3 Translational elongation 3 Pollen wall formation 2 Response to wounding 28
N-terminal protein
myristoylation
15 Sugar-mediated signaling 5 Translational elongation 3 Brassinosteroid-mediated
signaling
15
Regulation of translation 3 Gamma-aminobutyric acid
catabolic process
2 Base-excision repair 2 Root hair cell differentiation 4
Galactolipid biosynthetic
process
2 Response to high light intensity 3 Red light signaling pathway 2 Nucleotide metabolic process 2
Response to desiccation 9 Plant-type cell wall biogenesis 6 Red or far red light signaling
pathway
3 Regulation of membrane
potential
2
Response to cold 33 Toxin catabolic process 3 Focal adhesion formation 2 Cell morphogenesis 4
Trehalose biosynthetic
process
3 Response to cadmium ion 8 Detection of bacterium 3 Cytokinesis by cell plate
formation
6
Oxygen and reactive
oxygen species
metabolic process
10 Thylakoid membrane organisation
and biogenesis
3 Response to hypoxia 2 Proteolysis 15
Response to light
stimulus
22 Abscisic acid biosynthetic process 2 Response to insect 2 Defense response to
bacterium, incompatible
interaction
13
Seed germination 10 Plant-type primary cell wall
biogenesis
2 Defense response signaling
pathway, resistance gene
independent
2 Response to high light intensity 4
Histone phosphorylation 7 Protein ubiquitination during
ubiquitin-dependent protein
catabolic process
2 Auxin-mediated signaling
pathway
6 Protein amino acid
phosphorylation
26
Potassium ion import 5 Protein ubiquitination 4 Cation transport 2 Growth 3
Lipid metabolic process 4 Actin filament-based process 2 Response to ozone 2 Cell proliferation 4
Early endosome to late
endosome transport
3 Protein neddylation 2 Nitrate transport 2 Oligopeptide transport 5
Protein targeting 2 Cell cycle 2 Cell division 4 Actin cytoskeleton
organisation and biogenesis
5
Actin filament-based
movement
2 Anthocyanin biosynthetic process 4 Response to chitin 3 Actin filament-based process 3
Response to water
deprivation
23 Protein modification process 3 Detection of ethylene stimulus 2 Cytoskeleton organisation and
biogenesis
5
Base-excision repair 2 Response to reactive oxygen
species
3 Salicylic acid-mediated signaling
pathway
2 Root epidermal cell
differentiation
5
Funct Integr Genomics (2009) 9:231–242 239

number of occurrence reflects the possible involvement of
ABA-pathways in the smoke response. The co-suppressive
effect of ABA and smoke-water on maize germination
characteristics and seedling vigour further highlights this
proposed crosstalk (Figs. 1a and 2). It is established that a
single copy of ABRE is not sufficient for ABA-mediated
induction of transcription, but multiple ABREs or the
combination with coupling elements such as DRE can,
together with ABREs, establish a minimal ABA-responsive
complex (Narusaka et al. 2003) which is essential for ABA-
related gene expression. DRE element was also over-
represented after smoke-water treatment. DRE-related
motifs have been reported in promoter regions of cold-
and drought-inducible genes such as KIN, COR6 and
RAB17 (Wang et al. 1995). The present results suggest that
DRE-related motifs are involved in drought- and cold-
responsive but ABA-independent gene expression. The
Sp8b motif found in the promoters of genes involved in
glucose/ABA signaling interplay (Rook et al. 2001) was also
over-represented. Other motifs, such as the pathogenesis-
related Gt-box and MYB binding site type I confer to biotic
stress response which term could be found in the Gene
Ontology annotation list. However, the results of the
promoter analysis should be interpreted with caution and
we used it as a guideline for additional experiments.
Our data demonstrated that in the early post-germination
phase, smoke-water treatment resulted in the over-
expression of stress and ABA-related genes (Table 3). It
has been shown that a short time window exists during
which the germinating seeds are supposed to recapitulate at
least a part of a maturation program of seed development
(Lopez-Molina et al. 2002; Rajjou et al. 2004; Holdsworth
et al. 2008). This post-germination developmental arrest
checkpoint is mediated by ABA and requires the ABI5
transcription factor in Arabidopsis (Lopez-Molina et al.
2002). The maturation drawback implies the expression of
ABA-related genes (Rajjou et al. 2006) allowing the young
plantlets to reinforce the capacity to cope with environ-
mental stress factors. Besides, ABA-treated germinating
seeds accumulate high levels of sucrose from the break-
down of storage lipid suggesting also that ABA may have a
large effect on seedling development and vigour (Pritchard
et al. 2002). ABA is known as a stress hormone which
integrates cell stress responses in plant cells and as an
essential mediator in triggering plant responses to adverse
environmental stimuli (Chandler and Robertson 1994). This
role is further emphasised by the fact that exogenous appli-
cation of ABA is able to increase plant adaptive responses
to various environmental conditions (Smith-Espinoza et al.
2005). Osmopriming of young tomato seedlings promoted
vegetative growth during seedling establishment, which
was reflected in plant vigour, crop yield and seed quantity
(Albacete et al. 2008). Although ABA is recognised as a
growth retardant, some studies indicate that application of
exogenous ABA initially inhibits growth but after a short
latency period, it increases the growth rate (Hall and
McWha 1981). There are specific situations in which
ABA is associated with growth (Barrero et al. 2005) and
Table 3 (continued)
24 h 48 h
Terms in up-regulated genes Terms in down-regulated genes Terms in up-regulated genes Terms in down-regulated genes
Phenylpropanoid
biosynthetic process
9 Regulation of transcription, DNA
dependent
15 Abaxial cell fate specification 2 Response to fungus 21
ATP-dependent
proteolysis
5 Regulation of meristem
organisation
5 Proton transport 2 Coumarin biosynthetic process 4
Indole phytoalexin
biosynthetic process
3 Ubiquitin-dependent protein
catabolic process
10 Systemic acquired resistance,
salicylic acid-mediated
signaling pathway
3 Actin filament organisation 4
Inter-Golgi cisterna
vesicle-mediated
transport
3 Response to nutrient 2 Response to high light intensity 2 Sulphate assimilation 2
Negative regulation of
abscisic acid-mediated
signaling
6 Trichome branching 5 Circadian rhythm 4 Anastral spindle assembly
involved in male meiosis
2
Response to heat 16 Proanthocyanidin biosynthetic
process
5 Jasmonic acid and ethylene-
dependent systemic resistance
3 Mitochondrion organisation
and biogenesis
3
Response to other
organism
12 Actin cytoskeleton organisation
and biogenesis
3 DNA endoreduplication 3 MAPKKK cascade 4
The terms in italic forms are statistically less significant but seem to be relevant
240 Funct Integr Genomics (2009) 9:231–242

this is especially true when the growth is an aspect of a
stress response (De Smet et al. 2006). Tagetes erecta plants
exposed to ABA exhibited reduced growth (leaves, stem,
roots) but soon after plants exhibited increased growth in
leaves and stem diameter which finally resulted in
improved plant morphology and increased field survival
(Aguilar et al. 2000).
Smoke not only promotes germination, but its effect
extends beyond germination stimulation and can also act to
enhance seedling vigour and survival after stresses (Sparg
et al. 2005, 2006). These results, together with previous
findings, suggest that smoke has a dual effect. On the one
hand, it promotes a higher germination rate in an unknown
way, and on the other hand, the concerted over-expression
shows that smoke could induce stress and ABA-related
stress-like responses in the early post-germination phase.
This change in transcriptome is very similar to cold,
drought and several biotic responses which are mediated
by ABA and which may contribute to the higher seedling
vigour. Presumably, smoke application on young seedlings
may lead to better adaptation to environmental stress factors
occurring during radicle emergence resulting in higher
seedling vigour. Most of the transcripts affected by the
smoke-water are regarded as stress-related messages show-
ing that smoke indeed has a direct effect on gene expression
which is quite similar to the pattern experienced following
different stresses.
The hypothesis that formed the basis of the present study
was that smoke interferes with stress and/or ABA-related
signal transduction. This hypothesis is supported by the fact
that the joint application of smoke and ABA on chilled
stressed kernels resulted in decreased seedling vigour. The
high incidence of ABA-related promoter motifs in the
smoke responsive gene list also supported this finding. A
key conclusion is that smoke can act as a hardening factor
and its application can eventually lead to increased stress
tolerance and growth. Master genes orchestrating smoke
action in the early post-germination phase are still un-
known. In the near future, extensive transcriptome analysis
of kernels treated with smoke, ABA and exposed to
different stresses will be carried out in our laboratory to
draw the map of possible interactions of smoke- and stress-
related pathways. In addition, if the presumptive active
compounds will be available in sufficient quantity, an
extensive transcriptome analysis will be carried out to
distinguish the effects of the butenolide versus other
compounds.
Acknowledgments This work was supported by the Generation
Challenge Program (GCP), the Hungarian–South African Intergovern-
mental S&T Cooperation Programme, the Hungarian Scientific
Research Fund (OTKA—F16066), the Baross Gábor Projekt and the
National Research Foundation, Pretoria, South Africa. Thanks are due
to Ferenc Marincs for his kind advices.
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