Search tips
Search criteria 


Logo of plantsigLink to Publisher's site
Plant Signal Behav. 2016 May; 11(5): e1176654.
Published online 2016 April 26. doi:  10.1080/15592324.2016.1176654
PMCID: PMC4973791

Development of marker genes for jasmonic acid signaling in shoots and roots of wheat


The jasmonic acid (JA) signaling pathway plays key roles in a diverse array of plant development, reproduction, and responses to biotic and abiotic stresses. Most of our understanding of the JA signaling pathway derives from the dicot model plant Arabidopsis thaliana, while corresponding knowledge in wheat is somewhat limited. In this study, the expression of 41 genes implicated in the JA signaling pathway has been assessed on 10 day-old bread wheat seedlings, 24 h, 48 h, and 72 h after methyl-jasmonate (MeJA) treatment using quantitative real-time PCR. The examined genes have been previously reported to be involved in JA biosynthesis and catabolism, JA perception and signaling, and pathogen defense in wheat shoots and roots. This study provides evidence to suggest that the effect of MeJA treatment is more prominent in shoots than roots of wheat seedlings, and substantial regulation of the JA pathway-dependent defense genes occurs at 72 h after MeJA treatment. Results show that the expression of 22 genes was significantly affected by MeJA treatment in wheat shoots. However, only PR1.1 and PR3 were significantly differentially expressed in wheat roots, both at 24 h post-MeJA treatment, with other genes showing large variation in their gene expression in roots. While providing marker genes on JA signaling in wheat, future work may focus on elucidating the regulatory function of JA-modulated transcription factors, some of which have well-studied potential orthologs in Arabidopsis.

KEYWORDS: Jasmonate, marker genes, PR genes, transcription factor, wheat


allene oxide synthase
Hydroperoxyoctadeca-9Z, 11E-dienoic acid
jasmonic acid
methyl jasmonate
nonexpressor of pathogenesis-related (gene)
oxo phytodienoic acid
12-oxo-phytodienoic acid reductase
phenylalanine ammonia lyase
pathogenesis-related (genes)
salicylic acid
Triticum aestivum
wheat chemically induced (genes)
Zn transporter (gene).


Jasmonic acid (JA) is an oxylipin hormone derived from linolenic acid which is crucial for plants to regulate growth and development as well as to respond to biotic and abiotic stresses.1 The JA pathway has been better characterized in dicot models such as Arabidopsis and tobacco and includes JA biosynthesis followed by JA signal transduction, which starts in chloroplasts. Briefly, lipoxygenases (LOXs) which are encoded by LOX genes oxygenate the phospholipids of linolenic acid. Linolenic acid is then liberated from membrane lipids and forms hydroperoxy octadecadienoic acid (HPODE). Under the action of an allene oxide synthase (AOS) and an allene oxide cyclase (AOC), respectively, encoded by AOS and AOC genes, HPODE is converted into 12-OPDA,2,3 which is subsequently reduced to JAs via the catalysis of a peroxisome-localized enzyme, 12-oxo-phytodienoic acid reductase 3 (OPR3), followed by 3 cycles of β-oxidation in the peroxisome.4,5 Afterwards, JA-Ile, the JA bioactive form of JA, is formed through a conjugation of JA and isoleucine (Ile) under the action of a GH3 family amido synthetase. JA-Ile is subsequently recognized by CORONATINE INSENSITIVE 1 (COI1)-JASMONATE ZIM DOMAIN (JAZ) co-receptor complexes and activates a signaling cascade for the induction of a series of defense (e.g. PR) genes which are also expressed in response to wounding, insect herbivory and necrotrophic pathogens.6 A schematic presentation for JA biosynthesis and the JA pathway cascade is shown in Fig. 1.

Figure 1.
Schematic diagram of the JA signaling pathway in monocot plants with inclusion of some wheat genes used in this study (adapted from Lyons et al. 2013).

In comparison to Arabidopsis and other monocots such as rice and maize, knowledge on JA signaling and biosynthesis in wheat is limited and fragmented.7 However, JA-dependent responses to diseases, biotic and abiotic stresses have been increasingly investigated in wheat during the last 2 decades. The PR genes PR1.1 and PR1.2, as well as a lipase- and 2 chitinase- (CHI1, CHI4) encoding genes were highly induced in 3 weeks post-emergence wheat by JA application. The differential expression of these genes upon JA treatment was also potentiated by common bunt, a disease caused by the closely related fungi Tilletia tritici and Tilletia laevis.8 In 2 week-old wheat seedlings, application of JA induced 6 PR genes and 4 putative defense genes (TaGLP2a, TaPERO, WCI2, WCI3), which were also induced by Fusarium pseudograminearum infection, the causative agent of wheat crown rot disease.9 Similarly, using a transcriptome-based method, it was revealed that JA biosynthesis genes such as LOX, AOS, AOC and OPR3 and JA signaling transduction genes, including COI1, JAZ, MYC2, were induced in a fusarium head blight resistant wheat variety.10 These findings support the hypothesis that the JA pathway is greatly involved in the defense against wheat pathogens.11 The JA signaling pathway also mediates the response to biotic stress from pest attack in wheat. For instance, patterns and levels of genes involved in JA signaling, such as LOX, AOS, and AOC, were differentially affected in wheat in defense response to incompatible interactions with Russian wheat aphids, a serious pest of cereal crops worldwide.12 Functions which are unrelated to defense have been recently proposed for some genes involved in the JA signaling pathway. For example, overexpression of TaAOC1 enhanced salinity tolerance in wheat via a JA pathway-dependent manner.13 Furthermore, wheat genes belonging to the WRKY and MYB transcription factor family have been found to be differentially expressed in wheat under conditions of biotic and abiotic stress, such as Fusarium graminearum infection, extreme temperatures (3°C and 40°C), high salinity stress (10% NaCl), osmotic stress and treatment with SA.14,15

However, up-to-date information on plant gene expression during JA signaling in wheat is fragmented and is only presented for either shoots or roots. In this study, we systematically evaluated transcriptional levels of various genes that have been associated with JA signaling in wheat roots and shoots using quantitative real-time PCR. Our findings complement the current knowledge on marker genes for the JA pathway in wheat and will facilitate future studies on this pathway in wheat and other monocot plants.

Materials and methods

Plant growth conditions, treatments and sampling

A total of 180 wheat seeds (Crusader variety) were planted in a potting mix (Searles, Australia). After stratification at 4°C for 5 days, seeds were transferred to a controlled environment chamber (Percival Scientific, Boone, IA, USA) at 20–24°C with a light intensity of 150 µmol m−2 s−1. MeJA was applied for the induction of the JA signaling pathway as follows: 5 μL of neat MeJA was diluted in 995 μL absolute ethanol. As MeJA is a volatile, a volume of 300 μL of the MeJA solution was injected into a cotton ball attached on the lid of the tray. All trays were then immediately wrapped with 2 tightly sealed transparent plastic bags. Control plants were mock-treated with an equal amount of the solvent ethanol. Each treatment included 3 biological replicates, and each biological replicate contained a pool of 10 plants. During the plant exposure to treatments, tray positions were changed daily in the growth chamber to ensure randomization. To evaluate the transcript abundances of marker genes in response to MeJA treatments, plants were harvested 24 h, 48 h and 72 h after application of the MeJA-treatment. Roots and shoots were stored at −80°C prior to RNA extraction.

RNA extraction and qRT-PCR analysis

Wheat shoots and roots were ground to a fine powder in liquid nitrogen, and total RNA isolations were performed with the SV Total RNA Isolation Kit (Promega) according to the manufacturer's recommendations. The concentration of the obtained RNA samples was measured using a Nanodrop spectrophotometer (Thermo Scientific). The quality (integrity) of RNA samples was further confirmed by agarose gel electrophoresis (1%). The cDNA was generated by reverse transcription with the Superscript III kit (Life Technologies) from 1.5 μg of total RNA in a 20 μL reaction using both random hexamers and oligo dT primers. Relative quantification of gene expression was performed by using SYBR Green RT-PCR mixtures on a ViiA™ 7 sequence detection system (Applied Biosystems, USA). Targeted genes for quantitative analyses were selected based on previous studies involving JA biosynthesis, signaling and defense-related genes in wheat. Specific primers used in this study were either designed using the Primer Express Software v2.0 (Applied Biosystems) or selected from previous reports. All primers used in this study are listed in Table S1. Real-time quantitative reverse transcriptase PCR (qRT-PCR) assays were performed in a 10 μL reaction containing 5 μL SYBR® Green PCR Master Mix, 1 μL of a primer mix (0.3 μM for each primer), and 4 μL of cDNA templates (diluted 30 times from the original cDNA synthesis reaction). 18S rRNA was used as the housekeeping gene for normalization; cDNA for these primers were diluted 500 times prior to PCR reactions. PCR cycling included 95°C for 10 min (heat activation), 40 cycles at 95°C for 15 s, 60°C for 1 min (amplification); and then 95°C for 2 min, 60°C for 15 s, and 95°C for 15 s (melt curve analysis). Relative expression of each target gene was investigated using 3 biological replicates with 2 technical replicates, each. Data analysis was then performed with ViiA 7 RUO Software (Applied Biosystems) with the 18S rRNA gene as an endogenous reference for normalization.

Statistical analysis

Means and standard errors were calculated from 3 biological replicates. Two-tailed t-tests were performed to determine significant differences at the 5% significance level. Heatmaps and bar graphs were generated by the software R-3.2.2 and Graphpad Prism 6.0.1, respectively.

Results and discussion

Ten day-old wheat seedlings were used for the evaluation of potential marker genes of the JA signaling pathway. During wheat sampling, no evidence of a phytotoxic effect induced by MeJA treatment was observed. Expression levels (transcript abundances) of each gene in shoots and roots after MeJA treatment are summarized in Fig. 2 and Fig. 3, respectively. Corresponding bar graphs displaying relative expression profiles for each gene in both roots and shoots are shown in Fig. S1(a)~(v). The following section details the results and discussions of the genes examined.

Figure 2.
Heatmap summarizing variation in wheat gene expression between mock and MeJA-treated shoot samples. Significant differences are indicated by the asterisk(s) after the heatmap blocks of each gene (P < 0.05 (*), P < 0.01 (**), P < ...
Figure 3.
Heatmap summarizing variation in wheat gene expressions between mock and MeJA-treated root samples. Significant differences are indicated by the asterisks after each gene name (P < 0.05 (*)). The numbers in Fig. 3 show corresponding fold ...

JA biosynthesis-associated genes

Four genes whose functions have been associated with JA biosynthesis were chosen for the gene expression analysis, which include genes encoding a wheat allene oxide synthase (TaAOS), a wheat allene oxide cyclase (TaAOC1), and 2 wheat oxophytodienoate reductases (TaOPR1 and TaOPR3). TaAOS was induced in shoots but only at 24 h after MeJA treatment (Fig. 2; Fig. S1(q)). During JA biosynthesis, the enzyme allene oxide synthase (AOS) encoded by this gene catalyzes the conversion of hydroperoxyoctadecadienoic acid (HPODE) to 12-oxo-phytodienoic acid (OPDA). In a previous study, TaAOS was induced in wheat by Sitobion avenae attack.16 The expression of TaAOC1 in shoots was potentiated by 2.0-fold at 48 h after MeJA treatment (Fig. 2; Fig. S1(v)). TaAOC1 and TaAOS both catalyze the first step of the lipoxygenase pathway, and its encoded enzyme AOC mainly confers the unstable products of AOS with correct enantiomeric structure of natural JA in the α-linolenic acid metabolism pathway.7 Constitutive expression of this wheat gene in Arabidopsis thaliana and bread wheat led to a higher JA content in plants and shorter developed roots along with an enhanced tolerance to salinity.13

TaOPR1 (encoding wheat 12-oxo-phytodienoic acid reductase I) was significantly upregulated in shoots by 2.0- and 4.8-fold at 24 h and 72 h after MeJA treatment, respectively (Fig. 2; Fig. S1(s)). OPRs encoded by OPR genes catalyze the production of JA from its precursor of OPDA through the reduction of the double bond.7 In both Arabidopsis and wheat, OPR1 is involved in biological processes of plant growth and development, and can be stimulated by a variety of environmental and chemical stimuli, such as wounding, pathogen invasion and application of brassinosteroids.17-19 It has been recently revealed that TaOPR1 promoted wheat's salinity tolerance capability via increasing ABA signaling and scavenging reactive oxygen species (ROS), without involving the JA signaling pathway.20 TaOPR3 expression in shoots was increased by 1.5- and 6.8-fold at 24 h and 72 h after MeJA treatment, respectively (Fig. 2; Fig. S1(u)). F. gramineraum infection induced TaOPR3 together with another 13 genes associated with JA biosynthesis in the wheat landrace Wangshuibai.10 In Arabidopsis, among the 6 described OPRs, only OPR3 is involved in JA biosynthesis, which can be induced by touch, wind, UV light, application of detergent, wounding, and brassinosteroids.21 From what has been discussed above, it is clear that the genes of TaAOS, TaAOC1, TaOPR1 and TaOPR3 were greatly induced in wheat shoots after exogenous treatment with MeJA. Additionally, as these genes are not only essential for the synthesis of JA and its methyl ester but are also involved in plant response to biotic and (or) abiotic stresses, these genes are worth being assessed in future molecular studies on wheat.

Genes associated with JA signaling

COI1 expression was slightly but significantly repressed by 0.5-fold at 72 h after JA treatment in shoots (Fig. 2; Fig. S1(i)). In Arabidopsis and rice, COI1 forms a functional E3-type ubiquitin ligase complex that targets JAZ proteins (negative regulators for JA signaling) for degradation and COI1 was not induced after JA treatment.22 The functions of COI1 in wheat JA perception and/or signaling are currently unknown. However, in wheat roots, COI1 was induced within 6 h after inoculation with the Pseudomonas fluorescens biocontrol strain Q8r1-96,23 and our data show that COI1 was downregulated in the shoots of wheat seedlings (10-day old) by MeJA treatment.

Transcription factors

Transcription factors are key regulators for the expressions of many PR genes in monocot plants.7 For the 5 TFs tested in this study, TaWRKY72a/b, TaWRKY78 and ZAT11 changed in gene expressions after MeJA treatment, which indicates that these genes could be involved in the JA signaling pathway of wheat. Paralogous transcription factors (TFs) of the wheat WRKY family, such as TaWRKY72a/b, TaWRKY78 and ZAT11, are crucial components in regulating the expression of defense-related genes.24,25 The expression of TaWRKY72a/b in shoots increased 7.1-fold at 72 h after MeJA treatment (Fig. 2; Fig. S1(l)). TaWRKY72a/b has been shown to be expressed in leaves, roots, and crown and was up-regulated following the maturation and senescence of wheat leaves, which suggests that they may play important roles in regulating wheat leaf senescence.14 The expression of TaWRKY78 decreased significantly by 0.6-fold 72 h after MeJA application (Fig. 2; Fig. S1(m)). It has been demonstrated that TaWRKY78 and its Arabidopsis ortholog, AtWRKY20 are able to induce the promoter of wPR4e (coding for Wheatwin5) and the wPR4e Arabidopsis ortholog AtHEL, respectively. TaPR4 genes were induced by treatment with the SA analog benzothiadiazole (BTH) and MeJA, indicating that TaWRKY78 is involved in both SA- and JA-dependent defense response pathways.26 In Arabidopsis, WRKYs are most commonly associated with SA signaling, while wheat WRKY TFs examined in the present study have also been strongly influenced by the MeJA treatment (Fig. 2). This has also been reported to be the case in rice.27

ZAT11 belongs to the zinc transporter family protein. In wheat shoots, ZAT11 was significantly downregulated by 0.3-fold at 72 h after MeJA treatment (Fig. 2; Fig. S1(n)). ZAT11 (encodes zinc finger-C2H2 type family protein of Arabidopsis thaliana 11) is inducible by many stresses and regulates the expression of ascorbate peroxidase, which provides protection against hydrogen peroxide during oxidative stress.28 ZAT11 is also a dual-function transcriptional regulator that positively regulates primary root growth, but negatively regulates Ni2+ tolerance.29 The function of ZAT11 gene in wheat is still unknown but is likely to be different as its expression remained unchanged upon treatment with F. pseudograminearum CS3096.30 If TaWRKY72a/b, TaWRKY78 and ZAT11 are used as JA pathway marker genes in wheat, their involvement in other pathways should also be considered.

Pathogenesis-related genes

PR1.1 (pathogenesis-related 1 basic) was upregulated in shoots at 48 h and 72 h after MeJA treatment by 5.8- and 7.4-fold, respectively, and was downregulated in roots by 0.6-fold at 24 h post treatment (Fig. 2; Fig. S1(a)). Similarly, PR1.2 (pathogenesis-related 1 neutral) was induced in shoots by 1.9- fold at 72 h after MeJA treatment (Fig. 2; Fig. S1(b)). These two PR genes were also induced in wheat shoots by infection with the fungal pathogen Erysiphe graminis.31 The induction of PR1 in shoots suggests a cross-talk between the JA and SA signaling pathways, as wheat PR1 has also been reported to be typically induced during SA pathway activation.7 For example, the expression of PR1 was elevated in F. pseudograminearum-infected wheat spikes, which was accompanied by an accumulation of SA.32 In addition, PR2 (encoding β-1,3-endoglucanase) was upregulated by 28.4-fold in shoots at 72 h after MeJA treatment (Fig. 2; Fig. S1 (c)). PR3 was downregulated in roots by 0.6-fold and was upregulated at both 48 h and 72 h by MeJA treatment (Fig. 3; Fig. S1(d)). It also was induced by Fusarium asiaticum infection which causes head blight and seedling blight in both wheat spikes and seedlings.33 Interestingly, PR2 and PR3 are reported to be greatly induced in germinating wheat seeds upon infection with the hemibiotroph Fusarium culmorum.34 This pathogen has a short biotrophic stage and then changes to a necrotrophic stage, which is when the JA pathway is usually activated.35 At 72 h after MeJA treatment, PR4a (wheatwin1-4) expression in shoots increased by 12.8-fold (Fig. 2; Fig. S1(e)). The activation of PR4a genes has been reported to be involved in both JA and SA-dependent defense response pathways.26 Besides protecting wheat against fungal pathogens, wheatwin genes were developmentally regulated in the grain and may play a role in response to high temperatures.36 PR4 proteins show antifungal activity against several phytopathogenic fungi and have been demonstrated to possess ribonucleasic activity correlated to their antifungal capacity.37,38 PR5 encodes a thaumatin-like protein which exhibits antifungal activity against snow mold and Microdochium nivale. In shoots, PR5 (WAS-3a) was induced by 2.1-fold at 72 h post MeJA treatment (Fig. 2; Fig. S1(f)). PR5 encodes the major isoform of thaumatin-like protein in winter wheat cells and is markedly induced by treatment with abscisic acid (ABA) and by other elicitors, including chitosan and β-glucan.39 PR10 encodes a ribonuclease-like protein which is a pathogen-induced putative peroxidase from wheat. This gene was significantly induced in shoots by 7.1-fold at 72 h upon MeJA treatment (Fig. 2; Fig. S1(g)). This gene is induced by a range of pathogens and plays additional roles in development and enzymatic reactions.40 PR14 (LTP-2) which codes for a non-specific lipid transfer protein (ns-LTP), decreased by 0.7-fold at 24 h after MeJA treatment (Fig. 2; Fig. S1(j)). This ns-LTP has previously been isolated by Lu et al. (2005) 41 from a wheat suppression subtractive hybridization (SSH) cDNA library for common bunt (Tilletia tritici) infections. LTPs are widely known as ubiquitous proteins that are relevant to plant development and stress responses.41 Another study demonstrated a significant increase in LTP expression in 1 week-old seedlings after treatment with MeJA and SA.8 Collectively this shows that exogenous application of MeJA on wheat leads to the induction of a diverse range of PR genes which provide defensive functions. Prior upregulation of these genes may lead to a higher preparedness of wheat plants to subsequent pathogen attack and should be the subject of further investigation.

Other important plant defense genes

Besides genes that have previously been recognized as designated orthologs of PR genes in wheat, there is a number other potentially important defense genes involved in the wheat JA pathway. These include, for instance chitinase and lipase encoding genes, and those genes involved in the cross-talk with other signaling pathways, e.g., TaNPR1, linking JA and SA signaling. Chitinases are pathogenesis-related proteins that hydrolyze chitin, an essential structural component of fungal cell walls. CHI3 expression was significantly increased in shoots at 24 h, 48 h and 72 h after MeJA treatment by 2.0-, 2.7- and 16.5-fold, respectively (Fig. 2;Fig. S1(o)). CHI3 has been shown to be induced by F.graminearum.42 In shoots, the expression of the CHI4 precursor was downregulated 72 h post MeJA treatment by 0.2-fold (Fig. 2; Fig. S1(p)). This gene has been previously reported to be induced by both MeJA and common bunt infections (T. tritici) in wheat seedlings.8 WCI2 was significantly induced in shoots at 72 h post MeJA treatment by 2.2-fold (Fig. 2; Fig. S1(k)). WCI genes were involved in systemic acquired resistance (SAR), and a specific set of WCI genes have been induced by BTH. Induction of WCI genes was involved in increased wheat resistance to powdery mildew infection through affecting multiple steps of pathogen development.43 The LIPASE gene was isolated from a wheat SSH cDNA library for common bunt infections by Lu et al. (2005) 41 and was significantly induced in shoots 24 h, 48 h and 72 h post MeJA treatment (Fig. 2; Fig. S1(h)). The encoded lipases have been associated with pathogen resistance responses in plants through the SA signaling pathway but it also was reported to be strongly upregulated by MeJA in 2 and 3 week-old wheat seedlings.8

TaNPR1 (non-expressor of pathogenesis-related genes) is a key regulator of the SA signaling pathway, and as expected was found significantly repressed by 0.5-fold at 72 h post MeJA treatment (Fig. 2; Fig. S1(r)). AtNPR1 plays a center role in the onset of SA-mediated SAR.44 Importantly, AtNPR1 is involved in the cross-talk between SA- and JA-dependent pathways and assumes the key role in the suppression of JA-mediated defense responses by the SA pathway.45-47 In wheat, TaNPR1 in an HvSGT1-over-expressing line was greatly downregulated at 24 h post inoculation with biotrophic pathogen Blumeria graminis DC. f. sp. tritici compared to wild-type.48 The transcript abundance of TaPAL increased by 3.4-fold in shoots at 72 h after MeJA treatment (Fig. 2; Fig. S1(t)). Phenylalanine ammonia lyase (PAL) is involved in both SA and JA-dependent pathways and is essential for biosynthesis of phenylpropanoids. PAL is also associated with a variety of functions, including plant host defense against pathogens and response to abiotic stress like wounding.16,49 These genes as stated above may provide useful information for future studies on the effects of plant hormone or pathogen treatments on wheat. Their co-regulation by other defense pathways should be noted when used as marker genes for JA signaling in wheat.

Genes that were not differentially expressed by MeJA

Genes that are related to ROS production/scavenging systems, including CAT (catalase), SOD (superoxide dismutase) and APX (ascorbate-peroxidase), were not affected by MeJA treatment. The Jasmonate ZIM domain (JAZ) encoding gene did not respond to MeJA treatment within the examined period of time. Initially observed to be early upregulated by wounding or JA treatment, JAZ proteins are recognized as targets of the SCFCOI1 complex. The degradation of JAZ allows the release of positively acting TFs, such as MYC2 (encoding a basic-helix-loop-helix (bHLH) TF) and its homolog MYC3 that bind to JA-responsive elements occurring in promoters of JA-responsive genes via the mediator subunit MED25.50 MYC2 has emerged as a master regulator of most aspects of the jasmonate (JA) signaling pathway in Arabidopsis.7 However, a putative MYC2 ortholog in wheat was not induced in the current study.10 Additionally a C3H encoding C3H-type zinc finger TF, TaERF (wheat ethylene-responsive factor-like transcription factor), TaWRKY1, TaWRKY2, WCI3 (wheat chemical induced gene 3), Glu2 (neutral β-1,3; β-1,4-glucanase), Glu3 (basic β-1,4-glucanase) were not induced within 24-72 hours. It is possible that these genes may have responded to MeJA treatment at even earlier times than 24 h.

Late response JA signaling genes

In the present study, we found that most induced genes increased greatly in expression at 72 h-post MeJA treatment, especially those genes involved in plant defense (e.g., designated PR genes, CHI3, TaPAL). This result seems to contradict the notion that plants should respond to pest and pathogen invasions at an earlier stage than 72 h. JA marker gene expression studies in rice have also focused on early responses.22 However, chemical treatment with MeJA is different from pathogen and pest attacks and it has been reported that phase changes occurring during plant development can determine to what extent a plant responds to different signaling compounds (e.g. MeJA and SA). Defense-related genes of 1-, 2- and 3-week stage seedlings responded differentially to SA and MeJA treatment. Spraying MeJA solution on wheat shoots greatly induced CHI1, CHI3, CHI4, PR1.1 and Lipase genes 24 h after MeJA treatment on 3-week old wheat seedlings but not in one-week or 2-week old wheat seedlings.8 In our study, as 10-day old wheat seedlings were used, the early stage of these plants may have potentially led to the late responses of wheat plants to MeJA treatment. Additionally, incubation of wheat with MeJA vapors is different from the spraying method as previously reported.8,9,11,27,30 In comparison with the spraying method, incubating wheat with MeJA vapor of a relative low concentration (0.025 µL per liter) may have caused a delay of MeJA to reach wheat plants. Therefore, the treatment method used may have also contributed to the late response of wheat to MeJA treatment.

Large variances in gene expressions among replicates found in wheat roots

Many studies found differential expression in roots during plant defense signaling to be much less pronounced than in shoots.51 There are several trends that can be seen from the root heatmap data with only a few significantly differentially expressed genes (Fig. 3). These may serve as a basis for further studies, taking into consideration the factors that may contribute to larger variation in root samples. In comparison, the variance among shoot samples was much smaller than for root samples (Fig. 2; Fig. 3). Although not significant, there was a trend of an increase in expression of PR genes 72 h-post MeJA treatment (Fig. 3), which is consistent with the induced gene expression in shoots. As cDNA synthesis and RT-PCR were implemented in the same batch for root and shoot samples, we assumed that this experiment had been performed technically well. Nevertheless, traces of humic acid or other reverse transcriptase or PCR inhibiting compounds may have been present in root samples. We incubated wheat seedlings with MeJA vapors, which has possibly led to plant roots unevenly accessing MeJA molecules, considering that MeJA vapors first need to penetrate into the soil to reach the roots. In contrast, wheat shoots were evenly exposed to this volatile signaling compound.


In this study, using gene expression profiling by qRT-PCR, the expression of the genes potentially involved in JA biosynthetic and signaling pathway was examined at 3 time points post-MeJA treatment in wheat seedlings. Our results suggest that differential expression of JA-associated genes was more prominent at 72 h after MeJA application. These genes may serve as useful markers to further elucidate JA signaling in wheat or to confer resistance to pests and diseases. For example, the overexpression of JA-modulated PR genes may provide resistance against wheat pathogens. The observed differential expression of regulatory genes (including TFs) suggests a regulatory function during JA signaling. These genes may provide powerful tools for modulating JA signaling in wheat (e.g., toward disease resistance), as this has been highly successful for putative orthologs of these genes in Arabidopsis. While most of the present knowledge of the JA signaling pathway derives from the dicotyledonous model plant Arabidopsis thaliana, this study supports the notion that JA signaling in monocotyledonous plants could be used for similar functions.

Supplementary Material


Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.


The authors wish to thank Dr. Mark Dieters for providing the wheat seeds and the Australian Research Council for financial support. HL gratefully acknowledges funding from the China Scholarship Council (CSC), and the technical help from Dr. Max Chen (Yi-Chung Chen). The authors would also acknowledge Dr Jessica Dalton-Morgan for proof-reading the manuscript.


1. Creelman RA, Mullet JE Biosynthesis and action of jasmonates in plants. Annu Rev Plant Biol 1997; 48:355-81; PMID:15012267; [PubMed] [Cross Ref]
2. Laudert D, Pfannschmidt U, Lottspeich F, Holländer-Czytko H, Weiler E. Cloning, molecular and functional characterization of Arabidopsis thaliana allene oxide synthase (CYP 74), the first enzyme of the octadecanoid pathway to jasmonates. Plant Mol Biol 1996; 31:323-35; PMID:8756596; [PubMed] [Cross Ref]
3. Stenzel I, Hause B, Miersch O, Kurz T, Maucher H, Weichert H, Ziegler J, Feussner I, Wasternack C. Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana. Plant Mol Biol 2003; 51:895-911; PMID:12777050; [PubMed] [Cross Ref]
4. Stintzi A. The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis. Proc Natl Acad Sci USA 2000; 97:10625-30; PMID:10973494; [PubMed] [Cross Ref]
5. Strassner J, Schaller F, Frick UB, Howe GA, Weiler EW, Amrhein N, Macheroux P, Schaller A. Characterization and cDNA-microarray expression analysis of 12-oxophytodienoate reductases reveals differential roles for octadecanoid biosynthesis in the local versus the systemic wound response. Plant J 2002; 32:585-601; PMID:12445129; [PubMed] [Cross Ref]
6. Cheong JJ, Choi YD. Methyl jasmonate as a vital substance in plants. TIG 2003; 19:409-13; PMID:12850447; [PubMed] [Cross Ref]
7. Lyons R, Manners JM, Kazan K. Jasmonate biosynthesis and signaling in monocots: a comparative overview. Plant Cell Rep 2013; 32:815-27; PMID:23455708; [PubMed] [Cross Ref]
8. Lu ZX, Gaudet D, Puchalski B, Despins T, Frick M, Laroche A Inducers of resistance reduce common bunt infection in wheat seedlings while differentially regulating defence-gene expression. Physiol Mol Plant Pathol 2006; 67:138-48; [Cross Ref]
9. Desmond OJ, Edgar CI, Manners JM, Maclean DJ, Schenk PM, Kazan K Methyl jasmonate induced gene expression in wheat delays symptom development by the crown rot pathogen Fusarium pseudograminearum. Physiol Mol Plant Pathol 2006; 67:171-9; [Cross Ref]
10. Xiao J, Jin X, Jia X, Wang H, Cao A, Zhao W, Pei H, Xue Z, He L, Chen Q, et al. Transcriptome-based discovery of pathways and genes related to resistance against Fusarium head blight in wheat landrace Wangshuibai. BMC genomics 2013; 14:197; PMID:23514540; [PMC free article] [PubMed] [Cross Ref]
11. Li G, Yen Y Jasmonate and ethylene signaling pathway may mediate Fusarium head blight resistance in wheat. Crop Sci 2008; 48:1888-96; [Cross Ref]
12. Liu X, Meng J, Starkey S, Smith C. Wheat gene expression is differentially affected by a virulent Russian wheat aphid biotype. J Chem Ecol 2011; 37:472-82; PMID:21499720; [PubMed] [Cross Ref]
13. Zhao Y, Dong W, Zhang N, Ai X, Wang M, Huang Z, Xiao L, Xia G. A wheat allene oxide cyclase gene enhances salinity tolerance via jasmonate signaling. Plant Physiol 2014; 164: 1068-76; PMID:24326670; [PubMed] [Cross Ref]
14. Wu H, Ni Z, Yao Y, Guo G, Sun Q Cloning and expression profiles of 15 genes encoding WRKY transcription factor in wheat (Triticum aestivem L.). Prog Nat Sci 2008; 18:697-705; [Cross Ref]
15. Zhu X, Liu S, Meng C, Qin L, Kong L, Xia G WRKY transcription factors in wheat and their induction by biotic and abiotic stress. Plant Mol Biol Rep 2013; 31:1053-67; [Cross Ref]
16. Zhao LY, Chen JL, Cheng DF, Sun JR, Liu Y, Tian Z Biochemical and molecular characterizations of Sitobion avenae-induced wheat defense responses. Crop Prot 2009; 28:435-42; [Cross Ref]
17. Reymond P, Weber H, Damond M, Farmer EE. Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell 2000; 12:707-19; PMID:10810145; [PubMed] [Cross Ref]
18. Schaller F.. Enzymes of the biosynthesis of octadecanoid‐derived signalling molecules. J Exp Bot 2001; 52:11-23; PMID:11181709; [PubMed] [Cross Ref]
19. Tan CT, Carver BF, Chen MS, Gu YQ, Yan LL. Genetic association of OPR genes with resistance to Hessian fly in hexaploid wheat. BMC Genomics 2013; 14:369; PMID:23724909; [PMC free article] [PubMed] [Cross Ref]
20. Dong W, Wang M, Xu F, Quan T, Peng K, Xiao L, Xia G. Wheat oxophytodienoate reductase gene TaOPR1 confers salinity tolerance via enhancement of abscisic acid signaling and reactive oxygen species scavenging. Plant Physiol 2013; 161:1217-28; PMID:23321418; [PubMed] [Cross Ref]
21. Schaller F, Biesgen C, Müssig C, Altmann T, Weiler EW. 12-Oxophytodienoate reductase 3 (OPR3) is the isoenzyme involved in jasmonate biosynthesis. Planta 2000; 210:979-84; PMID:10872231; [PubMed] [Cross Ref]
22. Lee HY, Seo JS, Cho JH, Jung H, Kim JK, Lee JS, Rhee S, Do Choi Y Oryza sativa COI homologues restore jasmonate signal transduction in Arabidopsis coi1-1 mutants. PLoS ONE 2013; 8:p.e52802; PMID:23320078; [PMC free article] [PubMed] [Cross Ref]
23. Okubara PA, Call DR, Kwak Y-S, Skinner DZ Induction of defense gene homologues in wheat roots during interactions with Pseudomonas fluorescens. Biol. Control 2010; 55:118-25; [Cross Ref]
24. Riechmann JL, Meyerowitz EM The AP2/EREBP family of plant transcription factors. J Biol Chem 1998; 379:633-46; [PubMed] [Cross Ref]
25. Zhao J, Buchwaldt L, Rimmer SR, Sharpe A, McGregor L, Bekkaoui D, Hegedus D. Patterns of differential gene expression in Brassica napus cultivars infected with Sclerotinia sclerotiorum. Mol Plant Pathol 2009; 10:635-49; PMID:19694954; [PubMed] [Cross Ref]
26. Bertini L, Leonardi L, Caporale C, Tucci M, Cascone N, Di Berardino I, et al. Pathogen-responsive wheat PR4 genes are induced by activators of systemic acquired resistance and wounding. Plant Sci 2003; 164:1067-78; [Cross Ref]
27. Qiu D, Xiao J, Ding X, Xiong M, Cai M, Cao Y, Li X, Xu C, Wang S. OsWRKY13 mediates rice disease resistance by regulating defense-related genes in salicylate- and jasmonate-dependent signaling. MPMI 2007; 20:492-9; PMID:17506327; [PubMed] [Cross Ref]
28. Rizhsky L, Davletova S, Liang H, Mittler R. The zinc finger protein Zat12 is required for cytosolic ascorbate peroxidase 1 expression during oxidative stress in Arabidopsis. J Biol Chem 2004; 279:11736-43; PMID:14722088; [PubMed] [Cross Ref]
29. Liu XM, An J, Han H, Kim S, Lim C, Yun D-J, Chung WS ZAT11, a zinc finger transcription factor, is a negative regulator of nickel ion tolerance in Arabidopsis. Plant Cell Rep 2014; 33: 2015-2021; PMID:25163803; http;// [PubMed] [Cross Ref]
30. Desmond O. Wheat defence responses induced by the fungal pathogen Fusarium pseudograminearum. PhD Thesis, accessed on 12, December, 2014 2008.
31. Molina A, Görlach J, Volrath S, Ryals J Wheat genes encoding two types of PR-1 proteins are pathogen inducible, but do not respond to activators of systemic acquired resistance. Mol Plant Microb Interac 1999; 12:53-8; PMID:9885193; [PubMed] [Cross Ref]
32. Makandar R, Nalam VJ, Lee H, Trick HN, Dong Y, Shah J Salicylic acid regulates basal resistance to Fusarium head blight in wheat. Mol Plant Microb Interac 2012; 25:431-9; PMID:22112217; [PubMed] [Cross Ref]
33. Li X, Zhang J, Song B, Li H, Xu H, Qu B, Dang FJ, Liao YC. Resistance to Fusarium head blight and seedling blight in wheat is associated with activation of a cytochrome P450 gene. Phytopathology 2010; 100:183-91; PMID:20055652; [PubMed] [Cross Ref]
34. Caruso C, Chilosi G, Caporale C, Leonardi L, Bertini L, Magro P, et al. Induction of pathogenesis-related proteins in germinating wheat seeds infected with Fusarium culmorum. Plant Sci 1999; 140:87-97; [Cross Ref]
35. Bushnell W, Hazen B, Pritsch C, Leonard K Histology and physiology of Fusarium head blight. Fusarium head blight of wheat and barley 2003; 44-83
36. Altenbach SB, Kothari KM, Tanaka CK, Hurkman WJ Genes encoding the PR-4 protein wheatwin are developmentally regulated in wheat grains and respond to high temperatures during grainfill. Plant Sci 2007; 173:135-43; [Cross Ref]
37. Caporale C, Di Berardino I, Leonardi L, Bertini L, Cascone A, Buonocore V, Caruso C. Wheat pathogenesis-related proteins of class 4 have ribonuclease activity. FEBS Letters 2004; 575:71-6; PMID:15388335; [PubMed] [Cross Ref]
38. Bertini L, Caporale C, Testa M, Proietti S, Caruso C. Structural basis of the antifungal activity of wheat PR4 proteins. FEBS Letters 2009; 583:2865-71; PMID:19647737; [PubMed] [Cross Ref]
39. Kuwabara C, Takezawa D, Shimada T, Hamada T, Fujikawa S, Arakawa K. Abscisic acid- and cold-induced thaumatin-like protein in winter wheat has an antifungal activity against snow mould, Microdochium nivale. Physiol Plant 2002; 115:101-10; PMID:12010473; [PubMed] [Cross Ref]
40. Liu JJ, Ekramoddoullah AK The family 10 of plant pathogenesis-related proteins: their structure, regulation, and function in response to biotic and abiotic stresses. Physiol Mol Plant Path 2006; 68:3-13; [Cross Ref]
41. Wasternack C.. Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development. Ann Bot 2007; 100:681-97; PMID:17513307; [PMC free article] [PubMed] [Cross Ref]
42. Kong L, Anderson JM, Ohm HW. Induction of wheat defense and stress-related genes in response to Fusarium graminearum. Genome 2005; 48:29-40; PMID:15729394; [PubMed] [Cross Ref]
43. Görlach J, Volrath S, Knauf-Beiter G, Hengy G, Beckhove U, Kogel K-H, Oostendorp M, Staub T, Ward E, Kessmann H, et al. Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. Plant Cell 1996; 8:629-43; PMID:8624439; [PubMed] [Cross Ref]
44. Koornneef A, Pieterse CM. Cross talk in defense signaling. Plant Physiol 2008; 146:839-44; PMID:18316638; [PubMed] [Cross Ref]
45. Dong X.. NPR1, all things considered. Curr Opin Plant Biol 2004; 7:547-52; PMID:15337097; [PubMed] [Cross Ref]
46. Pieterse CM, Van Loon L. NPR1: the spider in the web of induced resistance signaling pathways. Curr Opin Plant Biol 2004; 7:456-64; PMID:15231270; [PubMed] [Cross Ref]
47. Spoel SH, Koornneef A, Claessens SM, Korzelius JP, Van Pelt JA, Mueller MJ, Buchala AJ, Métraux JP, Brown R, Kazan K, et al. NPR1 modulates cross-talk between salicylate-and jasmonate-dependent defense pathways through a novel function in the cytosol. Plant Cell 2003; 15:760-70; PMID:12615947; [PubMed] [Cross Ref]
48. Xing L, Qian C, Cao A, Li Y, Jiang Z, Li M, Jin X, Hu J, Zhang Y, Wang X, et al. The Hv-SGT1 gene from Haynaldia villosa contributes to resistances towards both biotrophic and hemi-biotrophic pathogens in common wheat (Triticum aestivum L.). PLoS ONE 2013; 8:e72571; PMID:24019872; [PMC free article] [PubMed] [Cross Ref]
49. Moran PJ, Thompson GA. Molecular responses to aphid feeding in Arabidopsis in relation to plant defense pathways. Plant Physiol 2001; 125:1074-85; PMID:11161062; [PubMed] [Cross Ref]
50. Kidd BN, Edgar CI, Kumar KK, Aitken EA, Schenk PM, Manners JM, Kazan K. The Mediator complex subunit, PFT1, is a key regulator of jasmonate-dependent defense in Arabidopsis. Plant Cell 2009; 21:2237-52; PMID:19671879; [PubMed] [Cross Ref]
51. Edgar CI, McGrath KC, Dombrecht B, Manners JM, Maclean DC, Schenk PM, Kazan K Salicylic acid mediates resistance to the vascular wilt pathogen Fusarium oxysporum in the model host Arabidopsis thaliana. Australasian Plant Pathol 2006; 35:581-91; [Cross Ref]

Articles from Plant Signaling & Behavior are provided here courtesy of Taylor & Francis