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. 2019 Jan 25;9(2):55. doi: 10.1007/s13205-019-1587-x

Expression analysis of the polyphenol oxidase gene in response to signaling molecules, herbivory and wounding in antisense transgenic tobacco plants

Ejaz Aziz 1,2, Riffat Batool 2, Wasim Akhtar 3, Shazia Rehman 4, Per L Gregersen 5, Tariq Mahmood 2,✉
PMCID: PMC6349263  PMID: 30729079

Abstract

Key message

We provide evidence that the expression of the PPO gene was significantly reduced in response to wounding, MeJ and herbivory in transgenic tobacco under wound-inducible OsRGLP2 promoter in an anti-sense orientation.

Abstract

Polyphenol oxidase (PPO) genes play an important role in plant defense mechanisms against biotic and abiotic stresses. In the present study, a 655 bp core sequence of the potato PPO gene was placed under the control of wound-inducible OsRGLP2 promoter in an anti-sense direction to evaluate its potential effects during biotic (Trialeurodes vaporariorum’s infestation) and various abiotic (wounding, MeJ, ABA) stresses. Transcriptional profiling of PPO gene by real-time PCR (qRT-PCR) in transgenic tobacco revealed a significant suppression (3.5-fold) of PPO in response to wounding than control plants after 24 h. In response to MeJ at different concentrations (100 µM and 200 µM), the PPO expression was greatly down-regulated by 4.7-fold after 6 h at 100 µM MeJ, and a non-significant expression was observed with ABA treatment. Moreover, significant levels of PPO reduction (sixfolds) was found in whitefly feeding assay indicating that expression of potato PPO in an anti-sense orientation had down-regulated the PPO activity. This down-regulation of PPO by wounding, MeJ and whitefly infestation clearly links the specific expression of PPO in biotic and abiotic stresses. In the future, PPO gene suppression in transgenic plants using anti-sense potato PPO gene construct can be used to inhibit enzymatic browning in fruits and vegetables, e.g., potato.

Electronic supplementary material

The online version of this article (10.1007/s13205-019-1587-x) contains supplementary material, which is available to authorized users.

Keywords: Polyphenol oxidase, Wounding, Herbivory, Elicitors, Silencing

Introduction

Plants are continuously exposed to various biotic and abiotic stresses. These stresses adversely affect plant growth and yield. Plants have evolved many different mechanisms to cope with stresses (Li and Steffens 2002). Among the biotic stresses, bacterial pathogens and insect pests are the most destructive for many crops such as tomato, sugar beet, legumes, cotton, maize, soybean, tobacco, pepper, alfalfa, potato, onion, sunflower, and citrus as well as many weeds (Bhonwong et al. 2009).

Polyphenol oxidases (PPO) are ubiquitous in plants and mainly related to defense (Constabel and Barbehenn 2008). They are present in all land plants except Arabidopsis (Tran et al. 2012). PPOs are plastid-localized nuclear-encoded enzymes that utilize molecular oxygen to oxidize phenols to quinones resulting in tissue browning, which is also observed during senescence, responses to pathogens and wounding (Mayer 2006). PPO-generated quinones alter dietary proteins in plants making them non-nutritive (indigestible) for insects (Constabel and Ryan 1998). Moreover, the induced PPO activity in many plants was found useful to resist bacterial, fungal and insect pathogens (Campos et al. 2004). Transgenic approaches provide an opportunity to study the involvement of PPO in plant defense, as its role has been validated in many plants using PPO over-expression or down-regulation tools. Down-regulation of StuPPO in transgenic tomato increased the susceptibility of plants against insects, which gained weight by feeding and more leaf area consumption (Thipyapong et al. 2004a; Mahanil et al. 2008; Bhonwong et al. 2009). However, over-expression of hybrid poplar PtdPPO hindered forest tent caterpillar from gaining weight and consuming more leaf area than control and under-expressed hybrid poplar plants (Wang and Constabel 2004a).

PPO was also found to be induced by wounding, linking its involvement in plant defense mechanisms (Aziz et al. 2017). Plants synthesize many hormones as signaling molecules which trigger defense against various stresses. Plant defense against chewing insects is modulated by jasmonic acid (MeJ) and ethylene (Stotz et al. 2000). PPO induction in aspen and hybrid poplar by injuries and MeJ showed a signal-induced PPO defense in plants (Haruta et al. 2001; Wang and Constabel 2004b). Recently, strawberry FaPPO over-expression conferring fungal resistance was found to be induced by MeJ and other stresses (Jia et al. 2016). In view of the role of PPO in plant defense, a study was designed to test the effects of herbivory, mechanical wounding and hormone treatments (MeJ, ABA) on transgenic tobacco plants harboring an anti-sense PPO gene under the regulation of a wound-inducible promoter.

Materials and methods

Plant material

Tobacco seeds (Nicotiana tabacum cv. Xanthi) obtained from National Agriculture Research Centre (NARC), Pakistan, grown in Murashige and Skoog (MS) medium (Murashige and Skoog 1962) were used in the present study. Tobacco plants were grown in a growth chamber under control conditions of 16:8 dark and light cycle at 27 °C.

Antisense polyphenol oxidase gene vector construction

PCR-amplified potato PPO copper-binding domain region (accession No. A27686.1) was directly ligated into the T/A cloning vector pTZ57R/T (Fermentas). The cloned PPO gene from T/A cloning system and the expression backbone vector p1391z_OsRGLP2 (Mahmood et al. 2013) were restricted with EcoRI and AvrII restriction enzymes. The recombinant plasmid (antisense PPO gene inserted in p1391Z_OsRGLP2) was prepared by ligating the digested and eluted PPO gene-specific insert downstream to wound-inducible OsRGLP2 promoter (already available in p1391Z) in antisense orientation and was named RGLP2P-AsPPO. The RGLP2P-AsPPO expression cassette (Fig. 1a) was transformed into E. coli (DH5α) by electroporation and the isolated plasmid was confirmed by PCR and EcoRI/AvrII double digestion. Further, the verified RGLP2P-AsPPO plasmid was elctroporated into the Agrobacterium tumefaciens strain EHA101. The plasmid of antibiotic (50 mg/L kanamycin)-resistant clones was again confirmed via PCR using gene-specific primers and restriction digestion.

Fig. 1.

Fig. 1

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a A schematic representation of the RGLP2P-AsPPO construct. p1391Z_OsRGLP2 vector with antisense-targeted region of potato PPO gene (under the control of OsRGLP2 promoter) b qRT-PCR primers representation from endogenous tobacco PPO gene. NtPPO1 qRT-PCR primers targeting the proximal coding region of the endogenous tobacco PPO gene, NtPPO2 qRT-PCR primers targeting the core region of the endogenous tobacco PPO gene, NtPPO3 qRT-PCR primers targeting the terminal region of the endogenous tobacco PPO gene

Tobacco transformation

Agrobacterium tumefaciens-mediated transformation methodology was applied to achieve genetic transformation (Horsch et al. 1989). The A. tumefaciens strain EHA101 harboring the RGLP2P-AsPPO expression cassette was used. The transformants that survived on selection medium (Hygromycin 50 mg/L) were cultured on simple 1/2 MS medium for root induction. Plant transformation was verified by PCR using PPO gene-specific primers and hygromycin primers.

Wound induction

To study the effect of wounding on the induction of PPO, the leaves of un-transformed and transgenic plants were mechanically injured (with the help of forceps) and placed for 12 and 24 h on solid MS medium. After wounding treatment, the wounded leaves were collected, frozen in liquid nitrogen and stored at − 80 °C for RNA isolation.

MeJ and ABA treatments

To examine the expression in response to hormone treatments, transgenic and non transgenic tobacco plants were treated with a solutions of ABA (Sigma-Aldrich) and MeJ (Sigma–Aldrich). Both ABA and MeJ were dissolved in ethanol (96%) and their final solution was prepared in distilled water. Transgenic and non-transgenic tobacco plants were sprayed with 100 µM or 200 µM of ABA and MeJ solutions, respectively, and harvested after 6 and 12 h. Control plants were also sprayed with Millipore water. The treated samples were stored at -80 °C till further use.

Biotic stress

Expression of the PPO gene was studied in response to insect infestation using adult whiteflies (Trialeurodes vaporariorum) on transgenic plants. The whiteflies were collected by aspiration from whitefly populations maintained on tobacco plants growing in controlled conditions (26 ± 2 °C and relative humidity 60–80%). Transgenic as well as control (non-transgenic) tobacco plants growing in pots with up to seven fully expanded leaves stage were exposed to whiteflies. The attack of whiteflies on tobacco plants was by their choice. For RNA isolation, fully expanded leaves from the middle region of the tobacco plants were harvested after 2, 3 and 5 days of infestation and stored at − 80 °C.

RNA isolation and cDNA synthesis

Total RNA was isolated from frozen samples, harvested from mechanical wounding, whitefly infestation assay, ABA and MeJ treatments along with controls using Spectrum™ Plant Total RNA isolation kit (Sigma, USA). The first-strand cDNA was obtained using 3 µg of RNA with nonamer and random oligonucleotide primers using the SuperScript II reverse transcriptase (Invitrogen).

qRT-PCR analysis

Real-time PCR (qRT-PCR) was performed in 384-well plates using the ViiATM 7 Real-Time PCR System (Applied Biosystems, USA). The PCR reaction was carried out using Power SYBR® Green PCR master mix (6 µL), 1.2 µL of each forward and reverse primer (1.5 µM), 0.5–1.0 µL of cDNA and 2.6–3.1 µL of nuclease-free water. Three different sets of primers (Fig. 1b) were designed from endogenous tobacco PPO cDNA sequences for qRT-PCR analysis and 18S rRNA and actin were selected as reference genes (Table 1). qRT-PCR for all primers was started with denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 60 s. Melting curve analysis consisting of 95 °C for 15 s, 55 °C for 1 min and 95 °C for 15 s. After successful amplification of targets, the cycle threshold (Ct) values were exported from the ViiATM 7 software and used as raw data for the analysis of qRT-PCR data. The R software and the HTqPCR (Dvinge and Bertone 2009) and Limma (Ritchie et al. 2015) add-on packages were used for the manipulation and analysis of the Ct values. The fold changes are shown on the log 2 scale.

Table 1.

List of primers used in qRT-PCR for expression analysis of endogenous NtPPO gene in transgenic tobacco plants harboring antisense PPO

Primers Primer sequence
18S

5′-GGTGGAGCGATTTGTCTGGT-3′

5′-CAGGCTGAGGTCTCGTTCGT-3′
Actin

5′-CCTGAGGTCCTTTTCCAACCA-3′

5′-GGATTCCGGCAGCTTCCATT-3′
NtPPO1

5′-ATGGACAGCGTTCCCTATTACA-3′

5′-GGAGTACATTGCTAAATACCAGTTAGC-3′
NtPPO2

5′-AACCCGTTCCGTGTGAAAGTCC-3′

5′-CTTCGATTACGCACCGATGCCA-3′
NtPPO3

5′-TCTCAAAGCTGGACAGAGCC-3′

5′-CCATCTTCGTCAAGGACCCA-3′
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18S 18S rRNA, NtPPO Nicotiana tabacum polyphenol oxidase, NtPPO1 qRT-PCR primers targeting the proximal coding region of the endogenous tobacco PPO gene, NtPPO2 qRT-PCR primers targeting the core region of the endogenous tobacco PPO gene, NtPPO3 qRT-PCR primers targeting the terminal region of the endogenous tobacco PPO gene

Statistical analysis

The data are the mean of three independent replicates. The statistical differences were compared with a t test at a significance level of P < 0.05 (*) and P < 0.01 (**).

Results and discussion

Generation of transgenic plants

Transgenic plants were developed through Agrobacterium-mediated transformation method. After co-cultivation, small bud-like calli appeared on cut edges which increased in mass up to 14 days (Fig. 2). Small shootlets started to develop after 28 days, which were cut individually from callus and shifted to simple MS media in jar for rooting. Mature plants were developed in about 45 days, shifted to the soil and maintained in greenhouse under control conditions of 16:8 dark and light cycle at 27 °C.

Fig. 2.

Fig. 2

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Regeneration of transgenic tobacco plants from leaf discs growing on selection media. a Calli emerging from cut edges of leaf discs. b 14 days old callus. c Regeneration of shoots from calli. d 28 days old shoot growing on selection media. e Shootlets growing on simple MS media for rooting. f Three-month-old transgenic plants at flowering stage growing in a greenhouse

Confirmation of transgenic plants

To confirm the insertion of transgene in transgenic plants, PCR was carried out with PPO gene-specific primers and hygromycin gene-resistant primers. High-quality PCR products of approximately 700 bp and 655 bp were obtained with Hyg and PPO primer sets, respectively (Supplementary Figs. 1 and 2).

Expression analysis in wounding

Plants being sessile continuously face environmental stresses including biotic and abiotic stresses. In abiotic stresses, wounding is common by attack of insect herbivory, wind hail or rain (Wang et al. 2014). Different defense-related genes are activated and up-regulated by wounding, pathogen infestation or predator attacks causing conspicuous changes in transcript accumulation and protein synthesis of those defense genes (Gulbitti-Onarici et al. 2009). Among these, PPO is considered important in plants defense mechanisms, as it is strongly induced by wounding, insect attacks and defense-related signals such as ABA, JA, MeJ and ethylene (Mayer 2006; Constabel and Barbehenn 2008). The role of PPO has been validated in transgenic plants revealing that PPO conferred resistance against bacterial, fungal and chewing insect herbivores (Thipyapong et al. 2007; Bhonwong et al. 2009; Jia et al. 2016).

In the present study, transgenic plants (PPO down-regulated) were mechanically wounded by forceps. Expression analysis of anti-sense PPO gene was carried out after 12 and 24 h intervals targeting proximal coding, core and terminal coding regions of the NtPPO gene (primers NtPPO1, NtPPO2 and NtPPO3) for wild and transgenic plants (Fig. 1b; Table 1). RT-PCR analysis showed a significant reduction (P < 0.05; P < 0.01) in the expression of endogenous tobacco PPO gene compared with control tobacco plants after 12 h (Fig. 3a; Table 2). The proximal coding region of PPO showed less down-regulation than core and terminal coding regions, while control plants showed an up-regulation of the endogenous PPO gene upon mechanical injury. However, after 24 h NtPPO2 showed greater down-regulation (3.5-fold) than NtPPO1 and NtPPO3. Overall, wounding depicted higher down-regulation of endogenous PPO gene over time (Fig. 3b; Table 2).

Fig. 3.

Fig. 3

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Relative expression analysis of NtPPO gene activity induced by wounding shown on the log2 scale. a After 12 h treatment. b After 24 h treatment. WT wild type, NtPPO1 qRT-PCR primers targeting the proximal coding region of the endogenous tobacco PPO gene, NtPPO2 qRT-PCR primers targeting the core region of the endogenous tobacco PPO gene, NtPPO3 qRT-PCR primers targeting the terminal region of the endogenous tobacco PPO gene. 18S and actin were used as reference genes. The data are the mean of three independent experiments and statistically significant difference is indicated by asterisks (*P < 0.05; **P < 0.01)

Table 2.

Difference in the expression level of the PPO gene in response to wounding stress in transgenic plants and WT

Stress treatment Target genes used Plants tested Expression level (mean ± SE) (12 h) Expression level (mean ± SE) (24 h)
Wounding NtPPO1 WT negative 0.00 ± 0.00 0.00 ± 0.00
WT wounded 1.41 ± 0.04 3.52 ± 0.04
Transgenic wounded − 0.90 ± 0.02* − 2.81 ± 0.02*
NtPPO2 WT negative 0.00 ± 0.00 0.00 ± 0.00
WT wounded 1.19 ± 0.05 3.52 ± 0.05
Transgenic wounded − 1.25 ± 0.01* − 3.54 ± 0.02**
NtPPO3 WT negative 0.00 ± 0.00 0.00 ± 0.00
WT wounded 1.20 ± 0.02 2.91 ± 0.02
Transgenic wounded − 1.37 ± 0.02** − 2.06 ± 0.01*
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WT wild type, NtPPO1 qRT-PCR primers targeting the proximal coding region of the endogenous tobacco PPO gene, NtPPO2 qRT-PCR primers targeting the core region of the endogenous tobacco PPO gene, NtPPO3 qRT-PCR primers targeting the terminal region of the endogenous tobacco PPO gene

Statistically significant difference is indicated by asterisks (*P <0.05 and **P <0.01)

Induced PPO expression is well studied in many environmental abiotic and biotic factors or stresses (Quarta et al. 2013). Other defense-related genes such as peroxidases and systemins also induced PPO up-regulation upon wounding indicating involvement of PPO in defense (Constabel et al. 1995). PPO induction and regulation in hybrid poplar, pineapple and rubber tree upon wounding were also reported earlier (Li et al. 2014), linking NtPPO down-regulation in transgenic tobacco in the present study. PPO down-regulation can weaken plant defense resulting in acquired leaf lesion necrosis accomplished by PPO-mediated cell death in walnut, leading to increased vulnerability against bacterial and insect attacks in tomato (Thipyapong et al. 2004a; Bhonwong et al. 2009; Araji et al. 2014). NtPPO down-regulation reached up to twofold after 24 h, showing a slightly delayed response to injuries as also reported after 36 h in Poplus trichocarpa (Haruta et al. 2001) and even after 2 days in pineapple (Zhou et al. 2003) and artichoke (Quarta et al. 2013).

Wound-induced NtPPO expression points PPO role in plant defense and can also be attributed to insect pest resistance due to its induction upon wounding and real herbivory (Haruta et al. 2001). Wound-induced NtPPO expression can be correlated to PPO over-expression which enhanced disease tolerance against Pseudomonas syringae in transgenic tomato (Li and Stiffen 2002) and transgenic Arabidopsis (Richter et al. 2012), in populus against insects (Wang and Constabel 2004a; Thipyapong et al. 2007; Bhonwong et al. 2009) and in transgenic strawberry with delayed fungal attack during fruit development (Jia et al. 2016). Soybean Gm PPO promoter exhibited an early response to Phytophthora sojae (Chai et al. 2013). Tran et al. (2012) reported that PPO is a part of defense in plants based on wound and pathogen-induced activities. The wound-induced down-regulation of NtPPO in our experiments can be attributed to a possible role in plant defense mechanism.

Expression analysis in response to ABA

To elucidate the activity of the suppressed PPO gene in response to exogenous ABA application, transgenic tobacco (anti-sense potato PPO gene) along with wild-type plants was treated with 100 or 200 µm of ABA for 6 and 12 h. The qRT-PCR analysis was carried out for wild-type and transgenic tobacco plants. Transcripts accumulation showed a significant down-regulation (P < 0.05; P < 0.01) of PPO gene in transgenic plants treated with different concentrations of ABA at different time intervals compared to WT plants. In the presence of 100 µm ABA, there was a slight up-regulation of 0.5-fold in WT plants, while a slight down-regulation of 0.3-fold was observed in transgenic plants after 6 h (Fig. 4a; Table 3). Twelve hours after using similar ABA concentration, wild-type treated plants showed an accumulation of PPO mRNA up to 0.6-fold and reduced expression of PPO gene in transgenic treated plants up to 0.5-fold (Fig. 4b; Table 3).

Fig. 4.

Fig. 4

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Relative expression analysis of NtPPO gene activity in response to 100 µM ABA treatment shown on the log2 scale. a After 6 h treatment. b After 12 h treatment. WT wild type, NtPPO1 qRT-PCR primers targeting the proximal coding region of the endogenous tobacco PPO gene, NtPPO2 qRT-PCR primers targeting the core region of the endogenous tobacco PPO gene, NtPPO3 qRT-PCR primers targeting the terminal region of the endogenous tobacco PPO gene. 18S and actin were used as reference genes. The data are the mean of three independent experiments, and statistically significant difference is indicated by asterisks (*P < 0.05; **P < 0.01)

Table 3.

Difference in the expression level of the PPO gene under different concentrations of ABA and MeJ in transgenic plants and wild type (WT)

Pytohormones Target genes used Plants tested Expression level at 100 µM concentration (mean ± SE) (6 h) Expression level at 100 µM concentration (mean ± SE) (12 h) Expression level at 200 µM concentration (mean ± SE) (6 h) Expression level at 200 µM concentration (mean ± SE) (12 h)
ABA NtPPO1 WT negative 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Transgenic negative 0.33 ± 0.02 0.38 ± 0.04 0.48 ± 0.02 0.39 ± 0.05
WT treated 0.43 ± 0.01 0.51 ± 0.01 0.72 ± 0.01 0.83 ± 0.04
Transgenic treated − 0.22 ± 0.01* − 0.43 ± 0.01* − 8.4 ± 0.03* − 0.54 ± 0.03*
NtPPO2 WT negative 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Transgenic negative 0.38 ± 0.01 0.48 ± 0.02 − 0.41 ± 0.04 − 0.43 ± 0.03
WT treated 0.44 ± 0.02 0.49 ± 0.01 0.79 ± 0.02 0.78 ± 0.05
Transgenic treated − 0.29 ± 0.01** − 0.45 ± 0.02* − 0.89 ± 0.01* − 0.93 ± 0.05**
NtPPO3 WT negative 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Transgenic negative 0.31 ± 0.02 0.45 ± 0.04 0.47 ± 0.03 0.45 ± 0.01
WT treated 0.48 ± 0.02 0.56 ± 0.03 0.86 ± 0.01 0.83 ± 0.02
Transgenic treated − 0.22 ± 0.01* − 0.48 ± 0.01* − 0.87 ± 0.02* − 0.83 ± 0.01**
MeJ NtPPO1 WT negative 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Transgenic negative 0.92 ± 0.23 0.132 ± 0.02 − 2.41 ± 0.07 − 0.153 ± 0.01
WT treated 2.44 ± 0.50 8.92 ± 0.34 5.80 ± 0.20 10.95 ± 0.25
Transgenic treated − 4.20 ± 0.06** 4.65 ± 0.27* − 4.70 ± 0.04* 7.13 ± 0.34*
NtPPO2 WT negative 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Transgenic negative 0.25 ± 0.01 − 0.77 ± 0.19 − 2.60 ± 0.01 − 0.48 ± 0.02
WT treated 2.80 ± 0.14 8.27 ± 0.21 5.93 ± 0.40 11.33 ± 0.32
Transgenic treated − 3.62 ± 0.03* 2.87 ± 0.05** − 5.37 ± 0.30** 7.34 ± 0.25*
NtPPO3 WT negative 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Transgenic negative 0.10 ± 0.03 0.35 ± 0.03 − 2.33 ± 0.05 − 0.70 ± 0.03
WT treated 0.95 ± 0.05 8.91 ± 0.28 6.11 ± 0.50 11.21 ± 0.19
Transgenic treated − 4.73 ± 0.02** 3.72 ± 0.04* − 4.59 ± 0.42* 7.47 ± 0.21*
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WT wild type, NtPPO1 qRT-PCR primers targeting the proximal coding region of the endogenous tobacco PPO gene, NtPPO2 qRT-PCR primers targeting the core region of the endogenous tobacco PPO gene, NtPPO3 qRT-PCR primers targeting the terminal region of the endogenous tobacco PPO gene

Statistically significant difference is indicated by asterisks (*P <0.05 and **P <0.01)

However, at 200 µM ABA after 6 h, the NtPPO gene showed transcript elevation level up to 0.8-fold in wild-type and down-regulation of 0.9-fold in transgenic plants (Fig. 5a). Similarly, after 12 h at 200 µM, PPO gene regulation remained unchanged after 6 h (Fig. 5b). Overall, all PPO gene regions showed similar transcript accumulation in response to ABA. Previously, Song et al. (2011) treated tomato plants with exogenous ABA and observed non-significant PPO activity between wild-type and transgenic tomato which supports our results. The antagonistic interaction between ABA and JA or salicylic acid (SA) showed resistance in Arabidopsis against F. oxysporum, in agreement with our study (Anderson et al. 2004). Likewise, elevated levels of ABA in tomato plants were negatively correlated with SA-dependent defense pathway (Audenaert et al. 2002).

Fig. 5.

Fig. 5

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Relative expression analysis of NtPPO gene activity in response to 200 µM ABA treatment shown on the log2 scale. a After 6 h treatment. b After 12 h treatment. WT wild type, NtPPO1 qRT-PCR primers targeting the proximal coding region of the endogenous tobacco PPO gene, NtPPO2 qRT-PCR primers targeting the core region of the endogenous tobacco PPO gene, NtPPO3 qRT-PCR primers targeting the terminal region of the endogenous tobacco PPO gene. 18S and actin were used as reference genes. The data are the mean of three independent experiments, and statistically significant difference is indicated by asterisks (*P < 0.05; **P < 0.01)

Furthermore, lack of significant PPO induction by exogenous ABA application in wild-type plant also supports the fact that ABA restrained the expression of several defensive genes such as PPO (Mohr and Cahill 2007). However, exogenous application of ABA resulted in induction of other defense-related genes such as POD, phenylalanine ammonia-lyase (PAL) and PPO that improved tomato’s resistance against A. solani (Song et al. 2011), whereas elevated level of ABA in Arabidopsis was linked with positive as well as negative role in disease resistance (Asselbergh et al. 2008) that might be involved in activation of other defense responses (Lee and Luan 2012).

Expression analysis in response to JA

MeJ, the volatile form of JA, has widely been used to study jasmonate signaling pathways and mechanisms of plant defense (Zhang et al 2015). In the present study, transgenic tobacco plants along with control were treated at 100 and 200 µM MeJ for 6 and 12 h. The transcript levels in transgenics along with control were analyzed by qRT-PCR for NtPPO1, NtPPO2 and NtPPO3. As shown in Fig. 6a, 100 µM MeJ after 6 h resulted in a differential expression pattern in both control and transgenic plants. However, there was an up-regulation (2.8-fold) of the target gene in WT plants at 100 µM MeJ applications. Interestingly, the expressions of NtPPO1, NtPPO2 and NtPPO3 were significantly down-regulated (P < 0.05; P < 0.01) up to 4.2-, 3.6- and 4.7-fold, respectively in transgenic plants after 6 h of 100 µM MeJ application (Fig. 6a; Table 3). The expression levels of NtPPO1, NtPPO2 and NtPPO3 in transgenics decreased by 4.6-, 2.8- and 3.7-fold, respectively, after 12 h with 100 µM MeJ, and it was significantly (P < 0.05; P < 0.01) lower than that of WT (Fig. 6b; Table 3). A similar suppression of NtPPO activity was also observed at 200 µM MeJ after 6 and 12 h (Fig. 7a, b; Table 3). However, with an increase in time period from 6 to 12 h, the transcript levels of NtPPO1, NtPPO2 and NtPPO3 increased in (non-transgenic) control plants following the treatment with 200 µM MeJ. In contrast, significant down-regulation of NtPPO was observed in transgenic plants at 6 and 12 h as compared with the control plants at 200 µM MeJ application. Overall, these results showed that silencing of PPO gene reduced the expression level of NtPPO in transgenic plants in response to both concentrations of MeJ (100 µM and 200 µM). However, pronounced reduction was observed at 100 µM MeJ after 12 h.

Fig. 6.

Fig. 6

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Relative expression analysis of NtPPO gene activity in response to 100 µM MeJ treatment shown on the log2 scale. a After 6 h treatment. b After 12 h treatment. WT wild type, NtPPO1 qRT-PCR primers targeting the proximal coding region of the endogenous tobacco PPO gene, NtPPO2 qRT-PCR primers targeting the core region of the endogenous tobacco PPO gene, NtPPO3 qRT-PCR primers targeting the terminal region of the endogenous tobacco PPO gene. 18S and actin were used as reference genes. The data are the mean of three independent experiments, and statistically significant difference is indicated by asterisks (*P < 0.05; **P < 0.01)

Fig. 7.

Fig. 7

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Relative expression analysis of NtPPO gene activity in response to 200 µM MeJ treatment shown on the log2 scale. a After 6 h treatment, b after 12 h treatment. WT wild type, NtPPO1 qRT-PCR primers targeting the proximal coding region of the endogenous tobacco PPO gene, NtPPO2 qRT-PCR primers targeting the core region of the endogenous tobacco PPO gene, NtPPO3 qRT-PCR primers targeting the terminal region of the endogenous tobacco PPO gene. 18S and actin were used as reference genes. The data are the mean of three independent experiments, and statistically significant difference is indicated by asterisks (*P < 0.05; **P < 0.01)

Mahanil et al. (2008) reported 1.5- to 7.3-fold lower PPO activity in anti-sense PPO transgenic tomato plants than non-transformed control and 40-fold decreased PPO activity was observed in PPO-silenced tomato plants than non-transformed control (Thipyapong et al. 2004a). Increased NtPPO expression by MeJ in WT tobacco may indicate MeJ defense signal against pathogens. As Constabel et al. (1995) reported, PPO activity increased rapidly after exposing wild-type tomato plants to MeJ vapors. Tobacco and hybrid poplar also strongly induced PPO in response to MeJ (Constabel and Ryan 1998). However, in case of hybrid poplar, PPO genes were found to be expressed differentially during development following MeJ treatment (Wang and Constabel 2004b; Tran and Constabel 2011). All these studies strongly support the involvement of MeJ in up-regulation of PPO gene under stress conditions. Moreover, failure to induce the PPO expression using anti-sense technique may help to overcome the browning reactions in important crops.

Expression analysis for whitefly infestation

In response to herbivory, plants have the ability to perceive signals rapidly and accurately against biotic attackers and activate the effective defense system (Mithöfer and Boland 2012). The plant must identify and respond to mechanical and chemical signals triggered upon insect attacks (Felton and Tumlinson 2008). Previously, different defense signaling pathways have been reported that were modulated after pest attack, such as JA and SA signaling pathways (Thaler et al. 2012). Trialeurodes vaporariorum is a kind of polyphagous whitefly that has caused serious losses in vegetable, horticultural and ornamentals crops worldwide (Lei et al. 1998).

The transcript level was analyzed using biotic stress caused by the whitefly (T. vaporariorum) infestation on the transgenic tobacco plants. Overall, transcript analysis by qPCR revealed a significant down-regulation (P < 0.05; P < 0.01) of PPO gene in transgenic plants after increasing the time period of white fly infestation. qRT-PCR analysis of the proximal coding (NtPPO1) region showed a slight up-regulation of 0.75-fold than the control plants after 2 days. However, the expression of NtPPO1 gene was down-regulated after 5 days of herbivory imposed by whiteflies feeding. This down-regulation was consistently followed and finally reached up to sixfold after 10 days (Fig. 8a; Table 4). Similarly, the NtPPO2 regions (core) also showed a slight up-regulation after 2 days and then followed by a gradual reduction of 2.7-fold in its expression after 5 days. After 10 days, down-regulation attained a level of fivefold in the NtPPO2 core region (Fig. 8b; Table 4). The terminal coding region (NtPPO3) showed very similar trend to the core coding (NtPPO2) region, showing an up-regulation after 2 days and a down-regulation after 5 and 10 days of whiteflies infestation (Fig. 8c; Table 4).

Fig. 8.

Fig. 8

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Relative expression analysis of the NtPPO gene activity in response to whitefly infestation shown on the log2 scale. a Expression analysis targeting the proximal coding region of the endogenous tobacco PPO gene. b Expression analysis targeting the core region of the endogenous tobacco PPO gene. c Expression analysis targeting the terminal coding region of the endogenous tobacco PPO gene. 18S and actin were used as reference genes. The data are the mean of three independent experiments, and statistically significant difference is indicated by asterisks (*P < 0.05; **P < 0.01)

Table 4.

Difference in the expression level of PPO gene in response to insect herbivory in transgenic plants

Stress treatment Target genes used Expression level (mean ± SE) (2 days) Expression level (mean ± SE) (5 days) Expression level (mean ± SE) (10 days)
Insect herbivory NtPPO1 0.74 − 2.60* − 6.0**
NtPPO2 0.90 − 2.70* − 5.04**
NtPPO3 0.32 − 1.02* − 5.67**
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NtPPO1 qRT-PCR primers targeting the proximal coding region of the endogenous tobacco PPO gene, NtPPO2 qRT-PCR primers targeting the core region of the endogenous tobacco PPO gene, NtPPO3 qRT-PCR primers targeting the terminal region of the endogenous tobacco PPO gene

Statistically significant difference is indicated by asterisks (*P <0.05 and **P <0.01)

However, comparatively greater down-regulation of PPO was shown by NtPPO1 (proximal coding region of PPO) which was ~ sixfold, indicating that the PPO gene is an important component of defense mechanism with the inducible nature of the PPO gene which was expressed in response to pathogen defense. PPO is found to be linked with the protection of plants against pathogens and herbivores. NtPPO suppression is consistent with tomato PPO anti-sense down-regulation up to 40-fold by P. syringae infestation without affecting the plant phenotype (Thipyapong et al. 2004a). Bhonwong et al. (2009) reported 1.5- to 2.9-fold reduction in PPO expression against cotton bollworm in PPO-suppressed tomato plants. Richter et al. (2012) also observed that PPO suppression enhanced the susceptibility to pathogens in dandelion. Ye et al. (2012) studied that phloem-feeding herbivorous insects can trigger transcript accumulation of PPO and POD genes that deter further insect damage in rice. Recently, induction of PPO activity was found in whitefly herbivory in wild-type tobacco plants that can be correlated with NtPPO regulation of the present results (Zhao et al. 2015) and PPO activity was recorded to be higher in whitefly-infested pepper genotypes than in non-infested genotypes (Latournerie-Moreno et al. 2015). Earlier, PPO expression was found to be reduced in cotton by silencing of the PPO gene in response to beet armyworm, which implicated PPO as a component of defensive system against insects (Bhonwong et al. 2009).

Similarly, PPO over-expression was found to enhance resistance against insect attack (Thipyapong et al. 2004a; Mahanil et al. 2008), and the anti-herbivory role of PPO was established in many transgenic plants (Wang and Constabel 2004b). PPO up-regulation engineered insect resistance in transgenic poplar against forest tent caterpillars (Wang and Constabel 2004a). Aspen PPO transcripts were also reported in elevations after forest tent caterpillar feeding (Haruta et al. 2001). The present inducible PPO gene suppression can indirectly be linked to the defensive role against herbivores as reported in several crops such as cotton (Kranthi et al. 2003), tomato (Thaler 2002) and soybean (Bi and Felton 1995). Moreover, the defensive role of PPO against insect attack was also confirmed in tomato due to the presence of herbivore-inducible signal systemin that was involved in PPO induction in response to herbivores (Constable et al. 1995).

Overall, the modification of the PPO gene in transgenic tobacco showed altered transcript levels of herbivore-induced PPO expression which is in accordance with a previous study on transgenic tobacco (Ren and Lu 2006). Herbivore-induced down-regulation of PPO gene in PPO-silenced plants confirmed the vital role of PPO in insect defense. Anti-sense down-regulation of PPO in transgenic tomato suggested enhanced vulnerability to Colorado potato beetle than control plants (Thipyapong et al. 2007). The previous works on over-expression or under-expression of PPO in transgenic plants are directly or indirectly in accordance with our results demonstrating that the PPO gene plays a significant role in insect resistance through herbivore-inducible mechanisms along with other defensive genes (Chakraborty and Chakraborthy 2005).

The present study clearly demonstrates that whitefly infestation on PPO-silenced tobacco plants repressed the PPO transcript level which might be induced in the initial stage of feeding. This down-regulation of PPO under herbivorous attack depicted the positive role of PPO during biotic stress. However, it is evident that only alteration in PPO gene expression is not sufficient for all plants against insect attacks due to complex insect resistance phenomenon. Anti-sense down-regulation of PPO in several important crops may provide insight into insect pest management programs, because over-expression of PPO in tomato and cotton was proved as a weak management strategy against lepidopterans (Hoover et al. 1998). On the other hand, insect resistance may be reduced in different plant–pest interactions if PPO activity is suppressed. Further, reduction in expression of PPO is found against insect herbivory through anti-sense technology using conserved copper-binding domain as a transgene under the control of wound-inducible promoter may be helpful in controlling browning in potato and apple (Coetzer et al. 2001; Thipyapong et al. 2004b). However, further detailed investigation will be required to assess the effects of PPO suppression and over-expression against pest–plant interactions.

Conclusion

In conclusion, our results have shown that the PPO gene was responsive to the applied stresses of wounding, MeJ and whitefly assay, but not to ABA. In response to mechanical wounding, transgenic tobacco harboring anti-sense potato PPO showed significant down-regulation of endogenous PPO after 24 h, depicting its potential role in plants during mechanical injury or biotic stress. However, ABA treatment showed no significant induction and down-regulation of NtPPO gene in wild-type and transgenic plants, respectively. Moreover, suppression of PPO gene has significantly reduced the NtPPO transcript level in response to 100 µM and 200 µM of MeJ treatments in transgenic tobacco plants that is linked with PPO induction in defense responses. Furthermore, whitefly infestation also showed down-regulation of the PPO gene in transgenic tobacco plants which indicates the critical role of PPO in biotic stress. These results indicate that the expression of potato PPO in anti-sense orientation inhibits PPO activity. Overall, PPO induction in the current study by biotic and abiotic stresses clearly links PPO to plant defense mechanism. Suppression of tobacco PPO by using the present construct harboring the anti-sense conserved copper-binding domain of potato PPO gene under the control of a plant origin wound-inducible promoter suggests that it can be a valuable tool for future analysis such as in potato to prevent enzymatic browning.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 111 KB) (111.5KB, pdf)

Acknowledgements

We acknowledge the financial support of the Higher Education Commission, Pakistan, for funding the International Research Initiative Program to conduct research work.

Author contributions

EA, TM and PLG conceived and designed the research. EA carried out the vector construction, plant transformation and expression analysis, performed data analyses and drafted the manuscript. RB, WA and TM contributed reagents and materials, participated in study design and coordination, and drafted the manuscript. SR and PLG contributed to data analyses and drafted the manuscript. All authors have read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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