Research Article - (2023) Volume 9, Issue 4
Received: 24-Mar-2023, Manuscript No. IPBMBJ-23-15957; Editor assigned: 27-Mar-2023, Pre QC No. IPBMBJ-23-15957 (PQ); Reviewed: 10-Apr-2022, QC No. IPBMBJ-23-15957; Revised: 24-May-2023, Manuscript No. IPBMBJ-23-15957 (R); Published: 31-May-2023
Proteinase Inhibitors (PIs) are distributed throughout the plant kingdom. They are characterized as natural plant defense proteins as their genes are induced by biotic and abiotic stresses and other stress prone circumstances (insect mastication, mechanical injuries, pathogen infestation, and drought and UV exposure). Expression of such stress responsive genes can be modulated with the help of signaling molecules. In this study, transgenic tobacco (Nico iana benthamiana) generated through leaf disc method harboring PI-II gene under OsRGLP2 promoter. The effect of Hydrogen Peroxide (H2O2) and Salicylic Acid (SA) were checked on PI-II gene expression in transgenic N. benthamiana. The maximum 10.4 fold induction was observed with 20 mM H2O2 and 8.05-fold with 15 mM SA. This H2O2 and SA signaling inducibility of PI-II is a strong indicative of its role in plant defense against abiotic stresses through SA and H2O2 dependent pathways. These results can be useful in studying a possible potential of PI-II gene to improve abiotic stress tolerance in plants.
Proteinase inhibitor-II; OsRGLP2 promoter; Hydrogen peroxide; Salicylic acid; Expression; Abiotic stress
PIs are naturally occurring proteins which are responsible for the inhibition of proteases. PIs have been characterized in microorganisms, animals and plants with a large number from storage organs of plants [1,2]. PIs families consist of serine PIs (serpin), kunitz type, bowman-birk and cysteine PIs (cystatins) [3]. The most widespread are serine PIs that are abundant in rhizomes, leaves and seeds of a large number of members of the Fabaceae, Solanaceae and Poaceae [4]. The plant’s defense against insects [5], bacterial and viral toxicities [6], fungal pathogens [7], as well as definite abiotic stresses [8] has been reported due to potential involvement of plant PI-II gene. Solanaceae is the only family, from which PI-II members have been reported [9]. It has been reported that PI-IIa and PI-IIb regulate the progressive Programmed Cell Death (PCD) and floral development [10]. Moreover, the metabolic rates and protein turnover is regulated via PIs to adjust the endogenous level of proteinases before and after seed germination [11]. It has been revealed that PI-II gene responds to system in, methyl jasmonate, polyethylene glycol, salt, Abscisic Acid (ABA), cold stress and electric current in numerous Solanaceae plants like Nicotiana, Capsicum annum and Solanum sp. [12]. In was also reported that the PI-II gene is induced by wounding in tomato (S. esculentum L.) [13]. While chymotrypsin, trypsin, elastase, pronase, oryzin and subtilisin are inhibited by PI-II gene [14]. Their maximum abundance has been observed in tobacco (N. naalata) flowers and tomato (S. lycopersicum) [15, 16].
Abiotic stress conditions induce ABA in plants while SA, Jasmonic Acid (JA) and ethylene are specially synthesized in biotic stresses [17]. In transgenic plants, PIs were found effective against abiotic factors explained by many research groups [18]. The level of many secondary metabolites was elevated in plants with SA and JA treatments [19]. Many physiological activities like respiration, cell development, stomata opening, seed propagation, seedlings growth and high temperature tolerance were signaled by SA in plants [20]. H2O2 mediates a suitable response to many stresses, a part of signaling molecules in plants which regulates the downstream gene expression during stress. The gene expression in plants is regulated through signal transduction pathway which can be tested by exogenous application of such signals leading to the stimulation or suppression of down-stream gene expression. PI-II gene is expressed in response to exogenous application of some signaling molecules in transgenic plants. Therefore, current study was carried out to observe the expression of PI-II gene (ligated downstream to OsRGLP2 promoter) in response to some signaling molecules (H2O2 and SA) in transgenic tobacco plant (N. benthamiana).
For the present study, seeds of T1 transgenic N. benthamiana plants developed through Agrobacterium mediated transformation harboring PI-II gene were used. In these plants the target PI-II gene was ligated to downstream of OsRGLP2 promoter in a pCAMBIA vector.
Plant Material
Transgenic seeds of N. benthamiana were sterilized, dried and spread on solid MS media for germination. Petri plates containing sterilized seeds were placed in growth chamber at conditions of 16/8 light and dark cycle at 25°C. Seeds began to germinate within a week and mature plants were obtained after 40 days which were multiplied using nodes and internodes. The well-established mature plants with proper vigor were used for expression analysis.
Expression Analysis
Expression analysis of PI-II gene was done by applying signaling molecules (SA and H2O2) on transgenic N. benthamiana through qRT-PCR. The fresh plants were used for each treatment while untreated transgenic plants were used as control.
H2O2 Stress
For experiment related to oxidative stress, two months old transgenic plants were shifted to solid MS media containing different concentrations of H2O2 (10 mM, 15 mM, and 20 mM) and plants were analyzed for expression analysis after different time intervals (6 h, 12 h and 24 h). Leaves were collected after 6, 12 and 24 h from each treatment and immediately stored in liquid nitrogen for total RNA isolation which was used for later experimentation.
SA Stress
Two months old transgenic plants were shifted to solid MS media containing 10 mM, 15 mM and 20 mM of SA. The leaves were collected from stressed plants after 6 h, 12 h and 24 h for each treatment and preserved in liquid nitrogen for further studies.
RNA Extraction and cDNA Synthesis
The harvested leaf tissue (100 mg) from untreated transgenic plants (control) and stressed transgenic plants with H2O2 and SA, was homogenized by grinding in liquid nitrogen. Afterwards, total RNA was extracted by manual method using DNase (RQ1 RNase-free DNase; Promaga M6101). The quality and quantity of isolated RNA was confirmed by Nanodrop (Nanodrop 1000 spectrophotometer, Thermoscientific). The cDNA was synthesized by adding Oligo (dT)18 primer 25 pM (Gene Link TM: e-oligos) and revert aid reverse transcriptase (15 U) using 1 μg of total RNA and cDNA samples were stored at -80°C.
Quantitative Real Time PCR (qRT-PCR)
Quantitative PCR was performed for each stress to analyze the expression of PI-II gene in comparison to house-keeping gene. The whole procedure comprised of the following steps; Using the MyGo-Pro RT-PCR System, ‘SYBR’ was used as a reporter dye. The total reaction volume was 12 μl comprising 2 μl cDNA (1:5 dilution), 6 μl (Invitrogen) SYBR Green master mix, 2 μl (Forward and Reverse) Primer mix (25 pM) and 2 μl Nuclease free water. A particular set of primers for gene of interest (PI-II) and housekeeping gene (Actin) were used. The RT-PCR reaction was carried with pre-denaturation at 95ᵒC for 10 minutes, 40 cycles of denaturation at 95 ᵒC for 10 seconds, annealing at 57.3ᵒC for 45 seconds and extension at 72ᵒC for 15 seconds steps.
Data Analysis
In terms of PI-II gene expression, raw data was collected for qRT-PCR data analysis. Mathematical model CT (2-ΔΔCT) using MyGo-Pro qRT-PCR software was used to analyze the data. For 2-ΔΔCT method, an internal control and calibrator was selected. In each reaction, the level of PI-II transcript driven by OsRGLP2 promoter as compared to control (untreated) was varied significantly by relative quantification of the samples. The fold change was calculated, and the graphical form of data generated in response to each stress.
Transgenic tobacco plants were used to check the expression of PI-II gene in response to H2O2 and SA.
Induction of PI-II gene in Response to H2O2
To evaluate the expression of PI-II gene, T1 transgenic plants were subjected to H2O2 stress. Overall the PI-II gene activity was found highly responsive with 20 mM H2O2 after 24 hours. The PI-II gene response showed an increase trend with the increase of H2O2 concentration. At 20 mM H2O2 after 6 hours, the induction of PI-II gene increased up to 7.36 fold and reached the maximum 10.4 fold after 24 hours. Furthermore, with the increase of H2O2 concentration (10 mM-20 mM), the PI-II gene expression also increased and reached to maximum at 20 mM. While the high expression (10.4 fold) was observed at 15 mM after 24 while with 10 mM the expression was not significant rather remained almost same near to 7 fold (Figure 1). It was observed that an overall increase in expression pattern of PI-II gene in response to H2O2 was remarkable.
Figure 1: Quantification of PI-II gene in response to 10, 15 and 20 mM H2O2 stress treatment. One-month old T1 transgenic Nicotiana lines were subjected to 10, 15 and 20 mM H2O2 growing on MS media and real-time PCR was performed after 6, 12 and 24 h to detect fold induction of PI-II gene. Figure data is mean ± of three independent readings (P<0.01, n=3).
Induction of P I-II Gene in Response to SA
Different concentrations of SA (10, 15 and 20 mM) were used to check the expression profile of PI-II gene which was found to be up-regulated up to maximum 8.05 fold with 15mM SA treatment (Figure 2). At 20 mM SA, the maximum induction of PI-II (6.9 fold) was significant after 12 h. Unlike H2O2, lower level of PI-II gene expression was observed with 10 mM SA treatment. The OsRGLP2 showed a response to SA signaling.
Figure 2: RT-PCR analysis of PI-II gene expression in response to 10, 15 and 20-mM SA application. Quantitative real-time PCR was carried out to detect fold induction of PI-II gene in one-month old T1 transgenic Nicotiana lines growing on MS media. Figure data is mean ± of three independent readings (P<0.01, n=3).
PIs are natural defense proteins and various signaling molecules such as JA, SA, ABA and ethylene are related to defense responses against phytopathogens, abiotic stresses and regulation of developmental stages, senescence and fertility. Both H2O2 and SA involve in Hypersensitive Response (HR), Systemic Acquired Response (SAR) and PAMP-Triggered Immunity (PTI) in plants. SA is categorized as biotic stressresponsive hormone in biochemical reactions and physiological responses. H2O2 is highly responsive to Pathogen-Associated Molecular Patterns (PAMPs), a part of recognition of pathogen infection as well as stable component of Reactive Oxygen Species (ROS). In the present study, the expression of PI-II gene in response to H2O2 and SA in T1 transgenic tobacco indicated that PI-II gene is highly responsive to these signaling molecules. The PIs expression was enhanced by abiotic stress factors such as ABA and H2O2. PIs induced defense related genes in excised tomato treated with glucose oxidase-glucose (G/GO), a H2O2 generating system, which contributes a continuous production of H2O2 within the applets leading to 80% accumulation of PI-I within 2 hour. H2O2 in bacterial pathogen infestation induced a plasma membrane intrinsic gene (At PIP1; 4) in A. thaliana leaf aquaporin. It was demonstrated that H2O2 may lead to enhance tolerance against pathogens, high temperature and oxidative stress in wild tomato (Solanum pennellii) cells. Furthermore, H2O2 also regulate the plant defense and HR related genes.
In transgenic tobacco, the PI-II expression after 6, 12 and 24 h with 10, 15 and 20 mM H2O2 concentrations which is in accordance with Arabidopsis suspension cultures in which 10 mM H2O2 resulted in cell death after 6 h reaching the maximum with 20 mM H2O2 after 24 h. Similarly, the Vacuolar Processing Enzymes Cysteine Proteinase (γVPECP) expression in Arabidopsis suspension cells was also increased with H2O2 exposure causing Programmed Cell Death (PCD) with higher PIs expression. Plant defense system produces GST (Glutathione S-transferase) mRNA in response of PCD which initiates defense mechanism by inducing the GSH (Glutathione) and ROS (H2O2, O2.) with maximum expression of defensive gene after 6 h and 24 h which is in consistent with the present up-regulation of PI-II gene in response to H2O2. The increased expression of Glycine soja cysteine (GsCPI14), Panax ginseng Cysteine (PgCPI) PI and Capsicum annum (CaPI-II) PI were observed against abiotic stresses.
In the present study, PI-II gene expression after 6 hours at 10 mM H2O2 which is similar to NtPI up-regulation with same concentration of H2O2. Salinity tolerance is also induced by activation of enzymatic ant oxidative defense system by exogenous application of H2O2. Alscher, Erturk reported that plant protection from superoxide, anion toxicity under oxidative stress and Superoxide Dismutase (SOD) activity plays a vital role through dismutation of superoxide to H2O2 and O2. The oxidative stress is controlled by SOD in peroxisomes, chloroplast and mitochondria organelles. For cell homeostasis, the control of H2O2 concentration is crucial in plants.
The SA perception in plants directly interacts with Pathogens Related (PR) defensive gene activation. In tomato and potato plants, both the HR and subsequent expression of SAR are required SA. In the current study, the PI-II gene was expressed the maximum after 12 h and then declined after 24 h because at very specific concentration of 20 mM SA. This upregulation of PI-II gene may occur due to the presence of some specific cis-regulatory elements in OsRGLP2 promoter. Exogenous SA also mediates ROS production enhancing the plant defense mechanism.
According to Miura and Tada, during plant development under abiotic stress such as chilling, heat, drought and salt stresses, SA being a phenolic compound confers a significant part. SA induced a variety of PR genes responsible for plant defense and in SA-dependent defense signaling, PR1a expression is often used as a reporter. In mature tobacco leaves and Gossypium hirsutum, the acidic PR-1 genes were identified and found highly expressed by SA after 12 h also a case in the present PI-II gene expression after 12 h of SA application at 15 mM.
Furthermore, S. lycopersicum Mitogen-Activated Protein Kinase (SlMAPK3) in immune responses were regulated by SA and Methyl Jasmonic Acid (MeJA). This SlMAPK3 expression by exogenous SA and MeJ increased the level of defenserelated genes (PR1, PR1b, SlPI-I and SlPI-II) after 12 hours which is in agreement with the present higher PI-II expression with SA after 12 h.
In the present study, the exogenous application of H2O2 and SA to T1 transgenic tobacco (N. benthamiana) increased the expression profile of PI-II gene showing that PI-II gene plays key role in plant defense against abiotic stresses. These results can be used for potential understanding of plant responses to such signaling molecules. Conclusively, the PI-II is a defenserelated gene that can be tested for developing transgenic crops tolerant to abiotic stresses.
This project is partially funded by Higher Education Commission (HEC) and Pakistan Sciences Foundation (PSF).
The authors declare that they have no conflict of interest.
TM conceived and designed research. NKB conducted experiments. NKB, WA, and SR and analyzed data, prepared figures and wrote the manuscript. SLS revised the manuscript. All authors read and approved the manuscript.
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Citation: Bangash NK, Rehman S, Akhtar W, Aziz E, Mahmood T, et al. (2023) Characterization of Tomato Proteinase Inhibitor-II Gene in Response to Salicylic Acid and H2O2 Signaling Molecules in Transgenic Tobacco. Biochem Mol Biol J. 9:31.
Copyright: © 2023 Bangash NK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
