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Agricultural Science Digest, volume 42 issue 6 (december 2022) : 756-759

Mechanical Wounding Induces Catalase Gene (VsCat) in Leaves of Common Vetch

Saeid Abu-Romman1,*, Tarek G. Ammari2
1Department of Biotechnology, Faculty of Agricultural Technology, Al-Balqa Applied University, Al-Salt 19117, Jordan.
2Department of Water Resources and Environmental Management, Faculty of Agricultural Technology, Al-Balqa Applied University, Al-Salt 19117, Jordan.
Cite article:- Abu-Romman Saeid, Ammari G. Tarek (2022). Mechanical Wounding Induces Catalase Gene (VsCat) in Leaves of Common Vetch . Agricultural Science Digest. 42(6): 756-759. doi: 10.18805/ag.D-337.
Background: In plants, wounding can result from mechanical injuries or from biotic factors induced by herbivores infestation and pathogen infection. Wounding enhances the production of reactive oxygen species (ROS). Enzymatic and nonenzymatic defense systems have been reported in plants to immediately combat increased levels of ROS. Plant catalases are encoded by a multigene family and are the major scavenging enzymes catalyzing the dismutation of toxic hydrogen peroxide to water and dioxygen. 

Methods: In the preasent work, a quantitative real-time PCR was used to quantify the expression level of a catalase gene from common vetch (Vicia sativa; VsCat) in response to mechanical wounding. 

Result: The results of the time course study showed that the transcript levels of VsCat were significantly increased in wounded leaves at all-time points examined with a peak expression of 7.6 folds at 2 h post wounding. The increased expression of VsCat might represent a direct defense against elevated H2O2 generated during wounding.
During their life cycle, plants are frequently exposed to different biotic and abiotic stress factors (Abu-Romman, 2016a; Salman et al., 2017; Abid et al., 2020). These stresses lead to a series of molecular and metabolic responses, adversely affecting crop growth, development, and productivity (Al-Momany and Abu-Romman, 2014; Ahanger et al., 2017; Sadder et al., 2019).
Under normal growth conditions, reactive oxygen species (ROS) are normally and continuously produced at minimal levels in different subcellular compartments. However, environmental stresses enhance the production threshold of ROS to the level that imposes oxidative damages to membranes and macromolecules (Apel and Hirt, 2004; Gill and Tuteja, 2010; Farooq et al., 2019). Under stress conditions, enzymatic and non-enzymatic scavenging mechanisms are operating to control elevated ROS levels. Superoxide dismutase, ascorbate peroxidase, catalase and glutathione reductase are the major detoxifying enzymes to combat ROS (Abu-Romman and Shatnawi, 2011). Whereas, the main non-enzymatic antioxidants are ascorbate and glutathione (Basu et al., 2010).
Leaf wounding can result from biotic factors by herbivores infestation and pathogen infection, or it can result from abiotic means in the form of mechanical injury like heavy precipitation, wind and harvesting (León et al., 2001). Wound responses in plants involve the activating of multiple signaling pathways responsible for healing and defense-responsive genes (Wasternack et al., 2006). ROS were reported to rapidly accumulate in wounded leaves and play a role in signaling (Suzuki and Mittler, 2012; Al-Momany and Abu-Romman, 2016; Lew et al., 2020).
Catalase (E.C. is the major scavenging enzyme which catalyzes the dismutation of toxic H2O2 to water and dioxygen (Asada, 1999). These heme containing enzymes are mainly peroxisomal and unlike animals, catalases in plants are encoded by a multigene family (Kamigaki et al., 2003; Mhamdi et al., 2010). Different catalase classes possess different expression patterns in different organs and in response to stresses (Purev et al., 2010; Su et al., 2014; Sun et al., 2018). A catalase gene was cloned and characterized from the forage legume common vetch (Vicia sativa), and this gene was up-regulated under different abiotic stresses and hormonal stimuli (Abu-Romman, 2016b). The molecular responses of this gene is not yet investigated in wounded leaves of common vetch. Therefore, the present study aims at investigating the expression pattern of catalase gene (VsCat) in response to wounding in common vetch.
Plant material and wounding
Seeds of common vetch (Vicia sativa cv. Mahali) were planted in a greenhouse as previously described (Abu-Romman, 2019). Mechanical wounding was carried out on intact three-week old common vetch plants by wounding 6 leaves per plant using scissors without damaging the main leaf vein. Collected tissues from wounded and unwounded leaves were quickly frozen in liquid nitrogen.
RNA isolation and VsCat expression
Total RNA was extracted from wounded leaves using Total RNA Purification Kit (Jena Bioscience, Germany), and cDNA from was synthesized from 3 μg of total RNA using PrimeScript™ RT Master Mix (TaKaRa, Japan).
The relative expression pattern of VsCat was explored in wounded leaves by quantitative real-time PCR (qRT-PCR) using KAPA SYBR® FAST qPCR Kit (KAPA BIO, USA). Primers used in qRT-PCR to amplify VsCat (forward: 5’-ATCCCCAGACTCACATCCAGG-3’; reverse: 5’-TTTCC CAGCCTTGTTGAGCAG-3¢) were designed based on VsCat coding sequence available in GenBank (Accession No. KX090583.1). Data of expression analysis were presented as fold change of VsCat transcripts normalized to V. sativa Actin (VsAct) reference gene (Accession No. GU946218) and set relative to that in unwounded plants, using the 2-DDCt method (Schmittgen and Livak, 2008). Primers used to amplify VsAct (forward: 5’-CAATCCA GGCCGTCTTGTCTC-3’; reverse: 5’-TCTGTTAAATCACG CCCAGCA-3’) yield 157 bp amplicon. Results were expressed as means of three biological replicates and were statistically analyzed using LSD.
Environmental stresses induce a drastic accumulation of reactive oxygen species (ROS) in different cellular compartments. These ROS cause oxidative damages to the major cellular macromolecules (Apel and Hirt, 2004; Gill and Tuteja, 2010; Farooq et al., 2019). Defense systems have been evolved in plants to immediately combat increased levels of ROS. Enzymatic and nonenzymatic antioxidants detoxify ROS in different organelles (Basu et al., 2010; Abu-Romman and Shatnawi, 2011). In this work, expression analysis was performed using qRT-PCR to explore the time course changes of transcript levels of Cat gene following mechanical wounding in common vetch.
Transcript levels of VsCat were significantly increased in wounded leaves at all-time points examined (Fig 1). The expression of VsCat was gradually up-regulated and scored 3.4- and 5.7-fold increase at 0.5 and 1 h post wounding, respectively. Afterward, the expression level further increased and reached a peak of 7.6 folds at 2 h post wounding treatment. On the other hand, drops in VsCat expression were recorded at 4 and 6 h of treatment and reached 3.9- and 3-fold, respectively. In plants, wounding caused from abiotic and biotic factors and induces the generation of ROS (León et al., 2001; Maffei et al., 2007). The increase in transcript levels of VsCat might represent a direct defense against elevated H2O2 generated during wounding. Moreover, different studies have shown increased levels of JA biosynthesis in wounded plants (Nyathi and Baker, 2006; Chen et al., 2019), which might contribute in increasing VsCat expression. In accordance to the present observation, different studies have showed increased Cat expression in response to wounding-associated H2O2 accumulation (Guan and Scandalios, 2000; Dong et al., 2015). Taking into account that VsCat encodes a peroxisomal Cat enzyme (Abu-Romman, 2016b), wounding was reported to induce the expression of peroxisome biogenesis genes (Lopez-Huertas et al., 2000).

Fig 1: Expression analysis of CAT in response to wounding in Vicia sativa.

Work conducted in the corresponding author’s laboratory is supported by Abdul Hameed Shoman Foundation. The authors would like to thank the National Agricultural Research Center (Jordan) for providing seeds of common vetch.
Saeid Abu-Romman and Tarek Ammari: Experimental design and article drafting.
Saeid Abu-Romman: Experimental operation.
I declare that the publication of this paper does not involve any conflict of interest between individuals or groups.

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