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Polyamine Oxidase 5 (pao5) Mediated Antioxidant Response to Promote Salt Tolerance in Arabidopsis Thaliana

Nur-E-Ferdousy
Department of Genetics & Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Tahjib-Ul-Arif
Department of Biochemistry & Molecular Biology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

G. H. M. Sagor
Department of Genetics & Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

Polyamines are aliphatic polycations ubiquitously found in plants, involved in growth, development and many other physiological processes. Polyamine oxidases are monomeric proteins of about 55 kDa that bear a non-covalently bound molecule of FAD as a cofactor which are responsible for polyamines catabolism. They play very crucial role in polyamine homeostasis. The present study was executed to know the physiological role of polyamine oxidases by using five polyamine oxidase mutants namely pao1, pao2, pao3, pao4, pao5 along with wild type Col-0. There was almost no difference at physiological condition, but the profound difference was found under salt stress condition. Among the plants pao5 mutants which is known to have two times higher thermospermine (T-Spm) content exhibited the maximum tolerance under salt stress compared to Col-0 and other mutants. The phenomenon was also confirmed using PAO5 promoter:GUS transgenic plants. At physiological condition, PAO5 promoter activity was observed throughout the plant but intensity is very low, likely, the intensity started to increase gradually once exposed to salt stress and after 9 hours the intensity was very high. Then to reveal the underlying mechanism of stress tolerance, ROS accumulation and antioxidant enzyme activity in pao5 mutants were measured. Atpao5 showed almost lower level of ROS accumulation and higher level of antioxidant enzyme activity both in salt stress and control compare to wild type plants. Thus, indicated that lack of AtPAO5 gene has crucial role in stress tolerance through maintaining the level of T-Spm in Arabidopsis thaliana.

Keywords: Polyamine Oxidase, T-spm, Salt stress, Antioxidant, Arabidopsis thaliana

Location: Department of Genetics & Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Start Date:

End Date:

Program Area: Risk Management in Agriculture

Commodity: Soil salinity

Objectives:

To determine the Polyamine Oxidase 5 (PAO5) mediated antioxidant response to promote salt tolerance in Arabidopsis thaliana

Methodology:

Seeds of Arabidopsis thaliana accession Col-0 (WT) and T-DNA insertion lines Atpao1 (SAIL_822_A11), Atpao2 (SALK_046281), Atpao3 (GK209F07), Atpao4 (SALK_133599), and Atpao5 (SALK_053110) were used in the study (Sagor et al., 2016). Two independent lines of AtPAO5 promoter::GUS transgenic plant were also used. Seeds of all lines were surface sterilized with 70% ethanol for 1 min and 1% sodium hypochloride with 0.1% Tween-20 for 15 min, followed by extensive washing with sterile distilled water. (Sagor et al., 2016). After sterilization, seeds were sown on ½ strength MS which was semi-solidified with plus 1.5% agar plates containing 1% sucrose and B5 vitamin (MP Biomedicals, Cat # 2625149). The plants were grown at 22°C under a 14 h light/10 h dark photocycle at around 60% relative humidity. Growth response to salt stress: Sterilized seeds of Arabidopsis were grown on ½ MS agar plates containing different concentration of NaCl (0, 25, 50 and 100 mM) and kept in a refrigerator at 4 ⁰C for 48 hours for seed stratification. Then, the plates were placed at a vertical position with an 85⁰ angle and incubated in plant growth room. Recording of data at different salt concentrated media: Data on germination percentage, root and shoot length, number of cotyledon and true leaf, leaf area and chlorophyll content were recorded as described by Sagor et al. (2016). Histochemical GUS assays: Histochemical GUS assays were performed as described by Jefferson et al. (1987) with slight modification according to Sagor et al. (2012). Sixteen days old PAO5 Promoter: GUS (line 9 and line 10) transgenic plants was detached from MS agar plate and incubated with or without 100mM NaCl solution for 0, 6 and 9 hours. After treatment the plant samples were incubated with GUS staining solution in the dark at 37 ⁰C for overnight. After incubation, stained plant cells were cleared by 70% ethanol to remove the chlorophyll. Samples were taken on light plate and photographed using digital camera (Canon R250, Japan). Antioxidant enzyme activity: Activities of catalase (EC: 1.11.1.6), Guaicol peroxidase (EC: 1.11.1.6) and Ascorbate peroxidase (EC: 1.10.3.3) were measured in both normal and 100 mM NaCl treated (12h) Col-0 and pao5 mutant plants using the standard protocol (Aebi, 1984; Nakano and Asada, 1981). Determination of H₂O₂ and MDA: Malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) contents were measured according to Heath and Packer (1968). Statistical analysis: All experiments were performed with at least three biological samples and three time repetition unless mentioned. Data analysis was performed using the Statistical tools (Student’s t-test) of Microsoft Excel software.

Source of Information: J Bangladesh Agril Univ 18(2): 203–211, 2020

Article Link (Online Journal): https://doi.org/10.5455/JBAU.103110

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The present study was executed to know the physiological role of polyamine oxidases by using five polyamine oxidase mutants namely pao1, pao2, pao3, pao4, pao5 along with wild type Col-0. There was almost no difference at physiological condition, but the profound difference was found under salt stress condition. Among the plants pao5 mutants which is known to have two times higher thermospermine (T-Spm) content exhibited the maximum tolerance under salt stress compared to Col-0 and other mutants. The phenomenon was also confirmed using PAO5 promoter::GUS transgenic plants. At physiological condition, PAO5 promoter activity was observed throughout the plant but intensity is very low, likely, the intensity started to increase gradually once exposed to salt stress and after 9 hours the intensity was very high. Then to reveal the underlying mechanism of stress tolerance, ROS accumulation and antioxidant enzyme activity in pao5 mutants were measured. Atpao5 showed almost lower level of ROS accumulation and higher level of antioxidant enzyme activity both in salt stress and control compare to wild type plants. Thus, indicated that lack of At PAO5 gene has crucial role in stress tolerance through maintaining the level of T-Spm in Arabidopsis thaliana.

Knowledge Management: Journal