ROS-dependent DNA damage and repair during germination of NaCl primed seeds

https://doi.org/10.1016/j.jphotobiol.2020.112050Get rights and content

Highlights

  • ROS production is reduced during priming with different concentration of NaCl resulting in reduced DNA damage.

  • Priming with 320 mM NaCl enhances germination by reactivating DNA repair mechanism.

  • Early reactivation of DNA repair and cell cycle activity is associated with early germination of seeds.

  • Priming with 320 mM NaCl enhances antioxidant machinery during germination to maintain required oxidative window.

Abstract

Reactive oxygen species (ROS) generated during rehydration of seeds is a major source of cellular damage. Successful germination depends on maintaining the oxidative window and ability of the cells to repair the DNA damage accumulated during seed developmental process, maturational drying, and germination. We explored the role of DNA damage, repair, cell cycle progression and antioxidant machinery in germination of seeds of Solanum melongena L. primed with 0, 320, 640 and 1200 mM sodium chloride (NaCl). The expression of antioxidant genes such as ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase2 (CAT2), and glutathione reductase (GR) was upregulated to maintain the oxidative window required for germination in seeds treated with 320 mM NaCl. ROS generated upon treatment with 320 mM NaCl resulted in minimal DNA damage and activated non-homologous end joining (NHEJ) and mismatch repair (MMR) pathway genes such as KU70 and mutS homolog 2 (MSH2) respectively. Treatment with higher concentrations of NaCl resulted in increased DNA damage despite lower ROS, without evoking DNA repair mechanisms. Uncontrolled rehydration resulted in higher levels of ROS and DNA damage, but activation of homologous recombination (HR) pathway gene, Nijmegen breakage syndrome 1 (NBS1), and genes involved in repairing oxidized guanine, such as oxoguanine DNA glycosylase (OGG1) and proliferating cell nuclear antigen (PCNA). In summary, controlled rehydration with 320 mM NaCl decreased the DNA damage, reactivated the antioxidant and DNA repair machinery, and cell cycle progression, thereby enhancing the seed germination.

Introduction

Seed germination is an intricate attribute of a plant with ecological and agricultural importance. Germination is one of the two major irreversible phase-transitions in plants, the commitment to flower being the other [1]. This complex process originates with the uptake of water and ends with the protrusion of radicle through the seed coat. The uptake of water is a triphasic event – the initial phase in which there is high influx of water due to relatively low water potential of the dry seeds, a middle plateau phase when the water uptake reaches a saturation point, and the emergence of radicle with an increased uptake of water in the final phase [2]. The viability of the seed is dependent on the stability of DNA during tissue-controlled maturational drying and the ability of the seed to repair DNA damage caused by free radicals during seed storage and germination [3]. The rehydration process elicits intricate cellular and metabolic activities leading to de novo synthesis of nucleic acids and proteins, initiation of DNA repair and activation of antioxidant mechanisms. Consequently, DNA repair during initial rehydration is critical for the successful emergence of radicle as the DNA damage accumulates in the cells due to poor repairing capacity during maturational drying and storage [4]. In plants, DNA strand breaks are repaired by the non-homologous end joining (NHEJ) and homologous recombination (HR) pathways, which play a pivotal role in double-strand break (DSB) repair [5]. The Nijmegen breakage syndrome 1 (NBS1) protein is an essential component of DNA damage signaling pathway and its role in HR mediated DNA repair and meiotic homologous recombination is well established in vertebrates and plants [6,7], whereas KU70 is the hallmark of NHEJ. KU70/80 heterodimer, a key protein in core-NHEJ, binds and processes the DNA damage as well as recruits other proteins to facilitate DNA break repair [8]. Two other important genes involved in overcoming the damage caused by the production of 8-oxoguanine due to excess of reactive oxygen species (ROS), 8-oxoguanine DNA glycosylase (OGG1) and proliferating cell nuclear antigen (PCNA) [9] were found to be upregulated in germinating seeds of Medicago truncatula in the presence of HDAC inhibitor [10]. However, DNA damage during imbibition can also delay the germination process [4]. For successful seed germination, the plant cell has to evoke an appropriate repair pathway depending on the type of DNA damage. In the majority of the plant species, most of the cells are arrested at the G0 or G1 phase, while a few cells are halted in S or G2 phase due to the suppression of metabolic activities in the dormant or quiescent seeds. However, there are ambiguous reports on cell cycle events during the different phases of germination. The accumulation of β-tubulin, initiation of DNA synthesis and increased cell cycle activity was observed in seeds of tomato, barley and gymnosperms during the initial period of imbibition [[11], [12], [13]].

ROS plays a dual role in the germination of seeds with its activity varying intensely during maturational drying and germination. In sunflower (Helianthus annuus), the loss of seed viability is associated with the accumulation of H2O2 in the embryonic axis [14]. On the contrary, H2O2 initiates the germination of pea (Pisum sativum) seeds by changing specific proteome, transcriptome and hormonal levels [15]. The carbonylation of protein reserve by ROS and the energy generated during lysis of carbonylated proteins were essential in the process of germination [16,17]. ROS was also shown to be a major signaling molecule in mediating dormancy release and germination, by mediating expression of genes encoding hydrolytic enzymes; peroxidases that weaken the testa and endosperm cap in seeds like tomato, Arabidopsis and lettuce [18,19]. Plants have developed well-orchestrated enzymatic and non-enzymatic free radical scavenging systems to regulate the ‘oxidative window’ – a fine balance between germination-promoting oxidative signaling and germination-delaying oxidative damage [20]. Several studies have shown the importance of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) during seed germination [[21], [22], [23]].

Solanum melongena L. is an important vegetable crop commonly known as brinjal, aubergine, or eggplant. A brinjal variety called Mattu Gulla (MG) growing in the Mattu village of Udupi district, Karnataka, India has immense commercial importance [24]. The fruits of MG are round, with green and white stripes on its skin and are known for their unique aroma and flavor. Recently, Geographical Indication Registry, Chennai, India has provided a Geographic Indication status to MG variety of brinjal. However, the commercial importance of the crop has been severely hindered by seed dormancy.

Seed priming, where seeds are exposed to organized rehydration to activate the pre-germinative metabolism, is one of the techniques generally employed to break the dormancy. The hydration treatment before sowing results in synchronous and quick germination, since phase I of water uptake is completed before planting [25]. We have earlier reported the positive effect of priming with single bondHe-Ne laser on seed germination in brinjal [26], while other studies have shown that NaCl as priming agent confers salinity stress tolerance [27]. Therefore, in the present study, we explored the role of NaCl-priming on DNA damage and reactivation of DNA repair, cell cycle activity and regulation of antioxidant system associated with the germination of S. melongena L. seeds.

Section snippets

Seed Treatment

Seeds of S. melongena L. var. MG were collected from ripe fruits and stored in airtight bottles at 25 °C as described earlier [28]. The healthy and morphologically uniform seeds were selected and primed in different concentrations of NaCl (320, 640 and 1200 mM) in dark at 25 °C for 48 h. Control seeds were imbibed in distilled water. Each treatment consisted of 25 randomly selected seeds and the experiment was carried out in triplicate. The control seeds and seeds primed with different

Effect of Priming on the Viability of Seeds and Moisture Content

The seeds were allowed to imbibe in different concentrations of NaCl for 48 h or distilled water and then tested for viability using tetrazolium chloride. The pink coloration of the control and primed seeds treated with 320, 640 and 1200 mM NaCl indicated that all the seeds were viable and the microscopic images of viable and heat-killed non-viable seeds are shown in Fig. S1. The seed moisture content, determined gravimetrically, increased exponentially during the initial 7 h of imbibition in

Discussion

Seed aging is a serious agronomic issue and has received quite a lot of attention from seed biologists across the world. ROS is one of the major factors responsible for seed aging by damaging the cell membrane and affecting various biological macromolecules (proteins, mRNA and DNA) besides other unknown factors [38]. The time-dependent accumulation of ROS in the embryonic axis is the main reason for the loss of viability in the stored seeds [[39], [40], [41]]. ROS is known to cause oxidation of

Conclusion

In the present study, brief priming with mild NaCl enhances the germination of S. melongena L. var. MG. An optimum level of ROS is maintained by activating antioxidant systems with controlled rehydration resulting in lowered DNA damage, and activating DNA repair machinery, thereby aiding in progression through the cell cycle and resulting in the early onset of germination. However, accumulation of DNA damage without activating the repair machinery at higher NaCl concentrations resulted in

Author's Contribution

AM, TSM, KS and KRK conceptualized and designed the study; KRK carried out the experiments, data acquisition, analysis and interpretation of data with the help of VBD, PSS, and KP; AM, TSM, SPK and KS analyzed the data; KRK wrote the manuscript, KRK, TSM, SPK, AM and KS revised the MS and all the authors gone through and approved.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of Competing Interest

We declare that there is no conflict of interest.

Acknowledgments

We thank Manipal Academy of Higher Education (MAHE), Manipal, Karnataka, India and TIFAC-CORE and FIST, DST, New Delhi, and K-FIST, VGST, Govt. of Karnataka for the facilities. We are grateful to MAHE for Dr. T.M.A. Pai Ph.D. scholarship to Kiran K.R., Deepika V.B. and Swathy PS and Council of Scientific and Industrial Research (CSIR), Govt. of India for Senior Research Fellowship to Kiran K.R. (09/1165(0006)/2018-EMR-I). We would like to thank Dr. B.S. Satish Rao, Dr. Shubhankar Das and Dr.

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