28
Figure 2.7 Effects of ascorbic acid (AsA) and glutathione (GSH) on intracellular ROS accumulation in BY-2 cells under selenate stress. Intracellular ROS levels in 4-day-old Se-unadapted tobacco BY-2 suspension cells under selenate stress in the presence or absence of AsA and GSH. The percentage of fluorescence intensity is shown as a ROS level. Averages from three independent experiments (n=3) per bar are shown. Bars with the same letters are not significantly different at P <0.05.
29
Amini et al. 2015). Therefore, the reduction of cytotoxic GSA is critical to the stress mitigation by exogenous proline, that is, inhibition of P5CDH activity by the stress can impair the mitigation by proline. The present results in this study suggest that selenate inhibits P5CDH, resulting in accumulation of cytotoxic GSA. However, it is difficult to quantitate GSA and P5C because they are equilibrated mixture.
Both salt stress and arsenate stress increased the number of Evans Blue-stained BY-2 cells (Banu et al. 2009, Nahar et al. 2017). However, the salt-induced cell death was suppressed by supplementation of proline (Banu et al. 2009) but the arsenate-induced cell death was aggravated by proline (Nahar et al. 2017). Proline induced cell death by ProDH activation, which leads to GSA accumulation (Amini et al. 2015). Hence, the difference in response to proline between salt-induced cell death and arsenate-induced cell death may be due to proline metabolism such as GSA accumulation. In this study, selenate stress induced cell death of BY-2 but the cell death was not affected by exogenous proline (Figure 2.2), which may be accounted for the proline metabolism.
Several studies have reported that selenate stress can increase intracellular ROS accumulation in plants (Tamaoki et al. 2008, Akbulut et al. 2010, Schiavon et al. 2012). Similarly, this study showed that intracellular ROS accumulation was increased by selenate stress (Figure 2.3A). In plants, low concentration of Se reduces the toxic effects of heavy metals by alleviating oxidative damages while high dose of Se induces the oxidative stress (Malik et al. 2012, Chen et al. 2014, Jozwiak et al. 2019). In this study, selenate stress increased antioxidant activities (Figure 2.4) and drastically induced selenate uptake (Figure 2.5), resulting in growth inhibition and cell death.
30
Islam et al. (2009) reported that Cd stress increased lipid peroxidation and supplementation of proline decreased Cd-induced lipid peroxidation in BY-2 cells.
Several studies have reported that salt-induced lipid peroxidation is decreased by the exogenous application of proline (Okuma et al. 2004, Demiral et al. 2004, Banu et al. 2009). The present study showed that selenate stress did not affect the MDA content and that exogenous proline also did not show any effect on MDA content under selenate stress (Figure 2.3B). These results indicate that the level of oxidative damage was not high enough to induce lipid peroxidation in selenate-treated BY-2 cells and that selenate inhibited BY-2 cell growth not via increasing oxidative damage.
It is well-known that proline plays an important role in protecting different enzymes against stresses such as salinity stress (Tsugane et al. 1999, Hong et al.
2000, Okuma et al. 2000, 2002). Some previous studies reported that the antioxidant defense system under salt and Cd stress was increased by exogenous proline application in BY-2 cells (Okuma et al. 2004, Hoque et al. 2007, Banu et al.
2009). Hossain et al. (2010) reported that the ascorbate peroxidase (APX) activity was increased in response to Cd stress on mung bean and supplementation of proline also increased APX activity under Cd stress. It has been reported that the APX, CAT, and POX activities are increased in response to salt stress and supplementation of proline also increases APX, CAT, and POX activities under salt stress on rice cultivars and suggest that proline detoxifies H2O2 by enhancing APX, CAT, and POX activities under salt stress (Hasanuzzaman et al. 2014, Bhusan et al. 2016). In this study, selenate stress increased activities of SOD, CAT, APX, and POX (Figure 2.4). Exogenous application of proline increased the APX and POX activities (Figure 2.4C, D) but did not affect either ROS accumulation (Figure 2.3A)
31
or the lipid peroxidation (Figure 2.3B) in the BY-2 cells under selenate stress. In this study, proline enhanced APX and POX activities (Figure 2.4C, D) and also accelerated selenate accumulation (Figure 2.5). Taken together with the result that proline did not mitigate selenate stress (Figure 2.1 and 2.2), these results suggest that the increment of APX and POX activities by exogenous proline is not sufficient to suppress selenate stress due to selenate accumulation.
Both AsA and GSH are known as a vital component for the detoxification of ROS in plants (Smirnoff et al. 2000, Foyer and Noctor 2011, Qian et al. 2014).
Exogenous application of AsA improves phytotoxic effects of selenium on rice by reducing oxidative damage (Sharma et al. 2014). Jung et al. (2019) reported that exogenous application of GSH alleviates heavy metal-induced oxidative damage and enhances heavy metal stress resistance in rice plants. In this study, the exogenous application of AsA and GSH significantly decreased the ROS accumulation in the selenate-stressed cells (Figure 2.7). However, GSH application significantly mitigated the growth inhibition by selenate (Figure 2.6) but AsA application did not significantly alleviate the growth inhibition (Figure 2.6). It has been reported that interference of GSH synthesis in plants by selenate or other Se compounds may diminish plant defense against hydroxyl radicals and oxidative stress (Terry et al. 2000). Hence, in this study, the exogenous application of GSH might increase the GSH level in BY-2 cells and the increased GSH might suppress the selenate toxicity. These results suggest that the application of proline does not always mitigate growth inhibition by heavy metals and suggest that the growth inhibition by selenate is not accounted for by ROS accumulation.
32 2.6 Conclusion
The presented results indicate that selenate induces cell death and inhibits growth in BY-2 cells while selenate elevates antioxidant enzyme activities and that proline enhances antioxidant enzymes such as APX and POX and also selenate accumulation. It is concluded that proline improves antioxidant activities and enhances selenate uptake and that the balance may account for growth inhibition and cell death.
33
CHAPTER 3
General Summary
The naturally occurring metalloid selenium (Se) has emerged as a potential health hazard for plants, animals, and humans, due to its extensive accumulation in the soils over the past decades. It is a micro-nutrient for animals but not for most plants and Se concentrations at higher than 100 µM can be hazardous for all biotas including plants and animals. Selenium plays dual role in plants, for instance, it reduces negative consequences of abiotic stresses and it can improve plant growth and development when applied at relatively low concentrations but Se at excessive concentrations leads to toxicity in plants, resulting in chlorosis and necrosis as well as restricted growth and reduced protein biosynthesis. Proline is accumulated as a compatible solute in plants under various stress conditions. Exogenous proline scavenges free radicles, improves plant metabolism and stimulates plant growth under stress conditions. It has been reported that exogenous application of proline can improve tolerance to a variety of environmental stresses in plants. It was reported that the growth inhibition by salt or cadmium (Cd) was alleviated by supplementation of proline in the BY-2 cells. However, whether proline mitigates selenate stress in BY-2 cells are to be investigated. In this study, I investigated the effects of exogenous proline on tobacco BY-2 cells cultured under selenate stress and I found that Selenate at 250 µM significantly inhibited the growth of BY-2 cells while exogenous proline at 10 mM did not mitigate the inhibition by selenate. I also found that selenate increased the number of Evans blue stained cells regardless of addition of 10 mM proline. However, it has been reported that salt-induced cell death was suppressed by supplementation of proline and arsenate-induced cell death was
34
aggravated by proline. Hence, the difference in response to proline between salt-induced cell death and arsenate-salt-induced cell death may be due to proline metabolism such as GSA accumulation. In this study, the cell death by selenate was not affected by exogenous proline which may be accounted for the proline metabolism. To insight into the issue that proline did not mitigate selenate stress, I further investigated the effects of proline on Se and ROS accumulation, antioxidant enzyme activities. I found that selenate stress led to an accumulation of Se, ROS, and increased antioxidant enzyme activities but did not significantly induce lipid peroxidation in the BY-2 cells. Supplementation of proline increased Se accumulation and APX and POX activities but did not affect either the ROS accumulation or the lipid peroxidation in the BY-2 cells under selenate stress. Taken together with the result that proline did not mitigate selenate stress, these results suggest that the increment of APX and POX activities by exogenous proline is not sufficient to suppress selenate stress due to selenate accumulation.
Further, I investigated whether the growth inhibition by selenate is accounted for ROS accumulation or not. I investigated the effects of ROS scavengers, GSH and AsA, I found that GSH reduced the ROS accumulation and significantly mitigated the growth inhibition whereas AsA significantly decreased the ROS accumulation in the selenate-stressed cells but did not affect the growth inhibition by selenate.
These results suggest that the application of proline does not always mitigate growth inhibition by heavy metals and suggest that the growth inhibition by selenate is not accounted for by ROS accumulation.
35
ACKNOWLEDGEMENTS
Thanks to the almighty Allah who keep me in good health to carry out my research and writing dissertation to complete my doctoral course.
I would like to express my deepest gratitude and sincere appreciation to my research supervisor Dr. Yoshiyuki Murata, Professor, Faculty of Agriculture, Okayama University, Japan for your valuable suggestions and scholastic guidance throughout the course of the study and writing of the thesis.
I also thankful to Dr. Yoshimasa Nakamura for your valuable discussion and suggestions during the course of this study.
It’s my pleasure to acknowledge Dr. Shintaro Munemasa for his help and valuable advice and intellectual suggestions during the research period that help me to set the experiment properly
I also thankful to Toshiyuki Nakamura for your suggestions regarding scientific meetings and use of laboratory equipment.
I would to extend my gratefulness to Professor Yoshinobu Kimura, Faculty of Agriculture for his valuable comments.
I would like to express my heartiest appreciation to Mohammad Saidur Rhaman for being the best companion, continuous support and suggestion for starting the experiment properly. I am also grateful to my beloved son Safwan Shahir for his special sacrifice.
I also thank all members of the laboratory of Chemistry of Bio-signaling, and laboratory of Food Biochemistry for their support and making my student life enjoyable.
Finally, I dedicate the thesis to my beloved parents Md. Motier Rahman and Mst.
Shamsunnahar.
36
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