Cross Protection by Cold-shock to Salinity and Drought Stress-induced Oxidative Stress in Mustard (Brassica campestris L.) Seedlings  

Mohammad Anwar Hossain1,2 , Mohammad Golam Mostofa1,3 , Masayuki Fujita1
1 Laboratory of Plant Stress Responses, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa 761-0795, Japan
2 Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh- 2202, Bangladesh
3 Department of Biochemistry, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh
Author    Correspondence author
Molecular Plant Breeding, 2013, Vol. 4, No. 7   doi: 10.5376/mpb.2013.04.0007
Received: 16 Jan., 2013    Accepted: 22 Jan., 2013    Published: 07 Feb., 2013
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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Mohammad et al., 2013, Cross Protection by Cold-shock to Salinity and Drought Stress-induced Oxidative Stress in Mustard (Brassica campestris L.) Seedlings, Molecular Plant Breeding, Vol.4, No.7 50-70 (doi: 10.5376/mpb.2013.04.0007)

Abstract

In the present study, cold-shock (6, 5.5 h) induced salinity and drought tolerance and involvement of antioxidative and glyoxalase systems were investigated in mustard (Brassica campestris L.) seedlings. Seven-day-old seedlings were subjected to salt (150 mmol/L NaCl, 48 h) and drought stress (induced by 20% PEG, 48 h) with or without cold pre-treatment. The results showed that both salt and drought stresses abruptly increased the hydrogen peroxide (H2O2) and lipid peroxidation (malondialdehyde, MDA) levels. Ascorbate (AsA), reduced glutathione (GSH) and oxidized glutathione (GSSG) contents, GSH/GSSG ratio and the activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPX), catalase (CAT), glyoxalase I (Gly I), and glyoxalase II (Gly II) showed both homogeneity and discrepancies in the responses of mustard seedlings to salinity and drought stresses. Drought stress treatment resulted in a significant increases in AsA content. The GSH and GSSG content increased in response to both salt and drought stresses, however, the GSH/GSSG ratio decreased significantly in response to drought stress. Salt stress treatment resulted in a significant increase of APX, MDHAR, GR, GST and Gly I activities, whereas, CAT and Gly II activities decreased. In contrast, drought stress treatment resulted in a significant increase in MDHAR, DHAR, GPX and Gly I activities; whereas, APX, CAT and Gly II activities decreased. Importantly, cold pre-treated salt and drought-stressed seedlings maintained higher level of AsA, GSH contents and GSH/GSSG ratio, higher activities of APX, DHAR, GR, GST, GPX, CAT, Gly I and Gly II, and lower the levels of GSSG, H2O2 and MDA as compared to the control as well as in most cases seedlings subjected to salt and drought stress without cold pre-treatment. Our findings showed that a retention of the imprint of previous stress exposure (short-term cold-shock), induces salt and drought-induced oxidative stress tolerance by modulating antioxidative and glyoxalase systems.

Keywords
Cross-adaptation; Cold-shock; Salt and drought stress; Antioxidative and glyoxalase system; Brassica campestris L.

Plants regularly face adverse growth conditions, such as drought, salinity, chilling, freezing, and high temperatures (Krasensky and Jonak, 2012). Soil water deficits and salinization are the most crucial abiotic stresses constraining crop yields worldwide (Munns, 2011; Cominelli et al., 2012). Both salinity and drought stresses become more problematic by the predicted forthcoming global changes in climate, foreseen extremization of environmental conditions, continuous increase of world population, ever- increasing deterioration of arable land, and scarcity of fresh water (Xiao et al., 2007). As a result, the development of improved levels of tolerance to these stresses has become an urgent concern for many crop breeding programs to ensure global food security to an increasing world population. In parallel, much research effort is being applied to gain a better understanding of the complex adaptive mechanisms used by plants to combat abiotic stress (Peng et al., 2009), although we are far from complete understanding of this complexity (Cominelli et al., 2012). Identification of key metabolic pathways, genes and proteins underlying abiotic stresses has thus become a priority in the research for improved crop stress tolerance (Janská et al., 2010; Hossain et al., 2011a; Kosová et al., 2011; Hossain and Fujita, 2012; Reguera et al., 2012). A deeper understanding of the regulation of these pathways and genes and their response to stress, would allow clarification of the ways in which plants adjust to a particular stress. Knowledge of this type widely accepted to provide opportunities for the manipulation of gene expression in crop plants, with a view of engineering higher level of salt, drought or cold stresses (Janská et al., 2010).

Salt and drought stresses invariably lead to oxidative stress in plant cell due to higher leakage of electrons towards O2 during photosynthetic and respiratory processes leading to enhancement of reactive oxygen species (ROS) and free radicals such as singlet oxygen (1O2), superoxide radicals (O2•−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH) (Cruz de Carvalho et al., 2008; Abogadallah, 2010; Hossain and Fujita, 2012). Recently, methylglyoxal (MG), a cytotoxic compound, was also found to increase in response to various stresses including salt and drought stresses (Hossain et al., 2009; Banu et al., 2010; Upadhyaya et al., 2011; Hossain et al., 2012a). An increase in MG level in plant cells further intensifies the production of ROS by interfering with different plant physiological and metabolic processes such as inactivation of the antioxidant defense system (Hoque et al., 2010; 2012; Hossain et al., 2012a) and interfering with vital plant physiological processes such as photosynthesis (Saito et al., 2010). The increase in ROS and MG exposes cells to oxidative stress leading to lipidperoxidation, chlorophyll destruction, biological macromolecule deterioration, membranedismantling, ion leakage, and DNA-strand cleavage and finally death of plants (El-Shabrawi et al., 2010; Hossain et al., 2011a; Hossain and Fujita, 2012; Hossain et al., 2012a).
To counter the deleterious effects of ROS, plants use an intrinsic mechanism known as the plant antioxidant system as a defense mechanism to regulate the ROS levels according the cellular needs at a particular time (Hossain et al., 2012a). These antioxidants includes the enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), catalase (CAT), glutathione peroxidase (GPX), glutathione S-transferase (GST) and water-soluble compounds such as ascorbate (AsA) and glutathione (GSH) (Apel and Hirt, 2004; Hasanu- zzaman et al., 2011a; Hossain et al., 2012a; Hossain and Fujita, 2012). Likewise, the glyoxalase system is an integral component and major pathway of cellular metabolism of cytotoxic MG, consists of two enzymes: glyoxalase I (Gly I) and glyoxalase II (Gly II). These enzymes act coordinately to convert MG and other 2-oxoaldehydes to their 2-hydroxyacids using GSH as a cofactor in a two-step reaction (Thornalley, 1990; Hossain et al., 2012a; 2012b). The spontaneous reaction between GSH and MG forms hemithioacetal, which is then converted to S-D-lactoylglutahione (SLG) by Gly I. The second reaction is the hydrolysis of SLG to D-lactate catalyzed by Gly II and GSH is recycled back into the system (Figure 1). The overexpression of the glyoxalase pathway enzymes in transgenic tobacco and rice plants has been found to lower the levels of ROS and MG under stress conditions by maintaining GSH homeostasis and increasing antioxidant enzyme activities (Yadav et al., 2005; Singla-Pareek et al., 2003; 2006; Hasanuzzaman et al., 2011b; Hossain et al., 2012a). Transgenic tobacco plants overexpressing both Gly I and Gly II genes also showed higher salinity tolerance and by additional increase of GSH metabolizing enzyme (GST, GPX and GR) activities further denoting the close interaction between the antioxidant system and the glyoxalase system in imparting stress tolerance in plants (Yadav et al., 2005; Hossain et al., 2012a). The results of numerous recent studies have shown that the alleviation of oxidative damage and increased resis- tance to abiotic stresses are often correlated with the more efficient antioxidative and glyoxalase systems (Figure 1; El-Shabrawi et al., 2010; Hasanuzzaman et al., 2011a; 2011b; Hossain et al., 2010, Hossain et al., 2011a; 2011b; Hossain et al., 2012a; 2012b).


Figure 1 Coordinated action of AsA- and GSH-based antioxidative system and GSH-based glyoxalase system in plant cells involved in ROS and MG detoxification (Hossain et al., 2011a)

Cold represents one of the most abiotic stresses influencing plant growth and development and plants have to cope with during their life cycle (Li et al., 2010). Cold temperature affects a broad spectrum of cellular components and metabolism, and temperature extremes impose stresses of variable severity that depend on the intensity and duration of the stress (Jan et al., 2009). Accumulating evidence suggest that cold acclimation is associated with complex biochemical and physiological changes, including protection and stabilization of cellular membranes, enhancement of antioxidant enzymes and higher contents of antioxidants (such as AsA and GSH) and synthesis and accumulation of cryoprotectant solutes in conjunction with dehydrin proteins, cold-regulated proteins (CORs) and heat shock proteins (HSPs) (Streb and Feierabend, 1999; Streb et al., 2003; 2008; Gomez et al., 2004; Janská et al., 2010; Li et al., 2010; Ao et al., 2012). Cold-acclimated plants also increase their photosynthetic activity, due to higher activities of Calvin cycle enzymes and higher rates of sucrose synthesis which serve as electron donor (Huner et al., 1993; Streb et al., 2008). Classical genetic studies have revealed that the ability of plants to cold acclimation is a quantitative trait involving the action of many genes with small additive effects (Thomashow, 1990; Jan et al., 2009). The exact molecular mechanism(s) of cross-adaptation is poorly known although a few hypotheses have been proposed underlying heat-shock induced abiotic stress tolerance with possible involvement of H2O2, GSH, AsA and HSPs (Gong et al., 2001; Volkov et al., 2006; Hsu and Kao, 2007; Chao et al., 2009, Cao et al., 2010; Chao and Kao, 2010; Ferreira-Silva et al., 2011). However, cold-shock induced cross-tolerance is very limited although few recent reports showed the possible physiological and biochemical mechanisms but the detailed data are poorly known. Low temperature pre-treatment induced thermotolerance in gape barry (
Vitis viniferaL.) was associated with the induction of HSP73, phospholipase D and salicylic acid (Wan et al., 2009). Cold pre-treatment (4℃, 36 h) enhances heavy metal (Pb) resistance in Arabidopsis thaliana by activating AtPDR12 gene which function as a pump to exclude Pb or Pb-containing toxic compounds from the cytoplasm to the exterior of the cell (Cao et al., 2010). Transgenic tobacco plants over-expressing cold regulated gene from Camellina sinensis, CsCOR1 enhances salt- and dehydration-tolerance (Li et al., 2010). Very recently, Ao et al (2012) showed that chill hardening induces chilling tolerance in Jatropha curcas seedlings by modulation of antioxidant enzymes such as SOD, APX, CAT, POD, GR and higher amount of AsA and GSH contents. Additionally, our recent studies showed an increase in Gly I activity, gene and protein expression and Gly II activity under cold stress (Hossain et al., 2009; Hossain and Fujita, 2009). Importantly, a series of our recent findings suggest that favorable modulation of AsA and GSH contents and their utilizing and regenerating enzymes is an important predominant factor controlling ROS an MG levels to ensure abiotic stress tolerance (Hossain et al., 2010, 2011b; Hasanuzzaman et al., 2011a, 2011b). However, it is unclear whether there is a cross-adaptation between cold, salt and drought stresses in Brassica and the possible involvement of antioxidant defense system and glyoxalase system. Considering the above facts, the present study was undertaken to explore the possible biochemical mechanisms of cold-shock induced salinity and drought tolerance in mustard seedlings. Our data showed the first experimental evidence that cold-shock enhances salt and drought induced oxidative stress tolerance in mustard seedlings by up-regulating the antioxidative and glyoxalase systems.
1 Results
1.1 Non-enzymatic antioxidant contents
The levels of non-enzymatic antioxidants varied significantly upon the imposition of salt or drought stress. Seedlings treated with salinity showed a non-significant (12%) increase in AsA content, whereas, drought-stressed seedlings showed a significant increase (37%) when compared with control (Figure 2A). Cold pre-treated salt and drought-stressed seedlings also showed a significant increase (29% and 60% by salt and drought stress, respectively) in AsA content when compared with control, however, the level was significantly higher (15% and 16% by salt and drought stress, respectively) than those of the seedlings subjected to salt and drought stress without cold pre-treatment.


Figure 2 Reduced Ascorbate (AsA) (A), reduced glutathione (GSH) (B), oxidized glutathione (GSSG) (C) and GSH/GSSG ratio (D) in mustard seedlings induced by cold-shock under salt and drought stress conditions

Seedlings treated with salt and drought stress showed a marked increase in GSH content (57% and 99% by salt and drought stress, respectively) when compared with control (Figure 2B). Cold pre-treated salt and drought-stressed seedlings also showed a 79% and 68% increase in GSH content when compared with control. Importantly, cold pre-treated salt-stressed seedlings showed a significant increase (14%), whereas, cold pre-treated drought-stressed seedlings showed a significant decrease (16%) in GSH content when compared with the seedlings subjected to salt and drought stress without cold pre-treatment.

Seedlings treated with salinity showed a significant increase (80%) in GSSG content but a sharp increase (385%) in GSSG content was observed in drought-stressed seedlings when compared with control (Figure 2C). Cold pre-treated salt-stressed seedlings showed a non-significant increase (12%) in GSSG content when compared with control, whereas, cold pre-treated drought-stressed seedlings showed a significant increase (197%) in GSSG content. However, cold pre-treated salt and drought-stressed seedlings showed a significantly lower level of GSSG content (38% and 39% by salt and drought stress, respectively) when compared with the seedlings subjected to salt and drought stress without cold pre-treatment.
The GSH/GSSG ratio decreased upon imposition of both salt and drought stress. Salt stress showed a non-significant decrease (13%) whereas drought stress showed a significant decrease (59%) when compared with control (Figure 2D). Importantly, cold pre-treated salt-stressed seedlings showed a significant increase (83%) in GSH/GSSG ratio when compared with the seedlings subjected to salt stress without cold pre-treatment.
1.2 Activities of antioxidant enzymes
The activity enzymes involved in ROS scavenging changed significantly upon imposition of stress treatments. Seedlings treated with salinity showed a significant increase (17%) in APX activity, whereas, a significant decrease (10%) was observed in response to drought stress when compared with control (Figure 3A).


Figure 3 Activities of APX (A), MDHAR (B), DHAR (C), and GR (D) in mustard seedlings induced by cold-shock under salt and drought stress conditions

Cold pre-treated salt and drought-stressed seedlings showed a significant increase (34% and 11% by salt and drought stress, respectively) in APX activity. Meanwhile, cold pre-treated salt and drought-stressed seedlings showed a significant increase (15% and 23% by salt and drought stress, respectively) in APX activity when compared with the seedling subjected to salt and drought stress without cold pre-treatment.
Seedlings treated with both salinity and drought stresses led to a significant increase in MDHAR activity (11% and 17% by salt and drought stress, respectively) when compared with control (Figure 3B). Cold pre-treated salt and drought stressed seedlings also showed a significant increase (13% and 20% by salt and drought stress, respectively) in MDHAR activity when compared with control. However, no significant variation in MDHAR activity was observed in cold pre-treated and untreated salt and drought-stressed seedlings.
Seedlings treated with salinity showed a non-significant increase in DHAR activity, whereas, a significant increase (31%) was observed in drought treated seedlings when compared with control (Figure 3C). Importantly, cold pre-treated salt and drought-stressed seedlings showed a significant increase in DHAR activity (39% and 50% by salt and drought stress, respectively). Cold pre-treated salt and drought-stressed seedlings showed significantly higher DHAR activity (24% and 15% by salt and drought stress, respectively) when compared with the seedling subjected to salt and drought stress without cold pre-treatment.
Salt stress resulted in a significant increase (27%) in GR activity, whereas, a non-significant decrease (12%) was observed under drought stress when compared with control (Figure 3D). A sharp increase in GR activity (63% and 56% by salt and drought stress, respectively) was also observed in cold pre-treated salt and drought-stressed seedlings when compared with control. Importantly, cold pre-treated salt stress seedlings showed a significant increase (28% and 78% by salt and drought stress, respectively) when compared with the seedlings subjected to salt and drought stresses without cold pre-treatment.
Seedlings treated with salt stress led to a significant increase (38%) in GST activity, whereas, drought-stressed led to a non-significant increase (18%) when compared with control (Figure 4A). Cold pre-treated salt and drought-stressed seedlings showed a significant increase in GST activity (57% and 42% by salt and drought stress, respectively). Importantly, cold pre-treated salt and drought-stressed seedlings maintained a significantly higher GST activity (14% and 20% by salt and drought stress, respectively) when compared with the seedlings subjected to salt and drought stress without cold pre-treatment.


Figure 4 Activities of GST (A), GPX (B) and CAT(C) in mustard seedlings induced by cold-shock under salt and drought stress conditions

Salt stress led to a slight increase (11%) in GPX activity, whereas, a significant increase (40%) in GPX activity was observed under drought stress when compared with control (Figure 4B). Cold pre-treated salt-stressed seedlings showed a non-significant increase (25%) in GPX activity when compared with the seedlings subjected to salt stress without cold pre-treatment. GPX activity remains unchanged in cold pre-treated and untreated drought-stressed seedlings.
The activity of CAT decreased significantly (22% and 19% by salt and drought stress, respectively) in response to salt and drought stress when compared with control (Figure 4C). Cold pre-treated salt- stressed seedlings maintained a similar level of CAT activity when compared with control, whereas, cold pre-treated drought-stressed seedlings showed a significant increase (14%). Importantly, cold pre- treated both salt and drought-stressed seedlings showed a significant increase in CAT activity (22% and 40% by salt and drought stress, respectively) when compared with the seedlings subjected to salt and drought stress without cold pre-treatment.
1.3 Glyoxalase pathway enzymes
Both salt and drought stresses significantly increased (16% and 26% by salt and drought stress, respectively) Gly I activity when compared with control (Figure 5A). Cold pre-treated salt and drought-stressed seedlings showed a 30% and 33% increase in Gly I activity when compared with control. Importantly, cold pre-treated salt-stressed seedlings showed a significant increase (13%), whereas, cold pre-treated drought stressed seedlings showed a non-significant increase in Gly I activity when compared with the seedlings subjected to salt and drought stresses without cold pre-treatment.
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