Excess boron responsive regulations of antioxidative mechanism at physio-biochemical and molecular levels in Arabidopsis thaliana

Highlights

• Physio-biochemical and molecular responses of Arabidopsis thaliana against boron (B) toxicity were investigated.

• Membrane damage occurred under 1 mM B supply but not under 3 mM B.

• Mainly, higher induced superoxide dismutase activity with CSD1 and MSD1 expressions prevented oxidative damage of 3 mM B.

• B toxicity induced ascorbate-glutathione cycle at transcriptional level.

Abstract

This work was aimed to evaluate the effect of boron (B) toxicity on oxidative damage level, non-enzymatic antioxidant accumulation such as anthocyanin, flavonoid and proline and expression levels of antioxidant enzymes including superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) and their respective activities as well as expression levels of miR398 and miR408 in Arabidopsis thaliana. Plants were germinated and grown on MS medium containing 1 mM B (1B) and 3 mM B (3B) for 14 d. Toxic B led to a decrease of photosynthetic pigments and an increase in accumulation of total soluble and insoluble sugars in accordance with phenotypically viewed chlorosis of seedlings through increasing level of B concentration. Along with these inhibitions, a corresponding increase in contents of flavonoid, anthocyanin and proline occurred that provoked oxidative stress tolerance. 3B caused a remarkable increase in total SOD activity whereas the activities of APX, GR and CAT remained unchanged as verified by expected increase in H₂O₂ content. In contrast to GR, the coincidence was found between the expressions of SOD and APX genes and their respective activities. 1B induced mir398 expression, whereas 3B did not cause any significant change in expression of mir408 and mir398. Expression levels of GR genes were coordinately regulated with DHAR2 expression. Moreover, the changes in expression level of MDAR2 was in accordance with changes in APX6 expression and total APX activity, indicating fine-tuned regulation of ascorbate-glutathione cycle which might trigger antioxidative responses against B toxicity in Arabidopsis thaliana.

Introduction

Boron (B), as a micronutrient, is essential for growth and development of plants (Warington, 1923). At physiological pH, main existing form of B is boric acid as an undissociated acid (Bolaños et al., 2004), which can form stable complexes with cishydroxyl groups (cis-diols) of many compounds (Loomis and Durst, 1992), such as ribose, sorbitol, serine and phenolics (Tate and Meister, 1978) including anthocyanins (Landi et al., 2014), and glycoproteins in the plasma membrane as well (Goldbach et al., 2001). Also, B cross-links to the apiose residue of rhamnogalacturonan II complex in plant primary cell wall (O'Neill et al., 2001). Although B is required for this structural role in addition to other suggested roles including plasma membrane integrity, phenol metabolism, sugar transport and ascorbate metabolism (Cakmak and Römheld, 1997), it can easily be toxic for plants when its concentration is slightly higher than that is necessary for normal growth (Mengel and Kirkby, 2001). Excess B causes significant problem by limiting crop yield in agricultural areas throughout the world including South Australia, California, Chile and Mediterranean countries i.e. Turkey (Aquea et al., 2012), especially central Anatolia (Ardıc et al., 2009). It occurs in soils because of over fertilization with minerals (Gupta et al., 1976) and the use of irrigation waters with high B (Branson, 1976) or can appear naturally in arid or semi arid areas worldwide due to evaporation of groundwater (Landi et al., 2012). Tolerant plants (e.g., Daucus carota) can overcome toxic effect of B in irrigation water with 2–4 mg L⁻¹ B while higher than 0.3 mg L⁻¹ B can lead to toxicity for sensitive plants (e.g., Phaseolus vulgaris) (Nable et al., 1997).

Some of known effects of excess B are deposition of lignin and suberin in roots (Ghanati et al., 2002) and reduced not only growth of shoots and roots (Lovatt and Bates, 1984, Nable et al., 1990), but also photosynthesis, stomatal conductance (Lovatt and Bates, 1984, Landi et al., 2013) and proton extrusion from roots (Roldan et al., 1992). These physiological disorders arised from B toxicity can be attributed to B-induced oxidative stress by enhancing overaccumulation of reactive oxygen species (ROS) such as superoxide radical (O₂·⁻), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH·). They induce cell death via oxidizing lipids, pigments, proteins and nucleic acids as well as inactivating enzymes (Blokhina et al., 2003). In order to overcome harmful effects of ROS, plants use ROS scavenging mechanisms including antioxidant enzymes such as superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POX; EC 1.11.1.7), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), glutathione reductase (GR; EC 1.6.4.2) and non-enzymatic antioxidants such as glutathione (GSH) and ascorbate (ASA) (Mathews et al., 1984). SOD decomposes O₂·^– radicals to molecular O₂ and H₂O₂ which is then catalyzed to O₂ and H₂O by CAT and POX. H₂O₂ is also detoxified in the ascorbate-glutathione cycle, which involves the oxidation and re-reduction of ASA and GSH through the APX and GR action (Noctor and Foyer, 1998).

There have been studies suggesting that the antioxidant response reduces oxidative damage of B toxicity in some plants (Cervilla et al., 2007, Gunes et al., 2006). For instance, enhanced activities of SOD, CAT and POX helped to protect chickpea from B toxicity induced oxidative stress (Ardıc et al., 2009). Similarly, higher B stress tolerance in soybean was provided by higher ROS scavenging capacity (Hamurcu et al., 2013). Also, Brassica seedlings exhibited increased activity of antioxidative enzymes and enhanced proline concentration against oxidative damage occurred by toxic B supply (Pandey and Archana, 2013). On the contrary, Karabal et al. (2003) suggested that antioxidant enzymes do not play a role in B tolerance mechanism against toxic B level in barley since toxic B concentrations led to membrane damage in a ROS independent manner. Likewise, lipid peroxidation level and H₂O₂ content did not decrease depending on the changes in antioxidant enzyme activities in maize (Esim et al., 2012). Moreover, Kayıhan (2014) supported this view by suggesting that total antioxidant enzyme activities may not involve in B toxicity tolerance mechanism in wheat plants due to the contradictory results of activities of APX, CAT and GR under B toxicity conditions. In short, studies focusing on the antioxidative reponses to B stress do not display consistence in plants (Liu et al., 2005). The antioxidative responses seemed to be regulated differentially in plants even in different cultivars of the same plant due to varying antioxidant capacities (Ardıc et al., 2009, Cervilla et al., 2007). Supportively, a high genotypic variation in B stress toleration ability of plants against excess B was identified (Hayes and Reid, 2004, Torun et al., 2006). Despite these studies, the antioxidant mechanisms at different levels of biological organization are not satisfactorily elucidated. Therefore, the aim of the study was to evaluate B responsive regulations of antioxidative mechanisms with an integrative approach including physiological, biochemical, transcriptional and post-transcriptional investigations in Arabidopsis thaliana. For this purpose, levels of H₂O₂ and malondialdehyde (MDA), an indicator of lipid peroxidation, and the contents of photosynthetic pigments such as chlorophyll a (chl a), chlorophyll b (chl b) and carotenoid were measured to determine the oxidative damage in high B treated-Arabidopsis thaliana. The concentration of total soluble proteins and the activities of CAT, APX, GR, SOD and POX and the contents of proline, anthocyanin, flavonoid, and total soluble and insoluble sugars were also measured in order to unravel the protective responses to B toxicity. Moreover, the changes in expression levels of enzymatic antioxidant genes including APX6, CAT1, CSD1 (Cu/ZnSOD), MSD1 (MnSOD), GR1 and GR2 and genes involved in ascorbate-glutathione cycle such as dehydroascorbate reductase (DHAR2) and monodehydroascorbate reductase (MDAR2) were investigated by quantitative real time PCR (qRT-PCR).

Besides, recent evidence exhibits that plant microRNAs (miRNAs), small endogenous RNAs of ∼22 nucleotides, have an important regulatory role in biotic and abiotic stress responses (Khraiwesh et al., 2012) by negatively affecting the expression of genes at post-transcriptional level (Kumar et al., 2015). For example, miR398 was found to target Cu/ZnSOD isoenzymes in Arabidopsis thaliana (Sunkar et al., 2006) whereas altered expression level of miR408 was suggested to affect abiotic stress responses by regulating genes encoding copper-containing proteins (Ma et al., 2015). Thus, in order to obtain the transcriptional regulation of Cu/ZnSOD under B toxicity in more detail, expression levels of miR398 and miR408 were also assessed in Arabidopsis thaliana. Overall, our study might provide a better insight into B responsive regulations of antioxidative mechanisms in plants.