Ameliorative Role of Silicon on Osmoprotectants, Antioxidant Enzymes and Growth of Maize Grown Under Alkaline Stress

Maize is a cereal crop which plays an important role in ensuring food security and social and economic stability. It is grown in different environments. Global agriculture is currently encountered with large scale alkalinity stress. Supplementing silicon, is an alternative way for overcoming the negative effects of sodicity on the plant growth and yield and is cheaper than other methods to reduce sodicity. Hence, a pot experiment was contemplated to examine the role of silicon in alleviating alkaline stress encountered by maize crop. Ameliorative role of silicon in alkaline stress soil was studied with four levels of alkaline stress (0, 25, 50, 75 mM) created through addition of sodium carbonate and three levels of silicon (0, 100 and 150 kg ha−1) applied to root using sodium meta silicate imposed on CO 8 maize as test crop. The experiment was conducted in factorial CRD with three replications. Maize crop experienced negative effect when grown in alkaline stressed soil. The growth (5 to 16 %), dry weight (28 to 59 %) and relative water content (5 to 23 %) reduced at various level of alkaline concentration. However, electrolyte leakage (6 to 49 %), proline (26 to 62 %), phenol (8 to 44 %), protein (6 to 19 %), anti-oxidant systems viz., peroxidase (30 to 52 %), SOD (4 to 16 %) and catalase activities (32 to 127 %) scaled up with levels of alkaline stress. Supplementing silicon in this soil ensured maize crop get a congenial environment to grow well and it was revealed by improved growth (5 to 10 %) and dry weight (17 to 30 %) of maize, relative water content (6 to 12 %) and antioxidant enzymes (25 to 52 %), water soluble protein (7 to10 %), phenol (10 to 18 %), while reduced electrolyte leakage (15 to 25 %) and proline (17 to 29 %). The study divulged the primary role played by silicon in overhauling the hostile alkaline environment by promoting better growth and dry weight of maize through its regulatory action on osmoprotectants and anti-oxidant enzymes and maximum effect was evident with 150 kg Si/ha.


Introduction
Salinization and alkalization are the major bottle necks to crop production. [18]. Salt causes slowing down of physiological and metabolic processes, excess ions leading to toxicity, oxidative damage, hormonal and nutrient breach and osmotic stress [12]. The extreme salt stress can give rise to demolition of cell membrane, low nutrient absorption, generation of toxic substances and very low level of photosynthetic efficiency, all these culminating to reduced growth and finally abysmal crop productivity and sometimes even death [4,43]. Alkaline stress accompanied by elevated pH value have choked to a greater extent on photosynthetic efficiency, carbohydrate and nitrogen metabolism, synthesis of amino acid and sugars in maize. The triggering action for the poor performance of crop and low yield in any stress environment has been the production of large quantity of reactive oxygen species (ROS) [14]. The malfunctioning of electron transport and photorespiration pathways in chloroplast and mitochondria has been the root cause for excess production of ROS and it resulted in damage to chlorophyll and cell membrane [14]. Prevalence of osmotic balance is the key to stabilization of metabolic activity and cell turgor and in turn helped in growth and yield [39]. Proline production in the plant has been associated with plants stress tolerance and level of production of proline in plant is related to intensity of salt stress [30]. The proline acts ROS scavenger through antioxidant activity thereby it protects photosynthetic apparatus and proteins and thus ensures normal growth in saline soil [43]. The phenolic substance also exhibit antioxidant activity through non enzymatic action by preventing lipid peroxidation through trapping lipid alkoxyl and thus protect the plants from possible ROS damage [22].The antioxidant enzymes viz., superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD exerts it influence under stress environment. by controlling ROS activity. Salt stress causes damage to cell membrane through electrolyte leakage and also reduced relative water content (RWC).
Maize is a major cereal crop next to rice and wheat in the world. Maize is a major silicon accumulating crop and are grown in diversified environment. Indian sub-continent, being tropical to subtropical region, the maize crop will encounter with alkalinity problem. It was observed that plant supplemented with silicon showed greater ability to protect itself from the negative impacts of salt stress. Silicon is a multifaceted element which imparts salt tolerance to plants through enhanced growth and biomass, maintenance of nutrient balance, forming structural rigidity, escalate photosynthetic efficiency, sustaining ion homeostasis, triggering anti oxidative system in plants, promoting specific secondary metabolites that are related to stress tolerance. [34,46]. Early research workers have demonstrated the unequivocal influence of exogenous application of silicon through seed priming [2,23] and soil application of 150 kg ha −1 [48] improved growth of maize and sorghum grown in alkaline stress, by the enhancement in leaf water content, and levels of photosynthetic pigments, soluble sugars, soluble proteins, total free amino acids and K +, as well as activities of SOD, CAT, and POD enzymes. Since there are only few literatures related to maneuvering of sodic soil through silicon addition and an attempt was made to study the role of silicon in enhancing maize growth when grown in alkaline stress soil.

Experimental Set Up
The pot experiment was conducted in pot culture yard located in the vicinity of Department of Soil Science and Agriculture Chemistry, Faculty of Agriculture, Annamalai University. The experimental area is geographically situated at latitude 11•24'N and of longitude of 79•44'E and at an altitude of +5.79 m above mean sea level. Bulk soil samples of 0-15 cm were collected from the university experimental farm. The experiment soil is clay loam in texture belong to Kondal series (Typic Haplusterts). The chemical characteristic of soil was pH-8.3, EC-0.67dSm −1 , SOC-5.2 g kg −1 , CEC-32.5 Cmol (+) kg −1 , KMnO 4 ¬N-265 kg ha −1 , Olsen-P-21.5 kg ha −1 , NH 4 OAc-K-196.5 kg ha and available silicon-37.9 mg kg −1 .

Treatment and Experimental Design
There were three levels of silicon (0, 100 and 150 kg ha −1 ) and four levels of alkaline stress (0, 25, 50 and 75 mM). There were 12 treatment combinations replicated thrice. The silicon source: sodium metasilicates (Na 2 O 3 Si.5H 2 O) and alkaline stress source: sodium carbonate The 36(4 × 3 × 3) pots were arranged in completely randomized design in factorial arrangement.

Pot Culture Preparation and Planting
Ten kilogram of processed bulk soil samples was transferred to thirty-six pots. Seeds of maize (Zea mays L. cv CO 8) were surface-sterilized with mercuric chloride (0.1 %) for 5 min, and then rinsed three times with distilled water. Calculated quantity of silicon through sodium metasilicates as per the treatments were applied to the soil. The seeds were sown in pots (five seeds/pot). at the time of sowing, the seeds were irrigated at field capacity with various alkaline salt concentrations of 0 (control), 25, 50, and 75 mM Na 2 CO 3 with each pot receiving 400 ml of a designated salt solution. The Na 2 CO 3 concentrations used were equivalent to 0 (control), 0.528, 1.056, and 1.584 g Na 2 CO 3 kg −1 soil, respectively. Leaching was avoided by maintaining soil water below field capacity at all times. The pots were then irrigated at field capacity with normal water through the whole experimental period. Thinning of maize plant was done to maintain two plants throughout the experiment. The duration of the trial was up to vegetative stage (30 days). Recommended dose of fertilizers (150:75:75 kg of N, P 2 O 5 , K 2 O /ha) using urea, superphosphate, and muriate of potash was applied basally uniformly to all the pots as solution culture.

Data Collection and Plant analysis
At the end of vegetative stage, height and dry weight of maize crop were recorded. Maize leaf was used to record relative water content, electrolyte leakage, proline, phenol, protein an anti-oxidant enzymes. The osmoregulators viz., proline [9] soluble protein [31], antioxidants viz., SOD activity [10], POD activity [33], CAT activity [22], relative water content [31] and electrolyte leakage [32] were assessed following standard procedure.

Statistical Analyses
The data was subjected to statistical analysis to get meaningful explanation for the variability obtained for various characters due to treatments following [19]. Regression analysis and correlation was worked out to find out the selective variation between variables.

Plant Height and Dry Weight
Graded dose of silicon and alkaline stress levels had significant effect on plant height and dry weight of maize (Figs. 1 and 2). The normal stature of maize crop was affected as it experienced alkaline stress. The percent reduction in height ranged from 5.0 (25 mM) to 16.72 (75 mM). At all varying levels of alkaline stress, height of maize increased progressively with soil application of silicon and maximum height observed with 150 kg Si/ha in non-stressed soil (58.9 cm). The percent improvement in plant height ranged from 5 to 10 %. Even in non -stressed soil, addition of graded concentration of silicon caused increase in shoot growth of maize and it was tune of 6.5 to 12.6 % over control. The dry weight of maize showed progressively decline with increasing level of alkali stress. The percent reduction in dry weight ranged from 26.8 (25 mM) to 58.0 (75 mM). At all alkali stress levels, soil application of silicon significantly improved dry weight. The maximum shoot dry weight was associated with 150 kg Si/ha. The percent improvement in dry weight ranged from 16.6 to 29.9.

Relative Water Content
Main effects of alkaline stress and silicon dose and interplay between them had significant influence on relative water content (RWC) in maize leaves (Fig. 3). The maize crop grown in alkali stress exhibited low relative water content as compared to crop grown in non-stress soil (control). The relative water content showed linear drop when maize crop was grown in soil containing sodium carbonate concentration ranging from 25 to 75mM. The percent reduction ranged from 5.0 to 23.1 %. The relative water content in maize leaves improved when grown in silicon fed soil containing varying level of alkali stress. The extent of improvement in RWC was spread over 6.9 to 12.12 %.

Electrolyte Leakage
Interactive effect between alkaline stress and silicon dose was significant (p < 0.05) with regard to electrolyte leakage (EL) in maize leaves (Fig. 4).Generally, electrolyte leakage increased with alkali stress and decreased with Si levels. The percent increase in electrolyte leakage ranged from 6.3 (25 mM) to 49.1 (75mM).The least electrolyte leakage occurred under 0 mM alkali stress level and decreased significantly with increasing Si levels. The percent reduction in electrolyte leakage ranged from 14.8 to 25.0 with silicon levels.

Osmoregulators
Distinctive effects of silicon soil application and alkali stress was observed on proline, protein and phenol contents in maize leaves compared to no silicon stress free soil (Table 1). Proline content in maize leaves increased linearly with concomitant increase in alkali stress level. It ranged from 16.9 (non-stress soil) to 27.4 µM/g tissue (stress soil). The percent increase in proline ranged from 26.6 to 62.1. At all alkali stress levels, proline accumulation decreased with silicon doses. It decreased from 26.4 to 18.9 µM/g tissue. The percent reduction in proline in maize leaves ranged from 18.9 to 28.4.
The phenol content showed an increasing trend as alkaline stress increased and ranged from 39.9 (non-stressed soil) to 57.5 µg/g (75 mM alkaline stress).The percent increase in phenol content ranged from 8.0 (25mM) to 44.1 (75 mM).At Alkali stress markedly improved on protein content in maize leaves over normal soil and non-silicon applied soil. In leaves of alkali stressed maize plants, protein content enhanced and it ranged from 26.3 to 31.5 mg/g DW. The percent improvement in protein content due to alkali stress ranged from 6.8 to 19.8.At all alkali stress levels, protein content increased with silicon levels. The value ranged from 27.5 to 30.2 mg/g DW. The impact was to the extent of 6.9 to 9.8 %.

Antioxidant Enzyme Activity
Addition of graded concentration of silicon at various level of alkaline stress significantly influenced antioxidants viz., SOD, CAT, and POD in the leaves of maize (Fig. 7). SOD activity in maize leaves increased linearly with concomitant increase in alkali stress level. It ranged from 0.345 U mg −1 FW (non-stress soil) to 0.400 U mg −1 FW (stress soil). The percent increase in SOD activity ranged Alkaline stress created by different concentration of sodium carbonate created an increase in catalase activity in maize leaf and it ranged from 6.3 to 14.3 U mg −1 FW. The per cent increase in catalase activity ranged from 31.7 to 126.9.At all alkali stress level, catalase activity improved further with silicon fertilization. The percent increase in catalase activity ranged from 24.7 to 49.4.

Discussion
As predictable, negative effects occurred for maize crop when grown in alkaline condition as compared to non-alkaline soil. The short stature of maize plant is associated with break in supply of nutrient and water for normal metabolic process on account of perturbation in stomatal functioning and root architecture [34]. The reduction in the growth of rapeseed cultivars grown in salt-stress conditions was associated with a reduction in the RWC and an increase in electrolyte leakage [25]. The low relative water content and increased electrolyte leakage in maize grown in alkaline stress soil was noticed in the present study. There was negative correlation between electrolyte leakage with plant height (r= -0.897**). The decrease in plant height of maize grown in alkaline soil as noticed in the present study was in agreement with earlier workers [34]. Soil application of silicon overrided the negative effect of alkali stress and it improved the plant height and the maximum effect was noticed with 150 kg Si ha −1 . The positive effect of silicon could be due to increased cell division, cell elongation and also deposition of silicon in plant tissue causing erectness of leaf and stem. [16] observed increase in plant height of wheat with silicon under drought stress.
The reduced dry weight of maize with alkali stress levels is associated with water stress, ion toxicity, nutritional disorder, oxidation stress, membrane disorganization, reduction in cell division and expansion [34,38].Decrease in plant biomass due to salt stress was reported [40]. This was confirmed by significant negative correlation between electrolyte leakage with dry weight (r= 0.830**). Addition of silicon to soil experiencing alkaline stress improved shoot dry weight. Improved relative water content, reduced electrolyte leakage, improved anti-oxidant systems on addition of silicon was noticed in the present study which would have increased shoot dry weight. Significant improvement in shoot dry weight of maize by silicon in salt stress was reported earlier by several workers [27].
Relative water content (RWC) in leaves is known as an alternative measure of plant water status reflecting the metabolic activity in tissues. Reduction in relative water content under alkaline stress is mainly attributed to osmotic stress that is posed by high pH environment. This results in  accumulation of proline which causes water deficit condition, and as a consequence slows down water uptake. Strong linear regressive model of the increasing alkaline concentration (independently variable) and the decrease of RWC in leaf (r 2 =0.9766) as dependent variable was noticed in the present study (Fig. 5).Exogenous silicon application triggered RWC recovery in alkaline stresses plant. Silicon promoted root growth and root hydraulic conductance thereby increased root water uptake and further improved leaf water content by regulating the activity of aquaporins under salt stress [20] as this constitutes an obstacle to water transpiration through stomata and cuticle helping to reduce alkalinity [26].The present outcome was corroborated by earlier researchers [2]. This was confirmed by a strong linear relationship between silicon levels with RWC (r2= 0.9895**) (Fig. 5).
Electrolyte leakage is widely used as a test for the stressinduced injury of plant tissues and a measure of plant stress tolerance. Alkali stress increased electrolyte leakage in maize leaf compared to non-stressed soil. It is mainly caused by the efflux of K + and so-called counter ions (Cl −, HPO 4 2−, NO 3 −, citrate 3−, malate 2− ) that move to balance the efflux of positively charged potassium ions [11].Strong linear regressive model of the increasing alkaline concentration (independently variable) and the increase of EL in leaf (r 2 = 0.9546) as dependent variable was noticed in the present study (Fig. 5).Application of silicon reduced electrolyte leakage .This may be explained by the fact that Si has the ability to maintain cells by improving the permeability of their plasma membranes, which improves access into the cell by antioxidative enzymes [3]. This was supported by significant positive linear relationship observed between silicon levels and EL (r 2 = 0.9926**) (Fig. 5). Amidst all stresses adaptation, osmotic adjustment is a part of salt stress mechanism to offset the loss of turgor by increasing and maintaining higher amount of intercellular compatible solutes in the cystol and vacuole [18]. Proline is one of the key osmolytes contributing to osmotic adjustment. Maize plant recorded a higher proline concentration when it was grown in alkali stress soil. In durum wheat seedlings proline can contribute for more than 39 % of the osmotic adjustment in the cytoplasmic compartments of old leaves [13]. Increase in proline in plant under stress environment is either associated with protein biosynthesis genes (P5CS, P5CR) or repression of the genes of its degradation pathway (PDH silencing) [36]. Addition of silicon reduced proline content in maize leaf grown in alkali stress soil. Si may provide a protective role helping to prevent lipid peroxidation induced by NaCl, because of this, proline content was significantly lower in the Si-treated maize seedlings under salt stress than those under salt stress without Si treatment [37]. Addition of silica reduced the proline content and increased protein content in soybean to counter balance the salt stress [21] strengthened the present findings.
Polyphenol cmpounds lend a hand in plant protection against ROS and it is produced in large quantity, whenever aerobic respiration or photosynthetic metabolism are disabled by environment stress [11]. The increase in phenolic contents in different plant tissues under increasing salinity has also been reported in a number of plants [41]. The present work also reports the increase in phenol content in maize plant grown in alkali stress soil. Silicon application improved phenol content in maize leaf grown in alkali stress. Application of silicon increased phenol by 36 % due to silicon in barley [24].
Maize leaf recorded more soluble protein grown in alkali stress compared to non -stressed soil Plant experiencing stress normally store small molecules of mass protein which is used as storage nitrogen that would be mobilized after stress relief [50]. Additionally, these proteins could also have a role in osmotic adjustment [7]. Increase in soluble protein in maize genotypes grown in salt stress soil was reported by [6]. The exogenous application of silicon improved soluble protein because silicon has pivotal role in binding amino acids to form specific proteins [45]. Silicon is actively engaged in the formation of DNA and functioning of mRNA [1]. Application of potassium silicate improved soluble protein [2]. Furthermore, through the regression test, it showed that alkaline levels and silicon doses determined significantly the levels of proline (r 2 = 0.9791**, 1**), phenol (r 2 = 0.9502**, 0.9897**) and protein (r 2 = 0.9883**, 0.9983**), respectively (Figs. 6 and 7).
Antioxidant protective enzymes exist in higher plants under different growth conditions. In plants, antioxidant enzymes mainly include catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) and its content is increased in stressed plant due production of ROS or increase in its activity in plants might be a protective mechanism to counter the oxidative damage [5]. SOD, POD and CAT activities in maize leaves increased linearly with concomitant increase in alkali stress level. SOD is the probably the key enzyme to defend against toxic ROS [47]. POD catalysed the reaction between H 2 O 2 and ROOH to H 2 O and ROH which ameliorated possible cell damage caused by ROS [50]. Catalase is a principal enzyme that scavenges active oxygen species and prevents lipid peroxidation, cell membrane damage and chlorophyll degradation. CAT controls H 2 O 2 level in plant cells and participates in the photosynthetic process. The significant positive correlation between the SOD and POD (r= 0.8736**), SOD and CAT (r= 0.8908**) and CAT and POD (r= 0.9446**) observed in this study suggested a synergistic effect of POD, CAT and SOD in resistance to alkali stress. Defence capability of antioxidant enzymes depended on cooperative actions of enzymes [49] lent support the present result. Silicon interventions through soil application improved SOD, POD and CAT enzymes activities in maize leaf grown in alkali stress soil. Silicon application strengthens the antioxidant defence system and maintains normal physiological processes [20]. Silicon enhanced the activity of antioxidative enzymes and reduced plasma membrane permeability [44]. Addition of silicon improved antioxidant enzymes in wheat seedling grown in salt stress [40].

Conclusions
From the present study, it can be concluded that silicon has ameliorative role through antioxidant mechanism in