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Beneficial Effects of Potassium on Growth, Water Relations, Mineral Accumulation and Oxidative Damage of Beta vulgaris in Sodic-alkaline Condition.

Byline: Liyun Liu, Hany A. El-Shemy and Hirofumi Saneoka

Abstract

Potassium (K+) plays a vital role for plant growth and therefore the amount of K+ seems to be a key of plant development. Therefore, this study investigated whether the sodic-alkalinity tolerance of Swiss chard (Beta vulgaris L. subsp. cicla) could be increased, via greater water uptake, higher concentrations of mineral elements and higher antioxidant enzyme activities in sodic-alkaline stress conditions with K+ supplementation. Forty seven day old uniform seedlings were pre-treated in hydroponic medium with or without K+ for 7 days, and then treated in non-alkaline or alkaline conditions for 9 days. The absence of K+ somewhat intensified the effect of alkalinity on reducing the growth of Swiss chard, the total dry weight (DW) was reduced by 34% in plants treated in sodic-alkaline conditions without K+ when compared with plants with K+. Relative water content (RWC) of leaves was increased by 10% by K+ supplementation in sodic-alkaline conditions.

Additionally, the osmotic adjustment was retorted; the contents of K+, Cl-, BO3, Fe3+ and Mn2+, and the activities of catalase, guaiacol peroxidase, and glutathione reductase were increased. On other hand, K+ supplementation in sodic-alkaline conditions helped to maintain guaiacol peroxidase activity similar to that of control plants, and reduced malondialdehyde content when compared with plants treated in sodic-alkaline conditions without K+. In conclusion, the K+ supplementation resulted in favorable changes in plants with higher micronutrient contents and a reduction of oxidative damage induced by sodicalkalinity in Swiss chard.

Keywords: Micronutrients; Osmotic adjustment; Potassium; Sodic-alkalinity; Swiss chard; Reactive oxygen species

Introduction

Potassium (K+) is major inorganic constituent of living cells and required in large amounts for plant growth and development, being taken from soil then translocated to various organs for many processes in plant cells (Kanai et al., 2011; Cherel et al., 2014). Plants need a high content of K+ for specific functions in the cytoplasm and 90% is localized in vacuoles (Wakeel et al., 2011). However, in conditions with high contents of Na+, Na+ reduce the activity of K+, and also compete with K+ for uptake sites at the plasma membrane (Shabala and Cuin, 2007). When positively charged Na+ crosses the plasma membrane, significant membrane depolarization makes passive K+ uptake through inward-rectifying K+ channels impossible, and K+ leakage increases through depolarization-activated outward-rectifying K+ channels (Shabala and Cuin, 2007).

For particular plant species of the Chenopodiaceae, K+ localized in vacuoles was substituted by Na+ to a high degree, and plants grow on soils with high K-fixing capacity have more potential for this substitution (Subbarao et al., 2000; Wakeel et al., 2010), and supplementation of Na+ in reduced amounts eliminated K+-deficient symptoms in conditions of limited K+ supply (Wakeel et al., 2011). Therefore, some species of Chenopodiaceae showed particularly strong stimulation by low Na+ application in either low- or high-K conditions (EI-Sheikh et al., 1967; Subbarao et al., 2003).

Many saline soils are also sodic-alkaline due to the presence of NaHCO3 and Na2CO3, which resulted in high soil pH, usually >8.5 (Lyubimova et al., 2009; Bui, 2013). Sodic-alkalinity is more deleterious to plant growth than salinity alone, and results in laboratory-bred NaCl-tolerant cultivars failing to achieve good yield in field conditions (Nishiuchi et al., 2010; Bui, 2013). Overproduced reactive oxygen species (ROS) can activate plasma membrane K+ and Ca2+- permeable conductance, which mediate K+ efflux and Ca2+ influx, respectively, resulted in a concomitant elevation of cytosolic Ca2+ activity and K+ loss (Demidchik et al., 2003; Demidchik et al., 2007). Alongside toxic Na+ influx, cytosolic ionic homeostasis is seriously impaired, and cellular tissue damage occurs via a number of destructive processes following a rise in ROS levels (Ashraf, 2009; Velarde-Buendia et al., 2012).

The most abundant types of ROS in plants are hydrogen peroxide (H2O2), superoxide radical, and hydroxyl radical. To detoxify ROS, plant cells and their organelles employ both enzymatic and non-enzymatic mechanisms (Zepeda-Jazo et al., 2011). Enzymatic antioxidants include superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), guaiacol peroxidase (GPX; EC 1.11.1.7), ascorbate peroxidase (APX; EC 1.11.1.1), and glutathione reductase (GR; EC 1.6.4.2) (Gill and Tutejia, 2010). APX, followed by CAT, is the key enzyme in detoxifying H2O2 (Ashraf, 2009). It plays a vital role in plant defense against oxidative stress (Ashraf, 2009; Hernandez et al., 2012). GPX not only scavenges H2O2, but also involves in the biosynthesis of cell wall components and tissue lignification (Cavalcanti et al., 2004). The common non-enzymatic antioxidants are glutathione, ascorbate, carotenoids, phenolic compounds and non-protein amino acids (Ashraf, 2009; Gill and Tuteja, 2010).

Swiss chard (Beta vulgaris L. subsp. cicla) is a foliage vegetable closely related to beets with a large leaf blade, thicker petiole, and no root enlargement (Welbaum, 2015). The leaf of Swiss chard contains nutritionally significant contents of magnesium, calcium and phosphorus, and has potential to protect humans against several chro nic diseases such as diabetes (Pyo et al., 2004; Maynard and Hochmuth, 2007; Sacan and Yanardag, 2010). However, the growth of Swiss chard is severely suppressed by alkaline conditions owing to serious impairment of intracellular Na+ and K+ homeostasis, and serious K+ starvation in tissues (Liu et al., 2013).

As mentioned above, although sodic-alkaline conditions and K+ starvation have been studied extensively, impact of the combination of these conditions on plants was not examined. The main objective of this study is to examine the influence of K+ on the responses of Swiss chard to sodic-alkaline constraints, and to explore differences in physiological parameters in Swiss chard seedlings subjected to sodic-alkaline conditions with or without K+ supplementation.

Materials and Methods

Plant Material and Treatment Conditions

The description of Swiss chard (Beta vulgaris L. subsp. cicla) seeds used in this study was the same as described by Liu et al. (2013). The seeds were sown in a plastic seed bed (96 wells per bed) and irrigated with tap water in a greenhouse of the Faculty of Applied Biological Sciences, Hiroshima University, Japan. Forty five days after sowing, uniform seedlings that have shed their cotyledons from the mother plants were transferred to deionized water for discarding nutrients on the root surface. After two days , seedlings were transplanted to water culture solution with or without K+ for 1 week (K+ pre-treatment). The nutrient medium with K+ contained the following macronutrients: 0.6 mM NH4NO3, 0.2 mM NH4H2PO4, 0.34 mM K2SO4, 0.34 mM KCl, 0.38 mM Ca(NO3)2, and 0.2 mM MgSO4, and the following micronutrients: 4 uM MnCl2*4H2O, 8 uM H3BO3, 0.12 uM CuSO4*5H2O, 0.3 uM ZnSO4*7H2O, 0.07 uM MoO3, and 20 uM Fe-EDTA.

For no K+ treatment, K2SO4 and KCl were omitted from the nutrient medium. 70 L plastic containers (12 plants for each container) were maintained under natural sunlight conditions of the greenhouse.

After K+ pre-treatment, the seedlings were transplanted to 20 L plastic containers (six plants for each container) filled with nutrient mediums as mentioned above, and treated by both sodic-alkalinity and non-sodic control, respectively (for 9 days). All 4 treatments were as follow: 0 mM K, 2 mM K, Alkalinity+0 mM K, and Alkalinity+2 mM K. The nutrient solutions of 2 mM K and Alkalinity+2 mM K treatments contained the same micronutrients and macronutrients with K+-pretreatment except for K+, which was simulated by adding 0.68 mM K2SO4 and 0.68 mM KCl. For 0 mM K and Alkalinity+0 mM K treatments, the omission of K2SO4 and KCl from the nutrient medium was continued. The sodic-alkalinity was simulated by a mixture of NaHCO3 and Na2CO3 (NaHCO3:Na2CO3 = 9:1 molar ratio) as described by our previous study (Liu et al., 2013).

The electrical conductivity (EC) of the nutrient medium was gradually increased from 186.8 mS m-1 to 340 mS m-1 to 493 mS m-1 for Alkalinity+2 mM K treatment, while from 208 mS m-1 to 354 mS m-1 to 506 mS m-1 for Alkalinity+0 mM K treatment, through the gradual increase of Na+ content from 20 mM to 40 mM to 60 mM every 3 days. 1 M NaOH was used to adjust the pH (5.5-6.0) of the basal nutrient solution, which was used for 0 mM K and 2 mM K treatments. For the Alkalinity+0 mM and Alkalinity+2 mM treatments, the pH ranges of the nutrient medium were increased to 8.8-9.1 after adding NaHCO3 and Na2CO3 to the basal nutrient solution. The nutrient medium was renewed every 3 days. The pots were still maintained under natural sunlight condition in the greenhouse.

The average temperature was 23degC, and the average of relative humidity was 58% during treatment period.

Physiological Parameters

At the end of the treatment period, plants were carefully separated into leaf blade, petiole and root, and each plant part was weighted and freeze-dried. The freeze-dried samples were used for the estimation of dry weight (DW), and young leaf blades were sampled for analyses. The relative water content (RWC) was measured as described by Turner (1981). The saturated osmotic potential (Ps 100) was calculated using the following equation (Saneoka et al., 1995): Ps 100 = (osmotic potential [RWC - apoplastic water content]/[1.0 - apoplastic water content]. The apoplastic water content is assumed as 0.15, and osmotic potential of the cell sap was measured by Wescor 5500 vapor pressure osmometer (Wescor Inc., Logan, UT, USA).

For mineral analysis, young leaf blades were freeze-dried and then ground into fine powder. Sample digestion for cations, and measurements of the Na+ and K+ contents were conducted as descripted by Liu et al. (2013), Mg2+ and Ca2+ contents were determined as described by Saneoka et al. (1999), and BO3, Cu, Fe, Mn, and Zn were determined using Inductively Coupled Plasma (iCAP 6000, Thermo Fisher Scientific Inc., UK). Anions were extracted in tightly closed micro tube with distilled water and finely ground powder at 100degC for 1 h and the supernatant obtained after centrifugation was used to determine anion concentration by ion chromatography (Dionex ICS-900, Nippon Dionex K.K., Osaka, Japan). The contribution of each ion to total osmotic potential at full turgor (Psp) was calculated as described by Ming et al. (2012).

The malondialdehyde (MDA) content was measured using a modified version of the thiobarbituric acid (TBA) procedure (Draper and Hadley, 1990). The fine powder (ground with liquid nitrogen) of young leaf blade was homogenized by an extraction buffer containing 3.3 mM HEPES (pH 7), 0.25% TBA, 10% trichloroacetic acid (TCA), 0.2 M HCl, 0.01% butylated hydroxytoluene, and 1.3% ethanol. The absorbance of the supernatant acquired after centrifugation was measured at 535 nm and 600 nm after incubating at 95degC, and the extinction coefficient (1.56x105 mM-1 cm-1) was used to calculate the MDA content.

The crude extract for measuring enzyme activity and protein concentration was prepared as described by Koca et al. (2007), with modifications. A frozen sample (0.5 g) was extracted with ice-cold 25 mM potassium phosphate buffer (pH 7.8) containing 0.5 mM ethylenediaminetetraacetic acid (EDTA), 2% polyvinylpolypyrrolidone, and 1 mM ascorbic acid. For CAT activity, it was assayed by measuring the initial rate of H2O2 disappearance at 240 nm (Aebi, 1984). For APX activity, the absorbance of ascorbic acid was determined at 290 nm (Nakano and Asada, 1981). For GPX activity, the absorbance of tetraguaiacol was measured at 470 nm (Velikova et al., 2000). For GR activity, the absorption of oxidized NADPH was monitored at 340 nm as described by Foyer and Halliwell (1976). The soluble protein content in the enzyme extract was determined as descripted by Assaha et al. (2015).

Statistical Analysis

All data were examined by one-way ANOVA using the IBM SPSS statistical package version 21. Test of significance was performed using Duncan's test at the 5% significance level. The values are means (+-SE) of six replicates.

Results

Plant Growth

The root DW of Swiss chard was 23% less, and shoot DW was not changed with Alkalinity+2 mM K treatment compared to those of 2 mM K treatment (Fig. 1). Additionally, the root DW was 43% less, shoot DW was 30% less with 0 mM K treatment, and root DW was 45% less, shoot DW was 33% less with Alkalinity+0 mM K treatments when compared with the root and shoot DW with 2 mM K treatment. Therefore, the reductions in plant growth caused by 0 mM K and Alkalinity+0 mM K treatments were >of the plants subjected to Alkalinity+2 mM K treatment. Also, leaf chlorosis phenomenon was observed in plants after treatment of Alkalinity+0 mM K.

Water Relations

The results indicated that the RWC was 6%, 8%, and 16% less, respectively, with 0 mM K, Alkalinity+2 mM K, and Alkalinity+0 mM K treatments when compared to 2 mM K treatment (Fig. 2). In addition, the Psp was 15% less with Alkalinity+2 mM K treatment, 18% more with 0 mM K treatment, and 13% more with Alkalinity+0 mM K treatments compared to 2 mM K treatment.

Cation and Anion Contents

Plants subjected to 0 mM K and Alkalinity+0 mM K treatments, Ca2+ content in young leaves were higher than in plants subjected to 2 mM K and Alkalinity+2 mM K treatments (Table 1). Ca2+ content was 75% more with 0 mM K, 48% more with Alkalinity+0 mM K; while 19% less with Alkalinity+2 mM K treatments when compared to 2 mM K treatment. Mg2+ contents in leaf tissues of Swiss chard was 90%, 107%, and 52% more with 0 mM K, Alkalinity+0 mM K, and Alkalinity+2 mM K treatments, respectively compared to 2 mM K treatment. The presence of a mixture of NaHCO3 and Na2CO3 in the culture medium resulted with 32- and 34-fold greater leaf Na+ content in Alkalinity+0 mM K and Alkalinity+2 mM K treatments, respectively. It is 4-fold greater in 0 mM K treatment compared to 2 mM K treatment. Potassium content was 82%, 57%, and 55% lower in young leaves of Swiss chard with Alkalinity+0 mM K, Alkalinity+2 mM K, and 0 mM K treatments, respectively compared to 2 mM K treatment.

Consequently, the lowest Na+/K+ ratio was observed with 2 mM K treatment. For example, the Na+/K+ ratio was 7-fold, 182-fold, and 79-fold higher in young leaves of Swiss chard with 0 mM K, Alkalinity+0 mM K, and Alkalinity+2 mM K treatments, respectively compared to 2 mM K treatment.

On the other hand, it was observed that Cl- content was significant lower with all treatments compared to 2 mM K treatments. It was 92%, 96% and 84% lower after treatments with Alkalinity+0 mM K, Alkalinity+2 mM K, and 0 mM K, respectively, compared to 2 mM K treatment. The NO - content was 7-fold, 4-fold and 1.6-fold greater in Alkalinity+0 mM K, Alkalinity+2 mM K, and 0 mM K treatments, respectively compared to 2 mM K treatment.

Table 1: Changes in the contents of cations and anions (mg g-1 DW), and Na+/K+ ratio in young leaves in response to alkaline and K+ treatments

Measurements###Treatments

###0 mM K###2 mM K###Alkalinity+0 mM K###Alkalinity+2 mM K

Cation

Ca2+###4.03+-0.22a###2.30+-0.28bc###3.4+-0.52ab###1.86+-0.21c

Mg2+###8.80+-0.66a###4.62+-0.46b###9.59+-1.31a###7.00+-0.74ab

Na+###8.35+-0.84b###2.25+-0.22c###73.58+-3.30a###79.18+-5.21a

K+###34.78+-0.93b###78.06+-4.43a###14.15+-1.36c###33.45+-1.79b

Na+/K+###0.24+-0.03c###0.03+-0.00d###5.50+-0.66a###2.39+-0.19b

Anion

Cl-###1.80+-0.15c###23.57+-2.09a###0.84+-0.09d###3.74+-0.41b

NO3###3.37+-0.90b###2.09+-0.86bc###15.32+-4.49a###8.29+-3.62ab

PO43-###23.68+-1.73a###27.19+-1.57a###5.79+-0.59b###3.46+-0.34b

Table 2: Changes in boron (BO3

Measurements###Treatments

###0 mM K###2 mM K###Alkalinity+0 mM K###Alkalinity+2 mM K

BO33-###27.53+-1.27b###41.97+-1.87a###17.40+-2.03c###27.75+-1.82b

Cu2+###2.91+-0.53ab###3.31+-0.39a###2.68+-0.32ab###1.59+-0.19b

Fe3+###169.63+-20.83a###79.56+-4.57b###43.24+-2.35c###52.89+-2.48bc

Mn2+###139.06+-12.22b###234.09+-29.11a###157.79+-14.28b###181.40+-16.67ab

Zn2+###13.41+-0.95b###21.93+-1.34a###9.23+-0.77c###10.19+-0.92c

PO4 content was unchanged by 0 mM K treatment, and significant lower with Alkalinity+2 mM K (79%) and Alkalinity+0 mM K (87%) treatments compared to 2 mM K treatment.

Micronutrient Contents

Borate (BO3 ) contents was 34%, 59% and 34% less in treatments of 0 mM K, Alkalinity+0 mM K and Alkalinity+2 mM K, compared to 2 mM K treatment (Table 3). Copper (Cu2+) content was 12%, 19%, and 52% less in 0 mM K, Alkalinity+0 mM K, and Alkalinity+2 mM K treatments, respectively, compared to 2 mM K treatment. Iron (Fe3+) content was 2-fold greater with 0 mM K treatment, while 0.46- and 0.34-fold lower with Alkalinity+0 mM K, and Alkalinity+2 mM K treatments, respectively, compared to 2 mM K treatment. Manganese (Mn2+) content was 0.41-, 0.33-, and 0.23-folds lower with 0 mM K, Alkalinity+0 mM K, and Alkalinity+2 mM K treatments, respectively compared to 2 mM K treatment. Zinc (Zn2+) content was 0.39-, 0.58-, and 0.54-fold lower with 0 mM K, Alkalinity+0 mM K, and Alkalinity+2 mM K treatments, respectively compared to 2 mM K treatment.

Contribution of Each Ion to Osmotic Potentials of Ions at Full Turgor

The percentage contribution of each ion at Psp from highest to lowest was as follows: K, Na, Mg, PO4, Ca2+, NO3 , Cl and micronutrients for 0 mM K plants (Table 3).

The order for 2 mM plants was K+, Cl-, PO4, Mg-, Na, Ca2+, NO3 , and micronutrients. However, the percentage 100 contribution of each ion at Psp from highest to lowest changed as follows: Na+, Mg2+, K+, NO3, Ca, PO4, Cl, and micronutrients for Alkalinity+0 mM K plants, and Na+, K, Mg, NO3, Cl, Ca, PO4, and micronutrients for Alkalinity+2 mM K plants.

Malondialdehyde Content and Antioxidant Enzyme Activities

The lowest value of MDA content was observed in leaf tissues of 2 mM K treatment and it was 8% more in Alkalinity+2 mM K treatment, 26% more in Alkalinity+0 mM K treatment, and 88% more in 0 mM K treatment compared to 2 mM K treatment (Fig. 3).

For the antioxidants enzymes, the CAT, APX, GPX, and GR activities decreased in treatments without K (Table 4). For example, CAT, APX, GPX and GR activities were 77%, 51%, 45% and 41% lower, respectively in 0 mM K treatment compared to 2 mM K treatment and were also 82%, 58%, 57%, and 87% less, respectively in Alkalinity+0 mM K treatment compared to 2 mM K treatment. With Alkalinity+2 mM K treatment, CAT, APX, and GR activities were 69%, 33%, and 75% lower, respectively, whereas GPX activity was 7% higher compared to the 2 mM K treatment.

Table 3: Percentage contribution of each ion to total osmotic potential at full turgor in young leaves in response to alkaline and K+ treatments

Measurements###Treatments

###0 mM K###2 mM K###Alkalinity+0 mM K###Alkalinity+2 mM K

Ca2+ (%)###4.84+-0.22a###1.72+-0.20bc###1.94+-0.39b###0.94+-0.05c

Mg2+ (%)###17.42+-1.49a###5.70+-0.52c###9.00+-1.37b###5.86+-0.67c

Na+ (%)###17.48+-1.78b###2.93+-0.08c###73.09+-1.45a###70.05+-1.66a

K+ (%)###42.81+-2.54b###59.85+-2.09a###8.27+-1.13d###17.40+-0.89c

Cl- (%)###2.45+-0.31b###19.92+-1.59a###0.54+-0.08c###2.15+-0.37b

NO3- (%)###2.61+-0.50b###1.01+-0.63bc###5.64+-2.02a###2.72+-1.52ab

PO43- (%)###12.00+-0.84a###8.58+-1.26b###1.39+-0.20c###0.74+-0.12c

Micronutrients (%)###0.40+-0.03a###0.30+-0.01b###0.12+-0.00c###0.14+-0.01c

Table 4: Changes in activity of catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and glutathione reductase (GR) in young leaves in response to alkaline and K+ treatments

Measurements###Treatments

###0 mM K###2 mM K###Alkalinity+0 mM K###Alkalinity+2 mM K

CAT (mM min-1 mg-1 protein)###54.86+-8.09b###130.62+-14.56a###23.30+-4.49c###39.86+-6.95bc

APX (mol min-1 mg-1 protein)###1096.10+-181.63b###2246.28+-152.61a###948.37+-127.47b###1505.08+-278.96b

GPX (mol min-1 mg-1 protein)###190.34+-40.03bc###346.49+-32.52a###148.95+-16.32c###371.31+-82.39a

GR (mol min-1 mg-1 protein)###494.55+-122.57b###843.21+-148.09a###113.25+-6.92c###214.07+-35.90bc

These results suggested that, reduction in enzymatic antioxidant capacity in Swiss chard seedlings caused by Alkalinity+0 mM treatment was higher than by 0 mM K and Alkalinity+2 mM K treatments.

Discussion

The effect of potassium on growth and antioxidant enzymes of Beta vulgaris L. in sodic-alkaline conditions seems to be an important study to investigate the role of K+ as a physiological parameter in plant cell. High pH and excessive Na+ in sodic-alkaline soil medium result in serious K+ deprivation in living plant cells (Liu et al., 2013). Several studies have reported that a combination of salinity and K+ starvation had a greater effect on the growth and productivity of barley and maize (Degl'Innocenti et al., 2009; Hafsi et al., 2010; Gong et al., 2011; Qu et al., 2012). This study showed that the lowest biomass production and chlorosis with Alkalinity+0 mM K treatment, which indicates that the absence of K+ partially intensified the effect of alkalinity on reducing the growth of Swiss chard.

As well, the higher biomass production with Alkalinity+2 mM K treatment than with Alkalinity+0 mM K suggest that pre-treated-plants with K+ could resist the detrimental effects of sodic-alkaline conditions. Therefore, K+ plays a crucial role in NaHCO3- and Na2CO3-treated Swiss chard. At the same time, excessive CO32- has its own toxic effect on plant growth because of adverse effects on protein synthesis, respiration, and the absorption of nutrients (Wu and Xing, 2012). High contents of HCO3- strongly affect the availability of several micronutrients, especially Fe, and it is often considered to be the primary factor inducing Fe chlorosis in leaves (Colla et al., 2010). Also, this study, with Alkalinity+0 mM K treatment, the combined stress caused by Na+ toxicity, CO32- and HCO3- toxicities, high pH, and absence of K+, induced the greatest adverse effects on plant growth when compared with other treatments.

Water deficit is the rapid and main consequence that plants experience when exposed to external excessive Na+, and plants can adapt to water deficit by accumulating osmolytes to maintain leaf turgor and physiological activities at relatively low leaf water potential (Ming et al., 2012). From the results here, young leaves of Swiss chard were exposed to water stress in all treatments when compared with 2 mM K treatment and with Alkalinity+0 mM K treatment water stress conditions were greater. Furthermore, osmotic adjustment was not achieved in young leaves of Swiss chard with Alkalinity+0 mM K treatment, indicating that the harmful effects caused by Alkalinity+0 mM K treatment were more serious than by Alkalinity+2 mM K treatment. Hence, the greater reduction in RWC, increase in Na+/K+ ratio, and lack of osmotic adjustment with Alkalinity+0 mM K treatment provides strong evidence that absence of K+ somewhat intensified the effect of sodic- alkalinity on reducing growth of Swiss chard.

The depletion of K in the cytoplasm was crucial for triggering programmed cell death (Shabala, 2009). Following K+ deprivation together with sodic-alkaline treatment, K+ deficiency in living cells of Swiss chard was significantly expedited owing to excessive Na+ accumulation, high pH, and reduction in cytoplasmic K+.

Plant cells of Swiss chard may have sensed the low content of K+ and initiated a series of severe physiological reactions, such as water deficit, ionic imbalance, and oxidative damage, and then seriously limited plant growth in Alkalinity+0 mM K treatment. In the other hand, with Alkalinity+2 mM K treatment, K+ content in living cells of Swiss chard just decreased in the sodic-alkaline treatment period, and maintained at a higher level than that of Alkalinity+0 mM K treatment. Additionally, in some species of Chenopodiaceae, such as sugar beet and red beet, Na+ can substitute for K+ localized in vacuoles for osmotic function to a high degree (Subbarao et al., 2000; Wakeel et al., 2010). For further clarification in this study, in the Alkalinity+2 mM K treatment, Na+/K+ ratio in young leaves of Swiss chard was increased significantly, however, shoot dry weight was not significantly affected, indicating that K+ ions in vacuoles might be substituted by Na+ ions for maintenance of osmotic adjustment.

Furthermore, K+ content in living cells were maintained at a higher level, and Na+ toxic effect in the cytoplasm was somewhat avoided. In terms of Na+ influx and K+ efflux from vacuoles, some studies reported that over-expression of tonoplast sodium/proton exchangers (NHXs) improved salt tolerance in Beta vulgaris (Blumwald and Poole, 1987; Xia et al., 2002; Adler et al., 2010), and tonoplast-located K+ channels or transporters facilitating K+ release from vacuoles into the cytoplasm (Wang and Wu, 2010; Cherel et al., 2014). The ability of Swiss chard to control the cytoplasmic K+ concentration in Alkalinity+2 mM K and Alkalinity+0 mM K conditions could possibly have been regulated by the over-expression of NHXs or tonoplast-located K+ channels and transporters, however, these phenomena need to be further investigated. Furthermore, Na + was taken up by high-affinity K+ transporters (HKTs) from the soil solution to reduce K+ requirements (Rodriguez-Navarro and Rubio, 2006).

Therefore, the higher value of Na+ content in 0 mM K treatment than in 2 mM K treatment indicates that Na+ influx component mediated by the HKT transporter into roots to maintain growth in Swiss chard in 0 mM K treatment.

Elevated cytoplasmic Ca2+ can stimulate NADPH oxidase-mediated production of ROS, and increased ROS levels in turn activate Ca2+-permeable ion channels leading to further Ca2+ influx under K+ deprival condition (Wang and Wu, 2010). Therefore, the higher Ca2+ content in Swiss chard in 0 mM K and Alkalinity+0 mM K treatments may have been caused by the hyperpolarization of cell membranes. This phenomenon was consistent with the significant increase in MDA content in 0 mM K and Alkalinity+0 mM K treatments in Swiss chard. Also, Mg2+ and Ca2+ in most tissues tends to be sequestered in vacuoles, where they contribute to turgor generation (Maathuis, 2009). The increased Ca2+ content together with Mg2+ was observed in 0 mM K and Alkalinity+0 mM K treatments to compensate the reduction in osmolarity induced by K+ deficiency in young leaves of Swiss chard.

Wakeel et al. (2009) reported that Ca2+ uptake and translocation through xylem sap was inhibited in Na+-treated sugar beet (Beta vulgaris L.). Hence, value of Ca2+ content in Alkalinity+0 mM K treatment was lower than in 0 mM K treatment, and value of Ca2+ content in Alkalinity+2 mM K treatment was lower than in 2 mM K treatment.

Although higher concentrations of Cl- is toxic to plants, it is considered as an essential nutrient that regulates enzyme activities in the cytoplasm, acts as a counter anion to stabilize membrane potential, and is involved in turgor and pH regulation (Teakle and Tyerman, 2010). Therefore, the reduction in Cl- in 0 mM K, Alkalinity+0 mM K, and Alkalinity+2 mM K treatments clearly contributed an adverse effect to the natural physiological metabolism processes of Swiss chard plants in these treatments. From a Cl- perspective, the efflux of Cl- could be balanced by the uptake of another anion. NO3 /Cl interactions are analogous to K+/Na+ interactions and many anion channels are more selective for NO - than Cl- (Teakle and Tyerman, 2010). In Alkalinity+2 mM K and Alkalinity+0 mM K treatments, the obvious inductions of NO- would likely offset the scarcity of anions caused by sharp reductions in Cl-.

Whereas, in the 0 mM K treatment, the scarcity of anions caused by sharp reductions in Cl- was offset by PO4 . In addition, NO3 is the predominant form of inorganic nitrogen in high-pH soils, and sodium enhances root-to-leaf nitrate translocation in Swiss chard (Maathuis, 2009; Emi et al., 2015).

The obvious inductions of NO3 in Alkalinity+2 mM K and Alkalinity+0 mM K treatments may also have affected due to high pH and excessive Na+, caused by NaHCO3 and Na2CO3. The PO4 contents decreased sharply, because of the precipitation of PO 3- at higher pH in the environment surrounding the roots under Alkalinity+2 mM K and Alkalinity+0 mM K treatments.

Micronutrients are essential for plant growth but are required in mu ch smaller amounts than macronutrients (Hansch and Mendel, 2009). They involve in all metabolic and cellular functions, and deficiency in any one of these trace elements reduces plant growth and crop yield (Hansch and Mendel, 2009; White and Brown, 2010). However, Cu2+, Fe3+, Mn2+, and Zn2+ contents in young leaves of Swiss chard decreased, because of the limited phytoavailability of these elements at high pH in Alkalinity+2 mM K and Alkalinity+0 mM treatments. Furthermore, BO3 can promote the structural integrity of bio-membranes (Brown et al., 2002; Hansch and Mendel, 2009). The significant decrease of BO3 content in Alkalinity+2 mM K and Alkalinity+0 mM K treatments might contribute to damage to the cell membrane in young leaves of Swiss chard in these treatments.

Osmotic adjustment helps plants to maintain turgor under water stress. Although organic components were the major components that contribute to osmotic adjustment in plants, inorganic components also involved (Kusaka et al., 2005). In this study, K+ was the major inorganic component contributing to osmotic adjustment in living cells of Swiss chard in 0 mM K and 2 mM K treatments. In contrast, in Alkalinity+0 mM K and Alkalinity+2 mM treatments, influx of excess Na+ causes ionic imbalance in the living cells, and Na+ was used to substitute for K+ for osmotic adjustment in young leaves of Swiss chard.

Oxidative stress occurs owing to the rapid accumulation of ROS in response to nutrimental starvation and salinity (Zhu, 2001; Shin et al., 2005), and results in the damage of cell membrane. From the results of this study, the greater increase in MDA content by 0 mM K treatment than that by Alkalinity+0 mM K and Alkalinity+2 mM K treatments, suggests that cell membrane damage was severe in 0 mM K treatment than in Alkalinity+0 mM K and Alkalinity+2 mM K treatments. This phenomenon is consistent with the results of Pi et al. (2014), who reported that MDA content was significantly lower in the replacement of K+ by Na+ groups than in K+-deficient groups in sugar beet with either a salt-sensitive genotype or a salt-tolerant one. They suggested that the substitution of K+ by Na+ in the living cells alleviates damage to the cytomembrane.

This phenomenon may prove the hypothesis that K+ ions in vacuoles are replaced by excessive cytoplasmic Na ions for the maintenance of cytoplasmic K+, and then the protection of cytomembrane in Swiss chard. Among all the treatments, the MDA content was the highest in 0 mM K treatment. Previous reports already suggested that the iron-catalyzed Haber-Weiss reaction is considered as the major mechanism for generating OH in biological systems, which together with H2O2 induces K+ efflux and Ca2+ influx from the plasma membrane (Kehrer, 2000; Demidchik et al., 2003; Demidchik et al., 2007; Zepeda-Jazo et al., 2011). Therefore, higher Fe3+ and Ca2+ contents, less K+ content, and lower enzyme activities (CAT, APX, GPX and GR) in 0 mM K treatment compared to 2 mM K treatment, provided a strong evidence for the overproduction of OH and H2O2 in Swiss chard in 0 mM K treatment.

APX, followed by CAT, are the key enzymes in detoxifying H2O2. However, the results here indicated the activities of APX, CAT and GR were decreased in 0 mM K, Alkalinity+0 mM K, and Alkalinity+2 mM K treatments compared to those of 2 mM K treatment. The reductions in their activities may be caused by the clear reductions in K+, Cl-, Fe3+, and Mn2+ contents in living cells. This is because K+ is involved in the activation of enzymes as the most abundant cation in the cytoplasm, and other elements are also involved with the oxidative enzymes. Among all the treatments, the integrality of cell membrane was most damaged in leaf tissues in 0 mM K treatment, while the lowest activities of CAT, APX, GPX and GR were observed in leaf tissues in Alkalinity+0 mM K treatment. It suggested that the significantly increased Fe3+ content may supply sufficient electrons for OH formation, which may cause major ROS, inducing cell membrane damage in Swiss chard in 0 mM K treatment.

In sugar beet and wild beet, increased GPX was found to protect the plants against oxidative stress during NaCl stress (Bor et al., 2003). In our study, the reduction in GPX activity coincided with the decrease in shoot DW in 0 mM K and Alkalinity+0 mM K treatments. Unaltered GPX activity in Alkalinity+2 mM K treatment coincided with unaltered shoot DW of Swiss chard. Also, GPX protected the plant against oxidative stress in Alkalinity+2 mM K treatment, while this protective function was lacking in 0 mM K and Alkalinity+0 mM K treatments. Therefore, GPX in Swiss chard may be involved in scavenging of H2O2.

In conclusion, K+ supplementation effectively alleviated tolerance to sodic-alkaline stress in Swiss chard seedlings, with its ability to increase cell extension in young leaves. Increased concentrations of K+, Cl-, BO3, Fe and Mn2+, and maintenance of GPX activity also contributed positively. In addition, the favorable changes of Ca2+ influx, K+ efflux, tissue water status, and greater enzyme activities (CAT, APX, GPX and GR) also contributed to the increased tolerance to sodic-alkaline stress conditions exhibited in plants with K+ supplementation.

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Author:Liu, Liyun; El-Shemy, Hany A.; Saneoka, Hirofumi
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