Comparative Effects of Salt Stress and Extreme pH Stress Combined ...

Rice Science, 2009, 16(4):274–282 Copyright © 2009, China National Rice Research Institute. Published by Elsevier BV. All rights reserved DOI: 10.1016/S1672-6308(08)60091-8

Comparative Effects of Salt Stress and Extreme pH Stress Combined on Glycinebetaine Accumulation, Photosynthetic Abilities and Growth Characters of Two Rice Genotypes Suriyan CHA-UM1, Kanyaratt SUPAIBULWATTANA2, Chalermpol KIRDMANEE1 (1National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Thailand Science Park, Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand; 2Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand) Abstract: Glycinebetaine (Glybet) accumulation, photosynthetic efficiency and growth performance in indica rice cultivated under salt stress and extreme pH stress were investigated. Betaine aldehyde dehydrogenase (BADH) activity and Glybet accumulation in the seedlings of salt-tolerant and salt-sensitive rice varieties grown under saline and acidic conditions peaked after treatment for 72 h and 96 h, respectively, and were higher than those grown under neutral pH and alkaline salt 2

stress. A positive correlation was found between BADH activity and Glybet content in both salt-tolerant (r =0.71) and 2

salt-sensitive (r =0.86) genotypes. The chlorophyll a, chlorophyll b, total chlorophyll and total carotenoids contents in the stressed seedlings significantly decreased under both acidic and alkaline stresses, especially in the salt-sensitive genotype. Similarly, the maximum quantum yield of PSII (Fv/Fm), photon yield of PSII ()PSII), non-photochemical quenching (NPQ) and net photosynthetic rate (Pn) in the stressed seedlings were inhibited, leading to overall growth reduction. The positive correlations between chlorophyll a content and Fv/Fm, total chlorophyll content and )PSII, )PSII and Pn as well as Pn and leaf area in both salt-tolerant and salt-sensitive genotypes were found. Saline acidic and saline alkaline soils may play a key role affecting vegetative growth prior to the reproductive stage in rice plants. Key words: rice; betaine aldehyde dehydrogenase; glycinebetaine accumulation; photosynthetic ability; chlorophyll a fluorescence; pigment; saline acidic soil; saline alkaline soil; pH stress

Rice (Oryza sativa L.) is one of the top five major carbohydrate crops for the world’s population, especially in Asia. It is a major staple food, which supports more than three billion people, and represents 50% to 80% of their daily calorie intake [1]. Rice has previously been reported to be salt-sensitive at the seedling and reproductive stages [2-3], leading to a reduction in crop productivity of more than 50% when exposed to 6.65 dS/m electrical conductivity soil salinity [4]. In saline soil, there are many environmental factors which interact with salt contamination, such as soil pH (acidic or alkaline), water deficit and nutrient deficiency [5-7]. Alkalinity and acidity in saline soil are two major factors that inhibit the growth and development of higher plants in both halophyte and glycophyte species [8-10]. In the case of rice, saline sodic and saline acidic soils are major factors in the induction of biochemical and physiological changes in plants, causing growth inhibition and yield loss [11-16]. Received: 9 April 2009; Accepted: 16 July 2009 Corresponding author: Suriyan CHA-UM ([email protected])

Glycinebetaine (Glybet) is a member of the quarterly ammonium compounds, which are accumulated in higher plants as compatible solutes [17] for abiotic defense mechanisms, e.g. against salt stress [18-19], drought stress [20], extreme temperature stress [20] and extreme pH stress [21]. In the Glybet biosynthesis pathway in higher plants, betaine aldehyde dehydrogenase (BADH) has been reported as a key enzyme in the final step, catalyzing betaine aldehyde to Glybet [22]. The BADH gene has been characterized and cloned in numerous experiments in halophyte species and is over-expressed in crop species such as rice [23], maize [24], carrot [25], tomato [26] and tobacco [27-28]. However, there are many plant species that have been identified as Glybet nonaccumulators when they were exposed to salt stress, including japonica rice [29-30], tomato [31], tobacco [32], Najas indica, N. gramenia [33], Alhagi sparsifolia, Calligonum caput-medusea, Populus eurphratica and Tamarix ramosissima [34]. In a previous study, the BADH activity and Glybet accumulation in indica rice under salt stress conditions were investigated [19, 35].

Suriyan CHA-UM, et al. Salt Stress and Extreme pH Stress on Glycinebetaine Accumulation of Two Rice Genotypes

The aim of this investigation is to evaluate the Glybet biosynthesis and the photosynthetic abilities and growth characters in two indica rice varieties, comparing their salt tolerance abilities under the conditions of salt stress and extreme pH stress.

MATERIALS AND METHODS Rice materials and treatments Rice seeds of the salt-sensitive (GS No. 4371) and salt-tolerant (GS No. 7032) genotypes were obtained from a germplasm bank [19]. Seeds were manually husked, sterilized once in 5% Clorox for 60 min, once in 30% Clorox for 30 min, and then rinsed three times with sterile distilled-water. Surface-sterilized seeds were germinated on 0.25% Phytagel-solidified MS media with 3% sucrose (photomixotrophic conditions) in a 250 mL glass vessel. The media were adjusted to pH 5.7 before autoclaving. Rice seedlings were cultured in vitro under the conditions of 25r2qC ambient temperature, 60%r5% relative humidity (RH) and 60r5 μmol/(m2·s) photosynthetic photon flux density (PPFD) provided by fluorescent lamps with a 16 h/d photoperiod. Seven-day-old seedlings were aseptically transferred to MS-liquid sugar-free media (photoautotrophic conditions). The pH in the culture medium was adjusted to 4.5 (acidic pH, 0.1 mol/L HCl), 7.0 (neutral pH, control) and 9.5 (alkaline pH, 0.1 mol/L KOH), and vermiculite was used as supporting material. The uncovered vessels containing photoautotrophic seedlings were transferred aseptically to a culture box chamber (Carry Box Model P-850, size 26 cm u 36 cm u 19 cm, Japan) with controlled RH at 65%r5% by 1.5 L saturated NaCl solution. The number of air exchanges in the culture box chambers was increased to 5.1r0.3 h-1 by punching the side of the plastic chambers with 32 holes and placing gas-permeable microporous polypropylene film (0.22 μm pore size) over each hole [35]. The chambers containing rice seedlings were acclimated for 14 days in a plant growth incubator under a temperature shift of 28r2qC/25r2qC (light/ dark), 500r100 μmol/mol CO2 concentration, 60%r 5% RH, and 120r5 μmol/(m2·s) PPFD provided by fluorescent lamps with a 12 h/d photoperiod. The culture media were adjusted to 0 (control) and 342 mmol/L NaCl (salt stress) for 0, 24, 48, 72, 96, 120 and

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144 h. Data, relating to the biochemical, physiological and growth characters of the rice seedlings were collected. The experiment was arranged as 3u2 factorials in a completely randomized design with ten replications and four seedlings per replication. The mean values of treatments were compared using the Duncan’s New Multiple Range Test (DMRT) and analyzed with the SPSS software. The correlations between BADH activity and Glybet content, chlorophyll a content and maximum quantum yield of PSII (Fv/Fm), total chlorophyll content and photon yield of PSII ()PSII), )PSII and net photosynthetic rate (Pn) as well as Pn and leaf area were evaluated using the Pearson’s correlation coefficients. Biochemical analysis Five hundred milligrams of fresh rice leaf tissue was ground in a mortar set on ice, along with 900 μL cold extraction buffer (50 mmol/L potassium phosphate buffer pH 6.5, containing 0.1 mmol/L EDTA and 20 mmol/L ȕ-mercaptoethanol). Plant debris was removed by centrifugation at 14 500 ug for 30 min at 4qC. The supernatant was mixed with 600 μL 20% sucrose and then applied to a Hitrap Q-FF ion exchange column (Q-Sepharose Fast Flow, Armersham, Sweden), equilibrated with buffer B. Eluted fractions displaying BADH enzyme activity were pooled, aliquoted and then stored at –20qC. The BADH activity in the mixture (0.5 mL) consisting of 1.0 mmol/L betaine aldehyde and 0.3 mmol/L NAD+, was assayed by a spectrophotometer at 340 nm (NADH formation) in a 100 mmol/L potassium phosphate buffer (pH 8.0), with 500 μL crude proteins. The reaction of betaine aldehyde and NAD+ was used as a blank. The initial steady-state rate was determined from the linear portions of reaction progress curves. Each determination was performed at least twice. One unit of activity is defined as the amount of enzyme that catalyzes the formation of 1 μmol of NADH per minute in a standard assay [36]. Total protein concentration was determined using the Bradford Protein Assay with bovine serum albumin as a standard [37]. Five hundred milligrams of rice leaf tissue was frozen in liquid nitrogen for sap extraction and then homogenized in methanol. Glybet was extracted with hot methanol (95qC) for 2 h. After the methanol was

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evaporated off in an air stream [38], the Glybet was then dissolved in water, centrifuged for 15 min at 9 000 ug, and the liquid phase was filtered with a 0.45 μm membrane filter (Minisart SRP15, Sartorius, Germany). The extracted clear solution was purified with a HiTrap Q-FF ion exchange column, followed by high performance liquid chromatography (HPLC, Water, US). The column was stainless steel (250 mm in length and 4.6 mm in width) packed with Partisil 10 SCX. The mobile phase was nanopure water containing 5% methanol buffered with 50 mmol/L KH2PO4 of pH 4.6. A water 2690 pump delivery system was set the flow rate at 1.0 mL/min. Detection was carried out using a Water 996 Photodiode Array (PDA) detector with the wavelength of 195 nm. Glybet (Merck, Germany) was used as a standard and the Glybet content was calculated using a standard curve equation. This compound was mixed with the leaf-extracted sample as an internal standard and the protocol was modified according to Gorham et al [39]. Physiological characteristics Chlorophyll a, chlorophyll b and total chlorophyll contents were analyzed following the methods of Shabala et al [40] and total carotenoids content was analyzed according to Lichtenthaler et al [41]. Net photosynthetic rate (Pn) was calculated by comparing the different concentrations of CO2 inside and outside of glass vessel containing rice seedlings. The CO2 concentrations inside and outside the glass vessel (Cin and Cout) at steady state were measured with a gas chromatography (Model GC-17A, Shimadzu Co.

Ltd., Japan). The Pn of in vitro cultivated seedlings was calculated according to the method of Fujiwara et al [42]. Chlorophyll a fluorescence emissions of the adaxial surface on the leaf were monitored using a fluorescence monitoring system (Hansatech Instruments Ltd, Norfolk, UK) in the pulse amplitude modulation mode, as previously described by Loggini et al [43] and Maxwell and Johnson [44]. Rice growth measurements Plant height, fresh weight, dry weight and leaf area of rice seedlings were measured as described by Lutts et al [45]. The leaf area of rice seedlings was measured using a leaf area meter DT-scan (Delta-Scan Version 2.03, Delta-T Devices, Ltd., UK).

RESULTS BADH activity The BADH activities in both salt-sensitive (Fig. 1-A) and salt-tolerant (Fig. 1-B) genotypes of indica rice treated with 342 mmol/L NaCl gradually increased in the initial exposure time (0–72 h) and then decreased during the late exposure time (96–144 h). The activities of BADH in both genotypes peaked after exposure to the salt stress for 72 h. In addition, the BADH activity in seedlings under the salt stress combined with acidic pH was expressed at a significantly higher level than that in seedlings under the salt stressed with neutral pH. Besides, the activities of BADH in both salt-sensitive and salt-tolerant genotypes cultured under 0 mmol/L NaCl combined with alkaline,

Fig. 1. BADH activities in salt-sensitive (A) and salt-tolerant (B) genotypes cultured photoautotrophically and subsequently exposed to 342 mmol/L NaCl or without NaCl combination with pH 4.5, 7.0 or 9.5 for 24, 48, 72, 96, 120 and 144 h. Error bars represent meanrSE.


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Fig. 2. Glycinebetaine contents in salt-sensitive (A) and salt-tolerant (B) genotypes cultured photoautotrophically and subsequently exposed to 342 mmol/L NaCl or without NaCl combination with pH 4.5, 7.0 or 9.5 for 24, 48, 72, 96, 120 and 144 h, respectively. Error bars represent meanrSE.

neutral and acidic pH conditions were initially expressed at very low levels [1.73–4.45 U/(min·mg)] and subsequently exhibited when seedlings were exposed to the salt stress [9.91–24.12 U/(min·mg)] combined with extreme pH stress (pH 4.5 or pH 9.5). Moreover, the BADH activities in both salt-sensitive and salt-tolerant genotypes grown in acidic or basic pH environments were exhibited at higher levels than those in neutral pH conditions (Fig. 1). The factors involved in the activity of BADH not only depended on the signals of salt stress and pH conditions, but were also related to rice genotype. Glybet accumulation The accumulation of Glybet in both salt-tolerant (Fig. 2-A) and salt-sensitive (Fig. 2-B) genotypes displayed a similar pattern to the BADH activity. It should be noted that the Glybet accumulation in rice seedlings was dependent on salt stress, rice genotype and extreme pH conditions (Fig. 2). The highest value (78.50 μg/g DW) was observed in the salt-tolerant genotype after exposure to the acidic salt stress for 96 h. The accumulation of Glybet in plants of different rice genotypes was stimulated by environmental factors, such as salt stress and acidic pH stress. Moreover, the results showed that the BADH activities in both saltsensitive (Fig. 3-A, r2 = 0.71) and salt-tolerant (Fig. 3-B, r2 = 0.86) genotypes were positively related to the Glybet accumulation. Photosynthetic pigment contents, physiological and growth characteristics Photosynthetic

pigment

contents,

including

chlorophyll a, chlorophyll b, total chlorophyll and total carotenoids contents in the salt-sensitive and salttolerant rice genotypes were significantly degraded when plants were exposed to acidic and alkaline stresses. The performances of the photosynthetic pigment contents in the salt-tolerant rice seedlings were maintained significantly better than those in the salt-sensitive seedlings when the seedlings were exposed to extreme pH and salt stresses (Table 1). Genotype, salt stress and extreme pH factors significantly affected the photosynthetic pigment stability. The chlorophyll a degradation in the salt stressed seedlings was positively related to the maximum quantum yield of PSII (Fv/Fm) in both salt-sensitive (r2 = 0.89) and salt-tolerant (r2 = 0.93) genotypes (Fig. 4). In addition, the total chlorophyll contents in the salt stressed seedlings were certainly correlated with the photon yield of PSII ()PSII) in both salt-sensitive (r2 = 0.91) and salt-tolerant (r2 = 0.86)

Fig. 3. Relationship between betaine aldehyde dehydrogenase (BADH) activity and glycinebetaine content in salt-sensitive (A) and salt-tolerant (B) genotypes cultured photoautotrophically and subsequently exposed to 342 mmol/L NaCl combination with pH 4.5, 7.0 or 9.5 for 144 h.


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Table 1. Chlorophyll a, chlorophyll b, total chlorophyll, and total carotenoids contents in salt-sensitive and salt-tolerant genotypes grown under 0 or 342 mmol/L NaCl with pH 4.5, 7.0 or 9.5 for 144 h. Genotype Salt-sensitive

Salt-tolerant

NaCl (mmol/L)

pH

Chlorophyll a (μg/g FW)

Chlorophyll b (μg/g FW)

0 0 0 342 342 342 0 0 0 342 342 342

4.5 7.0 9.5 4.5 7.0 9.5 4.5 7.0 9.5 4.5 7.0 9.5

570 f 813 d 731 e 184 i 270 h 185 i 882 c 1144 a 990 b 225 hi 359 g 226 hi

223 c 266 c 230 c 37 f 106 de 46 f 325 b 417 a 348 b 75 ef 158 d 78 ef

Total chlorophyll (μg/g FW) 793 f 1079 d 961 e 221 i 376 h 231 i 1207 c 1561 a 1338 b 300 hi 517 g 304 hi

Total carotenoids (μg/g FW) 191 c 222 ab 214 b 47 g 92 de 53 fg 220 ab 237 a 225 ab 65 fg 101 d 72 ef

Significant level Var ** ** ** ** NaCl ** ** ** ** pH ** ** ** ** VaruNaCl ** ** ** NS VarupH ** NS * NS NaClupH ** NS ** ** VaruNaClupH NS NS NS NS Data followed by different lowercase and uppercase letters in each column show significant difference at 0.05 and 0.01 levels, respectively, by the Duncan’s New Multiple Range Test (DMRT). *, P d 0.05; **, P d 0.01; NS, Non significant in statistical analysis.

genotypes (Fig. 5), leading to a reduction of net photosynthetic rate (Pn) (Fig. 6) (r2 = 0.83 and r2 = 0.81). The Fv/Fm and )PSII parameters in the stressed seedlings decreased significantly when the seedlings were exposed to the extreme pH stress and salt stress, while the non-photochemical quenching (NPQ) increased (Table 2). The reduction of pigment contents and chlorophyll a fluorescence parameters in the stressed seedlings directly restricted Pn. Moreover, the performances of the photosynthetic pigment contents and the chlorophyll a fluorescence parameter in the salt-tolerant seedlings under the extreme pH and salt stress were maintained better than those in the salt-

Fig. 4. Relationships between chlorophyll a content and maximum quantum yield of PSII (Fv/Fm) in the salt-sensitive (A) and salt-tolerant (B) genotypes cultured photoautotrophically and subsequently exposed to 342 mmol/L NaCl or without NaCl combination with pH 4.5, 7.0 or 9.5 for 144 h. Error bars represent meanrSE.

Fig. 5. Relationships between total chlorophyll content (TC) and photon yield of PSII ()PSII) in the salt-sensitive (A) and salttolerant (B) genotypes cultured photoautotrophically and subsequently exposed to 342 mmol/L NaCl or without NaCl combination with pH 4.5, 7.0 or 9.5 for 144 h. Error bars represent rSE.

Fig. 6. Relationships between photon yield of PSII ()PSII) and net photosynthetic rate (Pn) in the salt-sensitive (A) and salttolerant (B) genotypes cultured photoautotrophically and subsequently exposed to 342 mmol/L NaCl or without NaCl combination with pH 4.5, 7.0 or 9.5 for 144 h. Error bars represent meanrSE.


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Table 2. Maximum quantum yield of PSII (Fv/Fm), photon yield of PSII ()PSII), non-photochemical quenching (NPQ) and net-photosynthetic rate (Pn) in salt-sensitive and salt-tolerant genotypes grown under 0 or 342 mmol/L NaCl with pH 4.5, 7.0 or 9.5 for 144 h. Genotype Salt-sensitive

Salt-tolerant

NaCl (mmol/L)

pH

Fv/Fm

)PSII

NPQ

Pn [mol/(m2· s)]

0 0 0 342 342 342 0 0 0 342 342 342

4.5 7.0 9.5 4.5 7.0 9.5 4.5 7.0 9.5 4.5 7.0 9.5

0.86 ab 0.87 ab 0.86 ab 0.58 e 0.71 cde 0.59 de 0.88 a 0.89 a 0.89 a 0.66 cde 0.74 bc 0.72 cd

0.61 abc 0.74 a 0.66 ab 0.43 d 0.53 bcd 0.52 bcd 0.61 abc 0.68 ab 0.65 ab 0.47 cd 0.56 bcd 0.55 bcd

0.07 cd 0.03 d 0.05 d 0.14 a 0.07 bcd 0.11 abc 0.06 cd 0.03 d 0.04 d 0.12 ab 0.05 d 0.06 cd

2.48 d 2.68 c 2.57 cd 0.84 h 1.03 fg 0.91 gh 2.90 b 3.11 a 2.99 ab 1.18 ef 1.34 e 1.23 e

Significant level Var * NS * ** NaCl ** ** ** ** pH NS NS ** ** VaruNaCl NS NS NS ** VarupH NS ** NS NS NaClupH NS NS * NS VaruNaClupH NS NS ** NS Data followed by different lowercase and uppercase letters in each column show significant difference at 0.05 and 0.01 levels, respectively, by the Duncan’s New Multiple Range Test (DMRT). *, P d 0.05; **, P d 0.01; NS, Non significant in statistical analysis.

sensitive seedlings, resulting in high Pn. The growth characteristics, in terms of plant height, fresh weight, dry weight and leaf area in the salt-tolerant seedlings were exhibited to a significantly higher degree than those in the salt-sensitive seedlings when the seedlings were exposed to the extreme pH and salt stress. Also, the reduction of growth characters depended on rice genotype, salt stress and extreme pH conditions (Table 3).

DISCUSSION Glybet biosynthesis via BADH has been investigated in many plant species. The accumulation of Glybet is dependent on plant species [46-47], plant varieties [19, 48-49], plant organelles [48, 50] and environmental factors, such as salt [18, 51-52], drought [20, 49], alkaline stress [21, 53] and extreme temperature [20]. In the present study, the

Table 3. Growth characters of salt-sensitive and salt-tolerant genotypes grown under 0 or 342 mmol/L NaCl with pH 4.5, 7.0 or 9.5 for 144 h. Genotype Salt-sensitive

Salt-tolerant

NaCl (mmol/L)

pH

Plant height (cm)

Fresh weight (mg)

0 0 0 342 342 342 0 0 0 342 342 342

4.5 7.0 9.5 4.5 7.0 9.5 4.5 7.0 9.5 4.5 7.0 9.5

15.03 e 17.48 cd 16.23 de 11.59 g 13.45 f 12.03 fg 18.23 c 21.58 a 20.08 b 15.33 e 18.25 c 16.95 cd

123 de 137 c 131 cd 107 f 118 ef 109 f 138 c 165 a 150 b 117 ef 134 cd 127 cde

Dry weight (mg) 23 fg 54 c 51 c 18 h 38 e 36 e 26 f 66 a 59 b 22 gh 50 c 45 d

Leaf area (cm2) 5.05 d 5.46 bc 5.27 cd 2.81 f 3.07 f 2.85 f 5.60 bc 6.09 a 5.81 ab 4.15 e 4.28 e 3.96 e

Significant level Var ** ** ** ** NaCl ** ** ** ** pH ** ** ** ** VaruNaCl * * NS NS VarupH * * ** ** NaClupH NS NS ** * VaruNaClupH NS NS NS NS Data followed by different lowercase and uppercase letters in each column show significant difference at 0.05 and 0.01 levels, respectively, by the Duncan’s New Multiple Range Test (DMRT). *, P d 0.05; **, P d 0.01; NS, Non significant in statistical analysis.


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BADH activities and Glybet contents in rice plants cultured under the acidic (pH 4.5) stress for 72 and 96 h, respectively were enriched to a greater degree than those in the alkaline (pH 9.5) and neutral (pH 7.0) conditions. In Leymus chinensis, the BADH activity and Glybet content are regulated better in alkaline pH (9.3–9.6) than those in neutral conditions (pH 7.8) [21]. In addition, Glybet in the wheat genotype Giza 164 grown under alkaline conditions (pH 9.0) accumulated to a higher level than that in the genotype Gemmiza under control conditions [53]. The BADH activity and the Glybet accumulation in the salt-tolerant rice varieties were both greater than those in the salt-sensitive varieties under acidic and alkaline stresses. In a previous study, those parameters were investigated under neutral pH conditions [19]. In reed ecotypes, the BADH activity and the Glybet accumulation in dune and heavy salt meadow reeds were superior to those in swamp reeds [48]. Glybet accumulated to a greater degree in Atriplex halimus ‘Monastri’ than that in ‘Sbikha’ when grown under 160 mmol/L NaCl conditions for 10 days [49]. In addition, the mRNA expression of BADH in the leaf and root tissues of Avicennia marina ‘clone 2’ was regulated higher than that of ‘clone 13’ when grown under 400 mmol/L NaCl conditions [46]. Whereas the Glybet content and the BADH gene in Salicornia europaea and Suaeda maritima were positively expressed depending on salt stress treatments [47]. In Alternathera philoxeroides cell culture, sodium ions were enriched after exposure to salinity for 2 h and BADH protein peaked after exposure to salinity for 6 h. Subsequently, Glybet, proline and sucrose were accumulated as osmolytes after exposure to salinity for 12, 72 and 72 h, respectively [18]. The contents of photosynthetic pigments, including chlorophyll a, chlorophyll b and total carotenoids contents in rice plants grown under the extreme pH combined with salt stress declined significantly, being especially susceptible to salt. Similarly, the performances of the chlorophyll pigment contents in the flag leaves of tolerant rice varieties KS-282, Nona Bokra and NIAB-6 grown in saline sodic soil were maintained better than in the sensitive varieties Basmati-6129, Basmati-385, Basmati-370 and Basmati-4048 [11]. In addition, the chlorophyll a, chlorophyll b and total carotenoids contents in the salt resistant wheat

‘Xiaobingmai 33’ grown under alkaline salt stress (pH 9.9 and 60–75 mmol/L NaCl) were 2.5–5.0 times lower than those grown under salt stress only [54]. Water content in leaf tissues and water oxidation in the photosystem II of plants under salt stress decreased significantly in wheat [54] and rice [19]. In rice plants, Pns in the salt-sensitive varieties Jiyou 1 [15], Basmati6129, Basmati-385, Basmati-370 and Basmati-4048 [11] cultivated in saline alkaline soil were decreased significantly. In the present study, there was a positive relationship between chlorophyll a content and Fv/Fm, )PSII and Pn, Pn and leaf area in the stressed seedlings. These findings are similar to a previous study of rice [19]. Also, the growth performances in both salt-tolerant and salt-sensitive varieties were significantly reduced, depending on acidic and alkaline salt stresses. In conclusion, the BADH activity and the Glybet accumulation in the salt stressed rice seedlings peaked at 72 h and 96 h after being exposed to the acidic salt stress, respectively, maintained in the salt-tolerant genotype better than in the salt-sensitive genotype and displayed a positive correlation. The contents of photosynthetic pigments in the salt-tolerant genotype maintained at a higher level than those in the saltsensitive genotype, using Glybet as osmotic adjustment, leading to higher photosynthetic abilities in both light and dark reactions, identified by chlorophyll a fluorescence and Pn parameters. The photosynthetic abilities of salt-tolerant genotypes directly enhanced growth performance when exposed to extreme pH stress and salt stress.

ACKNOWLEDGEMENTS The authors are grateful to Prof. Dr. Selvaraj GOPALAN at the Plant Biotechnology Institute (PBI), the National Research Council (NRC), Canada for consulting on Glybet qualification by HPLC and Jonathan SHORE for grammatical proofing. This research was supported by the National Center for Genetic Engineering and Biotechnology (BIOTEC) (Grant No. BT-B-06-RG-14-4502) and partly funded by the International Atomic Energy Agency (IAEA) (Contract No. 12998/R0).

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