Survival percentage, photosynthetic abilities and growth characters of two indica rice (Oryza sativa L. spp. indica) cultivars in response to iso-osmotic stress

The aim of this study was to investigate pigment degradation, chlorophyll fluorescence diminution, photosynthetic ability and growth reduction in two rice cultivars, in response to either iso-osmotic salt stress or water-deficit stress. Seedlings of rice cultivars RD6 and KDML105 were photo-autotrophically grown in MS media and subsequently exposed to –0.23 (control), –0.40 or –0.67 MPa iso-osmotic NaCl (salt stress) or mannitol (water-deficit stress) for 14 days. The survival percentage of the two rice cultivars reduced dramatically when subjected to –0.67 MPa NaCl treatment. Chlorophyll a (Chla), chlorophyll b (Chlb), total carotenoids (Cx+c), maximum quantum yield of PSII (Fv/Fm) and photon yield of PSII (ΦPSII) in the stressed seedlings were significantly lower when compared to seedlings in the control group (without mannitol or NaCl), leading to low net-photosynthetic rate (Pn) and growth reduction. In addition, the growth characters of plantlets in the salt stress conditions were more sharply reduced, and the physiological changes greater than those in water-deficit stress conditions. On the other hand, non-photochemical quenching in the leaves of stressed plantlets increased significantly, especially in response to iso-osmotic salt stress. In the present study, overall growth performance and physiological characters of KDML105 grown under iso-osmotic stress were better than those of RD6. Additional key words: net-photosynthetic rate, photosynthetic pigments, salt stress, water deficit.


Introduction
Rice is a major source of carbohydrates in many regions of the world, especially Asian countries, feeding more than 3 billion people and providing 50-80% of their daily calorie intake (Khush, 2005). Rice has been identif ied as a salt susceptible and water def icit susceptible plant species, demonstrating negative effects, including, leaf necrosis, burning, stunting, flower sterility and yield loss (Shannon et al., 1998;Zeng and Shannon, 2000;Khan and Abdullah, 2003;Zeng et al., 2003). The trait of salt tolerance is a major issue, and has been a large factor in implementing breeding programs in rice for the development of novel crops, in order to provide food security Senadhira et al., 2002;Flowers and Flowers, 2005). In Thailand, jasmine rice or KDML105 is a premium quality grain, which is well known worldwide for its cooking quality, long grains, aroma, flavor and soft texture (Ariyaphanphitak et al., 2005;Laohakunjit and Kerdchoechuen, 2007). In addition, RD6, a gammairradiation mutant variety derived from KDML105, is a sticky or glutinous rice with high cooking quality, long grains, sticky texture and enriched aroma and flavor (Keeratipibul et al., 2008). These cultivars are widely cultivated in northeastern Thailand, which is a large area of rain fed lowland, affected by inland salinity and water deficit problems (Wongpokkhom et al., 2008). Hydroponic system has been well established for salt tolerant screening in large population of rice crop especially in IRRI (International Rice Research Institute) (Gregorio et al., 1997). Environmental control in plant tissue culture is an alternative protocol, which is a rapid, simple and reliable method to be screened more than 100 tree species (Kirdmanee and Mosaleeyanon, 2000), 1,000 lines of rice, 50 cultivars of sugarcanes and 40 clones of eucalypt for salt tolerant trait in one and a half months. A positive correlation between the data in the environmental control and salinity field trials has been investigated (Cha-um and Kirdmanee, 2008).
Water-deficit, and salt affected soil are two major abiotic stresses which reduce crop productivity, especially that of rice, by more than 50% world-wide (Mahajan and Tuteja, 2005). In salt-susceptible (glycophyte) plant species, biochemical, physiological and morphological characteristics are negatively affected, leading to abnormal growth and development, and eventual plant death (Hasegawa et al., 2000;Wang et al., 2001;Parida and Das, 2005). The effects of waterdeficit stress, caused by excessive salts or osmotic agents added to the media (sugar alcohol and polyethylene glycol), are well established in many crop species (Lutts et al., 2004;Wahid, 2004;Luo et al., 2005;Castillo et al., 2007). In halophyte, or tolerant plants, there are several defense mechanisms, such as osmoregulation, ion homeostasis, antioxidant and hormonal systems, which help plants to stay alive and continue developing, prior to their reproductive stages (Hasegawa et al., 2000;Wang et al., 2003;Reddy et al., 2004;Sairam and Tyagi, 2004;Mahajan and Tuteja, 2005). Mannitol is a member of the sugar alcohols which has been used to control the osmotic potential in nutrient solutions in order to induce water deficit conditions, especially in the root zone (Zang and Komatsu, 2007). It has been reported as an effective osmoticum which only controls the osmotic potential, whereas polyethylene glycol (PEG) has been established as a candidate osmoticum, being a membrane injury agent (Ahmad et al., 2007). Sodium chloride salt is a small molecule, quickly oxidized by water into Na + and Cl -, which cause damage to plant cells (Dionisio-Sese and Tobita, 1998;Sultana et al., 1999;Vaidyanathan et al., 2003;Cha-um et al., 2007a). Sodium chloride salts are quickly dissolved in the water and play as ionic effects in higher plant including rice crop. In contrast, the mannitol-induced osmotic stress is only functioned as osmoticum or water deficit effects (Castillo et al., 2007).
The aim of this work was to investigate pigment degradation, chlorophyll fluorescence diminution, photosynthetic ability and growth reduction in two rice cultivars, in response to either iso-osmotic salt stress or water-deficit stress.

Plant materials
Seeds of two rice cultivars, glutinous rice (cv. RD6 a KDML105-gamma irradiation mutant) and non-Osmotic stress responses in rice genotypes 263 glutinous rice (cv. KDML105), were obtained from the germplasm bank, Rice Research Center, Thailand. The seeds were manually dehusked, sterilized once in 5% Clorox ® for 60 min, once in 30% Clorox ® for 30 min, then rinsed three times with sterile distilled-water. Surface-sterilized seeds were germinated on 0.25% Phytagel ® -solidified MS media (Murashige and Skoog, 1962) with 3% sucrose (photomixotrophic condition) in a 250 mL glass vessel. The media were adjusted to pH 5.7 before autoclaving. Rice seedlings were cultured in vitro under conditions of 25 ± 2°C ambient temperature, 60 ± 5% relative humidity (RH) and 60 ± 5 µmol m -2 s -1 photosynthetic photon flux density (PPFD) provided by fluorescent lamps with 16 h d -1 photoperiod. Fourteen-day-old seedlings were aseptically transferred to MS-liquid sugar-free media (photoautotrophic conditions). The uncovered vessels containing photoautotrophic seedlings were transferred aseptically to a culture box chamber (Carry Box Model P-850, size 26 × 36 × 19 cm, Japan) with RH controlled at 65 ± 5% by 1.5 L saturated NaCl solution, in order to enhance realistic phenotypic expression in the aerial parts of the rice seedlings. The number of air exchanges in the culture box chambers was increased to 5.1 ± 0.3 µmol CO 2 h -1 by perforating the sides of the plastic chambers with 32 holes and placing gas-permeable microporous polypropylene film (0.22 µm pore size) over the holes (Cha-um et al., 2007a). The chambers containing the rice seedlings were acclimated for 14 days in a plant growth incubator under a temperature shift of 28± 2°C/ 25 ± 2°C (light/dark), 500± 100 µmol mol -1 CO 2 concentration, 60 ± 5% RH, 120 ± 5 µmol m -2 s -1 PPFD provided by fluorescent lamps with 16 h d -1 photoperiod. Mannitol (water-def icit stress) and sodium chloride (salt stress) in the culture media were adjusted to -0.23 (control), -0.40 (50 mM NaCl or 100 mM mannitol) or -0.67 MPa (100 mM NaCl or 200 mM mannitol) iso-osmotic pressure for 14 days. Survival percentage, photosynthetic pigments, chlorophyll fluorescence, net-photosynthetic rate (P n ) and growth characters were measured to determine physiological and biochemical changes.

Data collection
Chlorophyll a (Chl a ), chlorophyll b (Chl b ) and total chlorophyll (TC) concentrations were determined following the method of Shabala et al. (1998) and the total carotenoids (C x+c ) concentration was measured according to Lichtenthaler (1987). One hundred milligrams of leaf material was collected, placed in a 25 mL glass vial along with 10 mL 95.5% acetone, and blended using a homogeniser. The Chl a , Chl b , and C x+c concentrations were measured using an UV-visible spectrophotometer. A solution of 95.5% acetone was used as a blank.
Chlorophyll fluorescence emission from the adaxial surface of the leaf was measured using a fluorescence monitoring system in the pulse amplitude modulation mode, as previously described by Loggini et al. (1999). A leaf, adapted to dark conditions for 30 min using leaf-clips, was initially exposed to the modulated measuring beam of far-red light (LED source with typical peak at wavelength 735 nm). Original (F 0 ) and maximum (F m ) fluorescence yields were measured under weak modulated red light (< 0.5 µmol m -2 s -1 ) with 1.6 s pulses of saturating light (> 6.8 µmol m -2 s -1 PAR) and calculated using FMS software for Windows ® . The variable fluorescence yield (F v ) was calculated by the equation F m -F 0 . The ratio of variable to maximum fluorescence (F v /F m ) was calculated as maximum quantum yield of PSII photochemistry. The photon yield of PSII (Φ PSII ) in the light was calculated by Φ PSII = (F m ' -F)/F m ' after 45 s illumination, when steady state was achieved (Maxwell and Johnson (2000).
Net-photosynthetic rate (P n ) was calculated by measuring the different concentrations of CO 2 inside and outside the glass vessel containing the seedlings. The CO 2 concentrations (C in and C out ) at steady state were measured by gas chromatography (GC; Model GC-17A, Shimadzu Co. Ltd., Japan). The detector (TCD; thermal conductivity detector) and injector were set at 250°C. The temperature program of a GC capillary column (GS-Q, J&W Scientif ic ® , Germany) was set at 30°C for 1 min in the initial state and increased to 100°C at a rate of 20°C min -1 and held for 1 min (Fujiwara et al., 1987). The P n of in vitro cultivated seedlings was calculated according to the method of Fujiwara et al. (1987).
Survival percentage, fresh weight (FW), dry weight (DW), shoot height (SH), root length (RL) and leaf area (LA) of rice seedlings were measured, as described by Cha-um et al. (2006). Seedlings were dried at 80°C in a hot-air oven for 2 days, and then incubated in desiccators before the measurement of dry weight. The leaf area of rice seedlings was measured using a leaf area meter DT-scan. 264 T. Nishimura et al. / Span J Agric Res (2011) 9(1), 262-270

Experiment design and data analysis
The experiment was arranged as completely randomized design with ten replicates and four plantlets per replicate. The mean values obtained were compared by Tukey's HSD (honestly significant difference) test and analyzed using SPSS software. The correlations between biochemical, physiological and growth characters were evaluated using Pearson's correlation coefficients.

RD6 rice cultivar
The survival percentage of RD6 rice seedlings declined to 66.7 and 33.3% when exposed to -0.67 MPa mannitol induced stress and salt induced osmotic stress, respectively (Fig. 1). The level of photosynthetic pigments in the leaf tissues of rice seedlings cultivated under osmotic stress conditions decreased, and was correlated with the decrease in osmotic potential in the culture media (Table 1). Chl a , TC and C x+c in the osmotically stressed leaves of RD6 rice seedlings decreased significantly, related to the decrease in osmotic pressure in the culture media (Table 1). The Chl b content in the leaf tissues showed a dramatic decrease only under -0.67 MPa NaCl treatment. The Chl a :Chl b ratio began to decrease following exposure to -0.40 MPa NaCl. Those pigments were extensively damaged, by 71.9, 33.6, 58.2 and 80.7% respectively, when subjected to -0.67 MPa NaCl (Table 1). Moreover, the Chl a content in relation to osmotic stress was positively related to maximum quantum yield of PSII (F v /F m ) (R 2 = 0.65) (Fig. 2a). The F v /F m and photon yield of PSII (Φ PSII ) in RD6 rice seedlings grown under -067 MPa NaCl were significantly reduced when compared to the plantlets of the control group (-0.23MPa), while the same measurements in plantlets grown in -0.67 MPa mannitol were unchanged (Table 2). On the other hand, nonphotochemical quenching (NPQ) in osmotically stressed seedlings increased, especially in response to -0.67 MPa NaCl. The reduction of Φ PSII in response to osmotic stress was positively correlated with net-photosynthetic rate (P n ) (R 2 = 0.52) (Fig. 2c). P n reduced sharply in osmotically stressed seedlings exposed both Osmotic stress responses in rice genotypes 265  to extreme salt stress and extreme water-deficit stress (Table 2), leading to growth reduction. FW, DW, SH, RL and LA in osmotically stressed seedlings were significantly reduced, related to the osmotic pressure in the culture media (Table 3). In addition, biochemical, physiological and growth characters, including Chl a , TC, C x+c , F v /F m , Φ PSII , P n and DW were positively correlated with each other to a highly significant (p ≤ 0.01) level in statistical analysis (Table 4). In this study, iso-osmotic salt stress was found to inhibit the growth and development of RD6 rice seedlings to a greater extent than water-deficit stress.

KDML105 rice cultivar
In the case of KDML105, the survival percentage of rice seedlings declined to 81.7% and 58.3% when subjected to -0.67 MPa mannitol-induced stress and salt-induced osmotic stress, respectively (Fig. 1). The survival percentage of KDML105 rice under osmotic stress was maintained better than that of RD6 rice. Photosynthetic pigments in the leaf tissues of rice seedlings grown under osmotic stress were damaged, positively related to the decrease in osmotic potential in the culture media (Table 1). Chlorophyll a (Chl a ) and  (C x+c ) content in the leaf tissues dropped sharply, depending on the degree of mannitol-induced stress or salt-induced stress (Table 1). Chl a , TC and C x+c were signif icantly degraded, by 54.9, 35.7 and 68.7%, respectively when subjected to -0.67 MPa NaCl. The exception was Chl b , which was maintained (Table 1). In addition, the Chl a content in response to osmotic stress was positively related to maximum quantum yield of PSII (F v /F m ) (R 2 = 0.79) (Fig. 2b). The F v /F m and photon yield of PSII (Φ PSII ) in KDML105 seedlings grown under -0.67 MPa NaCl decreased significantly when compared to plantlets in the control group (-0.23MPa), while the Φ PSII in plantlets grown in -0.67 MPa mannitol were unchanged (Table 2). In contrast, NPQ in osmotically stressed seedlings increased, especially in response to -0.67 MPa NaCl. The reduction of Φ PSII in response to osmotic stress was positively correlated with net-photosynthetic rate (P n ) (R 2 = 0.62) (Fig. 2d). The P n in osmotically stressed seedlings reduced sharply during exposure to salt stress and water-deficit stress (Table 2), leading to growth reduction. FW, DW, SH, RL and LA in osmotically stressed seedlings were significantly reduced, related to osmotic pressure in the culture media (Table 3). The Chl a , TC, C x+c , F v /F m , Φ PSII , P n and DW showed a positive correlation with a highly significant level (p ≤ 0.01) ( Table 4).
Osmotic stress responses in rice genotypes 267 Table 4. Relationship between biochemical, physiological and growth characters of two rice cultivars grown under iso-osmotic water deficit (mannitol) and salt (NaCl) stress for 14 days. Highly significant levels at p ≤ 0.01 of all relationships are represented by Pearson's correlation coefficients

Discussion
The survival percentage of rice seedlings cultivated in salt-induced osmotic stress was greatly reduced, and to a higher degree than those under mannitol-induced stress, related to the genotype and degree of salt tolerance. RD6 and KDML105 rice cultivars have been reported as salt susceptible, showing toxic symptoms, including pigment degradation and growth reduction, in 171 mM NaCl or -0.42 MPa osmotic potential (Cha-um et al., 2007b(Cha-um et al., , 2010. The survival percentage of INIAP12 salt tolerant rice grown under iso-osmotic water deficit stress (200 mM mannitol) and salt stress (100 mM NaCl) was higher than that of CT6748 -8-CA-17, a salt sensitive cultivar (Morsy et al., 2007). Na + in culture media supplemented with NaCl may not only play a role as a toxic ion, but also have osmotic effects for the rice plant, especially in the seedling stage. In this study, KDML105 was found to be more tolerant to salt stress than RD 6. It is possible that Na + uptake and accumulation in KDML105 is lower than in RD6. Under 12 dS m -1 NaCl treatment, the Na + level in Pokkali rice (salt tolerant) was lower than that in IR28 (salt susceptible) (Dionisio-Sese and Tobita, 1998). The low Na + accumulation in salt tolerant cultivars of rice may depend on the activity of H + -ATPase, H + -PPase, Na + /H + antiporters and Na + /K + pumping (Fukuda et al., 2004;Martínez-Atienza et al., 2007). The photosynthetic pigments in osmotically stressed rice seedlings of different genotypes were damaged, depending on the increase in osmotic pressure. The degradation of TC and C x+c in the leaves of Koshihikari, a moderately salt tolerant genotype, when grown under salt stress, was demonstrated (Sultana et al., 1999). In addition, Chl a and Chl b in the Taipei 309 rice cultivar grown under sorbitol-induced water deficit were enriched, whereas under NaCl induced stress they dropped significantly (Bahaji et al., 2002). Enriched Na + in saltstressed seedlings badly damages the cells, organelles and tissues, prior to cell death (Ahmad et al., 2007;Castillo et al., 2007). Photosynthetic pigments and their activities are very sensitive to enriched Na + in the cells, identified by pigment degradation and chlorophyll fluorescence diminution, respectively (Dionisio-Sese and Tobita, 1998;Castillo et al., 2007). Pigment degradation in osmotically-stressed rice seedlings was a major factor in limiting photosynthetic activity, including low F v /F m , Φ PSII and P n, leading to overall growth inhibition. P n in salt-stressed Koshihikari rice (100 mM NaCl) dropped signif icantly, by 2.33 times, when compared to the control (0 mM NaCl) (Sultana et al., 1999). In most cases, the physiological characteristics, including levels of photosynthetic pigments, chlorophyll fluorescence and P n in salt stressed seedlings were higher than in seedlings grown under mannitolinduced stress and were directly related to growth inhibition and decreased productivity (Bahaji et al., 2002;Cha-um et al., 2010). Na + toxicity from the salt stress treatment is one of the most important factors in damaging the plant cells, and is greater than is found under mannitol-induced osmotic stress (Castillo et al., 2007). Growth characters, including SH, RL, LA, FW and DW, of rice grown under iso-osmotic water deficit and salt stress were reduced, related to the osmotic pressure in the culture media. This was especially the case under salt-induced osmotic stress and is similar to previous reports on the reduction of growth in rice exposed to iso-osmotic stress (Bahaji et al., 2002;Hien et al., 2003;Ahmad et al., 2007). In addition, fresh weight and dry weight in tolerant cultivars, including Basmati-370 and DR2, were maintained better than in the sensitive genotypes, Basmati-Kashmir and Cuom, when subjected to iso-osmotic stress derived from mannitol/PEG (polyethylene glycol) and NaCl salt (Hien et al., 2003;Ahmad et al., 2007). In this study, RD6 (waxy mutant line) was more sensitive to iso-osmotic stress than the original, KDML105. It is possible that the mutant line of RD6 rice is more susceptible to both salt and water deficit stress than the KDML105 cultivar. In contrast, salt tolerance in the Drew (Uddin et al., 2007), Dongjinbyeo (Lee et al., 2004) and IR-8 (Shereen et al., 2009) mutant lines of rice, derived using gamma ray irradiation, was superior to the original cultivars. The Homjan (HJ) rice variety has been classified as salt tolerant and water deficit tolerant, using in vitro rapid screening (Cha-um et al., 2010).
In conclusion, survival percentage, chlorophyll pigment levels and the photosynthetic abilities of rice seedlings grown under iso-osmotic salt stress, declined to a greater degree than in seedlings grown under mannitol-induced iso-osmotic stress, leading to a greater reduction in growth rate. Also, the RD6 rice cultivar was more sensitive to osmotic stress than the KDML105 cultivar when subjected to extreme conditions.