Effects of mannitol induced osmotic stress on proline accumulation, pigment degradation, photosynthetic abilities and growth characters in C₃ rice and C₄ sorghum

Abstract

Osmotic stress is one of the most important abiotic factors which inhibit growth and development in both the vegetative and reproductive stages of many plant species. The aim of this investigation was to compare the biochemical and physiological responses in C₃ rice and C₄ sorghum to water deficit. Chlorophyll a (Chl_a), chlorophyll b (Chl_b), total chlorophyll (TC) and total carotenoid (C_x+c) contents in both rice and sorghum seedlings under osmotic stress were adversely affected, related to increasing osmotic pressure in the culture media. In addition, the chlorophyll’s fluorescence parameters and net photosynthetic rate (P_n) decreased, leading to growth reduction. Also, a positive correlation was found between physiological and biochemical data, while proline accumulation showed a negative relationship. The Chl_b, P_n and fresh weight were maintained better in osmotic-stressed (-1.205 MPa) C₄ sorghum seedlings than those in C₃ rice seedlings. The growth and physiological responses of C₃ rice and C₄ sorghum decreased depending on the plant species, the osmotic pressure in the media and their interactions. Pigment content and P_n ability in C₄ sorghum grown under mannitol-induced osmotic stress increased to a greater degree than in C₃ rice, resulting in maintenance of growth.

Keywords

chlorophyll a fluorescence / net photosynthetic rate / Oryza sativa L. / pigment degradation / Sorghum bicolor (L.) Möench / water deficit stress

Introduction

Water deficit is a major problem worldwide, limiting plant growth and the productivity of crop species, especially in rain-fed agricultural areas (>1.2×10⁹ hm²) (Chaves and Oliveira, 2004; Kjine, 2006; Passioura, 2007). Water use efficiency (WUE) in the crops grown under water deficit stress is a target to be evaluated for use as water-deficit resistance criteria (Quarrie et al., 1999; Passioura, 2006; Zhang et al., 2007; Ali and Talukder, 2008). There has been a large amount of literature reviewing the effects of water deficit on the biochemical, physiological, morphological and anatomical changes in plants at the seedling stage, prior to the development of reproductive processes (Chaves et al., 2003; Flexas et al., 2004; Barnabás et al., 2008; Shao et al., 2008). Rice and sorghum crop species are two important grain crop species for which there are complete genome sequences (Goff et al., 2002; Yu et al., 2002; Paterson, 2008; Paterson et al., 2009). They are important as a carbohydrate source in people’s diets (Khush, 2005) and as an alternative source for bioethanol production (Xin et al., 2009). Rice, (C₃ photosynthesis (Choudhury, 2001)), has been identified as being susceptible to water deficit. There are many techniques that can be used to identify water-deficit tolerance traits from rice genetic resources which can then be used to produce transgenic rice in order to resist water limitation, especially in rain-fed areas (Fukai and Cooper, 1995; Lafitte et al., 2007; Kamoshita et al., 2008). In contrast, sorghum, (C₄ photosynthesis) (Choudhury, 2001), has been characterized as water deficit resistant and can grow well during under water deficit conditions (Buchanan et al., 2005; Sinclair et al., 2005; Sharma et al., 2006). In the case of rice, there have been several documents on changing photosynthesis from C₃ to C₄ using transgenic approaches, for the purpose of improving abiotic stress tolerance and crop productivity (Suzuki et al., 2000; Agarie et al., 2002; Chi et al., 2004; Suzuki et al., 2006; Bandyopadhyay et al., 2007; Wang and Li, 2008).

In water-deficit conditions, water availability in the nutrient solution is restricted by polyethylene glycol (PEG), mannitol or sorbitol in the growth medium or nutrient solution, causing low water use efficiency (WUE) in plants (Blum, 2005; Bloch et al., 2006; Costa et al., 2007; Shao et al., 2008). Low WUE is a primary effect of water deficit conditions on plants, and it leads to biochemical changes, including inhibition of Rubisco (ribulose-1,5-bisphosphatase carboxyase/oxygenase) activity, accumulation of osmolytes (proline, glycinebetaine, polyamine, glutathione, polyamines, sugars, sugar alcohols and a-tocolpherol) and increased levels of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) in order to reduce reactive oxygen species (ROSs) (Chaves et al., 2003; Nayyar, 2003; Selote et al., 2004; Nayyar and Gupta, 2006; Cha-um and Kirdmnaee, 2009). Physiological changes which also occur include the loss of membrane stability, reduced leaf water potential, pigment degradation, diminished chlorophyll a fluorescence, decreased stomatal conductance, reduced internal CO₂ concentration, net photosynthetic rate (P_n) reduction and growth retardation prior to plant death (Yordanov et al., 2003; Chaves and Oliveira, 2004; Reddy et al., 2004; Cattivelli et al., 2008; Shao et al., 2008). The aim of this study was to compare the photosynthetic pigments, chlorophyll a fluorescence, proline, P_n and growth of C₃ rice and C₄ sorghum in responses to water deficit stress.

Materials and methods

Plant materials

Seeds of rice (Oryza sativa L. spp. indica) cv. RD10 (sticky or glutinous rice) provided by Pathumthani Rice Research Center (Rice Research Institute, Department of Agriculture, Ministry of Agriculture and Cooperative, Thailand) and seeds of sorghum (Sorghum bicolor (L.) Möench) were de-husked by hand, sterilized once in 5% Clorox^Ò (5.25% sodium hypochlorite, The Clorox Co, Oakland, CA, USA) for 60 min and in 30% Clorox^Ò for 30 min, respectively, and then rinsed three times with sterile distilled-water. Surface-sterilized seeds were germinated on the 0.25% Phytagel^Ò-solidified MS medium (Murashige and Skoog, 1962) in a 250-mL glass jar vessel. The media were adjusted to pH 5.7 before autoclaving. Seedlings were cultured in vitro under conditions of 25 ± 2°C ambient temperature, with 60% ± 5% relative humidity (RH) and 60 ± 5 µmol∙m^-2∙s^-1 photosynthetic proton flux density (PPFD) provided by fluorescent lamps (TDL 36 W/84 Cool White 3350 Illumination, Phillips, Bangkok, Thailand) with 16 h∙d^-1 photoperiod. Fourteen-day-old rice seedlings were aseptically transferred to MS sugar-free liquid media (photoautotrophic conditions) using vermiculite as supporting material for 7 d (Cha-um et al., 2007). The number of air-exchanges in the glass vessels was adjusted to 2.32 µmol CO₂·h^-1 by punching a hole in the plastic cap (Æ 1 cm) and covering the hole with a microporous filter (0.20 µm pore size; Nihon Millipore Ltd., Tokyo, Japan). The seedlings were subsequently cultured in a plant growth incubator with CO₂ enrichment at (1000 ± 100) µmol∙mol^-1. Mannitol induced osmotic stress in the culture medium was adjusted to -0.238 MPa (control), -0.392 MPa (100 mmol∙L^-1), -0.674 MPa (200 mmol∙L^-1), -0.939 MPa (300 mmol∙L^-1) or -1.205 MPa (400 mmol∙L^-1) for 14 days. Photosynthetic pigments, proline content, chlorophyll a fluorescence, net photosynthetic rate (P_n) and growth characteristics were measured.

Data measurement

Chlorophyll a (Chl_a), chlorophyll b (Chl_b), total chlorophyll (TC) concentrations were determined following the method of Shabala et al. (1998) and total carotenoid (C_x+c) concentrations were determined following the method of Lichtenthaler (1987). One hundred milligrams of leaf material were collected, placed in a 25-mL glass vial, along with 10 mL 95.5% acetone, and blended using a homogenizer. The Chla, Chlb, and C_x+c concentrations were measured using a UV-visible spectrophotometer (DR/4000; HACH, Loveland, Colorado, USA). A solution of 95.5% acetone was used as a blank. The pigment degradation percentage was calculated following Cha-um and Kirdmanee (2008).

The proline content of the leaves was extracted according to the method of Bates et al. (1973). Leaf tissue (100 mg) was ground in liquid nitrogen. The homogenate was mixed with 1 mL aqueous sulfosalicylic acid (3% w/v) and filtered through filter paper (Whatman #1). The extracted solution was reacted with an equal volume of glacial acetic acid and ninhydrin reagent (1.25 mg ninhydrin in 30 mL glacial acetic acid and 20 mL 6 mol∙L^-1 H₃PO₄) and incubated at 95ºC for 1 h. The reaction was terminated by placing the container containing the reagents in an ice bath. The reaction mixture was vigorously mixed with 2 mL toluene. After warming to 25°C, the chromophore was measured using a spectrophotometer (DR/4000, HACH, Loveland, Colorado, USA) at 520 nm. L-proline (Fluka, Switzerland) was used as a standard.

Chlorophyll a fluorescence emission from the adaxial surface of the leaves was monitored with a Fluorescence Monitoring System (FMS 2; Hansatech Instruments Ltd., Norfolk, UK) in the pulse amplitude modulation mode, as previously described by Loggini et al. (1999) and Maxwell and Johnson (2000).

Net-photosynthetic rate (P_n) was calculated by comparing the different concentrations of CO₂ inside and outside of the glass vessel containing the seedlings. The CO₂ concentrations inside and outside the glass vessel (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 the GC capillary column (GS-Q, J & W Scientific^Ò, Germany) was set at 30°C for 1 min at the initial state and increased to 100°C at a rate of 20°C per min and held for 1 min (Fujiwara et al., 1987).

Shoot height, root length, number of leaves, leaf area, fresh weight and dry weight of rice and sorghum seedlings were measured. Seedlings were dried in a hot-air oven (Model 500, Memmert, Buchenbach, Germany) for 2 d and then incubated in desiccators before measurement of the dry weight (Cha-um et al., 2006). The leaf area of seedlings was measured using a Leaf Area Meter DT-scan (Delta-Scan Version 2.03, Delta-T Devices, Ltd., Burwell, Cambridge, UK).

Experiment design

The experiment was arranged as 2 ´5 factorials in a Completely Randomized Design (CRD) with ten replicates and two seedlings per replicate. The mean values were compared using Bonferroni’s correction analysis and analyzed with SPSS software (SPSS for Windows, SPSS Inc., Chicago, USA). The correlations between physiological and biochemical parameters were evaluated by Pearson’s correlation coefficients.

Results

Shoot height (SH), root length (RL), number of leaves (NL), leaf area (LA), fresh weight (FW) and dry weight (DW) of rice (C₃) and sorghum (C₄) seedlings cultured under reducing osmotic potential in the culture media showed a decreasing trend in relation to the osmotic potential. These parameters in osmotic-stressed rice seedlings (-1.205 MPa) dropped to lower levels than in the control seedlings (-0.238 MPa) by 19.55%, 13.53%, 31.51%, 35.94%, 51.12% and 23.74%, respectively, whereas in sorghum they were 23.64%, 23.23%, 21.42%, 51.15%, 31.89% and 29.50%, respectively (Table1). The different plant species, the osmotic potential in the culture media and their interactions strongly affected SH, RL, NL, LA, FW and DW (P≤0.05 or P≤0.01) (Table 1). Photosynthetic pigments, including chlorophyll a (Chl_a), chalorophyll b (Chl_b), total chlorophyll (TC) and total carotenoids (C_x+c), in osmotic stressed seedlings decreased significantly depending on plant species, osmotic pressure and their interactions. Photosynthetic pigments in -1.205 MPa osmotic stressed seedlings decreased by 28.86%, 72.56%, 47.98% and 29.85% respectively in rice, and by 49.22%, 54.76%, 50.73% and 34.80% respectively in sorghum, when compared to the control (-0.238 MPa) (Table 2). Degradation of Chla in osmotic stressed rice and sorghum was negatively related to the maximum quantum yield of PSII (F_v/F_m) with r²=0.62 (Fig. 1(a)) and r²=0.73 (Fig. 1(b)), respectively. In a similar way, the degradation of TC in osmotic stressed rice and sorghum was negatively related to the photon yield of PSII (F_PSII) with r²=0.95 (Fig. 2(a)) and r²=0.93 (Fig. 2(b)), respectively. The chlorophyll a fluorescence parameters, F_v/F_m and F_PSII, net photosynthetic rate (P_n) in osmotic stressed seedlings were drastically diminished according to plant species, osmotic stress and their interactions (Table 3). F_v/F_m and F_PSII in rice seedlings were reduced by 13.42% and 16.05%, respectively, and in sorghum by 8.94% and 20.86%, respectively, when exposed to osmotic stress (-1.205 MPa), while the proline content was enriched by 6.52 times in both rice and sorghum (Table 3). The reduction of P_n in osmotic stressed rice seedlings decreased sharply when compared to that in sorghum seedlings (Fig. 3). A correlation between physiological parameters i.e. Chl_a, Chl_b, TC, C_x+c, F_v/F_m, F_PSII, P_n and proline was demonstrated, except in the cases of C_x+c and F_PSII, as C_x+c and P_n were uncorrelated (Table 4). In addition, the diminished F_PSII in osmotic stressed seedlings was positively related to P_n in rice (r²=0.99) and sorghum (r²=0.96) crop species (Fig. 4), leading to dry mass reduction (r²=0.91 and r²=0.81, respectively) (Fig. 5).

Discussion

Mannitol induced osmotic stress has been widely investigated in many plant species, i.e., rice (Zang and Komatsu, 2007), sorghum (Sharma et al., 2006), sugarcane (Cha-um and Kirdmanee, 2008), Sesuvium portulacastrum (Slama et al., 2007), Fraxinus angustifolia (Tonon et al., 2004) and soybean (Neto et al., 2004). In the present study, the photosynthetic pigments in osmotic-stressed C₃ rice seedlings were damaged in relation to increased osmotic pressure in the culture media, especially in the case of Chl_b content, which was lower than in C₄ sorghum. The findings are quite similar to those of a previous investigation in which the chlorophyll pigments in the leaf tissues of C₃ wheat (cv. C306) grown under water deficit stress using PEG6000 dropped significantly depending on osmotic stress (-0.4 to -1.5 MPa) and to a greater extent than in C₄ maize (cv. Sartaj) (Nayyar and Gupta, 2006). Similarly, the chlorophyll a fluorescence parameters, F_v/F_m and F_PSII, in osmotic stressed leaves were significantly diminished, mainly in response to extreme osmotic stress (-1.203 MPa), resulting in low P_n. The F_v/F_m and F_PSII in the leaf tissues are the most popular parameters used to identify the water oxidation and photon yield harvesting in the light reaction of osmotic stressed plants (Ripley et al., 2007; Cha-um and Kirdmanee, 2008). In Alloteropsis semialata, the chlorophyll a fluorescence parameters including CO₂ assimilation (FCO₂) and F_PSII in drought-stressed C₄ types are better than those in C₃ types, leading to high P_n (Ripley et al., 2007). In addition, the growth performance of osmotic-stressed seedlings of C₃ rice declined significantly when compared to that of C₄ sorghum, demonstrated especially by fresh weight. There have been several reports comparing the growth characters of higher plants which have C₃ and C₄ photosynthetic pathways when cultivated under osmotic stress. The growth characteristics of the C₄ type are superior to those of the C₃ type when grown under osmotic stress (Nayyar, 2003; Nayyar and Gupta, 2006). On the other hand, the proline content was high in both C₃ rice and C₄ sorghum when exposed to osmotic stress. In the present study, the accumulation of proline in osmotic stressed C₄ sorghum occurred at a lower rate than in C₃ rice. Proline content, a biochemical stress indicator (Ashraf and Foolad, 2007), was regulated in C₃ wheat by -0.4 MPa PEG induced osmotic stress while in C₄ maize it was enhanced by -0.8 MPa PEG induced osmotic stress (Nayyar, 2003). Hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) generated in osmotic stressed C₄ maize were lower than in C₃ wheat whereas antioxidant substances (ascorbic acid and glutathione) and antioxidant enzyme activities (ascorbate peroxidase and glutathione reductase) in C₄ maize increased to a greater extent than in C₃ wheat (Nayyar and Gupta, 2006). It is possible that the osmotic-stress defense mechanisms in C₄ plants are more efficient than those in C₃ plants when exposed to stress. Also, a positive relationship between physiological and biochemical data was demonstrated, with the exception of proline content, which was negatively correlated with the other factors. These findings are similar to those of a previous publication which presented a positive correlation in the physiological data, especially in the photosynthesis system in C₃ and C₄ plants exposed to osmotic stress (Ripley et al., 2007).

Conclusions

In conclusion, the growth characteristics and physiological responses of C₃ rice and C₄ sorghum decreased, depending on plant species, osmotic pressure in the media and their interactions. The parameters of fresh weight, Chl_b and P_n in osmotic stressed rice seedlings decreased to a greater extent than those in sorghum. Pigment stabilization, P_n maintenance and growth performance in C₄ sorghum grown under osmotic stress were demonstrated to be greater than those in C₃ rice. These parameters may be employed as criteria for assessing osmotic stress tolerance (sorghum) or osmotic stress susceptibility (rice).

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