Abstract: The aim of this research is to investigate on the osmotic adjustment and pigment preservation by soluble sugar accumulation in salt stressed rice using exogenous glucose and abscisic acid (ABA) application, leading to plant growth and development. Soluble sugars including sucrose, glucose and fructose in the salt-stressed root tissues were continuously increased in the conditions of 111-222 mM glucose and 20-60 μ M ABA treatments and then drop in the extreme 333-444 mM glucose and 80 μ M ABA treatments. Osmolarity in the salt-stressed root tissues showed the similar pattern to the sugar responses and was negatively related to soluble sugar concentration (r = 0.91). Chlorophyll a, chlorophyll b and total carotenoid concentrations in the salt-stressed seedlings were significantly maintained by endogenous sugar osmotic adjustment. In addition to, the high osmolarity in salt-stressed seedlings was negatively related to total chlorophyll stabilization (r = 0.83). The total chlorophyll degradation in the salt-stressed leaf tissues was positively correlated with plant growth defined by shoot height (r = 0.81). Root length, root number and root cortex thickness of salt-stressed rice seedlings showed the highest at 222 mM glucose and 60 μ M ABA treatments for 146.1 cm, 17.3 and 1.3 μ m, respectively. An exogenous application of glucose and ABA in this investigation is an alternative way to acclimatize the rice crop before exposed to soil salinity and further applied for rice cultivation in salinity filed trial.
INTRODUCTION
Rice is an important carbohydrate crop to supply more than half of world’s population, especially in Asia. Rice crop is cultivated and consumed in Asian countries for 90%, which is a major food crop (Khush, 2005). Main barrier of rice production is abiotic stresses such as salinity soil, water deficit, flooding, nutrient deficiency/enrichment, acidic/basic soils, extreme temperature and UV irradiation (Cushman and Bohnert, 2000; Salekdeh et al., 2002; Lafitte et al., 2004). In case of soil salinity, there are several regions about 45.4 million hectares, including Asia, Africa, Latin America, North America, Europe and Australia, to be a serious problem, especially irrigated areas (Ghassemi et al., 1995; Singh and Chatrath, 2001). Rice crop has been reported as salt-sensitive species, especially in seedling and flowering stages, leading to yield reduction (Shannon et al., 1998; Zeng and Shannon, 2000; Khan and Abdullah, 2003; Zeng et al., 2002). Salt-tolerant breeding program in rice is a fruitful topic for plant breeders to solve the salinity toxic damage in both osmotic and ionic effects(Gregorio et al., 2002; Senadhira et al., 2002; Flowers and Flowers, 2005). It should be used an ntensive skill, time requirement and wide genetic resources. Alternatively, an exogenous application of osmotic solutes i.e., glycinebetaine (Cha-um et al., 2006; Demiral and Turkan, 2006), putrescine, spermine (Ndayiragije and Lutts, 2006) and spermidine (Roy et al., 2005; Ndayiragije and Lutts, 2006) has been reported. In addition to, ABA and sugar exogenous applications have been pretreated or acclimatized the plant before exposed to extreme environmental conditions, including salinity and drought (Gibson, 2000; Lin and Kao, 2001; Wang et al., 2003; Yin et al., 2004; Morsy et al., 2006). However, the function roles of exogenous sugar and ABA in salt defense responses are still unclear. Pathumthani 1 rice is a salt sensitive variety of indica subspecies, which is photoperiod insensitive, aroma flavor, high cooking quality and high yield (Ariyaphanphitak et al., 2005; Leohakunjit and Kerdchoechuen, 2007). In present study, the salt-tolerant defense mechanisms in indica subspecies using in vitro glucose and ABA application were intensively evaluated.
MATERIALS AND METHODS
Plant Material
Seeds of indica rice (Oryza sativa L. spp. indica cv.
Pathumthani 1) were obtained from the Pathumthani Rice Research Center (Rice
Research Institute, Department of Agriculture, Ministry of Agriculture and Cooperative,
Thailand). Seeds were dehusked by hand, sterilized once in 5% Clorox®
(5.25% sodium hypochlorite, The Clorox Co, US) for 60 min, once in 30% Clorox®
for 30 min and then rinsed thrice by sterile distilled-water. Surface-sterilized
seeds were germinated on 0.25% Phytagel®-solidified MS media
(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
condition of 25±2°C ambient temperature, 60±5% Relative Humidity
(RH) and 60±5 μmol m-2 s-1 Photosynthetic
Proton Flux (PPF) provided by fluorescence lamps (TDL 36 W/84 Cool White 3350
Im, Philips, Thailand) with 16 h d-1 photoperiod. Fourteen-day-old
rice seedlings were aseptically transferred to MS-liquid media, containing 0,
111, 222, 333 or 444 mM glucose combined with 0, 20, 40, 60 or 80 μM abscisic
acid (ABA) using vermiculite as supporting material. The number of air-exchanges
in the glass vessels was adjusted to 2.32 h-1 by punching a hole
on plastic cap (ø1 cm) and covering the hole with a microporous filter
(0.20 μm of pore size). All seedlings were continuously cultured under
the same conditions as during the seed germination and subsequently exposed
to 342 mM NaCl for a week (Fig. 1). Physiological characteristics,
soluble sugar content, root osmolarity, pigment concentration, morphological
and anatomical characteristics, root length, root number, shoot height and cortex
thickness were measured.
| Fig. 1: | Scheme of the experiment on in vitro photomixotrophic germination for 14 days, photoautotrophic acclimatization with various glucose and abscisic acid (ABA) pretreatments for 7 days and subsequently exposed to 342 mM NaCl for a week |
Physiological Characteristics
Endogenous soluble sugars, sucrose, glucose and fructose in root organ of
rice seedlings were evaluated following by Karkacier et al. (2003). A
hundred milligram frozen material was grinded in 1.5 mL micro tube with a small
pestle. One milliliter of nanopure water was added and then sonicated for 15
min. The extracted material was centrifuged at 12,000 rpm for 10 min and then
the supernatant was collected. The supernatant was directly filtered through
0.45 μm pore size prior to HPLC injection. Total soluble sugar, including
sucrose, glucose and fructose was analyzed using 410 differential refractometer
(RI) detector and Waters 600 gradient controller pump (Waters, Milford, MA,
USA) with Metacarb 87C analytical column (300x6.5 mm). Nanopure water was used
as mobile phase with 0.6 mL min-1 flow rate. The sucrose, glucose
and fructose sugar classes were used as standard.
Root osmolarity of rice seedlings was measured, according to Lanfermeijer et al. (1991). Fresh root tissues of rice were debris in 1.5 mL micro tube by glass rod. A twenty micro liter of extracted solution was directly dropped on a disc filter paper in osmometer chamber (Wescor, USA) and then measured.
Chlorophyll a (Chla), chlorophyll b (Chlb) and carotenoid (Cx+c) concentrations were analyzed following the methods of Shabala et al. (1998) and Lichtenthaler (1987), respectively. The Chla and Chlb concentrations were measured using an UV-visible spectrophotometer (DR/4000, HACH, USA) at wavelengths 662 nm and 644 nm. The Cx+c concentration was measured spectrophotometrically at 470 nm. A solution of 95.5% acetone was used as a blank. The Chla, Chlb and Cx+c (μg g-1 FW) concentrations in the leaf tissues were calculated according to the following equations:
where Di is the optical density at wavelength I.
Morphological and Anatomical Characteristics
The root length, root number and plant height of rice seedlings were measured
as described by Lutts et al. (1996). Excised roots in size 2-3 cm from
root cap were hand-sectioned and then cortex thickness was observed under light
microscope (200x) (Axiostar Plus, Carl Zeiss, Germany).
Experimental Design
The experiment was designed as 5x5 factorials in Completely Randomized Design
(CRD) with ten replicates and four plantlets per replication. The mean in each
treatment was compared by Dancan’s New Multiple Range Test (DMRT) at p≤0.01
and analyzed by SPSS software (SPSS for Windows, SPSS Inc., USA).
RESULTS AND DISCUSSION
Exogenous glucose and ABA pretreatments in the culture media of salt-stressed rice seedlings were directly enhanced on endogenous soluble sugars accumulation including sucrose, glucose and fructose. The results showed that sucrose, glucose and fructose concentration in salt-stressed root tissues were highest peak in the media supplemented with 222 mM glucose and 60 μM ABA for 86.94, 156.10 and 97.60 μg g-1 FW, respectively (Table 1). Sugar accumulation in salt-stressed seedlings trend to increase after exposed to low concentration of glucose (111-222 mM) and ABA (20-60 μM), while sharply drop in the high concentrations (Table 1).
| Table 1: | Sucrose, glucose and fructose concentrations in the root tissues of Indica rice seedlings pretreated by 0. 111, 222, 333 or 444 mM glucose and 0, 20, 40, 60 or 80 μM Abscisic Acid (ABA) and subsequently exposed to 342 mM NaCl for a week |
| Different letter(s) in each column show significant difference at p≤0.01 by Duncan’s New Multiple Range Test (DMRT), Highly significant in statistics is represented by ** | |
| Fig. 2: | Relationship between total sugar concentration and osmolarity in the root tissues of indica rice seedlings treated by 0. 111, 222, 333 or 444 mM glucose and 0, 20, 40, 60 or 80 μM abscisic acid (ABA) and subsequently exposed to 342 mM NaCl for a week. Error bars represent by±SE |
| Table 2: | Chlorophyll a, chlorophyll b and total carotenoid concentrations in Indica rice seedlings pretreated by 0, 111, 222, 333 or 444 mM glucose and 0, 20, 40, 60 or 80 μM abscisic acid (ABA) and subsequently exposed to 342 mM NaCl for a week |
| Different letter(s) in each column show significant difference at p≤0.01 by Duncan’s new Multiple Range Test (DMRT), Highly significant in statistics is represented by ** | |
An endogenous sugar concentration in salt-stressed seedlings was negatively related to osmolarity in the root tissues (r = 0.91) (Fig. 2). It means that soluble sugar accumulation in salt-stressed root tissues should be played a central role as defense mechanism in osmoregulation system to be preserved the water use efficiency. A low osmolarity or water available may be directly maintained by sugar in the root organ or root zone that directly attached to salinity in the media. In aerial zone, the chlorophyll a, chlorophyll b and carotenoid concentrations in the salt-stressed leaves were maintained to the highest in 222 mM glucose and 60 μM ABA for 159.47, 45.00 and 64.84 μg g-1 FW, respectively. The pigment concentration in salt-stressed leaves was a similar response to soluble sugar that increased in the low concentrations of glucose and ABA pretreatment and then reduced in a high dose application (Table 2). In addition to, a high osmolarity in salt-stressed roots was directly damaged on pigment concentration (r = 0.83) (Fig. 3). It should be noted that the exogenous glucose and ABA pretreatment before exposed to salt stress was an effective way to promote on soluble sugar accumulation for osmolarity control, leading to water use efficiency and pigment stabilization. The pigment stabilization in salt-stressed seedlings was positively correlated with growth performance in term of plant height (r = 0.81) (Fig. 4). The morphological and anatomical characters of salt-stressed root tissues were mentioned. Both exogenous glucose and ABA factors were directly affected on root length, root number and cortex thickness, while the glucose application does not influence on cortex thickness (Table 3). The root length, root number and cortex thickness of root organ in salt-stressed seedlings showed the highest in similar to previous pretreatment condition (222 mM glucose and 60 μM ABA) for 146.1 cm, 17.3 root and 1.3 μm, respectively (Table 3).
| Fig. 3: | Relationship between osmolarity and total chlorophyll concentration in indica rice seedlings treated by 0, 111, 222, 333 or 444 mM glucose and 0, 20, 40, 60 or 80 μM abscisic acid (ABA) and subsequently exposed to 342 mM NaCl for a week. Error bars represent by±SE |
| Fig. 4: | Relationship between total chlorophyll concentration and plant height in indica rice seedlings pretreated by 0, 111, 222, 333 or 444 mM glucose and 0, 20, 40, 60 or 80 μM abscisic acid (ABA) and subsequently exposed to 342 mM NaCl for a week. Error bars represent by ±SE |
Sugar is exogenously applied in the culture media for in vitro growth stimulation in higher plant species namely photomixotrophic growth (Schafer et al., 1992; Hdider and Desjardins, 1994; Ticha et al., 1998; Sima and Desjardins, 2001). Normally, the sugar concentration in the MS media is supplied on 88 mM as main carbon source for growth and development of rice crop (Cha-um et al., 2005) and rain tree (Mosaleeyanon et al., 2004). In this experiment, a high sugar concentration (111-444 mM glucose) is directly supplemented in media for osmotic regulation and energy source preservation, resulting in soluble sugar accumulation related to previous study on desert algal (Chen et al., 2003a) and rice crop (Cha-um et al., 2007). An osmolarity of the root tissues cultured on the media supplemented with a high concentration of sugar is increased, leading to plant water deficit as plant acclimatization before expose to extreme salt-stress (Chen et al., 2003a; Gupta and Kaur, 2005).
| Table 3: | Root length, root number and cortex thickness in root organ of Indica rice seedlings pretreated by 0, 111, 222, 333 or 444 mM glucose and 0, 20, 40, 60 or 80 μM abscisic acid (ABA) and subsequently exposed to 342 mM NaCl for a week |
| **Different letters in each column show significant difference at p≤0.01 by Duncan’s new Multiple Range Test (DMRT), Highly significant, significant and non-significant in statistics is represented by **, * and NS, respectively | |
There are many reports to mention on sugar accumulation in plant species as defensive response to salt stress in rice (Morsy et al., 2006), soybean (Liu and van Staden 2001), sorghum (de Lacerda et al., 2003; de Lacerda et al., 2005), kikuyu grass (Muscolo et al., 2003), wheat (Lutts et al., 2004), hexaploid triticale (Morant-Manceau et al., 2004), poplar (Watanabe et al., 2000), resurrection plant (Smith-Espinoza et al., 2003), salt-secretor mangrove (Parida et al., 2004) and citrus (Arbona et al., 2005). Salts, antioxidants and compatible solutes have been exogenously applied in the media to pretreated plants before exposed to salt stress (Ashraf and Foolad, 2007; Cha-um et al., 2006; Djanaguiraman et al., 2006; Cuin and Shabala, 2005; Yamane et al., 2004a and b). Root organ is firstly attached to salinity, which is selected and controlled the ion accumulation in plant cells using ion-pump or secrete to vacuole by ATP energy from sugar catabolism in mitochondria (Newmann et al., 1994; Vaughan et al., 2002; Bell and O’Leary, 2003; Zeng, 2005). Sugar accumulation in the plant cells is necessarily supported as energy source as well as controlled the cell osmotic pressure under soil salinity, especially root organ.
Abscisic acid or ABA is a member of endogenous hormonal regulations in higher plant, which is played an important role in signal transduction, gene(s) regulation and short-term defensive response to abiotic stresses, especially salinity (Hasegawa et al., 2000; Wilkinson and Davies, 2002; Seki et al., 2003; Sairam and Tyagi, 2004; Kaur and Gupta, 2005; Verslues and Zhu, 2005; Zhang et al., 2006). Generally, an ABA accumulation in plant species is progressively induced by salt stress treatment such as crop species (Degenhardt et al., 2000), brassica (He and Cramer, 1996), rice (Lin and Kao, 2001), maize (Jia et al., 2002), tomato (Chen et al., 2003b; Mulholland et al., 2003; Maggio et al., 2006) and barley (Fricke et al., 2006). Exogenous ABA treatment is an alternative way to enhance on accumulation and function as defensive response to salt stress via soluble sugar accumulation or osmotic adjustment in common bean (Khadri et al., 2006) and rice (Asch et al., 1995). Similarly, an ABA treatment in present study directly promotes on sucrose, glucose and fructose accumulation for osmolarity control in salt-stressed seedlings especially in the root tissues. It is similar to the previous publications that ABA applications in wheat is influence on turgor pressure and maintain on root osmolarity (Jones et al., 1987; Munns and Cramer, 1996), leading to root branching (Signora et al., 2001; de Smet et al., 2003; de Smet et al., 2006). In addition to, the endogenous sugar accumulation derived from ABA treated plants should be functioned as antioxidant (Yoshida et al., 2004) and reduced cell injuries (Arbona et al., 2006). Both sugar and ABA combinatorial functions for salinity defense mechanisms in higher plants are well established (Gazzarrini and McCourt, 2001; Knight and Knight, 2001; Ma et al., 2006; Rook et al., 2006). There is evident information on the regulation of ABA on carbohydrate metabolisms, relating to abiotic stress tolerance in term of water relation, cell/membrane stabilization, photosynthesis and overall growth performances.
In conclusion, the endogenous soluble sugar contents in the salt-stressed roots were alternatively accumulated by exogenous glucose and/or ABA application, resulting in root osmolarity control for water use efficiency in the roots as well as pigment stabilization in the leaves after exposed to salt stress. The osmolarity control and pigment stabilization in glucose and ABA pretreatment were directly stimulated on plant growth, especially the root tissues. The basic knowledge of this investigation will be further applied for rice cultivation in salinity filed trial.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Teeraporn Busaya-angoon at Pathumthani Rice Research Center, for providing of Pathumthani rice seeds. This research is supported by the National Center for Genetic Engineering and Biotechnology (BIOTEC; Grant number BT-B-06-RG-14-4502).