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Physiological evaluation of responses of rice (Oryza sativa L.) to water deficit
Plant Science 163 (2002) 815 /827 www.elsevier.com/locate/plantsci
Physiological evaluation of responses of rice (Oryza sativa L.) to water deficit Gloria S. Cabuslay a,*, Osamu Ito b, Arcelia A. Alejar c a
Plant Breeding, Genetics, and Biochemistry (PBGB) Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines b Crop Production and Environment Division, JIRCAS, 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan c UP Los Ban˜os, College 4031, Laguna, Philippines Received 12 February 2002; received in revised form 5 July 2002; accepted 5 July 2002
Abstract In view of the need to increase rice yields in rainfed areas, an experiment was done to (1) determine physiological traits that contribute to tolerance for water deficit at the seedling stage, (2) identify quick but reliable indices for selection for tolerance for water deficit, and (3) characterize rice cultivars popularly used for water-deficit studies in rainfed ecosystems. The study used 27 cultivars, which include those often used for drought studies in rainfed lowland and upland ecosystems. Mild water-deficit conditions were generated by adding polyethylene glycol 1500 to nutrient solution to provide an osmotic stress of /0.5 MPa to 3week-old seedlings. Water deficit caused a larger reduction in leaf area than in shoot dry matter, demonstrating the greater sensitivity of leaf enlargement to water stress than dry matter accumulation. Visual scoring to assess damage was found to be a reliable measure of tolerance for water deficit. Cultivars tolerant of mild water stress had a high relative transpiration (transpiration under stress compared with that under non-stressed conditions), low initial leaf area (leaf area before the onset of water stress), high carbon isotope discrimination in the leaf, and low specific leaf weight. These factors enabled tolerant cultivars to maintain high moisture in the leaf and to have high values of leaf area, shoot dry matter, and sugar and starch in tissues in stressed plants relative to the control. Mild water deficit increased water use efficiency in stressed plants, caused more degradation of starch than sugar in the leaf blade, and resulted in more accumulation of these carbohydrates in the leaf sheath. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Physiological traits; Water deficit; Water stress
1. Introduction Water deficit (commonly known as drought) can be defined as the absence of adequate moisture necessary for a plant to grow normally and complete its life cycle. The lack of adequate moisture leading to water stress is a common occurrence in rainfed areas, brought about by infrequent rains and poor irrigation. Rice yield is significantly reduced such that an average of only 1.5 t ha 1 is realized in the rainfed lowland ecosystem in South and Southeast Asia [1] compared with a range of
Abbreviations: PEG, polyethylene glycol; D , carbon isotope discrimination; WUE, water use efficiency; SLW, specific leaf weight. * Corresponding author E-mail addresses: [email protected] (G.S. Cabuslay), [email protected] (O. Ito).
3 /9 t ha 1 in areas where the water supply is sufficient [2]. In spite of numerous reports on water deficit and the recent advances in molecular biology techniques, drought tolerance remains poorly understood in comparison with grain quality and disease resistance, which are governed by major genes. This demonstrates the complexity of rainfed ecosystems, exacerbated by unpredictable moisture supply. In addition, high genotype by environment (G /E) interaction makes it difficult to identify consistently superior genotypes. For many studies, imposing water stress in fieldgrown crops is difficult because of the unpredictability of rainfall and the possibility of seepage from adjoining plots. Soil heterogeneity further complicates the interpretation of field data. The addition of non-ionic osmotic agents such as polyethylene glycol (PEG) to liquid nutrient media has been shown to closely mimic
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specific levels of soil water stress [3]. Because they are not readily taken up by the cells, they do not affect the ionic composition of the cell [4]. PEG cannot cross membranes (as can sucrose) such that the water potential gradient in the tissue is not altered [5]. Furthermore, drought escape by growing deep roots is not possible because the roots of all cultivars are uniformly exposed to the same osmotic stress. A deeper understanding of the mechanisms of rice tolerance for water deficit is necessary for breeders to be able to identify heritable traits that will make plants adapt to growth conditions in rainfed areas. Moreover, it is important to use a quick but reliable index for tolerance for water deficit that will enable mass screening of genotypes. Water use efficiency (WUE) (total dry matter produced per unit of water transpired) is regarded as a desirable trait under both irrigated and rainfed conditions. The use of cultivars with a high WUE increases the efficiency of irrigation under wellwatered conditions and that of rainfall use under rainfed environments [6]. Genotypic variation in WUE has been reported in rice [7], peanut [8], cowpea [9], and other crops. The use of WUE as a selection criterion in plant breeding is hampered by difficulty in taking measurements, especially in field studies. An indirect measure of WUE, termed carbon isotope discrimination (D ), was developed, which makes it possible to analyze a large number of plants. The technique is based on the ratio of the naturally occurring stable isotopes 13C and 12C. During photosynthesis, 13C is discriminated against resulting in plants having a smaller 13C/12C ratio than the CO2 of the surrounding air. It was predicted that D is negatively correlated with WUE [10], i.e. plants with a high WUE would have large ratios of 13C/12C (or less D ). The negative relationship between transpiration efficiency and D was shown as predicted in peanut [11] and in wheat [9] under both well-watered and water stress conditions. The objectives of this study were to (1) determine physiological traits that contribute to tolerance for water deficit at the seedling stage, (2) identify quick but reliable indices for selection for tolerance for water deficit, and (3) characterize rice cultivars popularly used for water-deficit studies in rainfed ecosystems.
2. Materials and methods 2.1. Plant material and growth conditions 3-week-old seedlings of 27 rice cultivars/lines of different adaptations (Table 1 [12]) were used. Some characteristics of these cultivars/lines are enumerated in Table 2 [13]. All studies were carried out in a phytotron glasshouse maintained at 29/21 8C (day/night) and 70%
relative humidity under natural light conditions. The experiment was laid out in a split-plot design using five replications, with PEG treatment as the main plot and cultivar as the subplot. Pregerminated seeds were sown in styrofoam seedbeds lined with nylon nets and floated over culture solution [14] maintained at pH 5.5. The culture solution was renewed once a week for 2 weeks and twice a week at the third week and thereafter. Seedlings (18-day-old) were transferred into 50 ml plastic centrifuge tubes containing normal nutrient solution. One tube contained a single plant, held in place by a styropor cover that fit the lid of the plastic tube and had a hole bored at the center. The level of the solution was maintained daily. 2.2. Treatment A water-deficit condition was imposed 3 days after transfer into the tubes by changing the nutrient solution to that containing PEG 1500 giving /0.5 MPa of stress. This was prepared by adding 130 g of PEG 1500 to 1 l of culture solution [15]. Solutions of PEG were purified by mixing with ion-exchange resin before adding nutrients. A control plot was maintained. The osmotic potential of the PEG solution was measured with a freezing point osmometer (Precision Systems, Inc., Natick, MA USA, Model 5004). The weight of tubes with plant material was measured daily and moisture lost was replenished. Moisture lost was taken as the amount of water transpired. The experiment was terminated after 6 days, when cultivars could be separated into tolerant and intolerant of water deficit according to a visual score. Average daily solar radiation was 18.5 MJ m 2. Before harvest, plants were scored visually for water stress damage, which included leaf rolling and leaf drying. A scale of 1, 3, 5, 7, and 9 was used where 1 refers to least damaged (tolerant of water deficit) and 9 pertains to most damaged (intolerant of water deficit). 2.3. Measurements Plants were sampled before (representing initial data) and after PEG treatment. Seedlings were washed with distilled water and blotted dry with a paper towel. The shoot was separated from the root and the leaf blade was detached from the leaf sheath. The area of green leaves was measured with a leaf area meter (LI-COR, Inc., Model LI-3100, Lincoln, NE). Immediately after determination of fresh weight, samples were immersed in liquid nitrogen. The frozen samples were temporarily stored in a deep-freezer ( / 20 8C) prior to freeze-drying. Freeze-dried samples were further oven-dried overnight to ensure total inactivation of enzymes. Dry weights were also taken.
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Table 1 Accession number, country of origin, adaptation, and isozyme grouping of 27 rice cultivars/lines used in this study Accession number
Cultivar
Country of origin
Adaptationa
Isozyme groupb
10929 11354 11355 11462 27748 28506 32583 38558 50634 53434 57020 67819 72931 76330 77181 77968
Mahsuri C4-137 IR20 Nam Sa-Gui 19 Khao Dawk Mali 105 IRAT9 IR442-2-58 IRAT104 UPLRi-5 IR52 IR6115-1-1-1 UPLRi-7 M55 IR72 ITA130 BPI RI 10 CT9993-5-10-1M IR52561-UBN-1-1-2 IR58821-23-B-1-2-1 IR62266-42-6-2 IR5178-1-1-4 ITA119 IRAT140 PI 163575 Lansaw Ifugao rice Qomenan
Malaysia Philippines Philippines Thailand Thailand Coˆte d’Ivoire Philippines Coˆte d’Ivoire Philippines Philippines Philippines Philippines Liberia Philippines Nigeria Philippines Colombia Philippines Philippines Philippines Philippines Nigeria Coˆte d’Ivoire Philippines Philippines Philippines Philippines
RL / IL DW RL U DW U U RL / U U IL U / U / / / / / U / / / /
I I I I I I I VI I I I I VI I VI I VI I I I I I VI I I I I
57011 80022 55684 5758 8134 8176 8144 a b
RL: rainfed lowland; IL: irrigated lowland; DW: deep water; U: upland. I: indica; VI: japonica; Classification according to Glaszmann [12].
Ethanol-soluble sugars and starch were analyzed by near-infrared spectrometry [16]. A wet assay for constructing the calibration curves for sugar and starch was done colorimetrically [17]. The carbon isotope composition (d13C) of dried ground leaf samples was analyzed using a continuous flow isotope ratio mass spectrometer (Europa Scientific Roboprep-CN (Europa Scientific Ltd., Crewe, Chesire, UK) coupled to VG Micromass (VG Isogas, Middlewich, Chesire, UK) 903). Carbon isotope discrimination (D ) was computed as shown below: D ()
13 d Cair d 13 Cplant
d 13 C ()
1 d 13 Cplant
3. Results and discussion 3.1. Morphological responses of cultivars grown under water deficit Water deficit at /0.5 MPa osmotic potential, simulated by adding PEG to the culture solution, caused only Table 2 Some phenotypic characteristics of 8 lines/cultivars included in this study Cultivar/line
1000;
R 1; RPDB
where R is the molar abundance ratio, 13C/12C, of a sample [18]. The value of d13Cair used for the calculation of D was /7.60 relative to PeeDee Belemnite. WUE for the period of PEG treatment was obtained by dividing shoot dry matter by water transpired. Specific leaf weight (SLW) was calculated as the ratio of green leaf weight to leaf area.
Phenotype description
CT9993-5-10
Deep roots, good seedling vigor, anther cultureresponsive, semi-upland IR52561-UBN-1- Shallow roots, improved, vigorous 1-2 IR58821-23-B-1- Deep roots, improved, semi-erect, sturdy 2-1 IR62266-42-6-2 Improved, semi-ground cover Khao Dawk Mali Intermediate to shallow root system, improved 105 traditional, high vigor, weak stems Mahsuri Poor roots, improved, high vigor, rainfed-lowland adapted Nam Sagui 19 Shallow roots, drought-adapted, high vigor, improved traditional IR20 Drought-susceptible, semidwarf From Samson et al. [13].
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slight leaf rolling and leaf drying in plants. This observation suggests that the treatment produced mild water stress. Development of leaf area was more severely affected by the stress than was shoot dry matter (Table 3), with leaf area declining by 71% on average and shoot dry matter declining by 27%. Except in IR20, green leaf area under stress declined to levels below those occurring before the imposition of the stress or initial leaf area, whereas positive growth was obtained in shoot dry matter (data not shown). These results demonstrate the more pronounced effect of water deficit on leaf expansive growth than on shoot dry matter, which is in line with the general observation that leaf expansion is more sensitive to water stress than photosynthesis [19,20]. A large decline in leaf area under mild water stress is disadvantageous to plants because it leads to reduced nutrient uptake, which is a consequence of reduced demand and reduced transpiration [21]. Reduced leaf area also decreases carbon assimilation, which is positively related to leaf area [22].
Table 3 The effect of 6-day duration of water deficit (with PEG at 0.5 MPa) on leaf area and shoot weight of 3-week-old seedlings grown inside the phytotrona Cultivar
Mahsuri C4-137 IR20 Nam Sa-Gui 19 Khao Dawk Mali 105 IRAT9 IR442-2-58 IRAT104 UPLRi-5 IR52 IR6115-1-1-1 UPLRi-7 M55 IR72 ITA130 BPI RI 10 CT9993-5-10-1M IR52561-UBN-1-1-2 IR58821-23-B-1-2-1 IR62266-42-6-2 IR5178-1-1-4 ITA119 IRAT140 PI 163575 Lansaw Ifugao rice Qomenan Mean % reduction a
Leaf area (cm2)
Shoot weight (mg)
Control
PEG
Control
80.4 92.7 55.1 78.7 75.8 64.2 103.2 99.7 68.5 107.8 93.5 103.2 122.4 63.7 89.8 79.2 91.5 71.0 70.5 118.9 86.4 89.7 96.9 113.0 73.5 72.1 82.6
25.8 23.4 29.3 22.9 38.3 16.2 34.9 17.8 12.4 50.5 30.8 31.9 19.6 26.8 16.3 24.8 18.8 26.3 26.1 46.9 31.6 18.2 21.7 18.8 16.8 20.5 20.6
86.8 70.6
(4.5) (3.4) (1.5) (6.7) (2.9) (1.2) (7.6) (1.2) (8.8) (2.6) (1.8) (4.5) (3.7) (1.8) (3.4) (4.2) (4.6) (3.7) (2.3) (6.6) (5.1) (1.7) (2.7) (4.9) (2.8) (3.7) (4.0)
PEG
(3.1) 644 (25) 481 (16) (3.8) 779 (42) 521 (17) (1.9) 456 (20) 399 (14) (2.1) 737 (54) 563 (15) (3.6) 691 (14) 594 (11) (3.0) 611 (20) 492 (24) (2.8) 772 (73) 522 (18) (1.4) 723 (12) 530 (7) (2.6) 496 (78) 308 (14) (3.9) 962 (18) 734 (14) (5.2) 767 (16) 587 (18) (3.1) 816 (38) 643 (17) (1.6) 906 (27) 619 (22) (3.9) 632 (8) 534 (19) (1.9) 758 (39) 504 (13) (4.4) 695 (17) 519 (12) (3.5) 706 (34) 456 (35) (2.0) 584 (32) 441 (9) (4.6) 610 (16) 479 (26) (4.6) 1020 (43) 697 (23) (5.1) 780 (41) 619 (39) (4.1) 648 (34) 396 (9) (5.3) 867 (28) 617 (5) (3.5) 860 (36) 575 (10) (1.7) 629 (23) 446 (37) (2.2) 644 (7) 436 (15) (3.9) 649 (29) 439 (6)
25.5
Values in parentheses are standard errors.
720 524 27
The water status of the leaf is usually monitored by measuring the leaf water potential. However, this process is slow and is not applicable for mass screening of cultivars. Visual scoring techniques, whether based on leaf rolling or leaf drying, were found to be highly correlated with maintenance of leaf water potential [23]. Desirable scores were the smaller numerical values, which refer to cultivars showing less damage from water stress. In this study, tolerance for water stress was assessed by visual scoring based on leaf rolling and leaf drying. Significant differences in visual score were found among the 27 cultivars tested (Table 4). Khao Dawk Mali 105 (KDML 105) and ITA119 were the most tolerant of water stress, while UPLRi-5 and PI 163575 were the most intolerant. KDML 105 is regarded as droughttolerant in field studies [24] with yield stability under low-input conditions [25], while ITA119 was reported to be highly drought-tolerant [21]. The visual score was positively correlated with a reduction in both leaf area (r /0.43*) and shoot dry matter (r /0.41*), indicating that cultivars with a high score, i.e. with less tolerance for water deficit, had a greater reduction in leaf area and in dry matter. Thus, visual scoring could be used effectively in mass screening of genotypes for tolerance for water deficit. 3.2. Water stress and transpiration Measurement of whole-plant rather than individualleaf transpiration would give more accurate information on how the plant responds to water stress. This takes into account differences in stomatal conductance among leaves of different ages and position in the canopy, which might be affected differently by stress. Aside from visual score, water stress tolerance of cultivars was also evaluated by comparing whole-plant transpiration under stress with that under non-stress conditions (herein referred to as relative transpiration). Thus, cultivars less affected by the stress will have transpiration values closer to those of plants grown under non-stress conditions. Relative transpiration data (Table 4) show that water stress caused more than 50% reduction in cumulative transpiration in all cultivars. Relative transpiration was highest in IR20 and KDML 105 while lowest values were obtained in M55 and PI 163575. Relative cumulative transpiration was highly and negatively correlated with visual score (r / /0.52**), indicating that, generally, cultivars with a high relative transpiration had a low visual score (having less damage from water stress). These results imply that cultivars with high relative transpiration are adapting to the stress and could possess genes for tolerance for water deficit. These genes are somehow working to prevent damage from water stress as manifested by the low visual score. Maintenance of transpiration is important in plants,
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Table 4 The effect of 6-day duration of water deficit (with PEG at 0.5 MPa) on visual scores and transpiration rates of 3-week-old seedlings grown inside the phytotron Cultivar
Visual score
Cumulative transpiration (g H2O/6 days) Control
PEG
Relative transpiration (PEG/control) 0.37 0.34 0.48 0.35 0.44 0.39 0.35 0.36 0.37 0.34 0.34 0.35 0.29 0.42 0.30 0.37 0.34 0.40 0.41 0.33 0.33 0.33 0.34 0.29 0.34 0.36 0.36
Mahsuri C4-137 IR20 Nam Sa-Gui 19 Khao Dawk Mali 105 IRAT9 IR442-2-58 IRAT104 UPLRi-5 IR52 IR6115-1-1-1 UPLRi-7 M55 IR72 ITA130 BPI RI 10 CT9993-5-10-1M IR52561-UBN-1-1-2 IR58821-23-B-1-2-1 IR62266-42-6-2 IR5178-1-1-4 ITA119 IRAT140 PI 163575 Lansaw Ifugao rice Qomenan
4.6 5.0 2.6 3.8 1.4 3.8 3.4 3.0 6.2 4.6 4.6 6.6 5.8 2.2 5.4 2.2 5.0 4.2 5.4 5.0 5.4 1.8 5.0 6.2 4.6 5.4 5.8
113.4 122.1 83.5 121.2 104.7 99.1 127.9 107.2 79.0 151.3 139.3 131.5 146.7 108.3 123.0 114.6 117.7 99.9 100.7 159.1 128.0 105.5 125.7 136.0 106.5 119.2 115.1
41.5 41.2 40.2 42.4 45.9 38.3 45.2 38.5 29.5 51.6 47.6 46.4 42.1 45.9 37.4 42.4 40.1 39.7 41.3 52.5 42.2 34.6 42.2 39.8 36.3 42.4 41.3
LSD0.05
2.1
15.9
4.0
especially during water stress, because it facilitates the dissipation of excess heat. This prevents photoinhibition, which decreases the efficiency of the photosynthetic system and enhances the production of toxic active oxygen species [26]. Transpiration also promotes water and nutrient absorption [21]. Relative transpiration was also strongly correlated with relative leaf area (r /0.70**), which demonstrates the importance of transpiration and water uptake in the maintenance of leaf area during stress. Maintenance of leaf area is desirable for a continued supply of photosynthates under stress and for regrowth upon relief of stress. Table 4 also shows that some shallow-rooted cultivars such as IR20 and IR72 had high relative transpiration and low visual score (less injury from water stress). IR20 is a semidwarf and shallow-rooted cultivar that is usually used as an intolerant check in screening for drought tolerance [21,24]. These findings demonstrate that when cultivars are compared based largely on shoot trait, and deep root character does not considerably contribute to water stress tolerance as when an osmoticum such as PEG is used, some shallow-rooted cultivars may exhibit tolerance for water deficit, probably because of shoot characters that are expressed. In
contrast, visual scoring in the field may be obscured by genotypic variability in the capacity to have access to water in the deeper soil horizons and by the ability to overcome the resistance provided by the soil against root penetration. Simulation of water stress with the use of PEG as was done in the present study differs from field experiments in that the osmotic potential of the medium is maintained, whereas, in the field, water is continuously being extracted by plants and is lost to the atmosphere by evapotranspiration. Thus, plants in field experiments where irrigation is withheld for some time experience a progression of water deficit from mild to severe. This consideration is important in comparing the results of the present experiment with those obtained in the field. In a field experiment [27], the high transpiration rate in IR72 resulted in greater stress than in RC14, a cultivar of similar phenology but released for the rainfed lowland ecosystem. The authors reported that IR72 was more sensitive to severe water stress, with higher values of leaf rolling and a greater reduction in height and yield attributes. Thus, having a high transpiration rate as in IR72 could be a desirable trait only under conditions of mild water deficit. A review [28] also pointed out the
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importance of water transpiration in increasing grain yield under the condition that all available soil water is not used up and is recharged each year. Initial leaf area (leaf area before water stress treatment) was negatively correlated (r / /0.68**) with relative transpiration. This shows that at the onset of water deficit and especially when solar radiation is high, having an initially large leaf area may be disadvantageous to plants because of more leaf surface exposed to solar radiation, bringing about increased photorespiration and water loss from the tissues. It is therefore possible to initially screen out drought-intolerant cultivars based on leaf area alone. Initial leaf area was also positively correlated (r /0.50**) with visual score, which further demonstrates that a large leaf area at the onset of stress leads to more severe injury from water deficit. These observations are in line with the report [29] that rice lines with large plant size were stressed more quickly and had high drought scores. 3.3. Water stress and WUE In this study, little variation in WUE was obtained among cultivars under non-stress conditions (Table 5). WUE ranged from 2.35 mg shoot dry matter/g H2O in UPLRi-5 to 4.21 mg shoot dry matter/g H2O in Lansaw. On the other hand, mild water stress widened variability among genotypes and increased WUE in all cultivars (twofold in some cultivars). An increase in WUE during mild water deficit was also reported in barley [30] and up to 100% in cowpea [31]. There was a slight variation in genotype ranking for WUE between control and stress conditions, although the correlation was high (r /0.80**). Variability in WUE under stress was not correlated with visual score. It is possible that in some cultivars, an increase in WUE was attained largely by stomatal closure, which decreased transpiration, rather than by maintenance of photosynthetic activity. Therefore, selection for high WUE per se under water-deficit conditions may not translate into tolerance, unless selection is done in tandem with other indices for tolerance such as visual scoring and relative growth. It is worth noting that KDML 105 and IR72 both had high WUE under stress conditions (6.69 and 6.25 mg shoot/g H2O, respectively) and tolerant visual scores (Table 4). It should be further explored how these cultivars maintain the balance between photosynthesis and transpiration under waterdeficit conditions. An overall assessment of WUE across genotypes under non-stress and water stress treatments for 6 days is shown in Fig. 1. The slopes of the graphs represent WUE and clearly show a higher WUE that can be achieved under stress (10.1 mg shoot dry matter/g H2O transpired) than under non-stress (4.1 mg shoot dry matter/g H2O transpired) conditions. However,
despite a high WUE, dry matter production was significantly depressed in cultivars grown under stress, which could be a consequence of stomatal and nonstomatal [32] inhibitions of photosynthesis under water deficit. Fig. 1 also shows the established linearity between dry matter production and transpiration [30,33], demonstrating the dependence of dry matter accumulation on transpiration rate. The relationship, though, is weaker under stress conditions, which could be attributed to genotypic variability in adapting to water deficit via modulation of stomatal aperture. 3.4. Water stress and carbon isotope discrimination Significant variation in D was found among genotypes grown under water stress (Table 5). Carbon isotope discrimination ranged from 20.1 for ITA130 to 21.8 for IR442-2-58. These values are comparable with the range of D (19.8 /21.5) obtained for 28 upland rice cultivars grown under mild water stress [7] and with the D (21.1 /22.3) obtained in leaves of 42 field-grown cowpea genotypes [34]. Although loosely, D was negatively correlated with visual score (r / /0.38*), which implies that D may also be an indicator of plant adaptation to water deficit. Under mild water deficit, plants having high D could be adapting to the stress. In this study, D was not correlated with WUE under stress conditions. It is possible that dependence of WUE on D is affected by several factors, one of which might be respiration rate, which is responsible for carbon losses from tissues. In general, up to 70% of the total carbon fixed during a plant’s lifetime is respired [35]. Whole plants respire 30/50% of the photosynthetic uptake over 24 h, even under the most favorable conditions [36]. A negative relationship was also not obtained between WUE and D among indica rice cultivars but was demonstrated only among japonica and aus rice [7]. Likewise, an absence of correlation was reported between WUE and D in three wheat cultivars measured in the field [6]. In peanut, only 68% of the variance of WUE was explained when WUE was regressed on D ; 32% of the variation in WUE remained unexplained [8]. With a decrease in stomatal conductance during water stress leading to a decrease in CO2 intake and decreased ci [37] and, most likely, an increase in leaf temperature [38], this loss in carbon by respiration is expected to increase. In this study, only 52% of the variance of shoot dry matter was explained when shoot dry matter under stress conditions was regressed on transpiration rate compared with 83% under non-stress conditions (Fig. 1). Increased respiratory loss in carbon during stress could have partly accounted for 48% of the variation in shoot dry matter. A deeper understanding of the link between WUE and D and the factors governing the relationship is needed to understand why the relationship is absent in
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Table 5 The effect of 6-day duration of water deficit (with PEG at 0.5 MPa) on WUE, carbon isotope discrimination (D ), and SLW of 3-week-old seedlings grown inside the phytotron, with cultivars arranged according to WUE under non-stress conditions (without PEG) Cultivar
WUE (mg shoot/g H2O)
D in PEG ()
Control
PEG
Lansaw IRAT104 IRAT140 ITA119 IR52 Khao Dawk Mali 105 BPI RI 10 ITA130 IR62266-42-6-2 PI163575 IRAT9 IR5178-1-1-4 IR442-2-58 IR6115-1-1-1 M55 IR72 IR52561-UBN-1-1-2 C4-137 UPLRi-7 IR58821-23-B-1-2-11 CT9993-5-10-1M IR20 Nam Sa-Gui 19 Ifugao rice Mahsuri Qomenan UPLRi-5
4.21 4.13 4.11 4.06 4.01 3.85 3.76 3.71 3.64 3.64 3.63 3.63 3.61 3.58 3.58 3.56 3.56 3.48 3.40 3.37 3.34 3.32 3.25 3.25 3.16 3.12 2.35
7.22 6.47 6.40 5.09 7.33 6.69 6.02 5.45 4.96 5.30 6.27 7.18 4.95 6.65 5.67 6.25 5.42 4.11 5.92 5.07 3.42 5.51 5.44 4.28 4.69 3.65 0.86
20.82 20.51 20.32 20.84 20.99 20.86 21.05 20.09 21.30 20.53 20.78 20.82 21.83 20.78 20.11 21.07 21.03 20.69 20.48 21.16 20.90 21.60 20.88 21.20 21.01 21.05 20.61
LSD0.05
0.30
0.69
0.26
some crops and under what environment the theory would be valid. Carbon isotope discrimination was positively correlated with relative transpiration (Fig. 2). High D and high transpiration in cultivars grown under moderate
Fig. 1. Relationship between shoot dry matter and cumulative transpiration within 6 days for seedlings of 27 rice cultivars grown under control (no PEG) and water-deficit conditions (with PEG at / 0.5 MPa) for 6 days.
SLW (g m 2) Control
PEG
36 36 41 34 36 40 35 42 35 36 40 44 32 33 34 43 38 37 34 36 37 37 42 40 35 37 32
96 126 111 99 52 58 74 119 52 129 102 82 51 65 124 73 61 87 70 70 99 50 94 78 71 87 105
1.4
12.4
water stress could be interpreted as an indication of high stomatal conductance. More positive D values indicate increased intercellular CO2 concentration, which is regulated by the extent of stomatal opening [39]. The positive correlation obtained between D and stomatal conductance in common bean supports the assumption that plants with high transpiration rates have more open stomates and can better discriminate against 13C [40]. Since stomatal closure is the main limitation to transpiration and photosynthesis during water stress, the ability to monitor stomatal conductance by D will give relevant information on how the plant is affected by water deficit. Further work is needed to establish the association between transpiration rate and D , and whether such a relationship could be attributed to variation in stomatal conductance. It was reported that, in addition to leaf conductance, other plant characters such as earliness and dry matter production tend to be correlated with D under field conditions, which imply that these characters represent specific adaptive combinations [41]. The application of D in plant breeding should not be limited to the prediction of WUE alone, since improved WUE may actually restrict growth, as in the case when WUE
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Fig. 2. Relationship between leaf carbon isotope discrimination (D ) and transpiration relative to the control for seedlings of 27 rice cultivars grown under water-deficit conditions (with PEG at /0.5 MPa) for 6 days.
is increased by partial closure of stomata [18]. Furthermore, D appears to be under genetic control and is a stable character across environments [41]. 3.5. Water stress and SLW Water deficit caused marked increases in SLW in all cultivars, with an increase of up to four times in M55 and PI 163575 (Table 5). This confirms the general observation that leaf thickness increases in plants subjected to water stress [42]. Increases in SLW with decreases in water potential were also reported in cotton [43]. A highly significant negative relationship was found between SLW after stress and D (Fig. 3), which was similar to that reported [8] using peanut cultivars under non-limiting water conditions. It is worth noting that, with the exception of IRAT104, cultivars located at the extreme right of the graph (which had high SLW but low D ) had high visual scores (see Table 4), indicating more injury by water stress. In this study, therefore, having low D and high SLW after water stress is an indication that the plant is suffering from water stress. High SLW in stressed plants could be a consequence of cell shrinkage following water deficit, resulting in a decrease in cell volume with the solutes within the cell becoming more concentrated [44]. This conclusion is further supported by the low moisture in leaves of cultivars with a high SLW after stress (Fig. 4). A high SLW was also found to be significantly and negatively correlated with relative water content, indicating that thicker leaves had low moisture in tissues [43]. A highly significant negative relationship was also found between SLW and cumulative transpiration after stress (Fig. 5). This indicates that the development of thick leaves after water stress reduces transpiration rate.
Fig. 3. Relationship between leaf carbon isotope discrimination (D ) and SLW for seedlings of 27 rice cultivars grown under water-deficit conditions (with PEG at /0.5 MPa) for 6 days.
It can be hypothesized that an increase in SLW in drought-intolerant cultivars is a strategy to conserve the already much reduced moisture in tissues, which is a consequence of reduced water uptake caused by a reduction in transpiration rate. Three types of transpirational control could have evolved in plants [45]. These can be categorized as reduced effective leaf surface area, reduced radiation absorption per unit effective surface area, and increased resistance to water vapor in the transport pathway. An increase in SLW could be a transpirational control related to the last two categories. However, this might increase leaf temperature and decrease carbon assimilation, leading to non-stomatal inhibition of photosynthesis such as decreased activity of many enzymes of the Calvin cycle (particularly rubisco) and degradation of cell membranes. Under the conditions imposed in this study, which, at /0.5 MPa osmotic concentration is considered mild stress, the thinner (low SLW) and more expanded leaves in cultivars tolerant of water deficit are adaptive mechanisms because they promote transpiration and photosynthetic activity. As pointed out earlier, high D could be an indication of high stomatal conductance,
Fig. 4. Relationship between % moisture and SLW for seedlings of 27 rice cultivars grown under water-deficit conditions (with PEG at /0.5 MPa) for 6 days.
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Fig. 5. Relationship between cumulative transpiration and SLW for seedlings of 27 rice cultivars grown under water-deficit conditions (with PEG at /0.5 MPa) for 6 days.
which could lead to the development of high shoot biomass relative to the control. It was recommended that selection for low SLW (in addition to early vigor) be done rather than for increased WUE because high WUE may be linked with reduced early growth and leaf area development [6]. It was also found that species with low values of SLW have higher relative growth rates, presumably because, for a given amount of leaf dry matter, plants with a low SLW possess greater leaf area and thus intercept more light [46]. 3.6. Effect of water stress on total sugars (ethanolsoluble) and starch For ease in discussion, leaf blade and leaf sheath will be referred to as leaf and sheath, respectively. Initial sugar and starch contents in leaf and sheath were not correlated with visual scores and relative shoot weight, indicating that the initial concentration of non-structural carbohydrates has no significant role in achieving tolerance for water deficit at the seedling stage. This is in contrast with the observations in submerged environments where initially high carbohydrate level in the tissues is considered one of the adaptive traits, by serving as source of metabolic activity during submergence [47]. It is possible that submergence and water deficit differ in degree of carbohydrate depletion. Since the stomates remain partially open, allowing continued assimilation under mild water deficit, dependence on initial carbohydrates may not be as crucial under water deficit as in submergence where the main effect on the plant tissues is carbohydrate depletion [48]. Reduction in sugar content in the leaf occurred in most cultivars when grown under stress (Table 6). In contrast, sugar in the sheath increased in most cultivars, with an almost 50% increase in IRAT9 and IRAT104. Starch content in the leaf was very low and nil in some cultivars, while that in the sheath was not as low and
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even increased in some cultivars, especially in IR20 (Table 7). A starch reduction in the leaf is a common observation in water-stressed plants [49]. A greater reduction in starch than in sucrose in the leaf could be a consequence of decreased total carbon flux, with a larger proportion going to sucrose than to starch [50]. Alternatively, a low carbon supply under water stress conditions could have remobilized chloroplastic starch to provide carbon in favor of more sucrose synthesis [37]. Work on excised bean leaves also suggested that wilting caused starch to be converted to sucrose [49]. Results also show some degree of accumulation of sugar and especially of starch in the sheath in some cultivars, notably IRAT9, IRAT104, IR72, and IR20 when grown under water stress. Starch in the sheath in stressed plants relative to the control was negatively correlated, although weakly, with score (r / /0.31), suggesting a protective role for the buildup of starch in the sheath against stress. Such an increase in starch in the sheath could be an indication of osmotic adjustment, which is the lowering of the water potential of the plant without an accompanying decrease in turgor, by an accumulation of solutes in cells [51]. Soluble sugars are among the compatible solutes involved in osmotic adjustment. Most studies point to the leaf and roots as the most likely sites for osmotic adjustment. Such an accumulation of sucrose, with more in the leaf sheath, can be attributed to a rice gene (sal T gene), which had its highest expression in the leaf sheath in response to salt stress and drought [4]. The authors explained that the leaf sheath is the organ most exposed to the stress because water is lost more readily from the sheath than elsewhere, or that water is rapidly drawn from the sheath to replace that lost from leaves or roots. This argument seems logical because water is being drawn simultaneously from the sheath by the root (exposed to low soil water potential) and by the leaf (exposed to vapor pressure deficit) during drought stress. Thus, the leaf sheath would most likely be the primary site for an adaptive response such as osmotic adjustment. An alternative explanation can be based on the observation that solutes usually accumulate in regions where growth is more rapid [52] and that phloem transport is relatively insensitive to low water potential [45]. Osmotic adjustment can therefore be seen as a strategy to protect the meristematic tissues during water stress. Since in rice and other grasses the meristematic tissue is enclosed in the leaf sheath, it is expected that the leaf sheath would exhibit a relatively higher rate of osmotic adjustment than the leaf blade or the root. It is therefore hypothesized that, in this study, increases in sugar and starch in the sheath reflect osmotic adjustment, and cultivars such as IRAT9, IRAT104, and IR20 could have osmotically adjusted. Solute accumulation
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Table 6 The effect of 6-day duration of water deficit (with PEG at 0.5 MPa) on sugar content in leaf blades and leaf sheaths of 3-week-old seedlings grown inside the phytotron Cultivar
Mahsuri C4-137 IR20 Nam Sa-Gui 19 Khao Dawk Mali 105 IRAT9 IR442-2-58 IRAT104 UPLRi-5 IR52 IR6115-1-1-1 UPLRi-7 M55 IR72 ITA130 BPI RI 10 CT9993-5-10-1M IR52561-UBN-1-1-2 IR58821-23-B-1-2-1 IR62266-42-6-2 IR5178-1-1-4 ITA119 IRAT140 PI 163575 Lansaw Ifugao rice Qomenan LSD0.05
Sugar in leaf blade (mg leaf 1)
Sugar in leaf sheath (mg sheath 1)
Control
PEG
PEG/control
Control
PEG
PEG/control
22.1 29.4 15.3 32.3 26.5 20.4 26.8 32.5 16.2 34.9 24.7 26.5 36.0 20.2 33.1 23.9 30.6 22.2 22.7 34.5 33.8 26.5 35.0 40.1 22.0 24.5 22.8
18.0 23.2 15.3 26.4 24.9 21.5 23.2 27.3 12.2 32.2 25.4 24.9 29.2 24.7 23.4 22.8 22.1 17.1 23.1 29.8 32.8 21.3 28.4 30.1 17.8 18.5 21.2
0.82 0.79 1.00 0.82 0.94 1.05 0.86 0.84 0.75 0.92 1.03 0.94 0.81 1.22 0.71 0.96 0.72 0.77 1.02 0.86 0.97 0.81 0.81 0.75 0.81 0.75 0.93
21.8 30.4 15.1 27.5 26.0 17.1 27.1 18.4 11.6 36.8 26.2 28.0 30.0 23.9 18.4 27.9 20.9 17.0 25.5 39.6 31.3 24.1 24.8 26.0 21.6 18.8 18.6
20.3 25.6 18.4 29.0 26.2 25.0 29.3 25.0 12.4 38.6 29.4 32.8 31.0 28.8 22.7 30.5 21.7 20.7 27.6 38.0 34.6 17.0 29.5 27.7 19.8 21.9 21.5
0.93 0.84 1.22 1.06 1.01 1.46 1.08 1.36 1.07 1.05 1.13 1.17 1.03 1.21 1.23 1.09 1.04 1.22 1.08 0.96 1.10 0.71 1.19 1.06 0.92 1.16 1.15
5.9
6.9
3.7
4.7
was also reported in the apices and enclosed leaves of wheat during water stress [53]. A negative correlation (r / /0.38*) was obtained between visual score and relative sugar in the leaf, indicating that, generally, cultivars with high sugar relative to the control (such as IR72 and KDML 105) had less severe leaf stress symptoms. Relative sugar in the leaf was strongly correlated with relative transpiration (Fig. 6) and relative shoot weight (r /0.76**), which suggests that sugar content was largely determined by photosynthetic activity and that cultivars tolerant of mild water deficit such as IR72 could have maintained fairly open stomates. This allowed physiological processes such as transpiration and CO2 assimilation to proceed. A similar trend was observed for starch (Fig. 6).
4. Conclusions In summary, this study demonstrated genotypic differences in the response of rice cultivars to water deficit. At the outset, it must be noted that we used 3-
week-old plants, which were treated with PEG 1500 for 6 days. Thus, the plants were still at the vegetative stage, at which point differences in growth stage could not have an effect. In general, water deficit had a more pronounced effect on leaf expansive growth than on shoot dry matter. Visual scoring proved to be a reliable index for tolerance for water stress, reflecting both a reduction in leaf area and in shoot dry matter after stress treatment. Maintenance of the transpiration rate during mild water stress enabled tolerant cultivars to minimize injury from the stress. A high transpiration rate in plants under stress relative to the control was highly associated with maintenance of leaf moisture and leaf area, which allowed photosynthetic activity to proceed and provide assimilates for continued growth. It was also significantly correlated with high carbohydrate production. Morphophysiological traits that allow maintenance of transpiration rate during mild water stress are low initial leaf area before the onset of stress, high D , and low SLW. Mild water stress also increased WUE in plants, which could have resulted from stomatal closure as indicated by reduced transpiration. More degradation of
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825
Table 7 The effect of 6-day duration of water deficit (with PEG at 0.5 MPa) on starch content in leaf blades and leaf sheaths of 3-week-old seedlings grown inside the phytotron Cultivar
Mahsuri C4-137 IR20 Nam Sa-Gui 19 Khao Dawk Mali 105 IRAT9 IR442-2-58 IRAT104 UPLRi-5 IR52 IR6115-1-1-1 UPLRi-7 M55 IR72 ITA130 BPI RI 10 CT9993-5-10-1M IR52561-UBN-1-1-2 IR58821-23-B-1-2-1 IR62266-42-6-2 IR5178-1-1-4 ITA119 IRAT140 PI 163575 Lansaw Ifugao rice Qomenan LSD0.05
Starch in leaf blade (mg leaf 1)
Starch in leaf sheath (mg sheath 1)
Control
PEG
PEG/control
Control
PEG
PEG/control
17.6 22.4 9.0 22.6 18.8 11.6 28.3 35.9 14.3 31.0 17.9 23.8 35.2 17.7 35.2 18.2 29.3 20.0 20.2 38.1 26.0 32.5 40.1 43.6 15.7 19.8 22.2
7.8 6.2 7.0 3.8 13.9 1.6 8.2 1.0 1.4 13.4 8.2 5.2 1.0 10.8 / 6.8 5.6 4.4 5.7 14.5 8.8 2.9 7.5 5.3 3.9 7.3 5.3
0.44 0.28 0.78 0.17 0.74 0.14 0.29 0.03 0.10 0.43 0.46 0.22 0.03 0.61 / 0.37 0.19 0.22 0.28 0.38 0.34 0.09 0.19 0.12 0.25 0.37 0.24
38.8 53.6 28.9 33.8 57.9 29.7 68.9 37.5 23.3 93.5 59.6 44.6 57.5 58.0 32.0 64.3 33.0 30.7 41.8 91.1 46.3 42.4 55.0 53.2 33.4 35.5 29.9
43.2 43.4 46.2 30.1 73.2 38.7 67.9 35.9 14.6 90.0 61.0 52.0 37.8 71.0 27.3 64.4 32.9 42.7 48.1 90.0 49.7 25.1 48.7 29.2 30.2 40.0 33.7
1.11 0.81 1.60 0.89 1.27 1.30 0.99 0.96 0.63 0.96 1.02 1.17 0.66 1.23 0.85 1.00 1.00 1.39 1.15 0.99 1.07 0.59 0.89 0.55 0.90 1.13 1.12
8.2
3.0
14.1
9.9
high transpiration rate might cause excessive moisture losses, which, during prolonged exposure, might lead to death of plants. It is therefore recommended that rainfed lowland areas be characterized according to the degree of stress that would likely develop, whether stored moisture would be available to support the plant until maturity or whether the duration of stress is not long enough to cause severe damage. In such a situation, the results of this study would apply.
Acknowledgements Fig. 6. Relationship between sugar and starch in leaf relative to control and transpiration relative to control for seedlings of 27 rice cultivars grown under water-deficit conditions (with PEG at /0.5 MPa) for 6 days.
starch than sugar occurred in the leaf blade. More carbohydrates accumulated in the leaf sheath, which could be an indication of osmotic adjustment. Since relatively high transpiration results in high dry matter productivity even under mild water stress, it is necessary to select cultivars that are able to maintain a high transpiration rate. However, under severe stress, a
The authors are grateful to Drs. Enrique P. Pacardo and Melinda F. Lumanta, Professors at the University of the Philippines, Los Ban˜os, Laguna, Philippines, for helpful suggestions on the paper. Sincere thanks are also accorded to Mr. Rogelio Reyes and Mr. Lamberto Licardo for technical assistance. This study was jointly funded by the Philippine Council for Advanced Science and Technology Research and Development (PCASTRD), Department of Science and Technology, Manila, Philippines, and the International Rice Research Institute, Los Ban˜os, Laguna, Philippines.
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Physiological evaluation of responses of rice (Oryza sativa L.) to water deficit Gloria S. Cabuslay a,*, Osamu Ito b, Arcelia A. Alejar c a
Plant Breeding, Genetics, and Biochemistry (PBGB) Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines b Crop Production and Environment Division, JIRCAS, 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan c UP Los Ban˜os, College 4031, Laguna, Philippines Received 12 February 2002; received in revised form 5 July 2002; accepted 5 July 2002
Abstract In view of the need to increase rice yields in rainfed areas, an experiment was done to (1) determine physiological traits that contribute to tolerance for water deficit at the seedling stage, (2) identify quick but reliable indices for selection for tolerance for water deficit, and (3) characterize rice cultivars popularly used for water-deficit studies in rainfed ecosystems. The study used 27 cultivars, which include those often used for drought studies in rainfed lowland and upland ecosystems. Mild water-deficit conditions were generated by adding polyethylene glycol 1500 to nutrient solution to provide an osmotic stress of /0.5 MPa to 3week-old seedlings. Water deficit caused a larger reduction in leaf area than in shoot dry matter, demonstrating the greater sensitivity of leaf enlargement to water stress than dry matter accumulation. Visual scoring to assess damage was found to be a reliable measure of tolerance for water deficit. Cultivars tolerant of mild water stress had a high relative transpiration (transpiration under stress compared with that under non-stressed conditions), low initial leaf area (leaf area before the onset of water stress), high carbon isotope discrimination in the leaf, and low specific leaf weight. These factors enabled tolerant cultivars to maintain high moisture in the leaf and to have high values of leaf area, shoot dry matter, and sugar and starch in tissues in stressed plants relative to the control. Mild water deficit increased water use efficiency in stressed plants, caused more degradation of starch than sugar in the leaf blade, and resulted in more accumulation of these carbohydrates in the leaf sheath. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Physiological traits; Water deficit; Water stress
1. Introduction Water deficit (commonly known as drought) can be defined as the absence of adequate moisture necessary for a plant to grow normally and complete its life cycle. The lack of adequate moisture leading to water stress is a common occurrence in rainfed areas, brought about by infrequent rains and poor irrigation. Rice yield is significantly reduced such that an average of only 1.5 t ha 1 is realized in the rainfed lowland ecosystem in South and Southeast Asia [1] compared with a range of
Abbreviations: PEG, polyethylene glycol; D , carbon isotope discrimination; WUE, water use efficiency; SLW, specific leaf weight. * Corresponding author E-mail addresses: [email protected] (G.S. Cabuslay), [email protected] (O. Ito).
3 /9 t ha 1 in areas where the water supply is sufficient [2]. In spite of numerous reports on water deficit and the recent advances in molecular biology techniques, drought tolerance remains poorly understood in comparison with grain quality and disease resistance, which are governed by major genes. This demonstrates the complexity of rainfed ecosystems, exacerbated by unpredictable moisture supply. In addition, high genotype by environment (G /E) interaction makes it difficult to identify consistently superior genotypes. For many studies, imposing water stress in fieldgrown crops is difficult because of the unpredictability of rainfall and the possibility of seepage from adjoining plots. Soil heterogeneity further complicates the interpretation of field data. The addition of non-ionic osmotic agents such as polyethylene glycol (PEG) to liquid nutrient media has been shown to closely mimic
0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 2 ) 0 0 2 1 7 - 0
816
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specific levels of soil water stress [3]. Because they are not readily taken up by the cells, they do not affect the ionic composition of the cell [4]. PEG cannot cross membranes (as can sucrose) such that the water potential gradient in the tissue is not altered [5]. Furthermore, drought escape by growing deep roots is not possible because the roots of all cultivars are uniformly exposed to the same osmotic stress. A deeper understanding of the mechanisms of rice tolerance for water deficit is necessary for breeders to be able to identify heritable traits that will make plants adapt to growth conditions in rainfed areas. Moreover, it is important to use a quick but reliable index for tolerance for water deficit that will enable mass screening of genotypes. Water use efficiency (WUE) (total dry matter produced per unit of water transpired) is regarded as a desirable trait under both irrigated and rainfed conditions. The use of cultivars with a high WUE increases the efficiency of irrigation under wellwatered conditions and that of rainfall use under rainfed environments [6]. Genotypic variation in WUE has been reported in rice [7], peanut [8], cowpea [9], and other crops. The use of WUE as a selection criterion in plant breeding is hampered by difficulty in taking measurements, especially in field studies. An indirect measure of WUE, termed carbon isotope discrimination (D ), was developed, which makes it possible to analyze a large number of plants. The technique is based on the ratio of the naturally occurring stable isotopes 13C and 12C. During photosynthesis, 13C is discriminated against resulting in plants having a smaller 13C/12C ratio than the CO2 of the surrounding air. It was predicted that D is negatively correlated with WUE [10], i.e. plants with a high WUE would have large ratios of 13C/12C (or less D ). The negative relationship between transpiration efficiency and D was shown as predicted in peanut [11] and in wheat [9] under both well-watered and water stress conditions. The objectives of this study were to (1) determine physiological traits that contribute to tolerance for water deficit at the seedling stage, (2) identify quick but reliable indices for selection for tolerance for water deficit, and (3) characterize rice cultivars popularly used for water-deficit studies in rainfed ecosystems.
2. Materials and methods 2.1. Plant material and growth conditions 3-week-old seedlings of 27 rice cultivars/lines of different adaptations (Table 1 [12]) were used. Some characteristics of these cultivars/lines are enumerated in Table 2 [13]. All studies were carried out in a phytotron glasshouse maintained at 29/21 8C (day/night) and 70%
relative humidity under natural light conditions. The experiment was laid out in a split-plot design using five replications, with PEG treatment as the main plot and cultivar as the subplot. Pregerminated seeds were sown in styrofoam seedbeds lined with nylon nets and floated over culture solution [14] maintained at pH 5.5. The culture solution was renewed once a week for 2 weeks and twice a week at the third week and thereafter. Seedlings (18-day-old) were transferred into 50 ml plastic centrifuge tubes containing normal nutrient solution. One tube contained a single plant, held in place by a styropor cover that fit the lid of the plastic tube and had a hole bored at the center. The level of the solution was maintained daily. 2.2. Treatment A water-deficit condition was imposed 3 days after transfer into the tubes by changing the nutrient solution to that containing PEG 1500 giving /0.5 MPa of stress. This was prepared by adding 130 g of PEG 1500 to 1 l of culture solution [15]. Solutions of PEG were purified by mixing with ion-exchange resin before adding nutrients. A control plot was maintained. The osmotic potential of the PEG solution was measured with a freezing point osmometer (Precision Systems, Inc., Natick, MA USA, Model 5004). The weight of tubes with plant material was measured daily and moisture lost was replenished. Moisture lost was taken as the amount of water transpired. The experiment was terminated after 6 days, when cultivars could be separated into tolerant and intolerant of water deficit according to a visual score. Average daily solar radiation was 18.5 MJ m 2. Before harvest, plants were scored visually for water stress damage, which included leaf rolling and leaf drying. A scale of 1, 3, 5, 7, and 9 was used where 1 refers to least damaged (tolerant of water deficit) and 9 pertains to most damaged (intolerant of water deficit). 2.3. Measurements Plants were sampled before (representing initial data) and after PEG treatment. Seedlings were washed with distilled water and blotted dry with a paper towel. The shoot was separated from the root and the leaf blade was detached from the leaf sheath. The area of green leaves was measured with a leaf area meter (LI-COR, Inc., Model LI-3100, Lincoln, NE). Immediately after determination of fresh weight, samples were immersed in liquid nitrogen. The frozen samples were temporarily stored in a deep-freezer ( / 20 8C) prior to freeze-drying. Freeze-dried samples were further oven-dried overnight to ensure total inactivation of enzymes. Dry weights were also taken.
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Table 1 Accession number, country of origin, adaptation, and isozyme grouping of 27 rice cultivars/lines used in this study Accession number
Cultivar
Country of origin
Adaptationa
Isozyme groupb
10929 11354 11355 11462 27748 28506 32583 38558 50634 53434 57020 67819 72931 76330 77181 77968
Mahsuri C4-137 IR20 Nam Sa-Gui 19 Khao Dawk Mali 105 IRAT9 IR442-2-58 IRAT104 UPLRi-5 IR52 IR6115-1-1-1 UPLRi-7 M55 IR72 ITA130 BPI RI 10 CT9993-5-10-1M IR52561-UBN-1-1-2 IR58821-23-B-1-2-1 IR62266-42-6-2 IR5178-1-1-4 ITA119 IRAT140 PI 163575 Lansaw Ifugao rice Qomenan
Malaysia Philippines Philippines Thailand Thailand Coˆte d’Ivoire Philippines Coˆte d’Ivoire Philippines Philippines Philippines Philippines Liberia Philippines Nigeria Philippines Colombia Philippines Philippines Philippines Philippines Nigeria Coˆte d’Ivoire Philippines Philippines Philippines Philippines
RL / IL DW RL U DW U U RL / U U IL U / U / / / / / U / / / /
I I I I I I I VI I I I I VI I VI I VI I I I I I VI I I I I
57011 80022 55684 5758 8134 8176 8144 a b
RL: rainfed lowland; IL: irrigated lowland; DW: deep water; U: upland. I: indica; VI: japonica; Classification according to Glaszmann [12].
Ethanol-soluble sugars and starch were analyzed by near-infrared spectrometry [16]. A wet assay for constructing the calibration curves for sugar and starch was done colorimetrically [17]. The carbon isotope composition (d13C) of dried ground leaf samples was analyzed using a continuous flow isotope ratio mass spectrometer (Europa Scientific Roboprep-CN (Europa Scientific Ltd., Crewe, Chesire, UK) coupled to VG Micromass (VG Isogas, Middlewich, Chesire, UK) 903). Carbon isotope discrimination (D ) was computed as shown below: D ()
13 d Cair d 13 Cplant
d 13 C ()
1 d 13 Cplant
3. Results and discussion 3.1. Morphological responses of cultivars grown under water deficit Water deficit at /0.5 MPa osmotic potential, simulated by adding PEG to the culture solution, caused only Table 2 Some phenotypic characteristics of 8 lines/cultivars included in this study Cultivar/line
1000;
R 1; RPDB
where R is the molar abundance ratio, 13C/12C, of a sample [18]. The value of d13Cair used for the calculation of D was /7.60 relative to PeeDee Belemnite. WUE for the period of PEG treatment was obtained by dividing shoot dry matter by water transpired. Specific leaf weight (SLW) was calculated as the ratio of green leaf weight to leaf area.
Phenotype description
CT9993-5-10
Deep roots, good seedling vigor, anther cultureresponsive, semi-upland IR52561-UBN-1- Shallow roots, improved, vigorous 1-2 IR58821-23-B-1- Deep roots, improved, semi-erect, sturdy 2-1 IR62266-42-6-2 Improved, semi-ground cover Khao Dawk Mali Intermediate to shallow root system, improved 105 traditional, high vigor, weak stems Mahsuri Poor roots, improved, high vigor, rainfed-lowland adapted Nam Sagui 19 Shallow roots, drought-adapted, high vigor, improved traditional IR20 Drought-susceptible, semidwarf From Samson et al. [13].
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slight leaf rolling and leaf drying in plants. This observation suggests that the treatment produced mild water stress. Development of leaf area was more severely affected by the stress than was shoot dry matter (Table 3), with leaf area declining by 71% on average and shoot dry matter declining by 27%. Except in IR20, green leaf area under stress declined to levels below those occurring before the imposition of the stress or initial leaf area, whereas positive growth was obtained in shoot dry matter (data not shown). These results demonstrate the more pronounced effect of water deficit on leaf expansive growth than on shoot dry matter, which is in line with the general observation that leaf expansion is more sensitive to water stress than photosynthesis [19,20]. A large decline in leaf area under mild water stress is disadvantageous to plants because it leads to reduced nutrient uptake, which is a consequence of reduced demand and reduced transpiration [21]. Reduced leaf area also decreases carbon assimilation, which is positively related to leaf area [22].
Table 3 The effect of 6-day duration of water deficit (with PEG at 0.5 MPa) on leaf area and shoot weight of 3-week-old seedlings grown inside the phytotrona Cultivar
Mahsuri C4-137 IR20 Nam Sa-Gui 19 Khao Dawk Mali 105 IRAT9 IR442-2-58 IRAT104 UPLRi-5 IR52 IR6115-1-1-1 UPLRi-7 M55 IR72 ITA130 BPI RI 10 CT9993-5-10-1M IR52561-UBN-1-1-2 IR58821-23-B-1-2-1 IR62266-42-6-2 IR5178-1-1-4 ITA119 IRAT140 PI 163575 Lansaw Ifugao rice Qomenan Mean % reduction a
Leaf area (cm2)
Shoot weight (mg)
Control
PEG
Control
80.4 92.7 55.1 78.7 75.8 64.2 103.2 99.7 68.5 107.8 93.5 103.2 122.4 63.7 89.8 79.2 91.5 71.0 70.5 118.9 86.4 89.7 96.9 113.0 73.5 72.1 82.6
25.8 23.4 29.3 22.9 38.3 16.2 34.9 17.8 12.4 50.5 30.8 31.9 19.6 26.8 16.3 24.8 18.8 26.3 26.1 46.9 31.6 18.2 21.7 18.8 16.8 20.5 20.6
86.8 70.6
(4.5) (3.4) (1.5) (6.7) (2.9) (1.2) (7.6) (1.2) (8.8) (2.6) (1.8) (4.5) (3.7) (1.8) (3.4) (4.2) (4.6) (3.7) (2.3) (6.6) (5.1) (1.7) (2.7) (4.9) (2.8) (3.7) (4.0)
PEG
(3.1) 644 (25) 481 (16) (3.8) 779 (42) 521 (17) (1.9) 456 (20) 399 (14) (2.1) 737 (54) 563 (15) (3.6) 691 (14) 594 (11) (3.0) 611 (20) 492 (24) (2.8) 772 (73) 522 (18) (1.4) 723 (12) 530 (7) (2.6) 496 (78) 308 (14) (3.9) 962 (18) 734 (14) (5.2) 767 (16) 587 (18) (3.1) 816 (38) 643 (17) (1.6) 906 (27) 619 (22) (3.9) 632 (8) 534 (19) (1.9) 758 (39) 504 (13) (4.4) 695 (17) 519 (12) (3.5) 706 (34) 456 (35) (2.0) 584 (32) 441 (9) (4.6) 610 (16) 479 (26) (4.6) 1020 (43) 697 (23) (5.1) 780 (41) 619 (39) (4.1) 648 (34) 396 (9) (5.3) 867 (28) 617 (5) (3.5) 860 (36) 575 (10) (1.7) 629 (23) 446 (37) (2.2) 644 (7) 436 (15) (3.9) 649 (29) 439 (6)
25.5
Values in parentheses are standard errors.
720 524 27
The water status of the leaf is usually monitored by measuring the leaf water potential. However, this process is slow and is not applicable for mass screening of cultivars. Visual scoring techniques, whether based on leaf rolling or leaf drying, were found to be highly correlated with maintenance of leaf water potential [23]. Desirable scores were the smaller numerical values, which refer to cultivars showing less damage from water stress. In this study, tolerance for water stress was assessed by visual scoring based on leaf rolling and leaf drying. Significant differences in visual score were found among the 27 cultivars tested (Table 4). Khao Dawk Mali 105 (KDML 105) and ITA119 were the most tolerant of water stress, while UPLRi-5 and PI 163575 were the most intolerant. KDML 105 is regarded as droughttolerant in field studies [24] with yield stability under low-input conditions [25], while ITA119 was reported to be highly drought-tolerant [21]. The visual score was positively correlated with a reduction in both leaf area (r /0.43*) and shoot dry matter (r /0.41*), indicating that cultivars with a high score, i.e. with less tolerance for water deficit, had a greater reduction in leaf area and in dry matter. Thus, visual scoring could be used effectively in mass screening of genotypes for tolerance for water deficit. 3.2. Water stress and transpiration Measurement of whole-plant rather than individualleaf transpiration would give more accurate information on how the plant responds to water stress. This takes into account differences in stomatal conductance among leaves of different ages and position in the canopy, which might be affected differently by stress. Aside from visual score, water stress tolerance of cultivars was also evaluated by comparing whole-plant transpiration under stress with that under non-stress conditions (herein referred to as relative transpiration). Thus, cultivars less affected by the stress will have transpiration values closer to those of plants grown under non-stress conditions. Relative transpiration data (Table 4) show that water stress caused more than 50% reduction in cumulative transpiration in all cultivars. Relative transpiration was highest in IR20 and KDML 105 while lowest values were obtained in M55 and PI 163575. Relative cumulative transpiration was highly and negatively correlated with visual score (r / /0.52**), indicating that, generally, cultivars with a high relative transpiration had a low visual score (having less damage from water stress). These results imply that cultivars with high relative transpiration are adapting to the stress and could possess genes for tolerance for water deficit. These genes are somehow working to prevent damage from water stress as manifested by the low visual score. Maintenance of transpiration is important in plants,
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Table 4 The effect of 6-day duration of water deficit (with PEG at 0.5 MPa) on visual scores and transpiration rates of 3-week-old seedlings grown inside the phytotron Cultivar
Visual score
Cumulative transpiration (g H2O/6 days) Control
PEG
Relative transpiration (PEG/control) 0.37 0.34 0.48 0.35 0.44 0.39 0.35 0.36 0.37 0.34 0.34 0.35 0.29 0.42 0.30 0.37 0.34 0.40 0.41 0.33 0.33 0.33 0.34 0.29 0.34 0.36 0.36
Mahsuri C4-137 IR20 Nam Sa-Gui 19 Khao Dawk Mali 105 IRAT9 IR442-2-58 IRAT104 UPLRi-5 IR52 IR6115-1-1-1 UPLRi-7 M55 IR72 ITA130 BPI RI 10 CT9993-5-10-1M IR52561-UBN-1-1-2 IR58821-23-B-1-2-1 IR62266-42-6-2 IR5178-1-1-4 ITA119 IRAT140 PI 163575 Lansaw Ifugao rice Qomenan
4.6 5.0 2.6 3.8 1.4 3.8 3.4 3.0 6.2 4.6 4.6 6.6 5.8 2.2 5.4 2.2 5.0 4.2 5.4 5.0 5.4 1.8 5.0 6.2 4.6 5.4 5.8
113.4 122.1 83.5 121.2 104.7 99.1 127.9 107.2 79.0 151.3 139.3 131.5 146.7 108.3 123.0 114.6 117.7 99.9 100.7 159.1 128.0 105.5 125.7 136.0 106.5 119.2 115.1
41.5 41.2 40.2 42.4 45.9 38.3 45.2 38.5 29.5 51.6 47.6 46.4 42.1 45.9 37.4 42.4 40.1 39.7 41.3 52.5 42.2 34.6 42.2 39.8 36.3 42.4 41.3
LSD0.05
2.1
15.9
4.0
especially during water stress, because it facilitates the dissipation of excess heat. This prevents photoinhibition, which decreases the efficiency of the photosynthetic system and enhances the production of toxic active oxygen species [26]. Transpiration also promotes water and nutrient absorption [21]. Relative transpiration was also strongly correlated with relative leaf area (r /0.70**), which demonstrates the importance of transpiration and water uptake in the maintenance of leaf area during stress. Maintenance of leaf area is desirable for a continued supply of photosynthates under stress and for regrowth upon relief of stress. Table 4 also shows that some shallow-rooted cultivars such as IR20 and IR72 had high relative transpiration and low visual score (less injury from water stress). IR20 is a semidwarf and shallow-rooted cultivar that is usually used as an intolerant check in screening for drought tolerance [21,24]. These findings demonstrate that when cultivars are compared based largely on shoot trait, and deep root character does not considerably contribute to water stress tolerance as when an osmoticum such as PEG is used, some shallow-rooted cultivars may exhibit tolerance for water deficit, probably because of shoot characters that are expressed. In
contrast, visual scoring in the field may be obscured by genotypic variability in the capacity to have access to water in the deeper soil horizons and by the ability to overcome the resistance provided by the soil against root penetration. Simulation of water stress with the use of PEG as was done in the present study differs from field experiments in that the osmotic potential of the medium is maintained, whereas, in the field, water is continuously being extracted by plants and is lost to the atmosphere by evapotranspiration. Thus, plants in field experiments where irrigation is withheld for some time experience a progression of water deficit from mild to severe. This consideration is important in comparing the results of the present experiment with those obtained in the field. In a field experiment [27], the high transpiration rate in IR72 resulted in greater stress than in RC14, a cultivar of similar phenology but released for the rainfed lowland ecosystem. The authors reported that IR72 was more sensitive to severe water stress, with higher values of leaf rolling and a greater reduction in height and yield attributes. Thus, having a high transpiration rate as in IR72 could be a desirable trait only under conditions of mild water deficit. A review [28] also pointed out the
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importance of water transpiration in increasing grain yield under the condition that all available soil water is not used up and is recharged each year. Initial leaf area (leaf area before water stress treatment) was negatively correlated (r / /0.68**) with relative transpiration. This shows that at the onset of water deficit and especially when solar radiation is high, having an initially large leaf area may be disadvantageous to plants because of more leaf surface exposed to solar radiation, bringing about increased photorespiration and water loss from the tissues. It is therefore possible to initially screen out drought-intolerant cultivars based on leaf area alone. Initial leaf area was also positively correlated (r /0.50**) with visual score, which further demonstrates that a large leaf area at the onset of stress leads to more severe injury from water deficit. These observations are in line with the report [29] that rice lines with large plant size were stressed more quickly and had high drought scores. 3.3. Water stress and WUE In this study, little variation in WUE was obtained among cultivars under non-stress conditions (Table 5). WUE ranged from 2.35 mg shoot dry matter/g H2O in UPLRi-5 to 4.21 mg shoot dry matter/g H2O in Lansaw. On the other hand, mild water stress widened variability among genotypes and increased WUE in all cultivars (twofold in some cultivars). An increase in WUE during mild water deficit was also reported in barley [30] and up to 100% in cowpea [31]. There was a slight variation in genotype ranking for WUE between control and stress conditions, although the correlation was high (r /0.80**). Variability in WUE under stress was not correlated with visual score. It is possible that in some cultivars, an increase in WUE was attained largely by stomatal closure, which decreased transpiration, rather than by maintenance of photosynthetic activity. Therefore, selection for high WUE per se under water-deficit conditions may not translate into tolerance, unless selection is done in tandem with other indices for tolerance such as visual scoring and relative growth. It is worth noting that KDML 105 and IR72 both had high WUE under stress conditions (6.69 and 6.25 mg shoot/g H2O, respectively) and tolerant visual scores (Table 4). It should be further explored how these cultivars maintain the balance between photosynthesis and transpiration under waterdeficit conditions. An overall assessment of WUE across genotypes under non-stress and water stress treatments for 6 days is shown in Fig. 1. The slopes of the graphs represent WUE and clearly show a higher WUE that can be achieved under stress (10.1 mg shoot dry matter/g H2O transpired) than under non-stress (4.1 mg shoot dry matter/g H2O transpired) conditions. However,
despite a high WUE, dry matter production was significantly depressed in cultivars grown under stress, which could be a consequence of stomatal and nonstomatal [32] inhibitions of photosynthesis under water deficit. Fig. 1 also shows the established linearity between dry matter production and transpiration [30,33], demonstrating the dependence of dry matter accumulation on transpiration rate. The relationship, though, is weaker under stress conditions, which could be attributed to genotypic variability in adapting to water deficit via modulation of stomatal aperture. 3.4. Water stress and carbon isotope discrimination Significant variation in D was found among genotypes grown under water stress (Table 5). Carbon isotope discrimination ranged from 20.1 for ITA130 to 21.8 for IR442-2-58. These values are comparable with the range of D (19.8 /21.5) obtained for 28 upland rice cultivars grown under mild water stress [7] and with the D (21.1 /22.3) obtained in leaves of 42 field-grown cowpea genotypes [34]. Although loosely, D was negatively correlated with visual score (r / /0.38*), which implies that D may also be an indicator of plant adaptation to water deficit. Under mild water deficit, plants having high D could be adapting to the stress. In this study, D was not correlated with WUE under stress conditions. It is possible that dependence of WUE on D is affected by several factors, one of which might be respiration rate, which is responsible for carbon losses from tissues. In general, up to 70% of the total carbon fixed during a plant’s lifetime is respired [35]. Whole plants respire 30/50% of the photosynthetic uptake over 24 h, even under the most favorable conditions [36]. A negative relationship was also not obtained between WUE and D among indica rice cultivars but was demonstrated only among japonica and aus rice [7]. Likewise, an absence of correlation was reported between WUE and D in three wheat cultivars measured in the field [6]. In peanut, only 68% of the variance of WUE was explained when WUE was regressed on D ; 32% of the variation in WUE remained unexplained [8]. With a decrease in stomatal conductance during water stress leading to a decrease in CO2 intake and decreased ci [37] and, most likely, an increase in leaf temperature [38], this loss in carbon by respiration is expected to increase. In this study, only 52% of the variance of shoot dry matter was explained when shoot dry matter under stress conditions was regressed on transpiration rate compared with 83% under non-stress conditions (Fig. 1). Increased respiratory loss in carbon during stress could have partly accounted for 48% of the variation in shoot dry matter. A deeper understanding of the link between WUE and D and the factors governing the relationship is needed to understand why the relationship is absent in
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Table 5 The effect of 6-day duration of water deficit (with PEG at 0.5 MPa) on WUE, carbon isotope discrimination (D ), and SLW of 3-week-old seedlings grown inside the phytotron, with cultivars arranged according to WUE under non-stress conditions (without PEG) Cultivar
WUE (mg shoot/g H2O)
D in PEG ()
Control
PEG
Lansaw IRAT104 IRAT140 ITA119 IR52 Khao Dawk Mali 105 BPI RI 10 ITA130 IR62266-42-6-2 PI163575 IRAT9 IR5178-1-1-4 IR442-2-58 IR6115-1-1-1 M55 IR72 IR52561-UBN-1-1-2 C4-137 UPLRi-7 IR58821-23-B-1-2-11 CT9993-5-10-1M IR20 Nam Sa-Gui 19 Ifugao rice Mahsuri Qomenan UPLRi-5
4.21 4.13 4.11 4.06 4.01 3.85 3.76 3.71 3.64 3.64 3.63 3.63 3.61 3.58 3.58 3.56 3.56 3.48 3.40 3.37 3.34 3.32 3.25 3.25 3.16 3.12 2.35
7.22 6.47 6.40 5.09 7.33 6.69 6.02 5.45 4.96 5.30 6.27 7.18 4.95 6.65 5.67 6.25 5.42 4.11 5.92 5.07 3.42 5.51 5.44 4.28 4.69 3.65 0.86
20.82 20.51 20.32 20.84 20.99 20.86 21.05 20.09 21.30 20.53 20.78 20.82 21.83 20.78 20.11 21.07 21.03 20.69 20.48 21.16 20.90 21.60 20.88 21.20 21.01 21.05 20.61
LSD0.05
0.30
0.69
0.26
some crops and under what environment the theory would be valid. Carbon isotope discrimination was positively correlated with relative transpiration (Fig. 2). High D and high transpiration in cultivars grown under moderate
Fig. 1. Relationship between shoot dry matter and cumulative transpiration within 6 days for seedlings of 27 rice cultivars grown under control (no PEG) and water-deficit conditions (with PEG at / 0.5 MPa) for 6 days.
SLW (g m 2) Control
PEG
36 36 41 34 36 40 35 42 35 36 40 44 32 33 34 43 38 37 34 36 37 37 42 40 35 37 32
96 126 111 99 52 58 74 119 52 129 102 82 51 65 124 73 61 87 70 70 99 50 94 78 71 87 105
1.4
12.4
water stress could be interpreted as an indication of high stomatal conductance. More positive D values indicate increased intercellular CO2 concentration, which is regulated by the extent of stomatal opening [39]. The positive correlation obtained between D and stomatal conductance in common bean supports the assumption that plants with high transpiration rates have more open stomates and can better discriminate against 13C [40]. Since stomatal closure is the main limitation to transpiration and photosynthesis during water stress, the ability to monitor stomatal conductance by D will give relevant information on how the plant is affected by water deficit. Further work is needed to establish the association between transpiration rate and D , and whether such a relationship could be attributed to variation in stomatal conductance. It was reported that, in addition to leaf conductance, other plant characters such as earliness and dry matter production tend to be correlated with D under field conditions, which imply that these characters represent specific adaptive combinations [41]. The application of D in plant breeding should not be limited to the prediction of WUE alone, since improved WUE may actually restrict growth, as in the case when WUE
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Fig. 2. Relationship between leaf carbon isotope discrimination (D ) and transpiration relative to the control for seedlings of 27 rice cultivars grown under water-deficit conditions (with PEG at /0.5 MPa) for 6 days.
is increased by partial closure of stomata [18]. Furthermore, D appears to be under genetic control and is a stable character across environments [41]. 3.5. Water stress and SLW Water deficit caused marked increases in SLW in all cultivars, with an increase of up to four times in M55 and PI 163575 (Table 5). This confirms the general observation that leaf thickness increases in plants subjected to water stress [42]. Increases in SLW with decreases in water potential were also reported in cotton [43]. A highly significant negative relationship was found between SLW after stress and D (Fig. 3), which was similar to that reported [8] using peanut cultivars under non-limiting water conditions. It is worth noting that, with the exception of IRAT104, cultivars located at the extreme right of the graph (which had high SLW but low D ) had high visual scores (see Table 4), indicating more injury by water stress. In this study, therefore, having low D and high SLW after water stress is an indication that the plant is suffering from water stress. High SLW in stressed plants could be a consequence of cell shrinkage following water deficit, resulting in a decrease in cell volume with the solutes within the cell becoming more concentrated [44]. This conclusion is further supported by the low moisture in leaves of cultivars with a high SLW after stress (Fig. 4). A high SLW was also found to be significantly and negatively correlated with relative water content, indicating that thicker leaves had low moisture in tissues [43]. A highly significant negative relationship was also found between SLW and cumulative transpiration after stress (Fig. 5). This indicates that the development of thick leaves after water stress reduces transpiration rate.
Fig. 3. Relationship between leaf carbon isotope discrimination (D ) and SLW for seedlings of 27 rice cultivars grown under water-deficit conditions (with PEG at /0.5 MPa) for 6 days.
It can be hypothesized that an increase in SLW in drought-intolerant cultivars is a strategy to conserve the already much reduced moisture in tissues, which is a consequence of reduced water uptake caused by a reduction in transpiration rate. Three types of transpirational control could have evolved in plants [45]. These can be categorized as reduced effective leaf surface area, reduced radiation absorption per unit effective surface area, and increased resistance to water vapor in the transport pathway. An increase in SLW could be a transpirational control related to the last two categories. However, this might increase leaf temperature and decrease carbon assimilation, leading to non-stomatal inhibition of photosynthesis such as decreased activity of many enzymes of the Calvin cycle (particularly rubisco) and degradation of cell membranes. Under the conditions imposed in this study, which, at /0.5 MPa osmotic concentration is considered mild stress, the thinner (low SLW) and more expanded leaves in cultivars tolerant of water deficit are adaptive mechanisms because they promote transpiration and photosynthetic activity. As pointed out earlier, high D could be an indication of high stomatal conductance,
Fig. 4. Relationship between % moisture and SLW for seedlings of 27 rice cultivars grown under water-deficit conditions (with PEG at /0.5 MPa) for 6 days.
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Fig. 5. Relationship between cumulative transpiration and SLW for seedlings of 27 rice cultivars grown under water-deficit conditions (with PEG at /0.5 MPa) for 6 days.
which could lead to the development of high shoot biomass relative to the control. It was recommended that selection for low SLW (in addition to early vigor) be done rather than for increased WUE because high WUE may be linked with reduced early growth and leaf area development [6]. It was also found that species with low values of SLW have higher relative growth rates, presumably because, for a given amount of leaf dry matter, plants with a low SLW possess greater leaf area and thus intercept more light [46]. 3.6. Effect of water stress on total sugars (ethanolsoluble) and starch For ease in discussion, leaf blade and leaf sheath will be referred to as leaf and sheath, respectively. Initial sugar and starch contents in leaf and sheath were not correlated with visual scores and relative shoot weight, indicating that the initial concentration of non-structural carbohydrates has no significant role in achieving tolerance for water deficit at the seedling stage. This is in contrast with the observations in submerged environments where initially high carbohydrate level in the tissues is considered one of the adaptive traits, by serving as source of metabolic activity during submergence [47]. It is possible that submergence and water deficit differ in degree of carbohydrate depletion. Since the stomates remain partially open, allowing continued assimilation under mild water deficit, dependence on initial carbohydrates may not be as crucial under water deficit as in submergence where the main effect on the plant tissues is carbohydrate depletion [48]. Reduction in sugar content in the leaf occurred in most cultivars when grown under stress (Table 6). In contrast, sugar in the sheath increased in most cultivars, with an almost 50% increase in IRAT9 and IRAT104. Starch content in the leaf was very low and nil in some cultivars, while that in the sheath was not as low and
823
even increased in some cultivars, especially in IR20 (Table 7). A starch reduction in the leaf is a common observation in water-stressed plants [49]. A greater reduction in starch than in sucrose in the leaf could be a consequence of decreased total carbon flux, with a larger proportion going to sucrose than to starch [50]. Alternatively, a low carbon supply under water stress conditions could have remobilized chloroplastic starch to provide carbon in favor of more sucrose synthesis [37]. Work on excised bean leaves also suggested that wilting caused starch to be converted to sucrose [49]. Results also show some degree of accumulation of sugar and especially of starch in the sheath in some cultivars, notably IRAT9, IRAT104, IR72, and IR20 when grown under water stress. Starch in the sheath in stressed plants relative to the control was negatively correlated, although weakly, with score (r / /0.31), suggesting a protective role for the buildup of starch in the sheath against stress. Such an increase in starch in the sheath could be an indication of osmotic adjustment, which is the lowering of the water potential of the plant without an accompanying decrease in turgor, by an accumulation of solutes in cells [51]. Soluble sugars are among the compatible solutes involved in osmotic adjustment. Most studies point to the leaf and roots as the most likely sites for osmotic adjustment. Such an accumulation of sucrose, with more in the leaf sheath, can be attributed to a rice gene (sal T gene), which had its highest expression in the leaf sheath in response to salt stress and drought [4]. The authors explained that the leaf sheath is the organ most exposed to the stress because water is lost more readily from the sheath than elsewhere, or that water is rapidly drawn from the sheath to replace that lost from leaves or roots. This argument seems logical because water is being drawn simultaneously from the sheath by the root (exposed to low soil water potential) and by the leaf (exposed to vapor pressure deficit) during drought stress. Thus, the leaf sheath would most likely be the primary site for an adaptive response such as osmotic adjustment. An alternative explanation can be based on the observation that solutes usually accumulate in regions where growth is more rapid [52] and that phloem transport is relatively insensitive to low water potential [45]. Osmotic adjustment can therefore be seen as a strategy to protect the meristematic tissues during water stress. Since in rice and other grasses the meristematic tissue is enclosed in the leaf sheath, it is expected that the leaf sheath would exhibit a relatively higher rate of osmotic adjustment than the leaf blade or the root. It is therefore hypothesized that, in this study, increases in sugar and starch in the sheath reflect osmotic adjustment, and cultivars such as IRAT9, IRAT104, and IR20 could have osmotically adjusted. Solute accumulation
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Table 6 The effect of 6-day duration of water deficit (with PEG at 0.5 MPa) on sugar content in leaf blades and leaf sheaths of 3-week-old seedlings grown inside the phytotron Cultivar
Mahsuri C4-137 IR20 Nam Sa-Gui 19 Khao Dawk Mali 105 IRAT9 IR442-2-58 IRAT104 UPLRi-5 IR52 IR6115-1-1-1 UPLRi-7 M55 IR72 ITA130 BPI RI 10 CT9993-5-10-1M IR52561-UBN-1-1-2 IR58821-23-B-1-2-1 IR62266-42-6-2 IR5178-1-1-4 ITA119 IRAT140 PI 163575 Lansaw Ifugao rice Qomenan LSD0.05
Sugar in leaf blade (mg leaf 1)
Sugar in leaf sheath (mg sheath 1)
Control
PEG
PEG/control
Control
PEG
PEG/control
22.1 29.4 15.3 32.3 26.5 20.4 26.8 32.5 16.2 34.9 24.7 26.5 36.0 20.2 33.1 23.9 30.6 22.2 22.7 34.5 33.8 26.5 35.0 40.1 22.0 24.5 22.8
18.0 23.2 15.3 26.4 24.9 21.5 23.2 27.3 12.2 32.2 25.4 24.9 29.2 24.7 23.4 22.8 22.1 17.1 23.1 29.8 32.8 21.3 28.4 30.1 17.8 18.5 21.2
0.82 0.79 1.00 0.82 0.94 1.05 0.86 0.84 0.75 0.92 1.03 0.94 0.81 1.22 0.71 0.96 0.72 0.77 1.02 0.86 0.97 0.81 0.81 0.75 0.81 0.75 0.93
21.8 30.4 15.1 27.5 26.0 17.1 27.1 18.4 11.6 36.8 26.2 28.0 30.0 23.9 18.4 27.9 20.9 17.0 25.5 39.6 31.3 24.1 24.8 26.0 21.6 18.8 18.6
20.3 25.6 18.4 29.0 26.2 25.0 29.3 25.0 12.4 38.6 29.4 32.8 31.0 28.8 22.7 30.5 21.7 20.7 27.6 38.0 34.6 17.0 29.5 27.7 19.8 21.9 21.5
0.93 0.84 1.22 1.06 1.01 1.46 1.08 1.36 1.07 1.05 1.13 1.17 1.03 1.21 1.23 1.09 1.04 1.22 1.08 0.96 1.10 0.71 1.19 1.06 0.92 1.16 1.15
5.9
6.9
3.7
4.7
was also reported in the apices and enclosed leaves of wheat during water stress [53]. A negative correlation (r / /0.38*) was obtained between visual score and relative sugar in the leaf, indicating that, generally, cultivars with high sugar relative to the control (such as IR72 and KDML 105) had less severe leaf stress symptoms. Relative sugar in the leaf was strongly correlated with relative transpiration (Fig. 6) and relative shoot weight (r /0.76**), which suggests that sugar content was largely determined by photosynthetic activity and that cultivars tolerant of mild water deficit such as IR72 could have maintained fairly open stomates. This allowed physiological processes such as transpiration and CO2 assimilation to proceed. A similar trend was observed for starch (Fig. 6).
4. Conclusions In summary, this study demonstrated genotypic differences in the response of rice cultivars to water deficit. At the outset, it must be noted that we used 3-
week-old plants, which were treated with PEG 1500 for 6 days. Thus, the plants were still at the vegetative stage, at which point differences in growth stage could not have an effect. In general, water deficit had a more pronounced effect on leaf expansive growth than on shoot dry matter. Visual scoring proved to be a reliable index for tolerance for water stress, reflecting both a reduction in leaf area and in shoot dry matter after stress treatment. Maintenance of the transpiration rate during mild water stress enabled tolerant cultivars to minimize injury from the stress. A high transpiration rate in plants under stress relative to the control was highly associated with maintenance of leaf moisture and leaf area, which allowed photosynthetic activity to proceed and provide assimilates for continued growth. It was also significantly correlated with high carbohydrate production. Morphophysiological traits that allow maintenance of transpiration rate during mild water stress are low initial leaf area before the onset of stress, high D , and low SLW. Mild water stress also increased WUE in plants, which could have resulted from stomatal closure as indicated by reduced transpiration. More degradation of
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825
Table 7 The effect of 6-day duration of water deficit (with PEG at 0.5 MPa) on starch content in leaf blades and leaf sheaths of 3-week-old seedlings grown inside the phytotron Cultivar
Mahsuri C4-137 IR20 Nam Sa-Gui 19 Khao Dawk Mali 105 IRAT9 IR442-2-58 IRAT104 UPLRi-5 IR52 IR6115-1-1-1 UPLRi-7 M55 IR72 ITA130 BPI RI 10 CT9993-5-10-1M IR52561-UBN-1-1-2 IR58821-23-B-1-2-1 IR62266-42-6-2 IR5178-1-1-4 ITA119 IRAT140 PI 163575 Lansaw Ifugao rice Qomenan LSD0.05
Starch in leaf blade (mg leaf 1)
Starch in leaf sheath (mg sheath 1)
Control
PEG
PEG/control
Control
PEG
PEG/control
17.6 22.4 9.0 22.6 18.8 11.6 28.3 35.9 14.3 31.0 17.9 23.8 35.2 17.7 35.2 18.2 29.3 20.0 20.2 38.1 26.0 32.5 40.1 43.6 15.7 19.8 22.2
7.8 6.2 7.0 3.8 13.9 1.6 8.2 1.0 1.4 13.4 8.2 5.2 1.0 10.8 / 6.8 5.6 4.4 5.7 14.5 8.8 2.9 7.5 5.3 3.9 7.3 5.3
0.44 0.28 0.78 0.17 0.74 0.14 0.29 0.03 0.10 0.43 0.46 0.22 0.03 0.61 / 0.37 0.19 0.22 0.28 0.38 0.34 0.09 0.19 0.12 0.25 0.37 0.24
38.8 53.6 28.9 33.8 57.9 29.7 68.9 37.5 23.3 93.5 59.6 44.6 57.5 58.0 32.0 64.3 33.0 30.7 41.8 91.1 46.3 42.4 55.0 53.2 33.4 35.5 29.9
43.2 43.4 46.2 30.1 73.2 38.7 67.9 35.9 14.6 90.0 61.0 52.0 37.8 71.0 27.3 64.4 32.9 42.7 48.1 90.0 49.7 25.1 48.7 29.2 30.2 40.0 33.7
1.11 0.81 1.60 0.89 1.27 1.30 0.99 0.96 0.63 0.96 1.02 1.17 0.66 1.23 0.85 1.00 1.00 1.39 1.15 0.99 1.07 0.59 0.89 0.55 0.90 1.13 1.12
8.2
3.0
14.1
9.9
high transpiration rate might cause excessive moisture losses, which, during prolonged exposure, might lead to death of plants. It is therefore recommended that rainfed lowland areas be characterized according to the degree of stress that would likely develop, whether stored moisture would be available to support the plant until maturity or whether the duration of stress is not long enough to cause severe damage. In such a situation, the results of this study would apply.
Acknowledgements Fig. 6. Relationship between sugar and starch in leaf relative to control and transpiration relative to control for seedlings of 27 rice cultivars grown under water-deficit conditions (with PEG at /0.5 MPa) for 6 days.
starch than sugar occurred in the leaf blade. More carbohydrates accumulated in the leaf sheath, which could be an indication of osmotic adjustment. Since relatively high transpiration results in high dry matter productivity even under mild water stress, it is necessary to select cultivars that are able to maintain a high transpiration rate. However, under severe stress, a
The authors are grateful to Drs. Enrique P. Pacardo and Melinda F. Lumanta, Professors at the University of the Philippines, Los Ban˜os, Laguna, Philippines, for helpful suggestions on the paper. Sincere thanks are also accorded to Mr. Rogelio Reyes and Mr. Lamberto Licardo for technical assistance. This study was jointly funded by the Philippine Council for Advanced Science and Technology Research and Development (PCASTRD), Department of Science and Technology, Manila, Philippines, and the International Rice Research Institute, Los Ban˜os, Laguna, Philippines.
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