Published online 27 October 2005
Published in Crop Sci 45:2517-2522 (2005)
© 2005 Crop Science Society of America
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CROP BREEDING, GENETICS & CYTOLOGY
Carbon Isotope Discrimination Accurately Reflects Variability in WUE Measured at a Whole Plant Level in Rice
S. M. Impa,
S. Nadaradjan,
P. Boominathan,
G. Shashidhar,
H. Bindumadhava and
M. S. Sheshshayee*
Department of Crop Physiology, University of Agricultural Sciences, GKVK Campus, Bangalore 560 065, INDIA
* Corresponding author (msheshshayee{at}hotmail.com)
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ABSTRACT
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Water use efficiency (WUE) is physiologically linked to discrimination of the stable isotope of carbon (
13C) in leaves of plant species. We determined the genetic variability in WUE by gravimetric approach and 13C among 34 diverse germplasm accessions of rice (Oryza sativa L.). The leaf 13C ranged between 18.7 and 21.6
, representing a significant variability and showed a strong inverse relationship with WUE. The gravimetrically determined WUE represents time integrated values, and hence its regression with 13C strongly proves the relevance of 13C as a surrogate for WUE in rice. For a trait to be successfully exploited for crop improvement, it should have low genotype x environment (G x E) interaction. Six contrasting genotypes selected and examined in a separate experiment showed good correspondence in both WUE and 13C between the experiments indicating that WUE is genetically controlled in rice and hence can be exploited through breeding. A prior knowledge on the constituent physiological factors controlling WUE is an important prerequisite for exploiting this trait in crop improvement programs. An inverse relationship between WUE and mean transpiration rate (MTR) indicates a stomatal control of WUE among rice genotypes. Although total biomass normally decreases while selecting for high WUE from among conductance types, a few promising genotypes with high WUE coupled with moderately high total biomass can still be identified for further crop improvement.
Abbreviations: BM, total biomass • CWT, cumulative water transpired • G x E, genotype x environment • LAD, leaf area duration • MTR, mean transpiration rate • NAR, net assimilation rate
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INTRODUCTION
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WITH MORE than 50% of the world's population consuming rice as a staple cereal, this crop is one of the most extensively cultivated crop species globally. To meet the forecasted demand from the world population by 2020, the global food production must increase by 1.6 to 2.6% annually (Araus et al., 2002; Braun et al., 1998). Although predominantly irrigated, a substantial area of rice cultivation is under water limited upland condition, especially in India where water limitation is the most overriding factor. The International Rice Research Institute (IRRI) estimates that it currently takes 5000 L of water to produce one kilogram of rice, which is twice that needed to grow other crops (http://www.irri.org; verified 17 July 2005). With decreasing fresh water resources for agriculture, enhancing the production potential under reduced water availability as well as technologies to save irrigation water are extremely critical.
Hence, improving drought tolerance of rice is important. Among several traits that impart drought tolerance, WUE, the amount of dry matter produced per unit amount of water transpired, seems to be most relevant. As per Passioura's growth and yield model (1986, 1996), WUE is an important yield-determining factor. Improving WUE would reduce the water requirement for a specific yield potential and thus can help save considerable amount of irrigation water. Further, an improvement in WUE can significantly enhance total biomass production as well as yield at a given level of soil water availability.
Though exploitable genetic variability in WUE was documented almost a century ago (Briggs and Shantz, 1913), significant progress in assessing genetic variability in WUE was achieved after the establishment of the physiological links between carbon isotope discrimination (13C) and WUE (Farquhar and Richards, 1984; Farquhar et al., 1989). Several container and field experiments have validated that 13C is a surrogate of WUE in many crop species [Condon et al., 1987, in Triticum aestivum L. and T. turgidum L. durum; Read et al., 1991, in Agropyron desertorum; Ismail and Hall, 1992, in Vigna unguiculata L. Walp; White et al., 1996 in Glycine max (L.) Merr.; Udayakumar et al., 1998a; Sheshshayee et al., 2003, in several field crops and perennial species].
In this manuscript, we examined the relationship between WUE measured at the whole plant level and 13C in two separate experiments using rice genotypes. Besides demonstrating that 13C is indeed a surrogate for WUE in rice, we show that stomatal conductance predominantly controls the variability in WUE among the rice genotypes.
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MATERIAL AND METHODS
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Two separate experiments were conducted to assess WUE at whole plant level at the field facility of the Department of Crop Physiology, University of Agricultural Sciences, Bangalore, India. The site is situated at 12°58' North latitude, 77°35' East longitude at an altitude of 930 m above mean sea level.
Experiment 1 (2001)
Zonal Agricultural Research Station, Mandya, India, and the Main Research Station, Hebbal, Bangalore, India, routinely evaluates rice germplasm accessions as well as genotypes developed by breeders under the All India Co-ordinated Rice Improvement Program (AICRIP). Promising cultures with relatively high harvest index suitable for both irrigated and upland conditions were identified for this investigation. The 34 selected genotypes were raised in carbonized rubber containers (60 L x 45 W x 30 D cm) filled with 25 kg of rooting mixture consisting of red sandy loam soil and farmyard manure in 3: 1 proportion (v/v). The containers were arranged randomly in open field conditions with two healthy and uniform seedlings in each container. Eight such containers were maintained for each genotype as replicates. The containers were irrigated twice daily to keep the soil at field capacity. Plastic pieces were spread on the soil surface to minimize direct soil evaporation. A set of four containers was maintained without plants ("bare" containers) to determine the surface evaporation losses.
Determination of Total Transpiration and WUE
Water use efficiency was determined gravimetrically as per Udayakumar et al. (1998b). Each of the containers was weighed daily with a load cell hanging balance (ATCO Balances, India). The difference in weight on subsequent days was corrected by adding equivalent volume of water (to maintain field capacity). The water thus added during the experimental period between 55 days after sowing (DAS) and 90 DAS was summed up to arrive at the total evapotranspiration. The cumulative water transpired (CWT) was computed by subtracting the water added to bare containers from the evapotranspiration. Total biomass was determined in a set of three containers representing each accession at the beginning of the experiment (55 DAS). The soil was washed carefully to remove the roots. And all the plant parts (leaves, stem, and roots) were separately oven dried at 70°C for 3 d. The total plant biomass was determined by recording the dry weights of all the plant parts. Before oven drying the samples, whole plant leaf area was determined with a leaf area meter (T Devices, Burwell, England, UK). Biomass and leaf area were once again recorded in plants from the remaining five containers of all the genotypes at the end of the experiment (90 DAS). Assuming a linear growth during the experimental period, WUE was computed as follows.
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where BM90 and BM55 are the total biomass (g pot–1) measured on 90 and 55 DAS, respectively. CWT is the cumulative water transpired during the same experimental period computed as follows
The other physiological parameters such leaf area duration (LAD), i.e., the functional leaf area during the experimental period was calculated as follows.
where LA90 and LA55 are the leaf area (in cm2 plant–1) measured on 90 and 55 DAS, respectively.
The Net assimilation rate (NAR) and the mean transpiration rate (MTR) were calculated as follows:
where BM90 and BM55 are the total biomass (in g plant–1) measured on 90 and 55 DAS, respectively.
Carbon Isotope Discrimination
Carbon isotope ratios were determined with an Isotope Ratio Mass Spectrometer (Delta-plus, ThermoFinnigan, Bremen, Germany) interfaced with an Elemental Analyzer (NA1112, CarloErba, Italy) via a continues flow device (Conflo–III, ThermoFinnigan, Bremen, Germany). A composite leaf sample comprising of 10 mature leaves representing all positions of the plant canopy were harvested and oven dried for 3 d at 70°C and homogenized to fine powder with a ball mill. Three replications for each of the 34 genotypes of rice were analyzed for 
13Clb with an analytical uncertainty of less than 0.1. Carbon isotope discrimination (13C expressed in notation) was computed as follows, assuming the isotopic composition of atmospheric air (13Ca) to be –8 (Farquhar et al., 1989):
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Experiment 2 (2002)
Six genotypes contrasting for WUE were selected from Exp. 1. Water use efficiency and other related traits were determined gravimetrically as described above. Leaf samples were separately harvested, oven dried, and used for carbon isotope ratio measurements.
Statistical Analysis
The data of gravimetry as well as carbon isotope ratios were analyzed for their statistical significance among the genotypes following a completely randomized design by MSTATC software (Anonymous, 1989).
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RESULTS AND DISCUSSION
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The total biomass and the cumulative water transpired by 34 genotypes of rice during the 30 d of peak vegetative growth was monitored gravimetrically and a significant genotypic variability was noticed in both the traits (Table 1). A strong positive correlation was noticed between total transpiration and total biomass (r = 0.77; p < 0.01; n = 34). The evaporation of water during transpiration as well as the CO2 entry for photosynthesis are controlled by stomatal diffusive factors, and hence total biomass production is often strongly linked to crop canopy transpiration (Angus and Harwaarden, 2001; Richards et al., 2002). Furthermore, a significant genotypic variability in the ratio of the total biomass to total transpiration (WUE) was also noticed (Table 1). As per the Passioura's yield model the two important physiological traits that determine total biomass are total transpiration and WUE (Passioura, 1986). Thus, attempts can be made to exploit this significant variability in WUE to improve total biomass.
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Table 1. Genotypic variation in total biomass (BM), cumulative water transpired (CWT), water use efficiency (WUE), and carbon isotope discrimination (13C) among 34 genotypes of rice of Exp. 1 (2001).
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Among several approaches, carbon isotope discrimination (13C) is the best-suited technique for the rapid assessment of the genetic variability in WUE. 13C varied significantly between 18.7 to 21.6 (Table 1). The rice germplasm accessions revealed a significant inverse relationship between 13C and WUE (Fig. 1) , which is consistent with the theoretical predictions (Farquhar et al., 1989). Such a relationship has been confirmed in several crop plants (Richards et al., 2002; Sheshshayee et al., 2003). Dingkuhn et al., (1991) and Peng et al., (1998) observed similar inverse relationships between 13C and WUE among indica and japonica rice varieties. In these studies, WUE was determined by the gas exchange approach. Although the ratio of assimilation to transpiration rates at single leaf level is a fairly good estimate of intrinsic WUE (Condon et al., 2002), gas exchange is a snapshot measurement and suffers because of its inability to detect diurnal and day-to-day variations. This important shortcoming is effectively overcome in the gravimetric approach that integrates the diurnal variations in transpiration (T) and photosynthesis, thus providing a more reliable and accurate measure of WUE. Our study offers the proof for the first time in rice that 13C is strongly related to the WUE measured at the whole plant level.
It is well known that WUE is a multigene controlled trait and is highly modulated by the prevailing environmental conditions (Martin et al., 1989). To develop "trait-based" breeding programs to exploit WUE, it becomes essential to initially assess the genetic stability of this parameter. Toward this end, six contrasting genotypes were selected, and their WUE was determined in a separate experiment. The selected genotypes varied significantly for total biomass, CWT, and WUE (Table 2). 13C consistently showed an inverse relationship with WUE (Fig. 2)
. Though both the experiments were conducted during the same months of two consecutive years, the range in the WUE values was smaller for Exp. 2. The leaf to air vapor pressure difference (VPD) was 2.4 Pa during April 2002 (Exp. 2), while it was 1.9 Pa during the same month in 2001 (Exp. 1). Because of a higher driving force for transpiration, the average CWT was higher for the Exp. 2. It is well known that the stomata close in response to increased VPD (El-Sharkaway et al., 1984). The reduced stomatal conductance would constrain the diffusion of CO2 for photosynthesis, and accordingly the average total biomass in the second experiment was significantly lower than that of the corresponding genotypes in the first experiment.
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Table 2. Genotypic variations in total biomass (BM), cumulative water transpired (CWT), water use efficiency (WUE), and carbon isotope discrimination (13C) among six selected genotypes of rice.
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Despite the environmental modulation of WUE, a strong positive regression of WUE values (Fig. 3a)
indicates a low G x E for this trait. A positive relationship between the 13C values of the two experiments further confirms this inference (Fig. 3b). Recently, Nadaradjan (2004) measured 13C in a mapping population of rice (CT9993 x IR62266) and found that 13C values of irrigated and nonirrigated plants were strongly related (r = 0.64; p < 0.01; n = 39), indicating a low G x E interaction for WUE in rice. A similar low G x E has also been reported in several other crop species (Hebbar et al., 1994; Ismail and Hall, 1992, 1993; Ismail et al., 1994; Ashok et al., 1999). These results indicate that WUE is a stable parameter, which is an essential criterion for exploiting this trait in breeding programs for crop improvement.
To achieve guaranteed success in breeding for WUE, it is essential to assess the intrinsic physiological factors that determine the observed differences in this trait. Water use efficiency can be increased either through a reduction in transpiration rate or an enhancement in the photosynthetic carbon assimilatory capacity. If increase in WUE is associated with reduced transpiration, such genotypes are often referred to as conductance types. On the other hand, if the variation in photosynthetic rate determines WUE, such genotypes can be categorized as capacity types (Farquhar and Lloyd, 1993; Udayakumar et al., 1998a; Scheidegger et al., 2000; Sheshshayee et al., 2003). Plants have naturally evolved to increase WUE through a reduction in transpiration associated with stomatal closure as a water conservation strategy. However, because of a strong link between transpiration and CO2 diffusion for photosynthesis, selection for high WUE among such conductance types would become counter productive.
To ascertain such controlling mechanisms in rice, the relationship of WUE with both mean transpiration rate and net assimilation rate (NAR) were examined. A significant inverse relationship was noticed between WUE and mean transpiration rate (Fig. 4)
, while no such association was found between WUE and NAR (Fig. 5) . This indicates that stomatal control of WUE predominates over that by carbon assimilation capacity among the rice genotypes examined in this study. Though selection for higher WUE from conductance type would normally associate with a reduced total biomass (Udayakumar et al., 1998a; Sheshshayee et al., 2003), genotypes such as IET16364 that had high total biomass coupled with a relatively high WUE could still be identified (Table 3). Such genotypes can serve as potential donors for further crop improvement.
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Fig. 5. Relationship between water use efficiency (WUE) and net assimilation rate (NAR) among 34 genotypes of rice (r = 0.14; n = 34, not significant)
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Table 3. Total biomass (BM), water use efficiency (WUE), net assimilation rate (NAR), mean transpiration rate (MTR), and carbon isotope discrimination (13C) of six selected genotypes of rice.
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ACKNOWLEDGMENTS
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Authors gratefully acknowledge the research grant provided by the Department of Science & Technology and Department of Biotechnology, Government of India (SP/IO/LF/01/98 and BT/IS/06/004/98). We also thank Prof. Udayakumar and Prof. Prasad, Department of Crop Physiology for the comments during the preparation of the manuscript and Mr. Nagabhushana, and Mr. Ramesha for the help during stable isotope analysis.
Received for publication February 5, 2005.
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ABSTRACT
INTRODUCTION
= IET 15963; 
= MRB-1; 
= IET 16364; 
= MRB-2).
