Plant Soil (2010) 335:349361 DOI 10.

1007/s11104-010-0423-1

REGULAR ARTICLE

Physiological and morphological traits related to water use by three rice (Oryza sativa L.) genotypes grown under aerobic rice systems
Naoki Matsuo & Kiyoshi Ozawa & Toshihiro Mochizuki

Received: 2 August 2009 / Accepted: 4 May 2010 / Published online: 4 June 2010 # Springer Science+Business Media B.V. 2010

Abstract We compared the plant growth, stomatal conductance (gs), leaf water content (LWC), and root length density (RLD) of Oryza sativa L. ssp. japonica cv. Sensho (traditional upland), ssp. indica cv. Beodien (traditional upland), and ssp. japonica cv. Koshihikari (improved lowland) under two aerobic rice systems [well-irrigated (WI) and water-saving (WS) treatments]. Irrigation water was applied every

Responsible Editor: Len Wade. N. Matsuo (*) Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 111 Harumachi, Kasuya-cho, Kasuya-gun, Fukuoka 811-2307, Japan e-mail: matsuon@affrc.go.jp K. Ozawa Japan International Research Center for Agricultural Sciences, 1091-1, Maezato-Kawarabaru, Ishigaki, Okinawa 907-0002, Japan T. Mochizuki Faculty of Agriculture, Kyushu University, 111 Harumachi, Kasuya-cho, Kasuya-gun, Fukuoka 811-2307, Japan Present address: N. Matsuo Lowland Crop Rotation Research Team, National Agricultural Research Center for Kyushu Okinawa Region, 496 Izumi, Chikugo, Fukuoka 833-0041, Japan

2 days from 21 to 68 days after sowing (DAS) and everyday thereafter in WI treatment and it was applied when soil water potential at 15 cm depth reached 15 kPa from 21 to 68 DAS and every 2 days thereafter in WS treatment to impose repetitive water stress. WS treatment used 35% less water than WI. Leaf area index (LAI) and shoot dry weight (SDW) were the lowest for Koshihikari in both treatments and the ratio of LAI and SDW in WS treatment to that in WI treatment was the lowest in Koshihikari. This indicates that aerobic cultivation was not suitable for Koshihikari even under well-irrigated conditions and that the effect of repetitive water stress was the most serious in Koshihikari. Midday gs of Sensho and Beodien in WS treatment were affected by irrigation, whereas that of Koshihikari was low and stable. LWC of Koshihikari was smaller than those of upland genotypes in both treatments. LWC of upland genotypes in WI and WS reached maximum and minimum values at predawn and evening, respectively, and recovered at night, but LWC of Koshihikari in WS treatment did not recover at night. RLD of upland genotypes was higher than that of Koshihikari, but no significant differences were observed among treatments. These results indicate that genotypic difference of physiological traits under aerobic conditions (both WI and WS) was caused by the genotypic difference of water uptake capacity, which can be partly caused by the RLD. In Koshihikari, however, the LWC difference between treatments can not be explained only by the RLD. Further studies will be needed to

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clarify physiological mechanism responsible for the water uptake capacity of roots in aerobic rice systems. Keywords Aerobic rice system . Lowland rice . Root systems . Stomatal conductance . Upland rice . Water-saving cultivation Abbreviations AWD alternate wetting and drying system DAS days after sowing gs stomatal conductance L0 root hydraulic conductance LA leaf area LAI leaf area index Lpr root hydraulic conductivity LWC leaf water content LWP leaf water potential RLD root length density RWD root weight density SDW shoot dry weight SMC volumetric soil moisture content SWP soil water potential WI well-irrigated WS water-saving WUE water-use efficiency

Introduction Rice is one of the worlds main staple crops and provides the necessary daily calories for millions of people (Kush 1997). More than 90% of the worlds rice is produced and consumed in Asia (FAO 1997), and rice production must be increased by an estimated 56% over the next 30 years to keep up with population growth and income-induced demand for food in most Asian countries (Hossain 1997), where about 75% of total rice production comes from irrigated lowlands (Maclean et al. 2002). Irrigated rice accounts for about 50% of the total amount of water diverted for irrigation, which itself accounts for 80% of the fresh water diverted (Guerra et al. 1998). However, an increasing scarcity of fresh water threatens the sustainability of irrigated rice ecosystems (Guerra et al. 1998; Tuong and Bouman 2003): this problem has been caused by population growth, increasing urban and industrial development, and decreasing availability of usable water due to pollu-

tion and resource depletion (Bouman and Tuong 2001). Therefore, the development of new rice cultivation techniques and cultivars are required to reduce water consumption in rice production systems. Various field techniques to save irrigation water have been explored. They include direct seeding, keeping the soil saturated, and alternate wetting and drying systems (AWD) in lowland fields. Bouman and Tuong (2001) concluded that, compared with continuously flooded conditions, small yield reductions (0 to 6%) occurred under saturated conditions, but larger reductions (10 to 40%) occurred under AWD, when soil water potential (SWP) during dry phase reached values between 10 and 40 kPa. A new water-saving technology is called aerobic rice system (Bouman 2001; Bouman et al. 2005). In aerobic rice systems, fields remain unsaturated throughout the growing season, as in wheat or maize cultivation. Water can be supplied by surface irrigation (e.g. flush or furrow irrigation) or by sprinklers, but in both cases, the goal is to keep the soil wet but not flooded or saturated. Aerobic rice systems could reduce water inputs by 11.5 to 50.7% in the Philippines (Belder et al. 2005; Bouman et al. 2005), by 29.2 to 65.3% in northern China (Bouman et al. 2006; Yang et al. 2005), and by 62.5 to 70.8% in Japan (Matsuo and Mochizuki 2009) compared with flooded paddy conditions. Matsuo and Mochizuki (2009) showed that aerobic rice systems could save more than 47% of irrigation water in comparison with AWD. Thus, aerobic rice systems have the potential to reduce irrigation requirements more than other techniques that have been developed. In practice, irrigation is applied to bring the soil water content up to field capacity after the water potential has reached a certain lower threshold, such as 15 or 30 kPa at a depth of 15 cm (Bouman et al. 2005; Matsuo and Mochizuki 2009). As a result, the soil in aerobic rice systems undergoes repetitive cycles of wet conditions and relatively mild drought stress. The most important concern is to save as much water as possible while maintaining yields at 70 to 80% of the yield for high-input flooded rice (Belder et al. 2005). Numerous reports have focused on drought resistance in rice under temporary or long-term water deficits (Cooper 1999; Fukai and Cooper 1995; Jackson et al. 1996; Lafitte et al. 2003; Ludlow and Muchow 1990; Turner 1986), and many traits that potentially contribute to drought resistance have been reviewed (Cooper 1999;

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Fischer et al. 2003; Fukai and Cooper 1995; Kamoshita et al. 2008; Lafitte et al. 2003; Nguyen et al. 1997; Price and Courtois 1999). However, the soil moisture conditions under aerobic rice systems should differ from those under temporary or long-term water deficits (i.e. alternately wet conditions and mild water stress vs. temporary mild or severe water stress). Despite this difference, many studies of aerobic rice systems have focused mainly on comparing agronomic traits such as shoot growth, yield, and yield components of aerobic rice with those observed under flooded paddy conditions (Bouman et al. 2005; Peng et al. 2006; Yang et al. 2005). This may be because the concept of aerobic rice is quite new and the difference in soil water conditions between aerobic rice system and temporary water stress conditions is not yet appreciated. Furthermore, there are no studies which have compared the agronomical, morphological and physiological traits between the two situations. This confusion may delay the development of a unified understanding of important traits for aerobic rice systems. Bouman et al. (2006) investigated root traits such as root length density (RLD), root weight density (RWD), and rooting depth and pattern for two aerobic rice genotypes and one lowland genotype under aerobic rice systems. They found no significant difference in root traits among the three genotypes, although the yields of the aerobic rice genotypes were higher than that of the lowland genotype. Matsuo et al. (2009) reported that repetitive water stress reduced the root hydraulic conductance (L0) more in a lowland rice genotype than it did in two upland rice genotypes. Matsuo and Mochizuki (2009) investigated the predawn leaf water potential (LWP) and bleeding rate of these three genotypes under aerobic rice systems and reported that these two parameters of a lowland genotype under aerobic rice systems were significantly lower than those under a flooded paddy cultivation, while no significant difference were observed among cultivation methods in two upland genotypes. Numerous studies proposed the importance of water-related traits, such as root traits (e.g. deep root and RLD), LWP and plant water conductivity in upland rice cultivations with or without drought (reviewed by Kamoshita et al. 2008). Because aerobic rice systems are partly similar cultivation methods with upland rice cultivation, these traits may also play important roles in aerobic rice systems. However, few studies verifies the importance of those traits under aerobic rice systems.

The aim of the present study was to analyze genotypic differences in morphological and physiological traits related to plant water status, such as leaf water content (LWC), stomatal conductance (gs), and RLD, by comparing two upland rice genotypes and one lowland rice genotype whose growth and yield responses to aerobic rice systems were previously shown to differ (Matsuo and Mochizuki 2009), under two aerobic rice systems.

Materials and methods Plant materials We used three rice (Oryza sativa L.) genotypes in this study: a traditional japonica upland genotype, Sensho; a traditional indica upland genotype, Beodien; and an improved lowland japonica genotype, Koshihikari. Sensho and Beodien can grow equally well under aerobic rice systems and flooded paddy conditions and their yields under aerobic rice systems are almost same as those under flooded paddy conditions. In contrast, Koshihikari cannot grow well under aerobic rice systems and its grain yield under aerobic rice systems decreases by 80% compared with that under flooded paddy conditions (Matsuo and Mochizuki 2009). Experimental design The lysimeter experiment was carried out in 2007 in a plastic greenhouse at the Japan International Research Center for Agricultural Sciences (JIRCAS), Tropical Agriculture Research Front, Okinawa, Japan. The soil contained 15.9% clay, 10.4% silt and 73.7% sand (i.e. it was a sandy clay loam). Its bulk density was 1.46 g cm3, its volumetric water content at field capacity (moisture remaining 24 h after irrigation) was 36.0%, and the permanent wilting point (1.5 MPa) occurred at 7.0% moisture content. We applied two water regimes [the well-irrigated (WI) and water-saving (WS) treatments] in a randomized complete block design with two replications under aerobic soil conditions. About 1 year before sowing the seeds, we installed cellulose acetate butyrate minirhizotron observation tubes (180 cm long by 5 cm in diameter; Bartz Technol. Co., Santa Barbara, CA, USA) in the central row of each replication in WS treatment at an angle of 45 from the vertical. A 90-cm section of

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the tube projected above the soil surface, and we wrapped it with aluminum foil and capped it with a rubber stopper. Culture details On 24 April, we sowed groups of three to four pregerminated seeds at a spacing of 3015 cm in a bottomless soil-filled container (180-cm length, 90-cm width, and 90-cm depth) buried in the soil. Seedlings were thinned to one plant per hill at 21 days after sowing (DAS). We supplied 13 mm of irrigation daily in both treatments until 21 DAS using line-source sprinklers. We then implemented the two water treatments. In WI treatment, we supplied 10 mm of irrigation water every 2 days from 22 to 67 DAS and everyday after 68 DAS not to impose water stress on plants. In WS treatment, we supplied 10 mm of irrigation water whenever the soil water potential (SWP) at a 15-cm depth reached 15 kPa from 22 to 67 DAS and every 2 days after 68 DAS to impose repetitive water stress on plants, which was often observed in aerobic rice systems (e.g. Yang et al. 2005). In this treatment, SWP was measured using tensiometers (as described in the next section). The decision to reinitiate irrigation in WS treatment therefore varied among the plots. We supplied chemical fertilizers at a rate of 4 g N m2, 12 g P m2, and 12 g K m2 as basal dressings 1 day before sowing, and then applied N fertilizer at a rate of 4 g m2 at 28 and 53 DAS as top dressings. Measurements and calculations The JIRCAS weather station recorded daily mean, maximum, and minimum temperatures and solar radiation. The soil temperature was monitored from 69 to 76 DAS at a depth of 15 cm from the soil surface. SWP was monitored from 28 DAS using tensiometers (DIK-3126, Daiki Rika Kogyo Co., Ltd, Saitama, Japan) installed at depths of 15, 22.5, 30, and 50 cm below the soil surface in WS treatment. It was only monitored at a depth of 15 cm in WI treatment. SWP was measured around 17:00 h every day and then irrigation water was applied whenever SWP at a depth of 15 cm decreased below 15 kPa. Before beginning our experiment, we determined the relationship between SWP and volumetric soil moisture content (SMC) (data not shown) and the results

of calculated SMC were shown. Because the tensiometers could not read the correct SWP values for Sensho in WS treatment at 65 DAS, we determined SMC gravimetrically thereafter. We measured the leaf area (LA) and leaf length of each genotype at 40 and 78 DAS. LA was measured with an AAM-8 leaf area meter (Hayashi Denko Co. Ltd., Tokyo, Japan). We performed simple curve linear regression to determine the relationship between leaf length and LA for each genotype. The correlation coefficients of the equations was significant (P<0.001) in all three genotypes, indicating good relationship between leaf length and LA. We measured the lengths of all green leaves, except dead leaves, in six medium-sized plants in each replication weekly from 36 DAS, and determined the LA of each plant by summing the individual LA values, which we calculated using the fitted equation described above. We then calculated the leaf area index (LAI). We obtained midday (11:0013:00 h) measurements of stomatal conductance (gs) from 69 to 76 DAS using an SC-1 leaf porometer (Decagon Devices Inc., Pullman, WA, USA). Growth stage of Sensho, Beodien and Koshihikari during this period was booting to heading, middle to late panicle development and booting to heading, respectively. We also recorded gs and LWC values from 0:00 to 24:00 h at intervals of 3 h at 75 DAS. We used the uppermost fully expanded leaves of the main culms of the six plants that were used for plant growth measurements for the midday gs and the daily change of gs measurements. For the LWC measurements, we excised leaf sections of 510 cm from the leaf tips of the uppermost fully expanded leaves of main culms or primary tillers that were not used for the gs measurements. We collected and stored the leaf sections in a cool and dark box, and transported the box immediately to a nearby laboratory so that the fresh weight could be measured with an electronic balance with an accuracy of 0.1 mg. We then determined the dry weight after oven-drying at 70C for at least 3 days. LWC was calculated as: LWC FW DW=FW 100% 1

where FW and DW represent the fresh weight and dry weight, respectively. We used four leaves for the LWC measurement in each replication. Simultaneously with the LWC measurements, we investigated the

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relationship between LWC and leaf water potential (LWP) that is often used as plant water status under water stress conditions. The sampling procedure was the same as in the LWC measurements, except that the length of the leaf sample was 10 cm (at a distance of 515 cm from the leaf tip). Excised leaves were subdivided into 2-cm pieces and placed into sample cups, then LWP was measured with a WP4 dewpoint psychrometer (Decagon Devices Inc.). LWC was then determined as described above. We performed simple linear regression to determine the relationship between LWC and LWP within each water treatment (WI vs. WS) and genotype. We used one leaf section from each replication at each sampling time. The correlation coefficients of the equations were significant (P<0.05), indicating that the LWP could be estimated from the LWC, instead of measuring LWP directly in this study. We harvested the aboveground biomass of the six plants used for the plant growth and gs measurements at the soil surface at the end of the experiment (79 DAS) and measured shoot dry weight (SDW) after oven-drying at 70C for at least 3 days. We also calculated water-use efficiency (WUE, g SDW kg1 input water) for each combination of genotype and treatment by dividing SDW by the total irrigation supplied during the study period. We observed the roots in WS treatment using a minirhizotron video camera system (Bartz Technol. Co.) at 53 and 78 DAS to observe root growth nondestructively. Scanned images (13.5 mm long by 19.6 mm wide) from the soil surface to the deepest layer were recorded by means of a personal computer and then root depth was analyzed. At the end of the experiment (79 DAS), we destructively sampled the root systems between hills from the soil surface to a 50-cm depth using stainless-steel sampling cores with a volume of 100 cm3 (50 mm inside diameter, 51 mm height; DIK-1801, Daiki Rika Kogyo, Co. Ltd.) at three positions per replication. We carefully washed soil away from the root systems using tap water, and then photographed the roots with a D70s digital camera (Nikon Co. Ltd., Tokyo, Japan). We measured the root length using Ver. 1.62 of the NIH Image software (http://rsb.info.nih.gov/nih-image/). Using this data, we converted the root length into the root length density (RLD, cm cm3). We determined root dry weights after oven-drying at 70C for at least 3 days and used these results to calculate root weight density (RWD).

Statistical analysis Effects of genotype and irrigation regime and the interaction between them on SDW and WUE were examined by a two-way analysis of variance (ANOVA). For LAI values, one-way ANOVA was conducted to analyze whether there were significant differences among means or not at each measurement time. If significant differences were observed, LSD test at 5% level was performed to separate the means. All statistical analyses were performed by using Unistat v. 5.6 software (Unistat Ltd., London, UK).

Results Weather Air temperature and solar radiation increased throughout the study period (Table 1). Mean air temperature, mean maximum air temperature, mean minimum air temperature, and solar radiation increased from 22.0 to 29.6C, from 24.3 to 32.2C, from 19.7 to 27.3C, and from 12.0 to 22.4 MJ m2 d1, respectively. Soil conditions We supplied a total of 327 mm of irrigation water in WI treatment for all genotypes. Less water was supplied in WS treatment: 227 mm in Sensho, 207 mm in Beodien, and 202 mm in Koshihikari. On average, this represents a 35% reduction in the total water input in WS treatment. Figure 1 shows the SMC values for the three genotypes in WS treatment at depths of 15, 22.5, 30, and 50 cm below the soil surface from 28 to 68 DAS. SMC responded directly to irrigation (i.e. increased after each irrigation) at a depth of 1530 cm, but not at a depth of 50 cm. In Sensho, SMC at a depth of 50 cm decreased gradually after the start of the measurements. Although the same tendency was observed in Beodien, the magnitude of the decrease was lower. In Koshihikari, SMC at a depth of 50 cm changed little during the experiment. Figure 2 shows SMC only at a depth of 15 cm in WS treatment from 69 to 76 DAS (during the gs measurements), because SMC at the other three depths (22.5, 30 and 50 cm) followed similar trend to that at 15 cm depth. Irrigation water was applied daily in WI treatment

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Table 1 Monthly mean air temperature, maximum air temperature, minimum air temperature, and solar radiation Mean temp. (C) April May June July 22.0 24.9 28.0 29.6 Mean maximum temp. (C) 24.3 27.7 30.5 32.2 Mean minimum temp. (C) 19.7 22.0 26.0 27.3 Solar rad. (MJ m2 day1) 12.0 17.6 18.8 22.4

and every 2 days in WS treatment during this period. SMC was highest in Koshihikari at all depths, followed by Beodien. SMC at a depth of 15 cm in WI treatment averaged 28.12.5% (meanSE) and 32.81.6% before and after 69 DAS, respectively
15 cm 30 cm 22.5 cm 50 cm

50 40 SMC (% v/v) 30 20 10 0 50 40 SMC (% v/v) 30 20 10 0 50 SMC (% v/v) 40 30 20 10 0 28

(data not shown). Figure 3 shows soil temperature of Sensho at a depth of 15 cm in both water treatments from 69 to 76 DAS. The soil temperature in WI treatment fluctuated between the same ranges of temperatures during this period. Although the soil temperature in WS treatment was the same as that in WI 1 day after irrigation, it increased to a higher daytime value 2 days after irrigation. The cumulative difference in soil temperature between WI and WS treatment totaled 34.7C on hourly basis 2 days after irrigation. The same trend was observed in the other genotypes (data not shown). Leaf and shoot growth Figure 4 shows LAI growth for the three genotypes in the two water treatments. Sensho showed faster LAI development than the other genotypes in both water treatments. The LAI of Beodien was the highest at 79 DAS, but did not differ significantly from that of Sensho. The LAI of Koshihikari was significantly lower than those of the other two genotypes at all
50 40 SMC (% v/v) 30 20 10 Sensho Beodien Koshihikari

(a)

(b)

(c)
36 44 52 60 68

0 69 70 71 72 73 DAS (day) 74 75 76

DAS (day)

Fig. 1 Soil moisture content (SMC) of the three genotypes in the water-saving treatment at depths of 15, 22.5, 30, and 50 cm from 28 to 68 DAS. (a) Sensho; (b) Beodien; (c) Koshihikari. Data for one replication are shown, because the timing of irrigation differed among the plots. SMC at a depth of 15 cm in the well-irrigated treatment during this period averaged 28.1 2.5% (meanSE)

Fig. 2 Soil moisture content (SMC) of the three genotypes in the water-saving treatment at a depth of 15 cm from 69 to 76 DAS. Because similar trends were observed at the other depths, SMC at a depth of only 15 cm is shown. Data are meansSE. SMC in the well-irrigated treatment at a depth of 15 cm during this period averaged 32.81.6% (meanSE)

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37 Soil temperature (oC) 35 33 31 29 27 68 69 70 71 72 73 74 75 76 77 DAS (day) Well-irrigated Water-saving

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WUE of Sensho was the highest, followed by Beodien in both water treatments. WUE of Sensho and Beodien were 20% or more higher in WS treatment than in WI treatment, whereas that of Koshihikari in WS treatment was only 86% of that in WI treatment. A two-way ANOVA revealed a significant genotypic effect on WUE. Stomatal conductance and leaf water content Figure 5 shows the midday gs values for the three genotypes from 69 to 76 DAS. The gs values of Sensho and Beodien were similar in WI treatment (about from 500 to 700 mmol m2 s1) and both were higher than those of Koshihikari (about from 400 to 550 mmol m2 s1). The gs values for Sensho and Beodien were strongly affected by irrigation in WS treatment: they were between 260 and 400 mmol m2 s1 1 day after irrigation and decreased to less than 170 mmol m2 s1 2 days after irrigation. The gs values for Koshihikari were relatively stable at 200300 mmol m2 s1 in WS treatment, regardless of irrigation. Figure 6 shows the daily changes in gs and LWC at 75 DAS. The gs values did not differ (less than 100 mmol m2 s1) among the water treatments and genotypes at night (from 0:00 to 6:00 h and from 21:00 to 24:00 h). The daytime (from 9:00 to 18:00 h) gs values of Sensho in WI treatment tended to be higher, followed by Beodien, and Koshihikari in this order. The daytime gs values of Sensho and Beodien averaged about 50% lower in WS treatment than in WI treatment. In Koshihikari, however, the daytime gs values in WS treatment was on average 21% lower than those in WI treatment. The LWC values were slightly lower (though almost insignificant) in WS treatment than in WI treatment throughout the day in all three genotypes and the LWC values in WS treatment were 1.5 to 6.9% lower than those in WI treatment. The LWC values of Sensho and Beodien were similar, and tended to be higher than those of Koshihikari in both water treatments. LWC reached its maximum and minimum values at 3:00 to 6:00 h and 15:00 to 18:00 h, respectively, in each water treatment and genotype, with one exception: LWC of Koshihikari in WS treatment reached its maximum at 3:00 h, but did not recover at night (after 18:00 h), and the LWC value at 24:00 h in WS treatment was significantly (P< 0.05) lower than that in WI treatment.

Fig. 3 Soil temperature of Sensho in the well-irrigated and the water-saving treatments at a depth of 15 cm from 69 to 77 DAS. Arrows show the timing of irrigation in the water-saving treatment. During this period, irrigation was applied daily in the well-irrigated treatment

times in both water treatments. LAI tended to be higher in WI treatment than in WS treatment until 65 DAS in Sensho and 72 DAS in Beodien, but thereafter the values did not differ between treatments. The LAI of Koshihikari was the same in both treatments until 65 DAS, but thereafter LAI was significantly higher in WI treatment, reaching nearly twice that in WS treatment by the end of the experiment. Table 2 shows the SDW and WUE values for the three genotypes at the end of the experiment. Sensho had the highest SDW and WUE in both water treatments, followed by Beodien. The SDW values for Sensho and Beodien in WS treatment were approximately 80% of those in WI treatment, whereas that of Koshihikari in WS treatment was only 54% of that in WI treatment. A two-way ANOVA detected the significant effects of genotype and water treatments on SDW, but their interaction was not detected.
5 4 3 2 1 0 36 44 51 58 DAS (day) 65 72 79
Sensho WI Beodien WI Koshihikari WI Sensho WS Beodien WS Koshihikari WS

Fig. 4 Transition of leaf area index (LAI) of the three genotypes in the well-irrigated (WI) and the water-saving (WS) treatments. Data are meansSE. Vertical bars are LSD at the 5% level

LAI

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Table 2 Shoot dry weight (SDW) and water-use efficiency (WUE) of the three genotypes in the well-irrigated and water-saving treatments at the end of the experiment (79 DAS) SDW (g m2) Well-irrigated Sensho Beodien Koshihikari ANOVA Genotype Water GW ns not significant
a

WUE (g m2 SDW kg1 water) Water-saving 48449 (83)a 3318 (79) 1788 (54) Well-irrigated 1.160.06 0.800.03 0.630.11 *** ns ns Water-saving 1.320.13 (120) 1.040.16 (127) 0.530.03 (86)

58233 42115 32742 *** *** ns

Values in parenthesis indicate the ratios of SDW and WUE in water-saving treatment to those in well-irrigated treatment

***P<0.001

Root traits In WS treatment, the root systems of Sensho and Beodien reached a depth of 80 cm by the end of the experiment, whereas root systems of Koshihikari were no longer present below 60 cm (data not shown). Figure 7 shows the RLD at 79 DAS in both water treatments. RLD values of Sensho and Beodien tended to be higher in WI treatment than in WS treatment, whereas the values did not differ between the water treatments in Koshihikari. A genotypic difference of RLD was observed in the shallow soil layers: RLD was highest for Beodien, followed by Sensho, in both water treatments. The same result was observed for RWD as for RLD (data not shown).

Discussion The LAI of Koshihikari was significantly lower than those of Sensho and Beodien in both water treatments (Fig. 4) and SDW of Koshihikari was tended to be lower than those of Sensho and Beodien (Table 2). The difference in plant growth among these three genotypes was observed at the early growth stage (from 36 DAS), as was previously reported (Matsuo and Mochizuki 2009). These results confirm that Koshihikari cannot well adapt to aerobic rice systems, even though it was grown under relatively wet aerobic conditions (i.e. WI treatment). This means that even

WI treatment was stressful for Koshihikari. Further the genotypic differences of plant growth in response to the amount of water input was also detected: significant difference of LAI between WI and WS treatments was observed in Koshihikari, but not in Sensho and Beodien. The WUE values for Sensho and Beodien in WS treatment were more than 20% higher than in WI treatment, whereas that in Koshihikari were only 86% of the values in WI treatment. The SMC of Sensho at a depth of 50 cm in WS treatment showed the fastest decrease (Fig. 1), probably due to the cultivars fast leaf growth (Fig. 4). Fast shoot growth during the early growth stage may be a desirable trait under aerobic rice systems from the viewpoint of competition between rice plants and weeds. Root systems of Sensho, Beodien, and Koshihikari had reached a depth of 80, 80, and 60 cm, respectively at 78 DAS (data not shown). The SMC at a depth of 50 cm in Koshihikari did not change throughout the experiment (Fig. 1), indicating that the root system of Koshihikari could not extract water at this depth, even though its roots had reached a depth of 60 cm by 78 DAS. We also observed genotypic differences in RLD (Fig. 7). Beodien had the highest RLD at shallow depths at the end of the experiment, followed by Sensho, but more than 70% of the total RLD was found in the top 25 cm of the soil in all three genotypes. Genotypic differences in RLD and root depth might have caused differences in the water uptake patterns among the genotypes. It was reported that Sensho and Beodien had thicker root than

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Well-irrigated

357

800
gs (mmol m -2s -1)

(a)

Water-saving

600 400 200 0 800

(b)

Well-irrigated Water-saving

600 400 200 0 800 600 400 200 0

(c)

Well-irrigated Water-saving

69

70

71

72 73 DAS (day)

74

75

76

Fig. 5 Midday stomatal conductance (gs) of the three genotypes in the well-irrigated and the water-saving treatments from 69 to 76 DAS. Arrows show the timing of irrigation in the water-saving treatment. During this period, irrigation was applied daily in the well-irrigated treatment. Data are means SE. (a) Sensho; (b) Beodien; (c) Koshihikari

Koshihikari in pot and hydroponic culture experiments (Matsuo et al. 2009). It is well known that genotypes with deeper, larger, thicker root can extract larger amount of soil water and maintain higher plant water status under upland conditions. (e.g. Lilley and Fukai 1994; Yoshida and Hasegawa 1982). Furthermore, Babu et al. (2003) demonstrated that root traits, such as root thickness and deep root weight measured in the glasshouse experiments were positively correlated with yield and yield components under upland condition with severe drought stress at prefloweing stage.

Because aerobic rice systems are partially or fully similar with irrigated upland or favorable rainfed upland conditions, deeper, larger and thicker root systems should be desirable traits under aerobic rice systems. Bouman et al. (2006), however, reported no significant genotypic differences in RLD, root depth, and root distribution between aerobic rice varieties (cv. Han Dao 297 and Han Dao 502) and a lowland variety (cv. Jin Dao 305) under aerobic rice systems, although the yields of the aerobic rice varieties were higher. Further detailed studies on root morphologies under aerobic rice systems will be needed to clarify the relationship between root traits and plant growth and/or yield. We observed a clear genotypic difference of midday gs in both water treatments (Fig. 5). Midday gs in WI treatment was higher in Sensho and Beodien than in Koshihikari throughout the measurement period, even though irrigation was applied everyday during this period. This result confirms again that even WI treatment was stressful for Koshihihkari. In WS treatment, midday gs values of Sensho and Beodien fluctuated greatly in response to irrigation, whereas those of Koshihikari remained low and stable regardless of irrigation. Smaller plant growth of Koshihikari in both WI and WS might be caused by the lower photosynthetic rate, resulting from lower gs. Stiller et al. (2003) demonstrated that under an upland condition with drought stress of 7 days, photosynthetic rate was limited by stomatal closure to some extent and photosynthetic rate of a lowland genotype was lower than that of an upland genotype. Their results may partially support this consideration. In the case of upland genotypes, when there is ample soil water (1 day after irrigation), they can open their stomata more fully, thereby increasing photosynthesis. But when water is insufficient, they close their stomata probably to prevent the development of excessive water stress. It may be possible that upland genotypes exhausted soil water within 1 day after irrigation and water uptake by roots did not meet the transpiration demand from their leaves at 2 days after irrigation. Lower SMC in upland genotypes than that in Koshihikari (Fig. 2) may support this consideration. The gs values of upland genotypes increased 1 day after irrigation and this phenomenon was observed at each irrigation. From these results, it can be conclude that upland genotypes used in this study can acclimatize to the fluctuation of soil water condition from the

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Fig. 6 Daily change in stomatal conductance (gs) and leaf water content (LWC) of the three genotypes in the well-irrigated and the water-saving treatments at 75 DAS. Data are meansSE. (a) and (b) Sensho; (c) and (d) Beodien; (e) and (f) Koshihikari

viewpoint of stomatal behavior. Although there are numerous studies on upland rice cultivations, most studies focus on the morphological and physiological traits under only one cycle of drought stress followed by recovery. However, rice plants under aerobic rice systems undergo several cycles of wetting and drying conditions (Matsuo and Mochizuki 2009; Yang et al. 2005). The only direct reference of acclimation of rice plants to the several cycles of drought stress was in the study of Srivalli et al. (2003). They imposed three cycles of water stress (for 10, 13 and 16 days) followed by recovery of 2 days on a pot-grown upland rice genotype (cv. Tulsi) and observed that leaf water status (i.e. LWP and relative water content) at the end of each water stress was lower than those of well-watered control plants, but this genotype could recover fully in terms of leaf water status after

irrigation. It is reported that there is a significant positive linear relationship between LWP and gs in an upland rice (cv. CT9993; Siopongco et al. 2009). These results support that upland genotypes have the ability to acclimatize to the fluctuant soil water conditions. Because only one upland genotype was used in the study of Srivalli et al. (2003), it is unclear whether genotypic difference of acclimation ability exist or not among rice genotypes. Further studies on genotypic difference of this ability will help to clarify acclimation mechanism to several cycles of wetting and drying conditions We also detected genotypic differences in the daily change in LWC values in response to the two water regimes (Fig. 6). We analyzed the relationship between LWC and LWP and confirmed significant (p<0.05) positive correlation (data not shown). LWC

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Fig. 7 Root length density (RLD) of the three genotypes in the well-irrigated and the water-saving treatments at 79 DAS. (a) Sensho; (b) Beodien; (c) Koshihikari

of Koshihikari tended to remain at lower values than those of upland genotypes throughout the measurement in both WI and WS treatments, indicating Koshihikaris lower LWP. It is reported that leaf hydraulic conductivity decreases as LWP decreases under an upland field condition (Stiller et al. 2003). It may be possible that leaf hydraulic conductivity of Koshihikari was lower than those of upland genotypes. Although LWC recovered at nighttime in most treatments and genotypes, LWC of Koshihikari in WS treatment did not recover after 18:00 h and instead kept decreasing. The reasons for this phenomenon should be lower water uptake capacity caused by its smaller and shallower root systems in Koshihikari. Although there was no significant difference in RLD in Koshihikari between the two water treatments

(Fig. 7), the LWC recovery behavior differed between treatments. These results indicate the difference in LWC recovery of Koshihikari between water treatments cannot be explained only by the difference in RLD. It is possible that water uptake capacity of roots can be physiologically affected by the water treatments. Stiller et al. (2003) revealed that irrigation regimes affected root pressure (an index of root activity) in rice plants: root pressure of plants rewatered after drought of 7 days was 7-fold higher than those of well-irrigated control plants. Our previous report (Matsuo and Mochizuki 2009) revealed that bleeding rate (also an index of root activity) of Koshihikari in aerobic rice systems was significantly lower than that in a continuously flooded paddy condition, but the bleeding rates of Sensho and Beodien did not differ between two water regimes. These reports indicate that water regimes affect the root activity and genotypic differences of root activity in response to water regimes exist in rice. Araki and Iijima (2005) revealed that upland rice (cv. IRAT 109) had the ability to change the position of water uptake in response to soil water conditions: when soil water content at shallow layer (025 cm depth) was sufficient, more than 90% of soil water was extracted from the shallower layer, but when soil water at shallow layer dried, about 42% of soil water was extracted from deeper layer (2550 cm depth). This adaptive mechanism may be a desirable one to save surface irrigation water in aerobic rice systems. The water uptake capacity of roots is known to be reflected in root hydraulic conductance (L0) or hydraulic conductivity (Lpr) (e.g. Matsuo et al. 2009; Miyamoto et al. 2001; Ranathunge et al. 2003, 2004, 2005). We have previously investigated root L0 for the three genotypes used in the present study under well-irrigated aerobic conditions and repetitive water stress conditions and reported that root L0 of Koshihikari was lower than those of the upland genotypes even under well-irrigated conditions. This result suggests that Koshihikaris water uptake capacity is lower than those of the upland genotypes under aerobic soil conditions and supports the idea that WI treatment is a stressful condition for Koshihikari, as described above. Root L0 of Koshihikari under repetitive water stress conditions was 69% lower than that under wellirrigated conditions, whereas those of Sensho and Beodien under repetitive water stress conditions

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Plant Soil (2010) 335:349361 Acknowledgements We thank Mr. Masashi Takahashi and Ms. Emiko Takama, JIRCAS, for their technical support during the data collection, and Dr. Satoshi Shimamura, National Institute of Crop Science, for his critical review of this manuscript.

were 31% lower and 36% higher, respectively, than under well-irrigated conditions, indicating that the effects of repetitive water stress on water uptake capacity differed among the genotypes and that Koshihikari was more strongly affected. The fact that LWC of Koshihikari in WS treatment did not recover at night may partially result from its low root L0 reduced by repetitive water stress. Further studies on physiological response of water uptake capacity to soil water conditions will help to reveal the optimal irrigation schedule in aerobic rice systems. The irrigation pattern clearly affected the surface soil (15 cm depth) temperature: there were no differences between WI and WS 1 day after irrigation, while soil temperature in WS was higher than that in WI 2 days after irrigation (Fig. 3). It is reported that soil temperature significantly affects gas exchange and water-related traits in plant species, including cotton (Radin 1990), olive (Pavel and Fereres 1998), oak (Cochard et al. 2000) and soybean (Ziska 1998). The values of LWP, hydraulic conductivity and gas exchange parameters increase with increasing soil temperature (from about 10 to 30C, depending on plant species). However, high soil temperature (over 35C) is reported to decrease plant water status, root hydraulic conductivity, photosynthesis parameters, chlorophyll accumulation and consequently decreases both shoot and root growth (Kuroyanagi and Paulsen 1988; Radin 1990; Tahir et al. 2009; Udomprasert et al. 1995; Xu and Huang 2000). It was found that soil temperature in WS treatment was over 35C 2 days after irrigation. Thus, it is possible that soil temperature affected the physiological behaviors to some extent in this experiment, especially in Koshihikari. Although there are numerous studies on upland rice cultivations, most studies mainly focused on the physiological and morphological traits in response to soil water conditions. As far as we know, there are no studies on the effect of high soil temperature on plant growth or physiological behavior in rice. Soil temperature should affect the root growth, root activity and root Lpr or L0, because root systems directly contact with the soil. Researchers working on aerobic rice systems had better address not only the effects of soil moisture conditions but also the effects of high soil temperature on the plants and genotypic difference of the tolerance to high soil temperature. This will lead to develop a new research area in rice.

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