Shoot and Root Development in Rice (Oryza sativa L.) Genotypes during Progressive Drying in Soils with Varying Moisture Regimes

Philippine Journal of Crop Science (PJCS) August 2012, 37 (2):1-12 Copyright 2012, Crop Science Society of the Philippines

Shoot and Root Development in Rice (Oryza sativa L.) Genotypes during Progressive Drying in Soils with Varying Moisture Regimes Roel R. Suralta1*, Nonawin B. Lucob1 and Loida M. Perez2

1Agronomy, Soils and Plant Physiology Division; 2Genetic Resources Division, Philippine Rice Research Institute, Maligaya, Science City of Muñoz, 3119 Nueva Ecija. *Corresponding author, [email protected]

Rainfed lowland rice productivity is negatively affected by drought. Root plasticity is a key trait for plant adaptation, and an important consideration in breeding high-yielding rice suited to drought-prone environments. This study was conducted to characterize shoot and plastic root developmental responses of the parents of doubled-haploid lines (DHL), and identify the drought intensity that can induce greater differences in root plasticity between parents. Rice genotypes CT9993 (upland japonica) and IR62266 (lowland indica) were grown in greenhouse and subjected to continuously waterlogged (CWL) and transient waterlogged-to-droughted (W-D) conditions with different soil moisture contents (SMC). Under W-D, CT9993 had progressive reductions in shoot biomass with decreasing SMC down to 20%, before it further reduced to 5% SMC. In contrast, IR62266 had progressive reductions in shoot biomass with decreasing SMC down to 15%. Furthermore, CT9993 also maintained stomatal conductance and transpiration, unlike IR62266, during the progressive drying. On the other hand, photosynthesis increased in IR62266 unlike in CT9993, which was maintained, thereby improving its water use efficiency under W-D down to 5% SMC. Compared with IR62266, CT9993 under W-D at 10% SMC had higher ratio of nodal roots, almost equal proportion of root lengths among soil depths, and greater promotion of L-type lateral roots at deeper soil layer where moisture is relatively higher than at shallow soil layer. Under W-D, key traits for drought adaptations include greater root plasticity in terms of nodal root production and elongation, and branching in CT9993 and shoot response for improved photosynthesis in IR62266. Furthermore, the drought intensity at 10% SMC induced greater differences in root plastic development between parents during progressive soil drying.

Keywords: doubled-haploid lines, drought, drought intensity, progressive soil drying, root plasticity, water

uptake, water use efficiency

INTRODUCTION

Rainfed rice systems occupy 37% of the total rice production area in the Philippines (PhilRice 2007). Rainfed upland and lowland ecosystems are both highly drought-prone because of their uneven topography and heavy dependence on rainfall as source of water. Rainfed upland field has poor accumulation of water due to uneven upper toposequence, absence of bunds, and lower water-holding capacity of the soil. In a rainfed lowland rice ecosystem, the area has bunded fields, complete reliance on rainfall or drainage from higher lands for a water source, and variable soil water-table depth depending on the field toposequence (Serraj et al. 2009). Furthermore, rainfed lowland rice is usually exposed to soil moisture that fluctuates between anaerobic (O2-deficient) and aerobic (drought) to various extents during the cropping season. Fluctuating soil moistures are crucial for plant productivity due to their marked effects on soil condition, availability of nutrients and water, as well as root development and functions (Kato et al. 2007; Siopongco et al. 2008; Suralta and Yamauchi 2008; Suralta et al. 2008a, 2008b, 2010; Suralta 2010).

The shoot and root developments of rice vary to some extent under drought conditions. The reduction in shoot dry matter is always relatively greater than the reduction in root dry matter under drought, which results in the increase in root to shoot dry matter ratio (Blum 2005). Shoot dry matter decreases under drought due to the reduction in number of tillers, leaf area and plant height (Asch et al. 2005). Depending on the genotypes, the roots have the ability to undergo developmental changes at different extents to better adapt to drying soil conditions. The root morphological adaptations under drought include the development of deep and extensive root systems (Kato et al. 2007), which include thick roots (Price et al. 2000) and increased root length (RL) density (Siopongco et al. 2005), as a result of the plasticity in lateral root (LR) development (Kano et al. 2011; Kano-Nakata et al. 2011; Suralta et al. 2010). These adaptations are perceived to be associated with increased water extractions (Kato et al. 2007; Siopongco et al. 2005, 2006) and increased nutrient uptake (Suralta 2010). Thus, the capacity of a root system for water and nutrient uptake has been the major determinant of grain yield in rainfed lowland rice (Niones et al. 2012). Hence, the elucidation of the genetic control of root

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system morphology under water deficit is essential for breeding rice suitable for drought-prone environments. Recent progress in DNA markers and their linkage maps has provided powerful tools for mapping quantitative trait loci (QTL) (Yano 2001) and the ultimate goal of identifying genes controlling root system morphology. Many studies have reported QTL controlling rice root system characteristics but these were mostly limited to traits like mass and depth (Kamoshita et al. 2008; Horii et al. 2006; Wang et al. 2005) that are still compounded with several component root traits that can respond differently to drought stress. Another study analyzed QTL associated with LR production regardless of the types of LR (Zheng et al. 2003). The L- and S-type LRs generally differ in length, diameter and histological structure as well as response to drought stress (Bañoc et al. 2000b; Suralta et al. 2010). On a detailed morphological level, studies on QTL associated with root system growth either limit their measurements on partitioning of root mass under droughted soil conditions (Horii et al. 2006) or direct measurements of component root traits under hydroponics condition (Wang et al. 2005). The judicious evaluation of specific component root traits and the understanding of soil moisture regimes in both rainfed and upland rice systems are important to successful QTL studies on root system morphology and its application in breeding and development of drought tolerant rice cultivars. Two contrasting genotypes, CT9993-5-10-1-M (abbreviated as CT9993) and IR62266-42-6-2 (abbreviated as IR62266), were used to develop a novel mapping population of anther culture-derived doubled-haploid lines (DHLs) for QTL studies. CT9993 is an upland japonica type with specific adaptation to some drought conditions, while IR62266 is a lowland indica type with general adaptation over environments with stable yields. These two genotypes genetically differ in their mechanism of responses to drought (Babu et al. 2003; Kamoshita et al. 2002a, b; Salekdeh et al. 2002; Siopongco et al. 2009; Zhang et al. 2001; Zheng et al. 2003). The DHLs are proven to be useful for precise evaluation of root traits under drought with reduced effects of genetic confounding of other traits (Siopongco et al. 2005, 2006). The availability of DHLs derived from CT9993 and IR62266, one of the most widely used materials for drought studies in rice, provides an opportunity for mapping QTL for specific component root traits under progressive drought stress. Although the two rice genotypes differ in their adaptation to drought stress, information on the extent of their genotypic differences on which specific component root traits and at which intensity of soil moistures during progressive drought is still lacking. This information is crucial in the subsequent establishment of the appropriate phenotyping environments and screening of specific

component root traits under progressive drought for root QTL study. This study was conducted to determine the suitability of the DHLs derived from CT9993 and IR62266 for QTL analysis of plastic component root traits under progressive drought. Specifically, this study: 1) characterized shoot and plastic root developmental responses of the DHLs parents; and 2) identified the drought intensity during progressive drought stress that can induce greater differences in shoot and root plastic developmental responses between the two DHL parents. MATERIALS AND METHODS Time and Place of Study

This greenhouse study was conducted from August to September 2011 at the Philippine Rice Research Institute-Central Experiment Station (PhilRice-CES), Muñoz, Nueva Ecija, Philippines (15°40’N, 120°53’E, 57.6 masl). The average daily solar radiation, minimum and maximum temperatures, and pan evaporation were 17.7 MJ m-2 d-1, 24.6°C, 31.2°C, and 4.0 mm d-1, respectively. Experimental Design and Treatments

To analyze more precisely the trend of responses to varying intensities of soil moistures within each genotype, the treatments were arranged in split-plot based on completely randomized design (CRD) with three replications (Figure 1). The genotypes were assigned in the mainplot, while the soil moistures were assigned in the subplots. The data were analyzed using IRRISTAT program (version 4.1), and means of the treatments were compared using the least significant difference (LSD) test at 5% level of significance. Plant Materials Two rice genotypes, lowland adapted indica IR62266 and upland adapted japonica CT9993 were used. Three pre-germinated seeds from each genotype were grown in each box (25 cm x 2 cm x 40 cm, L x W x H) filled with 2.8 kg soil (mixture of 1.7 kg garden and 1.1 kg sandy loam soils) following the method of Kono et al. (1987). The seedlings were thinned to one seedling per box at 3 days after sowing (DAS). The soil in each box was pre-mixed with 730 mg ammonium sulfate (21-0-0), 460 mg solophos (0-18-0) and 140 mg muriate of potash (0-0-60). Soil Moisture Treatments and Plant Sampling

Six soil moisture treatments used in the study were: continuously waterlogged (CWL) condition as control, and five transient waterlogged-to-droughted (W-D) conditions as the other treatments. In CWL treatment, water level was increased and maintained at 2 cm above the soil surface from 3 DAS until the end of the experiment (40 DAS). In W-D treatments, the soil

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inside the box was first waterlogged during 3-14 DAS. Thereafter, watering was withheld and the soil was allowed to dry, with the SMC maintained at varying levels (25, 20, 15, 10 and 5% w/w) until 40 DAS. Boxes were weighed daily using a digital balance to record the wet mass of the soil. The SMC (% by mass) in each box was calculated as the ratio between water mass (difference between the wet mass of the soil excluding the box on a given day and the dry mass of the soil at 2.8 kg) and the dry mass (2.8 kg) of soil. Once the target SMC of the W-D treatments was reached, watering was done to replace the amount of water lost and maintain the desired SMC. Drought treatments imposed through progressive soil drying were terminated at 40 DAS. Sampling was made at 40 DAS, i.e. after 40 d of continuously waterlogged or 26 d of progressive drought conditions. Three plants were collected from each treatment combination. Data Gathered Physiological Measurements The stomatal conductance (SC), transpiration (Tr) and photosynthesis (Ps) of the second-youngest fully expanded leaf were measured using a portable photosynthesis system (Li-6400XT, LiCOR Inc., Lincoln, Nebraska, USA) between 1000 h to 1200 h at 40 DAS, the day the experiment terminated. Shoot and Root Growth Measurements The shoots were cut and oven dried at 70°C for 48 h before weighing. The whole root system was sampled using a pinboard following the methods of Kono et al. (1987). The extracted root systems embedded in plastic sheets were temporarily stored in alcohol for further measurements. Prior to measurements, alcohol was removed and root systems were stained in 0.25% Coomassie Brilliant Blue R 250 aqueous

solution for at least 24 h. This staining procedure was indispensable for taking high-resolution digital scans of the entire root system, including the fine lateral roots. After staining, the root samples were washed gently with running water to remove excess stains. The total number of nodal roots (NR) was manually counted. The length of each NR was measured using a meter stick. Thereafter, the root system was divided into four portions corresponding to four soil depths (0-10 cm, 10-20 cm, 20-30 cm and 30-40 cm below the soil surface). From each portion, three randomly selected NRs were sampled and cut into 3-cm segments, keeping the LRs intact. In this way, the number of each type of LRs was determined. The L-type LRs are long, thick and branched into high order LRs, while S-type LRs are short, slender and non-branching. The total number of LRs and specific type of LR were expressed as linear frequency (the number of LRs per unit length of root axis; (Ito et al. 2006). After manual measurements, root samples was from each portion scanned at 600 dpi (EPSON 4990). Scanned images were analyzed for root length (RL) using WinRhizo v. 2007d (Régent Instruments, Québec, Canada). A pixel threshold value of 175 was set for the RL analysis. RESULTS Shoot Growth At vegetative stage, shoot dry matter production is an indicator of the plant’s performance under drought stress. In this study, shoot dry weight and tillering were significantly reduced by W-D in both genotypes (Table 1). In IR62266 under W-D, shoot dry matter production was progressively reduced from 25 to 15% SMC, while no reduction was observed when SMC was further reduced from 15 to 5%. In CT9993, on the other hand, shoot dry matter production under W-D conditions (20, 15 and 10% SMC) did not differ significantly while generally lower than 25% SMC. Shoot dry matter production in CT9993 was reduced when SMC was further reduced to 5% under W-D condition. Under W-D conditions, tillering was more inhibited in IR62266 than in CT9993 with a decrease in SMC (Table 1). Stomatal Conductance, Transpiration, Photosynthesis and Water Use Efficiency

Transpiration and photosynthesis are regulated by the size of stomatal openings depending on the prevailing environmental factors such as soil water status. Thus, stomatal conductance is a good indicator of root development and water uptake capacity under drying soil conditions. The SC in IR62266 was significantly reduced by W-D regardless of SMC (Table 2). On the other hand, the SC of CT9993 was significantly reduced by W-D at 5% SMC only. The Tr in IR62266 was significantly reduced by W-D at 10 and 5% SMC,

Figure 1. The experimental set up at 15 days after sowing (DAS), when watering in the transient waterlogged-to-drought (W-D) treatments was stopped to commence with the progressive soil drying for another 26 days.

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while significantly reduced at 5% SMC only in CT9993 (Table 2). The Ps in IR62266 was significantly increased by W-D in most SMC, while significantly reduced at 5% SMC (Table 2). On the other hand, the Ps in CT9993 was maintained under W-D at most SMC, except at 5% where a reduction was observed. Water use efficiency (WUE) is an indicator of the efficiency of carbon dioxide intake (Ps) per unit amount of water lost (Tr) in the leaf. The WUE in IR62266 was significantly increased under W-D with a decrease in SMC (Table 2). In CT9993, WUE was significantly increased only at 5% SMC. Root Growth and Development Root growth and development is considered very important for plants to adapt to soil undergoing progressive drought stress. The root system profiles of IR62266 and CT9993 grown under CWL and W-D conditions are shown in Figure 2. Relative to their CWL controls, root system development (RSD) of both genotypes appeared to be inhibited by W-D, with IR62266 showing greater inhibitions as SMC were decreased. The quantitative data for these root system profiles are shown in Table 3 and Figures 2-6. Nodal and Lateral Root Productions Nodal root production of IR62266 under W-D was not significantly different with SMC reductions from 25 to 10%, but generally lower than the values obtained from CWL control (Table 3). Further decrease in SMC to 5% also reduced the NR production. On the other

Table 1. Shoot dry weight and tillering of rice genotypes IR62266 and CT9993 grown under 40 days of continuous waterlogging (CWL) or transient waterlogged for 14 days followed by progressive drought for 26 days (W-D). Values in W-D treatments refer to the soil moisture content (%) maintained after progressive soil drying.

Genotype (G)/ Soil Moisture Content (SMC)

Shoot dry weight (g per plant)

Tillers per plant

IR62266

CWL 3.47 a 6.3 a

W-D25 1.84 b 4.3 b

W-D20 1.45 c 4.0 b

W-D15 1.15 d 2.7 d

W-D10 1.23 d 3.3 c

W-D5 1.21 d 3.3 c

CT9993

CWL 3.16 a 2.3 a

W-D25 1.56 b 0.3 b

W-D20 1.20 c 0.3 b

W-D15 1.14 c 0.3 b

W-D10 1.17 c 0.3 b

W-D5 0.85 d 0.3 b

G ** **

SMC ** **

G x SMC ns ns *and** significant at 5, 1% level; ns, not significant. In a column within each genotype, means followed by a common letter are not significantly different at 5% level by LSD.

Table 2. Stomatal conductance, transpiration, photosynthesis and water use efficiency of rice genotypes IR62266 and CT9993 grown under 40 days of continuous waterlogging (CWL) or transient waterlogged for 14 days followed by progressive drought for 26 days (W-D). Values in W-D treatments refer to the soil moisture content (%) maintained after progressive soil drying.

Genotype (G)/ Soil Moisture Content

(SMC)

Stomatal Conductance (mmol m-2 s-1)

Transpiration (mmol m-2 s-1)

Photosynthesis (µmol m-2 s-1)

Water Use Efficiency

IR62266 CWL 0.84 a 14.3 a 21.6 b 0.00153 d

W-D25 0.62 b 12.2 ab 20.6 a 0.00170 d

W-D20 0.54 b 11.9 b 26.9 a 0.00233 b

W-D15 0.60 b 12.5 ab 25.8 a 0.00207 c

W-D10 0.53 b 10.8 b 27.1 a 0.00250 b

W-D5 0.11 c 3.03 c 12.2 c 0.00403 a

CT9993

CWL 0.69 ab 10.3 ab 23.5 a 0.00230 b

W-D25 0.85 a 11.9 ab 24.2 a 0.00203 b

W-D20 0.88 a 11.6 ab 24.2 a 0.00210 b

W-D15 0.75 ab 12.4 a 25.1 a 0.00220 b

W-D10 0.61 b 9.8 b 21.8 ab 0.00227 b

W-D5 0.18 c 4.8 c 18.2 b 0.00380 a

G ** * ns ns

SMC ** ** ** **

G x SMC * ** ** ** * and **, significant at 5 and 1% level, respectively; ns, not significant. In a column within each genotype, means followed by a common letter are not significantly different at 5% level by LSD. Water use efficiency was computed as the ratio between photosynthesis and transpiration for each experimental treatment.


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hand, NR production in CT9993 was similar from 25 to 15% SMC but values were generally lower than CWL. Further reductions in NR production were observed at 10 and 5% SMC. The total NR length in both genotypes was similarly inhibited under W-D at any SMC (Table 3). Mean NR length in both genotypes was not affected by W-D in most SMC, except at 5% wherein it was significantly promoted (Table 3). Regardless of SMC, the total RL was significantly reduced by W-D in both genotypes at similar extents (Table 3). Furthermore, the total LR length in IR62266 was significantly reduced by W-D at 15% SMC only, while the total LR length was maintained in CT9993 at at 10% SMC only (Table 3). Root Length Distribution at Different Soil Depths The extent of root length distribution at varying soil depths is functionally important for plants under drought because it determines the volume of contact with soil, and plant’s capability for water and nutrient uptake. The RL distribution at different soil depths under varying levels of soil moisture treatments in both genotypes is presented in Figure 3. In IR62266, the RL distribution was similar across soil moisture treatments, wherein their distributions were generally more concentrated within 10-20 and 20-30 cm depths than at shallow (0-10 cm depth) and deepest soil layers (30-40 cm depth). Similar trends were observed

Figure 2. Root system profiles of CT9993 and IR62266

grown either under 40 days of continuous

waterlogging (CWL) or transient waterlogged for

14 days followed by progressive drought for 26

days (W-D). Values in W-D treatments refer to

the soil moisture content (%) maintained after

progressive soil drying. The intact root system

was extracted from root box using a pinboard as

described by Kono et al. (1987). Prior to taking

of digitized photographs in a scanner, the root

systems were stained with 0.25% of Coomassie

Brilliant Blue R solution for at least 24 h.

Table 3. Root system development of rice genotypes IR62266 and CT9993 grown under 40 days of continuous waterlogging (CWL) or transient waterlogged for 14 days followed by progressive drought for 26 days (W-D). Values in W-D treatments refer to the soil moisture content (%) maintained after progressive soil drying.

Genotype (G)/ Soil Moisture

Content (SMC)

Nodal Roots per plant

Total Nodal Root Length

(cm per plant)

Mean Nodal Root Length

(cm per plant)

Total Root Length

(cm per plant)

Total Lateral Root Length

(cm per plant)

IR62266

CWL 135.0 a 2669.3 a 19.94 b 12385.4 a 9719.4 a

W-D25 51.0 b 952.1 b 20.28 b 9116.6 b 8164.4 ab

W-D20 50.0 b 963.1 b 19.39 b 9025.0 b 8061.9 ab

W-D15 48.0 b 827.9 bc 17.30 b 8076.0 b 7248.2 b

W-D10 46.3 b 812.3 bc 17.53 b 8401.9 b 7589.7 ab

W-D5 22.3 c 610.6 c 28.21 a 8238.7 b 7718.1 ab

CT9993

CWL 83.0 a 1835.5 a 22.14 bc 10270.4 a 8434.9 a

W-D25 41.3 b 881.5 b 21.25 c 6936.5 b 6055.0 b

W-D20 27.7 bc 700.4 bc 25.30 abc 6477.8 b 5777.4 b

W-D15 25.0 bc 582.8 bc 23.00 bc 6505.4 b 5922.6 b

W-D10 22.3 c 600.7 bc 27.31 a 7316.1 b 6715.5 ab

W-D5 14.7 c 419.1 c 28.50 a 6212.4 b 5793.3 b

G ** ** ** ** **

SMC ** ** ** * **

G x SMC * ns ns ns ns

* and **, significant at 5 and 1% level, respectively; ns, not significant. In a column within each genotype, means followed by a common letter are not significantly different at 5% level by LSD.

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in CT9993, although the distribution of RLs was closer among soil depths compared with those in IR62266 (Figure 3). Frequency Distribution of Nodal Root Lengths The nodal root is one of the key root traits that influence the vertical distributions of LR along the soil profiles. The ratio of NRs classified according to their lengths relative to the total NRs, expressed as percentage was also determined (Figure 4). In IR62266, most NRs had lengths ranging 0-10 cm. The NR length distribution among soil depths had similar patterns between CWL and W-D at 15% SMC, and changed minimally at 10% or lower SMC. Furthermore, the proportion of NRs of IR62266 with length longer than 40 cm was also small, and did not vary much under W-D even with the reduction in SMC. On the other hand, most of the NRs in CT9993 were in the range of 0-10 cm under relatively wetter soil conditions (CWL and W-D at 25 and 20% SMC). However, under relatively drier soil conditions i.e., W-D with 15% SMC and below, the distribution of NRs (0

-40 cm lengths) especially at 10% SMC did not differ among range of lengths. Furthermore, the proportion of NRs with more than 40 cm in lengths progressively increased with the reductions in SMC. Nodal root elongation in CT9993 was largely promoted at 10% SMC. Linear Frequency of Lateral Roots along Nodal Root Axis The total root length of the whole rice root system is mainly composed of LRs. The plasticity in LR production, especially the L-types, is one of the key traits for rice adaptation under drought stress. The total number of LRs along NR axis regardless of types was highest at 10-20 cm soil depths, while similar magnitudes although lower, were observed for the rest of the soil depths regardless of genotypes and soil moisture treatments (Figure 5). Furthermore, the dynamics in S-type LRs along nodal root axis was not significantly different regardless of soil depths and soil moisture treatments in both genotypes (Figure 6). On

Figure 3. Root length distribution at different soil depths of rice genotypes IR62266 and CT9993 grown either under 40 days of continuous waterlogging (CWL) or transient waterlogged for 14 days followed by progressive drought for 26 days (W-D). Values in W–D treatments refer to the soil moisture content (%) maintained after progressive soil drying. Data shown are means ± SD of three replications.

Figure 4. Histogram showing the distribution of nodal roots (expressed percentage of the total nodal roots, NR) classified according to their lengths of rice genotypes IR62266 and CT9993 grown under 40 days of continuous waterlogging (CWL) or transient waterlogged for 14 days followed by progressive drought for 26 days (W-D). Values in W-D treatments refer to the soil moisture content (%) maintained after progressive soil drying. Data shown are means ± SD of three replications.


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the other hand, the dynamics of L-type LR among soil depths showed an interesting response to soil moisture treatments (Figure 7). Compared to the CWL control, the L-type LR in IR62266 at 30-40 cm soil depths was significantly promoted by W-D, even at 25% SMC. No significant increases in L-type LRs in IR62266 with further decrease in SMC from 25-5% under W-D were observed. The L-type LRs in IR62266 at other soil depths (0-10, 10-20, and 20-30 cm) were also promoted under W-D at the same extent, except at 25% SMC. On the other hand, the L-type LRs in CT9993, especially at deeper soil layers (20-30 and 30-40 cm soil depths), were progressively promoted under W-D with the reduction in SMC. This indicates that the promotion of L-type LRs in CT9993 was triggered when soil was already experiencing mild to severe drought stress. Furthermore, the L-type LRs in CT9993 in shallow soil layers (0-20 cm) were not affected by W-D at any level of SMC. DISCUSSION

Several studies have validated genotypic differences between the CT9993 and IR62266 under drought (Babu et al. 2003; Kamoshita et al. 2002a; Kamoshita et al. 2002b; Siopongco et al. 2009; Zhang et al. 2001). However, most of the root traits examined are only compounded root related traits such as thickness, depth and mass (Kamoshita et al. 2008). This is the first study that quantified genotypic differences between CT9993 and IR62266 at the level of component root traits under progressive drought in soil, which are crucial for the QTL analysis of root plasticity using their corresponding DHLs. Soil Moisture Effects on Shoot Growth Wide variation of the phenotypic traits between potential parents is important for QTL analysis studies. The present study indicates that CT9993 and IR62266 had wide genotypic differences in response to soil moisture deficit. The dry matter production of

Figure 5. Linear frequency of LRs along nodal root (NR) axis at different soil depths of rice genotypes IR62266 and CT9993 grown under 40 days of continuous waterlogging (CWL) or transient waterlogged for 14 days followed by progressive drought for 26 days (W-D). Values in W-D treatments refer to the soil moisture content (%) maintained after progressive soil drying. Data shown are means ± SD of three replications.

Figure 6. Linear frequency of S-type LRs along nodal root (NR) axis at different soil depths of rice geno-types IR62266 and CT9993 grown under 40 days of continuous waterlogging (CWL) or transient waterlogged for 14 days followed by progressive drought for 26 days (W-D). Values in W-D treat-ments refer to the soil moisture content (%) main-tained after progressive soil drying. Data shown are means ± SD of three replications.

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both genotypes was generally reduced under W-D condition although reduction in dry matter production was more abrupt in IR62266 than in CT9993 with decreasing SMC from 25 to 5% (Table 1). Overall however, the degree of reductions in shoot dry matter of both genotypes was almost similar, relative to their CWL controls. While CT9993 had better ability than IR62266 in terms of plastic RSD (Figures 2, 3, 4 and 7), the latter genotype had better ability than the former in terms of shoot response for improving WUE under W-D especially at 10% SMC (Table 2). This further confirmed that both parents have drought adaptation mechanisms, even though different, which can be considered in selecting potential DHLs that have both traits to mitigate the effect of drought on rice productivity. Dry matter production, especially under water-limited condition, is determined by WU and WUE (Kobata et al. 1996) of the plant. Using selected chromosome segment substitution lines (CSSL), Suralta et al.

(2010) have shown that dry matter production in rice is a function of WU, indicating that adaptation of rice under water-limited conditions in terms of growth and productivity could be achieved mainly through dehydration avoidance i.e., by maintaining water uptake rather than by tolerance to desiccation (Blum 2005). Water use efficiency may be poor indicator of improved drought resistance especially when its variation is driven mainly by variation in water use (or transpiration) rather than by variation in plant production (or photosynthesis) or assimilation per given amount of water use (Blum 2005). In this study, the improvement of WUE in IR62266 was due to combined effect of reduction in transpiration (WU) and the parallel increase in photosynthesis with decreasing SMC under W-D condition (Table 2). One of the traits that can influence WUE is functional stay green (FSG) character, resulting to a relatively higher photosynthesis or longer photosynthetic duration of the plant (Thomas and Smart 1993). This trait was found to be effective in reducing the effect of drought in sorghum (Mahalakshmi and Bidinger 2002) and in wheat (Christopher et al. 2004). Current study is undergoing to evaluate the efficiency of FSG in maintaining WUE during reproductive stage drought in rice genotypes with high root plastic ability during vegetative stage drought. Soil Moisture Effect on Root Growth and Development Water use is associated with maintained root function brought about by root plastic responses under water-limited soil conditions. In this study, RSD responses such as the absolute number and length (total and mean) of NRs, total root and LR lengths have similar trend for both genotypes in response to W-D (Table 3). In this situation, solid conclusions cannot be drawn with regards to the contribution of root plasticity to the observed genotypic variations in dry matter production in response to W-D at different SMC during progressive drought. Thus, RSD was further analyzed in terms of the distribution of total root and NR growth and the development and intensity of LR branching to detect clear differences at different soil depths in response to progressive drought (Figures 2-6). Root Length Distribution at Different Soil Depths The distribution of RLs plays critical role for exploration of available water under progressive soil drying. Interestingly, while both genotypes showed similar pattern of total root growth under W-D condition, CT9993 had higher ability than IR62266 for plastic root growth at deeper soil layers (Figure 4), where soil moisture tended to be higher during progressive drought (data not shown). This clearly explains the higher maintenance of Tr in CT9993 than in IR62266 under W-D (Table 2). Nodal Root Length Distribution

Figure 7. Linear frequency of L-type LRs along nodal root (NR) axis at different soil depths of rice genotypes IR62266 and CT9993 grown under 40 days of continuous waterlogging (CWL) or transient waterlogged for 14 days followed by progressive drought for 26 days (W-D). Values in W-D treatments refer to the soil moisture content (%) maintained after progressive soil drying. Data shown are means ± SD of three replications.


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The analysis of NR according to their lengths showed that CT9993 has the ability to progressively increase the proportion of NRs having more than 40 cm in lengths especially under W-D at 10% SMC (Figure 4). On the other hand, IR62266 has also 40 cm NRs, but their proportion relative to the total NRs did not respond to decreasing SMC. Most of the NRs in IR62266 were confined only at the shallow soil layer in most of the SMC, except at 5% (Figure 4). The height of the root box used in this study is 40 cm only, thus NRs of CT9993 that elongated beyond 40 cm in length were accumulated at the bottom of the boxes. The SMC along soil profile within the box was relatively higher at deeper soil layers (data not shown). Assuming that the height of the box and depth of soil profile was more than 40 cm, we expected that this deep root development in CT9993 should have contributed more for water uptake, hence much less reductions in dry matter production than IR62266 under W-D at 10% SMC. In previous studies, deep root development tended to increase as water stress progresses especially in controlled pot experiments (Asch et al. 2004; Price et al. 2002; Singh et al. 2000; Trillana et al. 2001). Under rainfed lowland condition, the genotypic variations in deep root development and water uptake from deeper soil layers contributed to the variations in growth and yield under progressive drought (Henry et al. 2011). Furthermore, the genotypic variations in root development at the shallow soil layers in response to re-watering (or periods of rainfall events in the field) after drought also contributed to the growth and development in rice (Nakata et al. pers. comm.). The above findings indicate that root plasticity in response to re-watering is one of the important mechanisms for drought recovery as has been shown earlier by Bañoc et al. (2000a, 2000b). Rainfed lowland rice areas generally differ in basic characteristics such as the presence and depth of hardpan layers, timing and intensity of drought, and magnitude of increase in mechanical impedance during periods of declining soil moistures, which may affect the root penetrating ability into deeper soil layers (Cairns 2004; Kato et al. 2007; Samson et al. 2002). Current studies are being conducted to clarify the effect of prevailing soil and climatic environment in the rainfed lowland and upland areas such as soil moisture and hardness distribution with depths to be coupled with root distribution, and precipitation and their interactions in relation to the genotypic differences in growth and yield for each site. Lateral Root Production In addition to higher proportion of NRs that penetrated into deeper soil layers, LR development along these NRs is also important for more functional deep root development of rice (Bañoc et al. 2000a). In this study, the linear frequency of LRs along NR axis regardless of types was highest at shallow soil layers

(10-20 cm depth) and was consistent for both genotypes (Figure 5). Unlike S-type LRs (Figure 6), the L-type LRs at the deepest soil layer in both genotypes responded plastically with a decrease in SMC under W-D condition (Figure 7). Between the two genotypes, L-type LRs in CT9993, especially at the lower half (20-30 and 30-40 cm depths) of soil profiles, progressively increased with a decrease in SMC down to 10%. In IR62266, L-type LRs at deepest soil layer was significantly promoted during the onset of progressive drought, i.e., only when soil was relatively wet (Figure 7). No further increase in L-type LRs in IR62266 was observed with further decrease in SMC. The L-type LRs along NRs of IR62266 from other soil depths increased with a reduction in SMC (Figure 7). The above findings suggest that the compounded RSD responses (e.g., total RL) may not be enough to detect the differences in rice adaptation in terms of dry matter production under drought. There is still a need to further analyze how these RLs were distributed among soil depths, as there are spatial differences of soil moistures relative to soil depth. Overall, the distribution of RL especially in CT9993 was particularly affected by the proportion of NRs that elongated into the deeper soil layers and the simultaneous promotion of L-type LRs. These responses contributed to the maintenance of Tr (WU) of CT9993 (Table 2) and thus, gradual reductions of its dry matter production under W-D condition (Table 1). IR62266, on the other hand, lacked the ability to promote root growth at deeper soil layers where the soil moisture is available. Despite such limitation in root plastic development under drought, IR62266 has drought adaptation mechanism other than root plasticity, particularly its ability to increase Ps efficiency (WUE) under water deficit conditions (Table 2). Limitation of the Study

This study limited its scope on the shoot and root developmental responses of DHL parents to progressive drought stress during the vegetative stage growth only. The genotypic variations in root developmental analysis at different intensities of soil moistures was focused more on the response of root branching and distribution rather than potential deep rooting. CONCLUSION We have quantified the specific component root traits of DHL parents that showed plastic RSD responses to progressive drought. Compared with IR62266, CT9993 has greater ability for promoted root growth and distribution at deeper soil layers where soil

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moisture is relatively higher during progressive drought. This resulted in a maintained Tr (WU) and dry matter production in CT9993. This confirmed the dehydration avoidance ability of CT9993 under water deficit soil conditions as attributed by root plasticity. Root plasticity was expressed fully under W-D and differential genotypic responses were observed at 10% SMC. The root traits in CT9993 that specifically influenced the distribution of root growth under deeper soil were the promoted elongation that led to greater proportion of NRs and the promotion of L-type LRs along nodal root axis. On the other hand, IR62266 has shoot trait response, such as the ability to increase Ps efficiency under progressive drought and WUE. Both genotypes have key traits that would be of consideration for phenotyping of their corresponding DHLs under W-D at 10% SMC. RECOMMENDATIONS

Based on the findings of this study, the DHLs derived from CT9993 and IR62266 are suitable genetic materials for mapping QTL associated with plastic component root traits under progressive drought stress. Another set of experiment using their DHLs is under way to analyze QTL associated with WU and WUE through phenotyping of component root trait plasticity and Ps efficiency, respectively. Selection of potential DHLs will also be done for further gene level expression studies. ACKNOWLEDGMENT

We acknowledge research funding from the Department of Agriculture-Biotechnology Project Implementation Unit (DA-Biotech PIU). We thanked the International Rice Research Institute (IRRI) for providing the seeds of CT9993 and IR62266 and their DHLs. LITERATURE CITED Asch F, Dingkuhn M, Sow A, Audebert A. 2004.

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Shoot and Root Development in Rice (Oryza sativa L.) Genotypes during Progressive Drying in Soils with Varying Moisture Regimes

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