Introduction

Water and nitrogen are major limiting factors for plant growth and production (Evans 1983; Tezara et al. 1999; Lawlor 2002; Passioura 2007). Water and N uptake are generally associated with higher plants; for example, it has been demonstrated that nitrate uptake is positively related to water uptake (Guo et al. 2002; Gorska et al. 2008a). Decreased hydraulic conductance as well as water deficits will reduce the leaf elongation rate, leaf potential, stomatal conductance (gs), and nutrient uptake (Tanguilig et al. 1987; Saliendra et al. 1995).

Numerous studies have reported that water uptake into roots and transport to the shoot are often affected by root/rhizosphere water potential and oxygen deficiency, transpiration rate, and biotic or abiotic perturbation (Steudle 2000a; Maurel et al. 2008; Vandeleur et al. 2009). Root hydraulic conductance (L r ), an important parameter demonstrating water transport ability through root xylem, is plant species-dependent, which is mainly affected by the root morphology, anatomy and aquaporin (AQP) activity (Bramley et al. 2009). The formation of aerenchyma and the impermeable barrier to oxygen potential poses a large resistance to radial flow, which inevitably decreases the L r (Colmer 2003). Inhibition of the root cortical cell hydraulic conductivity (L c ) could account for the decrease in L r of wheat, while L c may be related to AQP activity under the regulation of cytoplasmic pH, free Ca2+, phosphorylation and H2O2 (Bramley et al. 2010; Bramley and Tyerman 2010). Vandeleur et al. (2005) reported that the water permeability of root plasma membrane vesicles was also strongly inhibited by reductions in the pH.

Low N can significantly reduce the effective leaf area as a result of reductions in the leaf size, chlorophyll content and CO2 assimilation, leading to a subsequent decrease in the crop yield (Bennett et al. 1989; Dordas and Sioulas 2008). Conversely, increased N supply can improve the water use efficiency (WUE) and plant productivity (Lajtha and Whitford 1989; Green and Mitchell 1992; Livingston et al. 1999; Hobbie and Colpaert 2004). Many studies have focused on the effect of N on stomatal conductance or mesophyll conductance. For example, increasing the photosynthetic rate (A) following increased N investment in Rubisco with little or no effect on stomatal conductance (gs) (Wang et al. 1998; Welander and Ottosson 2000) or with an increase in mesophyll conductance (gm) (Warren and Adams 2006; Flexas et al. 2008) lead to an increase in WUE. Other work has also shown that A and gs were directly proportional to the plant hydraulic conductance, suggesting that changes in the plant hydraulic conductance may affect the plant carbon gain (Hubbard et al. 2001). Whether N affected the plant hydraulic conductance by altering characteristics of the roots or leaves remains unclear, although changes induced by N nutrition to each of these attributes can potentially affect L r .

Previous studies have shown that low N can decrease the plant hydraulic conductance (Radin and Boyer 1982; Radin 1983). Low phosphorus (P) and low potassium (K) also showed effects that paralleled those of low N (Dordas and Sioulas 2008; Oddo et al. 2011). Plants grown on low P had a reduced leaf water potential, transpiration rate, and L r , leading to reductions in the leaf area and shoot dry weight. The decrease in L r was caused by a reduced radial conductivity, which could result from decreased root cell membrane permeability and from changes in apoplastic or symplastic pathways (Augé and Stodola 1990; Cui and Nobel 1992). Recent findings have suggested that the N availability potentially affected L r via changes in AQP expression (Ishikawa-Sakurai et al. 2014). Another study reported that there was a rapid increase in L r associated with the addition of NO3 −, but similar effects were not observed for other anions, such as SO4 2− and H2PO3 −. This response could be attributed to changes in root cell membrane hydraulic conductance by NO3 −, which was related to a hydraulic signal and AQP activity but not to AQP expression or de novo protein synthesis (Gorska et al. 2008a, b). It is possible that the rapid increase in L r in the presence of increased NO3 − availability is an important trait that enhances the ability of plants to compete for the NO3 − in soil (Gloser et al. 2007). Although these studies provided some explanation for the increase in L r observed when the plant is supplied with N, how N uptake is affected by water uptake and the relationship between these two factors are still not completely understood.

Based on our previous cultivar selection experiment, we chose the two rice cultivars of ‘Shanyou63’ (hybrid indica, China) and ‘Yangdao6’ (conventional indica, China), which were widely planted in China and showed different responses of water uptake ability and photosynthesis to nitrogen forms and/or concentrations (Yang et al. 2012; Li et al. 2013). Our previous studies had shown that under well watered conditions, physiological effects of moderate N supply were independent of the applied N form (Yang et al. 2012). In consideration of the field environment, lowland rice roots are actually exposed to a mixed nitrate and ammonium supply in the rhizosphere (Briones et al. 2003; Kirk and Kronzucker 2005). In addition, aggravating water shortage, conventional flood irrigation methods should be replaced with intermittent irrigation or dry cultivation in rice production. There will be an increase in nitrate content in these conditions. So, in the present study, we supplied even concentration of nitrate and ammonium, and assessed growth, leaf gas exchange, WUE, L r , and factors affecting water absorption under various N supplies in two rice cultivars of Shanyou63 and Yangdao6. We discuss the effects of N on root anatomy and water transport pathways as well as the contribution of AQP to water uptake; we also comment on the differences between these two cultivars.

Materials and methods

Plant materials and growth conditions

Rice seeds (Oryza sativa L., cv. “Shanyou63” hybrid indica China, and “Yangdao6” conventional indica China) were sterilized in 10 % H2O2 for 30 min and transferred to moist filter paper for germination. When the seedlings developed an average of 2.5 visible leaves in distilled water, they were transplanted into 6-L rectangular containers (30 × 20 × 10 cm) with quarter-strength nutrient solution (for composition, see below) for 2 days. Then, the seedlings were transferred to half-strength nutrient solution for 3 days. Later, the seedlings were supplied with full-strength nutrient solution for 4 days. Next, the seedlings were supplied with low-N (20 mg·L−1), moderate-N (40 mg·L−1) and high-N (100 mg·L−1) nutrient solutions. The nutrient solutions included the following components: macronutrients (mg·L−1): N as equal moles of (NH4)2SO4 and Ca(NO3)2, 10 P as KH2PO4, 40 K as K2SO4 and KH2PO4, 40 Mg as MgSO4·7H2O, 57 Ca as CaCl2·2H2O and micronutrients (mg·L−1): 2.0 Fe as Fe-EDTA (FeSO4·7H2O), 0.5 Mn as MnCl2·4H2O, 0.2 B as H3BO3, 0.05 Mo as (NH4)6Mo7O24·4H2O, 0.01 Zn as ZnSO4·7H2O, 0.01 Cu as CuSO4·5H2O, and 2.8 Si as Na2SiO3·9H2O. A nitrification inhibitor dicyandiamide (DCD, 1 mg·L−1) was added to each nutrient solution to prevent ammonium oxidation. The nutrient solutions were changed every 3 days, and the pH was adjusted to 5.50 ± 0.05 daily with 0.1 M HCl and 0.1 M NaOH. Each treatment included five replicates in a completely randomized design. The spatial arrangement of the treatments was randomized to avoid edge effects in the greenhouse.

Plants were grown in the greenhouse at a temperature of 25 ± 5 °C. The maximum light intensity at midday was more than 1,500 μmol·m−2·s−1 at the leaf level. Physiological and biochemical parameters were measured at tillering stage about 35–45 days after transplanting.

Biomass measurements

Plant samples were harvested and separated into root, stem, and leaf fractions at tillering stage. All samples were oven-dried at 105 °C for 30 min and then at 70 °C to a constant weight. The leaf area was photographed and then analyzed using Image-Pro Plus 6.0 software.

Gas exchange measurements

At tillering stage, the photosynthetic rate of the newly expanded leaves was measured using a Li-Cor 6400 portable photosynthesis open system with red-blue light from 09:00 to 11:00 AM. During the measurements, the leaf temperature was maintained at 28 °C, and the photosynthetic photo flux density (PPFD) was 1,500 μmol photons·m−2·s−1. In addition, the CO2 concentration in the cuvette was adjusted to the atmospheric concentration of CO2 (390 μmol·mol−1), and the relative humidity was maintained at 40 %. After equilibration to a steady state, the gas exchange parameters, including light-saturated photosynthetic rate (A), stomatal conductance (gs), intercellular CO2 concentration (Ci) and transpiration rate (Tr), were recorded. More details about measurement can be found in our previous studies or other studies (Long et al. 1996; Long and Bernacchi 2003; Li et al. 2009). Water use efficiency (WUE) was calculated as the ratio of A to Tr (A/Tr), and the stomata limitation (Ls) was calculated from the equation of Ls = (A i -A)/A i (Farquhar and Sharkey 1982), where A i is the light-saturated photosynthetic rate when Ci was 390 μmol·mol−1.

Leaf water potential measurements

The leaf water potential was determined at 00:00–04:00 AM at tillering stage (Han et al. 2013). Newly expanded leaves were sampled, immediately followed by cutting into small pieces and spread evenly on the bottom of the sample cup to determine water potential using a WP4 Dewpoint Potential Meter (Decagon devices, Pullman, WA, USA).

Nitrogen absorption measurements

At tillering stage, each rice plant was transferred to a medium containing 20, 40 or 100 mg·L−1 N as equal moles of NO3 − and NH4 + for 8 h. Nitrogen absorption was assessed by the amount of NO3 − and NH4 + remaining in the medium after the measurement period. The levels of NO3 − and NH4 + were determined using an Auto Analyzer 3 digital colorimeter (BRAN+LuEBBE). The reagents used for NO3 − determination were 1 % sulfanilamide (C6H8N2O2S), 0.05 % N-(1-naphthyl) ethylenediamine dihydrochloride (NEDD), 10 % (V/V) H3PO4, 4 % NaOH, 0.3 % (V/V) H3PO4, 0.001 % CuSO4·5H2O, 0.01 % ZnSO4·7H2O and 0.2 % hydrazine sulphate (N2H4·H2SO4). The reagents used for NH4 + determination were 4 % tri-sodium citrate (C6H5Na3O7·2H2O), 4 % sodium salicylate (Na2C7H5O3), 0.1 % sodium nitroprusside {Na2[Fe(CN)5NO]·2H2O}, 4 % NaOH and 0.3 % dichloro-isocyanuric acid sodium salt dehydrate (DCI).

Water uptake rate and AQP activity measurements

The water uptake by intact plants with or without the addition of 0.1 mM HgCl2 to hydroponic solutions was determined by the depletion of nutrient solution (by weight) between 10:30 to 11:30 AM (1 h) at tillering stage. The contribution of Hg-sensitive AQP to the water uptake was calculated from the difference in the depletion of nutrient solution between the control and HgCl2-treated plants, and the water uptake of HgCl2-treated plants was generally regarded as an apoplastic contribution. The water uptake per leaf area (LWU) was calculated from the ratio of total water uptake to total leaf area per plant.

Root hydraulic conductance measurements

Hydraulic conductance was measured using a high-pressure flow meter (HPFM, Decagon Devices) at 10:00–12:00 AM at tillering stage. The HPFM was attached to the detached root with an Omnifit connector. Positive pressure (P i ) was applied at a constant rate of 3–7 kpa·s−1, allowing the slope of the relationship between flow (F) and pressure (P i ) to be obtained as a transient measurement (K). After measuring K, the root was oven-dried to a constant weight (W). The root hydraulic conductance (L r ; kg·s−1·g−1·MPa−1) was calculated as the following: L r = K/W; for method details, see Yang et al. (2012).

Root anatomy observations

At tillering stage, the root aerenchyma tissue was studied using fresh free-hand sections (Lux et al. 2005). The sections were cut from 6–7 cm segments above the root tip with a razor blade, transferred to a drop of water on a microscope slide and then observed using an Olympus BX51 light microscope. Images were recorded using a high-sensitivity CCD color camera system.

Lignin measurements

At tillering stage, the lignin content of the roots was estimated using the acetyl bromide procedure (Iiyama and Wallis 1990; Vander et al. 1998). The cell walls were prepared from frozen rice roots by grinding in liquid nitrogen, followed by three sequential extractions with 95 % ethanol, ethanol: n-hexane (1:2) twice and then distilled water. Lyophilized and vacuum oven-dried cell walls (20 mg) were transported into glass tubes with 2.5 ml acetyl bromide reagent [25 %(v/v) acetyl bromide in acetic acid] and then incubated in a 70 °C water bath for 30 min. Ice-cooled samples were then added to 2 ml of 2 M NaOH, 5 ml of acetic acid, and 0.2 ml of 7.5 M hydroxylamine hydrochloride, and the volume was adjusted with acetic acid to 20 ml. The absorbance of the resulting solution was measured using an ultraviolet (UV) spectrophotometer at 280 nm. The lignin content was calculated using the molar extinction coefficient of 17 L·g−1·cm−1 (Hatfield and Fukushima 2005).

Root porosity measurements

At tillering stage, the porosity of the roots was determined according to previous methods (Visser et al. 2000; Fan et al. 2003). Briefly, 0.8 g of fresh roots was cut into approximately 1–2 cm length segments and weighed (RFW, root fresh weight). A 25 ml Pyrex pycnometer flask filled with degassed water was weighed before (W1) and after (W2) the addition of these segments. Next, the root segments were transferred to a vial, which was also filled with degassed water and vacuum-infiltrated until no air bubbles appeared. These vacuum-infiltrated root segments were retransferred to the above degassed water-filled pycnometer, and the pycnometer was weighed again (W3). The root porosity was calculated as the following:

$$ \mathrm{Porosity}\left(\%\right)=\left[\left({\mathrm{W}}_3-{\mathrm{W}}_2\right)/\left(\mathrm{R}\mathrm{F}\mathrm{W}+{\mathrm{W}}_1-{\mathrm{W}}_2\right)\right]\times 100 $$

RNA extraction and semi-quantitative RT-PCR

At tillering stage, tissues of the rice roots and newly expanded leaves were sampled and quickly frozen in liquid nitrogen at 10:00–11:00 AM, then ground in a pre-cooled mortar with a pestle. RNA was extracted from the frozen tissue powder with TRIZOL reagent (Invitrogen). For reverse transcription-polymerase chain reaction (RT-PCR), first-strand cDNA was synthesized using M-MLV reverse transcriptase (Promega, Madison, WI, USA). The PCR conditions used were 95 °C for 3 min followed by 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. The PCR products were subjected to agarose gel electrophoresis and stained with ethidium bromide. The signal intensity of the stained bands was imaged using a charge-coupled device (CCD) camera. All of the primers and cycle numbers used for PCR are listed in Table 1.

Table 1 Sequences of primer pairs and cycles used for semi-quantitative PCR
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Statistical analysis of the data

Differences among treatments were analyzed with the SAS 9.0 statistical software package, using the analysis of variance (ANOVA) program. Significant differences (P < 0.05) among treatments are indicated in the tables and figures by different letters.

Results

Effects of the various N supplies on plant growth

The shoot dry mass was increased with increasing N supply in both cultivars, while the root dry mass was decreased, resulting in a reduced root/shoot ratio under high N supply (Table 2). Compared with low N supply, the root/shoot ratios under high N supply were decreased by 56 % and 57 % in Shanyou63 and Yangdao6, respectively. The leaf area was increased significantly in Shanyou63, but there was no difference between moderate-N-supplied plants and high-N-supplied plants in Yangdao6.

Table 2 Effects of various N supplies on the plant dry weight (g·plant−1), root/shoot ratio, leaf area (cm2·plant−1) and the number of tillers (No.·plant−1) in rice seedlings
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Effects of the various N supplies on photosynthetic parameters

Light-saturated photosynthetic rate (A) was increased significantly under high N supply in both rice cultivars. Under low N and moderate N supplies, A was lower in Yangdao6 than in Shanyou63, while there was no significant difference between the two cultivars under high N supplies (Table 3). This finding indicated that, compared with the low N supply, the increase in A under high N supply was more pronounced in Yangdao6 than in Shanyou63.

Table 3 Effects of various N supplies on photosynthetic parameters and WUE in rice seedlings. Net photosynthetic rate (A, μmolCO2·m−2·s−1), stomatal conductance (gs, molCO2·m−2·s−1), intercellular CO2 concentration (Ci, μmol·mol−1), transpiration rate (Tr, mmolH2O·m−2·s−1), water use efficiency (WUE, μmolCO2·mmol−1H2O), and stomata limitation (Ls)
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Stomatal conductance (gs) showed no significant difference under the three N supplies in Shanyou63, while it was increased in Yangdao6 supplied with high N (Table 3). The intercellular CO2 concentration (Ci) was decreased from 319 to 303 μmol mol−1 in Shanyou63 and was approximately 310 μmol mol−1 under all three N supplies in Yangdao6. The leaf transpiration rate (Tr) showed no significant differences in Shanyou63, while an increase was observed in Yangdao6. A comparison of the two cultivars revealed that the Tr was lower in Yangdao6 than in Shanyou63, particularly under low N and moderate N supplies. WUE was increased with increasing N supplies in both cultivars, and this effect was comparable under all three of the N supplies between both cultivars (Table 3 and Fig. 1). Yangdao6 had lower stomatal limitation (Ls) than that of Shanyou63 under high N supply.

Fig. 1
figure 1

The relationship between WUE and the water uptake rate (a), WUE and the N uptake rate (b). Filled squares and open squares represent Shanyou63 and Yangdao6, respectively. The lines represent linear regression, and the regression equations are (a) y = 0.0249x + 1.5876, R 2 = 0.9817, P = 0.000; (b) y = 1.0995x + 1.1019, R 2 = 0.8789, P = 0.006

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Effects of the various N supplies on plant water status and related parameters

The root hydraulic conductance (L r ) was also increased under high N supply in both rice cultivars (Fig. 2). Compared with the low N supply, a 168 % increase was observed in Shanyou63 and a 324 % increase was observed in Yangdao6 under high N supplies. There was no difference in the low-N-supplied plants between the two cultivars, but higher levels of L r were observed in Yangdao6 compared with Shanyou63 under both moderate and high N supplies.

Fig. 2
figure 2

Effects of various N supplies on the root hydraulic conductance of rice seedlings. Plant growth conditions are the same as those shown in Table 2. Data are represented as the mean of five replicates, and the bars indicate the SD. Significant differences (P < 0.05) are indicated by different letters

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The leaf water potential (ψleaf) was decreased significantly in Shanyou63 supplied with high N, but no significant difference was observed in Yangdao6. Under low N supply, ψleaf was similar between the two cultivars, but it was lower in Shanyou63 than in Yangdao6 under both moderate and high N supplies (Fig. 3).

Fig. 3
figure 3

Effects of various N supplies on the leaf water potential of newly expanded leaves in rice seedlings. Plant growth conditions and data representations are the same as those shown in Fig. 2

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The root water uptake rate was increased with increases in the N supply in both rice cultivars (Table 4). Compared with low N supply, this parameter was increased by 292 % and 255 %, respectively, in Shanyou63 and Yangdao6 under high N supply. Under low or moderate N supply, the root water uptake rate was similar in both cultivars, while under high N supply, it was higher in Yangdao6 than in Shanyou63.

Table 4 Effects of various N supplies on the water uptake rate (with or without HgCl2) based on per unit root dry weight for 1 h [gH2O·(g root DW)−1·h−1] and the decrease in response to the addition of 0.1 mM HgCl2 to rice plants. The total water uptake per plant normalized to leaf area (LWU) under control condition was also calculated as the ratio of total water uptake to total leaf area [gH2O·(m Leaf)−2·s−1]
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The addition of 0.1 mM HgCl2 significantly suppressed the root water uptake rate in both cultivars, but it also increased with an increased N supply. Under high N supply, Yangdao6 had an increased water uptake rate than that of Shanyou63 in response to HgCl2 treatment. When the HgCl2 percentage of inhibition was calculated, it was shown that the magnitude of the decrease was greater and this effect was more pronounced in Yangdao6 than in Shanyou63 (Table 4). Compared with moderate N supply, there was a significant increase in the water uptake per leaf area (LWU) under high N supply in Yangdao6, but not in Shanyou63 (Table 4).

Effects of the various N supplies on root aerenchyma, lignin and porosity

To study the effects of root anatomy and morphology on the water uptake, the aerenchyma tissue was observed, and the root porosity and lignin content were also measured. The transverse freehand sections from the root showed evidence of aerenchyma and fine intercellular spaces (Fig. 4). High N visibly decreased the formation of aerenchyma in both rice cultivars. In addition, Yangdao6 had less aerenchyma than did Shangyou63 under high N supply.

Fig. 4
figure 4

Observation of aerenchyma tissue (arrows) and fine intercellular spaces (arrowheads) in rice root by free-hand section using bright field microscopy. SY and YD represent Shanyou63, Yangdao6; L, M and H represent low-N, moderate-N and high-N, respectively. Scale bars = 200 μm

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Root porosity was also significantly decreased in both cultivars, but there was no difference between moderate and high N supplies in Shanyou63 (Fig. 5). Compared with a low N supply, root porosity was decreased by 19 % and 56 % in Shanyou63 and Yangdao6, respectively, under high N supply.

Fig. 5
figure 5

Effects of various N supplies on the root porosity of rice seedlings. Plant growth conditions and data representations are the same as those shown in Fig. 2

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The lignin content of the roots exhibited a similar tendency to that of the root porosity. Compared with low N supply, the former parameter was decreased by 24 % and 26 % under high N supply in Shanyou63 and Yangdao6, respectively. There was no difference in the lignin content between two cultivars at each N supply (Fig. 6).

Fig. 6
figure 6

Effects of various N supplies on the root lignin content of rice seedlings. Plant growth conditions and data representations are the same as those shown in Fig. 2

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Effects of the various N supplies on the expression of the leaf and root AQP genes

The mRNA levels of the plasma membrane intrinsic proteins (PIP2s) genes were analyzed by reverse transcription-PCR (RT-PCR) in the leaves and roots. Three replicates rendered qualitatively similar results. Representative examples are shown in Figs. 7 and 8. The expression levels of OsPIP2;4 increased with increasing N supplies in the leaves of both rice cultivars. The leaf mRNA levels of OsPIP2;1, OsPIP2;2 and OsPIP2;3 were increased in Yangdao6, while there was no difference in Shanyou63. The root mRNA levels of OsPIP2;1 and particularly OsPIP2;4, OsPIP2;5 were significantly increased with increasing N supplies in both rice cultivars. The other PIPs expression in roots was nearly the same among the various N supplies.

Fig. 7
figure 7

Effects of various N supplies on leaf AQP expression in rice seedlings. Total RNA fractions were prepared from the roots and newly expanded leaves as described in the Materials and Methods section. The mRNA levels were analyzed by semi-quantitative RT-PCR using specific primers as shown in Table 1. The PCR products were separated on agarose gel and imaged after ethidium bromide staining. Photographic images are shown at the bottom of each graph. The numbers in parentheses indicate the number of PCR cycles. SY and YD represent Shanyou63, Yangdao6; L, M and H represent low-N, moderate-N and high-N, respectively. The OsActin (ACT) gene was used for an internal standard

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Fig. 8
figure 8

Effects of various N supplies on root AQP expression in rice seedlings. The procedure and data representations are the same as those shown in Fig. 7

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Discussion

Water absorption, photosynthetic rate and WUE are increased under high N supply

Plant growth or crop productivity is always correlated with the N supply, and high N supply can improve photosynthesis and the shoot dry weight but inhibit root growth, resulting in a decreased root/shoot ratio (Linkohr et al. 2002; Tian et al. 2005; Li et al. 2009). In the present study, we observed similar patterns in both rice cultivars, with an increased shoot weight and lower root/shoot ratio under high N supply (Table 2). Analysis of the gas exchange parameters illustrated that the photosynthetic rate (A) was increased with increasing N supply in both rice cultivars, but the magnitude of the increase varied. Compared with low N supply, A was increased by 17 % and 43 % in Shanyou63 and Yangdao6, respectively, under high N supply. This finding may have been caused by a lower stomatal limitation (Ls) in Yangdao6 compared with Shanyou63 under high N supply (Table 3).

Compared with low N treatment, the instantaneous WUE was increased by 22 % and 26 % under high N supply in Shanyou63 and Yangdao6, respectively; these findings are consistent with those obtained in previous studies (Guehl et al. 1995; Livingston et al. 1999; Hobbie and Colpaert 2004). Livingston et al. (1999) indicated that the higher WUE of white spruce with increased N supply was due to a decrease in gs. In our study, there was an increase in A, while gs was constant or slightly increased with increasing N supply, particularly in Yangdao6 (Table 3). Thus, we suggest that gs was not responsible for the higher WUE in rice. We found a strong positive correlation not only between WUE and the N uptake rate but also between WUE and the water uptake rate (Fig. 1), indicating that the N uptake or water uptake might be a crucial factor influencing changes in WUE.

It is well known that the roots are the most important factor in water uptake for plant water relations. The root hydraulic conductivity (L r ) reflects the ability of the root to absorb water and may vary in response to external or internal factors, such as the water status due to drought or salinity or the demand for water from the shoot due to transpiration, the nutritional state, changes in the anatomy and structure of the root and AQP activity (Steudle 2000a). The decreased L r temporarily decreased the xylem hydrostatic potential and consequently the leaf water potential (Else et al. 2001), which significantly affected plant water relations.

The present study showed that with an increasing N supply, L r was increased (Fig. 2), which was accompanied by increased water absorption (Table 4). There was a significant positive correlation (R 2 = 0.75) between water absorption and the hydraulic conductance. The leaf water potential was also affected by the various N supplies, while each of the two cultivars showed a distinct response (Fig. 3). Yangdao6 had an increased leaf water potential compared with that of Shanyou63 under high N supply; these results suggested that Yangdao6 had a more favourable water status due to the higher L r .

Root anatomy, morphology and AQPs are responsible for the ability to absorb water

In the root cylinder, there are three parallel pathways for water movement, i.e., the apoplastic, symplastic and transcellular pathways, with the latter two representing cell-to-cell pathways (Steudle 2000b). The apoplastic pathway resistance is strongly associated with the formation of the aerenchyma (Yang et al. 2012) and suberized and/or lignified cell walls (North and Nobel 1995; Hose et al. 2001). Many studies have indicated that N-, P- or sulfur (S)-deficiency induces aerenchyma formation in the root (Drew et al. 1989; Bouranis et al. 2003; Fan et al. 2007; Postma and Lynch 2011) and an increase in the lignin and/or suberin of the root or stem (Entry et al. 1998; Fritz et al. 2006; Pitre et al. 2007). The formed aerenchyma and lignin will inevitably inhibit L r and water uptake via the apoplastic pathway. Taken together, our results illustrated that with an increasing N supply, aerenchyma formation, porosity and lignin were significantly decreased in the root (Figs. 4, 5 and 6), thereby improving the apoplastic water uptake (Table 4). Both porosity and lignin showed significant negative correlations with the water uptake rate, which was consistent with the results of previous studies (Yang et al. 2012). Under high N supply, the aerenchyma and root porosity of Yangdao6 were significantly lower than those of Shanyou63, which might underlie the increased ability to absorb water in Yangdao6.

In the cell-to-cell pathway, the water molecules need to diffuse through the plasma membrane into the cytoplasm. During this process, the AQPs embedded in the membrane play an important role in water transport (Steudle and Peterson 1998; Steudle 2001; Kaldenhoff and Fischer 2006). Under well-watered conditions, the contribution of AQP to L r was approximately 35 %–75 % (Maggio and Joly 1995; Lu and Neumann 1999; Wan and Zwiazek 1999; Barrowclough et al. 2000; North and Nobel 2000; Martre et al. 2001). Hacke et al. (2010) showed that seven AQPs were upregulated in the stem xylem of hybrid poplar under high N conditions. It is also possible that N or P deficiency may have decreased the activity and/ or content of AQPs, further reducing the L r (Carvajal et al. 1996; Clarkson et al. 2000). In the present study, Hg-sensitive AQP activity was determined by the discrepancy in the water uptake rate with and without the addition of 0.1 mM HgCl2, an AQP inhibitor (Javot and Maurel 2002; Wan et al. 2004; Beaudette et al. 2007). This finding showed that independent of the N supply and compared with the control (without HgCl2), the water uptake rates in all HgCl2 treatments decreased in both rice cultivars (Table 4). The decreased percentage represents the Hg-sensitive AQP activity, which was higher under high N supply, demonstrating that the high-N-supplied plants had an increased ability to take up water via the cell-to-cell pathway. We also investigated the transcription levels of OsPIPs that is a subfamily of AQPs using RT-PCR. The root mRNA expression levels of OsPIP2;1, OsPIP2;4 and OsPIP2;5 were significantly increased with increasing N concentration in both rice cultivars, which may also result in an increased ability to take up water via the cell-to-cell pathway of high-N plants. Upon comparison of the two rice cultivars, the mRNA expression levels of Shanyou63 were similar or slightly higher than those of Yangdao6, but the contribution of Hg-sensitive AQPs to water uptake was higher in Yangdao6 than in Shanyou63 regardless of the N supply (Table 4); these findings suggest that the root AQP activity plays a more important role in water uptake in Yangdao6 than in Shanyou63, which might account for the increased ability to take up water in Yangdao6 than in Shanyou63 under high N supply.

Differential response of water absorption under the various N supplies among the rice cultivars

Although the ability to take up water and the L r both increased with the N supply, they were significantly higher in Yangdao6 than in Shanyou63 under high N supply, and the WUE was lower in Shanyou63 (Table 3). As previously described, this effect was caused by differences in the aerenchyma, root porosity and root AQP activity between Yangdao6 and Shanyou63. In addition, although the root/shoot ratio decreased with the N supply, and proportion of the decrease was consistent from Shanyou63 to Yangdao6 (Table 2), the transpiration rate (Tr) in Shanyou63 was higher than that in Yangdao6, suggesting that Shanyou63 had a much higher water loss than that of Yangdao6. In addition, Shanyou63 had a reduced ability to absorb water than that of Yangdao6 under high N supply. When we calculated the total water uptake per leaf area, the results also showed the significant lower values in Shanyou63 (Table 4). This indicates relative water absorption ability on the base of leaf area under high N supply was lower in Shanyou63 compared with Yangdao6. Some studies reported that the decrease in leaf water potential was correlated with the changes in structure of cell or tissue of plants (Morgan 1986; Shangguan et al. 2000) or osmolytes, such as proline (Nayyar 2003). The changed leaf structure and/or osmolytes of Shanyou63 under high N condition might be the potential candidate for the decreased leaf water potential. Moreover, the mRNA expression levels in the leaves of Yangdao6 were higher than those of Shanyou63, particularly OsPIP2;3 and OsPIP2;6 under high N supply. Parent et al. (2009) showed that water-deprived maize plants with high AQP expression and activity had an increased rate of recovery of leaf water potential after re-watering. Higher AQP expression in the leaf may be used for fine water regulation, which decreases water loss and increases WUE, thereby contributing to the maintenance of a favorable plant water status. In this study we provided even concentrations of nitrate and ammonium, we do not explore which N form may dominate the physiological effects observed here. But, in our previous studies there were no significant differences in L r , root xylem sap flow rate, aerenchyma formation and root porosity between the nitrate fed plants and ammonium fed plants under non-water stress condition (Yang et al. 2012). The differences in nitrogen form effects on water relations were mainly observed under water stress condition. By varying nitrate and ammonium concentrations, the contribution of the different N forms to the physiological effects will be explored in a future study.

It can be concluded that under high N supply, decreased aerenchyma, porosity and lignin content of the roots, as well as increased AQP expression, were responsible for the high water absorption rate. Compared with the characteristics of Shanyou63, the greatly reduced aerenchyma and porosity of the root, higher hydraulic conductivity, and higher AQP activity in Yangdao6 resulted in increased WUE and water potential. These findings indicated that increasing N fertilization might increase the water absorption, WUE and production of rice plants via changes in the root properties.