Low O2 supply is involved in the poor growth in root-restricted plants of tomato (Lycopersicon esculentum Mill.)

Environmental and Experimental Botany 61 (2007) 181–189

Low O2 supply is involved in the poor growth in root-restricted plants of tomato (Lycopersicon esculentum Mill.) Kai Shi a , Wen-Hai Hu a , De-Kun Dong a , Yan-Hong Zhou a , Jing-Quan Yu a,b,∗ a

Department of Horticulture, Huajiachi Campus, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, PR China b Key Laboratory of Horticultural Plant Growth, Development and Biotechnology, Agricultural Ministry of China, Kaixuan Road 268, Hangzhou 310029, PR China Received 8 January 2007; accepted 26 May 2007

Abstract To investigate whether O2 deficit is involved in the depressed shoot growth under root restriction condition, tomato (Lycopersicon esculentum Mill.) plants were grown hydroponically and subjected to root restriction with or without supplemental aeration. Plant biomass, leaf nutrient concentration, dissolved O2 concentration in nutrient solution, root respiration, root hydrolytic ATPase activities, xylem sap abscisic acid (ABA) concentration, water relations, leaf gas exchange, and root cell viability were investigated throughout the experiment. Root restriction significantly depressed root and shoot growth as early as 15 days after treatment and this depressive effect was alleviated by vigorous aeration around the restricted root zone. Growth suppression by root restriction occurred concomitantly with sharp decreases in dissolved O2 concentration in solution together with significant decreases in root total respiration, cytochrome pathway capacity, hydrolytic ATPase activities, and root cell viability. However, no such decreases were found in well aerated root restriction plants. Root restriction-induced growth suppression was independent of nutrients level in leaves and was not primarily related to the decline of leaf water potential or the gas exchange. Root restriction resulted in an increase in xylem sap ABA concentration from day 15 to the end of the experiment but no such effect was observed in well aerated plants. It is likely therefore, that O2 was one of the main limiting factors to the reduced shoot growth under root restriction condition. © 2007 Elsevier B.V. All rights reserved. Keywords: Root restriction; Low oxygen; Lycopersicon esculentum; Root respiration; Photosynthesis; Xylem sap ABA; Plasma membrane H+ -ATPase; Root viability

1. Introduction Growing plants in a limited rooting volume, root restriction, is a powerful technique to improve the utilizing efficiency of agricultural resources such as space, water and nutrition. It can also be used to improve the fruiting efficiency and fruit qualities and to solve the problems caused by soil sickness or soil contaminations (Bar-Tal and Pressman, 1996; Mataa and Tominaga,

Abbreviations: ABA, abscisic acid; Asat , light-saturated rate of the CO2 assimilation; Ci , intercellular CO2 concentration; COX, cytochrome c oxidase; Cyt pathway, cytochrome pathway; FDA, fluorescein diacetate; gs , stomatal conductance; PI, propidium iodide; PM-H+ -ATPase, plasma membrane H+ -ATPase; RR, root restriction plants; RR + O2 , root-restriction plants with aeration; SHAM, salicylhydroxamic acid; V-H+ -ATPase, vacuolar H+ -ATPase ∗ Corresponding author at: Department of Horticulture, Huajiachi Campus, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, PR China. Tel.: +86 57186971120; fax: +86 57186049815. E-mail address: [email protected] (J.-Q. Yu). 0098-8472/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2007.05.010

1998). Several techniques such as potted culture, trough culture with a limited substrate or soil and nutrient film technique have also been developed for commercial production (Spensley et al., 1978; Chen et al., 1999). However, disadvantages such as the reduction of leaf area, plant height and biomass production as well as root death also arise from root restriction both in soil culture and soilless culture (Peterson et al., 1991a; Goto et al., 2002). Although reduced shoot growth by root restriction has been well recognized, the detailed mechanism involved remains unclear. Hameed et al. (1987) presented strong evidence of the involvement of drought stress in the decrease of leaf growth and net CO2 assimilation rate even though the plants were grown in nutrient solution. Carmi and Heuer (1981) showed that the reduced plant growth under root restriction condition was not due to water or nutrient deficit and proved that exogenous application of gibberellins and cytokinins partially overcame the suppression of shoot growth. Choi et al. (1997), however, observed that root restriction of tomato plants

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strongly suppressed transport of Ca2+ ions from roots to new leaves and apices. Additionally, several studies showed that root-synthesized ABA was also involved in the inhibition of shoot growth (Ternesi et al., 1994; Liu and Latimer, 1995), while others argued that the slight increase of xylem sap ABA concentration in root-restricted plants could not fully account for the large reductions in shoot dry mass production (Ismail and Davies, 1998). This discrepancy is largely attributed to differences in species, cultural methods, growth conditions and others (Ismail and Davies, 1998). Furthermore, it remains unclear whether these effects were the primary effects or the secondary ones. While the role of O2 has been widely investigated in stressed plants (Szal et al., 2003; Geigenberger, 2003; Fukao and BaileySerres, 2004), there is little information on the influence of root restriction on O2 level and associated physiological changes. O2 is essential for the oxidative phosphorylation, which provides ATP as the primary source of energy for cellular metabolism. Low O2 restricts respiration, energy metabolism and leads to a decrease in adenylate energy state (Geigenberger, 2003). Kharkina et al. (1999) also observed that a decrease in root respiration paralleled the shift in shoot:root ratio for the rootrestricted plants. When roots grow in a confined rooting volume, the root system is densely matted and leads to O2 diffusion and supply being inevitably altered. However, there are no direct evidences to support the hypothesis that O2 deficiency is one of the primary limiting factors responsible for the inhibited plant growth by root restriction. To test the hypothesis that depressed plant growth in root-restricted plants is related to O2 deficiency, tomato (Lycopersicon esculentum Mill.) plants were grown hydroponically either subjected to root restriction or not at different rhizosphere O2 levels. Plant biomass, leaf nutrient assimilation, dissolved O2 concentration in nutrient solution, root respiration, root hydrolytic ATPase activities, xylem sap abscisic acid (ABA) concentration, water relations, leaf gas exchange, and root cell viability were investigated accordingly. 2. Materials and methods 2.1. Plant material Tomato (Lycopersicon esculentum Mill. cv. Hezuo 903) seeds were sown in growth medium containing a mixture of vermiculite and perlite (1:1, v:v) in trays in a greenhouse. Twenty days later, groups of six seedlings were transferred into 15 l tanks (40 cm × 25 cm × 15 cm) filled with half-strength Enshi nutrient solution (Yu and Matsui, 1997). The solution was continuously aerated with an air-pump and the temperature was controlled at 25 ◦ C/20 ◦ C (day/night) using thermostats. The mean daily maximum and minimum air temperature in the greenhouse was maintained at 29 and 17 ◦ C, respectively. After 1 week of preculture, roots of each plant were moved into a small cylinder for the root restriction treatment (RR). The

small cylinders were made from nylon bags (mesh < 0.05 mm in diameter) with a capacity of 100 ml (3 cm in diameter, 14 cm in depth) and was sustained with a stainless steel skeleton, thus allowing solution exchanges but not root penetration. Meanwhile, for root restriction plants with supplemental aeration (RR + O2 treatment), half of the cylinders were aerated by an air pump. Plants grown directly in the 15 l tanks served as controls, and preliminary experiment showed that growth for plants grown in well-aerated 15-l tanks was not different from that grown in the 30- and 40-l tanks for a period of 30 days. In the experimental system, each tank included one treatment, and there were 15 tanks (six plants per tank) for every treatment. Vigorous aeration was supplied to all tanks in order to ensure adequate circulation of the nutrient solution in the 15 l containers. 2.2. Growth measurements and leaf element concentration determinations Plant materials were harvested on days 0, 5, 10, 15, 20, and 25 after start of treatment and separated into leaves, stems and roots. Dry mass of shoot and roots were determined after 3 days in a forced draught oven at 80 ◦ C. On day 25, the dried leaf materials were ground into powder to pass through a 40-mesh screen for later element concentration analysis. Leaf samples of 300 mg were digested with concentrated sulphuric acid at 250 ◦ C. The total N and P concentrations in the digested solutions were analyzed using Kjeldahl method and the molybdovanadate procedure (Boltz and Lueck, 1958) with a spectrophotometer (Model UV-2401PC; Shimadzu, Tokyo, Japan), respectively. K, Ca and Mg concentrations were assayed with an atomic absorption spectrophotometer (Model AA-6300, Shimadzu, Tokyo, Japan). 2.3. Analysis of dissolved O2 concentration in solution, root respiration and cytochrome c oxidase activity O2 concentration in the nutrient solution within the cylinder was measured after calibration was executed with oxygensaturated and sodium dithionite-saturated solution at standard pressure and constant 25 ◦ C. Root respiration was measured following the method of Millenaar et al. (2002) and Atkin et al. (2002) with some modifications. Generally, roots were cut into 2-mm long segments and 0.1 g was placed into 2 ml of air-saturated 20 mM potassium phosphate buffer (pH 6.8) in a Clark-type electrode (Hansatech, King’s Lyn, UK). Cytochrome pathway (Cyt pathway) capacity was measured at 25 ◦ C by the addition of 20 mM salicylhydroxamic acid (SHAM) to inhibit alternative pathway respiration. Cytochrome c oxidase (COX) activity (EC 1.9.3.1) was measured according to Millenaar et al. (2002). In brief, sample (0.3 g fresh weight) was ground in liquid N2 , using a mortar and pestle and then suspended in a total volume of 1.2 ml with 0.1 M potassium phosphate buffer (pH 7.2) and 0.1% (v/v) Triton-X 100. The extract was centrifuged at 13,000 × g for 5 min at 4 ◦ C and the supernatant was used for the spectrophotometrical assay. COX activity was

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measured at 550 nm (25 ◦ C) in the presence of 20 ␮M reduced cytochrome c in 10 mM phosphate buffer (pH 7.2). Prior to the assays, cytochrome c (in phosphate buffer pH 7.2) was reduced with sodium dithionite and the excess dithionite was then removed by a gentle flow of normal air in the solution for a few minutes.

2.4. Plasma and tonoplast-enriched membrane isolation and the measurements of plasma membrane H+ -ATPase and vacuolar H+ -ATPase activities Plasma membrane (PM) and tonoplast-enriched membrane fractions were isolated on sucrose step gradients according to Garbarino and DuPont (1988) with some modifications. In brief, 5 g of fresh roots was homogenized using a mortar and pestle in 10 ml of ice-cold buffer containing 250 mM sucrose, 10% (w/v) glycerol, 1% (w/v) polyvinylpyrrolidone40, 5 mM EDTA, 0.5 mM EGTA, 0.5% (w/v) bovine serum albumin, and 60 mM HEPES-Tris buffer (pH 7.2). Just before use, 2 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonylfluoride were added to the buffer. The homogenate was filtered through four layers of cheesecloth and centrifuged at 10,000 × g for 30 min. The resulting supernatant was then centrifuged at 85,000 × g for 40 min. The microsomal pellet was first suspended in an ice-cold buffer containing 10 mM HEPESTris (pH 7.2) with 250 mM sucrose, 2 mM DTT, 5 ␮g ml−1 chymostation, and then layered on a 15%/33%/45% (w/w) discontinuous sucrose gradient that contained 20 mM HEPESTris buffer (pH 7.2), 2 mM DTT, and 5 mM EDTA, 0.5 mM EGTA. The gradient was centrifuged at 100,000 × g for 2.5 h. The tonoplast-enriched fraction was collected at the 15%/33% interface and the plasma membrane-enriched fraction was collected at the 33%/45% interface. The tonoplast-enriched and the plasma membrane-enriched fraction was then diluted to 1 and 3 volumes, respectively, with ice-cold buffer consisting of 20 mM HEPES-Tris buffer (pH 7.2), 2 mM DTT, 5 mM EDTA, 0.5 mM EGTA, and centrifuged at 100,000 × g for 1 h. The pellet was resuspended in 1 ml buffer containing 20 mM HEPES-Tris buffer (pH 7.2), 3 mM MgCl2 • 6H2 O, 0.5 mM EGTA 300 mM sucrose. Protein was quantified according to the method of Bradford (1976) using bovine serum albumin as a standard. Hydrolytic ATPase activities were determined by measuring the release of Pi colorimetrically (Fiske and Subbarow, 1925). The reaction was initiated by the addition of 50 ␮l 30 mM disodium-ATP. After 30 min of incubation at 37 ◦ C, the reaction was stopped with 1 ml of stopping regents [2% (v/v) concentrated H2 SO4 , 5% (w/v) SDS, and 0.7% (w/v) (NH4 )2 MoO4 )] followed immediately by 50 ␮l of 10% (w/v) ascorbic acid. After color development was completed, A660 was measured by means of a spectrophotometer. In the measurement, plasma membrane H+ -ATPase (PM-H+ -ATPase) (EC 3.6.1.35) and vacuolar H+ -ATPase (V-H+ -ATPase) (EC 3.6.1.34) activities were measured with and without Na3 VO4 and KNO3 respectively because Na3 VO4 and KNO3 were the effective inhibitors to PM-H+ -ATPase and V-H+ -ATPase respectively. The difference between them was attributed to the enzyme activities.

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2.5. Xylem sap ABA analysis In order to collect sap from the plants, the main stem was cut 2 cm above the root-shoot interface below the cotyledons then followed the procedure of Bahrun et al. (2002) using root pressure. Sap from 3 or 4 plants was bulked for each sample and stored at −80 ◦ C. Prior to further analysis for ABA concentration, the sap samples were thawed, purified and dried as described by Yu and Matsui (1994). The fraction containing ABA was added with fresh N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine before they were subjected to gas chromatograph (Shimazu GC-14B, Kyoto, Japan) analysis. A DB-1 capillary column (30 m × 0.25 mm, J&W Scientific) was used for the GC with N2 as carrier gas. The injection temperature was set at 280 ◦ C. The column temperature was elevated from 140 to 255 ◦ C at a rate of 5 ◦ C min−1 . 2.6. Leaf water potential and gas exchange measurements Leaf water potential was measured on the second fully developed leaves according to Bahrun et al. (2002). Leaf discs (5.0 mm in diameter) were sampled and incubated in a C-52 chamber (Wescor Inc., Logan, Utah, USA) for at least half an hour before reading the water potential using a dew-point microvoltmeter (HR-33T, Wescor Inc., Logan, Utah, USA). Gas exchange measurements were made using a CO2 and H2 O gas exchange system (LI-6400; LI-COR, Inc., Lincoln, NE, USA) mounted with a red/blue LED light source (640002B, LI-COR) on the second fully developed leaves. The leaf temperature, leaf-to-air vapor pressure deficit, reference CO2 concentration and PPFD were maintained at 25 ◦ C, 1–1.3 kPa, 390 ␮mol mol−1 and 1000 ␮mol m−2 s−1 respectively. 2.7. Measurement of root viability Viability of root tips was determined by staining the cells with fluorescein diacetate (FDA)-propidium iodide (PI) by using the method of Jones and Senet (1985) with some modifications (Ishikawa and Wagatsuma, 1998). Briefly, the roots were stained for 10 min with a mixture of FDA (12.5 ␮g ml−1 )–PI (5 ␮g ml−1 ), then the root tips were observed with a confocal microscope (LSM510, Carl-Zeiss, Germany) equipped with an argon–He Ne laser. The wavelengths for excitation and emission for FDA were 488 nM and 505–530 nm, respectively, and those for PI was 543 and 585 nm, respectively. Image processing was completed using the software supplied by the confocal microscope manufacturer. 2.8. Statistical methods Throughout the experiment, all measurements were performed randomly using four replicates. SAS 8.0 (SAS Institute, Cary, NC) for windows was used for statistical analysis. The data were analyzed with a one-way analysis of variance and differences between treatments were separated by the Tukey’s HSD test at the P < 0.05 level.

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3. Results 3.1. Effects of root restriction and aeration on plant growth and nutrient level in leaves Root restriction treatment (RR) had no significant effects on the shoot and root growth during the first 10 days, but significantly decreased shoot and root dry mass from 15 days (Fig. 1). At final harvest, root biomass, shoot biomass and total plant biomass of RR plants were reduced by 65.9, 24.6 and 30.2%, respectively, as compared with the control. In contrast, shoot biomass for well aerated root-restriction plants (RR + O2 treatment) was not significantly different from that of the control. However, suppressed root growth was found in RR + O2 plants. Higher shoot:root ratio were observed in root-restricted plants, especially in RR treatment (Fig. 1C). Analysis of nutrient level in leaves showed that RR treatment significantly increased N concentration but reduced Mg concentration in leaves. In addition, there was also a slight decrease of K concentration in RR + O2 leaves (Table 1). However, no significant quantitative changes were observed among different treatment for other elements analyzed. 3.2. Effects of root restriction and aeration on dissolved O2 concentration in solution, intact root respiration and COX activity Dissolved O2 concentration in nutrient solution kept constant at about 250 ␮M for control plants. After RR treatment, however, it declined to 158 ␮M at 20 days. Supplemental aeration increased the O2 concentration to about 230 ␮M, a value close to that of control (Fig. 2A). Analysis of root respiration showed that the roots of RR treatment exhibited a significant reduction in the total respiration after 15 days of treatment. In contrast, this reduction was not observed in RR + O2 plants (Fig. 2B). RR treatment also resulted in significant decreases of Cyt pathway capacity and COX activity at 20 days (Fig. 3). However, Cyt pathway capacity and COX activity in RR + O2 treatment plants were close to the control levels (Fig. 3). 3.3. Effects of root restriction and aeration on PM-H+ -ATPase and V-H+ -ATPase activities in roots RR treatment dramatically inhibited the activities of PMH+ -ATPase and V-H+ -ATPase from 15 days to the end of the experiment (Fig. 4). At 25 days, PM-H+ -ATPase and V-H+ -

Fig. 1. Changes in shoot and root dry mass (A), total plant dry mass (B) and shoot:root ratio (C) for control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2 , filled triangles) plants. Data are the means of four replicates with standard deviations shown by vertical bars. Different letters are significantly different between the treatments at 5% level according to Tukey tests.

ATPase activities for the RR treatment were only 81.3 and 47.4% of the control, respectively. By comparison, no decreases in the activities of these enzymes were found in RR + O2 treatment.

Table 1 Effects of root restriction and aeration on N, P, K, Ca, and Mg concentration in tomato leaves after 25 days of treatment Treatment

Control RR RR + O2

Nutrient concentration in leaves (mg g−1 DW) N

P

K

Ca

Mg

30.0 ± 1.1 b 47.7 ± 5.3 a 30.6 ± 3.4 b

7.3 ± 0.1 a 7.1 ± 0.8 a 6.2 ± 0.1 a

95.1 ± 4.8 a 88.0 ± 4.7 ab 84.1 ± 2.7 b

19.2 ± 0.3 a 16.3 ± 2.1 a 19.4 ± 1.8 a

8.2 ± 0.1 a 5.8 ± 0.6 b 7.1 ± 0.5 a

Data are shown as the means of four replicates ± S.D. Values followed by same letters in a column are not significantly different according to Tukey tests at 5% level.

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Fig. 2. Changes in the dissolved O2 concentration in nutrient solution (A) and the root total respiration (B) of control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2 , filled triangles) plants. Data are the means of four replicates with standard deviations shown by vertical bars. Different letters are significantly different between the treatments at 5% level according to Tukey tests.

with time. However, no such difference was observed in RR + O2 plants relative to control. 3.5. Effects of root restriction and aeration on leaf water potential and gas exchange

Fig. 3. Effects of root restriction and aeration on cytochrome pathway (Cyt pathway) capacity and cytochrome c oxidase (COX) activity after 20 days of treatment. Data are the means of four replicates with standard deviations shown by vertical bars. Different letters are significantly different between the treatments at 5% level according to Tukey tests.

3.4. Effects of root restriction and aeration on ABA concentration in xylem sap Xylem sap ABA concentration was also analyzed to examine whether root restriction-induced changes in plant growth was related to root-sourced chemical signals. As shown in Fig. 5, RR treatment resulted in a significant increase in xylem sap ABA concentration from days 15 to 25. ABA concentration increased by 75.1% 15 days after treatment and this difference increased

There was no significant difference in leaf water potential among all treatments before 15 days (Fig. 6A). However, from 15 days onward, leaf water potential decreased significantly in RR plants and declined to a value of −1.28 MPa at 20 days. In RR + O2 treatment, however, the value was close to the control level and little variation was found throughout the experiment (Fig. 6A). RR treatment significantly reduced net photosynthetic rates at saturated PPFD (Asat ) from 20 days to the end of the experiment (Fig. 6B). The reduction in Asat was accompanied with reductions in stomatal conductance (gs ) (Fig. 6C) and intercellular CO2 concentration (Ci ) (Fig. 6D). However, these three parameters for RR + O2 plants were not significantly different from those of controls. 3.6. Effects of root restriction and aeration on root cell viability Measurements were also carried out to examine the effects of root restriction on root cell viability (Fig. 7). After staining

Fig. 4. Changes in root plasma membrane H+ -ATPase (PM-H+ -ATPase) (A), vacuolar H+ -ATPase (V-H+ -ATPase) (B) for control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2 , filled triangles) plants. Data are the means of four replicates with standard deviations shown by vertical bars. Different letters are significantly different between the treatments at 5% level according to Tukey tests.

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with FDA-PI, viable live cells usually exhibit green color while dead cells exhibit red color. At 20 days, most root cells for the control plants were viable whilst most of root-tip cells in RR treatment plants had lost viability, especially in the root apex and elongation zone. In comparison, the root tip cells for RR + O2 treatment were as viable as controls. 4. Discussion

Fig. 5. Changes in xylem sap ABA concentration for control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2 , filled triangles) plants. Data are the means of four replicates with standard deviations shown by vertical bars. Different letters are significantly different between the treatments at 5% level according to Tukey tests.

We found that root restriction resulted in a significant inhibition of plant growth in terms of shoot and root dry matter production. The reduction in plant growth became apparent from 15 days and the differences between RR and control plants increased with time (Fig. 1). Reduced plant growth by root restriction has been well observed in early studies and our finding is generally in agreement with these previous reports (Ruff et al., 1987; Yeh and Chiang, 2001). It is important to elucidate the primary factor responsible for the reduced plant growth by root restriction. Nutrient deficit, water balance and chemical signals such as ABA from restricted

Fig. 6. Changes in leaf water potential (A), light-saturated rate of CO2 assimilation (Asat , B), stomatal conductance (gs , C), and intercellular CO2 concentration (Ci , D) for control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2, filled triangles) plants. Leaf temperature was maintained at 25 ◦ C with 390 ␮mol mol−1 CO2 and 1000 ␮mol m−2 s−1 incident PPFD for the gas exchange measurement. Data are the means of four replicates with standard errors shown by vertical bars. Different letters are significantly different between the treatments at 5% level according to Tukey tests.

Fig. 7. Confocal microscopic observation of root viability after 20 days of treatment. Viable cells show green fluorescence while non-viable cells show red. (A): Control; (B): root restriction; (C): root restriction + O2 .

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roots all have been proposed as the possible factors responsible for the suppressive effects of root restriction on plant growth (Choi et al., 1997; Ismail and Noor, 1996; Hurley and Rowarth, 1999). In our study, plant growth reduction appeared in RR plants from 15 days (Fig. 1), but significant difference in leaf water potential between control and RR treatment was found only from 20 days (Fig. 6A). Accordingly, reduced plant growth was not induced primarily by water stress as suggested by Carmi and Heuer (1981). Similarly, reduced plant growth could not be attributed to deficiency of a specific element since the rootrestricted plants did not display any visible signs of nutrient deficit and it is unlikely that the slight difference in Mg concentrations of leaves induced such a large reduction in biomass production (Fig. 1, Table 1). This is in agreement with an early study by Bar-Tal et al. (1995) who found that N nutrient or water stress did not play major role in the growth reduction induced by root restriction. Although several studies have indicated the importance of O2 level in root-restricted plants, no conclusive evidences have been obtained (Kharkina et al., 1999; Peterson et al., 1991b). Here we found that RR treatment resulted in a gradual decrease in dissolved O2 concentration in nutrient solution within the cylinder and this reduction was accompanied by significant reduction in root respiration (Fig. 2). It is likely that the low O2 available within the nylon cylinder in our study resulted from both the relatively higher requirement for O2 for root respiration and the physical barrier due to increased root density. A number of authors have observed reductions in plant growth and decreases of metabolic activities when O2 became deficient (Liu et al., 2005; Rolletschek et al., 2003; Ismond et al., 2003). Tsuji et al. (2000) suggested that low O2 may restrict root respiration by decreasing the transcript levels of nuclear-encoded respiratory genes. In our studies, only the RR treatment plants exhibited a significant reduction in root total respiration from 15 days to the end of the experiment (Fig. 2B). Furthermore, plant growth was significantly improved by the aeration of the nutrient solution, and changes in root respiration were also in agreement with those of plant growth (Figs. 1 and 2). There were positive relationships between plant growth and root respiration. Meanwhile, root restriction was also accompanied by root cell death in our experiment (Fig. 7). All these results suggest that O2 deficit was one of the important limiting factors related to the growth inhibition in root-restricted plant, and also suggested that root respiration was a major metabolic process affected by root restriction stress. Both Cyt pathway and cyanide-resistant alternative pathway respiration exist in roots. Cyt pathway respiration (terminating at COX) couples electron transport to the generation of a proton motive force for the synthesis of ATP (Millar et al., 1998; Millenaar et al., 2002; Izabela et al., 2003). This study clearly demonstrated that the Cyt pathway capacity and COX activity were dramatically decreased in RR plants (Fig. 3). It is likely that this decrease was triggered by decreased O2 concentration in root since Cyt pathway capacity and COX are suppressed under low O2 condition (Ricard et al., 1994). When COX activity becomes O2 -limited, the efficiency of ATP formation and the energy status are sharply reduced, and this will impair cellular metabolism and function (Geigenberger,

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2003). In this case, the PM-H+ -ATPase and the V-H+ -ATPase are presumed to be restricted (Drew, 1997). In this study, both PM-H+ -ATPase and V-H+ -ATPase activities decreased from 15 days in RR plants but not in RR + O2 treatment (Fig. 4). PM-H+ ATPase is responsible for the export of protons to apoplast while the V-H+ -ATPase is responsible for pumping H+ into the vacuole and both of them are essential for intracellular pH regulation (Palmgren, 2001; Drew, 1997). Decrease in PM-H+ -ATPase and V-H+ -ATPase activities would bring about cytoplasmic acidosis in root cells and loss of root tip viability (Drew, 1997). This was also observed in the present study (Figs. 4 and 7). In addition to the role of modulation of pH in cytoplasm, PM-H+ -ATPase also drives essential processes such as active nutrient transport by enhancing the electrochemical proton gradient which constitutes a driving force for solutes to enter the cell (for review, see Sze, 1985; Morsomme and Boutry, 2000). Water uptake might also be related with this enzyme activity since osmotic flows driven by gradients in osmotic pressure play an important role in water movement through the roots (Steudle and Peterson, 1998; Hejl and Koster, 2004). Accordingly, the reduction in nutrient concentration (Fig. 6A) and water potential in leaves for the RR treatment (Table 1) may be one of the secondary effects in response to root restriction but not the primary change. ABA is well known for its role in the regulation of the stomatal behaviour in water stressed plants (Zhang and Davies, 1990a,b; Bahrun et al., 2002). In our study, RR treatment resulted in a sharp increase in ABA concentration in xylem sap and this increase occurred coincidently with the significant reduction in stomatal conductance (gs ) (Figs. 5 and 6C), However, decreases in CO2 assimilation rates (Asat ) and leaf water potential occurred later than that of gs (Fig. 6). It is likely that the decrease in Asat of RR plants was induced by the secondary water stress, and the decreased gs (Fig. 6C) and transpiration rate (data not shown) seemed to be effective in preventing large loss of water during the early stage. During the process, the elevated xylem sap ABA concentration might participate in regulating the stomatal conductance (Figs. 5 and 6C) (Zhang and Davies, 1990a,b; Bahrun et al., 2002). The discrepancy in the changes of biomass accumulation and Asat also suggest this possibility since biomass accumulation is not only influenced by photosynthesis, but also by factors such as leaf expansion (Figs. 1 and 6B). Several studies indicated that root restriction might suppress shoot growth primarily by root-synthesized ABA (Ternesi et al., 1994; Liu and Latimer, 1995). However, the great increase of ABA concentration in xylem sap for RR plants may be induced by the root-zone hypoxia as found by other researchers (Zhang and Davies, 1987; Olivella et al., 2000; Nan et al., 2002). This is supported by our study where xylem sap ABA concentration for RR + O2 plants was not significantly different from that of control (Fig. 5). Similar results were also observed by Ismail and Davies (1998). Through manipulation of O2 level in rootrestricted plants, we conclude that aeration condition rather than ABA concentration is the primary factor limiting shoot growth in root-restricted plants. It is worth to note that all plants were raised in nutrient solutions in our experiment. For the root restriction experiments carried out with soil or substrate culture systems, several mech-

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