Journal of Experimental Botany, Vol. 53, No. 375, pp. 1765-1770,
August 1, 2002
© 2002 Oxford University Press
Does shoot water status limit leaf expansion of nitrogen-deprived barley?
Received 8 November 2001; Accepted 26 April 2002
1 Department of Botany, University of Queensland, Brisbane, QLD 4072, Australia
2 CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia
3 To whom correspondence should be addressed at Department of Biological Sciences, IENS, University of Lancaster, Lancaster LA1 4YQ, UK. Fax: +44 (0)1524 843854. E-mail: I.Dodd{at}lancaster.ac.uk
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Abstract |
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The role of shoot water status in mediating the decline in leaf elongation rate of nitrogen (N)-deprived barley plants was assessed. Plants were grown at two levels of N supply, with or without the application of pneumatic pressure to the roots. Applying enough pressure (balancing pressure) to keep xylem sap continuously bleeding from the cut surface of a leaf allowed the plants to remain at full turgor throughout the experiments. Plants from which N was withheld required a greater balancing pressure during both day and night. This difference in balancing pressure was greater at high (2.0 kPa) than low (1.2 kPa) atmospheric vapour pressure deficit (VPD). Pressurizing the roots did not prevent the decline in leaf elongation rate induced by withholding N at either high or low VPD. Thus low shoot water status did not limit leaf growth of N-deprived plants.
Key words: Key words: Barley, evaporative demand, leaf expansion, nitrogen, water status.
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Introduction |
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Reduction in nitrogen supply to the roots can reduce leaf expansion within 24 h (Palmer et al., 1996). How this happens is not clear. There is evidence that N deprivation may reduce root cell hydraulic conductivity (Radin and Matthews, 1989) and the overall hydraulic conductance of the plant and thence leaf turgor (Radin and Boyer, 1982) which may, in turn, reduce growth rate. However, others have shown that plants grown at different N supplies showed no differences in leaf turgor (Palmer et al., 1996; Fricke et al., 1997).
Leaf growth can be restricted by increased evaporative demand even when the soil is well-watered (Ben Haj Salah and Tardieu, 1997) as a result of decreased leaf turgor (Serpe and Matthews, 2000). Thus the effects of a limited nitrogen supply on leaf turgor, and thence possibly on growth, may depend on the evaporative demand.
Previous experiments on the relationship between turgor and leaf growth under N deprivation (Radin and Boyer, 1982; Palmer et al., 1996; Fricke et al., 1997) have imposed different nitrogen treatments, then measured leaf turgor. A different approach was used here. Plants were grown under high and low VPDs, and turgor maintained while imposing different nitrogen treatments. This was done by growing plants in root pressure chambers, which maintain xylem sap continuously on the verge of bleeding from a cut leaf, so that they remain fully turgid at all times (Passioura and Munns, 1984). At low VPD, it was hypothesized that N deprivation may inhibit leaf growth principally due to a lack of N, and maintenance of leaf turgor by pressurizing the roots would have little or no effect on leaf elongation rate. At high VPD, it was predicted that maintenance of leaf turgor by pressurization would increase leaf growth by overriding a significant hydraulic effect that would otherwise decrease leaf water status.
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Materials and methods |
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Barley (Hordeum vulgare L. cv. Himalaya) plants were grown in a controlled environment chamber at a photosynthetic photon flux density of 400 µmol m–2 s–1 and a 9 h photoperiod. Two different daytime VPDs were imposed in sequential experiments by adjusting the cabinet temperature. The low VPD experiment (Experiment 1) had a 20/16 °C day/night temperature, giving average ambient day and night VPDs of 1.2 kPa and 0.65 kPa, respectively. Plants for the high VPD experiments were also grown under these conditions until high VPD was imposed on day 1. The high VPD experiments (Experiments 2, 3) had a 28/18 °C day/night temperature, giving average ambient day and night VPDs of 2.0 kPa and 1.0 kPa, respectively.
Plants were grown in a 4:1 sand:perlite mix in 220 cm3 stainless steel cylindrical pots (45 mm diameter, 150 mm long) designed to fit in small pressure chambers (Termaat et al., 1985). A piece of wire mesh was taped over the base of the pot to retain the growth medium and assist drainage. Plants were irrigated by siphoning 100 cm3 of nutrient solution into the top of the pots. Following irrigation, pots were placed on cotton towels for 10 min to assist drainage.
Plants were sealed into the top of the pots with silicone rubber (Sylgard 184, Dow Corning, Midland, USA). The pots were placed in pressure chambers that were connected to a pressure controller that operated cylinders of air and N2. Each pressure controller was connected to six pots in series representing a single N treatment. The controller mixed the two gases to maintain O2 partial pressure at 21 kPa, its value in normal air (Termaat et al., 1985; Passioura, 1988). The chambers were bled continuously to prevent the build-up of gases such as ethylene and CO2 in the pots.
Seeds were surface-sterilized in 1% (v/v) sodium hypochlorite for 2 min before being placed on distilled water-saturated filter paper in the dark for 36 h prior to planting. Plants were irrigated with complete nutrient solution [composition (in mM) KNO3, 2; Ca(NO3)2.4H2O, 1.5; KH2PO4, 1; MgSO4.7H2O, 2; (in µM) NaFeEDTA, 71.4; H3BO3, 5; MnCl2.4H2O, 0.5; ZnSO4.7H2O, 0.2; (NH4)4Mo7O24.4H2O, 0.1; CuSO4.5H2O, 0.2] at 10 (planting day), 3 and 0 d before imposition of pressure and N treatments.
On day 1 of each experiment, 2 h before commencement of the photoperiod, all plants were flushed with 100 cm3 of water to remove residual nutrient solution. Then half the plants received 100 cm3 of complete nutrient solution while the other half received an iso-osmotic, nitrate-free solution in which nitrate was replaced by chloride and the cation concentrations were unchanged. Following drainage, plants were placed into the pressure chambers. Six plants per N treatment remained unpressurized throughout each experiment, while pressurization of another six plants per N treatment commenced at the beginning of the photoperiod. Balancing pressure was manually established and maintained by ensuring that the xylem of leaf 1 was on the verge of bleeding at all times. Individual plants varied slightly in the pressure required to induce bleeding, thus sufficient pressure was applied to keep three of the six plants bleeding at all times. Since balancing pressure varies diurnally according to the transpiration rate and hydraulic resistance of the plant, manual adjustment of the pressure was required throughout the photoperiod and during the first 2 h of the dark period. Balancing pressures remained constant overnight until 2 h before commencement of the photoperiod, when pressure was released to allow irrigation of all pots. Irrigation took no more than 1.5 h, after which balancing pressure was restored prior to the start of the next photoperiod.
Leaf length measurements commenced on day 0 (1 d before the application of nitrogen and pressure treatments). A piece of graph paper photocopied onto acetate was used to measure leaf length at 09.30 h and 18.00 h, to partition leaf elongation rate (LER) into light and dark periods. Parallel measurements of both leaves 2 and 3 were made for 1 d on emergence of leaf 3, then only leaf 3 was measured.
Additional experiments determined the balancing pressure and root hydraulic conductance of individual plants grown at low VPD (18 °C, 0.6 kPa day and night) in a controlled environment chamber at a photosynthetic photon flux density of 200 µmol m–2 s–1 and a 9 h photoperiod. Plants (Hordeum vulgare L. cv. Gilbert) were grown as described previously (Stirzaker and Passioura, 1996) in 1270 cm3 plastic cylindrical pots (90 mm diameter, 200 mm long) capped with an aluminium plate, designed to fit in a pressure chamber. Plants were watered weekly with 200 cm3 of complete nutrient solution until leaf 3 had emerged. All plants were then flushed with 200 cm3 of water to remove any residual nutrient solution. Then half received 200 cm3 of complete nutrient solution while the remainder received an iso-osmotic, nitrate-free solution.
After 3–6 d of N deprivation, the balancing pressure of individual plants was determined 4–8 h after the commencement of the photoperiod. Two sequential light intensities were used to alter the VPD around the leaves. Plants of similar leaf area from each N treatment were paired and placed in two separate pressure chambers. The lights above the pressure chambers were dimmed at 11.45 h, and a balancing pressure determined between 12.30–14.00 h. All lights in the growth chamber were then switched on, and a balancing pressure determined between 14.45–15.45 h. After placement of the plants in the pressure chambers, the tip of leaf 2 was removed to give a uniform leaf 2 length. Pressure (supplied by a cylinder of compressed air) was initially raised to 100 kPa, then increased by 20 kPa min–1 in 10 kPa increments to determine the balancing pressure. Once xylem sap appeared at the cut surface, the plants were allowed to bleed for 1.5 h (low light) or 1 h (high light) and balancing pressure was adjusted if necessary.
After 4–6 d of N deprivation, the root hydraulic conductance of individual plants was determined 2–5 h after the commencement of the photoperiod, in two separate experiments. Each plant was placed in a pressure chamber and the shoot removed 10 mm above the seed base. After excision, xylem sap was allowed to exude for 5 min and collected into a preweighed Eppendorf tube. Pressure was then applied and xylem sap was collected for 5 min at each pressure. Six to seven pressures (at intervals between 50 and 400 kPa) were applied in an increasing series, then five pressures were applied in a decreasing series. Each set of measurements took 1 h. There was no significant hysteresis in the flow versus pressure relationship when comparing increasing and decreasing pressures (data not shown). The slope of the relationship between exudate flow rate (in mm3 s–1) and applied pressure over the linear part of the curve gave the root hydraulic conductance (Lpr). In all cases, the linear correlation coefficients (r2) were >0.95.
Significant differences between treatments at each measurement occasion were discriminated using Student’s unpaired t-test.
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Results |
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Growth at low (1.2 kPa) VPD
In plants supplied with N, no difference was found in leaf elongation rate (LER) between those grown with and without pressure, in all experiments (data not shown). The absence of effect of balancing pressure on growth of well-watered and well-fertilized plants has been shown previously (Passioura, 1988). Thus data for the LER of N-supplied, pressurized plants are not presented in Fig. 1.
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N deprivation had a small inhibitory effect on the growth of leaf 2 (Fig. 1b). Growth of leaf 3 was inhibited 40% by N deprivation when it first emerged on day 2, before recovering overnight (Fig. 1c). For the remainder of the experiment, day and night growth of leaf 3 of N-deprived plants was similar (Fig. 1c). LER was approximately halved by day 4.
Applying balancing pressure did not affect the growth of N-deprived plants throughout the experiment (Fig. 1b, c). On day 5, all plants were flushed with complete nutrient solution and pressurization ceased. By day 7, LERs of N-deprived and N-supplied plants were once again similar. Another cycle of N deprivation and root pressurization was initiated, applying the same N and pressure treatments to each plant as before. Again in leaf 4, which was now the growing leaf, growth of N-deprived plants was not enhanced by the application of balancing pressure during both day and night. N deprivation reduced LER by 35% after a further 3 d (data not shown).
Growth at high (2.0 kPa) VPD
The high daytime VPD caused by increased cabinet temperature produced a pronounced diurnal variation of LER (Fig. 2b, c) in both N-supplied and N-deprived plants, due to the dependence of LER on temperature. In plants supplied with N, balancing pressure again had no effect on LER in experiments lasting 4–6 d (data not shown).
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N deprivation caused a 20% inhibition on the growth of leaf 2, during the photoperiod of day 2 (Fig. 2b). During the photoperiod of day 3, the effect of N deprivation was more severe in leaf 2 than in the newly emerged leaf 3 (Fig. 2c), but by day 4, the LER of leaf 3 of N-deprived plants was reduced by about 30% (Fig. 2c).
Applying balancing pressure did not affect the growth of N-deprived plants except during the photoperiod of day 3 and the dark period of day 4. Plants under balancing pressure grew faster than unpressurized plants during the photoperiod of day 3, but slower during the dark period of day 4 (Fig. 2b, c).
Since the application of balancing pressure to N-deprived plants prevented the decline in photoperiod LER on day 3, but not day 4, a further experiment was conducted at high VPD (Fig. 3). N deprivation reduced growth by more than 40% at the end of the experiment. There was no effect of balancing pressure on LER of N-deprived plants except on day 4. On this day, pressurized, N-deprived plants grew slightly faster than unpressurized plants during the photoperiod, but slower during the ensuing dark period. There was no effect of pressurization on LER when integrated over a 24 h period. These transient differences in LER between pressurized and unpressurized N-deprived plants were not seen on days 5 and 6.
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Experiments conducted at different VPDs showed no consistent differences in the proportional response of leaf growth to N deprivation (data not shown).
Balancing pressures and hydraulic conductance
In experiments where the pressure chambers were connected in series, the balancing pressure varied with time of day and was greater at high VPD (Figs 1a, 2a). The N-deprived plants appeared to have a higher balancing pressure than N-supplied plants on days 2–4. To confirm this, individual plants were measured in a separate experiment where the VPD was altered by changing the light intensity. At low light intensity, the balancing pressure of N-deprived plants (0.19±0.01 MPa, n=4) was significantly (P <0.05) higher than that of N-supplied plants (0.15±0.01 MPa, n=3). Similarly, at high light intensity, the balancing pressure of N-deprived plants (0.32±0.02 MPa, n=4) was significantly (P <0.05) higher than that of N-supplied plants (0.27±0.01 MPa, n=3).
Since whole plant transpiration rates were not determined simultaneously with the balancing pressures, whole plant hydraulic conductance could not be calculated. Instead, hydraulic conductance of detached root systems was measured. There was a linear relationship between sap flow rate and applied pressure for both N treatments, but the slope of the relationship (the root hydraulic conductance) was reduced by about 40% in N-deprived plants (Fig. 4).
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Discussion |
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That N deprivation decreased shoot water potential is indicated by the higher balancing pressure of N-deprived plants. The simplest interpretation of balancing pressure is that it is equal and opposite to the hydrostatic pressure in the xylem of the unpressurized plant. Measurements of root hydraulic conductance, as determined from the slope of the pressure-flow relationship (Fig. 4), are consistent with similar measurements in sunflower (Radin and Boyer, 1982), and have the possible implication that N deprivation leads to low leaf water status and thence slower growth.
Decreased shoot water status in response to N deprivation is not universal, as instantaneous measurements of cell turgor using the pressure probe have not detected decreased leaf turgor in both N-deprived barley (Fricke et al., 1997) and sunflower (Palmer et al., 1996). However, these measurements of epidermal cell turgor may not reflect the water status of the rest of the growing tissue, as epidermal cells are comparatively insensitive to changes in xylem water potential (Nonami et al., 1997). Further, they may not always detect transient differences in leaf turgor, which may influence growth. For this reason, maintenance of plants at full turgor (via root pressurization) throughout the diurnal cycle was necessary to confirm that reduced turgor did not limit growth of N-deprived plants.
The root pressurization experiments were conducted at two different VPDs, as higher transpiration rates at high VPD may have had greater effects on shoot turgor and leaf growth. However, raising the shoot water status did not overcome the growth reduction due to N deprivation, even at high VPD (Figs 2, 3).
The lack of growth response to elevated water status in N-deprived barley may reflect the species chosen. Radin (1983) suggested that leaf elongation of dicotyledonous species is more sensitive to N deprivation than that of cereals, because the growing tissue of dicotyledons is exposed and subject to evaporation or transpiration whereas that of cereals is not. It would be interesting to repeat such pressurization experiments in a dicotyledon that might be more responsive than barley to a possible low N-induced water deficit.
Reductions in leaf growth without reductions in leaf turgor have been interpreted as evidence for the existence of root-derived chemical signals moving to the shoots and influencing growth (Passioura, 1988; McDonald and Davies, 1996). In the case of N deprivation, these ‘signals’ may simply be a lack of resources (N) for growth, which would obviate the need to invoke more complex explanations such as the action of hormonal signals which specifically limit leaf growth (Kuiper et al., 1988). Certainly N deprivation reduces the N content of expanding cells, however, there is no simple relationship between tissue N content and local growth rates (Gastal and Nelson, 1994). Rather, it is the delivery of N to the growing cells that is likely to determine growth rates.
Leaves of N-deprived plants have fewer and smaller cells, implying effects on both cell division and cell expansion. The relative effects on each process depends on the stage of leaf development when N is withheld (Roggatz et al., 1999). Effects on cell division may be particularly important in monocotyledonous species such as barley, which show large reductions in cell flux through the elongation zone in response to N deprivation (Fricke et al., 1997).
Ultimately, understanding the mechanisms of effects of N limitation on both cell division and cell expansion will explain the observed changes in leaf growth. The total N status of the tissue may be less important in regulating growth than metabolic signals derived from nitrate, which could trigger changes in gene expression that affect both the uptake and assimilation of nitrate, as well as initiating changes in carbon metabolism (Stitt, 1999). Such feed-forward signals may prove to be important controlling factors in plant growth.
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Acknowledgements |
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We thank Dr Jianmin Guo for help in the use of the pressure chambers. ICD thanks the Australian Research Council for financial support, and Mr Peter Tam (Grainco Ltd., Toowoomba, Australia) for donation of barley seed.
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References |
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