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Keywords:

  • Lactuca sativa (lettuce);
  • nitrate;
  • photosynthesis;
  • relative growth rate (RGR);
  • stomatal index;
  • stomatal conductance;
  • nitrogen deficiency

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    C assimilation (A) has been shown to limit the growth of young Lactuca sativa (lettuce) plants following an interruption in their external N supply. Further data from these plants were used to test two hypotheses: that N-limited growth of lettuce is associated with lower stomatal conductance (gs); and that reductions in gs result from adjustments to stomatal frequency or distribution.
  • • 
    The photosynthetic characteristics, nitrate and organic N-concentrations, as well as epidermal and stomatal distributions, were determined in leaves of hydroponically grown lettuce plants supplied continuously with N or with N removed for up to 14 d.
  • • 
    Although N-limited plants had lower maximum rates of A, comparisons at equivalent values of gs showed that A was not directly limited by organic-N but by gs. Reductions in gs under N-limiting conditions did not associate with adjustments to stomatal frequency or distribution.
  • • 
    Associations between plant N and A could arise either through stomata responding directly to signals induced by N deprivation or to increased CO2 partial pressure at the sites of carboxylation.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

There is a large body of literature relating leaf N (in particular organic-N) content to C assimilation rates (A) (Nátr, 1972; Field & Mooney, 1986; Evans, 1989). This dependence of A on plant N concentration is often attributed to the effects of N-availability on the amount of photosynthesis-related enzymes in the leaves (Evans & Seemann, 1989; Hikosaka & Terashima, 1995). However, this relationship may not be independent of other factors. For example, environmental stresses can reduce photosynthesis by direct effects on the photosynthetic capacity of the mesophyll, or by CO2 limitation caused by increases in stomatal or mesophyll resistances (Nátr, 1972; Jones, 1998). Indeed, Jones (1998) emphasizes that stomatal effects should not be ignored when differences in A between two plants or treatments are measured.

In previous studies, the growth of crisphead lettuce (Lactuca sativa) became disrupted following an interruption in N supply (Burns et al., 1997; Broadley et al., 2000). This occurred through changes in growth components, for example there was a decrease in rates of A under N-limiting conditions. Although Broadley et al. (2000) reported a positive relationship between plant N and A in lettuce, this effect was confounded by a similar relationship between plant N and stomatal conductance (gs). In other words, growth may have been limited by the partial pressure of CO2 at the sites of carboxylation, and not directly by a lack of N required for the production of photosynthesis-related enzymes.

The primary aim of this study was to examine these relationships further and test the hypotheses that: N-limited growth of lettuce is associated with lower gs; and that reductions in gs result from adjustments to stomatal frequency or distribution. A secondary aim of this paper was to describe the dynamics of leaf morphology (whole-leaf area, and the production and expansion of epidermal cells) during the onset of N deficiency. Through addressing these two aims, this paper attempts to synthesize how a reduction in N supply limits growth in a particular crop of commercial importance.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The experimental procedure is outlined in detail elsewhere (Broadley et al., 2000). A brief description follows.

Plant material and environmental conditions

Lettuce plants (Lactuca sativa L. cv. Saladin R100), supplied as pelleted seed (Elsoms Seeds Ltd, Spalding, UK), were raised in perlite and irrigated with dilute nutrient solution (Table 1). At the four-to-five-leaf stage, the plants were transferred into the hydroponic units containing full-strength nutrient solution. Two treatments were subsequently imposed: one in which the nutrient solution was continually supplied (control), and one in which the N was removed from the nutrient solution at day 47. The experiment was carried out from December 1998 to February 1999 in a glasshouse compartment set to 25/12°C day/night. Natural daylight was supplemented using high-pressure 400 W Na-vapour lamps to provide a 15-h d−1-light period.

Table 1.  Composition of nutrient solution used in the hydroponic growth of lettuce
MacronutrientsmMMicronutrientsµM
  1. The pH was adjusted to 5.5–6.0 using Ca(OH)2 or H2SO4 as required. The solution was used at quarter strength during growth of plants in perlite. 15NH415NO3 was used at an atom percentage enrichment of 2.75%.

NH4NO34.00H3BO330.00
KH2PO40.25MnSO4·4H2O10.00
KOH0.50ZnSO4·7H2O 1.00
MgSO4·7H2O0.75CuSO4·5H2O 3.00
CaCl2·2H2O0.03Na2MoO4·2H2O 0.50
FeNaEDTA0.10  
CaSO4·2H2O4.00  

Experimental design

There were eight separate hydroponic units (tanks) arranged in a 4 × 2 array with treatments systematically allocated to pairs of tanks in a 2 × 2 design. At each sampling date, one plant was removed at random from each tank. All measurements were made on this plant (i.e. four plants per treatment). ANOVA were performed to assess the effects of treatment, and also effects of sampling date within treatment, assuming a split-plot type design: treatments applied to main plots (tanks), and sampling date applied to subplots (plants within tanks). No estimate of variation was made across the experimental area although the treatments were arranged to minimize this. Statistical analyses were performed using Genstat 5 (Genstat 5 Committee, 1997).

Gas exchange measurements

Measurements of A and gs were made using a portable infra-red gas analyser open system fitted with a 625-mm2 broad-leaf stirred cuvette and a light-unit attached to the cuvette (LCA-4, Analytical Development Co., Hoddesdon, UK). The air flowing through the cuvette was pumped from the outside of the glasshouse. Two types of gas exchange measurements were performed: instantaneous ‘snapshot’ readings and the response of A to light (Parsons et al., 1997).

Instantaneous gas-exchange measurements were made on all unfolded leaves following N interruption, on every leaf with sufficient area to fill the leaf cuvette. The response of A and gs to light was measured on two or three mature leaves per plant (including the last fully expanded leaf) at 9 and 11 d following the imposition of treatments. In all, 16 leaves were characterized. Recordings of A and gs in response to light were made under 11 PPFR levels, 35 ± 1 Pa of CO2, 21 kPa of O2 and 25°C. During steady-state measurements, the leaf-to-air vapour pressure deficit was c. 1.4 kPa. PPFR at the leaf surface was varied between 29 and 1253 µmol photon m−2 s−1, using a series of neutral density filters fitted to the light-unit attached to the leaf cuvette (Parsons et al., 1997). Irradiances at the leaf surface were determined by positioning the LCA-4 PAR sensor at the centre of the cuvette and measuring the radiation transmitted through each filter. Gas exchange rates were recorded every 30 s for at least 3 min once steady-state CO2 exchange was attained. At each PPFR, the arithmetic mean of between four and seven readings was calculated.

The following equation was fitted to the light response curves for each leaf,

inline image

(A, the C assimilation rate (µmol C m−2 s−1); I, the PPFR (µmol photons m−2 s−1); Amax, the maximum A (as I tends to infinity); Km, the value of I when A is ½Amax; and Imin, the value of I at which A reaches 0 (the light compensation point).) In order to estimate the time needed to reach maximum gs, stomatal closure was induced in one plant from each treatment by maintaining them in the dark for 1 h before measurements (Parsons et al., 1997).

Morphological measurements

Measurement of leaf area, number of stomata, and number and measurement of epidermal cells were performed on every leaf with an area greater than 625 mm2. Leaf areas were obtained using a flatbed scanner and HP Deskscan II software (ScanJet 4p, Hewlett Packard Co., Palo Alto, CA, USA). Images were digitally measured using Sigma Scan 3.02 software (Jandel Scientific Software, Erkrath, Germany).

Epidermal cell data and stomatal numbers were obtained from images of the adaxial and abaxial surfaces of every leaf on each plant sampled. To obtain these images, impressions were taken by smearing a thin layer of adhesive (‘Copydex’, Henkel Products, Winsford, UK) over a 50–100 mm2 portion of the leaf, and allowing the adhesive to dry. A thin layer of adhesive was also smeared over a half of one side of a microscope slide and allowed to dry, and the leaf and slide pressed together. Digital images of surfaces of epidermal cell layers were captured from the microscope slide using a video camera (DXC-151P, Sony, Japan) mounted on a light microscope and connected to a computer using WinTV 1.6 software (Hauppauge Computer Works, Inc., Hauppauge, NY, USA). Epidermal cells and stomatal numbers were counted, and images prepared for subsequent digital measuring in Sigma Scan 3.02 using Paint Shop Pro 5.01 (JASC Software Inc., Minnetonka, MN, USA). From these data, stomatal frequency (number of stomata per unit area) and stomatal index (SI, the ratio of stomata to epidermal cells per unit area) were calculated (Salisbury, 1927; Tichá, 1982). From these images, mean cell areas and total number of cells in the epidermal layer were estimated. Previous experimental work revealed that estimates of total number of cells in the epidermal layer is strongly correlated with total numbers of cells in whole leaves (IG Burns, unpublished). We assume that shrinkage is similar for stomata and epidermal cells.

Plant N measurements

15NH415NO3 (at 15N percentage enrichment of 2.75%) was used as a N source for all plants from sowing until day 47. Total-N analyses were performed by mass spectrometry on subsamples of 0.5–1.5 mg dry plant tissue (Mylnefield Research Laboratories, Dundee, UK). Nitrate-N was measured following the procedure of Hunt & Seymour (1985). A maximum of 0.2 g of dry leaf tissue was shaken for 30 min in a 50-ml flask containing 50 ml deionized water and 200–300 mg of activated charcoal. The solution was filtered through Whatman no. 1 filter paper, with the initial 2 ml of filtrate discarded. The filtrate was then analysed for NO3 using a continuous flow colourmetric method on the FIA (FIASTAR 5012, FOSS Tecator, Sweden). Measurements were made on all leaves of sufficient d. wt. Where the dry leaf weight was < 0.05 g, the amount of deionized water used was adjusted accordingly; for samples < 0.05 g, 25 ml of deionized water was added and for samples < 0.02 g, 10 ml of water was added. Organic-N was estimated as the difference between the total and NO3-N components, assuming that the amount of NH4+-N was negligible.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Whole plant responses

There were effects of treatment on all components of growth (Broadley et al., 2000). On a whole plant basis, A decreased in N-limited plants, a response paralleled by differences in gs between treatments (Table 2).

Table 2.  The C assimilation rate (A) and stomatal conductance (gs) of Lactuca sativa after 47 d growth (± SEM, n = 4). +N, control plants continuously supplied with N, −N, plants whose supply of N was removed on day 47. Values are expressed on a per-whole plant basis, estimated from instantaneous values for individual leaves, weighted by leaf area
Days after sowing+N treatment−N treatment
A (µmol C m−2 s−1)SEMgs (mol H2O m−2 s−1)SEMA (µmol C m−2 s−1)SEMgs (mol H2O m−2 s−1)SEM
474.4470.6480.0570.0115.3180.7340.0600.009
495.9520.1040.1060.0075.8120.2710.0800.005
514.5950.4370.0410.0064.5390.7570.0480.010
545.0860.9950.0520.0122.3060.3270.0250.002
565.3090.5820.0510.0091.9140.3420.0180.002
585.9000.4990.1040.0103.2930.4690.0580.020
614.0321.0380.0500.0181.4350.2130.0180.004

Gas exchange data

The photosynthetic responses of mature lettuce leaves to PPFR are presented in Fig. 1. The curves presented are from one control, and one N-limited plant on day 56, 9 d after imposition of treatments, and were typical of the total of 16 leaves sampled (data not shown for all leaves). Eqn 1 was fitted to all of the 16 curves; all 16 fits had a r2 of at least 98.4%. No signs of photoinhibition were observed at high irradiance. Leaves from control plants had a higher Amax than leaves from N-limited plants (t, 3.42; 14 df; P = 0.004). Mean values for Amax from leaves of control and N-limited plants were 13.09 ± 0.72 (n = 9) and 9.06 ± 0.97 (n = 7), respectively (mean ± SEM). There were no significant differences in Km or Imin between treatments (P > 0.05). Mean values of Km were 242.2 ± 10.45 and 218.6 ± 18.33 and values of Imin were 35.05 ± 1.53 and 36.61 ± 1.62 for control and N-limited plants, respectively.

image

Figure 1. The photosynthetic responses of selected Lactuca sativa leaves to PPFR. (a) Response of C assimilation to PPFR. (b) Response of stomatal conductance to PPFR. Filled symbols, control plants supplied continuously with N; open symbols, N-limited plants whose supply of N was removed on day 47. For control plants circles, triangles and squares, represent leaves 8, 10 and 11, respectively (youngest fully expanded leaf). For N-limited plants, circles and triangles represent leaves 9 and 10 (youngest fully expanded leaf). The curves were obtained on day 56. Each point represents the mean of between four and seven replicate IRGA readings (± SEM).

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Maximum gs in plants previously darkened for 1 h was attained after 50 min under the leaf cuvette conditions. The initial time taken for gs to reach steady-state (i.e. where the stomata are at their maximum potential aperture) was similar for the two treatments. A plot of the relationship between gs and A, for the five leaves of contrasting N-status, shown in Fig. 1, reveals no significant differences in the shape of the initial response of (Fig. 2). However, gs of leaves of N-limited plants did not reach the same levels as control leaves. At a time when N was already limiting growth (between seven and 14 d following the imposition of treatments) there was no significant relationship between A or gs and either leaf NO3-N or leaf organic-N, expressed on a leaf area basis (Fig. 3).

image

Figure 2. The relationship between stomatal conductance and C assimilation in selected Lactuca sativa leaves at PPFR > 900 µmol photons m−2 s−1. Filled symbols, control plants supplied continuously with N; open symbols, N-limited plants whose supply of N was removed on day 47. For control plants circles, triangles and squares, represent leaves 8, 10 and 11, respectively (youngest fully expanded leaf). For N-limited plants, circles and triangles represent leaves 9 and 10 (youngest fully expanded leaf). The curves were obtained on day 56. Each point represents an individual IRGA reading.

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image

Figure 3. Plots of NO3-N and organic-N, expressed on leaf-area basis, and gas exchange characteristics in Lactuca sativa. Carbon assimilation rates (a, b) and stomatal conductance values (c, d) are presented on an instantaneous basis, and represent data for all leaves greater than 625 mm2. Filled symbols, control plants supplied continuously with N; open symbols, N-limited plants whose supply of N was removed on day 47. Measurements were taken at 54, 56, 58 and 61 days.

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The dependence of C assimilation on N

There was a positive relationship between the estimated Amax values and leaf organic-N concentration for all 16 leaves (Fig. 4). The mean values of Amax and leaf organic-N concentration show this relationship for leaves of a similar developmental state from the two treatments.

image

Figure 4. Maximum rate of photosynthesis as a function of organic-N concentration in selected Lactuca sativa leaves. Two or three leaves were sampled per plant (including the most recent fully expanded leaf) on days 56 and 58. Filled symbols, control plants supplied continuously with N; open symbols, N-limited plants whose supply of N was removed on day 47. Error bars in both directions represent SEMs. The line is a fit through the mean leaf values (y = 13.3 − 102.5 × 0.3042x; r2 = 0.98; P = 0.001).

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Carbon assimilation is presented as a function of organic-N, at three selected values of gs, 0.05, 0.1 and 0.15 mol H2O m−2 s−1, spanning a range of values of gs at which measurements were obtained for several leaves from each treatment (Fig. 5). Leaves contained more organic-N in the control plants than in the N-limited plants (P < 0.001; 1, 21 df). The mean organic-N level for control and N-limited plants was 5.44% and 2.65% d. wt, respectively (0.59 LSD at P = 0.05). This illustrates that organic-N was not limiting A directly.

image

Figure 5. Carbon assimilation as a function of organic-N in selected Lactuca sativa leaves. These data represent individual IRGA measurements (± SEM) when obtained at three rates of gs (0.05, 0.1 and 0.15 mol H2O m−2 s−1). Two or three mature leaves were sampled per plant (including the most recent fully expanded leaf) on days 56 and 58. Filled circles, control plants supplied continuously with N; open circles, N-limited plants, whose supply of N was removed on day 47.

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Leaf morphology and leaf N data

Total leaf area increased faster in control plants than in N-limited plants (Fig. 6a). There were no differences in the number of total stomata per leaf between adaxial and abaxial leaf surfaces (Fig. 6b). Linear regression between adaxial and abaxial stomatal numbers, constrained through the origin, was highly significant (y = 0.983x; P < 0.001; r2 = 0.90). Therefore, all subsequent analyses on stomatal density were restricted to adaxial epidermal layers.

image

Figure 6. Leaf morphological characteristics in Lactuca sativa following treatment imposition after 47 d. Filled circles, control plants supplied continuously with N; open circles, N-limited plants, whose supply of N was removed on day 47. (a) Total leaf area per plant (b) total number of stomata on adaxial and abaxial leaf surfaces (c) adaxial stomatal density (d) adaxial stomatal index.

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Although total leaf area increased during control plant development, stomatal frequency and SI remained constant. Further, there were no differences in stomatal frequency or SI between control and N-limited plants. Approximately 100 stomata per mm2, with an SI of 8–14% were maintained (Fig. 6b,c). Thus, the reduction in gs observed under N-limited conditions did not result from adjustments in stomatal frequency or from a change in the production rates of stomata.

The dynamics of leaf morphology were analysed in the plants. The areas of individual leaves are presented at 2, 4, 7, 9, 11 and 14 d following imposition of treatments (Fig. 7). The maximum expanded leaf at the onset of treatments was leaf seven. Leaf areas above this insertion level continued to increase in control plants, and had larger final areas than corresponding leaves in N-limited plants.

image

Figure 7. Mean leaf area (± SEM; n = 4) of individual leaves in Lactuca sativa after (a) 49 (b) 51 (c) 54 (d) 56 (e) 58 and (f) 61 d growth. Filled circles, control plants supplied continuously with N; open circles, N-limited plants, whose supply of N was removed on day 47.

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To determine which processes are associated with changes in leaf area, the mean area of an epidermal cell for each leaf and the total number of epidermal cells per leaf were calculated at 2, 4, 7, 9, 11 and 14 d following imposition of treatments. Nitrogen-limited leaves had smaller cell areas than leaves from control plants in older and intermediate leaves (Fig. 8). There were no treatment differences in cell areas from leaves which expanded following the imposition of treatments (leaf 12 and above). The opposite response occurred in total numbers of epidermal cells per leaf. Leaves which expanded before the imposition of treatments (leaf 11 and below), showed no further changes in number of epidermal cells per leaf during subsequent plant development, irrespective of N treatment (Fig. 9). However, leaves that expanded after this time had significantly fewer epidermal cells per leaf.

image

Figure 8. Mean adaxial epidermal cell area (± SEM; n = 4) in Lactuca sativa after (a) 49 (b) 51 (c) 54 (d) 56 (e) 58 and (f) 61 d growth. Filled circles, control plants supplied continuously with N; open circles, N-limited plants, whose supply of N was removed on day 47.

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image

Figure 9. Mean number of adaxial cells per leaf (± SEM; n = 4) in Lactuca sativa after (a) 49 (b) 51 (c) 54 (d) 56 (e) 58 and (f) 61 d growth. Filled circles, control plants supplied continuously with N; open circles, N-limited plants, whose supply of N was removed on day 47.

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The NO3-N (Fig. 10) and organic-N (Fig. 11) concentrations were recorded in all leaves at 2, 4, 7, 9, 11 and 14 d following imposition of treatments. In the control plants, NO3-N concentration was highest in intermediate leaves. Differences in leaf NO3-N concentration between the two treatments occurred after 2 d of N starvation, particularly in the younger leaves. After 4 d, the differences became pronounced, and after 7 d, virtually all NO3-N had disappeared in N-limited plants. However, leaf NO3-N concentration also appeared to decrease with time in all leaves in the control plants.

image

Figure 10. Mean leaf NO3-N concentration (± SEM; n = 4) in Lactuca sativa after (a) 49 (b) 51 (c) 54 (d) 56 (e) 58 and (f) 61 d growth. Filled circles, control plants supplied continuously with N; open circles, N-limited plants, whose supply of N was removed on day 47.

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image

Figure 11. Mean leaf organic-N concentration (± SEM; n = 4) in Lactuca sativa after (a) 49 (b) 51 (c) 54 (d) 56 (e) 58 and (f) 61 d growth. Filled circles, control plants supplied continuously with N; open circles, N-limited plants, whose supply of N was removed on day 47.

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Organic-N concentration was higher in new leaves than in older leaves. Differences in organic-N concentration between control and N-limited plants occurred in leaves 7-to-10 after 7 d. In younger leaves, there were no differences in organic-N between control and N-limited plants, indicating that even under severe N-deficiency, organic-N concenetrtion is maintained in the new leaves.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Although the impact of high NO3 consumption on human health is far from clear (Addiscott, 2000), EC legislation has defined maximum permissible limits on NO3- levels in several vegetable crops, including spinach and lettuce (EC Commission Regulation no. 466/2001). Managing the NO3-content of crops can be achieved through low-NO3 production systems (Andersen & Nielsen, 1992), through improved predictions of crop-response to applied NO3 in conventional production systems (Greenwood et al., 1996), or by exploiting more efficient NO3-assimilation traits for selection purposes (Zhang & Forde, 2000). All of these approaches require a detailed understanding of the processes by which N influences plant growth.

Previous data from this experiment on the relationship between RGR and total-N concentration indicate that the growth of these lettuce approached zero when N had declined to approx. 2% of dry matter (Broadley et al., 2000). Further, instantaneous measures of gas exchange also revealed that the principal growth component causing reductions in RGR was A, when A is expressed on a whole-plant basis. The current paper was designed to investigate this relationship more closely. One of the approaches adopted was to characterize the parameters of steady-state photosynthesis, derived from light-response curves (Parsons et al., 1997). This showed that N-limited plants had lower light-saturated rates of photosynthesis on a leaf area basis (Amax), with no treatment differences in photosynthetic efficiency (Km) or light compensation point (Imin) (Fig. 1). As with RGR, there was a significant association between leaf organic-N and Amax with Amax approaching zero at 2% N in the dry matter (Fig. 4).

From these results, it is tempting to conclude that Amax and leaf organic-N are associated, leading to the subsequent conclusion that N-limited growth of plants is directly caused by a lack of photosynthetically related enzymes. For example, up to 80% of leaf organic-N is allocated to photosynthetic proteins (Evans & Seemann, 1989), and there is a strong correlation between Amax and N concentration across a wide number of species (Field & Mooney, 1986). Furthermore, many studies have shown Amax to be highly correlated with the amount or activity of photosynthetic components such as Rubisco, cytocrome-f and other coupling factors (Hikosaka & Terashima, 1995).

However, parameters describing the response of photosynthesis to light may not always be physiologically meaningful. For example, at low irradiances photosynthesic is not limited by Amax but by photon availability (Farquhar & Sharkey, 1982). Therefore, photosynthesis rates should not be considered solely in terms of Amax. In this study, N-limited plants did not attain the same level of stomatal conductance as control plants (Fig. 2). Indeed, comparisons made at equivalent values of gs showed no differences in A between leaves of 5.44% and 2.65% d. wt organic-N (Fig. 5). Furthermore, when N was limiting growth significantly (between 7 and 14 d following the imposition of treatments), instantaneous measurements of A from all leaves on a leaf area basis revealed that there was no significant relationship with either leaf NO3-N or leaf organic-N (Fig. 3). These observations support the hypothesis that A was not directly limited by organic-N, but rather by lower values of gs. In other words, photosynthesis was more likely to have been limited by CO2 partial pressure at the sites of carboxylation than by direct effects of enzyme activity. Analyses based on measurements of A at different substomatal CO2 partial pressures (‘A/Ci’ curves) would allow such hypotheses to be tested directly (cf. Parsons et al., 1997).

If gs limits growth during periods of N deficiency, then there are two hypotheses which might explain this: that N supply affects the frequency or distribution of stomata; or that N limits gas exchange across stomata. This second hypothesis may occur via a direct plant response to a lack of N, or through an indirect compensatory response to lower rates of carboxylation and thus an increased partial pressure of CO2 at these sites.

Lettuce has equivalent numbers of adaxial and abaxial stomata; reductions in gs under N-limiting conditions were not associated with adjustments to stomatal distribution between leaf surfaces (Fig. 6b). Stomatal frequency is variable and influenced by environmental effects (Tichá, 1982). A more stable method of describing the epidermal-stomatal complex, developed by Salisbury (1927), is SI. However, there was no evidence that either the stomatal frequency or index changed during N-limited growth. Thus, it is unlikely that N affected the development of the epidermal-stomatal complex in this study. Although observations were made on leaves formed before onset of N starvation, these leaves make up the bulk of a plant's photosynthetic apparatus and will therefore dominate any observed growth responses.

The second hypothesis, that N limits gas exchange across stomata, could not be tested directly using the approaches outlined here. However, indirect evidence from measurements of leaf morphology suggest that may have occurred. The mean numbers and areas of epidermal cells indicate that the final size of young leaves in N-limited plants was reduced by cell division in early development, and cell expansion as the leaves mature (Figs 7, 8, 9). These data are consistent with data reported from sunflower (Helianthus annus) (Trápani et al., 1999) and Ricinus communis (Roggatz et al., 1999). Therefore, in the leaves that form the bulk of the photosynthetic apparatus, cell expansion rate is the dominant growth component compromised during N deficiency. The distribution of NO3-N and organic-N in individual leaves suggests that the onset of N-deficient responses is associated with a loss of NO3-N in all leaves. Since the lack of cell and leaf expansion in N-limited plants will be caused by osmotic disruptions in the leaf, it is possible that NO3 is involved, either directly or indirectly, in this response. Nitrate may function as an osmolite (Cárdenas-Navarro et al., 1999) or as a signalling molecule (Zhang & Forde, 2000) in plants. In tomato, leaf-growth declines in response to N-stress before reductions in total plant weight gains or photosynthetic potential, possibly through the production of ABA which can cause stomatal closure (Chapin, 1990). Future studies on lettuce will help to determine if reductions in gs are an independent response to nutrient deprivation, or a compensatory response to lower rates of carboxylation.

Nitrogen-limited growth, reported previously for lettuce (Broadley et al., 2000), was caused through a reduction in gs. By describing the dynamics of leaf morphology, N-concentration and N-form in all leaves of plants grown under N-limiting conditions, we conclude that associations between plant N and A may be caused by a stomatal response to reductions in plant NO3-N. Stomata might be directly responding to signals induced by N deprivation, or in response to carboxylation rates.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors thank Mr A. Mead, Dr V. Valdes Ruiz and Dr P. J. White for their helpful suggestions. The Ministry of Agriculture, Fisheries and Food, UK supported this work.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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