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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.
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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.