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- Materials and Methods
The understanding of nitrate uptake and assimilation in plants and its regulation has advanced considerably during the past decade (Forde & Clarkson, 1999; Kaiser & Huber, 2001; Forde, 2002; Stitt et al., 2002; Foyer et al., 2003). Two of the main environmental factors of importance for the regulation of nitrate uptake and assimilation are nitrate and light (Forde & Clarkson, 1999). Nitrate induces both the transcription of nitrate transporters and of nitrate reductase (NR), the primary nitrate reducing enzyme (Forde & Clarkson, 1999; Forde, 2000). When nitrate is available in excess to plant demand, the transporters and nitrate reductase are feedback regulated by products of nitrate assimilation and the transporters possibly also by nitrate itself (Sivasankar & Oaks, 1996; Gojon et al., 1998; Forde, 2000; Orsel et al., 2002). Light affects nitrate uptake both as a signal activating transporters and nitrate reductase and as a resource affecting plant photosynthesis and growth (Cheng et al., 1992; Lillo et al., 1998; Forde, 2000; Klein et al., 2000; Kaiser & Huber, 2001).
The effect of short-term changes in irradiance on nitrate uptake has been most intensely studied in micro-algae, where NO3− uptake as a function of irradiance has been shown to follow a saturation curve similar to the light–photosynthesis curve (Priscu et al., 1991; Muggli & Smith, 1993; Kristiansen et al., 1998). The increased NO3− uptake at increased irradiance is believed to be related to the increased photosynthetic rates that enhance the plants demand for nitrogen and at the same time provide the energy and carbon skeletons needed (Priscu et al., 1991). Long-term effects of irradiance on nitrate uptake appear to be mediated through the effect of irradiance on plant growth rates and plant demand for nitrogen (Imsande & Touraine, 1994). Thus, plants grown at high irradiance have higher growth rates and higher nitrate uptake rates and reduction capacity, and vice versa.
In terrestrial plants, nitrate is taken up by the root and can be assimilated by both root and shoot (Gojon et al., 1994). Thus, it has been suggested that the effect of photon flux density on root NO3− uptake is mediated through phloem-transported compounds, of which sugars are considered the most important in stimulating nitrate uptake, whereas amino acids such as glutamine and asparagine are considered important factors for downregulating uptake (Delhon et al., 1996; Sivasankar & Oaks, 1996; Gojon et al., 1998; Forde, 2000, 2002). In aquatic macrophytes both roots and shoots possess the ability to take up nutrients, including nitrate, although the relative importance of the two uptake routes differs with growth conditions and nutrient ion species (Barko & Smart, 1981; Thursby & Harlin, 1984; Cedergreen, 2002). The mechanisms regulating the balance between root and shoot uptake is not known in any details, although it appears that the relative availability of nutrients to root and shoot is of importance. However, effects of other environmental factors have only attracted little, if any, attention (Barko & Smart, 1981; Thursby & Harlin, 1984; Cedergreen, 2002; Cedergreen, 2003).
If aquatic macrophytes respond to increased irradiance in the same way as terrestrial plants do, it is to be expected that both short- and long-term changes in irradiance and an associated change in nitrogen demand by the plants, will be reflected in adjustments in shoot nitrate uptake and assimilation rates that are proportional to the change in photosynthesis. For root nitrate uptake two opposite-directed scenarios can be suggested. Uptake and assimilation may either increase in response to increased irradiance, as a consequence of increased flux of photosynthates from shoot to root, or decrease, as a consequence of increased flux of reduced N-compounds following an increased NO3− uptake and assimilation by the shoot. Nitrate reduction can potentially take place in both roots and shoots of aquatic macrophytes (Cedergreen, 2003). Considering the dependence of nitrate reduction on energy and carbon skeletons, however, shoot reduction is expected to be predominant, at least in situations of high shoot uptake.
It was the aim of this study to investigate the effect of irradiance on the division of nitrate uptake and reduction between root and shoot of an aquatic macrophyte. We wished to assess both short-term responses and the long-term acclimatizations to changes in irradiance. As a model organism we used Lemna minor; a small floating macrophyte with a simple morphological organization consisting of one leaf-like frond and one unbranched root. Lemna minor is a cosmopolitan inhabitant of stagnant, nutrient-rich waters (Hillman, 1961). The floating nature of the plants allows easy manipulation of nitrate availability to root and frond while still keeping the plants under near-natural growth conditions. The long-term acclimatization of nitrate uptake and assimilation was assessed by growing the plants at combinations of high and low irradiance and two nitrate availabilities.
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A significant result of this study is the observation that changes in light regime alone can affect the balance between root and frond NO3− uptake of L. minor, even though the external NO3− concentration is the same for the two tissues. These results show that the balance between root and frond (or leaf) uptake by aquatic macrophytes might be regulated in a complex way and not only by the relative nutrient availability to roots and leaves, although the latter is of significance (Carignan, 1982). It is not clear from our study why L. minor responds by changing the relative uptake of nitrate through root and frond while maintaining total uptake, but the data suggest that the observed changes are coupled to photosynthesis. For fronds, the change in uptake capacity in response to short-term irradiance increase was closely coupled to frond chlorophyll and N content (Fig. 2b,c) and thereby, presumably, to the ability of the plant to absorb light energy and use it in carbon assimilation (Behaeghe & Impens, 1995). The regulation of root uptake is probably mediated by reduced N-compounds as root uptake decreased with increasing frond uptake, and vice versa (Fig. 2a), as is commonly observed for the regulation of root uptake in terrestrial plants (Cooper & Clarkson, 1989; Sivasankar & Oaks, 1996; Gojon et al., 1998; Forde, 2002). The fact that the whole plant uptake was relatively constant under both irradiance regimes (Fig. 1, Table 3), indicates that whole plant demand for N did not change significantly within the timeframe of the experiment.
The changes in nitrate reductase activity in roots and fronds in response to changed irradiance did not reflect the changes in uptake of the two tissues, although NRA have been shown to be regulated by the same compounds as the high-affinity nitrate uptake system (Sivasankar & Oaks, 1996; Gojon et al., 1998; Forde, 2000; Kaiser & Huber, 2001; Orsel et al., 2002). On a whole-plant basis, nitrate reductase activity did increase with whole-plant uptake, indicating that the enzyme activity balanced plant uptake (or vice versa). Looking at root and frond NRA separately, however, revealed, on average, 16 times higher root NRA compared with frond NRA on a dry weight basis, comprising about 78% of total plant NRA, which contrasts with the 52% average contribution of roots to whole-plant uptake. This, combined with the correlation between root NRA and frond uptake (Fig. 3c), suggests that NO3− reduction in L. minor mainly takes place in the root. This is also supported by the close coupling between NRA and internal NO3− concentration, which corresponds well with the various findings of NO3− induction of nitrate reductase activity (Li & Oaks, 1993, 1995; Samuelson et al., 1995). The lack of correlation between NRA and sugar content, which was approximately constant compared with the variations in NRA (Table 2), does not eliminate the possibility that sugars played a role in regulating NRA, as it is the flux, rather than the content, which is of importance (Delhon et al., 1996; Kaiser & Huber, 2001). It must also be kept in mind that the NRA measured in vitro indicates the potential for nitrate reduction, but that nitrate reduction in situ can be substrate limited and is therefore not necessarily reflected in the in vitro measurements (Kaiser et al., 2000). Studies of both in vitro and in situ nitrate reduction in Lemna gibba have, however, shown in vitro measurements to reflect in situ measurements relatively well (Ingemarsson, 1987).
The finding of considerably higher NRA in roots compared with fronds and the indications that NO3− taken up by fronds is at least partly reduced in roots are interesting, as the proximity of photosynthetically derived energy and carbon skeletons in fronds was expected to favour frond NO3− reduction over root reduction (Raven, 1985), at least as long as NO3− was taken up by fronds. This pattern was found for lettuce, where application of N to leaves was shown to increase leaf NRA while decreasing root NRA, even when grown at ample root NO3− supply (Hufton et al., 1996). There are, to our knowledge, no studies documenting a transport of NO3− from shoots to roots, but reversed xylem flow is known from both roots and fruits exhibiting low transpiration rates (Lang & Thorpe, 1989; Sakuratani et al., 1999). Reversed xylem flow could take place in L. minor, where leaf transpiration potentially can be covered by leaf water uptake. In submerged aquatic plants, where the transpiration is absent, acropetal water transport does take place and is closely coupled to light (Pedersen & Sand-Jensen, 1993). Further knowledge of the water movement in floating macrophytes is, however, needed to confirm the existence of reversed xylem flow. High root NRA was also observed in another Lemna species, L. gibba, under both natural conditions and when grown with 500 µmol NO3− m−3 (Cedergreen, 2003). The ecological significance of reducing NO3− in roots as opposed to shoots is unclear, but it seems, judging from the NRA distribution, to be a common trait among several aquatic species capable of taking up NO3− by shoots, and might be a plesiomorphic character of the terrestrial ancestors (Cedergreen, 2003).
The long-term acclimatizations of L. minor to the growth conditions showed that nitrate uptake was rate-saturated at the growth conditions being closely coupled to population growth rate. This corresponds well to previous studies showing a balanced response by the plants to ensure optimal use of the resources available (Cooper & Clarkson, 1989; Imsande & Touraine, 1994; Bazzaz, 1997). In addition to acclimatization of the nitrate uptake apparatus, L. minor acclimatized to the light and nitrate regimes through various morphological and physiological changes. These acclimatizations also followed the patterns known from terrestrial plants, where growth under low irradiance generally results in larger specific leaf area and less biomass allocation to roots, increased chlorophyll content on a dry weight basis and a smaller accumulation of starch than growth under high irradiance (McDonald et al., 1986; Minotta & Pinzauti, 1996; Reich et al., 1998), whereas acclimatizations to low nitrogen availability generally result in lower specific leaf area and more biomass allocated to roots, lower N, NO3− and chlorophyll content and higher starch reserves (Tables 1 and 2) (McDonald et al., 1986; Rufty et al., 1988; Skillman & Osmond, 1998; Peterson et al., 1999). We consider the functional significance of these acclimatizations to be analogous to those of terrestrial plants, although the functional significance of an increased allocation of biomass to roots in response to either low N availability or high irradiance might seem less obvious for a plant such as L. minor that can take up nutrients through both root and frond. However, considering the investment needed to obtain a particular nutrient-assimilating surface by L. minor, this would be lower when biomass and energy are invested in root tissue rather than frond tissue (Cedergreen, 2002). Thus, in a situation of increased demand for assimilating surfaces for nutrient uptake, investment in root tissue would be more beneficial than investment in frond tissue.
In conclusion, the present study showed that short-term changes in irradiance changed the balance between root and frond NO3− uptake of Lemna minor, but that whole plant uptake remained approximately balanced to population growth rates. The high nitrate reductase activities of roots compared with fronds, suggests, together with the measured uptake rates by roots and fronds, that NO3− is transported from fronds to roots for reduction. This contrasts what is observed for terrestrial plants (Gojon et al., 1994) and the division of NO3− uptake and reduction between roots and fronds of L. minor might represent an uptake and assimilation pattern unique to floating aquatic plants.