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- Materials and Methods
The nitrogen (N) supply to plants is heterogeneous in both space and time, varying with site and season and through depletion. This supply is also increasingly influenced by human activity, through N deposition (Vitousek, 1994; Sala et al., 2000) and through changes in the availability of N through mineralization in warmer soils (Melillo et al., 2002). N availability can influence species composition (Carroll et al., 2003), rates of decomposition (Melillo et al., 2002) and N mineralization (Carroll et al., 2003; Throop et al., 2004). Nitrogen is also an important determinant of plant productivity (Aerts et al., 1995; Reich et al., 1997), tissue N concentrations (Oren et al., 2001) and carbon:nitrogen (C:N) ratios (Throop et al., 2004). Tissue N concentration, in turn, can be an important determinant of the rate of key physiological processes in the plant, such as photosynthesis and respiration (R).
There is a strong correlation between the rate of photosynthesis and foliar N concentration (e.g. Field & Mooney, 1986; Evans, 1989; Reich et al., 1998) because of the high N investment in the photosynthetic apparatus (Evans, 1989). The proportion of photosynthetic N allocated to Rubisco and electron transport in the thylakoid membranes is particularly important in determining rates of photosynthesis and, subsequently, photosynthetic nitrogen use efficiency (PNUE; Poorter & Evans, 1998; Westbeek et al., 1999). There is also a correlation between R and N concentration in leaves (Ryan, 1995; Reich et al., 1998; Mitchell et al., 1999; Tjoelker et al., 1999; Griffin et al., 2001; Loveys et al., 2003; Noguchi & Terashima, 2006). Although coupling between leaf R and N tends to be maintained irrespective of the origins of the species (Reich et al., 1996, 2006; Tjoelker et al., 1999), environment-mediated changes in the relationship between leaf R and leaf N can occur. For example, in a comparison of 70 Australian perennial species, Wright et al. (2001) showed that, whilst the slope of log–log plots of leaf R (on a dry mass basis) vs leaf N concentration was constant across sites, there were differences in the intercept for sites differing in nutrient availability and rainfall.
In contrast to the abundance of studies investigating the relationship between N and metabolism in leaves, relatively few studies have focused on coupling between root N concentration and root metabolism. Rates of R are positively correlated with the concentration of N in fine roots (Pregizter et al., 1998; Reich et al., 1998; Burton et al., 2002; Tjoelker et al., 2005; Bahn et al., 2006), and as roots age, root R declines in parallel with a sharp decline in root N concentration (Volder et al., 2005). The availability of N in soil can also affect root respiration, with rates of root R being lower in plants growing on N-deficient soils compared with plants well supplied with N (Van der Werf et al., 1992). The linkage between root R and N probably reflects the respiratory costs associated with the uptake and assimilation of N, protein turnover and maintenance of solute gradients (Scheurwater et al., 1998). Bahn et al. (2006) showed that the relationship between root R and N concentration for temperate mountain grasslands varied between sites, suggesting that root R–N relationships are not fixed. Whether differences in the relationship between root R and N concentration vary systematically with environmental factors such as growth temperature is not known.
In addition to playing an important role in determining rates of photosynthesis, N supply can also affect the extent of cold acclimation of the photosynthetic apparatus (Martindale & Leegood, 1997), a phenomenon that requires additional N investment in chloroplastic and cytosolic proteins (Stitt & Hurry, 2002). Cold acclimation can be defined as adjustments in metabolic processes in such a way that plant performance is improved in the cold (Körner & Larcher, 1988); full acclimation can result in complete metabolic homeostasis, i.e. identical rates of metabolism in plants growing at contrasting temperatures (Stitt & Hurry, 2002; Atkin & Tjoelker, 2003). Although the impact of N supply on respiratory acclimation is not known (either for leaves or for roots), there is strong a priori evidence that N availability could influence respiratory acclimation. In leaves, growth in the cold increases both R and N concentrations (e.g. Ryan, 1995). Similarly, species adapted to the cold often exhibit higher tissue N concentrations and R (Körner & Larcher, 1988). Tjoelker et al. (1999) found greater cold acclimation of leaf R in conifers than in broad-leaved trees, with foliar N concentrations increasing with decreasing growth temperature in the conifers but not in the broad-leaved trees. Cold acclimation of leaf R is dependent on increases in the capacity for mitochondrial respiration (Armstrong et al., 2006), which in turn would probably require an increase in N investment in respiratory proteins (and thus organic N); limitations in N supply could therefore restrict the extent to which R acclimates to low growth temperatures. Increases in R per unit organic N could occur if cold treatment increases respiratory flux via increases in substrate supply and/or reductions in adenylate restriction of electron transport (Atkin et al., 2000). Alternatively, higher rates of R in cold-acclimated roots might reflect an increase in demand for respiratory energy associated with a higher organic N concentration.
N supply might also impact on the short-term temperature dependence of R [i.e. the proportional change in R per 10°C change in temperature (Q10)]. Atkin & Tjoelker (2003) highlighted how variations in respiratory capacity, adenylates and substrate supply could influence Q10 values. If respiratory capacity is altered by changes in N supply and/or changes in N supply alter the fixation of CO2 and the production of sugars, then one might expect that the Q10 of R would vary with N availability and N concentration in the tissues. Alternatively, the Q10 of root R may vary in response to the turnover of adenylates associated with N uptake, transport and assimilation. Support for the suggestion that nutrient availability and the Q10 of R are linked comes from recent work by Turnbull et al. (2005), who found that the Q10 of leaf R in a temperate rainforest varied with nutrient availability (determined via analyses of extractable soil nitrate, ammonium and phosphorus concentrations; Richardson et al., 2004) along a soil chronosequence in New Zealand; leaf R Q10 values were highest at sites with the greatest soil nutrient concentration. The importance of N supply per se (as opposed to variations in other nutrients) in determining the Q10 of R in leaves or roots was not, however, established. Understanding how N supply impacts on the temperature response of root R is necessary for predicting future rates of R in environments where both N availability and temperature regimes are altered; in addition to an increase in mean annual temperatures, increases in variation about the mean temperature are expected, with potential for an increase in seasonal differences and an increase in the frequency of extreme events (IPCC, 2001).
In this study, we investigated the role external N supply plays in determining the response of root R of several herbaceous plant species after 7 d at a lower growth temperature. The specific questions addressed were as follows.
To what extent do variations in N supply and N tissue concentration affect rates of root R of several herbaceous plant species?
Does the short-term temperature sensitivity of root respiration (Q10) vary with N concentration?
Is the degree of acclimation of roots to a decrease in temperature greater at high N supply than at low N supply?
Is cold acclimation of root R associated with an increase in root N concentration (with higher R being required to meet the increased energy demands associated with higher protein concentrations, etc.) or are higher rates of R in cold-acclimated plants achieved via increased rates of R per unit organic N?