DOI: 10.25911/r8ja-sz61
title: Source data used in Zhai et al. (Functional Ecology, 2025) Nutrient-induced changes in root respiration in 10 woody plant species

description: Nitrogen (N) and phosphorus (P) are soil macronutrients that influence ecosystem productivity through strong impacts on plant metabolism. The influence of nutrient supply on the relationships between leaf respiration rate (R) and leaf N concentration ([N]) has been widely investigated. By contrast, how root R responds to variations in nutrient availability and whether there remains a general response across a wide range of species is less well known. Here, we conducted an experiment assessing the effects of N and P supply on root R in 10 woody plant species, with root R being determined by the in vivo rate of O2 consumption. Maximum R (Rmax) was also quantified by O2 uptake in the presence of exogenous substrate and respiratory uncoupler.
We investigated whether the response of root R to nutrient availability (N or P) varies with the other nutrient (P or N) supply in woody plants. Our first hypothesis was that root R would be higher in plants grown in high-N or high-P supply compared with low-nutrient supply, with the promoting effect of high-N or high-P supply being more effective when the other nutrient (P or N) is not limiting (Hyp. 1). Second, because respiration can be significantly impacted by the concentration of key metabolic enzymes which is associated with the level of tissue [N], we hypothesized that the effect of nutrient supply on root R-[N] (or [P]) relationships will be similar to those reported for leaves (Crous et al., 2017 – New Phytologist, 215, 992-1008), with low-nutrient availability resulting in an increase in the proportionality coefficient (i.e. intercept of log root R-root nutrient relationships), but not the scaling exponent (i.e. slope of log root R-root nutrient relationships) (Hyp. 2). We also assessed whether contrasting N and P supply affected the regulatory control (adenylate/substrate) of root and leaf R in woody species, with our study focussing on the effect of nutrient supply on maximum R (Rmax) and the proportion of R engaged in vivo (i.e. R/Rmax)]. Here, we tested a third hypothesis that Rmax would be lower in plants grown on low-N or low-P supply and that R/Rmax would be lower in low-P grown plants (due to greater adenylate restriction of respiration when P is limiting), but not in low-N grown plants (Hyp. 3). Our experiments used a subset of 10 woody species from the same sand/hydroponic-grown plants in a previously published study that reported on leaf-level CO2 exchange (Crous et al., 2017)
For this study, 10 woody species were used for growth treatments, see Crous et al. (2017). Briefly, seedlings were transplanted into 3.18 L plastic cylinders (50 cm in height and 9 cm in diameter) containing sterilized sand in November 2008 and were measured in June and July 2009. All the seedings were less one year old when they were transplanted. Plants were divided into four treatment groups, with each group receiving modified Hoagland No. 1 solution containing specific concentrations of N and P. Here, our aim was to achieve changes in foliar chemistry that were also reflected in rates of leaf metabolism, rather than necessarily provide field-relevant levels of nutrient availability for any of the species in question. N and P were provided at nominally ‘high’ and ‘low’ levels in four treatment combinations consisting of high N and high P (HNHP), high N and low P (HNLP), low N and high P (LNHP), and low N and low P (LNLP). The HN solution contained 5 mM KNO3 and LN solution 0.4 mM KNO3. The ‘high P’ solutions contained 1 mM KH2PO4 whereas ‘low P’ had 2.0 uM KH2PO4 to limit storage of P in the vacuole as a buffer. Thus, N:P supply ratios varied from 5:1 for HNHP to 2500:1 for HNLP, 0.4:1 for LNHP and finally 200:1 for LNLP. In addition to N and P, the modified Hoagland solution also contained 0.07 mM Ca2Cl and 0.45 mM MgSO4, as well as several micronutrients (4.2 µM boron, 1.2 µM manganese, 0.8 µM zinc, 0.03 µM copper, 0.04 µM molybdenum and 0.01 µM cobalt). Each nutrient solution was balanced for cations and iron (Fe) was added as ferric EDTA to a level of approximately 8 µM Fe. These micronutrient concentrations were one-tenth of those in the recommended Hoagland solution because full strength can result in toxic symptoms. Immediately after transplanting, between 120 and 150 mL of nutrient solution was applied to individual plants each day at the rate of 20 mL min-1, pumped from 200-L storage containers which were refilled regularly. 
Respiration (R) measurements were made after seven months of growth following the commencement of nutrient treatments. Oxygen (O2) uptake by detached roots was measured polarographically in cuvettes containing 30-40 mL of aerated modified Hoagland’s nutrient solution (pH 5.8) buffered with 10 mM MES (2-(N-Morpholino) ethanesulfonic acid) using Clark-type O2 electrodes (Dual Digital Model 20; Rank Brothers, Cambridge, UK) coupled to a computer-based data acquisition system (NI-DAQ for Windows 2000, National Instruments, Berkshire, UK). The cuvette was maintained at a constant measurement temperature of 25°C in a water bath. Measurements were made in solutions containing the same N and P concentrations in which each plant was grown. Whole roots were removed from the growth cylinders and carefully washed in water. Uniformly metabolically active young roots were sub-sampled for the R measurements, as these are responsible for active exploration and nutrient uptake (see cited publications in Zhai et al. 2025, Functional Ecology). Following 10 min stabilization, O2 depletion was recorded over a 10 min period, with all measurements terminating before the oxygen concentration was 40% of air saturation. Following measurements, roots were oven-dried (2 days at 70 °C) and the dry mass was recorded. Root [N] and [P] were analysed on dried and ground root samples after Kjeldahl digestion (see cited publications in Zhai et al. 2025, Functional Ecology), using a flow injection analyser (Lachat Instruments, Loveland, CO, USA) for N and P using the indophenol blue and ammonium molybdate methods, respectively. It should be noted that, for those woody species that formed proteoid root systems (cluster roots), only non-proteoid roots were selected to evaluate the R rate. Leaf O2 uptake was measured using a Hansatech Oxygraph (Norfolk, England) O2 electrode system, using 2 mL cuvettes. Intact leaf discs (cut with a sharpened 0.7 cm2 leaf corer) from the lamina region of the most recently fully expanded leaves were sliced into 2 mm thick slides while immersed in a measurement buffer [10 mM Hepes, 10 mM MES and 0.2 mM CaCl2.2H2O (pH 7.2)]; leaf slices in buffer solution were kept in darkness for 30 mins to overcome post-illumination transients and wounding effects (see cited publications in Zhai et al. 2025, Functional Ecology). Following the measurements of respiratory O2 uptake (10 min stabilization, followed by O2 depletion being recorded for several minutes, with measurements terminating before O2 concentration was 40% of air saturation), samples were oven-dried at 70°C for at least two days to determine dry mass. To assess the impact of nutrient treatments on the proportional engagement of respiratory capacity (R/Rmax), we measured the rates of O2 uptake by roots and leaves of woody plants in the combined presence of an uncoupler and exogenous substrate. These were done at the same time as the above measurements using a matched sample from the same plants. We adopted a similar approach to that adopted previously (see cited publications in Zhai et al. 2025, Functional Ecology), with exogenous substrate (50 mM glucose from a 2 M stock) and uncoupler (3 µM carbonyl cyanide m-chlorophenyl hydrazone) prepared as previously described (see cited publications in Zhai et al. 2025, Functional Ecology). The proportional engagement of respiratory capacity was indicated by the ratio of root R to Rmax (R/Rmax).
Oxygen (O2) uptake by detached roots was measured polarographically in cuvettes containing 30-40 mL of aerated modified Hoagland’s nutrient solution (pH 5.8) buffered with 10 mM MES (2-(N-Morpholino) ethanesulfonic acid) using Clark-type O2 electrodes (Dual Digital Model 20; Rank Brothers, Cambridge, UK) coupled to a computer-based data acquisition system (NI-DAQ for Windows 2000, National Instruments, Berkshire, UK). The cuvette was maintained at a constant measurement temperature of 25°C in a water bath. Root [N] and [P] were analysed on dried and ground root samples after Kjeldahl digestion (see cited publications in Zhai et al. 2025, Functional Ecology), using a flow injection analyser (Lachat Instruments, Loveland, CO, USA) for N and P using the indophenol blue and ammonium molybdate methods, respectively. Leaf O2 uptake was measured using a Hansatech Oxygraph (Norfolk, England) O2 electrode system, using 2 mL cuvettes. Relevant indophenol blue and ammonium molybdate methods standard were used for the flow injection analyser (Lachat Instruments, Loveland, CO, USA) for N and P, respectively. All plants and nutrient treatments were randomly assigned to six replicate blocks spread across two adjacent glasshouses (with three replicate blocks in each glasshouse). The glasshouses employed natural light and the growth temperature was maintained at 25°C/18°C day/night. The experimental approach used was previously shown to be effective in generating a range of leaf-nutrient and functional phenotypes between ‘deficient’ and ‘adequate-abundant’ (Crous et al., 2017) and tissue N and P contents were consistent with the range of previously published values (see cited publications in Zhai et al. 2025, Functional Ecology). There was no evidence from leaf physiological responses (Crous et al., 2017) of toxicity effects in the HNHP treatments. Additionally, leaf P contents (Crous et al., 2017) in the two most likely P-sensitive Hakea species did not reach levels previously reported as critical for toxicity effects (see cited publications in Zhai et al. 2025, Functional Ecology), with the exception of Hakea multilineata in the HNHP treatment only. Roots from this species in this treatment were not included in the analysis.