Plant and Soil 221: 13–24, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
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Effects of rhizospheric bicarbonate on net nitrate uptake and partitioning between the main nitrate utilising processes in Populus canescens and Sambucus nigra Wolfgang Wanek∗ and Marianne Popp Institute of Plant Physiology, University of Vienna, Althanstraße 14, A-1091 Vienna, Austria Received 30 March 1999. Accepted in revised form 19 October 1999
Key words: assimilation, dissolved inorganic carbon, nitrate, uptake, xylem transport
Abstract Increased levels of rhizospheric dissolved inorganic carbon have repeatedly been demonstrated to enhance plant growth by up to 80%, although carbon from dark fixation accounts for only 1–3% of total plant carbon gain. This study, therefore, aimed at investigating the effects of bicarbonate on nitrate uptake, assimilation and translocation to shoots. Clonal saplings of poplar (Populus canescens (Ait.) Sm.) and elder (Sambucus nigra L.) were grown hydroponically for 35 days in a nutrient solution containing 0, 0.5 and 1 mM bicarbonate and 2 mM nitrate as the sole nitrogen source at pH 7.0. Net nitrate uptake, root nitrate accumulation and reduction, and export of nitrogenous solutes to shoots were measured after incubating plants with 15 N-labelled nitrate for 24 h. Net nitrate uptake increased non-significantly in plant species (19–61% compared to control plants) in response to 1 mM bicarbonate. Root nitrate reduction and nitrogen export to shoots increased by 80 and 95% and 15 and 44% in poplar and elder, respectively. With enhanced root zone bicarbonate, both species also exhibited a marked shift between the main nitrate utilising processes. Poplar plants increasingly utilised nitrate via nitrate reduction (73– 88% of net nitrate uptake), whereas the proportions of export (20–9%) and storage in roots (7–3%) declined as plants were exposed to 1 mM external bicarbonate. On the other hand, elder plants exhibited a significant increase of root nitrate reduction (44–66%) and root nitrate accumulation (6–25%). Nitrate translocation to elder shoots decreased from 50 to 8% of net nitrate uptake. The improved supply of nitrogen to shoots did not translate into a significant stimulation of growth, relative growth rates increased by only 16% in poplar saplings and by 7% in elder plants.
Introduction Terrestrial plants assimilate most of their carbon via ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) using atmospheric carbon dioxide as a substrate, but may additionally use rhizospheric inorganic carbon by dark fixation via phosphoenolpyruvate carboxylase (PEP carboxylase) (Cramer et al., 1993; Popp and Summons, 1983; Vuorinen et al., 1992). This carbon accounts for only 1–3% of total plant carbon gain (Cramer and Lips, 1995; Pelkonen et al., 1985). Nevertheless, increased levels of root∗ FAX No: + 43 1 31336 776.
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available inorganic carbon were repeatedly mentioned to stimulate plant biomass production of trees and cultivated plants by up to 80% (Baron and Gorski, 1986; Pelkonen et al., 1985; Vuorinen et al., 1995). However, there is still considerable controversy about whether increased rhizospheric inorganic carbon stimulates growth. Higher concentrations of bicarbonate often significantly retard growth and may perturb plant trace element nutrition (White and Robson, 1990; Yang et al., 1993). Root carbon fixation plays a key role in root nitrogen assimilation and amino acid synthesis by replenishing tricarboxylic acid cycle intermediates (Popp and Summons, 1983); it also maintains charge and
14 pH balance during ion uptake and assimilation (Davies, 1973; Latzko and Kelly, 1983; Martinoia and Rentsch, 1994). Rhizospheric inorganic carbon may thus directly or indirectly affect root ion uptake and nitrogen metabolism via dark fixation of bicarbonate through PEP carboxylase (Cramer et al., 1996; Gao and Lips, 1997; Vuorinen, 1997). This study thus aimed at investigating the effects of enhanced rhizospheric bicarbonate on uptake, root assimilation and allocation to shoots of nitrate-N and its implications for productivity in two tree species.
Materials and methods Plant material Clonal material of elder (Sambucus nigra L.) was obtained from a local tree nursery. Poplar plants (Populus canescens (Ait.) Sm.; P. alba × P. tremula /INRA clone 717 1-B4) were kindly supplied by the Institute of Forest Botany and Tree Physiology (Freiburg, Germany, Prof. Heinz Rennenberg) and were cloned by micropropagation. Saplings, at 4 months of age, were cut back and re-rooted in nutrient solution. After 2 months of preadaptation, 3–6 plants per species were transferred to nutrient solution containing KHCO3 at 0, 0.5 and 1 mM for a total experimental period of 35 days. The hydroponic medium (adjusted to pH 7.0 with KOH) contained 2 mM KNO3 , 1 mM KH2 PO4 , 0.5 mM CaCl2 , 0.5 mM MgSO4 , 60 µM Fe-EDTA, 6 µM H3 BO3 , 1 µM MnSO4 , 1 µM ZnCl2 , 0.1 µM Na2 MoO4 and 0.1 µM CuSO4 . The K+ concentration was kept constant at 4 mM by adding K2 SO4 . The hydroponic system was set up in a growth chamber with a light/dark period of 12 h/12 h (photosynthetic active radiation at plant level 450 µmol m−2 s−1 ) at 26/22 ◦ C and a relative humidity of 60/80%. Plants of the same species and treatment were combined in the same hydroponic system and each plant was provided with 2 L of culture medium, which was aerated with 100 mL ambient air min−1 and renewed every 2 days. The concentration of dissolved inorganic carbon (carbon dioxide electrode Model 95-02, Orion) and the pH value of the nutrient solution were monitored regularly. These did not change significantly over 2 days of plant exposure, inorganic carbon concentration lay within ±10% of the initial concentration, the pH value was within ±0.1 units of the initial value.
Growth analysis and measurement of transpiration Transpiration rates were calculated from weight loss of the nutrient medium after correction for the average volume loss of 3 aerated control hydroponic systems without plants. Fresh weights of individual plants were measured weekly during the experimental phase, and, as water availability did not change during the experiment, relative growth rates were calculated according to Poorter (1989). 15 N
Experiment
After the experimental period of 35 days, plants were exposed to 2 mM KNO3 (13 at% 15 N) for 24 h to quantify net nitrate uptake. Thereafter plant roots were washed two times (1 min each) in 1 mM CaSO4 and blotted dry. Roots, stems and leaves were fractionated, dried at 80 ◦ C for 24 h and homogenized in a ball mill (MM2, Retsch, Austria). Xylem sap was collected from lower shoot axes under mild vacuum (250 mbar). Anion and amino acid analysis Plant material was extracted with hot water (4% w/v) for 30 min to analyze the anion and amino acid composition of samples. The organic acid and inorganic anion content of hot water extracts and xylem sap was quantified by chemically suppressed anion chromatography (DX 500 chromatography system, Dionex, Austria). Anions were separated on an anion exchange column (AS11, 250×4 mm i.d., Dionex) using an NaOH gradient (2.5 min isocratic at 0.05 mM, linear increase from 0.05 to 37.5 mM over 15.5 min) at 32 ◦ C and a flow rate of 2 mL min−1 . Amino acids were prepurified by adsorbing to cation exchange columns (DOWEX 50WX8, H+ form; Fluka, Austria; 1 mL volume), eluting with 6 N NH3 , drying in vacuo and redissolving in deionized water. Amino acids were separated with an amino acid analyzer (Alpha Plus, Pharmacia LKB, Austria) on an Ultropac 7 column (Lithium form, 200×4.6 mm i.d., Pharmacia) using a complex step gradient of lithium citrate buffers and temperature (0.20–1.65 M, pH 2.80–3.55, 35–75 ◦ C) at a flow rate of 0.4 mL min−1 and detected after post column derivatization with ninhydrin/hydrindantin. Fractionation of nitrogen species Soluble nitrogen forms in xylem sap and roots were analyzed by fractionating reduced nitrogen forms (ammonium plus amino acids) and nitrate on cation exchange columns (DOWEX 50WX8, H+ form, Fluka,
15 1 mL volume). Nitrate was washed through the column with 3 mL deionized water. Reduced nitrogenous compounds were eluted with 3 volumes of 1 N HCl, taken to dryness and dissolved in a minimum volume of deionized water. Aliquots of the flowthrough (nitrate) and of the desorbed nitrogen fraction (ammonium and amino acids), containing 5–50 µg N, were pipetted into tin capsules, dried overnight in a vacuum concentrator and analyzed for their 15 N content. Isotopic analysis The nitrogen isotopic composition of bulk material and nitrogen fractions, as well as the carbon isotopic composition of bulk leaf material, was analyzed by continous flow gas isotope ratio mass spectrometry. The elemental analyzer (EA 1110, CE Instruments, Italy) was interfaced via a ConFlo II system (Finnigan MAT, Germany) to the gas isotope ratio mass spectrometer (DeltaPLUS, Finnigan MAT). High purity CO2 and N2 reference gases were run with each analysis. Reference gases were calibrated to Vienna Pee Dee Belemnite (V-PDB) and atmospheric nitrogen (at-air) international standards using IAEA-CH-6, IAEA-CH7 for δ 13 C and IAEA-N-1, IAEA-N-2 and IAEANO-3 for δ 15 N (reference materials were supplied by IAEA, Austria). The standard deviation of repeated measurements of a laboratory standard was 0.1 and 0.15‰ for δ 13 C and δ 15 N, respectively. The 15 N content of plant material was expressed as atom% 15 N = 15 N / (14 N + 15 N) and APE (atom% 15 N excess) = atom% 15 Nsample − atom% 15 Ncontrol. The natural abundance of 13 C was calculated as follows: δ 13 C = (R sample /R standard − 1) × 1000[‰ vs. V-PDB] where R is the ratio of mass 45 to mass 44 (carbon). Additionally, compound-specific 15 N-labelling of amino acids was determined by derivatization of prepurified amino acids with N-methyl-N-(tert.butyldimethylsilyl) trifluoroacetamide and capillary GC-MS analysis of the derivatives (MacKenzie and Tenaschuk, 1985). Calculation of nitrate fluxes Calculations were performed for individual plants each. Relative rates of nitrate fluxes are expressed in% of net nitrate uptake. ‘Net nitrate uptake’ was derived from 15 N nitrate incorporation by whole plants within 24 h. ‘N retention in roots’ was measured as total
amount of 15 N label retained in roots after 24 h. Isolation of root nitrate and measurement of its 15 N content led to the calculation of ‘nitrate retention in roots’. The difference between total N and nitrate retention represents ‘retention of reduced N in roots’. ‘N Export to shoots’ was determined as the amount of 15 N transported to shoots within 24 hours; retranslocation of 15 N from shoots to roots was not considered as phloemxylem cycling of N was reported to be low for trees in the short-term (Gojon et al., 1991). The contribution of 15 N nitrate to total 15 N in xylem saps was used to calculate the rates of ‘nitrate export to shoots’ and of ‘export of reduced N to shoots’ from the data on N export to shoots. ‘Root nitrate reduction’ represents the sum of retention of reduced N in roots and export of reduced N to shoots. Statistical analysis All statistical tests were performed with Statgraphics Plus Version 4.0 (Statistical Graphics Corporation, USA). Results were subjected to one-way analysis of variance (ANOVA) to test for significant differences between means with rhizospheric bicarbonate as the single categorial predictor variable. Tukey HSD test, a multiple comparison test, was used to determine significant differences between group means in the ANOVA setting. Tukey HSD was selected as it is generally more conservative than the Fisher LSD test but less conservative than Scheffe‘s test. Additionally, data were tested for statistically significant relationships between bicarbonate as single continuos predictor variable and plant performance with simple regression analysis (least square analysis) for Populus canescens. Sambucus nigra results were subjected to polynomial regression analysis (2nd order) as elder performance showed a curvilinear relationship to bicarbonate.
Results Net nitrate uptake and leaf gas exchange Net nitrate uptake of poplar saplings increased insignificantly by 61% at the 1.0 mM bicarbonate level compared with control plants (Figure 1A). Nonetheless, a weak linear relationship was found between bicarbonate and net nitrate uptake for poplar (r2 =0.25, P=0.11; Table 2). At 1 mM bicarbonate, transpiration rates of poplar increased 12-fold above control levels. However, its statistical significance could not
16 be evaluated as water loss was measured integratively over all plants of a species per treatment (see ‘Material and methods’). Elder plants exhibited only a small, insignificant increase in nitrate incorporation and transpiration up to 0.5 mM bicarbonate (Figure 1B). Above this concentration, transpiration leveled off and nitrate uptake decreased to nearly control rates. Net nitrate uptake and bicarbonate did not show any significant relationship in elder (Table 2). Carbon isotope discrimination did not change in elder leaves, but showed a significant shift of 2‰ towards lower values as a response to bicarbonate in poplar leaves (Figure 2).
Figure 1. Effect of rhizospheric bicarbonate on net nitrate uptake and transpiration rates of (A) Populus canescens and (B) Sambucus nigra plants. Saplings of poplar and elder were grown in hydroponics with 2 mM nitrate as a nitrogen source and 0 (), 0.5 (3) and 1 mM bicarbonate () at pH 7.0 for 35 d. Net nitrate uptake was monitored as incorporation of K15 NO3 (2 mM, 13 at% 15 N) within 24 h. Transpiration rates were calculated from weight loss of the nutrient solution. Bars indicate SE of the mean (n=3–6). Different letters represent significant differences within each species (one-way ANOVA, Tukey HSD), P-levels are given in the figure.
Figure 2. Effect of rhizospheric bicarbonate on the δ 13 C signature of bulk leaf material of (A) Populus canescens and (B) Sambucus nigra plants. Saplings of poplar and elder were grown in hydroponics with 0 (), 0.5 (3) and 1 mM bicarbonate () at pH 7.0 for 35 d. Bars indicate SE of the mean (n=3–6). Different letters represent significant differences within each species (one-way ANOVA, Tukey HSD), P-levels are given in the figure.
Nitrogen fractions in root tissue and xylem saps Total root nitrogen content, ranging from 2.01 to 2.13% DW in poplar plants and 2.34 to 2.64% in elder plants (data not shown), was not affected by bicarbonate (Table 2). Both species contained significant amounts of nitrate in roots (Figure 3), with elder showing enhanced accumulation of nitrate in roots as root bicarbonate levels increased. Total amino acid content of roots was much lower than nitrate levels, but with the exception of poplar at 0.5 mM bicarbonate did not respond significantly to enhanced root zone bicarbonate (Figure 3). Only the aspartate and asparagine content increased from 7.8 to 16.2 µmol N g−1 DW in poplar roots and to a lesser extent in elder (data not shown). On the other hand, root levels of glutamate and glutamine did not change significantly (7.2–7.6 µmol N g−1 DW, data not shown) in poplar roots. Incorporation of 15 N into oxaloacetate-based amino acids (aspartate and asparagine) increased in roots as well as in xylem sap (Table 3), while label incorporation into the 2-oxoglutarate-based amino acid pool (glutamate and glutamine) remained fairly constant or decreased in xylem sap. This translated into a significant increase in the labeling ratio of (aspartate plus asparagine) to (glutamate plus glutamine). Malate and citrate contents increased in elder roots by 75% but only insignificantly by 36% in poplar roots (Figure 3). In xylem sap, the ratio of reduced nitrogen to nitrate was reversed compared to that found in roots, amino acids being the dominant nitrogenous solutes exported to shoots (Figure 3). The concentration of reduced nitrogen in xylem sap of poplar and elder plants increased with rising bicarbonate levels, but nitrate increased only in elder xylem saps (Figure 3).
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Figure 3. Effect of rhizospheric bicarbonate on root tissue and xylem sap composition of Populus canescens and Sambucus nigra plants. Saplings of poplar and elder were grown in hydroponics with 0 (), 0.5 (3) and 1 mM bicarbonate () at pH 7.0 for 35 d. Xylem sap, which was collected from lower shoot axes under mild vacuum, and hot water extracts from root tissues were analyzed for nitrate and organic acids (malate, lower bar stack; citrate, upper bar stack) by anion chromatography and for amino acids using a commercial amino acid analyzer. Different letters represent significant differences within each species (one-way ANOVA, Tukey HSD), P-levels are given in the figure.
18 Table 1. Influence of rhizospheric bicarbonate on net nitrate uptake and nitrate-utilizing processes in Populus canescens and Sambucus nigra (absolute rates in mmol plant−1 d−1 ; relative rates in percent of net nitrate uptake). Label in nitrate, reduced N and total nitrogen of roots and xylem sap was measured separately after fractionation and used for the calculation of fluxes. Data were calculated from 3 to 6 replicate plants from the 15 N-experiment. SD, standard deviation. Different letters represent significant differences within each species (ANOVA, Tukey HSD); P
