Nitrate dynamics in natural plants: insights based on the concentration and natural isotope abundances of tissue nitrate

Abstract

The dynamics of nitrate (NO(-) 3), a major nitrogen (N) source for natural plants, has been studied mostly through experimental N addition, enzymatic assay, isotope labeling, and genetic expression. However, artificial N supply may not reasonably reflect the N strategies in natural plants because NO(-) 3 uptake and reduction may vary with external N availability. Due to abrupt application and short operation time, field N addition, and isotopic labeling hinder the elucidation of in situ NO(-) 3-use mechanisms. The concentration and natural isotopes of tissue NO(-) 3 can offer insights into the plant NO(-) 3 sources and dynamics in a natural context. Furthermore, they facilitate the exploration of plant NO(-) 3 utilization and its interaction with N pollution and ecosystem N cycles without disturbing the N pools. The present study was conducted to review the application of the denitrifier method for concentration and isotope analyses of NO(-) 3 in plants. Moreover, this study highlights the utility and advantages of these parameters in interpreting NO(-) 3 sources and dynamics in natural plants. We summarize the major sources and reduction processes of NO(-) 3 in plants, and discuss the implications of NO(-) 3 concentration in plant tissues based on existing data. Particular emphasis was laid on the regulation of soil NO(-) 3 and plant ecophysiological functions in interspecific and intra-plant NO(-) 3 variations. We introduce N and O isotope systematics of NO(-) 3 in plants and discuss the principles and feasibilities of using isotopic enrichment and fractionation factors; the correlation between concentration and isotopes (N and O isotopes: δ(18)O and Δ(17)O); and isotope mass-balance calculations to constrain sources and reduction of NO(-) 3 in possible scenarios for natural plants are deliberated. Finally, we offer a preliminary framework of intraplant δ(18)O-NO(-) 3 variation, and summarize the uncertainties in using tissue NO(-) 3 parameters to interpret plant NO(-) 3 utilization.

Keywords: atmospheric nitrate; denitrifier method; isotopic enrichment; isotopic fractionation; nitrate reductase; oxygen isotope; plant nitrate; soil nitrogen availability.

Figure 1

Assigned isotopic ratios (A: δ¹⁵N; B: δ¹⁸O) of NO⁻₃ standards (IAEA NO₃, USGS-32, USGS-34, and USGS-35) shown against corresponding isotope values measured in MQ (Millipore) water and in plant extracts (the initial NO⁻₃ in plant extracts was removed using the same protocol as that described in Liu et al., 2012a).

Figure 2

Schematic map showing major NO⁻₃ sources and processes in leaves and roots of natural plants.

Figure 3

Tissue NO⁻₃ concentrations in natural plants growing under disturbed conditions (acidic irrigation and liming; Gebauer et al., 1988), in N-polluted forest plants (Stams and Schipholt, 1990), in natural and crop plants with artificial NO⁻₃ supply (data of natural plants were cited from Gebauer et al., ; Stadler and Gebauer, ; Robe et al., ; Simon et al., . Data of crop plants were cited from Evans et al., ; Yoneyama and Tanaka, ; Prasad and Chetty, and references cited therein).

Figure 4

(A) Relation between NO⁻₃ concentrations in soil and natural plants. Plant NO⁻₃ data in the left panel are shown for individual samples in Guiyang, southwestern China and western Tokyo, Japan reported by Liu et al. (2012a, 2013a). Plant NO⁻₃ data in the right panel show organ-specific and whole-plant concentrations (averages of different species) in ecosystems of Central Europe (see details in Gebauer et al., 1988), and leaf NO⁻₃ of different species (H. hirta, P. japonica, L. stellipilum, L. triloba) in a temperate forest of central Japan (Koyama et al., 2013). (B) Relations between total N, C/N, and tissue NO⁻₃ concentration in natural plants. Mosses include different species in different habitats of Guiyang, Southwestern China, and Western Tokyo, Japan (cited from Liu et al., 2012a,c). Vascular leaves I, petioles and roots were reported for a coniferous and a broadleaved plant in western Tokyo, Japan (cited from Liu et al., 2013a). Vascular leaves II included fern, oak, and pine species at the Camp Paivika and Camp Osceola forest sites in the San Bernardino Mountains of southern California, USA (cited from Fenn et al., 1996).

Figure 5

Preliminary relation between δ¹⁸O and Δ¹⁷O values of NO⁻₃ in mosses and vascular plants. The δ¹⁸O and Δ¹⁷O values were considered respectively, as −5 to 5‰ and 0‰ for soil NO⁻₃ (black and solid line), 70 and 25‰ for atmospheric NO⁻₃ (red square). Dashed lines show the isotopic range of mixing between atmospheric and soil sources. Dashed lines with arrows show the vectors of δ¹⁸O enrichments because of NR reduction.

Figure 6

Schematic showing δ¹⁸O-NO⁻₃ variations in plants under different uptake (from soil and or atmospheric sources: distinct in the δ¹⁸O value), translocation (from soil and or root to shoot), and reduction (potentially inducible by increasing [NO⁻₃] or no reduction and no isotopic enrichment with NO⁻₃ accumulation, depending on species). Long and short solid lines with arrows respectively show the vectors of δ¹⁸O-NO⁻₃ and [NO⁻₃] variations. Dashed lines with arrows show the uptake, transportation, and translocation of NO⁻₃ from the soil to roots and or to leaves, from atmosphere to leaves, during which isotope effects were regarded as negligible. Shaded areas (gray for roots, green for leaves) show isotopic enrichment during the mixing of different sources (the δ¹⁸O-NO⁻₃ in plants should be distributed between the δ¹⁸O values of sources, depending on the fraction of each source) and or the occurrence of NR reduction activities (the δ¹⁸O-NO⁻₃ in plants would be higher than the δ¹⁸O of sources but the magnitude of enrichment depends on in situ NR dynamics; presumably less than that presented in Table 1). For scenarios that occurred, leaf uptake of atmospheric NO⁻₃ was assumed to be homogeneous. The shaded area, the spatial distance, and length of lines had no quantitative implications. S1–S12 correspond to scenarios 1–12 in the main text. Briefly, S1, no occurrence of NO⁻₃ reduction in roots; S2, (inducible) root NO⁻₃ reduction; S3, no NO⁻₃ was transported from soil to leaves and leaf NO⁻₃ was derived from the atmosphere, but no reduction occurred; S4, no NO⁻₃ was transported from soil to leaves and leaf NO⁻₃ was from atmosphere and (inducible) reduction occurred; S5, leaf NO⁻₃ was taken up directly from the soil, but no reduction occurred; S6, leaf NO⁻₃ was taken up from the soil and reduction occurred therein; S7, leaf NO⁻₃ is completely or partially transported from the root where it has experienced reduction, but no further reduction in the leaf; S8, leaf NO⁻₃ is completely or partially transported from the root where it has experienced reduction, and is further reduced in the leaf; S9, leaf NO⁻₃ was from both atmosphere and soil but no reduction occurred in the leaf; S10, leaf NO⁻₃ was from both atmosphere and soil, and reduction occurred in the leaf; S11, leaf NO⁻₃ is a mixture of atm-NO⁻₃ and root NO⁻₃ but no reduction occurred; S12, leaf NO⁻₃ is a mixture of atm-NO⁻₃ and root NO⁻₃, and reduction occurred in the leaf; S13, leaf NO⁻₃ is a mixture of soil NO⁻₃, atm-NO⁻₃, and root NO⁻₃, but no reduction occurred in the leaf; S14, leaf NO⁻₃ is a mixture of soil NO⁻₃, atm-NO⁻₃, and root NO⁻₃, and reduction occurred in the leaf. The δ¹⁸O differences between S13 and S11, between S12 and S14 depend on the fraction of soil NO⁻₃ in the mixed pool of leaves.