Environ. Sci. Technol. 2008, 42, 1630–1637
Determination of Ammonium Turnover and Flow Patterns Close to Roots Using Imaging Optodes NIKLAS STRÖMBERG* Department of Chemistry, Göteborg University, Göteborg SE-412 96, Sweden
Received June 12, 2007. Revised manuscript received October 22, 2007. Accepted November 07, 2007.
The physical effect of nitrogen upon plants has been studied thoroughly; however, direct studies of nitrogen turnover close to roots have been limited by analytical techniques with low spatial and temporal resolution. Thus, little is known about differences in turnover taking place along and between intact root structures over time as well as how root arrangement, root cell type, plant age, microbial activity, and the dark/light cycle influence uptake and supply of nutrients to root structures. In this study an imaging (planar) optode was used to quantify ammonium over time close to an intact root system of a large fruit bearing tomato plant (Lycopersicon esculentum). Images throughout the experiment made it possible to define the ammonium depletion zone and active turnover potential as well as determine turnover rate and flow patterns around the root system over time. The results indicated that ammonium uptake for tomato plants proceeds over the entire root structure but transverse thin peripheral roots are about twice as efficient as the main root and that the uptake process might influence nutrient availability. The flow patterns close to the root structure revealed that apical regions seem to have a central role in ammonium acquisition.
Introduction Most plants use nitrogen compounds such as ammonium or nitrate available in the soil pore water for protein and nucleic acid anabolism (1). With respect to these dissolved nutrients, ammonium is the most easily metabolized and often preferred nitrogen species under mixed conditions (2, 3). Generally, most plants (however, exceptions occur) cannot accumulate nitrogen compounds that later on could be released on demand due to lack of storage facilities (4). Therefore, optimal plant growth relies on a continuous supply through diffusion or mass flow to the roots. Under natural conditions, however, plant growth often becomes nitrogen limited and plants adapt to nitrogen deficiency by altering the root architecture through proliferation of new root structures into nitrogen rich areas and increase the root surface area (5). Examples include the development of root hairs, micrometer-sized epidermal outgrowths located in the elongation zone of the roots (1), that can constitute more than 70% of the root surface (5). Root hairs have been shown to be vital for phosphate uptake for tomato (6), but their relative importance for ammonium acquisition compared with other root structures is less known (7, 8). Moreover, the processes of how ammonium is incorporated in roots * Corresponding author phone: +46 31 772 27 84; fax: +46 31 772 27 85; e-mail:
[email protected]. 1630
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are not completely understood. However, kinetic studies of
13NH + in rice roots indicated that uptake might be dependent 4
on both active and passive transport mechanisms (9). The complexity of the uptake processes for ammonium in roots is further illustrated for tomato by the presence of two recently characterized high-affinity transporters which are differentially regulated by N and contribute to root-hair-mediated ammonium acquisition from the rhizosphere (10). Finally, microbes that concurrently or symbiotically consume/release ammonium or convert it to other nitrogen species over time affect the turnover. Due to all these processes and effects, studies of ammonium turnover in this ever-changing environment require measurements with both high spatial and temporal resolution. The overall objective of this study was to provide a method with good coverage and spatial and temporal resolution, as a complement to microelectrodes, to study ammonium turnover in the close vicinity of undetached root structures. Techniques for Ammonium Screening in Complex Environments over Time. The only technique to study ammonium turnover over relatively large parts of root structures has been based on the use of a fast-scanning microelectrode in combination with microscopy (8). The technique provided relatively high spatial resolution but to the expense of coverage and was restricted to roots grown in solution. To fulfill the requirements for both high temporal and spatial resolution as well as coverage in the measurements, a new tool, i.e., imaging (planar) optodes, was developed in 1995 and used for studies of oxygen distribution in sediments (11). Later on also, pH measurements were made in sediments using a similar technique (12). The general detection principle for imaging optodes is based on changes in the luminescence properties from immobilized solute specific indicators onto/within thin-layered plastic films when exposed to the analyte. The sensor film in contact with the sample is illuminated, and usually the fluorescence intensity or fluorescence lifetime from the plastic film is detected with a camera (Figure 1). Direct quantification using the intensities should be avoided due to various artifacts such as, e.g., uneven dye distribution and variations in the excitation light source ,and therefore normalization procedures are usually required before quantification. Recently, a promising ratiometric sensing scheme (13) was applied on a fast, selective, and pH-independent ammonium optode (14). The normalization procedure removed artifacts associated with imaging, such as uneven illumination and instrument fluctuations. The optode was thoroughly tested for imaging during 10 days and the associated calibration technique, time-correlated pixel-bypixel calibration (TCPC), not only removed time dependent drift in response (15) but also facilitated control of the sensor performance throughout measurements (16). This technique together with the ratiometric optode is used in this study for complete characterization of ammonium turnover close to roots.
Experimental Section Plant Cultivation. The about 6-month-old plant (Lycopersicon esculentum (L. esculentum)) was precultivated in commercial plant soil and had several large fruits before it was replanted in a specially designed pot with removable glass sides (Figure 1). The plant was adapted for 1 week to a 12 h light cycle in 18 °C in this new environment. The artificial light source gave a maximum illumination (0.3 m from the light source) of 2 × 2700 lx (top; OSRAM L20W/20S) and 4700 lx (side; 2x Philips TLD 18W/95) to cover the large 10.1021/es071400q CCC: $40.75
2008 American Chemical Society
Published on Web 01/31/2008
FIGURE 1. Optical system and sensor assembly used during the experiment. The illustration describes the Xenon light source (A), the dual bandpass filter changer (B), the liquid light guide (C), the focusing lenses (D), the sensor assembly in the pot (E), the macrolens (F), the infinity region with bandpass filters for wavelength selection (G), the CCD camera (H), the dual filter control unit (I), and the image data transfer to computer (J). The close up of the sensor assembly (E) shows the transparency film (I) that isolated the studied root structure from the rest of the plant; the reservoir with 500 × 10-6 M ammonium solution (II), the optical isolation (III), the transparency film with ammonium sensor and alignment spots (IV), and the front removable glass (V) facing the illumination and the macrolens. canopy of the plant. The rhizosphere was kept in darkness throughout the experiment. A transparency film isolated a single root from the rest of the plant and soil, and a 2 mm thick sheet of paper acted as a reservoir for a 500 × 10-6 M ammonium solution. The isolated root was gently washed with tap water in order to remove soil particles before an optical isolation (colorit 090) with excellent tear strength in water was attached to the surface of the sensor facing the root structure. Sensing Principles of the Imaging Ammonium Optode. The sensing scheme for ammonium is based on a phase transfer of an ammonium-nonactin (ammonium ionophore) complex together with the solvent sensitive merocyanine540 in a hydrogel-ether emulsion (14). The fluorescent dye changes excitation–emission maxima upon shift of solvent (17–19) induced by altered ammonium concentration. Quantification of ammonium was made through a dual excitation dual emission image ratio (excitation:emission/excitation: emission (nanometer values), 572:592/511:572) coupled to a recently developed calibration method (15). Imaging System. The imaging system (Figure 1), thoroughly described in earlier work (15), was used with a modified excitation wavelength representing the dye in the hydrogel, 511 nm instead of 520 nm, and bandpass with a full width at half-maximum of 2 nm instead of 20 nm. The decreased excitation light intensity due to the smaller bandpass was compensated for by an increased integration time (84 s). The image resolution determined by the edgeresponse approach (20) was 210 × 10-6 m. However, the diffusion error reduces the effective sensor resolution in direct proportion to the overall thickness of the sensor film and optical isolation. Time-Correlated Pixel-by-Pixel Calibration and Signal Quality Control. The sensor film assembled in a flow cell was exposed to ammonium calibration solutions (0, 125 × 10-6, 250 × 10-6, 375 × 10-6, and 500 × 10-6 M; ionic strength (NaCl + NH4+) ) 3.0 × 10-3 M) for 20 min (per solution) before imaging. Each concentration was represented by 10 images captured at both wavelengths. To
reduce the systematic noise associated with the multiple image capture, dark noise was subtracted at each wavelength before images were aligned to the same position. Images (10) were averaged at each wavelength in order to suppress shot noise and subsequently filtered (3 × 3 median) before and after the image ratio. At the end of the calibration procedure, the cell was flushed with blank solution for at least 60 min to minimize possible carryover from ammonium bound in the sensor film. The optode film was removed and assembled in the pot and thereafter exposed to the root structure and reservoir (Figure 1). After capturing the experimental images, the sensor was recalibrated in the flow cell according to the procedure described above. Finally the analyte responses in each pixel from the two time-separated calibrations were interconnected using a linear response function dependent on time (15). The concentration in the experimental images from the root is determined and visualized in each pixel using a unique calibration curve calculated from the response function at the time of image capture. Statistical parameters throughout measurements such as analytical sensitivity, limit of detection, and relative signal variation were predicted using the time correlated pixel-by-pixel calibration protocol and the individual images captured for the ensemble averaging (16). Determination of the Ammonium Turnover. Images of the concentrations (Figure 2) were calculated as described above, and the turnover rates were calculated using these images and the time for image capture according to eq 1 (C1 and C2 refers to first and subsequent concentration images; t1 and t2 refers to first and subsequent times of image capture). Turnover rates were also calculated in the same manner using the values from boxes A-C. To increase the visibility of the small changes in turnover, the root structure was cropped from the turnover images (Figure 3). The images were complemented with average and standard deviations retrieved from pixels (441) in the three boxes (A-C) representing different areas in the images. The resolution of the color bar (Figure 3) was adjusted to 4 × 10-6 M/h, thus showing a valid VOL. 42, NO. 5, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Ammonium distributions after about 40 h with outlined root position. Boxes A-C define regions used to characterize the complete experiment. At the time of capture the predicted LOD, the smallest detectable change in concentration, relative signal variation (relative standard error of the mean), and operational lifetime of the three boxes were on average 3 × 10-6 M, 12 × 10-6 M, 2%, and 11 days, respectively. The outlined root structure is constructed by both automatic and manual digitalization in Adobe Photoshop and is overlaid on the experimental image calculated in Matlab. (P ) 0.95) turnover rate, using the predicted analytical sensitivity at the time for image capture from the calibration protocol. C2 - C1 ⁄ t2 - t1
(1)
Determination of the Depletion Zone and the Active Turnover Potential. A typical phenomenon associated with root uptake of ammonium and potassium is the gradual development of a depletion zone close to active root structures due to a higher consumption than supply (21). The depletion zone was determined according to an arbitrarily defined criterion where each pixel, reduced to about half of its initial value in the matrix, was visualized by a color from yellow (low consumption) to red (high consumption). The nutrient uptake potential of a root system (roots per number of plants or roots per unit soil surface) generally by far exceeds the nutrient requirement of the plant (21). However, it does not account for roots being active or inactive. The imaging technique was used to calculate a new parameter that could be used to estimate the active ammonium turnover potential of a root system or a particular root structure. The term turnover rate is used to include the effect from eventual microbes (the root was not sterilized); however, the method still assumes that turnover is mainly linked to root uptake. It was defined by the area of the depletion zone (colored pixels in Figure 4a) per root cross-section area within the depletion zone. The images were constructed by a ratio of a binary concentration images (>300 µm, 0;
