Plant growth conditions
Zea mays L. hybrid CG60 X CG102 [29] seed was used for all experiments. Seeds were surface-sterilized by soaking 4 min in 70 % ethanol solution, 2 min in 4 % NaClO, followed by washing five times in sterile double distilled (dd) H2O. Seeds were germinated in 18-cell (two per cell, 8.5×8.5×9 cm) growth trays of Turface® (Profile Products, Buffalo Grove, USA), a baked-clay gravel with extremely low background levels of nitrogen (N). In previous experiments [30, 31] the gravel was found to contain 0.053 % N, of which only a fraction is available for plant uptake; N-free nutrient solution soaked with the Turface® gravel for 24 h was found to contain only 1.42 mg/L total N, equivalent to 0.1 mM. Growth flats were placed into plastic sub-irrigation trays (51×25.5×6 cm) containing 2 L ddH2O with no additional nutrients. For germination, the trays were initially placed in darkness in a laboratory cabinet at room temperature until plant emergence, thinned to one plant per cell, and arranged (completely randomized design, CRD) in a greenhouse with the following growing conditions: 28 °C/20 °C day/night (16 h/8 h), with 1000 W high pressure sodium and 1000 W metal halide lamps supplemented with GroLux bulbs, resulting in an average light intensity range of 803–1026 μmol m−2 s−1 (canopy level at noon). Plants were randomized daily and watered with ddH2O as needed.
Relative measurements of glutamine from leaf disk extracts
Twelve days after sowing (DAS), sub-irrigation trays were emptied of remaining ddH2O. Plants were supplied with one of six different modified Hoagland’s nutrient solutions consisting of 0.1 mM K2SO4, 1.0 mM KCl, 2 mM KH2PO4, 1 mM MgSO4·7H2O, 0.03 g/L chelated micronutrients (10046, Plant Products, Leamington, Canada) and either 0, 2, 5, 10, 15, or 20 mM total N provided as NH4NO3. Each nutrient solution (1.5 L) was poured into the sub-irrigation trays, with an additional 30 ml applied near the base of each plant.
At various time-points after nutrient application (1, 6, 18, 12, and 24 h; starting at 9:30 AM, 2:30 PM, 8:30 PM, 2:30 AM, and 8:30 AM respectively), sampling was performed on leaves 1, 2, and 3, as defined by their order of emergence. Leaf tissue disks (6.35 mm in diameter) were harvested with a hand punch tool (235270975, Fiskar’s Brands Inc., Middleton, USA) at equally spaced intervals along the mid-vein, extending from the ligule region to the leaf tip in leaves 1 and 2. Because leaf 3 was still expanding, harvest of leaf disks extended from where the leaf exited the whorl to the tip. All tissue was frozen immediately in liquid N2, and stored at −80 °C. Three, five, and four different positions were harvested from leaves 1, 2 and 3 respectively. Four plants (replicates) were sampled for each time/nitrogen combination, and the most informative treatments were repeated in an independent trial.
Leaf tissue disks were analyzed for glutamine (Gln) content with the GlnLux biosensor as previously described [12] with some modifications (Fig. 1a). Leaf disks were homogenized with a pellet pestle in a mixture of sterile sand and 20 μl 0.1 % chilled protease inhibition cocktail (PIC) (P9599-1ml, Sigma-Aldrich, St. Louis, USA), and centrifuged (model 5415R, Eppendorf, Hauppauge, USA; 4 °C, 20 min, 13 200 rpm). The resulting plant tissue extract supernatant was diluted 100-fold in 0.1 % PIC and stored overnight at −20 °C until analysis.
Concurrently, GlnLux biosensor cells were cultured for 16 h in Luria Broth (LB) (37 °C) with shaking (245 rpm). Biosensor cells were then pelleted (2500 rpm, 10 min) and washed with M9 minimal growth media (DF0485170, BD, USA) three times. Cells were re-suspended in M9 media (OD595 = 0.025) and incubated for 16 h (37 °C, 245 rpm) to deplete endogenous Gln. All media were supplemented with 50 μg/ml kanamycin and 100 μg/ml carbenicillin, as GlnLux contains Kanr and Ampr resistance genes [12].
Each leaf disk extract (10 μl) was combined with 10 μl prepared GlnLux cells and 80 μl M9 in white, flat bottom 96-well plates (07-200-589, Corning Inc., Corning, USA). A negative control of 10 μl 0.1 % PIC in place of extract was also included on each 96-well plate for subtractive normalization of the luminescence data. Plates were incubated for 2 h to allow biosensor activation, then luminescence output was quantified using a 96-well luminometer (MicroLumatPlus, Berthold Technologies, Bad Wildbad, Germany) (37 °C, 1 s photon capture per well).
Normalized luminometer data (raw outputs – negative control) was plotted against the duration of N uptake/assimilation, and against the N application rate. Outliers were identified and removed with ROUT, Q = 1 % [32]. Means were compared with the Holm-Šídák method [33–35], or Dunnett’s multiple means comparison [36] at P < 0.05 as indicated in the figure legends. Kruskal-Wallis tests with Dunn’s multiple means comparisons were used where data displayed non-normality, as identified with Bartlett’s test [37–39]. All statistical analyses were performed in GraphPad Prism 6 (GraphPad Software Inc., San Diego, USA).
Generating whole-leaf in situ images of free glutamine
As above, at 12 DAS, sub-irrigation trays were emptied of remaining ddH2O. Plants were then supplied with 0 or 20 mM total N (NH4NO3) provided as modified Hoagland’s nutrient solution (as described above). Again, 1.5 L of nutrient solution was poured into each sub-irrigation tray, and 30 ml near the base of each plant.
Leaves were harvested after 1 h (starting at 9:30 AM), 12 h (8:30 PM) and 24 h (8:30 AM) of N uptake/assimilation. Harvesting of leaf 1 consisted of removing the entire leaf at the ligule. For the younger leaves, as the ligules had not yet developed, leaves 2 and 3 were cut from the plant where the leaf blade curled in upon itself to meet the stem. Three replicates were harvested per treatment combination, frozen immediately in liquid N2, and stored at −80 °C until imaging.
Images of free Gln within maize leaf tissue were generated with GlnLux solid agar media as previously described [12] with modifications (Fig. 1b). Briefly, GlnLux biosensor cells were cultured for 16 h (37 °C, 245 rpm) in LB broth supplemented with 0.2 mM Gln, 4.0 mM glucose, 50 μg/ml kanamycin and 100 μg/ml carbenicillin. Cells were then centrifuged (2500 rpm, 10 min), re-suspended in 0.01 M potassium phosphate buffer (pH 7.0) and washed two more times. Cells were suspended in M9 medium (OD595 of 1.0). GlnLux solid agar media was prepared by combining the GlnLux culture (10 % v/v) with concentrated M9 medium containing 10 g/L Bacto agar pre-cooled (to 42 °C), and pouring this mixture into sterile 150×15 mm Petri dishes. GlnLux solid agar media plates were stored at 4 °C overnight prior to use. Frozen leaves were thawed at room temperature for 30 s and pressed into the GlnLux agar (pre-incubated at room temperature). Plates were inverted, incubated (37 °C, 2.5 h), and imaged with a charge-coupled-device (CCD) chip camera (7383–0007, Princeton Instruments, Trenton, USA) pre-cooled to −100 °C for a 1000 s exposure. Incubation and imaging of plates were staggered to ensure that conditions across replicates were constant. However, to negate the potential effects of slight incubation length differences (on the scale of seconds) in situ image standardization was performed across plates in WinView (version 2.5.16.5, Princeton Instruments, Trenton, USA) by adjusting image intensity according to the signal produced by a disk of agar (2.4 % agar in water, radius = 3 mm) containing 1 × 10−2 M Gln pressed into each plate at the time of leaf placement. This effect was examined for its potential to confound results by comparison of the standard disk image intensity to that of leaves pooled across N treatments, with F tests at P < 0.05 (GraphPad Prism 6, GraphPad Software Inc.)
Investigating the effect of Gln diffusion on whole-leaf in situ images
To examine Gln diffusion through GlnLux agar, luminescence output from leaves was visualized over multiple, consecutive incubation intervals. Plants were initially germinated and grown with only ddH2O in Turface® gravel until they were at the same growth stage as the main experiments. Hoagland’s solution containing 20 mM N was then provided for 2 h, after which plants were moved back to N-free solution for a further 10 h. Leaves 1, 2, and 3 were harvested and placed on GlnLux agar alongside disk standards of Gln (0, 3.125 × 10−4, 6.250 × 10−4, 1.250 × 10−3, 2.500 × 10−3, 5.000 × 10−3, 1 × 10−2 M, left to right; volume = 51 μl, radius = 3 mm). Plates were imaged once before incubation, and then incubated at 37 °C for intervals of 1000 s with imaging following each interval. Plates were incubated a further 6.5 h and imaged. All images were captured with a 1000 s exposure and standardized to a range of 1000–6000 light intensity units in WinView (version 2.5.16.5, Princeton Instruments, Trenton, USA).
To determine the effect that Gln diffusion through the GlnLux agar imposes on vein-level resolution, the diameters of midveins, longitudinal, and transverse leaf vein tissues were quantified with NIS-Elements (version 4.51, Nikon Instruments, Tokyo, Japan) following 4x brightfield microscopy (Nikon Eclipse 50i, Nikon Instruments). Diameters of longitudinal and transverse leaf vein tissues from whole-leaf in situ images were quantified with ImageJ (version 1.50i, NIH, Bethesda, USA) for comparison against microscopy with the Holm-Šídák test at P < 0.05 (GraphPad Prism 6, GraphPad Software Inc.). The veins of three biological replicates were quantified using both microscopy and in situ images.
It was postulated that differing tissue thicknesses may impact the luminescence output of in situ images. Two experiments were conducted to investigate this possibility:
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i)
Three sets of agar Gln disks with different heights/volumes were prepared, scaling linearly (h = 1.8, 3.6, 5.4 mm; V = 51, 102, 153 μl. Radius was held constant at 3 mm). The molarity of Gln within the standards was held constant across the three different height/volume levels (0, 3.125 × 10−4, 6.250 × 10−4, 1.250 × 10−3, 2.500 × 10−3, 5.000 × 10−3, 1 × 10−2 M Gln). Image standardization was applied using WinView software (version 2.5.16.5, Princeton Instruments) (1000 s exposure, 1000–6000 light intensity units) after 2.5 and 6 h.
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ii)
Three sets of agar Gln disks with different heights/volumes were prepared, scaling linearly as above. However, total moles of Gln within the standards was held constant across the three different height/volume levels (0, 15.94, 31.87, 63.75, 127.5, 255.0, 510.0 nmol). Image standardization was applied using WinView software (version 2.5.16.5, Princeton Instruments) (1000 s exposure, 1000–6000 light intensity units) after 2.5 and 6 h.