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To elucidate the stimulation of leaf growth by atmospheric nitrogen dioxide (NO2), we performed a kinematic analysis of the eighth leaves of Arabidopsis thaliana (accession C24) plants grown for 17–35 days after sowing in the presence or absence of 50 ppb NO2 (designated +NO2 plants and –NO2 plants, respectively). We found that the peak and mean values of the relative rates of leaf expansion, cell division and cell expansion were always greater in +NO2 plants than in –NO2 plants. No evidence for prolonged duration was obtained. Thus, NO2 treatment increased the rates of both cell proliferation and enlargement to increase leaf size. Furthermore, a fold-change analysis showed that cell proliferation and enlargement differentially regulated NO2-induced leaf expansion.
Leaf growth consists of 2 components: an increase in volume – the expansive growth – and an increase in dry matter – the structural growth.1 Atmospheric nitrogen dioxide (NO2), as a positive growth regulator, stimulates both of these components of leaf growth. We previously reported that NO2 increases leaf size and shoot biomass by 2.5-fold each.2-4 These increases were attributable to stimulation of both cell proliferation and cell enlargement by NO2.2 This was based on a static analysis of 35-day-old plants grown in the presence or absence of NO2; however, the kinematics of cell proliferation and enlargement induced by NO2 have not been explored. Here, we conducted a kinematic analysis of the 8th leaves of Arabidopsis thaliana (Arabidopsis) grown in the presence or absence of NO2 to understand the mechanisms underlying stimulation of leaf growth by NO2. Kinematic analysis showed that NO2 treatment increased the rates of both cell proliferation and enlargement to increase leaf size, and that cell proliferation and enlargement differentially regulated NO2-induced leaf expansion.
Plants were grown and treated with NO2 as reported previously.2 Briefly, Arabidopsis thaliana (L.) Heynh. accession C24 seeds were surface-sterilized with 1.0% sodium hypochlorite, rinsed in pure water (18.0 MΩ), sown in a rectangular plastic tray (22 × 5 × 20 cm width, height and depth, respectively) containing vermiculite and perlite (1:1, v/v) and held in a glass-walled NO2-exposure chamber (1.3 × 1.0 × 0.65 m width, height and depth, respectively; NOX-1000-SCII, Nippon Medical & Chemical Instruments Co., Osaka, Japan) in a growth room. The temperature, CO2 concentration and relative humidity in the chamber were set at 22 ± 0.1°C, 360 ± 30 ppm and 70 ± 1.5%, respectively. Air entering the chamber (at 1 L min−1) from outside was stripped of NO, NO2 and O3 (to 0 ppb) using activated charcoal and NaMnO4 (PureliteE30; Nippon Puretec Co., Tokyo, Japan). The tray was placed under fluorescent light (40 µmol photons m2 s−1) in a 16:8 h light:dark cycle and seeds were allowed to germinate and grow for 1 week in air lacking NO2. NO2 was then added to air entering the exposure chamber at concentrations of 0 or 50 ± 0.3 ppb (designated –NO2 control plants and +NO2 treated plants, respectively). Seedlings were irrigated twice weekly with half-strength inorganic salts in Murashige and Skoog medium5 and grown for an additional 4 weeks in the chamber.
Every two days starting from 17 days after sowing (DAS), the 8th leaves were harvested from –NO2 control and +NO2 treated plants and the leaf area and the number and size of paradermal leaf cells were determined as reported previously.2 Briefly, leaves were fixed in formalin–acetic acid–alcohol (FAA) and cleared using a chloral hydrate solution following Tsuge et al.6 Whole leaves and leaf cells were observed under a stereoscopic microscope (MZ FLIII ; Leica Microsystems, Wetzlar, Germany) and a Nomarski differential interference contrast compound microscope (ECLIPSE 80i; Nikon, Tokyo, Japan), respectively, as described in Horiguchi et al.7 Microphotographs were captured and leaf area, cell density and cell size were determined using ImageJ software (http://rsb.info.nih.gov/ij/). No leaves from plants younger than 17 DAS were used because their 8th leaf samples were too small to be analyzed using the current methods.
From these microscopic data, the relative leaf expansion rate (RLER) or increase in leaf area per leaf per unit time (mm2 mm−2 day−1), relative cell division rate (RCDR) or increase in cell number per cell per unit time (cell cell−1 day−1) and relative cell expansion rate (RCER) or increase in cell area per cell per unit time (µm2 µm−2 day−1) were determined. Results were statistically analyzed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA) and are depicted in Figure 1. Numerical details of leaf area, cell number and cell size determinations are provided in Table S1.
In both +NO2 treated and –NO2 control plants, the area of the 8th leaves expands in a sigmoidal way as a function of DAS. Both curves showed a distinct difference around 21 DAS onward by NO2 treatment and were about 2-fold different at 35 DAS (Fig. 1A, see also below). Leaf area in +NO2 plants and in –NO2 plants was highly correlated with their respective calculated leaf area and the product of cell number and cell size (R2 = 0.9998, P < 0.0001 and R2 = 0.9977, P < 0.0001, respectively). This result agrees with the general idea that the size of an organ is determined by the number and size of its constituent cells.8-10
The peak values of the RLER, RCDR and RCER were always greater in +NO2 plants compared to corresponding values in –NO2 plants (Fig. 1D, E and F, respectively); respective values were 1.75 and 1.45 (mm2 mm−2 day−1), 0.83 and 0.62 (cell cell−1 day−1) and 0.51 and 0.45 (µm2 µm−2 day−1).
The mean values of RLER, RCDR and RCER, where the sum of all RLER (RCDR or RCER) values was divided by 7 (for RLER and RCDR) or 8 (for RCER), were always greater in +NO2 plants than in –NO2 plants. The mean RLER, RCDR and RCER values in +NO2 and –NO2 plants were 0.74 and 0.65 (mm2 mm−2 day−1), 0.27 and 0.20 (cell cell−1 day−1) and 0.26 and 0.22 (µm2 µm−2 day−1), respectively. We therefore conclude that NO2 treatment increased the rates of cell proliferation and enlargement, leading to increased leaf size. Numerical details of relative rates are provided in Table S2.
According to the leaf expansion model,1,11 the time courses of the RLER, RCDR and RCER curves in dicots are divided into 3 phases. The first phase is characterized by maximum and constant RLER and RCDR and by low and constant RCER. During the second phase, RLER remains constant and RCDR decreases, while RCER increases and peaks at the end of the second phase. RLER and RCER decrease, but cell division almost stops during the third phase.1,11
In –NO2 plants, the RLER and RCDR curves appeared to be almost constant at 19–21 DAS and slightly decreased at 21–23 DAS (Fig. 1D and E), which correspond to the characteristics of the first and second phases, respectively, of the leaf expansion model described above. The RCER curve showed a peak around 23 DAS (Fig. 1F), which corresponds to the end of the second phase. All three curves are close to, but distinctly above, zero at 33 DAS (Fig. 1, see also Table S2), showing that the third phase continued up to 33 DAS. Therefore, the time courses of the curves can be divided into the first (19–21 DAS), second (21–23 DAS) and third (23–33 DAS) phases in –NO2 plants.
In contrast, in +NO2 plants, the RLER and RCDR curves decreased almost monotonically from 19 to 33 DAS (Fig. 1D and E), and therefore lacked features characteristic of the first phase of the model.1,11 Thus, the first phase may have started earlier than 19 DAS in +NO2 plants, which is at least 2 days earlier than in –NO2 plants. In addition, the RCER curve exhibited a peak around 21 DAS (Fig. 1F) corresponding to the end of the second phase, which is 2 days earlier than in –NO2 plants. All three curves were close to zero at 33 DAS, as in –NO2 plants (Fig. 1), and the RCDR curve was below zero at 29 DAS (Table 2S), indicating that the third phase may have ended earlier in +NO2 plants than in –NO2 plants. Together, these results suggest that NO2 treatment accelerated the appearance of the 8th leaf (by about 2 days), which is consistent with our previous result12 that NO2 treatment increased the rate of leaf appearance. Nonetheless, the timing and rate of leaf appearance is not directly related to leaf size. In addition to the rate of cell division or expansion, the duration of the period during which a cell divides or expands determines leaf size.13 However, no experimental evidence showing that NO2 treatment prolonged cell division or cell expansion duration was obtained by the present kinematic analysis.
Fold changes in leaf area, cell number and cell size following NO2 treatment (designated FCLA, FCCN and FCCS, respectively) were calculated using the following equations: FCLA = (leaf area in +NO2 plants)/(leaf area in –NO2 plants), FCCN = (cell number in +NO2 plants)/(cell number in –NO2 plants) and FCCS = (cell size in +NO2 plants)/(cell size in –NO2 plants). Results are plotted against DAS (Fig. 2).
The FCLA and FCCN curves bear a remarkable resemblance to each other except that the peak value of the latter (4.9) was almost half that of the former (8.5; see Fig. 2). This suggests the following: First, the high resemblance and coincidence of the timing of the peaks show that cell proliferation is an important factor determining increased leaf size in response to NO2. Second, the distinct difference in peak value between FCLA and FCCN provides quantitative evidence that cell proliferation is not the sole contributor to the increase in leaf area, but that cell enlargement in response to NO2 is also important.
The FCCS curve exhibited a small, but distinct peak (a value of 4.0) at 23 DAS, which was 4 days later than the FCLA or FCCN peaks (see Fig. 2). This marked delay in the FCCS peak from FCCN implies a time lag between the peak of the contribution of cell enlargement to NO2-induced leaf expansion and the peak of the contribution of cell proliferation to NO2-induced leaf expansion. These findings agree with the general idea that cell division precedes cell expansion, although these processes largely overlap.9,11,14
The FCCS curve crossed the FCCN curve around 22 DAS and the 3 curves merged around 29 DAS (see Fig. 2). The crossing of the FCCS and FCCN curves also implies temporally differential regulation of leaf expansion by cell proliferation and enlargement following NO2 treatment. Numerical details of fold change values are provided in Table S3. Supporting the above discussion are the following results: FCLA was highly correlated with FCCN (R2 = 0.94, P < 0.001), and the correlation between FCLA and FCCS was low (R2 > 0.20), but was high when the peak and second peak FCLA values were omitted (R2 = 0.89, P < 0.01).
In conclusion, our findings show that NO2 treatment increased the rates, but not the duration, of both cell proliferation and enlargement to increase leaf size, and that cell proliferation and enlargement differentially regulate NO2-induced leaf expansion in Arabidopsis.
No potential conflicts of interest were disclosed.
This work was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (24580477, 21580403 to MT).