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A circadian and an ultradian rhythm are both evident in root growth of rice
Journal of Plant Physiology 168 (2011) 2072–2080
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A circadian and an ultradian rhythm are both evident in root growth of rice Morio Iijima a,∗ , Naofumi Matsushita b a b
School of Agriculture, Kinki University, Nara 631-8505, Japan Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
a r t i c l e
i n f o
Article history: Received 30 March 2011 Received in revised form 21 June 2011 Accepted 21 June 2011 Keywords: Circumnutation Endogenous rhythm Oryza sativa Periodicity Root elongation
a b s t r a c t This paper presents evidence for the existence of both a circadian and an ultradian rhythm in the elongation growth of rice roots. Root elongation of rice (Oryza sativa) was recorded under dim green light by using a CCD camera connected to a computer. Four treatment conditions were set-up to investigate the existence of endogenous rhythms: 28 ◦ C constant temperature and continuous dark (28 DD); 28 ◦ C constant temperature and alternating light and dark (28 LD); 33 ◦ C constant temperature and continuous dark (33 DD); and diurnal temperature change and alternating light and dark (DT-LD). The resulting spectral densities suggested the existence of periodicities of 20.4–25.2 h (circadian cycles) and 2.0–6.0 h (ultradian cycles) in each of the 4 treatments. The shorter ultradian cycles can be attributed to circumnutational growth of roots and/or to mucilage exudation. The average values across all the replicate data showed that the highest power spectral densities (PSDs) corresponded to root growth rhythms with periods of 22.9, 23.7, and 2.1 h for the 28 DD, 28 LD, and 33 DD treatments, respectively. Accumulation of PSD for each data set indicated that the periodicity was similar in both the 28 DD and 33 DD treatments. We conclude that a 23-h circadian and a 2-h ultradian rhythmicity exist in rice root elongation. Moreover, root elongation rates during the day were 1.08 and 1.44 times faster than those during the night for the 28 LD and DT-LD treatments, respectively. © 2011 Elsevier GmbH. All rights reserved.
Introduction Crop root growth is often determined by the increase in length per unit time, i.e., the root elongation rate (RER). Traditionally, RERs have been based on the total root elongation per day or week. However, only in a few studies, RERs were estimated by measuring root elongation every hour for the evaluations of root growth (Gordon et al., 1992), root tropisms (Ishikawa et al., 1991), and responses of roots to plant growth regulators (Tanimoto and Watanabe, 1986). Head (1965) was the first to demonstrate a diurnal fluctuation in RER of cherry trees grown in field soil; however, environmental factors, including the periodical illumination of roots by two reflector lamps, might have affected the estimated RERs. A previous study showed diurnal fluctuations in RERs calculated every hour under constant growth conditions for sorghum and upland rice (Iijima et al., 1998). The maximum RERs were 1.4–4.4 times higher than the minimum RERs. However, details of the fluctuations could not be obtained in that study because of the limitation of the experimental
Abbreviations: RERs, root elongation rates; DD, continuous dark; LD, alternating light and dark; DT, diurnal temperature change; PSD, power spectral density. ∗ Corresponding author. Tel.: +81 742 43 7209; fax: +81 742 43 1155. E-mail address: [email protected] (M. Iijima). 0176-1617/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2011.06.005
set-up. In this study, we attempted to analyze the fluctuations in the RER in rice. Here there seems to be a basic circadian rhythm on which shorter rhythms were superimposed. The circadian rhythm, an endogenous rhythm observed in plants, has been studied extensively in relation to plant shoot growth (Walter et al., 2009). Daily rhythmic patterns for plant root growth parameters have also been found with respect to various physiological phenomena such as nitrate uptake (Pearson and Steer, 1977), root respiration (Hansen, 1980), xylem sap exudation (Vaadia, 1960), hydraulic conductivity, root pressure (Henzler et al., 1999), water uptake (Nakanishi et al., 2001), and mucilage exudation (Iijima et al., 2003c). Recently, a diurnal growth pattern of root elongation was reported in Arabidopsis thaliana (Yazdanbakhsh and Fisahn, 2011; Yazdanbakhsh et al., 2011), although the endogenous rhythmicity itself was not analyzed. Root growth patterns of shorter duration, such as oscillation, were also analyzed in previous studies (Walter et al., 2003). The existence of an endogenous rhythm, such as a circadian and/or an ultradian rhythm, however, has never been analyzed as a parameter of root growth. A detailed mathematical analysis of the RER under continuous dark or light conditions is necessary to test predictions derived from the hypothesis that endogenous rhythms exist in root elongation. Therefore, this study aimed to analyze, for the first time, the endogenous rhythmicity of root elongation by using hourly growth measurements.
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Materials and methods Plant material and treatment Upland rice (Oryza sativa L., cv. Norin 11) was used for RER measurements, because it has a single, positively gravitropic seminal root. To analyze the rhythmicity of root elongation, 4 treatments with different temperatures and light conditions were used: (1) 28 ◦ C constant temperature and continuous dark (28 DD), (2) 28 ◦ C constant temperature and alternating light and dark (28 LD), (3) 33 ◦ C constant temperature and continuous dark (33 DD), and (4) diurnal ambient temperature and alternating light and dark (DT-LD). The 28 DD treatment allowed observation of spontaneous rhythmicity in roots under constant darkness without any external rhythmic factors, and the 28 LD allowed observation of rhythms in a day–night environment (light:dark = 12 h:12 h). By comparing the observations under these 2 treatment conditions, the rhythmicity can be judged as either endogenous or exogenous. Unfortunately, our experimental set-up did not allow testing under continuous light, but DD conditions can be used to assess rhythmicity. In the 33 DD treatment, the temperature was 5 ◦ C higher than that in the 28 DD treatment. By comparing the observations under these 2 treatment conditions, temperature compensation of the endogenous rhythmicity can be determined. The DT-LD treatment simulates a typical field environment for upland rice growth in summer in Nagoya, Japan, with an average temperature of 28 ◦ C. The treatment enables estimation of the daily changes in RER under natural field environment. The day length was adjusted to 12 h to allow comparisons with other treatments. Phase shifting was not tested because of the limitation imposed by the experimental periods. The root elongation measurements were conducted for a maximum of 3 days, and this duration was insufficient for conducting phase shifttests.
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Table 1 Pre-germination and pre-observation treatments that applied to plants of the four sets of experimental observations.
28 DD 28 LD 33 LD DT-LD
Pre-germination
Pre-observation
28 ◦ C constant dark (0–48 h) 28 ◦ C constant dark (0–48 h) 33 ◦ C constant dark (0–48 h) 28 ◦ C constant dark (0–48 h)
28 ◦ C constant dark (48–72 h) 28 ◦ C LD = 12:12 (48–120 h) 33 ◦ C constant dark (48–72 h) 28 ◦ C LD = 12:12 (48–120 h)
28 DD, 28 ◦ C constant temperature and constant dark; 28 LD, 28 ◦ C constant temperature and LD = 12 h:12 h; 28 DD, 33 ◦ C constant temperature and constant dark; DT-LD, diurnal temperature change and LD = 12 h:12 h. In parentheses time since the start (0 h) of germination.
Seed germination and pre-treatment Rice seeds were surface-sterilized by immersing them in 1% sodium hypochlorite solution for 10 min and washing them with deionized water for 60 min. Surface-sterilized caryopses were germinated at either 28 ◦ C or 33 ◦ C (depending on the subsequent experiment) in darkness for 48 h in deionized water, which was replaced twice daily. The duration of the germination and the temperature and light conditions before germination and before root observations are summarized in Table 1. From approximately 50 germinated seeds, 3–5 seedlings that had 5–10mm-long straight seminal roots were selected. The selected roots were grown in growth pouches moistened with deionized water for 24–72 h for pre-adjustment to the different temperatures and light conditions in each treatment. For the treatments that involved day–night cycles, i.e., the 28 LD and the DT-LD treatments, the pre-adjustment period was 3 days, because the shoots need to be exposed to light (380 mol m−2 s−1 ) for at least 24 h. The roots were not exposed to light during the pre-adjustment period.
Fig. 1. Schematic diagram of the root observation chamber. (a) Root observation dark box (depth × width × height: 0.9 m × 0.6 m × 0.9 m). Light supplied from the top of the chamber did not penetrate into the dark box in which the roots grew. (b) The root box (0.01 m × 0.25 m × 0.60 m) and camera system. The CCD camera position was controlled precisely by 2 industrial actuators. Roots were continuously illuminated with dim green light (520 ± 10 nm; 1.33 × 10−3 W m−2 ) which does not affect the root growth. (c) Side view of the root box. The roots were sandwiched between an acrylic plastic board and a loamy sand soil firmly. Contact between roots and soil was prevented by a sheet of filter paper. One-millimeter squares printed on the filter paper allowed comparative assessments of hourly increments in root growth.
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Root growth observation system Four seedlings were transplanted to a root box (depth × width × height: 0.01 m × 0.25 m × 0.60 m) suspended over a root observation dark box (0.9 m × 0.6 m × 0.9 m), which was placed in a chamber with controlled environment (Fig. 1). The experimental set-up was similar to the one described previously (Iijima et al., 1998). Light supplied from the top of the chamber did not penetrate into the dark box in which the roots grew. The average photosynthetic flux density was 380 mol m−2 s−1 at the level of the topmost leaf. Aerated, one-quarter-strength Hoagland’s solution was supplied continuously at a rate of 0.6 L h−1 to the root box by a peristaltic pump, and the solution was drained from an outlet. Plant root tips exhibit nutational movement. To minimize this movement, the roots were sandwiched between an acrylic plastic board and loamy sand soil that was packed in the box to achieve a dry bulk density of 1.3 Mg m−3 . The soil mechanical impedance, measured by a penetrometer device, was 0.093 ± 0.004 MPa. Contact between the roots and soil was prevented by a sheet of filter paper. One-millimeter squares printed on the filter paper allowed comparative assessments of hourly increments in root growth. Air was allowed to circulate freely around the roots by means of a small fan and an air vent. The fan also enabled the temperature inside the darkened box to be similar to the temperature outside the box (i.e., the growth chamber). Temperatures were recorded automatically at several points in the root box by means of a thermocouple, as shown in Fig. 2. The temperature was precisely controlled near the root tip. Daily fluctuations were ±0.2 ◦ C for 28 DD and 33 DD and ± 0.6 ◦ C for 28 LD. For the DT-LD treatment, the temperature in the growth chamber was adjusted according to the outside air temperature. RER measurements RER has been measured using several techniques: the transducer-based root auxanometer (Evans, 1976), rhizometer (Tanimoto and Watanabe, 1986), digitizer system (Ishikawa et al., 1991), time-lapse video (Gordon et al., 1992; Iijima et al., 1998; Yazdanbakhsh and Fisahn, 2011), and image sequence analysis (Walter et al., 2002). In this study, we used a CCD camera connected to a computer system to monitor the RER. Root images were first captured 7–9 h after transplanting. The seminal root axis was photographed using a mono-color chilled CCD camera (C5985; Hamamatsu Co. Ltd., Japan), which ensures high resolution under dim light conditions. The minimum light intensity required for image capture with this camera was approximately 1 × 10−4 lux. Root images were captured at hourly intervals and stored directly on a computer for 3 days (2 days for DT-LD). The CCD camera position was controlled precisely by 2 industrial actuators (movement accuracy, ±0.05 mm; IA-03WX-35; THK Co. Ltd.) and did not cause any alteration in the light level at the root surface. To minimize the parallax effect, the roots were positioned just on the grid line of the filter paper (1-mm squares were printed on the filter paper), and the eye of the camera was moved automatically. Theoretically, the parallax effect should be less than 0.01 mm, considering the camera eye position and the distance to the root tips. Therefore, for the analysis, we considered the parallax effect to be negligible. Root growth is affected by exposure to bright light (Feldman, 1984). Illumination of roots by dim green light (520 ± 10 nm; 1.20 × 10−2 W m−2 ) does not affect root growth (Pilet and Ney, 1978; Pilet, 1979). In our study, roots were continuously illuminated with dim green light (520 ± 10 nm; 1.33 × 10−3 W m−2 ). The mean root elongation when the roots were exposed to this light intensity for 6 days (82.8 ± 2.6 mm) did not differ statistically from that when the roots were grown in the dark (82.3 ± 5.9 mm). Temperature; light; and supply of nutrients, water, and air around the roots were precisely
controlled to minimize fluctuations in the RER measurements. For measuring the hourly RER, the root image was displayed on the computer monitor at a 150× magnification, and the RER was measured using image analysis software (IP Lab Spectrum; Signal Analytics Co. Ltd.). The starting point at the edge of the root cap was picked up by eye, and the distances between the hourly starting points were calculated. The reading error in the measurement, which is determined by the resolution of the image, dot size in the monitor, and cursor movement, was less than ±0.007 mm. Data analysis Hourly RER data were analyzed by the computing program “MemCalc” (Suwa Trust Co. Ltd., Japan). This program allows a time series analysis, which is a linearized version of the nonlinear least squares method combined with the maximum entropy spectral analysis method. Frequency-domain analysis and time-domain analysis were used to acquire the highest peak of PSD from the time series data of the RER (mathematical details in Ohtomo et al., 1994). First, an optimum fitting curve, computed with the least square fitting function, was determined from the original time series data of the RER. Subsequently, the residual time series data (deviation) were obtained by subtracting the least square fitting from the original time series. The deviation from the least square fitting was finally analyzed to acquire the peak in spectral density. This peak indicates the most likely period for the rhythm (Gorton et al., 1989). The highest peaks from the individual plant data and the average value of all the replicates, as well as the accumulated PSD from individual roots, were used to analyze the periodicity of root elongation in each treatment. Replication Four plants were grown for each experiment. Each experiment was replicated 10 times for 28 DD, 4 times for 28 LD and 33 DD, and only once for DT-LD. Altogether, 76 roots were examined over 3 days, and their elongation was recorded. Of these 76 plants, data of 32 plants (54%) were selected. These 32 plants, for which complete and unambiguous observations were recorded, were used for the determination of rhythmicity by maximum entropy spectral analysis. This group comprised 12 replicates for 28 DD, 8 for 28 LD, 9 for 33 DD, and 3 for DT-LD. Results Seedling growth under different conditions Upland rice was grown in a root observation dark box (Fig. 1) under 4 different lights and temperature conditions (Table 1 and Fig. 2). The characteristics of rice seedling growth are shown in Table 2. The average RER was highest in the diurnal conditions with different day and night temperatures and the light/dark cycle (DT-LD). This average RER was approximately twice that recorded in 28 DD. The RERs in the 28 LD treatment were 44% higher than the RERs in the 28 DD treatment. However, when the temperature was increased by 5 ◦ C, i.e., in the 33 DD treatment condition, the RERs were only 24% higher. Because the seedlings grown under DD conditions could not conduct photosynthesis, the physiological state of DD seedlings was probably different from that of seedlings grown under LD conditions. By the end of the experiments, the age of seedlings was 4 days after the pre-germination period, for both 28 DD and 33 DD. A continuous dark environment for 4-day-old rice seedling is not unusual, because seedling emergence in the field sometimes takes more than 4 days, depending on the sowing depth and temperature.
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Fig. 2. Temperature changes in the root zone and dark box. (a) 28 ◦ C constant temperature – continuous dark (28 DD), (b) 28 ◦ C constant temperature – alternating light and dark (28 LD), (c) 33 ◦ C constant temperature – continuous dark (33 DD) and (d) diurnal ambient temperature – alternating light and dark (DT-LD). Underline indicates dark period.
Analysis of the RER from individual plants RER fluctuations showed different patterns in individual roots. Representative patterns of 28 DD condition are shown in Fig. 3 and are as follows: (a) a cycle lasting approximately 1 day was evident and shorter growth cycles were superimposed on this cycle; (b) the RER gradually increased with short growth cycles, and then decreased rapidly at the end of observation period; (c) the RER showed a sharp initial increase during the first half of the observation period with short growth cycles, and decreased gradually during the second half. The highest PSD value for RER, which is an indication of the strength of the periodicity, corresponded to cycles of 25.2 h, 2.4 h, and 68.2 h for patterns (a), (b), and (c), respectively.
The long cycle of 68.2 h in pattern (c) was regarded as a growth trend over the 3-day observational period rather than an indication of an infradian (more than 28 h) endogenous rhythmicity of root growth. To diminish trends of root growth, optimum fitting curves, computed with the least square fitting function, were determined from the original time series data of the RER, and then the residual time series data were used for the analysis. The most probable cycles, indicated by the highest PSD in each plant, are shown in Table 3. Eleven plants (38%), derived from all 3 treatments (28 DD, 28 LD, and 33 DD), showed circadian cycles (20 h to 28 h), and the remaining 18 plants showed ultradian (less than 20 h) cycles. The majority of the plants (71%) in the 2 different DD treatments showed the short ultradian cycles, and those in the LD treatment
Table 2 Root and shoot elongation rates and plant growth during whole experimental period.
Root elongation rates (mm/h) Shoot elongation rates (mm/h) Plant height (mm) Number of leaves
28 DD
28 LD
33 DD
DT-LD
1.03 ± 0.05 0.28 ± 0.03 21.6 ± 2.0 1
1.48 ± 0.08 0.76 ± 0.09 66.1 ± 2.8 2–3
1.28 ± 0.04 0.60 ± 0.03 44.9 ± 2.4 2
2.02 ± 0.08 0.73 ± 0.06 50.0 ± 0.7 2–3
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Averaged RER analysis
Fig. 3. Three examples of typical root growth rates from individual roots of 10 replicate experiments under 28 ◦ C constant temperature – continuous dark (28 DD) conditions. (a) a cycle lasting approximately 1 day was evident and shorter growth cycles were superimposed on this cycle; (b) the RER gradually increased with short growth cycles, and then decreased rapidly at the end of observation period; (c) the RER showed a sharp initial increase during the first half of the observation period with short growth cycles, and decreased gradually during the second half.
The periodicity described in the previous paragraph was based on the strongest PSD, using data from single roots. The strength of the PSDs differed significantly among plants. For example, the strengths of the PSD values of the ultradian cycles in 28 LD were 1.6 (2.2 h), 2929.3 (2.1 h), and 15.4 (4.0 h). The simple average value for the cycle time from these values (2.8 h) cannot be regarded as a representative of the 28 LD treatment because of the significantly different strengths of PSD in individual roots. The normal statistical procedures such as averaging of several observations and standard error explanation are not applicable in this case. Therefore, the increment of measured root length per hour was averaged among the replicate plants. The composite RER was analyzed mathematically, and the characteristics of each treatment were emphasized (Gorton et al., 1989). The average value of the periodicity in each treatment is shown in Figs. 4 and 5. The RERs in both the 28 DD and 33 DD cycles increased gradually with a small cycle of a few hours and then decreased at the end of the experiment. The long cycle over the 3-day growth period was evident. In contrast, diurnal rhythmicity was clearly demonstrated in 28 LD. Here, again, the optimum fitting curve was determined from the original time series data of RER (a), and subsequently, the residual time series data (b) were used for the spectral analysis. A single, strong peak was clearly observed in all the treatments, although the relative strengths of the PSD were different. The cycles were nearly 23 h for 28 DD, 24 h for 28 LD, and 2 h for 33 DD. The periodicity of RER under 28 LD conditions coincided with the circadian cycle; however, in 28 DD conditions, at the same temperature, the cycle differed by 1 h. This result shows that root elongation exhibits a 23-h endogenous circadian rhythm. The relative strength of the PSD became weaker under constant dark conditions. Amplitude of RER differences among each hour became much smaller under constant dark condition. Temperature compensation of the endogenous rhythmicity was not found by this analysis. The 33 DD treatment showed only the 2-h ultradian cycle, which clearly differed from the cycles in the other 2 treatments. It seems that the higher temperature masked the circadian rhythm but did not affect the ultradian rhythm.
Accumulated PSD analysis tended to show circadian cycles (63%), in which the short cycles were also observed. Most of the short cycles (78%) lasted between 2 h and 4 h. The ultradian cycle was also evident under the LD conditions. This is interesting because, generally, diurnal LD conditions lead to the strong 24-h cycle enforced by the external stimulus.
Table 3 Periodicity (h) of root growth corresponding to the greatest power spectrum density for plants in each experiment. 28 DD (72 ha ) b
Circadian cycles (20–28 h)
25.2 (38.7 ) 23.8 (8.5) 21.8 (19.2) 22.9 (9.5)
Ultradian cycles (<20 h)
3.4 (14.1) 5.0 (2.7) 4.1 (3.2) 2.8 (7.2) 2.7 (25.2) 4.0 (4.3) 2.4 (14.7) 6.0 (43.5)
a b
28 LD (72 h)
33 DD (53 h)
24.4 (8.5) 23.6 (4.4) 21.8 (5.4) 23.3 (91.6) 24.0 (11.0)
21.0 (22.8) 20.4 (9.5)
2.2 (1.6) 2.1 (2929.3) 4.0 (15.4)
Duration of root elongation rates measurement. Power spectral density of the period.
2.3 (11.0) 3.7 (33.7) 4.5 (33.7) 3.2 (75.0) 5.8 (19.6) 2.0 (42.2) 3.4 (104.1)
As stated previously, individual plants showed various PSD intensities for RER periodicity. To evaluate the different PSD intensity equally, accumulation of relative PSD by replacing the maximum PSD with 1 for each plant data is shown in Fig. 6. By doing so, not only the individual plant data are evaluated but also all the possible cycle would not be cancelled. Moreover, the possible cycles will be strengthened by accumulating individual peaks. A cluster of peaks around 24–22 h was observed in all of the 3 treatments. When all the plant data accumulated, the maximum value was found to be more than 1, in cases where the frequency (Hz) for each PSD was the same. The highest peaks in the clustered area were 23.2 h, 23.8 h, and 21.5 h for 28 DD, 28 LD, and 33 DD, respectively. PSD values of cycles outside this clustered area were relatively small in the LD conditions, whereas those in the DD conditions could be clearly observed. All the ultradian cycles could be recognized by the accumulation of the PSD. The peak patterns in the 2 different DD treatments, compared to the peak patterns in the LD treatment, were relatively similar. Therefore, the accumulated PSD analysis showed that the cycle patterns under different temperature conditions were highly similar. Temperature is a stronger entraining stimulus than light in Neurospora (Liu et al., 1998; McClung, 2000). In the CAM plants Kalanchoë daigremontiana (Lüttge and Beck, 1992) and Bryophyllum fedtschenkoi (Anderson and Wilkins, 1989), a high temperature (more than 30 ◦ C) causes an
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Fig. 4. Power spectral analysis of average values of root growth rates in continuous dark (28 DD) and day-night cycle (LD 12:12) at 28 ◦ C constant temperature. (a) Original time series data and optimum least square fitting curve. (b) Residual time series data from the fitting curve. (c) Spectral analysis of (b) by maximum entropy method. Underline indicates dark period.
arrhythmic behavior in net CO2 exchange. The higher temperature may also cause slight changes in the cyclic root growth pattern. RER during day and night As shown in Table 4, the average RER during the day was significantly higher than that during the night in both the LD treatments. For the 28 LD treatment, temperatures during the day slightly differed from those during the night, and these temperatures were 28.4 ◦ C and 27.6 ◦ C, respectively. Admittedly, this is a small change; Table 4 Root elongation rates (mm/h) during day and night in two experimental sets of plants growing at 28 ◦ C. The data from the second day of observation period were used for the comparison of 28 LD. Day–night 12 h
28 LD DT-LD
Day Night Day Night
Mid day 6 h (0900–1500 h) and mid night 6 h (2100–0300 h) 1.97 1.82 2.15 1.49
± ± ± ±
0.01*** 0.03 0.07*** 0.04
1.99 1.76 2.31 1.48
± ± ± ±
0.01*** 0.04 0.05*** 0.05
Values are means ± SE of eight and three replicates for 28 LD and DT-LD, respectively. *** Significant difference at P < 0.001 by ANOVA.
however, it may be large enough to affect periodic growth behavior. To test whether these temperature differences affected the RER, total root elongation under constant dark condition for 6 days of growth were compared. The total elongation was 99.7 ± 3.1 mm at 28.4 ◦ C and 99.6 ± 1.9 mm at 27.6 ◦ C. This difference was insignificant; therefore, the results indicate that temperature was not the cause of the RER difference between day and night. In the DT-LD treatment, midday RER was 1.56 times faster than the midnight RER (Table 4). In this case, the RER changes do correspond to the different root zone temperatures, as shown in Fig. 7.
Discussion This paper provides the first evidence that rice root elongation growth shows both circadian and ultradian rhythms: a 23-h circadian cycle and a shorter 2-h ultradian cycle. Unlike root elongation, stem elongation has been studied extensively, perhaps because shoot measurements can be much easily obtained (Walter et al., 2009). Moreover, shoot growth is less affected by the light and dry air conditions required for the observations. Shoot elongation has been reported to show cycles of 23 h in wild spinach stems (Lecharny and Wagner, 1984), of less than 24 h in rose stems (Tutty et al., 1994), of nearly 24 h in oat coleoptiles (Ball and Dyke, 1954), of 21.4–23.5 h in Arabidopsis inflorescence stems (Jouve et al., 1998),
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Fig. 6. Accumulation of the relative power spectral density (PSD) by replacing the maximum PSD with a value of 1 for the analyzed data of each plant. (a) 28 DD with 12 replicate plants, (b) 28 LD with eight replicate plants and (c) 33 DD with nine replicate plants.
Fig. 5. Power spectral analysis of average values of root growth rates in continuous dark at 33 ◦ C constant temperature (33 DD). (a) Original time series data and optimum least square fitting curve. (b) Residual time series data from the fitting curve. (c) Spectral analysis of (b) by maximum entropy method. Underline indicates dark period.
and of 25.2 h in hypocotyls in Arabidopsis (Dowson-Day and Millar, 1999). These periods, except the last one, are in agreement with our results of root elongation. It is not surprising that shoot and root elongation rhythms are similar at this seedling stage, because both organs form a continuum along the plant axis. Diurnal rhythmicity in elongation has been examined in leaves (Christ, 1978a,b; Walter et al., 2009). However, an endogenous rhythm of leaf elongation seems not to have been found so far, though many studies have recorded up-and-down nastic leaf movements (Pedersen et al., 1993). Circadian cycles have also been described in relation to the physiology of higher plants. For example, stomatal opening/closure occurs over a period of 22 h (Gorton et al., 1989) or approximately 24 h (Hennessey et al., 1993). Photosynthetic rates show a 24.5h period (Hennessey and Field, 1991). Thus, a 22–25-h circadian rhythmicity is the most common cycle duration for growth and physiological phenomena examined so far. Regarding the ultradian cycles, nutational movement and growth rates have been extensively studied in shoots. For example, a 100-min (Millet et al., 1984) or a 2–4-h (Koukkari, 1994) period of circumnutational movement is observed in shoots of climbing bean; moreover, cycles of 45–120 min in shoot elongation rates in radish, cucumber, and sunflower (Kristie and Jolliffe, 1986) and cycles of 112–133 min in soybean (Kurt et al., 1998) have been reported. In roots, circumnutational cycles of 1–2 h have been
reported in Kentucky blue grass (Fisher, 1964), and cycles ranging from 12 min to 1 h have been reported in maize (Walter et al., 2003). Recently, Yazdanbakhsh et al. (2011) also reported cycles of 8–9 h in Arabidopsis root growth. The ultradian cycle observed in the present study are most probably related to the root circumnutational growth. Although roots with visible nutational growth were excluded from the analysis, several others, with apparently insignificant movements, were included in the RER measurement. Shorter ultradian cycles of less than 1 h seem to exist in shoot elongation growth (Kristie and Jolliffe, 1986). In the course of the present study, we confirmed a less than 1 h cycle of root tip circumnutation (Iijima et al., 1998), though the roots in question were not included in the present RER analysis. This short cycle was visible when the root tip was not fixed properly between the panel and the filter paper. The ultradian cycle of root elongation found in this study might be related to the production of both mucilage and root border cells. The resolution of the camera system was such that rhythmic swelling and contraction of the extreme root tip (root cap) could be detected. This type of movement would be indistinguishable from rhythmic pulses of elongation of the main root axis. The roots grown in a mechanically impeded soil release both mucilage (Iijima and Kono, 1992) and root border cells (Iijima et al., 2000, 2003a,b, 2004) from the root cap, which covers the meristematic region of the roots. Under the experimental conditions in this study, the roots were not in direct contact with the soil. Thus, the cells could be retained for a longer duration in the cap region than when they are allowed to be removed by abrasion with the growth medium. The accumulation of the border cells on the tip portion of the cap could result in small differences in RER, because relatively large number of these cells accumulates around the tip. So far, cycles of 2–3 h have been detected for the droplet size distribution of mucilage (Mollenhauer et al., 1975), Golgi apparatus development (Jones and Morre, 1973), and release of water-soluble exudates (Pearson et al., 1981). The cycle of mucilage exudation and release may, in turn, influence the release of border cells, which then adhere to the
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Fig. 7. Root elongation rates under daily temperature fluctuation in the LD 12:12 environment. (a) Average value of three replicates. (b) The corresponding temperature fluctuations (same data as Fig. 2 d). Underline is the night period.
extending flank of the roots and move away from the tip by further tip growth. The rhythmicity in mucilage exudation, together with the release of border cells, should be analyzed to clarify the contribution of these to the ultradian cycles in root growth. Root elongation was slightly greater during the day than during the night when the temperature was constant (28 LD in Table 4). This is in agreement with results of a study on monocot leaf elongation (Christ, 1978a; Poiré et al., 2010), which showed a lower growth rate at night than during the day. This observation might be related to the water status within the plant (Christ, 1978b) and perhaps to diurnal changes in the root sap pressure (Vaadia, 1960). During daytime, when transpiration is active, the water potential gradient will continuously lead to drawing of water present near the root tip towards the roots. This may increase the efficiency of water supply to the elongation zone of roots and hence contribute to greater cell expansion during the daytime. Indeed, the water distribution pattern in the shoot during the day differs from that during the night (Iijima et al., 2011). Another explanation for the higher growth rate during the day is the slight increase in the translocation of photosynthetically fixed carbon into the root tip during the day. Diurnal regulation of the accumulation of starch (Li et al., 1992) and total sugars (Geiger et al., 2000) in leaves suggests a diurnal pattern in carbon allocation to the root elongation zone and in the utilization of the carbon for growth. Evidence for the effects of carbon supply on the rhythmicity of root growth was reported recently in Arabidopsis thaliana (Yazdanbakhsh et al., 2011). Whether diurnal rhythms in mitotic activity in the root meristem (Bishop and Klein, 1973) are translated into rhythmic elongation patterns remains an open question. In this study, we used various environmental conditions to analyze the endogenous rhythms of root elongation growth. Compared to DD conditions, light–dark 24 h cycle, especially with daily temperature fluctuations, induced a clear 24-h pattern. Environmental conditions, including light and temperature, are thought to regulate RER. The reason for the existence of various rhythms under constant light condition is currently unknown. Relatively young age of the seedlings may be associated with the variation in the rhythms. For
example, the cell population in the root apical region (meristem and elongation zone) during the first 3–4 days would be changing from the cells formed in the embryo to cells formed after the continuous light condition; the embryonic elongating cell population would be swept from the growth zone day by day. Similarly, the dimensions of the growth zone may change during this early stage of post-germination root growth, and changes in growth rate may occur as the roots and their cells emerge from the immediate postgermination phase. An extended time-course analysis, which is not applicable at the moment due to the experimental difficulties, may provide further information regarding the root elongation rhythms. Although biological rhythms have been studied mostly under constant environmental conditions, an understanding of how the circadian system functions in the real world will require more complex experimental conditions (Nozue et al., 2007). Actual root elongation in the field soil, at least in the case of young roots near the soil surface, will probably be highly similar to that in the DT-LD treatment, with diurnal changes in temperature and light. Under such a condition, the shoot and root growth patterns will be strongly affected by the physiological processes responsible for the interaction between internal gene control and environmental impact (Walter and Schurr, 2005; Walter et al., 2009). Acknowledgement This study was funded by a grant-in-aid for basic research (B212460010) from the Japan Society for the Promotion of Science. References Anderson CM, Wilkins MB. Period and phase control by temperature in the circadian rhythm of carbon dioxide fixation in illuminated leaves of Bryophyllum fedtschenkoi. Planta 1989;177:456–69. Ball NG, Dyke IJ. An endogenous 24-hour rhythm in the growth rate of the Avena coleoptile. J Exp Bot 1954;5:421–33. Bishop RC, Klein RM. 1973 Innate and photo-controlled mitotic rhythms in onion root-tip cells. Can J Genet Cytol 1973;15:667–70. Christ RA. The elongation rate of wheat leaves. I. Elongation rates during day and night. J Exp Bot 1978a;29:603–10.
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Christ RA. The elongation rate of wheat leaves. II. Effect of sudden light change on the elongation rate. J Exp Bot 1978b;29:611–8. Dowson-Day MJ, Millar AJ. Circadian dysfunction causes aberrant hypocotyl elongation patterns in Arabidopsis. The Plant J 1999;17:63–71. Evans ML. A new sensitive root auxanometer. Preliminary studies of the interaction of auxin and acid pH in the regulation of intact root elongation. Plant Physiol 1976;58:599–601. Fisher JE. Evidence of circumnutational growth movements of rhizomes of Poa pratensis L. that aid in soil penetration. Can J Bot 1964;42:293–9, 1964. Feldman LJ. Regulation of root development. Annu Rev Plant Physiol 1984;35:223–42. Geiger DR, Servaites JC, Fuchs MA. Role of starch in carbon translocation and partitioning at the plant level. Aust J Plant Physiol 2000;27:571–82. Gordon D, Hettiaratchi DRP, Bengough AG, Young IM. Non-destructive analysis of root growth in porous media. Plant Cell Environ 1992;15:123–8. Gorton HL, Williams WE, Binns ME, Gemmell CN, Leheny EA, Shepherd AC. Circadian stomatal rhythms in epidermal peels from Vicia faba. Plant Physiol 1989;90:1329–34. Hansen GK. Diurnal variation of root respiration rates and nitrate uptake as influenced by nitrogen supply. Physiol Plant 1980;48:421–7. Head GC. Studies of diurnal changes in cherry root growth and nutational movements of apple root tips by time-lapse cinematography. Ann Bot 1965;29:219–24. Hennessey TL, Field CB. Circadian rhythms in photosynthesis: oscillations in carbon assimilation and stomatal conductance under constant conditions. Plant Physiol 1991;96:831–6, 1991. Hennessey TL, Freeden AL, Field CB. Environmental effects on circadian rhythms in photosynthesis and stomatal opening. Planta 1993;189:369–76. Henzler T, Waterhouse RN, Smyth AJ, Carvajal M, Cooke DT, Schaffner AR, et al. Diurnal variations in hydraulic conductivity and root pressure can be correlated with the expression of putative aquaporins in the roots of Lotus japonicus. Planta 1999;210:50–60. Iijima M, Kono Y. Development of Golgi apparatus in the root cap cells of maize (Zea mays L.) as affected by compacted soil. Ann Bot 1992;70:207–12. Iijima M, Oribe Y, Horibe Y, Kono Y. Time lapse analysis of root elongation rates of rice and sorghum during day and night. Ann Bot 1998;81:603–7. Iijima M, Griffith B, Bengough AG. Sloughing of cap cells and carbon exudation from maize seedling roots in compacted sand. New Phytol 2000;145:477–82. Iijima M, Barlow PW, Bengough AG. Root cap structure and cell production rates of maize (Zea mays L.) roots in compacted sand. New Phytol 2003a;160:127–34. Iijima M, Higuchi T, Barlow PW, Bengough AG. Root cap removal increases root penetration resistance in maize (Zea mays L.). J Exp Bot 2003b;54:2105–9. Iijima M, Sako Y, Rao TP. A new approach for the quantification of root-cap mucilage exudation in the soil. Plant Soil 2003c;255:399–407. Iijima M, Higuchi T, Watanabe A, Bengough AG. Method to quantify root border cells in sandy soil. Soil Biol Biochem 2004;36:1517–9. Iijima M, Yoshida T, Kato T, Kawasaki M, Watanabe T, Somasundaram S. Visualization of lateral water transport pathways in soybean by a time of flight-secondary ion mass spectrometry cryo-system. J Exp Bot 2011;62:2179–88. Ishikawa H, Hasenstein KH, Evans ML. Computer-based video digitizer analysis of surface extension in maize roots: kinetics of growth rate changes during gravitropism. Planta 1991;183:381–90. Jones DD, Morre DJ. Golgi apparatus mediated polysaccharide secretion by outer root cap cells of Zea mays. III. Control by exogenous sugars. Physiol Plant 1973;29:68–75. Jouve L, Greppin H, Agosti RD. Arabidopsis thaliana floral stem elongation: evidence f or an endogenous circadian rhythm. Plant Physiol Biochem 1998;36:469–72. Koukkari WL. Movement of a bean shoot: an introduction to chronobiology. Chronobiol Int 1994;11:85–93. Kristie DN, Jolliffe PA. High-resolution studies of growth oscillations during stem elongation. Can J Bot 1986;64:2399–405. Kurt A, Robert S, Willard K. Ultradian movements of shoots of two species of soybeans: Glycine soja (Sieb. and Zucc.) and Glycine max (L.) Merr. Chronobiol Inter 1998;15:1–11.
Lecharny A, Wagner E. Stem extension rate in light-grown plants: evidence for an endogenous circadian rhythm in Chenopodium rubrum. Physiol Plant 1984;60:437–43. Li B, Geiger DR, Shieh WJ. Evidence for circadian regulation of starch and sucrose synthesis in sugar beet leaves. Plant Physiol 1992;99:1393–9. Liu Y, Merrow M, Loros JJ, Dunlap JC. How temperature changes reset a circadian oscillator. Science 1998;281:825–9. Lüttge U, Beck F. Endogenous rhythms and chaos in crassulacean acid metabolism. Planta 1992;188:28–38. McClung CR. Circadian rhythms in plants: a millennial view. Physiol Plant 2000;109:359–71. Millet B, Melin D, Bonnet B, Ibrahim CA, Mercier J. Rhythmic circumnutation movement of the shoots in Phaseolus vulgaris L. Chronobiol Int 1984;1:11–9. Mollenhauer HM, Morre DJ, Vanderwoude WJ. Endoplasmic reticulum–Golgi apparatus associations in maize root tips. Mikroskopie 1975;31:257–72. Nakanishi T, Yokota H, Tanoi K, Furukawa J, Ikeue N, Ookuni Y, et al. Circadian rhythm in 15 O-labeled water uptake manner of a soybean plant by PETIS (Positron Emitting Tracer Imaging System). Radioisotopes 2001;50:163–8. Nozue K, Covington MF, Duek PD, Lorrain S, Fankhauser C, Harmer SL, et al. Rhythmic growth explained by coincidence between internal and external cues. Nature 2007;448:358–63. Ohtomo N, Terachi S, Tanaka Y, Tokiwano K, Kaneko N. New method of time series analysis and its application to Wolf’s sunspot number data. Jpn J Appl Phys 1994;33:2821–31. Pearson CJ, Steer BT. Daily changes in nitrate uptake and metabolism in Capsicum annuum. Planta 1977;137:107–12. Pearson CJ, Volk RJ, Jackson WA. Daily changes in nitrate influx, efflux and metabolism in maize and pearl millet. Planta 1981;152:319–24. Pedersen M, Johnsson A, Mæhle J, Dalløkken R. Short period leaf movements in Oxalis regnellii. Physiol Plant 1993;89:277–84. Pilet PE. Kinetics of the light-induced georeactivity of maize roots. Planta 1979;145:403–4. Pilet PE, Ney D. Rapid localized light effect on root growth in maize. Planta 1978;144:109–10. Poiré R, Wiese-Klinkenberg A, Parent B, Mielewczik M, Schurr U, Tardieu F, et al. Diel time-courses of leaf growth in monocot and dicot species: endogenous rhythms and temperature effects. J Exp Bot 2010;61:1751–9. Tanimoto E, Watanabe J. Automated recording of lettuce root elongation as affected by auxin and acid pH in a new rhizometer with minimum mechanical contact to roots. Plant Cell Physiol 1986;27:1475–87. Tutty JR, Hicklenton PR, Kristie DN, McRae KB. The influence of photoperiod and temperature on the kinetics of stem elongation in Dendranthema grandiflorum. J Am Soc Hortic Sci 1994;119:138–43. Vaadia Y. Autonomic diurnal fluctuations in rate of exudation and root pressure of decapitated sunflower plants. Physiol Plant 1960;13:701–17. Walter A, Schurr U. Dynamics of leaf and root growth – endogenous control versus environmental impact. Ann Bot 2005;95:891–900. Walter A, Spies H, Terjung S, KD usters R, Kirchgeßner N, Schurr U. Spatio-temporal dynamics of expansion growth in roots: automatic quantification of diurnal course and temperature response by digital image sequence processing. J Exp Bot 2002;53:689–98. Walter A, Feil R, Schurr U. Expansion dynamics, metabolite composition and substance transfer of the primary root growth zone of Zea mays L. grown in different external nutrient availabilities. Plant Cell Environ 2003;26:1451–66. Walter A, Silk WK, Schurr U. Environmental effects on spatial and temporal patterns of leaf and root growth. Annu Rev Plant Biol 2009;60:279–304. Yazdanbakhsh N, Fisahn J. Stable diurnal growth rhythms modulate root elongation of Arabidopsis thaliana. Plant Root 2011;5:17–23. Yazdanbakhsh N, Sulpice R, Graf A, Stitt M, Fisahn J. Circadian control of root elongation and C partitioning in Arabidopsis thaliana. Plant Cell Environ 2011., doi:10.1111/j.1365-3040.2011.02286.x.
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A circadian and an ultradian rhythm are both evident in root growth of rice Morio Iijima a,∗ , Naofumi Matsushita b a b
School of Agriculture, Kinki University, Nara 631-8505, Japan Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
a r t i c l e
i n f o
Article history: Received 30 March 2011 Received in revised form 21 June 2011 Accepted 21 June 2011 Keywords: Circumnutation Endogenous rhythm Oryza sativa Periodicity Root elongation
a b s t r a c t This paper presents evidence for the existence of both a circadian and an ultradian rhythm in the elongation growth of rice roots. Root elongation of rice (Oryza sativa) was recorded under dim green light by using a CCD camera connected to a computer. Four treatment conditions were set-up to investigate the existence of endogenous rhythms: 28 ◦ C constant temperature and continuous dark (28 DD); 28 ◦ C constant temperature and alternating light and dark (28 LD); 33 ◦ C constant temperature and continuous dark (33 DD); and diurnal temperature change and alternating light and dark (DT-LD). The resulting spectral densities suggested the existence of periodicities of 20.4–25.2 h (circadian cycles) and 2.0–6.0 h (ultradian cycles) in each of the 4 treatments. The shorter ultradian cycles can be attributed to circumnutational growth of roots and/or to mucilage exudation. The average values across all the replicate data showed that the highest power spectral densities (PSDs) corresponded to root growth rhythms with periods of 22.9, 23.7, and 2.1 h for the 28 DD, 28 LD, and 33 DD treatments, respectively. Accumulation of PSD for each data set indicated that the periodicity was similar in both the 28 DD and 33 DD treatments. We conclude that a 23-h circadian and a 2-h ultradian rhythmicity exist in rice root elongation. Moreover, root elongation rates during the day were 1.08 and 1.44 times faster than those during the night for the 28 LD and DT-LD treatments, respectively. © 2011 Elsevier GmbH. All rights reserved.
Introduction Crop root growth is often determined by the increase in length per unit time, i.e., the root elongation rate (RER). Traditionally, RERs have been based on the total root elongation per day or week. However, only in a few studies, RERs were estimated by measuring root elongation every hour for the evaluations of root growth (Gordon et al., 1992), root tropisms (Ishikawa et al., 1991), and responses of roots to plant growth regulators (Tanimoto and Watanabe, 1986). Head (1965) was the first to demonstrate a diurnal fluctuation in RER of cherry trees grown in field soil; however, environmental factors, including the periodical illumination of roots by two reflector lamps, might have affected the estimated RERs. A previous study showed diurnal fluctuations in RERs calculated every hour under constant growth conditions for sorghum and upland rice (Iijima et al., 1998). The maximum RERs were 1.4–4.4 times higher than the minimum RERs. However, details of the fluctuations could not be obtained in that study because of the limitation of the experimental
Abbreviations: RERs, root elongation rates; DD, continuous dark; LD, alternating light and dark; DT, diurnal temperature change; PSD, power spectral density. ∗ Corresponding author. Tel.: +81 742 43 7209; fax: +81 742 43 1155. E-mail address: [email protected] (M. Iijima). 0176-1617/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2011.06.005
set-up. In this study, we attempted to analyze the fluctuations in the RER in rice. Here there seems to be a basic circadian rhythm on which shorter rhythms were superimposed. The circadian rhythm, an endogenous rhythm observed in plants, has been studied extensively in relation to plant shoot growth (Walter et al., 2009). Daily rhythmic patterns for plant root growth parameters have also been found with respect to various physiological phenomena such as nitrate uptake (Pearson and Steer, 1977), root respiration (Hansen, 1980), xylem sap exudation (Vaadia, 1960), hydraulic conductivity, root pressure (Henzler et al., 1999), water uptake (Nakanishi et al., 2001), and mucilage exudation (Iijima et al., 2003c). Recently, a diurnal growth pattern of root elongation was reported in Arabidopsis thaliana (Yazdanbakhsh and Fisahn, 2011; Yazdanbakhsh et al., 2011), although the endogenous rhythmicity itself was not analyzed. Root growth patterns of shorter duration, such as oscillation, were also analyzed in previous studies (Walter et al., 2003). The existence of an endogenous rhythm, such as a circadian and/or an ultradian rhythm, however, has never been analyzed as a parameter of root growth. A detailed mathematical analysis of the RER under continuous dark or light conditions is necessary to test predictions derived from the hypothesis that endogenous rhythms exist in root elongation. Therefore, this study aimed to analyze, for the first time, the endogenous rhythmicity of root elongation by using hourly growth measurements.
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Materials and methods Plant material and treatment Upland rice (Oryza sativa L., cv. Norin 11) was used for RER measurements, because it has a single, positively gravitropic seminal root. To analyze the rhythmicity of root elongation, 4 treatments with different temperatures and light conditions were used: (1) 28 ◦ C constant temperature and continuous dark (28 DD), (2) 28 ◦ C constant temperature and alternating light and dark (28 LD), (3) 33 ◦ C constant temperature and continuous dark (33 DD), and (4) diurnal ambient temperature and alternating light and dark (DT-LD). The 28 DD treatment allowed observation of spontaneous rhythmicity in roots under constant darkness without any external rhythmic factors, and the 28 LD allowed observation of rhythms in a day–night environment (light:dark = 12 h:12 h). By comparing the observations under these 2 treatment conditions, the rhythmicity can be judged as either endogenous or exogenous. Unfortunately, our experimental set-up did not allow testing under continuous light, but DD conditions can be used to assess rhythmicity. In the 33 DD treatment, the temperature was 5 ◦ C higher than that in the 28 DD treatment. By comparing the observations under these 2 treatment conditions, temperature compensation of the endogenous rhythmicity can be determined. The DT-LD treatment simulates a typical field environment for upland rice growth in summer in Nagoya, Japan, with an average temperature of 28 ◦ C. The treatment enables estimation of the daily changes in RER under natural field environment. The day length was adjusted to 12 h to allow comparisons with other treatments. Phase shifting was not tested because of the limitation imposed by the experimental periods. The root elongation measurements were conducted for a maximum of 3 days, and this duration was insufficient for conducting phase shifttests.
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Table 1 Pre-germination and pre-observation treatments that applied to plants of the four sets of experimental observations.
28 DD 28 LD 33 LD DT-LD
Pre-germination
Pre-observation
28 ◦ C constant dark (0–48 h) 28 ◦ C constant dark (0–48 h) 33 ◦ C constant dark (0–48 h) 28 ◦ C constant dark (0–48 h)
28 ◦ C constant dark (48–72 h) 28 ◦ C LD = 12:12 (48–120 h) 33 ◦ C constant dark (48–72 h) 28 ◦ C LD = 12:12 (48–120 h)
28 DD, 28 ◦ C constant temperature and constant dark; 28 LD, 28 ◦ C constant temperature and LD = 12 h:12 h; 28 DD, 33 ◦ C constant temperature and constant dark; DT-LD, diurnal temperature change and LD = 12 h:12 h. In parentheses time since the start (0 h) of germination.
Seed germination and pre-treatment Rice seeds were surface-sterilized by immersing them in 1% sodium hypochlorite solution for 10 min and washing them with deionized water for 60 min. Surface-sterilized caryopses were germinated at either 28 ◦ C or 33 ◦ C (depending on the subsequent experiment) in darkness for 48 h in deionized water, which was replaced twice daily. The duration of the germination and the temperature and light conditions before germination and before root observations are summarized in Table 1. From approximately 50 germinated seeds, 3–5 seedlings that had 5–10mm-long straight seminal roots were selected. The selected roots were grown in growth pouches moistened with deionized water for 24–72 h for pre-adjustment to the different temperatures and light conditions in each treatment. For the treatments that involved day–night cycles, i.e., the 28 LD and the DT-LD treatments, the pre-adjustment period was 3 days, because the shoots need to be exposed to light (380 mol m−2 s−1 ) for at least 24 h. The roots were not exposed to light during the pre-adjustment period.
Fig. 1. Schematic diagram of the root observation chamber. (a) Root observation dark box (depth × width × height: 0.9 m × 0.6 m × 0.9 m). Light supplied from the top of the chamber did not penetrate into the dark box in which the roots grew. (b) The root box (0.01 m × 0.25 m × 0.60 m) and camera system. The CCD camera position was controlled precisely by 2 industrial actuators. Roots were continuously illuminated with dim green light (520 ± 10 nm; 1.33 × 10−3 W m−2 ) which does not affect the root growth. (c) Side view of the root box. The roots were sandwiched between an acrylic plastic board and a loamy sand soil firmly. Contact between roots and soil was prevented by a sheet of filter paper. One-millimeter squares printed on the filter paper allowed comparative assessments of hourly increments in root growth.
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Root growth observation system Four seedlings were transplanted to a root box (depth × width × height: 0.01 m × 0.25 m × 0.60 m) suspended over a root observation dark box (0.9 m × 0.6 m × 0.9 m), which was placed in a chamber with controlled environment (Fig. 1). The experimental set-up was similar to the one described previously (Iijima et al., 1998). Light supplied from the top of the chamber did not penetrate into the dark box in which the roots grew. The average photosynthetic flux density was 380 mol m−2 s−1 at the level of the topmost leaf. Aerated, one-quarter-strength Hoagland’s solution was supplied continuously at a rate of 0.6 L h−1 to the root box by a peristaltic pump, and the solution was drained from an outlet. Plant root tips exhibit nutational movement. To minimize this movement, the roots were sandwiched between an acrylic plastic board and loamy sand soil that was packed in the box to achieve a dry bulk density of 1.3 Mg m−3 . The soil mechanical impedance, measured by a penetrometer device, was 0.093 ± 0.004 MPa. Contact between the roots and soil was prevented by a sheet of filter paper. One-millimeter squares printed on the filter paper allowed comparative assessments of hourly increments in root growth. Air was allowed to circulate freely around the roots by means of a small fan and an air vent. The fan also enabled the temperature inside the darkened box to be similar to the temperature outside the box (i.e., the growth chamber). Temperatures were recorded automatically at several points in the root box by means of a thermocouple, as shown in Fig. 2. The temperature was precisely controlled near the root tip. Daily fluctuations were ±0.2 ◦ C for 28 DD and 33 DD and ± 0.6 ◦ C for 28 LD. For the DT-LD treatment, the temperature in the growth chamber was adjusted according to the outside air temperature. RER measurements RER has been measured using several techniques: the transducer-based root auxanometer (Evans, 1976), rhizometer (Tanimoto and Watanabe, 1986), digitizer system (Ishikawa et al., 1991), time-lapse video (Gordon et al., 1992; Iijima et al., 1998; Yazdanbakhsh and Fisahn, 2011), and image sequence analysis (Walter et al., 2002). In this study, we used a CCD camera connected to a computer system to monitor the RER. Root images were first captured 7–9 h after transplanting. The seminal root axis was photographed using a mono-color chilled CCD camera (C5985; Hamamatsu Co. Ltd., Japan), which ensures high resolution under dim light conditions. The minimum light intensity required for image capture with this camera was approximately 1 × 10−4 lux. Root images were captured at hourly intervals and stored directly on a computer for 3 days (2 days for DT-LD). The CCD camera position was controlled precisely by 2 industrial actuators (movement accuracy, ±0.05 mm; IA-03WX-35; THK Co. Ltd.) and did not cause any alteration in the light level at the root surface. To minimize the parallax effect, the roots were positioned just on the grid line of the filter paper (1-mm squares were printed on the filter paper), and the eye of the camera was moved automatically. Theoretically, the parallax effect should be less than 0.01 mm, considering the camera eye position and the distance to the root tips. Therefore, for the analysis, we considered the parallax effect to be negligible. Root growth is affected by exposure to bright light (Feldman, 1984). Illumination of roots by dim green light (520 ± 10 nm; 1.20 × 10−2 W m−2 ) does not affect root growth (Pilet and Ney, 1978; Pilet, 1979). In our study, roots were continuously illuminated with dim green light (520 ± 10 nm; 1.33 × 10−3 W m−2 ). The mean root elongation when the roots were exposed to this light intensity for 6 days (82.8 ± 2.6 mm) did not differ statistically from that when the roots were grown in the dark (82.3 ± 5.9 mm). Temperature; light; and supply of nutrients, water, and air around the roots were precisely
controlled to minimize fluctuations in the RER measurements. For measuring the hourly RER, the root image was displayed on the computer monitor at a 150× magnification, and the RER was measured using image analysis software (IP Lab Spectrum; Signal Analytics Co. Ltd.). The starting point at the edge of the root cap was picked up by eye, and the distances between the hourly starting points were calculated. The reading error in the measurement, which is determined by the resolution of the image, dot size in the monitor, and cursor movement, was less than ±0.007 mm. Data analysis Hourly RER data were analyzed by the computing program “MemCalc” (Suwa Trust Co. Ltd., Japan). This program allows a time series analysis, which is a linearized version of the nonlinear least squares method combined with the maximum entropy spectral analysis method. Frequency-domain analysis and time-domain analysis were used to acquire the highest peak of PSD from the time series data of the RER (mathematical details in Ohtomo et al., 1994). First, an optimum fitting curve, computed with the least square fitting function, was determined from the original time series data of the RER. Subsequently, the residual time series data (deviation) were obtained by subtracting the least square fitting from the original time series. The deviation from the least square fitting was finally analyzed to acquire the peak in spectral density. This peak indicates the most likely period for the rhythm (Gorton et al., 1989). The highest peaks from the individual plant data and the average value of all the replicates, as well as the accumulated PSD from individual roots, were used to analyze the periodicity of root elongation in each treatment. Replication Four plants were grown for each experiment. Each experiment was replicated 10 times for 28 DD, 4 times for 28 LD and 33 DD, and only once for DT-LD. Altogether, 76 roots were examined over 3 days, and their elongation was recorded. Of these 76 plants, data of 32 plants (54%) were selected. These 32 plants, for which complete and unambiguous observations were recorded, were used for the determination of rhythmicity by maximum entropy spectral analysis. This group comprised 12 replicates for 28 DD, 8 for 28 LD, 9 for 33 DD, and 3 for DT-LD. Results Seedling growth under different conditions Upland rice was grown in a root observation dark box (Fig. 1) under 4 different lights and temperature conditions (Table 1 and Fig. 2). The characteristics of rice seedling growth are shown in Table 2. The average RER was highest in the diurnal conditions with different day and night temperatures and the light/dark cycle (DT-LD). This average RER was approximately twice that recorded in 28 DD. The RERs in the 28 LD treatment were 44% higher than the RERs in the 28 DD treatment. However, when the temperature was increased by 5 ◦ C, i.e., in the 33 DD treatment condition, the RERs were only 24% higher. Because the seedlings grown under DD conditions could not conduct photosynthesis, the physiological state of DD seedlings was probably different from that of seedlings grown under LD conditions. By the end of the experiments, the age of seedlings was 4 days after the pre-germination period, for both 28 DD and 33 DD. A continuous dark environment for 4-day-old rice seedling is not unusual, because seedling emergence in the field sometimes takes more than 4 days, depending on the sowing depth and temperature.
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Fig. 2. Temperature changes in the root zone and dark box. (a) 28 ◦ C constant temperature – continuous dark (28 DD), (b) 28 ◦ C constant temperature – alternating light and dark (28 LD), (c) 33 ◦ C constant temperature – continuous dark (33 DD) and (d) diurnal ambient temperature – alternating light and dark (DT-LD). Underline indicates dark period.
Analysis of the RER from individual plants RER fluctuations showed different patterns in individual roots. Representative patterns of 28 DD condition are shown in Fig. 3 and are as follows: (a) a cycle lasting approximately 1 day was evident and shorter growth cycles were superimposed on this cycle; (b) the RER gradually increased with short growth cycles, and then decreased rapidly at the end of observation period; (c) the RER showed a sharp initial increase during the first half of the observation period with short growth cycles, and decreased gradually during the second half. The highest PSD value for RER, which is an indication of the strength of the periodicity, corresponded to cycles of 25.2 h, 2.4 h, and 68.2 h for patterns (a), (b), and (c), respectively.
The long cycle of 68.2 h in pattern (c) was regarded as a growth trend over the 3-day observational period rather than an indication of an infradian (more than 28 h) endogenous rhythmicity of root growth. To diminish trends of root growth, optimum fitting curves, computed with the least square fitting function, were determined from the original time series data of the RER, and then the residual time series data were used for the analysis. The most probable cycles, indicated by the highest PSD in each plant, are shown in Table 3. Eleven plants (38%), derived from all 3 treatments (28 DD, 28 LD, and 33 DD), showed circadian cycles (20 h to 28 h), and the remaining 18 plants showed ultradian (less than 20 h) cycles. The majority of the plants (71%) in the 2 different DD treatments showed the short ultradian cycles, and those in the LD treatment
Table 2 Root and shoot elongation rates and plant growth during whole experimental period.
Root elongation rates (mm/h) Shoot elongation rates (mm/h) Plant height (mm) Number of leaves
28 DD
28 LD
33 DD
DT-LD
1.03 ± 0.05 0.28 ± 0.03 21.6 ± 2.0 1
1.48 ± 0.08 0.76 ± 0.09 66.1 ± 2.8 2–3
1.28 ± 0.04 0.60 ± 0.03 44.9 ± 2.4 2
2.02 ± 0.08 0.73 ± 0.06 50.0 ± 0.7 2–3
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Averaged RER analysis
Fig. 3. Three examples of typical root growth rates from individual roots of 10 replicate experiments under 28 ◦ C constant temperature – continuous dark (28 DD) conditions. (a) a cycle lasting approximately 1 day was evident and shorter growth cycles were superimposed on this cycle; (b) the RER gradually increased with short growth cycles, and then decreased rapidly at the end of observation period; (c) the RER showed a sharp initial increase during the first half of the observation period with short growth cycles, and decreased gradually during the second half.
The periodicity described in the previous paragraph was based on the strongest PSD, using data from single roots. The strength of the PSDs differed significantly among plants. For example, the strengths of the PSD values of the ultradian cycles in 28 LD were 1.6 (2.2 h), 2929.3 (2.1 h), and 15.4 (4.0 h). The simple average value for the cycle time from these values (2.8 h) cannot be regarded as a representative of the 28 LD treatment because of the significantly different strengths of PSD in individual roots. The normal statistical procedures such as averaging of several observations and standard error explanation are not applicable in this case. Therefore, the increment of measured root length per hour was averaged among the replicate plants. The composite RER was analyzed mathematically, and the characteristics of each treatment were emphasized (Gorton et al., 1989). The average value of the periodicity in each treatment is shown in Figs. 4 and 5. The RERs in both the 28 DD and 33 DD cycles increased gradually with a small cycle of a few hours and then decreased at the end of the experiment. The long cycle over the 3-day growth period was evident. In contrast, diurnal rhythmicity was clearly demonstrated in 28 LD. Here, again, the optimum fitting curve was determined from the original time series data of RER (a), and subsequently, the residual time series data (b) were used for the spectral analysis. A single, strong peak was clearly observed in all the treatments, although the relative strengths of the PSD were different. The cycles were nearly 23 h for 28 DD, 24 h for 28 LD, and 2 h for 33 DD. The periodicity of RER under 28 LD conditions coincided with the circadian cycle; however, in 28 DD conditions, at the same temperature, the cycle differed by 1 h. This result shows that root elongation exhibits a 23-h endogenous circadian rhythm. The relative strength of the PSD became weaker under constant dark conditions. Amplitude of RER differences among each hour became much smaller under constant dark condition. Temperature compensation of the endogenous rhythmicity was not found by this analysis. The 33 DD treatment showed only the 2-h ultradian cycle, which clearly differed from the cycles in the other 2 treatments. It seems that the higher temperature masked the circadian rhythm but did not affect the ultradian rhythm.
Accumulated PSD analysis tended to show circadian cycles (63%), in which the short cycles were also observed. Most of the short cycles (78%) lasted between 2 h and 4 h. The ultradian cycle was also evident under the LD conditions. This is interesting because, generally, diurnal LD conditions lead to the strong 24-h cycle enforced by the external stimulus.
Table 3 Periodicity (h) of root growth corresponding to the greatest power spectrum density for plants in each experiment. 28 DD (72 ha ) b
Circadian cycles (20–28 h)
25.2 (38.7 ) 23.8 (8.5) 21.8 (19.2) 22.9 (9.5)
Ultradian cycles (<20 h)
3.4 (14.1) 5.0 (2.7) 4.1 (3.2) 2.8 (7.2) 2.7 (25.2) 4.0 (4.3) 2.4 (14.7) 6.0 (43.5)
a b
28 LD (72 h)
33 DD (53 h)
24.4 (8.5) 23.6 (4.4) 21.8 (5.4) 23.3 (91.6) 24.0 (11.0)
21.0 (22.8) 20.4 (9.5)
2.2 (1.6) 2.1 (2929.3) 4.0 (15.4)
Duration of root elongation rates measurement. Power spectral density of the period.
2.3 (11.0) 3.7 (33.7) 4.5 (33.7) 3.2 (75.0) 5.8 (19.6) 2.0 (42.2) 3.4 (104.1)
As stated previously, individual plants showed various PSD intensities for RER periodicity. To evaluate the different PSD intensity equally, accumulation of relative PSD by replacing the maximum PSD with 1 for each plant data is shown in Fig. 6. By doing so, not only the individual plant data are evaluated but also all the possible cycle would not be cancelled. Moreover, the possible cycles will be strengthened by accumulating individual peaks. A cluster of peaks around 24–22 h was observed in all of the 3 treatments. When all the plant data accumulated, the maximum value was found to be more than 1, in cases where the frequency (Hz) for each PSD was the same. The highest peaks in the clustered area were 23.2 h, 23.8 h, and 21.5 h for 28 DD, 28 LD, and 33 DD, respectively. PSD values of cycles outside this clustered area were relatively small in the LD conditions, whereas those in the DD conditions could be clearly observed. All the ultradian cycles could be recognized by the accumulation of the PSD. The peak patterns in the 2 different DD treatments, compared to the peak patterns in the LD treatment, were relatively similar. Therefore, the accumulated PSD analysis showed that the cycle patterns under different temperature conditions were highly similar. Temperature is a stronger entraining stimulus than light in Neurospora (Liu et al., 1998; McClung, 2000). In the CAM plants Kalanchoë daigremontiana (Lüttge and Beck, 1992) and Bryophyllum fedtschenkoi (Anderson and Wilkins, 1989), a high temperature (more than 30 ◦ C) causes an
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Fig. 4. Power spectral analysis of average values of root growth rates in continuous dark (28 DD) and day-night cycle (LD 12:12) at 28 ◦ C constant temperature. (a) Original time series data and optimum least square fitting curve. (b) Residual time series data from the fitting curve. (c) Spectral analysis of (b) by maximum entropy method. Underline indicates dark period.
arrhythmic behavior in net CO2 exchange. The higher temperature may also cause slight changes in the cyclic root growth pattern. RER during day and night As shown in Table 4, the average RER during the day was significantly higher than that during the night in both the LD treatments. For the 28 LD treatment, temperatures during the day slightly differed from those during the night, and these temperatures were 28.4 ◦ C and 27.6 ◦ C, respectively. Admittedly, this is a small change; Table 4 Root elongation rates (mm/h) during day and night in two experimental sets of plants growing at 28 ◦ C. The data from the second day of observation period were used for the comparison of 28 LD. Day–night 12 h
28 LD DT-LD
Day Night Day Night
Mid day 6 h (0900–1500 h) and mid night 6 h (2100–0300 h) 1.97 1.82 2.15 1.49
± ± ± ±
0.01*** 0.03 0.07*** 0.04
1.99 1.76 2.31 1.48
± ± ± ±
0.01*** 0.04 0.05*** 0.05
Values are means ± SE of eight and three replicates for 28 LD and DT-LD, respectively. *** Significant difference at P < 0.001 by ANOVA.
however, it may be large enough to affect periodic growth behavior. To test whether these temperature differences affected the RER, total root elongation under constant dark condition for 6 days of growth were compared. The total elongation was 99.7 ± 3.1 mm at 28.4 ◦ C and 99.6 ± 1.9 mm at 27.6 ◦ C. This difference was insignificant; therefore, the results indicate that temperature was not the cause of the RER difference between day and night. In the DT-LD treatment, midday RER was 1.56 times faster than the midnight RER (Table 4). In this case, the RER changes do correspond to the different root zone temperatures, as shown in Fig. 7.
Discussion This paper provides the first evidence that rice root elongation growth shows both circadian and ultradian rhythms: a 23-h circadian cycle and a shorter 2-h ultradian cycle. Unlike root elongation, stem elongation has been studied extensively, perhaps because shoot measurements can be much easily obtained (Walter et al., 2009). Moreover, shoot growth is less affected by the light and dry air conditions required for the observations. Shoot elongation has been reported to show cycles of 23 h in wild spinach stems (Lecharny and Wagner, 1984), of less than 24 h in rose stems (Tutty et al., 1994), of nearly 24 h in oat coleoptiles (Ball and Dyke, 1954), of 21.4–23.5 h in Arabidopsis inflorescence stems (Jouve et al., 1998),
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Fig. 6. Accumulation of the relative power spectral density (PSD) by replacing the maximum PSD with a value of 1 for the analyzed data of each plant. (a) 28 DD with 12 replicate plants, (b) 28 LD with eight replicate plants and (c) 33 DD with nine replicate plants.
Fig. 5. Power spectral analysis of average values of root growth rates in continuous dark at 33 ◦ C constant temperature (33 DD). (a) Original time series data and optimum least square fitting curve. (b) Residual time series data from the fitting curve. (c) Spectral analysis of (b) by maximum entropy method. Underline indicates dark period.
and of 25.2 h in hypocotyls in Arabidopsis (Dowson-Day and Millar, 1999). These periods, except the last one, are in agreement with our results of root elongation. It is not surprising that shoot and root elongation rhythms are similar at this seedling stage, because both organs form a continuum along the plant axis. Diurnal rhythmicity in elongation has been examined in leaves (Christ, 1978a,b; Walter et al., 2009). However, an endogenous rhythm of leaf elongation seems not to have been found so far, though many studies have recorded up-and-down nastic leaf movements (Pedersen et al., 1993). Circadian cycles have also been described in relation to the physiology of higher plants. For example, stomatal opening/closure occurs over a period of 22 h (Gorton et al., 1989) or approximately 24 h (Hennessey et al., 1993). Photosynthetic rates show a 24.5h period (Hennessey and Field, 1991). Thus, a 22–25-h circadian rhythmicity is the most common cycle duration for growth and physiological phenomena examined so far. Regarding the ultradian cycles, nutational movement and growth rates have been extensively studied in shoots. For example, a 100-min (Millet et al., 1984) or a 2–4-h (Koukkari, 1994) period of circumnutational movement is observed in shoots of climbing bean; moreover, cycles of 45–120 min in shoot elongation rates in radish, cucumber, and sunflower (Kristie and Jolliffe, 1986) and cycles of 112–133 min in soybean (Kurt et al., 1998) have been reported. In roots, circumnutational cycles of 1–2 h have been
reported in Kentucky blue grass (Fisher, 1964), and cycles ranging from 12 min to 1 h have been reported in maize (Walter et al., 2003). Recently, Yazdanbakhsh et al. (2011) also reported cycles of 8–9 h in Arabidopsis root growth. The ultradian cycle observed in the present study are most probably related to the root circumnutational growth. Although roots with visible nutational growth were excluded from the analysis, several others, with apparently insignificant movements, were included in the RER measurement. Shorter ultradian cycles of less than 1 h seem to exist in shoot elongation growth (Kristie and Jolliffe, 1986). In the course of the present study, we confirmed a less than 1 h cycle of root tip circumnutation (Iijima et al., 1998), though the roots in question were not included in the present RER analysis. This short cycle was visible when the root tip was not fixed properly between the panel and the filter paper. The ultradian cycle of root elongation found in this study might be related to the production of both mucilage and root border cells. The resolution of the camera system was such that rhythmic swelling and contraction of the extreme root tip (root cap) could be detected. This type of movement would be indistinguishable from rhythmic pulses of elongation of the main root axis. The roots grown in a mechanically impeded soil release both mucilage (Iijima and Kono, 1992) and root border cells (Iijima et al., 2000, 2003a,b, 2004) from the root cap, which covers the meristematic region of the roots. Under the experimental conditions in this study, the roots were not in direct contact with the soil. Thus, the cells could be retained for a longer duration in the cap region than when they are allowed to be removed by abrasion with the growth medium. The accumulation of the border cells on the tip portion of the cap could result in small differences in RER, because relatively large number of these cells accumulates around the tip. So far, cycles of 2–3 h have been detected for the droplet size distribution of mucilage (Mollenhauer et al., 1975), Golgi apparatus development (Jones and Morre, 1973), and release of water-soluble exudates (Pearson et al., 1981). The cycle of mucilage exudation and release may, in turn, influence the release of border cells, which then adhere to the
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Fig. 7. Root elongation rates under daily temperature fluctuation in the LD 12:12 environment. (a) Average value of three replicates. (b) The corresponding temperature fluctuations (same data as Fig. 2 d). Underline is the night period.
extending flank of the roots and move away from the tip by further tip growth. The rhythmicity in mucilage exudation, together with the release of border cells, should be analyzed to clarify the contribution of these to the ultradian cycles in root growth. Root elongation was slightly greater during the day than during the night when the temperature was constant (28 LD in Table 4). This is in agreement with results of a study on monocot leaf elongation (Christ, 1978a; Poiré et al., 2010), which showed a lower growth rate at night than during the day. This observation might be related to the water status within the plant (Christ, 1978b) and perhaps to diurnal changes in the root sap pressure (Vaadia, 1960). During daytime, when transpiration is active, the water potential gradient will continuously lead to drawing of water present near the root tip towards the roots. This may increase the efficiency of water supply to the elongation zone of roots and hence contribute to greater cell expansion during the daytime. Indeed, the water distribution pattern in the shoot during the day differs from that during the night (Iijima et al., 2011). Another explanation for the higher growth rate during the day is the slight increase in the translocation of photosynthetically fixed carbon into the root tip during the day. Diurnal regulation of the accumulation of starch (Li et al., 1992) and total sugars (Geiger et al., 2000) in leaves suggests a diurnal pattern in carbon allocation to the root elongation zone and in the utilization of the carbon for growth. Evidence for the effects of carbon supply on the rhythmicity of root growth was reported recently in Arabidopsis thaliana (Yazdanbakhsh et al., 2011). Whether diurnal rhythms in mitotic activity in the root meristem (Bishop and Klein, 1973) are translated into rhythmic elongation patterns remains an open question. In this study, we used various environmental conditions to analyze the endogenous rhythms of root elongation growth. Compared to DD conditions, light–dark 24 h cycle, especially with daily temperature fluctuations, induced a clear 24-h pattern. Environmental conditions, including light and temperature, are thought to regulate RER. The reason for the existence of various rhythms under constant light condition is currently unknown. Relatively young age of the seedlings may be associated with the variation in the rhythms. For
example, the cell population in the root apical region (meristem and elongation zone) during the first 3–4 days would be changing from the cells formed in the embryo to cells formed after the continuous light condition; the embryonic elongating cell population would be swept from the growth zone day by day. Similarly, the dimensions of the growth zone may change during this early stage of post-germination root growth, and changes in growth rate may occur as the roots and their cells emerge from the immediate postgermination phase. An extended time-course analysis, which is not applicable at the moment due to the experimental difficulties, may provide further information regarding the root elongation rhythms. Although biological rhythms have been studied mostly under constant environmental conditions, an understanding of how the circadian system functions in the real world will require more complex experimental conditions (Nozue et al., 2007). Actual root elongation in the field soil, at least in the case of young roots near the soil surface, will probably be highly similar to that in the DT-LD treatment, with diurnal changes in temperature and light. Under such a condition, the shoot and root growth patterns will be strongly affected by the physiological processes responsible for the interaction between internal gene control and environmental impact (Walter and Schurr, 2005; Walter et al., 2009). Acknowledgement This study was funded by a grant-in-aid for basic research (B212460010) from the Japan Society for the Promotion of Science. References Anderson CM, Wilkins MB. Period and phase control by temperature in the circadian rhythm of carbon dioxide fixation in illuminated leaves of Bryophyllum fedtschenkoi. Planta 1989;177:456–69. Ball NG, Dyke IJ. An endogenous 24-hour rhythm in the growth rate of the Avena coleoptile. J Exp Bot 1954;5:421–33. Bishop RC, Klein RM. 1973 Innate and photo-controlled mitotic rhythms in onion root-tip cells. Can J Genet Cytol 1973;15:667–70. Christ RA. The elongation rate of wheat leaves. I. Elongation rates during day and night. J Exp Bot 1978a;29:603–10.
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Christ RA. The elongation rate of wheat leaves. II. Effect of sudden light change on the elongation rate. J Exp Bot 1978b;29:611–8. Dowson-Day MJ, Millar AJ. Circadian dysfunction causes aberrant hypocotyl elongation patterns in Arabidopsis. The Plant J 1999;17:63–71. Evans ML. A new sensitive root auxanometer. Preliminary studies of the interaction of auxin and acid pH in the regulation of intact root elongation. Plant Physiol 1976;58:599–601. Fisher JE. Evidence of circumnutational growth movements of rhizomes of Poa pratensis L. that aid in soil penetration. Can J Bot 1964;42:293–9, 1964. Feldman LJ. Regulation of root development. Annu Rev Plant Physiol 1984;35:223–42. Geiger DR, Servaites JC, Fuchs MA. Role of starch in carbon translocation and partitioning at the plant level. Aust J Plant Physiol 2000;27:571–82. Gordon D, Hettiaratchi DRP, Bengough AG, Young IM. Non-destructive analysis of root growth in porous media. Plant Cell Environ 1992;15:123–8. Gorton HL, Williams WE, Binns ME, Gemmell CN, Leheny EA, Shepherd AC. Circadian stomatal rhythms in epidermal peels from Vicia faba. Plant Physiol 1989;90:1329–34. Hansen GK. Diurnal variation of root respiration rates and nitrate uptake as influenced by nitrogen supply. Physiol Plant 1980;48:421–7. Head GC. Studies of diurnal changes in cherry root growth and nutational movements of apple root tips by time-lapse cinematography. Ann Bot 1965;29:219–24. Hennessey TL, Field CB. Circadian rhythms in photosynthesis: oscillations in carbon assimilation and stomatal conductance under constant conditions. Plant Physiol 1991;96:831–6, 1991. Hennessey TL, Freeden AL, Field CB. Environmental effects on circadian rhythms in photosynthesis and stomatal opening. Planta 1993;189:369–76. Henzler T, Waterhouse RN, Smyth AJ, Carvajal M, Cooke DT, Schaffner AR, et al. Diurnal variations in hydraulic conductivity and root pressure can be correlated with the expression of putative aquaporins in the roots of Lotus japonicus. Planta 1999;210:50–60. Iijima M, Kono Y. Development of Golgi apparatus in the root cap cells of maize (Zea mays L.) as affected by compacted soil. Ann Bot 1992;70:207–12. Iijima M, Oribe Y, Horibe Y, Kono Y. Time lapse analysis of root elongation rates of rice and sorghum during day and night. Ann Bot 1998;81:603–7. Iijima M, Griffith B, Bengough AG. Sloughing of cap cells and carbon exudation from maize seedling roots in compacted sand. New Phytol 2000;145:477–82. Iijima M, Barlow PW, Bengough AG. Root cap structure and cell production rates of maize (Zea mays L.) roots in compacted sand. New Phytol 2003a;160:127–34. Iijima M, Higuchi T, Barlow PW, Bengough AG. Root cap removal increases root penetration resistance in maize (Zea mays L.). J Exp Bot 2003b;54:2105–9. Iijima M, Sako Y, Rao TP. A new approach for the quantification of root-cap mucilage exudation in the soil. Plant Soil 2003c;255:399–407. Iijima M, Higuchi T, Watanabe A, Bengough AG. Method to quantify root border cells in sandy soil. Soil Biol Biochem 2004;36:1517–9. Iijima M, Yoshida T, Kato T, Kawasaki M, Watanabe T, Somasundaram S. Visualization of lateral water transport pathways in soybean by a time of flight-secondary ion mass spectrometry cryo-system. J Exp Bot 2011;62:2179–88. Ishikawa H, Hasenstein KH, Evans ML. Computer-based video digitizer analysis of surface extension in maize roots: kinetics of growth rate changes during gravitropism. Planta 1991;183:381–90. Jones DD, Morre DJ. Golgi apparatus mediated polysaccharide secretion by outer root cap cells of Zea mays. III. Control by exogenous sugars. Physiol Plant 1973;29:68–75. Jouve L, Greppin H, Agosti RD. Arabidopsis thaliana floral stem elongation: evidence f or an endogenous circadian rhythm. Plant Physiol Biochem 1998;36:469–72. Koukkari WL. Movement of a bean shoot: an introduction to chronobiology. Chronobiol Int 1994;11:85–93. Kristie DN, Jolliffe PA. High-resolution studies of growth oscillations during stem elongation. Can J Bot 1986;64:2399–405. Kurt A, Robert S, Willard K. Ultradian movements of shoots of two species of soybeans: Glycine soja (Sieb. and Zucc.) and Glycine max (L.) Merr. Chronobiol Inter 1998;15:1–11.
Lecharny A, Wagner E. Stem extension rate in light-grown plants: evidence for an endogenous circadian rhythm in Chenopodium rubrum. Physiol Plant 1984;60:437–43. Li B, Geiger DR, Shieh WJ. Evidence for circadian regulation of starch and sucrose synthesis in sugar beet leaves. Plant Physiol 1992;99:1393–9. Liu Y, Merrow M, Loros JJ, Dunlap JC. How temperature changes reset a circadian oscillator. Science 1998;281:825–9. Lüttge U, Beck F. Endogenous rhythms and chaos in crassulacean acid metabolism. Planta 1992;188:28–38. McClung CR. Circadian rhythms in plants: a millennial view. Physiol Plant 2000;109:359–71. Millet B, Melin D, Bonnet B, Ibrahim CA, Mercier J. Rhythmic circumnutation movement of the shoots in Phaseolus vulgaris L. Chronobiol Int 1984;1:11–9. Mollenhauer HM, Morre DJ, Vanderwoude WJ. Endoplasmic reticulum–Golgi apparatus associations in maize root tips. Mikroskopie 1975;31:257–72. Nakanishi T, Yokota H, Tanoi K, Furukawa J, Ikeue N, Ookuni Y, et al. Circadian rhythm in 15 O-labeled water uptake manner of a soybean plant by PETIS (Positron Emitting Tracer Imaging System). Radioisotopes 2001;50:163–8. Nozue K, Covington MF, Duek PD, Lorrain S, Fankhauser C, Harmer SL, et al. Rhythmic growth explained by coincidence between internal and external cues. Nature 2007;448:358–63. Ohtomo N, Terachi S, Tanaka Y, Tokiwano K, Kaneko N. New method of time series analysis and its application to Wolf’s sunspot number data. Jpn J Appl Phys 1994;33:2821–31. Pearson CJ, Steer BT. Daily changes in nitrate uptake and metabolism in Capsicum annuum. Planta 1977;137:107–12. Pearson CJ, Volk RJ, Jackson WA. Daily changes in nitrate influx, efflux and metabolism in maize and pearl millet. Planta 1981;152:319–24. Pedersen M, Johnsson A, Mæhle J, Dalløkken R. Short period leaf movements in Oxalis regnellii. Physiol Plant 1993;89:277–84. Pilet PE. Kinetics of the light-induced georeactivity of maize roots. Planta 1979;145:403–4. Pilet PE, Ney D. Rapid localized light effect on root growth in maize. Planta 1978;144:109–10. Poiré R, Wiese-Klinkenberg A, Parent B, Mielewczik M, Schurr U, Tardieu F, et al. Diel time-courses of leaf growth in monocot and dicot species: endogenous rhythms and temperature effects. J Exp Bot 2010;61:1751–9. Tanimoto E, Watanabe J. Automated recording of lettuce root elongation as affected by auxin and acid pH in a new rhizometer with minimum mechanical contact to roots. Plant Cell Physiol 1986;27:1475–87. Tutty JR, Hicklenton PR, Kristie DN, McRae KB. The influence of photoperiod and temperature on the kinetics of stem elongation in Dendranthema grandiflorum. J Am Soc Hortic Sci 1994;119:138–43. Vaadia Y. Autonomic diurnal fluctuations in rate of exudation and root pressure of decapitated sunflower plants. Physiol Plant 1960;13:701–17. Walter A, Schurr U. Dynamics of leaf and root growth – endogenous control versus environmental impact. Ann Bot 2005;95:891–900. Walter A, Spies H, Terjung S, KD usters R, Kirchgeßner N, Schurr U. Spatio-temporal dynamics of expansion growth in roots: automatic quantification of diurnal course and temperature response by digital image sequence processing. J Exp Bot 2002;53:689–98. Walter A, Feil R, Schurr U. Expansion dynamics, metabolite composition and substance transfer of the primary root growth zone of Zea mays L. grown in different external nutrient availabilities. Plant Cell Environ 2003;26:1451–66. Walter A, Silk WK, Schurr U. Environmental effects on spatial and temporal patterns of leaf and root growth. Annu Rev Plant Biol 2009;60:279–304. Yazdanbakhsh N, Fisahn J. Stable diurnal growth rhythms modulate root elongation of Arabidopsis thaliana. Plant Root 2011;5:17–23. Yazdanbakhsh N, Sulpice R, Graf A, Stitt M, Fisahn J. Circadian control of root elongation and C partitioning in Arabidopsis thaliana. Plant Cell Environ 2011., doi:10.1111/j.1365-3040.2011.02286.x.