Statistical interferometric investigation of nano-scale root growth: effects of short-term ozone exposure on ectomycorrhizal pine (Pinus densiflora) seedlings
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- Rathnayake, A.P., Kadono, H., Toyooka, S. et al. J For Res (2007) 12: 393. doi:10.1007/s10310-007-0040-x
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This study presents the effects of short-term ozone exposure on the nano-scale growth behavior of the fine roots of Pinus densiflora (Japanese red pine) seedlings. Root elongation measurements were obtained in nanometers for very short (sub-second) time intervals by using the optical interference method called statistical interferometry, developed by the authors. Three categories of P. densiflora seedlings were investigated; two categories were infected with ectomycorrhiza of Pisolithus sp. (Ps) and Cenococcum geophilum (Cg), while the third was without any fungal infection. In experiments, two points on a root with a separation of 3 mm were illuminated by laser beams and the elongation was measured continuously by analyzing speckle patterns successively taken by a CCD camera. The ectomycorrhizal fungi-infected and uninfected seedlings were exposed to ozone at concentrations of 120 and 240 ppb for periods of 1, 3, or 5 h in separate treatments. The root elongations of P. densiflora seedlings were measured before and immediately after the each ozone treatment and then the root elongation rates (RER) were determined for growth-measurement periods of 5.5 s and 9.5 min. From the measurements obtained for 9.5 min, we found that the RERs of uninfected and Cg-infected seedlings were reduced by 42 and 18%, respectively, after 5 h of exposure to 120 ppb ozone compared with that before exposure, while the reduction in RER of Ps-infected seedlings was not significant. When the concentration of ozone was increased to 240 ppb, the RERs of Ps-infected and Cg-infected seedlings were reduced by 32 and 44%, respectively, after exposure for 5 h, while the reduction in RER of uninfected seedlings was 59%. These observations prove that the non-mycorrhizal seedling roots are more sensitive to ozone stress. From this study, we found that the RERs of both mycorrhizal and non-mycorrhizal seedlings apparently fluctuated throughout the measurements, even within a few minutes.
KeywordsEctomycorrhizaOzonePinus densifloraRoot elongationStatistical interferometry
Air pollution is one of the consequences of expanding population and rapid growth of industrial activities. The basic components causing air pollution are the oxides of nitrogen (NOx) and volatile organic compounds (VOCs) emitted by vehicles and industries. Ozone is a secondary air pollutant, generated by a series of reactions of NOx and VOCs in the presence of UV radiation from strong sunlight (Dohrmann and Tebbe 2005). The impact of ozone on plant growth was first identified in the 1950s (Ashmore 2005), and it is now well known as a harmful photochemical oxidant, affecting the life of plants in natural ecosystems.
The growth of most vascular plants in ecosystems is supported by the mycorrhizal fungi, which serve as the main organ for uptake of nutrients. Mycorrhizal fungi form symbiotic associations with plant roots (Smith and Read 1997) facilitating their growth in a new, nutrient poor, and dry soil environment, benefitting the smooth growth and survival of plants (Simon et al. 1993). Ectomycorrhizal fungi, which colonize roots of various species of woody plants, such as eucalyptus, pine, oak, and hazel, forms a mantle around fine roots gaining carbon and other essential organic substances from plants in return for helping the plants to take up water, mineral salts, and metabolites. Ectomycorrhizal fungi play an important role in the carbon dynamics of forest ecosystems and it has been found that these fungi stimulate the photosynthetic process of host plants (Wu et al. 2002), caused by the increased carbon sink strength of ectomycorrhizal roots (Smith and Read 1997).
In general, the impact of ozone on plant growth has been investigated basically in two categories, based on the period of exposure and the concentration of ozone (Morgan et al. 2003). Chronic or long-term exposure is examined for many years or throughout the growth period of plants while acute or short-term exposure is examined on a daily or hourly basis for ozone concentration peaks of >120 ppb. Although ambient ozone concentrations typically range between 20 and 60 ppb, a peak of 100–150 ppb has been found in the Mediterranean area (Pasqualini et al. 2001) causing immediate impact on plant growth. Studies conducted in North America, Europe, and Japan have revealed that exposure to ambient ozone levels below 100 ppb for several months is sufficient to inhibit dry matter production and the physiological functions of sensitive forest species (Izuta et al. 2001). Moreover, acute ozone dosages can affect the plant species severely causing alteration of physiological activity such as photosynthesis (Pasqualini et al. 2001). These alterations can lead to foliar injury, accelerated senescence, and reduced shoot and root growth (Peel and Dann 1991), while causing initial loss of photosynthesis followed by cell death (Sandermann 1996).
Assessment of the impacts of ozone on plant shoots or leaves is comparatively easy under slightly or highly elevated ozone concentrations, since the above-ground plant components are easily accessible (Anttonen and Kärenlampi 1996; Yoshida et al. 2001). However, investigation of the effects of ozone on below-ground processes or root growth still remains a challenge (Yoshida et al. 2001), since the plant roots and associated symbionts are covered by soil, precluding direct access. Ozone may have direct effects on fine root dynamics and mycorrhizal associations or indirect effects caused by reduced photosynthesis and below-ground carbon allocation (Andersen and Rygiewicz 1995). Since ozone does not penetrate through the soil, direct impact on roots and mycorrhiza does not seem to be present (Manninen et al. 1998). Further, the symbiotic relationship between mycorrhizal fungi and host plants leads to a large impact on root growth, since symbionts contribute to several physiological and metabolic changes in plants (Varma and Hock 1995). Although the effects of ozone on growth of roots and associated mycorrhizal fungi have been investigated widely (Andersen 2003), it was not possible to find evidence that mycorrhizal and non-mycorrhizal seedlings respond differently to ozone stress (Andersen and Rygiewicz 1995).
The impact of acute ozone exposure on root growth has been investigated in many studies (Andersen 2003), and it has been found that ozone affects the below-ground processes of plants by altering the growth behavior of the roots and the associated symbionts. Most of these studies were based on daily or weekly exposure to ozone (Coleman et al. 1996; Hofstra et al. 1981). However, alterations in the natural growth process of plant roots can happen even after short periods, such as 1, 3, or 5 h of ozone exposure. The impact on root growth of short-period exposure to ozone would be on the micro or nano-scale; therefore, very high-sensitivity techniques are needed to evaluate these effects. Further, the immediate effects of ozone on fine root dynamics are still unknown, since the investigations were limited by the lack of experimental tools capable of detecting immediate impacts on biological activities. Moreover, when evaluating the impact of short-period ozone exposure, the level of measurements or the sampling period is also very important, as it reflects the sensitivity of the technique and the accuracy of the results. With conventional experimental techniques for root dynamics studies, the achievable minimum sampling time was limited to a daily or hourly basis. Therefore, monitoring of very short-period dynamics such as in seconds or minutes was not possible.
In this study, we apply an optical interference technique called statistical interferometry (Kadono and Toyooka 1991) to investigate very short-period dynamic behavior of fine roots, facilitating measurement of sub-nanometer growth in sub-seconds, which is not possible by any other method. Since the technique offers very high sensitivity, we can detect immediate changes of biological activities, such as root elongation, after short-period ozone exposure, in real-time, and in a non-contact manner. Here, we examine the immediate response of Pinus densiflora Sieb. and Zucc. seedling roots to short-term exposure to ozone, where nano-scale variations of fine root elongations are investigated. P. densiflora is the dominant conifer in East Asian countries, mainly Japan, China, and Korea. It is particularly known to be associated with ectomycorrhizal symbionts such as Pisolithus sp. and Cenococcum geophilum Fr., and prefers well-drained soil; it can, however, grow in nutritionally poor soil. P. densiflora has been used for forest rehabilitation practices under various disturbances, since it grows rapidly in infertile granite regions, and on ridges and mountains (Choi et al. 2005).
In this paper, we investigate the impact of ozone on root elongations of P. densiflora seedlings infected with two ectomycorrhizal fungi; Pisolithus sp. (Ps) and C. geophilum (Cg), in comparison with uninfected seedlings. The nano-scale elongations in fine roots of P. densiflora seedlings were obtained for several seconds and fluctuations of root elongation rates (RER) were monitored for several minutes. The root growth dynamics were obtained before and immediately after exposure to 120 and 240 ppb ozone for periods of 1, 3, or 5 h in separate treatments.
Materials and methods
The Pinus densiflora seedlings infected with Pisolithus sp. (Ps) and Cenococcum geophilum (Cg), which are ectomycorrhizal fungi, were produced by the following procedure. Inoculums of Ps and Cg were initially prepared by incubating their hyphae in autoclaved soil (peat-moss:vermiculite = 1:3 (v/v)) with added modified Melin–Norkrans (MMN) solution. Next, using the prepared inoculums, either Ps or Cg fungi was inoculated into uninfected P. densiflora seedlings in separate treatments. Then, a few seedlings infected with Ps and Cg were transplanted in separate plastic cases (diameter:depth = 170:120 mm) packed with sterilized soil (pH 5.25, average total carbon and nitrogen contents were 4.78 and 0.28%, respectively). The sterilized soil was prepared by autoclaving a mixture (1:1 v/v) of Shibanome soil (volcanic sand) and Kuroboku soil (black soil rich in humus content). After these seedlings had been grown in a growth chamber (KG-50HLAW-S, Koito Industries, Japan) for about two months, in order to enable the spreading of extraradical mycelia of the inoculated fungi in the soil, P. densiflora seeds sterilized with 1% sodium hypochlorite solution were sowed in the cases and grown for about two more months. In addition, in order to produce the uninfected seedlings, the sterilized seeds were directly sowed in sterilized soil and grown in the growth chamber for about two more months. Thus, three kinds of seedling, i.e., Ps-infected, Cg-infected, and uninfected seedlings, were produced.
The day/night condition was 12/12 h.
The light intensity was about 600 μmol m−2 s−1 at the top of the seedling.
The day/night temperature conditions were 25/18°C.
For the ozone experiment, ozone was generated by a custom-built system consisting of an air compressor, reaction tubes, and UV lamp and controlled by industrial type pressure and flow regulators. The P. densiflora seedlings cultivated as described above were exposed to 120 or 240 ppb concentrations of ozone for periods of 1, 3, or 5 h in separate treatments.
In most cities in Japan, urban photochemical oxidant, ozone, warnings are issued when the concentration of ozone continues at a level of 120 ppb or above, and a serious ozone warning is issued when the concentration exceeds the level of 240 ppb, as a preventive action (Acid Deposition and Oxidant Research Center 2006). In our experiments, we aimed to investigate the effects of ozone on root growth, when the ozone concentration was at the threshold value (120 ppb) of the urban ozone warning and, further, at twice this threshold (240 ppb). These levels of ozone are also important for understanding the critical ozone concentrations in forest environments, since ozone levels of many mountains in the Kanto district, Japan, have been reported as 100 ppb or higher (Hatakeyama and Murano 1996; Matsumura 2001).
Principle and experimental arrangement of statistical interferometry
Statistical interferometry is a new technique, developed to measure the deformation of an object having an optically rough surface (Kadono and Toyooka 1991). This method is far more accurate than conventional deterministic interferometric techniques and the measurements can be obtained with sub-nanometer accuracy (Kadono et al. 2001). When a rough surface is illuminated by a laser light, the light is randomly scattered by the rough surface and the scattered light amplitudes are superposed coherently. This randomly scattered wavefront generates a granular pattern with high contrast, called a speckle pattern.
The light field of the speckle pattern generated from the rough surface, satisfying the condition that the roughness of the object is higher than half of the wavelength of the light used, is called a fully developed speckle field. The statistical properties of such a speckle field are very stable and independent of the scattering characteristics of the object, the quality of the optical components employed, and the parameters of the optical system. In the fully developed speckle field the speckle phase can take any value from −π to π with an equal probability, resulting in a uniform distribution of the probability density function (PDF). In other words, the PDF of the speckle phase takes a rectangular function which has a constant value of 1/2π from −π to π. In statistical interferometry, the uniformity of the PDF of the speckle phase together with its stability is utilized as the standard in the determination of the object phase.
Reliability of measurements depends only on the stability of statistics of the fully developed speckle field (Kadono et al. 2001), which can be easily satisfied for most natural objects, including living matter. In contrast to conventional optical interferometry, there is no requirement for a complex optical system in order to prepare an accurate reference wavefront. Since the principle of statistical interferometry is based on the statistics of the speckle phase, the accuracy of measurements depends only on the number of data samples used to calculate the PDF of the speckle phase, which is limited by the number of pixels of the CCD camera. The availability, at fairly low cost, of the CCD cameras having millions of pixels makes it easier to obtain a large number of data samples. According to a computer simulation, an accuracy of λ/1000 can be attained with 30,000 data samples, where λ is the wavelength of the light used.
By analyzing the interference speckle patterns according to the algorithms of statistical interferometry (Kadono et al. 2001), corresponding PDFs of the speckle phase were derived, and the object phases were determined by use of software developed to be compatible with Matlab (Release 13, The MathWorks, MA, USA). From the calculated object phase data, corresponding root elongation values were obtained.
Root elongation was measured using the experimental setup shown in Fig. 1. For the measurements, root samples were selected randomly from the lateral fine roots (diameter ≈ 1 mm, Satomura et al. 2003). In mycorrhizal fungi-infected seedlings we observed that ectomycorrhiza colonized both short and long lateral roots at the location where they extend from the main root. In our measurements only long roots were considered, as we needed to simply hold the root sample and illuminate two points on it. The two illuminating points selected were very close to the tip of the root, where the mycorrhiza were not present, and we aimed to obtain the elongation measurements by targeting the elongation zone (Taiz and Zeiger 2002) of the roots. Before the measurements, the selected root sample was taken out of the plastic case, with the lid off, while the whole plant remained in the case, and smoothly attached to a simple holder. On the root sample two selected points were covered with white wheat flour over the points before illuminating, in order to avoid the biospeckle (Oulamara et al. 1989) effect. The biospeckle effect is the dynamic change of the speckle pattern caused by light scattering due to moving scatterers inside the root sample. This biospeckle effect has to be suppressed in our measurements, as it reduces the correlation of the speckle patterns. After ensuring stable environmental conditions, root elongation data were obtained in the form of interference speckle patterns observed through the CCD camera and transferred to a computer through a frame grabber. At the same time, the interference speckle patterns from the illuminating points on the root were displayed on a monitor connected to the frame grabber. The measurements were carried out in a dark environment, where only the laser illumination could be visible.
Initially, two illuminating points were set to be 3 mm apart, in such a way that elongation of the selected 3-mm length segment of the root sample was monitored continuously throughout the period of measurement. During the elongation measurements, speckle interference images corresponding to a particular growth period of a root sample, such as 5.5 s, were captured in 0.5-s time intervals by the CCD camera and stored in computer memory.
The above measurements were carried out for different roots, which were similar in physical appearance and on the same seedling both before and after ozone exposure. We avoided measuring the same root when monitoring the growth before and after ozone exposure, since measurement of same root might cause mechanical stress. RER were calculated by linear regression of corresponding root elongation curves using commercially available scientific graphing and analysis software, Origin 7 SRI for Windows (Origin Lab, USA).
A one-way analysis of variance (ANOVA) followed by the Dunnet test was conducted to distinguish the effects of ozone exposure on RERs of mycorrhizal and non-mycorrhizal P. densiflora seedlings before and after ozone exposure. This statistical testing was carried out on the RERs obtained for 5.5 s and 9.5 min of growth measurement. In both cases, the level of statistical significance was established at P ≤ 0.05 and the variation of RERs after each ozone treatment was tested in comparison with that before exposure (control). All data were initially tested for normality and homogeneity and the statistical calculations were performed using GraphPad Prism (Version 4.00 for Windows; GraphPad Software, San Diego, CA, USA).
Root elongation rates (RER) of ectomycorrhizal fungi-infected and uninfected seedlings in 5.5-s periods before and immediately after ozone exposure
Root elongation rate (nm s−1 mm−1)
9.82 ± 0.28
9.50 ± 0.40 ns
9.18 ± 0.61 ns
7.96 ± 0.90 ns
9.33 ± 0.49 ns
8.17 ± 0.25*
7.76 ± 0.37*
7.40 ± 0.29
7.28 ± 0.27 ns
7.25 ± 0.33 ns
6.08 ± 0.23*
6.56 ± 0.17 ns
4.98 ± 0.36**
4.23 ± 0.18**
6.17 ± 0.32
5.58 ± 0.42 ns
5.09 ± 0.68*
3.59 ± 0.44**
5.60 ± 0.41 ns
4.06 ± 0.52**
2.50 ± 0.23**
Root elongation rates (RER) of ectomycorrhizal fungi-infected and uninfected seedlings in 9.5-min periods before and immediately after ozone exposure
Root elongation rate (nm s−1 mm−1)
9.87 ± 2.49
9.80 ± 2.09 ns
9.37 ± 1.85 ns
8.54 ± 1.52 ns
9.32 ± 2.00 ns
8.20 ± 1.64*
6.76 ± 1.28**
7.71 ± 2.25
7.32 ± 1.89 ns
7.26 ± 1.72 ns
6.29 ± 1.35*
6.73 ± 1.91 ns
5.07 ± 1.01**
4.33 ± 0.83**
6.31 ± 1.72
5.79 ± 1.70 ns
5.05 ± 1.56*
3.63 ± 1.09**
5.14 ± 1.57*
4.53 ± 1.42**
2.58 ± 0.80**
In this study we found that the root elongations and RERs of both mycorrhizal and non-mycorrhizal seedlings were reduced by exposure to ozone (Figs. 3 and 4, Tables 1 and 2). In the experiments, the shoots of the P. densiflora seedlings were exposed to ozone. Therefore, the reductions of root elongations after exposure to ozone were probably mediated by the shoot physiology (Anttonen and Kärenlampi 1996). The development of roots is often dependent on the availability of photosynthate, while carbon-limiting factors such as ozone affect the root growth rapidly and significantly (Andersen 2003). Therefore, a possible reason for the reductions of root elongations is the stress caused by ozone exposure, since ozone stress affects the photosynthetic activity of plants causing a reduction in carbon allocation to roots (Cooley and Manning 1987; Spence et al. 1990; Gorissen et al. 1994; Rennenberg et al. 1996). Further, the ozone stress can reduce carbon fixation, alter leaf and root respiration rates, shift the partitioning of carbon into different chemical forms, and disrupt carbon and nutrient allocation patterns (Chappelka and Samuelson 1998). The reduction of root elongation implies that ozone exposure affects root growth resulting in a decrease in root biomass. Recent studies using open-top chamber experiments revealed that exposure for long periods to ambient concentrations of ozone can lead to reduction in the root dry weight of beech seedlings to 64% of the control (Takeda and Aihara 2007).
In this study, we observed that reductions of RER in mycorrhizal seedlings were not significant after 1 or 3 h of exposure to 120 ppb ozone. But after exposure of non-mycorrhizal seedlings to 120 ppb ozone for 3 h the RER were reduced significantly by 20% of that before the exposure (Table 2). When we increased the concentration of ozone to 240 ppb, the RER of non-mycorrhizal seedlings were reduced significantly after 1 h exposure by 18% of that before the exposure, while the reduction in the RER of mycorrhizal seedlings was not significant (Table 2). These results indicate that the roots of non-mycorrhizal seedlings are more sensitive to ozone stress than the roots of mycorrhizal seedlings.
In the experiments, initially we obtained root elongations and RERs for 5.5-s measurement periods to observe the root elongation behavior over a period of a few seconds. Next, in order to observe the root elongation behavior over a period of a few minutes, we extended the measurement period to 9.5 min, in 30-s intervals. We observed similar root elongation behavior in both measurement periods (5.5 s and 9.5 min), but the RERs obtained in the 9.5-min fluctuated with time. This fluctuation of RERs was on the nano-scale and occurred even within a few minutes (Fig. 5). Our measurements were carried out for steady-state root elongation, where roots are growing at a constant rate (Sharp et al. 1998), as observed from Figs. 3 and 4. It was confirmed that the error of the measurements using statistical interferometry is less than 0.01 nm s−1 mm−1, which is much below the typical RER values obtained in the present experiments. Therefore, we suspect that this type of fluctuation is probably due to some biological phenomenon which is not fully understood with the present progress of our studies.
In the statistical interferometry, the root sample has to be illuminated before the measurements. Part of the illuminating beam can penetrate the root and be dynamically scattered, which degrades the accuracy of the measurements. To solve this problem we tested various methods to cover the surface of the root sample to prevent the laser beam entering the root. In the experiments, the surface of the root sample was covered with white wheat flour over the points to be illuminated, and it was confirmed that this preparation is very effective for suppressing the penetration of laser beams into the root. Further, in order to illuminate the root sample, it has to be uncovered from the plastic case, while the whole plant remains in the case. Although the preparations described above restrict the use of the present version of the statistical interferometry experimental system, the growth measurement period is very short (several seconds or a few minutes), ensuring minimum disturbance of the natural growth process of the roots. Since the measurements are obtained in a non-destructive and non-contact manner, growth monitoring will not affect the life of the plants.
The greatest advantage of the proposed statistical interferometry is that very accurate and reliable measurements can be performed just by illuminating a living object, such as a plant root, with a laser; using a simple optical system. The study presented in this paper suggests possible applications of statistical interferometry for investigating the immediate impact of environmental factors such as ozone exposure, acid rain, soil acidification, and UV radiation, on the dynamic growth behavior of plants in very short periods of several seconds or a few minutes, which is not possible with conventional experimental methods. Because of the statistical basis of the method, improvement of the accuracy is inherently assured, by taking a larger number of data samples into account, while maintaining robustness with great promise in the real-time exploration of eco-biological phenomena in a non-destructive and non-contact manner.
Our study verifies that indirect exposure to photochemical oxidants (ozone) affects the root growth of P. densiflora seedlings, even though the period of exposure is a few hours only, and that the ectomycorrhizal fungi-infected seedlings are less susceptible than the uninfected seedlings. However, additional experiments are required to evaluate the effect of combined stresses and limited nutrient conditions, as the actual growing conditions of plants are much different in forest environments. Since the influence of ozone on an individual seedling is a complex process which varies in response to environmental factors, complete assessment of the effects of ozone on forest plants requires continuous development of experimentation on different scales focusing on the possible causes and their impacts on the natural root growth process.
This work was partly supported by the Grant-in-Aid for Scientific Research B (16310020) of the Ministry of Education, Culture, Sports, Science and Technology, Japan.