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The photoreceptors for chloroplast photorelocation movement have been known, but the signal(s) raised by photoreceptors remains unknown. To know the properties of the signal(s) for chloroplast accumulation movement, we examined the speed of signal transferred from light-irradiated area to chloroplasts in gametophytes of Adiantum capillus-veneris. When dark-adapted gametophyte cells were irradiated with a microbeam of various light intensities of red or blue light for 1 min or continuously, the chloroplasts started to move towards the irradiated area. The speed of signal transfer was calculated from the relationship between the timing of start moving and the distance of chloroplasts from the microbeam and was found to be constant at any light conditions. In prothallial cells, the speed was about 1.0 µm min−1 and in protonemal cells about 0.7 µm min−1 towards base and about 2.3 µm min−1 towards the apex. We confirmed the speed of signal transfer in Arabidopsis thaliana mesophyll cells under continuous irradiation of blue light, as was about 0.8 µm min−1. Possible candidates of the signal are discussed depending on the speed of signal transfer.
Organelle movement is essential for plant growth and development and tightly regulated by environmental conditions.1 It is well known that light regulates chloroplast movement in various plant species. Chloroplast movement can be separated into three categories, (1) photoperception by photoreceptors, (2) signal transduction from photoreceptor to chloroplasts and (3) movement of chloroplasts and has been analyzed from a physiological point of view.2 We recently identified the photoreceptors in Arabidopsis thaliana, fern Adiantum capillus-veneris, and moss Physcomitrella patens. In A. thaliana, phototropin 2 (phot2) mediates the avoidance movement,3,4 whereas both phototropin 1 (phot1) and phot2 mediate the accumulation response.5 A chimeric photoreceptor neochrome 1 (neo1)6 was identified as a red/far-red and blue light receptor that mediates red as well as blue light-induced chloroplast movement in A. capillusveneris.7 Interestingly, neo1 mediated red and blue light-induced nuclear movement and negative phototropic response of A. capillus-veneris rhizoid cells.8,9 On the mechanism of chloroplast movement, we also found a novel structure of actin filaments that appeared between chloroplast and the plasma membrane at the front side of moving chloroplast.10 Recent studies using the technique of microbeam irradiation have revealed that chloroplasts do not have a polarity for light-induced accumulation movement and can move freely in any direction both in A. capillus-veneris prothallial cells and in A. thaliana mesophyll cells.11 However, the signal that may be released from photoreceptors and transferred to chloroplasts remains unknown.
To understand the properties of the signal for the chloroplast accumulation response, we examined the speed of signal transfer in dark-adapted A. capillus-veneris gametophyte cells and A. thaliana mesophyll cells by partial cell irradiation with a red and/or blue microbeam of various light intensities for 1 min and the following continuous irradiation, respectively.12
As shown in Figure 1, the relation between the distance of chloroplasts from the microbeam and the timing when each chloroplast started moving toward the microbeam irradiated area (shown as black dots in Fig. 1) was obtained and plotted. The lag time between the onset of microbeam irradiation and the timing of start moving of chloroplasts is the time period needed for a signal to reach each chloroplast. To obtain more accurate data many chloroplasts at various positions were used. The slope of the approximate line indicates the average speed of the signal transfer. Shown with a protonemal cell at the left side of this figure is an instance where the speed of signal transfer from basal-to-apical (acropetal) direction is obtained.
In protonemal cells, which are tip-growing linear cells, the average speed of signal transfer was about 2.3 µm min−1 from basal-to-apical (acropetal) and about 0.7 µm min−1 from apical-to-basal (basipetal) directions. These values were almost constant irrespective of light intensity, wavelength, irradiation period, and the region of the cell irradiated.12 The difference of speed between basipetal and acropetal directions may be depending on cell polarity. The signal transfer in prothallial cells of A. capillus-veneris and mesophyll cells of A. thaliana was about 1.0 µm min−1 to any direction, probably because they may not have a polarity comparing to protonemal cells or have a weak polarity if any. Thus, the speed of signal transfer must be conserved in most land plants,12 if not influenced by strong polarity. Table 1 summarizes the speeds of signal transfer under different light conditions in A. capillus-veneris and A. thaliana.
Calcium ions have been proposed as one of the candidates of the signal. Calcium is reported to be necessary for chloroplast movement in some plants.13,14 Chloroplast movement under polarized light could not be induced in the existence of EGTA in protonemal cells of A. capillus-veneris, although chloroplasts show slight movement in random direction.13 In Lemna trisulca, chloroplast movement correlates with an increase of cytoplasmic calcium levels and is inhibited by antagonists of calcium homeostasis.14 The speed of intracellular transfer of calcium ions in plant cells was measured only in moss Physcomitrella patens by microinjection of a calcium indicator into protonemal cells.15 The speed of calcium waves in the cytoplasm of protonemal cell was about 3.4 µm sec−1. The speed of substance transfer as signals is not known in plant cells except for the above instance, as far as we know, but in animal cells various experimental data has been accumulated.16–21
The transfer speed of calcium waves visualizing cytoplasmic free calcium by microinjection of aequorin was about 8 µm sec−1 in Xenopus eggs.16 Calcium ion expands as a spherical wave and the wave speed in plane is 50 µm sec−1 in rat cardiac myocytes when measured by loading a membrane-permeable indicator of calcium into the cell. The maximum velocity was 112 µm sec−1.17 Calcium waves could also be observed in the SR-free single isolated rabbit cardiac myofibrils with a propagation velocity of 15.5 µm sec−1.18 The propagation velocity of the calcium wave was about 65–100 µm sec−1 by calciuminduced calcium release (CICR) in pig heart muscle cells.19–21 Comparing these values to our data in A. capillus-veneris, the speed of signal transfer in chloroplast movement in fern gametophytes was 100–200 times slower than those measured for calcium ion transfers in animal cells, suggesting that the calcium might not be the signal involved in chloroplast movement.
Intracellular transport is depended on the cytoskeleton systems in many cases. So the speed of movement of the cytoskeleton itself has been examined. When motor-proteins (such as 22s dynein, 14s dynein, kinesin) were anchored on a slide glass microtubules overlaid moved with a speed of about 4.52, 4.29, 0.422 µm sec−1, respectively. In similar ways, actin filaments placed over myosin-coated glass moved at about 5.21 µm sec-1.22 On the other hand, the motor domain of the Centromere Binding Factor (CBF) protein complex moves at 4.04 µm min−1 on microtubules.23 In A. capillus-veneris protonemal cells, the speed of cytoplasmic streaming depending on the actomyosin system was calculated from the speed of oil drop movement.24 The speed was dependent upon the position of long protonemal cells and was about 2 µm min−1 in the apical region and gradually increased to 10 µm min−1 in the basal region. In comparison to the data cited here, the speed of signal transfer involved in chloroplast accumulation was 30–120 times slower than the speed of the actomyosin system or the microtubule-kinesin/dynein system, but it is similar to the moving speed of a protein complex on a microtubule23 and oil droplets in a protonemal cell.24
Polymerization rates of cytoskeletal proteins have been measured using in vitro systems. For instance, the plus end of microtubules from bovine brains grew at 1.04–1.88 µm min−1.25,26 Polymerization rate of actin filaments from rabbit muscle was about 0.13–0.49 µm min−1 and depended on the G-actin concentration.27 Live BHK21 fibroblasts, mouse melanoma cells and Dictyostelium amoebae expressing GFP-actin fusion proteins move on glass by using three-dimensional F-actin bands. These structures propagate throughout the cytoplasm at rates ranging between 2–5 µm min−1 in each cell type and produce lamellipodia or pseudopodia at the cell boundary.28 The extending speed of these cytoskeletons is roughly equal to the speed of signal transfer for the chloroplast accumulation response. We therefore aim to measure the speed of extension of these filaments when a method of gene transformation has been established for A. capillus-veneris.
This work was partly supported by the Japanese Ministry of Education, Sports, Science, and Technology (MEXT 13139203, 17084006 to M.W.), the Japan Society of Promotion of Science (JSPS 13304061, 16107002, 20227001 to M.W.), and a Research Fellowship for Young Scientists (to H.T.).
Previously published online: www.landesbioscience.com/journals/psb/article/11338