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Chloroplast actin filaments organize meshwork on the photorelocated chloroplasts in the moss Physcomitrella patens

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Abstract

Cytoskeleton dynamics during phototropin-dependent chloroplast photorelocation movement was analyzed in protonemal cells of actin- and microtubule-visualized lines of Physcomitrella patens expressing GFP- or tdTomato-talin and GFP-tubulin. Using newly developed epi- and trans-microbeam irradiation systems that permit fluorescence observation of the cell under blue microbeam irradiation inducing chloroplast relocation, it was revealed that meshwork of actin filaments formed at the chloroplast-accumulating area both in the avoidance and accumulation movements. The structure disappeared soon when blue microbeam was turned off, and it was not induced under red microbeam irradiation that did not evoke chloroplast relocation movement. In contrast, no apparent change in microtubule organization was detected during the movements. The actin meshwork was composed of short actin filaments distinct from the cytoplasmic long actin cables and was present between the chloroplasts and plasma membrane. The short actin filaments emerged from around the chloroplast periphery towards the center of chloroplast. Showing highly dynamic behavior, the chloroplast actin filaments (cp-actin filaments) were rapidly organized into meshwork on the chloroplast surface facing plasma membrane. The actin filament configuration on a chloroplast led to the formation of actin meshwork area in the cell as the chloroplasts arrived at and occupied the area. After establishment of the meshwork, cp-actin filaments were still highly dynamic, showing appearance, disappearance, severing and bundling of filaments. These results indicate that the cp-actin filaments have significant roles in the chloroplast movement and positioning in the cell.

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Abbreviations

BDM:

2,3-Butanedion monoxime

cp-actin filament:

Chloroplast actin filament

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Acknowledgments

This work was supported by the Grant-in-Aid for Scientific Research (19039027, 19570045 and 22570047 to AK; 13139203, 13304061, 16107002 and 20227001 to MW; 20570041 to TK) from the Ministry of Education, Sports, Science and Technology of Japan. Initial studies of this work were done by Ms. M. Nagai and Ms. T. Ashizawa (Tokyo Metropolitan University). We thank Dr. M. Hasebe (NIBB) for the gift of several vector constructs for P. patens transformation, Dr. R. Y. Tsien (UCSD) for the gift of tdTomato cDNA and Mr. N. Yamada (Tokyo Metropolitan University) for the illustration of microbeam irradiation systems.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Correspondence to Akeo Kadota.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Appearance and disappearance of cp-actin filaments on the chloroplast. These movies show the actin meshwork formation on the chloroplasts under microbeam irradiation with strong blue light (Movie S4) and the disappearance of the meshwork in the dark (Movie S5). Cell was treated with both 0.25 mM oryzalin and 25 mM BDM for 2 h and then irradiated with a strong blue light microbeam (20 μm in width, 175 W m−2). An area next to the microbeam (shown in Fig. 7a) was observed. Several images of these movies are presented in Fig. 7b, c (MOV 2975 kb)

See caption of Supplementary Movie S4 (MOV 1009 kb)

Actin dynamics in the meshwork. These movies show the dynamics of actin meshwork. Protonemal cell was irradiated with blue light microbeam (175 W m−2) for 60 min and the dynamics of the actin meshwork formed next to microbeam area (shown in Fig. 8a) were observed every 5 s under microbeam irradiation. Several images of these movies are presented in Fig. 8b (MOV 256 kb)

See caption of Supplementary Movie S6 (MOV 214 kb)

Fig. S1

Scheme of epi-microbeam irradiation system. In addition to the optical path for ordinal transmission observation, the epi-microbeam irradiation system is equipped with two optical paths, one for epifluorescence microscopy and the other for microbeam irradiation with the stimulus light. The system permitted time-lapse recording of fluorescence image at defined intervals in epi-fluorescence mode, as well as continuous microbeam irradiation of cells with defined wavelengths and intensities in epi-microbeam mode excluding the period of fluorescence image acquisition. Switching between the two modes was performed by turning a mirror connecting the two optical systems (PPT 52 kb)

Fig. S2

Scheme of trans-microbeam irradiation system. The trans-microbeam irradiation system is equipped withtwo optical paths, one for epifluorescence microscopy and the other for ordinal transmission microscopy. For trans-microbeam irradiation, slit and interference filter was placed in the latter light path to illuminate the part of the cell with defined wavelengths and intensities. Microbeam irradiation with the stimulus light was performed continuously, being independent from fluorescence image acquisition (PPT 36 kb)

Movie S1

Actin meshwork locates between plasma membrane and chloroplasts. These movies show the Z-scanned fluorescence images of the cell irradiated with a microbeam (10 μm in width) of weak blue light (2.4 W m−2) for 1 h (Movie S1) and with that of strong blue light (96 W m−2) for 1 h thereafter (Movie S2). Note the actin meshwork is seen only on the plasma membrane side of chloroplasts. Parts of these movies were presented in Fig. 1b (MOV 1619 kb)

Movie S2

See caption of Supplementary Movie S1 (MOV 1962 kb)

Movie S3

Reorganization of actin filaments and microtubules during blue light-induced accumulation and avoidance movements of chloroplasts. This movie shows the reorganization of actin filaments and microtubules during chloroplast photomovement. Protonemal cell was irradiated with a microbeam of weak blue light (1 W m−2) for 50 min and then of strong blue light (100 W m−2) for 65 min. Area of microbeam irradiation is indicated with blue lines at the beginning of each treatment. Note the actin meshwork appears on the chloroplasts at microbeam area under weak light irradiation and at both sides of microbeam under strong light irradiation. Selected images of this movie are presented in Fig. 2 (MOV 4599 kb)

Movie S4

Appearance and disappearance of cp-actin filaments on the chloroplast. These movies show the actin meshwork formation on the chloroplasts under microbeam irradiation with strong blue light (Movie S4) and the disappearance of the meshwork in the dark (Movie S5). Cell was treated with both 0.25 mM oryzalin and 25 mM BDM for 2 h and then irradiated with a strong blue light microbeam (20 μm in width, 175 W m−2). An area next to the microbeam (shown in Fig. 7a) was observed. Several images of these movies are presented in Fig. 7b, c (MOV 2975 kb)

Movie S5

See caption of Supplementary Movie S4 (MOV 1009 kb)

Movie S6

Actin dynamics in the meshwork. These movies show the dynamics of actin meshwork. Protonemal cell was irradiated with blue light microbeam (175 W m−2) for 60 min and the dynamics of the actin meshwork formed next to microbeam area (shown in Fig. 8a) were observed every 5 s under microbeam irradiation. Several images of these movies are presented in Fig. 8b (MOV 256 kb)

Movie S7

See caption of Supplementary Movie S6 (MOV 214 kb)

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Yamashita, H., Sato, Y., Kanegae, T. et al. Chloroplast actin filaments organize meshwork on the photorelocated chloroplasts in the moss Physcomitrella patens . Planta 233, 357–368 (2011) doi:10.1007/s00425-010-1299-2

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Keywords

  • Actin filament
  • Chloroplast photorelocation
  • Cp-actin filament
  • Microtubule
  • Photomovement
  • Phototropin