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Two Kinesin-Like Proteins Mediate Actin-Based Chloroplast Movement in Arabidopsis Thaliana

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Two Kinesin-Like Proteins Mediate Actin-Based Chloroplast Movement in Arabidopsis Thaliana Two kinesin-like proteins mediate actin-based chloroplast movement in Arabidopsis thaliana Noriyuki Suetsugua, Noboru Yamadab, Takatoshi Kagawac,1, Hisashi Yonekurad, Taro Q. P. Uyedad, Akeo Kadotab, and Masamitsu Wadaa,b,2 aDepartment of Biology, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan; bDepartment of Biological Sciences, Graduate School of Science and Engineering, Tokyo Metropolitan University, Tokyo 192-0397, Japan; cGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan; and dResearch Institute for Cell Engineering, National Institute for Advanced Industrial Science and Technology (AIST), Tsukuba 305-8562, Japan Edited by Peter H. Quail, University of California, Albany, CA, and approved March 31, 2010 (received for review November 6, 2009) Organelle movement is essential for efficient cellular function in other components involved in cp-actin filament regulation during eukaryotes. Chloroplast photorelocation movement is important chloroplast movement, we screened mutants deficient in chlo- for plant survival as well as for efficient photosynthesis. Chloro- roplast photorelocation movement using a previously developed plast movement generally is actin dependent and mediated by blue band assay (4, 8, 11). Here, we characterized a kinesin-like protein light receptor phototropins. In Arabidopsis thaliana, phototropins for actin-based chloroplast movement 1 (kac1)mutant. mediate chloroplast movement by regulating short actin filaments on chloroplasts (cp-actin filaments), and the chloroplast outer Results envelope protein CHUP1 is necessary for cp-actin filament accumu- Positional Cloning of KAC1 Gene and Characterization of kac Mutants. lation. However, other factors involved in cp-actin filament regula- Two independent kac1 alleles, kac1-1 and kac1-2, were isolated tion during chloroplast movement remain to be determined. Here, (Fig. 1A). A map-based approach revealed that the KAC1 gene is we report that two kinesin-like proteins, KAC1 and KAC2, are At5g10470 (Fig. S1 A and B). Analyses of a transfer DNA (T-DNA) essential for chloroplasts to move and anchor to the plasma mem- insertion line of KAC1 (kac1-3)(Figs. S1B and S2 A and B)and brane. A kac1 mutant showed severely impaired chloroplast accu- genetic complementation of kac1-1 with an At5g10470 genomic mulation and slow avoidance movement. A kac1kac2 double construct confirmed that KAC1 is At5g10470. Moreover, Arabi- mutant completely lacked chloroplast photorelocation movement dopsis thaliana has one gene that is highly similar to KAC1; this and showed detachment of chloroplasts from the plasma mem- gene is named “KAC2” (At5g65460) and is located on the lower brane. KAC motor domains are similar to those of the kinesin-14 arm of chromosome 5 (Fig. S1B). Note that KAC1 was identified subfamily (such as Ncd and Kar3) but do not have detectable mi- previously as a protein interacting with various proteins implicated crotubule-binding activity. The C-terminal domain of KAC1 could in cell-cycle and microtubule function: CDKA1-interacting protein interact with F-actin in vitro. Instead of regulating microtubules, [designated “KLP2/KCA1” (12, 13)], the geminivirus protein AL1- KAC proteins mediate chloroplast movement via cp-actin filaments. interacting protein [designated “GRIMP” (14)], and the katanin- We conclude that plants have evolved a unique mechanism to reg- interacting protein [designated “KSN1” (15)]. In this paper we ulate actin-based organelle movement using kinesin-like proteins. refer to KLP2/KCA1/GRIMP/KSN1 as “KAC1” and KCA2 as “KAC2.” Although we cannot exclude completely the possibility cp-actin | blue light | organelle movement | phototropin that KAC proteins regulate cell-cycle or microtubule-dependent cell growth, under our growth conditions we could not detect ob- rganelle movement is pivotal for efficient cellular functions in vious growth or developmental defects even in the kac1kac2 Oplants as well as in animals and fungi (1). Chloroplast pho- double mutant. Moreover, observations of microtubule dynamics torelocation movement is essential for the adaptation of plants using GFP-TUA6 showed that no gross or obvious defect could be under the fluctuating natural light conditions (2). Chloroplasts observed without a more quantitative analysis (Movie S1 and accumulate toward light under low-light conditions (accumulation Movie S2). However, we cannot exclude the possibility that these response) to maximize light capture for photosynthesis, and they KAC1 interactive proteins are involved in chloroplast movement. escape from intense light (avoidance response) to avoid chloro- To investigate the function of both KAC1 and KAC2 in chlo- plast photodamage (2, 3). These responses are mediated by blue roplast photorelocation movement, single and double mutants light receptor phototropins in green algae, mosses, and ferns as (Figs. S1B and S2A) were examined in detail. If not specified well as in seed plants (2). Arabidopsis thaliana expresses two otherwise, kac1-1 and kac2-1 were used in all experiments as phototropins (4–6); phot1 and phot2 are required for the accu- representative lines for kac1 and kac2, respectively, because sim- mulation response whereas phot2 alone is essential for the ilar results were obtained using other kac1 or kac2 alleles. When avoidance response. dark-adapted wild-type and kac mutant plants were irradiated fl fl Like other plant organelle movements, chloroplast photo- with a low- uence (LL) or high- uence (HL) white light for 2 h, relocation movement generally is dependent on actin filaments typical chloroplast photorelocation movement was observed in rather than on microtubules (2). Our recent study suggested that mesophyll cells of both the wild type and kac2. Chloroplasts chloroplast photomovement and anchorage to the plasma membranes are mediated by short actin filaments on chloroplasts (cp-actin filaments); formation of cp-actin filaments is associated Author contributions: N.S., N.Y., T.K., H.Y., T.Q.P.U., A.K. and M.W. designed research; N.S., N.Y., T.K., H.Y., T.Q.P.U., and A.K. performed research; N.S., N.Y., T.K., H.Y., T.Q.P.U., with phototropin-mediated chloroplast movement (7). The A.K., and M.W. analyzed data; and N.S. wrote the paper. chloroplast outer membrane protein, chloroplast unusual posi- The authors declare no conflict of interest. tioning 1 (CHUP1), which can bind to both F-actin and G-actin This article is a PNAS Direct Submission. – fi in vitro (8 10), is implicated in cp-actin lament formation (7). 1Present address: National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305- The correlation of cp-actin signal with the velocity of chloroplast 8602, Japan. fi 2 movement suggests that cp-actin laments play a role in gener- To whom correspondence should be addressed. E-mail: [email protected]. ating the motive force (7), but it remains to be determined if cp- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. actin filaments actually provide the motive force. To identify 1073/pnas.0912773107/-/DCSupplemental. 8860–8865 | PNAS | May 11, 2010 | vol. 107 | no. 19 www.pnas.org/cgi/doi/10.1073/pnas.0912773107 Downloaded by guest on September 27, 2021 A B area (Movie S3), but kac1kac2 chloroplasts did not (Movie S4), WT kac1-1 kac2-1 kac1-1kac2-1 indicating that kac1kac2 is deficient in the avoidance response WT kac1-1 as well as the accumulation response. GBA D KAC1 and KAC2 Genes Are Functionally Equivalent but Have Different Expression Levels. WBA When the velocity of chloroplast avoidance and LL accumulation were measured and plotted as the function of the fluence rate of the irradiated microbeam, as described previously C (16) (see legend of Fig. 1C for details), the velocity in the wild- type chloroplasts increased proportionally to the fluence rate; 1 HL 3 the relationship was clear for the avoidance movement and slight 2 20 µm for the accumulation movement (Fig. 1D). However, in the kac1 D mutant, the chloroplast accumulation response was hardly de- avoid accumulate R 1.5 0 tected (Fig. 1D and Movie S5). Interestingly, the velocity of the D B D WT kac1 avoidance movement was the same as that of the wild type in 20 1 kac2 -0.5 response to the lower fluence rates of the strong blue light (11, 22, − − − − and 49 μmol m 2 s 1) but saturated at around 49 μmol m 2 s 1. 0.5 -1 WT Hence, the velocity of avoidance in wild-type chloroplasts was 10 kac1 2 avoid fl 3 kac2 twice or more than that in kac1 at the higher uence rates (188 1 0 -1.5 −2 −1 accumulate 1030 100 300 10 10030300 and 376 μmol m s ), indicating that kac1 shows partial im- 0 0306090 pairment of the avoidance response. The kac2 mutant plants Time (min) showed almost normal chloroplast photorelocation movement (Fig. 1D). RT-PCR analyses revealed that both KAC1 and KAC2 Fig. 1. kac mutants are deficient in chloroplast photorelocation movement. transcripts were expressed in various tissues (Fig. S2C). However, (A) Green (GBA) and white band assay (WBA) for detection of chloroplast the microarray data showed that KAC1 was more highly expressed accumulation and avoidance responses, respectively. The irradiated area is than KAC2, especially in chloroplast-containing tissues. The KAC indicated on both sides of the leaves by black bars (ca. 1 mm). (B) Charac- terization of chloroplast photorelocation movement in mesophyll cells of protein level was examined in wild-type, kac1, kac2, and kac1kac2 kac mutants. Plants that were dark-adapted for about 12 h (D) were irra- using KAC antiserum (which can detect KAC1 and KAC2 equally − − diated with white light at 10 (LL) or 100 μmole m 2 s 1 (HL) for 2 h. All in immunoblotting after SDS/PAGE) (Fig. S3 A and B). A band of micrographs were taken at the focal plane of the anticlinal wall closest to approximately 150 kDa, corresponding to the size of full-length the epidermis.
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