1358 Plant Physiology®, November 2018, Vol. 178, pp. 1358–1369, www.plantphysiol.org © 2018 American Society of Plant Biologists. All Rights Reserved.

CM

Plant photosynthetic performance is affected by var-ious environmental factors, including light, tempera-ture, and CO2 concentration (Kaiser et al., 2015; Yamori, 2016). In particular, light is not only an essential energy source for photosynthesis but also is a key factor in the

coordination of plant growth and development that helps to maximize photosynthetic performance (Christie, 2007; Kami et al., 2010).

The blue light (BL) receptor, phototropin (phot), mediates various BL responses that facilitate photosyn-thesis; these include phototropism, chloroplast move-ment, stomatal opening, and leaf flattening (Christie, 2007). Light-induced chloroplast movement is one of the most important responses for the utilization of photosynthetic light (Suetsugu and Wada, 2013). Chlo-roplasts move toward weak light-irradiated areas to efficiently absorb light (the accumulation response), whereas they move away from excess light to avoid photodamage (the avoidance response; Suetsugu and Wada, 2013). In Arabidopsis (Arabidopsis thaliana), the accumulation response is regulated by phot1 and phot2 (Sakai et al., 2001), whereas the avoidance response is regulated mainly by phot2 (Jarillo et al., 2001; Kagawa et al., 2001).

The importance of phototropin-mediated responses in photosynthetic performance and plant growth has been examined using mutant Arabidopsis plants defective in phototropin-mediated responses. Under a condition in which weak BL is superimposed on red light, phot1 mutant plants exhibit reduced photosyn-thetic performance and growth because of attenuated chloroplast accumulation, weak stomatal opening, and curled leaves (Takemiya et al., 2005; Inoue et al., 2008). The nonphototropic hypocotyl3 (nph3) mutants also are

Chloroplast Accumulation Response Enhances Leaf Photosynthesis and Plant Biomass Production1

Eiji Gotoh,a,2,3,4 Noriyuki Suetsugu,b,c,2 Wataru Yamori,d Kazuhiro Ishishita,a Ryota Kiyabu,a Masako Fukuda,a Takeshi Higa,e,5 Bungo Shirouchi,a and Masamitsu Wadae

aFaculty of Agriculture, Kyushu University, Fukuoka 812-8581, JapanbInstitute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United KingdomcGraduate School of Biostudies, Kyoto University, Kyoto 606-8502, JapandGraduate School of Science, University of Tokyo, Tokyo 113-0033, JapaneGraduate School of Science and Engineering, Tokyo Metropolitan University, Tokyo 192-0397, JapanORCID IDs: 0000‑0002‑8952‑987X (E.G.); 0000‑0002‑3328‑3313 (N.S.); 0000‑0001‑7215‑4736 (W.Y.); 0000‑0002‑3196‑2160 (T.H.); 0000‑0001‑6672‑7411 (M.W.)

Under high light intensity, chloroplasts avoid absorbing excess light by moving to anticlinal cell walls (avoidance response), but under low light intensity, chloroplasts accumulate along periclinal cell walls (accumulation response). In most plant species, these responses are induced by blue light and are mediated by the blue light photoreceptor, phototropin, which also regu-lates phototropism, leaf flattening, and stomatal opening. These phototropin-mediated responses could enhance photosynthesis and biomass production. Here, using various Arabidopsis (Arabidopsis thaliana) mutants deficient in chloroplast movement, we demonstrated that the accumulation response enhances leaf photosynthesis and plant biomass production. Conspicuously, phototropin2 mutant plants specifically defective in the avoidance response but not in other phototropin-mediated responses displayed a constitutive accumulation response irrespective of light intensities, enhanced leaf photosynthesis, and increased plant biomass production. Therefore, our findings provide clear experimental evidence of the importance of the chloroplast accumulation response in leaf photosynthesis and biomass production.

1This work was supported in part by the Grant-in-Aid for Sci-entific Research Grants (15K18713 and 18K14491 to E.G.; 26840097 and 15KK0254 to N.S.; 16H06552 and 18H02185 to W.Y.; 20227001, 23120523, 25120721, and 25251033 to M.W.) from the Japan Society for the Promotion of Science, Grant-in-Aid for JSPS Research Fellow (17J06717 to T.H.), and the Research Grant for Young Investigators of the Faculty of Agriculture, Kyushu University (to E.G.).

2These authors contributed equally to the article.3Author for contact: eiji.gotoh@agr.kyushu-u.ac.jp.4Senior author.5Current address: Institute for Protein Research, Osaka University,

Osaka 565-0871, Japan.The author responsible for distribution of materials integral to

the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Eiji Gotoh (eiji.gotoh@agr.kyushu-u.ac.jp).

E.G. and N.S. conceived and designed the research; E.G. per-formed most of the experiments; N.S. generated all mutants; E.G., N.S., W.Y., and M.W. analyzed the data; W.Y. analyzed photoinhi-bition, K.I., T.H., and M.F. contributed to microscopy analysis; R.K. contributed to analysis of plant biomass production; B.S. contributed to statistical analysis; E.G. and N.S. wrote the article; all authors read and edited the article.

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Plant Physiol. Vol. 178, 2018 1359

impaired in photosynthesis and plant growth under the same weak BL condition (Inoue et al., 2008). Be-cause nph3 is defective in leaf flattening but is normal in chloroplast accumulation and stomatal opening, leaf flattening should have a greater contribution in the photosynthetic performance and plant growth than in other phototropin-mediated responses, at least un-der weak BL conditions (Inoue et al., 2008). However, chloroplast accumulation and stomatal opening still could contribute to photosynthesis and plant growth, because phot1 phot2 exhibits more severe growth de-fects than phot1 and nph3 (Takemiya et al., 2005; Inoue et al., 2008). Nevertheless, blue light signaling1 mutants, which specifically lack BL-induced stomatal opening, exhibit slight defects in photosynthesis only under CO2-limiting conditions (Takemiya et al., 2013). Previ-ous studies have shown that the chloroplast avoidance response is essential for escaping photodamage and, thus, for the survival of plants under strong light con-ditions (Kasahara et al., 2002; Sztatelman et al., 2010; Davis and Hangarter, 2012). It has been suggested for many years that the accumulation response might be required for efficient light capture under weak light conditions (Senn, 1908; Zurzycki, 1955; Brugnoli and Björkman, 1992). However, the importance of the chlo-roplast accumulation response remains unclear.

Here, we found that the chloroplast accumulation response is essential for efficient photosynthetic per-formance and growth in Arabidopsis.

RESULTS

The Chloroplast Accumulation Response Ensures Efficient Light Capture But Is Apparent Only under Very Weak Light

To examine the effect of the chloroplast accumu-lation response on the absorption of light by leaves, we compared the absorption in wild-type and phot1 phot2 leaves irradiated with weak or strong BL. The phot1 phot2 mutants lack both the accumulation and avoidance responses; thus, their chloroplasts are unre-sponsive to BL with regard to chloroplast movement (Sakai et al., 2001). The absorption of light by wild- type leaves exposed to weak BL, where the chloro-plast accumulation response was induced, was much higher than the absorption of strong BL, where the avoidance response was induced (Supplemental Fig. S1, A and C). In accordance with the light absorp-tion spectra of photosynthetic pigments in plants, the wavelength bands near the red and blue regions were highly absorbed in the weak BL-exposed leaves (Fig. 1A). In contrast, no significant difference in leaf absorption was detected between the weak and strong BL-induced leaves of phot1 phot2 (Fig. 1A; Supplemen-tal Fig. S1, B and D). Consistent with previous studies (Inoue and Shibata, 1973; Kasahara et al., 2002; Davis et al., 2011; Davis and Hangarter, 2012), these results

indicated that the chloroplast accumulation response enhanced the utilization of photosynthetically active radiation (350–750 nm).

We then assessed the relationship between chlo-roplast movement and photosynthetic activity at var-ious intensities of white light. In the wild type, the CO2 assimilation rate increased largely up to a light intensity of approximately 200 μmol m−2 s−1, and the rate of assimilation saturated at a light intensity of 400 μmol m−2 s−1 (Fig. 1B). The chloroplast accumula-tion response was induced at 10 μmol m−2 s−1 and was most evident at 20 μmol m−2 s−1, which is near the com-pensation point of the light response curve of pho-tosynthesis (Fig. 1, B, inset, and C). Unexpectedly, at light intensities above 20 μmol m−2 s−1, the chloroplast accumulation response was rapidly attenuated and the chloroplast avoidance response was induced instead (Fig. 1, B and C). When exposed to 160 μmol m−2 s−1 light, almost all the chloroplasts moved to the side wall as a result of the chloroplast avoidance response (Fig. 1C). Thus, the accumulation response was induced only under very weak light conditions even though photosynthetic activity increases under stronger light (up to approximately 400 μmol m−2 s−1). In other words, the avoidance response occurs under relatively weak light (of about 40 μmol m−2 s−1) conditions even though photodamage is not apparent at these light intensities (Kasahara et al., 2002).

Although we demonstrated previously that the chlo-roplast avoidance response is essential for survival under continuous excess light conditions (Kasahara et al., 2002), natural light conditions frequently fluc-tuate because of sunflecks within the canopy or be-cause of the presence of clouds; thus, plants often are exposed to unanticipated strong light. Recent studies reveal that fluctuating light could induce much stron-ger photoinhibition than constant light illumination (Külheim et al., 2002; Suorsa et al., 2012). Thus, the avoidance response under relatively weak light (Fig. 1, B and C) should be induced to escape from the unan-ticipated fluctuating light-induced photoinhibition at the cost of light capture for photosynthesis. Therefore, we examined fluctuating light-induced photoinhibi-tion in the wild type and phot2 mutant lines defective in the chloroplast avoidance response (Kagawa et al., 2001). Under the conditions of our experiment, the extent of photoinhibition of PSII and PSI by exposure to fluctuating light was much stronger than that under constant high-light treatment (Fig. 2; Supplemental Fig. S2). When exposed to fluctuating light (15 or 40 µmol m−2 s−1 for 5 min and 500 µmol m−2 s−1 for 2 min) for 2 h, the wild-type plants showed the chloroplast avoidance response but phot2 mutant plants exhibit-ed a constitutive chloroplast accumulation response even after exposure to fluctuating light (Fig. 2, D and E, for 15 µmol m−2 s−1; Supplemental Fig. S2, D and E, for 40 µmol m−2 s−1). Although photoinhibition of PSII and PSI was observed in all plant lines after both fluc-tuating light treatments, phot2 plants showed much stronger photoinhibition than wild-type plants (Fig. 2,

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A–C; Supplemental Fig. S2, A–C), indicating that the constitutive accumulation response in the phot2 plants is detrimental to the photosynthetic apparatus under fluctuating light conditions, similar to that under the con-stant light conditions (Kasahara et al., 2002; Sztatelman et al., 2010; Davis and Hangarter, 2012). These results suggest that, when a weak light environment is inter-rupted frequently by strong light, photoinhibition in the chloroplasts could occur easily if chloroplasts are positioned on the cell surface. This might be why the chloroplast accumulation response occurs only partially even under relatively weak light (Fig. 1, B and C).

The Chloroplast Accumulation Response Enhances Photosynthesis in Leaves

In addition to the role in avoiding photodamage, the avoidance response induced in the uppermost palisade layer could facilitate light penetration into deeper cell layers, thereby increasing total photosynthetic activity in the leaves (Vogelmann, 1993; Terashima and Hikosaka, 1995; Evans et al., 2009; Kume, 2017). However, the contribution of light penetration to deeper cells might

be minimal, because the avoidance response only occurred partially below a white light intensity of 160 μmol m−2 s−1 (Fig. 1C). Thus, we hypothesized that, under these moderate light (ML) conditions (neither weak nor strong light intensity), wild-type plants could not efficiently use the light because the accumu-lation response was not complete and the avoidance response had already been induced. To test the impor-tance of the accumulation response, wild-type and mutant plants defective in light-induced movements and showing different chloroplast distribution patterns were grown at ML intensity (120 μmol m−2 s−1; Fig. 1B, orange narrow line). Under ML conditions, the accu-mulation response was not complete in the wild-type plants; thus, the chloroplasts in the palisade cell layer were only partially located on the cell surface (Fig. 3A). The chloroplasts in the J-domain protein required for chloroplast accumulation response1 (jac1) mutant were constitutively located on the side wall of palisade cells because of the defect in the accumulation response (Suetsugu et al., 2005; Fig. 3A; Supplemental Fig. S3). In the leaves of the chloroplast unusual positioning1 (chup1)

Figure 1. The chloroplast accumulation response enhances photosynthetic light utilization. A, Difference spectra of light ab-sorption between weak and strong BL-irradiated leaves. The leaves of wild-type (WT) and phot1 phot2 plants were exposed to weak (3 μmol m−2 s−1) or strong (50 μmol m−2 s−1) BL for 3 h. The difference in the absorption was calculated as the absorption of light by the strong BL-irradiated leaf subtracted from that of the weak BL-irradiated leaf. Data show means ± se (n = 9). B, Light-response curve of CO2 assimilation rate and chloroplast distribution in wild-type plants. The area covered with chloro-plasts (chloroplast area) was determined from the images of mesophyll cell surfaces. The inset shows the magnification of the compensation point of the light response curve of photosynthesis. Data show means ± se (n = 3). Green, orange, and gray nar-row lines indicate the light intensities of 50, 120, and 300 μmol m−2 s−1, respectively. C, Chloroplast distribution in wild-type plants irradiated with various intensities of white light. Photographs show the upper surface of palisade tissue cells (top row) and the cross sections (bottom row). Before the light exposure, plants were dark adapted for 14 h (dark sample). The plants were irradiated with various intensities of white light for 3 h. The chloroplasts are indicated by chlorophyll autofluorescence (red). Bars = 20 μm.

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mutant (Oikawa et al., 2003), most of the chloroplasts were not localized on the upper cell surface but aggre-gated at the bottom of the cell (Fig. 3A; Supplemental Fig. S3). Conversely, chloroplasts in the phot2 mutants were constitutively located on the cell surface because these mutants lack the avoidance response (Kagawa et al., 2001; Fig. 3A). The distribution pattern of chlo-roplasts in the phot2 jac1 double mutant was interme-diate between that in the phot2 and jac1 mutants and was similar to that in the wild-type plants because the phot2 jac1 mutant was defective in both the accumula-tion and avoidance responses (Suetsugu et al., 2005). The area covered with chloroplasts per whole cell sur-face was similar in the wild-type and phot2 jac1 plants (Fig. 3B). The chloroplast area in the phot2 plants was approximately 2-fold larger, whereas that in the jac1 plants was much smaller compared with the chloro-plast area in the wild type (Fig. 3B). Unexpectedly, the chloroplasts in the phot2 chup1 plants also aggregated, but more chloroplasts were situated on the upper cell

surface compared with that in the chup1 plants (Fig. 3, A and B; red arrowheads in Supplemental Fig. S3).

The amount of light absorbed was related to the chloroplast distribution patterns (Fig. 3C). The amount of light absorbed in the leaves was higher in the phot2 plants and lower in the jac1 and chup1 plants compared with the wild-type plants, as described previously (Davis et al., 2011; Davis and Hangarter, 2012). The absorption of light in the leaves of the phot2 jac1 and phot2 chup1 mutants was intermediate between that in the phot2 and jac1 or chup1 mutants. However, the absorption in phot2 jac1 was slightly lower than that in the wild-type plants (Fig. 3C), although the area covered with chloroplasts on the upper surface of palisade cells was similar in wild-type and phot2 jac1 plants (Fig. 3B). Observation of the cross sections of wild-type and mutant leaves showed some space free of chloroplasts on the periclinal walls of sponge cells in phot2 jac1 plants (red arrows in Supplemental Fig. S3) but not in wild-type and phot2 plants (Supplemen-tal Fig. S3), which explains the lower absorption in the

Figure 2. Photoinhibition in wild-type (WT) and various mutant plants. All plants were grown for 4 weeks under 14-h-daylight (120 μmol m−2 s−1) conditions and exposed to constant high light (HL; 500 μmol m−2 s−1) for 2 h or to fluctuating light (alternating between HL at 500 μmol m−2 s−1 for 2 min and low light [LL] at 15 μmol m−2 s−1 for 5 min) for 2 h. A, Photoinhibition before (left) and after (right) treatment with fluctuating light. The maximum quantum yield of PSII (Fv/Fm), which is an indicator of functional PSII reaction centers, was measured before and after treatment with fluctuating light. B and C, Degree of photoinhibition of PSII and PSI after treatment with constant high-intensity or fluctuating light. B, Degree of photoinhibition in PSII. Fv/Fm was measured before and after the light treatment. C, Degree of photoinhibition in PSI. The maximum level of the P700 signal (full oxidation of P700), which is an indicator of the functional PSI reaction center, was measured before and after the light treatment. The degree of photoinhibition of PSII and PSI is relative to the initial Fv/Fm and maximum P700 values (%). Data show means ± se (n = 3). Statistical significance was evaluated by one-way ANOVA followed by the Tukey-Kramer multiple comparison test. Different letters indicate significant differences (P < 0.05). D and E, Chloroplast distribution patterns in wild-type and various mutant plants treated with fluctuating light. The chloroplast distribution pattern is shown before (D) and after (E) exposure to fluctuating light. Leaves were detached from plants exposed to constant or fluctuating light. Bars = 20 μm.

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phot2 jac1 leaves versus the wild-type leaves. The order of light absorption from the highest to the lowest was phot2 > wild type > phot2 jac1 ≥ phot2 chup1 > chup1 ≥ jac1. Thus, the phot2 mutation partially alleviated the defective absorption of light in the leaves of the jac1 and chup1 mutants by changing the chloroplast distri-bution pattern. Overall, there was a direct relationship between the chloroplast distribution patterns and the light absorbance through leaves.

To address the role of the chloroplast distribution patterns in leaf photosynthesis, we determined pho-tosynthetic CO2 fixation in the wild-type and mutant plants under ML conditions. Consistent with the chlo-roplast distribution patterns, the net CO2 assimilation rate was reduced significantly in the jac1 (2.1 ± 0.07) and chup1 (1.91 ± 0.04) plants and was increased (3.58 ± 0.05) in the phot2 plants compared with the wild-type plants (2.96 ± 0.05; Fig. 4A). In the phot2 jac1 and phot2 chup1 double mutants, the CO2 assimilation rate was intermediate (2.67 ± 0.03 and 2.25 ± 0.05, respectively) between that in the phot2 and jac1 or chup1 mutants, respectively, and was decreased slightly compared with that in the wild-type plants (Fig. 4A). This corre-sponds with the order of light absorption in the plant lines described above.

Phototropins affect leaf photosynthesis through sto-matal opening (Kinoshita et al., 2001), leaf flattening, leaf positioning (Inoue et al., 2008), and palisade cell development (Kozuka et al., 2011). However, under the ML conditions used in this study, stomatal conduc-tance, an indicator of stomatal aperture, was similar in the wild-type and mutant plants (Fig. 4B). Moreover, leaf flattening and palisade cell development were normal in the mutants used in this study under ML conditions (Figs. 3A and 5A). Thus, the altered photo-synthesis in the phot2, jac1, and chup1 mutants should be attributable to the altered chloroplast distribution patterns. To confirm this, we further examined pos-sible defects in photosynthesis in the mutant lines. Chlorophyll content in all mutants examined was com-parable to that in the wild-type plants (Fig. 4C). The value of Fv/Fm, an indicator of PSII damage, was not significantly different in the different plants (Fig. 4D). Additionally, the amounts of proteins involved in the photosystems and that of the large subunit of Rubisco were similar in the wild type and the mutants (Sup-plemental Fig. S4). Thus, the photosynthetic activity of the chloroplasts in each mutant line should be normal and the same. We examined oxygen evolution from the isolated leaf protoplasts obtained from the wild-type

Figure 3. Chloroplast distribution patterns and light absorbance of wild-type (WT) and mutant plants under ML conditions. A, Views of the upper surface of palisade tissue cells (top row) and of the cross sections (bottom row) of wild-type and various mutant plants. Plants were grown under ML for 45 d. Leaves were detached 3 h after light onset of the final light/dark cycle. Bars = 20 μm. B, Area occupied by chloroplasts on the palisade cell surface of wild-type and mutant plants. Data show means ± se (n = 30 cells). Statistical significance was evaluated by one-way ANOVA followed by the Tukey-Kramer multiple comparison test. Different letters indicate significant differences (P < 0.05). C, Difference spectra of light absorption by the leaves of wild-type and mutant plants grown under ML conditions. The difference in the absorption over the range of wavelengths (350–750 nm) was calculated as the absorption of light by the leaves of the wild type subtracted from the light absorbed by the leaves of each mutant. Data show means ± se (n = 9) of three independent experiments.

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and mutant plants. The differences in the chloroplast distribution between the wild type and the mutant lines could be ignored in the protoplast preparation; thus, we could measure the photosynthetic activity at the cellular level. There was no significant difference in oxygen evolution among the different plants (Fig. 4E). These findings clearly indicate that the chloroplast accumulation response enhances photosynthesis in leaves through more efficient light utilization.

The Chloroplast Accumulation Response Increases Plant Biomass Production

Consistent with the phenotypes associated with photosynthetic performance under the ML condition, the jac1 plants were smaller and exhibited lesser pro-duction of plant biomass than the wild-type plants, as reflected in plant leaf area and dry and fresh weights (Fig. 5). Notably, the phot2 plants were larger than the wild-type plants and showed an increase in plant biomass (Fig. 5). The values of leaf area and the abo-veground weight in the phot2 plants increased more than 1.5-fold over the respective values in the wild-type plants (Fig. 5, B–D). The growth and biomass of the phot2 jac1 plants were intermediate between those of the phot2 and jac1 plants and were slightly lower than those of the wild-type plants (Fig. 5); these results were consistent with lower photosynthetic performance by the leaves of the phot2 jac1 plants compared with the absorption by the leaves of the wild-type plants (Figs. 3C, 4A, and 5, B–D). Consequently, the chup1 mutant plants were smaller than the wild-type plants, and this growth defect was alleviated in the phot2 chup1 plants, as was the case in the jac1 and phot2 jac1 plants (Fig. 5). When the plants were observed every other day from 4 d after germination, a small but statistically signifi-cant difference between the wild type and phot2, jac1, or chup1 mutant plants occurred 8 d after germination (Supplemental Fig. S5). Similar results were obtained from long day-grown plants, although the difference between wild-type and phot2 plants appeared 12 d after germination (Supplemental Fig. S6). Thus, the effect of chloroplast positioning in plant growth was detectable even during early seedling growth, and the cumulative effects were apparent over 2 weeks irre-spective of daylength (Supplemental Figs. S5 and S6).

The phot1 mutants are more defective in leaf flat-tening and stomatal opening than the phot2 mutants (Takemiya et al., 2005; Inoue et al., 2008), and previous studies have demonstrated that phot1 mutants exhibit less plant biomass production than wild-type plants, mainly because of the leaf-flattening defects (Takemiya et al., 2005; Inoue et al., 2008). However, under the ML conditions used in this study, the chloroplast distribu-tion pattern was similar between wild-type and phot1 plants, and the aboveground fresh weight and leaf area in the wild-type and phot1 plants were not significantly different (Supplemental Fig. S7). The order of biomass production among plant lines from high to low was phot2 > wild type ≠ phot1 > phot1 phot2. These results

Figure 4. The chloroplast accumulation response enhances leaf photo-synthesis. A and B, Net CO2 assimilation (A) and stomatal conductance (B) in the wild type (WT) and various mutant plants under white light at 120 μmol m−2 s−1. Prior to the measurements, plants grown for 4 to 5 weeks were kept in the dark for 5 h. Data show means ± se (n = 9) of three independent experiments. C, Chlorophyll content of rosette leaves in the wild type and each mutant plant. Chlorophyll content was determined in the leaves of plants grown for 5 weeks. Data show means ± se (n = 9) of three independent experiments. FW, Fresh weight. D, Fv/Fm of rosette leaves in the wild type and each mutant plant. Data show means ± se (n = 9) of three independent experiments. E, Oxygen evolution from isolated mesophyll protoplasts. The mesophyll proto-plasts were dark adapted for at least 3 h; oxygen evolution then was measured at 120 μmol m−2 s−1 white light. Data show means ± se (n = 9) of three independent experiments. Statistical significance in all graphs was evaluated by one-way ANOVA followed by the Tukey-Kramer multiple comparison test. Different letters indicate significant differ-ences (P < 0.05); NS indicates no significant difference.

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further confirmed that the chloroplast accumulation response in the palisade cells was essential for the increase in plant biomass under ML conditions.

The Chloroplast Accumulation Response Increases Plant Biomass Production across Various Light Intensities

To further confirm the positive role of the chloro-plast accumulation response in plant biomass pro-duction, plants were cultured under two additional conditions, namely LL and HL (50 and 300 μmol m−2 s−1, respectively; Fig. 1B, green and gray narrow lines, re-spectively). Under LL and HL conditions, the distri-bution patterns of chloroplasts were similar across all mutant plants and were similar to those in the plants under the ML condition (Fig. 6B; Supplemental Fig. S8, A and B). The difference in chloroplast distribution be-tween the wild-type and mutant plants under the LL conditions was the same as that under the ML condi-tion. However, under the HL condition, fewer chloro-plasts were located on the cell surface in the wild-type plants than under the LL and ML conditions as a re-sult of the avoidance response (Supplemental Fig. S8). Consequently, the area covered with chloroplasts per whole cell surface in the wild-type plants was similar to that in the jac1 plants but was less than that in the phot2 jac1 plants under the HL condition (Fig. 6B). All the plants showed increased biomass with the increas-ing intensity of light (Fig. 6, A and C). The biomass in the phot2 mutants showed an especially steep increase with light. The jac1 plants were smaller than the wild-

type and other mutant plants under the LL condition but were similar in size to the wild-type plants under the HL condition (Fig. 6, A and C). Notably, the phot2 jac1 plants were slightly larger than the wild-type plants under the HL condition (Fig. 6, A and C). Al-though biomass production in the jac1 plants under the HL condition was similar to that in the wild-type plants, the light-induced increase in plant biomass was severely attenuated in the chup1 mutant. In the phot2 chup1 mutant, biomass production was partially recovered under both the light conditions compared with that in the chup1 plants but was still much lower than that in the wild-type, phot2, and phot2 jac1 plants (Fig. 6, A and C). Unlike the chloroplasts in the phot2 and jac1 mutant leaves, those in the chup1 mutants were aggregated as a result of detachment from the plasma membrane (Oikawa et al., 2003, 2008; Fig. 3A; Supplemental Figs. S3 and S8). Thus, not only the dis-tribution pattern (periclinal or anticlinal) but also the proper attachment to the plasma membrane should be important in leaf photosynthesis and biomass produc-tion. The order of biomass production under the HL condition was phot2 ∼ phot2 jac1 > wild type ≥ jac1 > phot2 chup1 > chup1 (Fig. 6, A and C). Overall, the chlo-roplast-occupied area correlated strongly with plant biomass production under all examined light condi-tions (Fig. 6D). These results clearly indicate that the chloroplast accumulation response in the palisade cells is essential for increased plant biomass in a wide range of light intensities.

Figure 5. Growth and biomass production in wild-type (WT) and various mutant plants grown under ML conditions. A, Photographs of plants grown under ML conditions for 45 d. Representative wild-type and mutant plants are shown. Bar = 2 cm. B, Leaf area of wild-type and mutant plants grown under ML conditions for 45 d. Data show means ± se (n = 24) of eight independent experiments. C and D, Aboveground fresh weight (C) and aboveground dry weight (D) of plants grown under ML conditions for 45 d. Data show means ± se (n = 24) of eight independent ex-periments. Statistical significance in B to D was evaluated by one-way ANOVA followed by the Tukey-Kramer multiple comparison test. Different letters indicate significant dif-ferences (P < 0.05).

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DISCUSSION

Chloroplast Movement Regulates Tradeoffs between Biomass Production and Photoprotection under a Fluctuating Light Environment

In this study, we provide clear evidence that chlo-roplast movement is indispensable for plant growth and survival under fluctuating light conditions and that the chloroplast accumulation response greatly en-hances photosynthetic performance and plant biomass production under constant light conditions.

Nevertheless, the accumulation response was induced only under very LL conditions in wild-type plants (Fig. 1). The accumulation response, which occurred only around the photosynthetic compensation point, also was observed in the leaves of the shade plant

Tradescantia albiflora (Terashima and Hikosaka, 1995). Our findings indicate that the avoidance response can be induced under relatively weak light conditions. Considering that leaves consist of multiple mesophyll cell layers, the avoidance response induced in the uppermost palisade layer could allow light to pene-trate into the deeper cell layers under weak light con-ditions (Vogelmann, 1993; Terashima and Hikosaka, 1995; Evans et al., 2009; Kume, 2017). Moreover, the avoidance response could function as a guard against sudden exposure to strong light, such as sunflecks under the canopy. Indeed, the phot2 plants were severely dam-aged by strong light under both constant and fluctu-ating light conditions but experienced much stronger damage under the fluctuating light conditions than under the constant light conditions (Fig. 2). Therefore, our findings add chloroplast movement to a list of efficient photoprotective mechanisms under fluctuating

Figure 6. The chloroplast accumulation response enhances plant biomass production. A, Photographs of plants grown for 35 d under various light intensities. Plants were grown for 35 d under white light at 50, 120, and 300 μmol m−2 s−1. Bars = 2 cm. B, Area occupied by chloroplasts on the palisade cell surface of wild-type (WT) and mutant plants. The chloroplast area and whole cell area were measured using the surface image of mesophyll cells of each plant. Data show means ± se (n = 30 cells). C, Aboveground fresh weight of plants grown for 35 d under various light intensities. Data show means ± se (n = 24) of eight independent experiments. D, Correlation between the chloroplast-occupied area per whole cell and plant biomass. Values of the chloroplast-occupied area and the fresh weight were derived from B and C, respectively. Green, orange, and gray symbols indicate data from plants grown under white light at 50, 120, and 300 μmol m−2 s−1, respectively. The relationship coefficients of each growth light condition are indicated. Statistical significance in B and C was evaluated by one-way ANOVA followed by the Tukey-Kramer multiple comparison test. Different letters indicate significant differences (P < 0.05).

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light conditions in addition to mechanisms in the chloroplasts, including nonphotochemical quenching (NPQ) and cyclic electron transport (Külheim et al., 2002; Suorsa et al., 2012; Yamori and Shikanai, 2016). A previous study also showed that phot1 phot2 double mutants lacking light-induced chloroplast movement are susceptible to fluctuating light conditions (Dutta et al., 2015). However, even though light-induced chlo-roplast movement was not apparent, the jac1 plants resisted strong light and the chup1, phot2 jac1, and phot2 chup1 plants exhibited weaker photoinhibition com-pared with the phot2 plants under fluctuating light con-ditions (Fig. 2). Notably, the chup1 mutant plants were more susceptible to both continuous excess light and fluctuating light conditions than the wild-type plants, but the susceptibility was weaker than in phot2 mutant plants (Fig. 2; Kasahara et al., 2002). Although chlo-roplasts accumulated on the cell surface in the phot2 mutant plants, most chloroplasts were positioned on the cell bottom in chup1 mutant plants (Fig. 2; Supple-mental Fig. S3). These findings indicate that the consti-tutive accumulation of chloroplasts on the cell surface is harmful to plant photosynthesis and growth under the strong light conditions. Therefore, even though the greater accumulation response could be beneficial for plant biomass production, this trait in phot2 mutants has not been selected under natural conditions. Collectively, the movement and distribution should be tightly reg-ulated by light, and this tight regulation is essential for plant growth and survival under fluctuating light conditions. In other words, the balance between chlo-roplast accumulation and avoidance regulates the tradeoff between light harvesting and photoprotec-tion. Given that, under natural conditions, the chloro-plast distribution constantly changes (Williams et al., 2003), sophisticated monitoring of light conditions and the control of chloroplast movements by phototropins are essential for plant survival under the ever-changing natural light conditions.

Periclinal Positioning of Chloroplasts by the Chloroplast Accumulation Response Ensures Efficient Photosynthesis and Biomass Production in a Wide Range of Light Intensities

Our analysis with mutant plants clearly showed that the chloroplast accumulation response enhances leaf photosynthesis and biomass production under contin-uous white light conditions in a wide range of light intensities. Leaf photosynthesis and biomass produc-tion were enhanced prominently in the phot2 mutants (Figs. 4–6). The phot2 mutants exhibit normal phototro-pism, leaf flattening, and stomatal opening (Kinoshita et al., 2001; Sakai et al., 2001; Sakamoto and Briggs, 2002; Kozuka et al., 2011) but exhibited a stronger chloroplast accumulation response than the wild type because of the defective avoidance response (Fig. 3; Supplemental Figs. S3, S7, and S8; Jarillo et al., 2001; Kagawa et al., 2001). Thus, the constitutive chloroplast accumulation response may be a main contributor to

enhanced photosynthesis and biomass production in phot2 mutant plants.

Although phot2 mutants are susceptible to excess continuous light (Kasahara et al., 2002) and fluctuating light stress (this work), they exhibited enhanced pho-tosynthesis and biomass production even under con-tinuous HL conditions (Fig. 6). Besides the chloroplast avoidance response, there are various long-term pho-toacclimation responses to strong light stress, including gene expression (Li et al., 2009). Previously, Dutta et al. (2015) showed that phot1 phot2 double mutants are susceptible to fluctuating light conditions, particu-larly on the first day of exposure, but could acclimate to prolonged fluctuating light conditions, indicating that phototropins are not involved in long-term pho-toacclimation responses. Indeed, phot2 mutant plants showed only minor defects in the gene expression re-sponse induced by strong BL (Lehmann et al., 2011). Moreover, there were no differences in NPQ and the activities of scavenging enzymes, including catalase, ascorbate peroxidase, and superoxide dismutase, between the wild type and phot2 after HL treatment (500 μmol m−2 s−1 for NPQ measurement and 1,000 μmol m−2 s−1 for enzyme assays; Kasahara et al., 2002). Therefore, the phot2 mutant plants are normal in other photoacclimation responses. Furthermore, our HL condition (300 μmol m−2 s−1) is much less light than natural sunlight (500–2,000 μmol m−2 s−1; Külheim et al., 2002; Williams et al., 2003), which likely explains the enhanced biomass production under our HL condi-tions in phot2 mutant plants.

The jac1 and chup1 mutant plants defective in the chloroplast accumulation response displayed reduced photosynthesis and biomass production. In partic-ular, the jac1 mutant plants are defective specifically in the accumulation response (Suetsugu et al., 2005) and showed the reverse phenotype in photosynthesis and biomass production from the phot2 mutant plants, further verifying the positive role of the chloroplast accumulation response in photosynthesis and bio-mass production (Figs. 4–6). Furthermore, the phot2 mutation partially suppressed the reduced photo-synthesis and biomass production in jac1 and chup1 mutants by changing the chloroplast distribution. In phot2 jac1 and phot2 chup1, more chloroplasts occu-pied the upper cell surface of the palisade cells com-pared with jac1 and chup1, respectively (Fig. 3, A and B; Supplemental Fig. S3). The mechanism by which phot2 mutations rendered more periclinal positioning of chloroplasts might be different between jac1 and chup1. Chloroplast movement and positioning are reg-ulated by the specialized actin structure on the chloro-plasts, chloroplast-actin (cp-actin) filaments (Kadota et al., 2009; Kong et al., 2013). The cp-actin filaments can regulate all aspects of chloroplast movement and positioning, including the direction and speed of chloroplast movements and the anchoring of chloro-plasts to the plasma membrane (Kadota et al., 2009; Kong et al., 2013). Phot2 has a pivotal role in these regulations, particularly under strong light conditions

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(Kong et al., 2013). The jac1 mutant plants exhibit a stronger avoidance response than wild-type plants, even under weak light (Suetsugu et al., 2005), and show normal regulation of cp-actin filaments during the avoidance response (Ichikawa et al., 2011). Thus, the phot2 mutation should suppress the cp-actin fil-ament-mediated avoidance response in phot2 jac1 plants; therefore, phot2 jac1 mutants have more chlo-roplasts on the periclinal walls than jac1.

Conversely, it is difficult to explain why the phot2 mutation changed the chloroplast distribution pat-tern in phot2 chup1 mutant plants, because cp-actin filaments have never been detected in chup1 mutant plants (Kadota et al., 2009; Kong et al., 2013). Thus, it is unlikely that the difference in the chloroplast posi-tioning between chup1 and phot2 chup1 is attributable to the regulation of cp-actin filaments. One possibility is that phot2 might regulate cp-actin-independent chloroplast positioning in chup1 mutant plants. Indeed, the measurement of light-induced changes in leaf transmittance caused by chloroplast photorelocation revealed that chup1 mutant plants exhibited very slight but significant increases in the transmittance in response to BL and phot2 chup1 plants lacked this light-induced change (Supplemental Fig. S9). Double mutant plants defective in two homologous kinesin- like proteins, kinesin-like protein for actin-based chloroplast movement1 (kac1) kac2, are defective in light-induced chloroplast movements and lack cp-actin filaments, similar to chup1 mutant plants (Suetsugu et al., 2010). Importantly, chup1 kac1 kac2 triple mutant plants show almost no light-induced chloroplast movement, and chloroplast positioning is different between chup1 and chup1 kac1 kac2 triple mutant plants (Suetsugu et al., 2016). Therefore, the difference in chloroplast position-ing between chup1 and phot2 chup1 could be regulated by KAC proteins. Moreover, the residual chloro-plast movement in kac1 kac2 also is phot2 depen-dent (Suetsugu et al., 2016), indicating that phot2 could regulate both cp-actin-dependent (i.e. CHUP1- and KAC-dependent) and -independent chloroplast movements. Whatever the underlying mechanisms, the phot2 mutation changed the chloroplast posi-tioning and enhanced photosynthesis and biomass production in phot2 jac1 and phot2 chup1, further reinforcing that the chloroplast accumulation response to the periclinal walls is required for efficient biomass production.

In conclusion, our findings experimentally demonstrate the significance of the chloroplast accumulation response, which has been suggested for many years (Senn, 1908; Zurzycki, 1955; Brugnoli and Björkman, 1992). Therefore, in addition to the chloroplast avoidance response (Kasahara et al., 2002; Sztatelman et al., 2010; Davis and Hangarter, 2012) and cold-induced chloroplast positioning (Fujii et al., 2017), the chlo-roplast accumulation response has an important role in the adaptation to ever-changing environmental conditions.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) wild-type Columbia-0 gl1 and the phot1-5 (Huala et al., 1997), phot2-1 (Kagawa et al., 2001), phot1-5 phot2-1 (Kinoshita et al., 2001), jac1-2 (Suetsugu et al., 2005), chup1-3 (Oikawa et al., 2003), and phot2-1 jac1-2 (Kodama et al., 2010) mutant plants were used in the study. The phot2-1 chup1-3 plants were generated by genetic crossing. The plants were grown in soil in a growth chamber (LPH-350S; NK System) and were main-tained at 22°C under 55% relative humidity. Plants were grown under 8-h daylight conditions, except for the experiments shown in Figure 2 and Supple-mental Figures S2 and S6, where they were grown according to measurement range and research purpose.

Light Sources

For plant growth, fluorescent lamps (FL20SS and FL40SS; Toshiba) were used as the white light source. For the analysis of chloroplast movement, BL with an emission maximum at 470 nm was provided by light-emitting photo-diodes (IS-big and Is-mini; CCS). The photon flux density was measured using a light meter (LI-250; Li-Cor) attached to a light sensor (LI-190; Li-Cor).

Analysis of Light Absorption Spectra and Transmittance Changes

The light absorption spectrum and transmittance of an entire leaf was measured with a microplate reader (Multiskan GO; Thermo Fisher) accord-ing to previous studies (Kodama et al., 2010; Gotoh et al., 2018). Detached leaves (from 4- to 5-week-old plants) were placed on the surface of solidified 1% (w/v) gellan gum and irradiated with weak (3 μmol m−2 s−1) or strong (50 μmol m−2 s−1) BL for 3 h. After 3 h, the absorption of light in the 300- to 800-nm wavelength range was measured every 1 nm.

Observation of Chloroplast Distribution Patterns

The intracellular distribution of chloroplasts was observed in 35-d-old plants. The plants were irradiated with light of different qualities and inten-sities, as indicated for each experiment in the respective figure legend. The leaves of the plants were fixed with 2.5% (v/v) glutaraldehyde (Wako). The cross sections, except for those shown in Supplemental Figure S3, were obtained with a vibrating microtome (VT1200 S; Leica). The distribution of chloroplasts on the upper surface of the palisade cells and in the cross sections was observed with two laser scanning confocal microscopes (either FV10i [Olympus] or SP5 [Leica]). The projection images were constructed from z-stacks by the software supplied by the manufacturer. To obtain the cross sections shown in Supplemental Figure S3, leaves fixed with 2.5% (v/v) glu-taraldehyde solution and then 1% (w/v) OsO4 were dehydrated with a graded acetone and embedded in epoxy resin. Specimens were sectioned with an ultramicrotome (AG Reichert Division, Leica)

Analysis of CO2 Assimilation and Stomatal Conductance

CO2 assimilation and stomatal conductance in intact leaves were analyzed with an open gas-exchange system (LI-6400; Li-Cor) attached to a normal open chamber (Li-Cor). The plants were kept in the dark for at least 3 h prior to the measurements, and then the measurements were made under controlled air conditions (temperature of 22°C, relative humidity of 50%–60%, and CO2 concentration of 400 μL L−1). The net CO2 assimilation was denoted as the dif-ference in the values of CO2 assimilation between the light (120 μmol m−2 s−1) and dark treatments.

Measurement of Oxygen Evolution Rate

The protoplasts of mesophyll cells were prepared from 20 leaves of 4- to 5-week-old plants, following the procedure described previously (Riazunnisa et al., 2007). The intactness of the protoplasts was determined by fluorescein diacetate staining, and a preparation containing 90% intact protoplasts was

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used. The assay for oxygen evolution was carried out using an oxygen elec-trode (OXYG1-PLUS; Hansatech) at 22°C under white light (120 μmol m−2 s−1).

Estimation of Chlorophyll Content

Chlorophyll was extracted from four or five fully expanded leaves with 80% acetone. After centrifugation of the acetone extracts, the absorbance (OD 645 nm and 663 nm) of the supernatant was measured. The OD values obtained were used to calculate the chlorophyll content as described previously (Arnon, 1949).

Analysis of Photoinhibition

The plants were placed in an IMAGING-PAM device (Heinz Walz) and exposed to constant HL or to fluctuating light. The Fv/Fm and the maximum level of the P700 signal (full oxidation of P700) after dark incubation for 30 min were measured before and after the light treatment by an IMAGING-PAM device and a Dual-PAM-100 system (Walz) as described previously (Yamori et al., 2016).

Immunoblot Analysis

Total protein was extracted from the rosette leaves of 4- to 5-week-old plants in a buffer that contained 50 mm Tris-HCl (pH 7.5), 100 mm NaCl, 5 mm EDTA, 0.5% (v/v) Triton X-100, 1 mm DTT, and 1 mm phenylmethylsulfonyl fluoride, as described previously (Yamasaki et al., 2009). The proteins were separated using SDS-PAGE on a 12% (w/v) acrylamide gel and transferred to a polyvinylidene fluoride membrane. Antibodies for Rubisco large subunit, PsaA (a core protein of PSI), PsbB (CP47 protein of PSII), Cyt f (cytochrome f protein of the thylakoid Cyt b6/f complex), and plastocyanin (Agrisera) were used.

Quantification of Plant Biomass

The fresh weights of all the rosette leaves of 35-d-old plants were measured with an electronic balance, and photographs of those leaves were taken im-mediately after the measurement. The dry weight was measured using leaves that were dried at 75°C for 4 d. Leaf area measurements were conducted using ImageJ (National Institutes of Health).

Statistical Analysis

Since it was possible to estimate the interval of the mean value of each parameter in each group using se, all values are expressed as means ± se. The comparisons among the groups were performed using one-way ANOVA fol-lowed by the Tukey-Kramer multiple comparison test. Differences were con-sidered significant at P < 0.05. Statistical analysis was performed using IBM SPSS Statistics version 22.0 for Windows (IBM Japan). Logarithmic transfor-mation was used to normalize data with skewed distribution (Supplemental Fig. S9) before statistical analysis.

Accession Numbers

Accession numbers are as follows: PHOT1 (AT3G45780), PHOT2 (AT5G58140), JAC1 (AT1G75100), and CHUP1 (AT3G25690).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Light absorption curves of weak and strong light- irradiated leaves of wild-type and phot1 phot2 mutant plants.

Supplemental Figure S2. Photoinhibition in wild-type and various mutant plants.

Supplemental Figure S3. Chloroplast distribution pattern of wild-type and mutant plants under ML.

Supplemental Figure S4. Protein expression levels of photosynthesis- related proteins in wild-type and various mutant plants.

Supplemental Figure S5. Time course of plant biomass production under short-day conditions in wild-type and various mutant plants.

Supplemental Figure S6. Time course of plant biomass production under long-day conditions in wild-type and various mutant plants.

Supplemental Figure S7. Plant biomass production in wild-type and phototropin mutant plants.

Supplemental Figure S8. Chloroplast distribution pattern of plants grown under different light intensities.

Supplemental Figure S9. Analysis of chloroplast photorelocation move-ment through a measurement of light-induced changes in leaf trans-mittance.

ACKNOWLEDGMENTS

We thank Tomonao Matsushita, Toshihiro Kumamaru, and Susumu Shiraishi (Kyushu University) for research support and Aeri Choung, Yuki Hirose, and Shinho Goto (Kyushu University) for technical assistance.

Received April 24, 2018; accepted September 23, 2018; published September 28, 2018.

LITERATURE CITED

Arnon DI (1949) Copper enzymes in isolated chloroplasts: polyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1–15

Brugnoli E, Björkman O (1992) Chloroplast movements in leaves: influence on chlorophyll fluorescence and measurements of light-induced absor-bance changes related to ΔpH and zeaxanthin formation. Photosynth Res 32: 23–35

Christie JM (2007) Phototropin blue-light receptors. Annu Rev Plant Biol 58: 21–45

Davis PA, Hangarter RP (2012) Chloroplast movement provides photopro-tection to plants by redistributing PSII damage within leaves. Photosynth Res 112: 153–161

Davis PA, Caylor S, Whippo CW, Hangarter RP (2011) Changes in leaf optical properties associated with light-dependent chloroplast movements. Plant Cell Environ 34: 2047–2059

Dutta S, Cruz JA, Jiao Y, Chen J, Kramer DM, Osteryoung KW (2015) Non- invasive, whole-plant imaging of chloroplast movement and chlorophyll fluorescence reveals photosynthetic phenotypes independent of chloroplast photorelocation defects in chloroplast division mutants. Plant J 84: 428–442

Evans JR, Kaldenhoff R, Genty B, Terashima I (2009) Resistances along the CO2 diffusion pathway inside leaves. J Exp Bot 60: 2235–2248

Fujii Y, Tanaka H, Konno N, Ogasawara Y, Hamashima N, Tamura S, Hasegawa S, Hayasaki Y, Okajima K, Kodama Y (2017) Phototropin per-ceives temperature based on the lifetime of its photoactivated state. Proc Natl Acad Sci USA 114: 9206–9211

Gotoh E, Suetsugu N, Higa T, Matsushita T, Tsukaya H, Wada M (2018) Palisade cell shape affects the light-induced chloroplast movements and leaf photosynthesis. Sci Rep 8: 1472

Huala E, Oeller PW, Liscum E, Han IS, Larsen E, Briggs WR (1997) Arabidop-sis NPH1: a protein kinase with a putative redox-sensing domain. Science 278: 2120–2123

Ichikawa S, Yamada N, Suetsugu N, Wada M, Kadota A (2011) Red light, Phot1 and JAC1 modulate Phot2-dependent reorganization of chloroplast actin filaments and chloroplast avoidance movement. Plant Cell Physiol 52: 1422–1432

Inoue Y, Shibata K (1973) Light-induced chloroplast rearrangements and their action spectra as measured by absorption spectrophotometry. Planta 114: 341–358

Inoue S, Kinoshita T, Takemiya A, Doi M, Shimazaki K (2008) Leaf positioning of Arabidopsis in response to blue light. Mol Plant 1: 15–26

Jarillo JA, Gabryś H, Capel J, Alonso JM, Ecker JR, Cashmore AR (2001) Phototropin-related NPL1 controls chloroplast relocation induced by blue light. Nature 410: 952–954

Kadota A, Yamada N, Suetsugu N, Hirose M, Saito C, Shoda K, Ichikawa S, Kagawa T, Nakano A, Wada M (2009) Short actin-based mechanism for light-directed chloroplast movement in Arabidopsis. Proc Natl Acad Sci USA 106: 13106–13111

Gotoh et al.

www.plantphysiol.orgon November 3, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.

Plant Physiol. Vol. 178, 2018 1369

Kagawa T, Sakai T, Suetsugu N, Oikawa K, Ishiguro S, Kato T, Tabata S, Okada K, Wada M (2001) Arabidopsis NPL1: a phototropin homolog con-trolling the chloroplast high-light avoidance response. Science 291: 2138–2141

Kaiser E, Morales A, Harbinson J, Kromdijk J, Heuvelink E, Marcelis LF (2015) Dynamic photosynthesis in different environmental conditions. J Exp Bot 66: 2415–2426

Kami C, Lorrain S, Hornitschek P, Fankhauser C (2010) Light-regulated plant growth and development. Curr Top Dev Biol 91: 29–66

Kasahara M, Kagawa T, Oikawa K, Suetsugu N, Miyao M, Wada M (2002) Chloroplast avoidance movement reduces photodamage in plants. Nature 420: 829–832

Kinoshita T, Doi M, Suetsugu N, Kagawa T, Wada M, Shimazaki K (2001) Phot1 and phot2 mediate blue light regulation of stomatal opening. Nature 414: 656–660

Kodama Y, Suetsugu N, Kong SG, Wada M (2010) Two interacting coiled-coil proteins, WEB1 and PMI2, maintain the chloroplast photorelocation move-ment velocity in Arabidopsis. Proc Natl Acad Sci USA 107: 19591–19596

Kong SG, Arai Y, Suetsugu N, Yanagida T, Wada M (2013) Rapid severing and motility of chloroplast-actin filaments are required for the chloroplast avoidance response in Arabidopsis. Plant Cell 25: 572–590

Kozuka T, Kong SG, Doi M, Shimazaki K, Nagatani A (2011) Tissue-auton-omous promotion of palisade cell development by phototropin 2 in Arabi-dopsis. Plant Cell 23: 3684–3695

Külheim C, Agren J, Jansson S (2002) Rapid regulation of light harvesting and plant fitness in the field. Science 297: 91–93

Kume A (2017) Importance of the green color, absorption gradient, and spec-tral absorption of chloroplasts for the radiative energy balance of leaves. J Plant Res 130: 501–514

Lehmann P, Nöthen J, von Braun SS, Bohnsack MT, Mirus O, Schleiff E (2011) Transitions of gene expression induced by short-term blue light. Plant Biol (Stuttg) 13: 349–361

Li Z, Wakao S, Fischer BB, Niyogi KK (2009) Sensing and responding to ex-cess light. Annu Rev Plant Biol 60: 239–260

Oikawa K, Kasahara M, Kiyosue T, Kagawa T, Suetsugu N, Takahashi F, Kanegae T, Niwa Y, Kadota A, Wada M (2003) Chloroplast unusual po-sitioning1 is essential for proper chloroplast positioning. Plant Cell 15: 2805–2815

Oikawa K, Yamasato A, Kong SG, Kasahara M, Nakai M, Takahashi F, Ogura Y, Kagawa T, Wada M (2008) Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement. Plant Physiol 148: 829–842

Riazunnisa K, Padmavathi L, Scheibe R, Raghavendra AS (2007) Preparation of Arabidopsis mesophyll protoplasts with high rates of photosynthesis. Physiol Plant 129: 679–686

Sakai T, Kagawa T, Kasahara M, Swartz TE, Christie JM, Briggs WR, Wada M, Okada K (2001) Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation. Proc Natl Acad Sci USA 98: 6969–6974

Sakamoto K, Briggs WR (2002) Cellular and subcellular localization of pho-totropin 1. Plant Cell 14: 1723–1735

Senn G (1908) Die Gestalts- und Lageveränderung der Pflanzen-Chromato-phoren. Wilhelm-Engelmann, Leipzig, Germany

Suetsugu N, Wada M (2013) Chloroplast photorelocation movement: a sophis-ticated strategy for chloroplasts to perform efficient photosynthesis. In MM Najafpour, ed, Advances in Photosynthesis: Fundamental Aspects. InTech, Eijieka, Croatia,

Suetsugu N, Kagawa T, Wada M (2005) An auxilin-like J-domain protein, JAC1, regulates phototropin-mediated chloroplast movement in Arabidop-sis. Plant Physiol 139: 151–162

Suetsugu N, Yamada N, Kagawa T, Yonekura H, Uyeda TQP, Kadota A, Wada M (2010) Two kinesin-like proteins mediate actin-based chloro-plast movement in Arabidopsis thaliana. Proc Natl Acad Sci USA 107: 8860–8865

Suetsugu N, Higa T, Gotoh E, Wada M (2016) Light-induced movements of chloroplasts and nuclei are regulated in both cp-actin-filament-depen-dent and -independent manners in Arabidopsis thaliana. PLoS ONE 11: e0157429

Suorsa M, Järvi S, Grieco M, Nurmi M, Pietrzykowska M, Rantala M, Kangasjärvi S, Paakkarinen V, Tikkanen M, Jansson S, (2012) PROTON GRADIENT REGULATION5 is essential for proper acclimation of Arabi-dopsis photosystem I to naturally and artificially fluctuating light condi-tions. Plant Cell 24: 2934–2948

Sztatelman O, Waloszek A, Banaś AK, Gabryś H (2010) Photoprotective func-tion of chloroplast avoidance movement: in vivo chlorophyll fluorescence study. J Plant Physiol 167: 709–716

Takemiya A, Inoue S, Doi M, Kinoshita T, Shimazaki K (2005) Phototropins promote plant growth in response to blue light in low light environments. Plant Cell 17: 1120–1127

Takemiya A, Sugiyama N, Fujimoto H, Tsutsumi T, Yamauchi S, Hiyama A, Tada Y, Christie JM, Shimazaki K (2013) Phosphorylation of BLUS1 kinase by phototropins is a primary step in stomatal opening. Nat Com-mun 4: 2094

Terashima I, Hikosaka K (1995) Comparative ecophysiology of leaf and cano-py photosynthesis. Plant Cell Environ 18: 1111–1128

Vogelmann TC (1993) Plant tissue optics. Annu Rev Plant Physiol Plant Mol Biol 44: 231–251

Williams WE, Gorton HL, Witiak SM (2003) Chloroplast movements in the field. Plant Cell Environ 26: 2005–2014

Yamasaki H, Hayashi M, Fukazawa M, Kobayashi Y, Shikanai T (2009) SQUAMOSA promoter binding protein-like7 is a central regulator for copper homeostasis in Arabidopsis. Plant Cell 21: 347–361

Yamori W (2016) Photosynthetic response to fluctuating environments and photoprotective strategies under abiotic stress. J Plant Res 129: 379–395

Yamori W, Shikanai T (2016) Physiological functions of cyclic electron trans-port around photosystem I in sustaining photosynthesis and plant growth. Annu Rev Plant Biol 67: 81–106

Yamori W, Makino A, Shikanai T (2016) A physiological role of cyclic electron transport around photosystem I in sustaining photosynthesis under fluctu-ating light in rice. Sci Rep 6: 20147

Zurzycki J (1955) Chloroplast arrangements as a factor of photosynthesis. Acta Soc Bot Pol 24: 27–63

Chloroplast Movement Increases Plant Biomass

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