PLASTID MOVEMENT IMPAIRED1 and PLASTID MOVEMENT IMPAIRED1-RELATED1 Mediate Photorelocation Movements of Both Chloroplasts and Nuclei^,

Two C2 domain proteins regulate light-mediated movements of plastids and nuclei in both mesophyll and pavement cells.

Abstract

Organelle movement and positioning play important roles in fundamental cellular activities and adaptive responses to environmental stress in plants. To optimize photosynthetic light utilization, chloroplasts move toward weak blue light (the accumulation response) and escape from strong blue light (the avoidance response). Nuclei also move in response to strong blue light by utilizing the light-induced movement of attached plastids in leaf cells. Blue light receptor phototropins and several factors for chloroplast photorelocation movement have been identified through molecular genetic analysis of Arabidopsis (Arabidopsis thaliana). PLASTID MOVEMENT IMPAIRED1 (PMI1) is a plant-specific C2-domain protein that is required for efficient chloroplast photorelocation movement. There are two PLASTID MOVEMENT IMPAIRED1-RELATED (PMIR) genes, PMIR1 and PMIR2, in the Arabidopsis genome. However, the mechanism in which PMI1 regulates chloroplast and nuclear photorelocation movements and the involvement of PMIR1 and PMIR2 in these organelle movements remained unknown. Here, we analyzed chloroplast and nuclear photorelocation movements in mutant lines of PMI1, PMIR1, and PMIR2. In mesophyll cells, the pmi1 single mutant showed severe defects in both chloroplast and nuclear photorelocation movements resulting from the impaired regulation of chloroplast-actin filaments. In pavement cells, pmi1 mutant plants were partially defective in both plastid and nuclear photorelocation movements, but pmi1pmir1 and pmi1pmir1pmir2 mutant lines lacked the blue light-induced movement responses of plastids and nuclei completely. These results indicated that PMI1 is essential for chloroplast and nuclear photorelocation movements in mesophyll cells and that both PMI1 and PMIR1 are indispensable for photorelocation movements of plastids and thus, nuclei in pavement cells.

In plants, organelles move within the cell and become appropriately positioned to accomplish their functions and adapt to the environment (for review, see Wada and Suetsugu, 2004). Light-induced chloroplast movement (chloroplast photorelocation movement) is one of the best characterized organelle movements in plants (Suetsugu and Wada, 2012). Under weak light conditions, chloroplasts move toward light to capture light efficiently (the accumulation response; Zurzycki, 1955). Under strong light conditions, chloroplasts escape from light to avoid photodamage (the avoidance response; Kasahara et al., 2002; Sztatelman et al., 2010; Davis and Hangarter, 2012; Cazzaniga et al., 2013). In most green plant species, these responses are induced primarily by the blue light receptor phototropin (phot) in response to a range of wavelengths from UVA to blue light (approximately 320–500 nm; for review, see Suetsugu and Wada, 2012; Wada and Suetsugu, 2013; Kong and Wada, 2014). Phot-mediated chloroplast movement has been shown in land plants, such as Arabidopsis (Arabidopsis thaliana; Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001), the fern Adiantum capillus-veneris (Kagawa et al., 2004), the moss Physcomitrella patens (Kasahara et al., 2004), and the liverwort Marchantia polymorpha (Komatsu et al., 2014). Two phots in Arabidopsis, phot1 and phot2, redundantly mediate the accumulation response (Sakai et al., 2001), whereas phot2 primarily regulates the avoidance response (Jarillo et al., 2001; Kagawa et al., 2001; Luesse et al., 2010). M. polymorpha has only one phot that mediates both the accumulation and avoidance responses (Komatsu et al., 2014), although two or more phots mediate chloroplast photorelocation movement in A. capillus-veneris (Kagawa et al., 2004) and P. patens (Kasahara et al., 2004). Thus, duplication and functional diversification of PHOT genes have occurred during land plant evolution, and plants have gained a sophisticated light sensing system for chloroplast photorelocation movement.

In general, movements of plant organelles, including chloroplasts, are dependent on actin filaments (for review, see Wada and Suetsugu, 2004). Most organelles common in eukaryotes, such as mitochondria, peroxisomes, and Golgi bodies, use the myosin motor for their movements, but there is no clear evidence that chloroplast movement is myosin dependent (for review, see Suetsugu et al., 2010a). Land plants have innovated a novel actin-based motility system that is specialized for chloroplast movement as well as a photoreceptor system (for review, see Suetsugu et al., 2010a; Wada and Suetsugu, 2013; Kong and Wada, 2014). Chloroplast-actin (cp-actin) filaments, which were first found in Arabidopsis, are short actin filaments specifically localized around the chloroplast periphery at the interface between the chloroplast and the plasma membrane (Kadota et al., 2009). Strong blue light induces the rapid disappearance of cp-actin filaments and then, their subsequent reappearance preferentially at the front region of the moving chloroplasts. This asymmetric distribution of cp-actin filaments is essential for directional chloroplast movement (Kadota et al., 2009; Kong et al., 2013a). The greater the difference in the amount of cp-actin filaments between the front and rear regions of chloroplasts becomes, the faster the chloroplasts move, in which the magnitude of the difference is determined by fluence rate (Kagawa and Wada, 2004; Kadota et al., 2009; Kong et al., 2013a). Strong blue light-induced disappearance of cp-actin filaments is regulated in a phot2-dependent manner before the intensive polymerization of cp-actin filaments at the front region occurs (Kadota et al., 2009; Ichikawa et al., 2011; Kong et al., 2013a). This phot2-dependent response contributes to the greater difference in the amount of cp-actin filaments between the front and rear regions of chloroplasts. Similar behavior of cp-actin filaments has also been observed in A. capillus-veneris (Tsuboi and Wada, 2012) and P. patens (Yamashita et al., 2011).

Like chloroplasts, nuclei also show light-mediated movement and positioning (nuclear photorelocation movement) in land plants (for review, see Higa et al., 2014b). In gametophytic cells of A. capillus-veneris, weak light induced the accumulation responses of both chloroplasts and nuclei, whereas strong light induced avoidance responses (Kagawa and Wada, 1993, 1995; Tsuboi et al., 2007). However, in mesophyll cells of Arabidopsis, strong blue light induced both chloroplast and nuclear avoidance responses, but weak blue light induced only the chloroplast accumulation response (Iwabuchi et al., 2007, 2010; Higa et al., 2014a). In Arabidopsis pavement cells, small numbers of tiny plastids were found and showed autofluorescence under the confocal laser-scanning microscopy (Iwabuchi et al., 2010; Higa et al., 2014a). Hereafter, the plastid in the pavement cells is called the pavement cell plastid. Strong blue light-induced avoidance responses of pavement cell plastids and nuclei were induced in a phot2-dependent manner, but the accumulation response was not detected for either organelle (Iwabuchi et al., 2007, 2010; Higa et al., 2014a). In both Arabidopsis and A. capillus-veneris, phots mediate nuclear photorelocation movement, and phot2 mediates the nuclear avoidance response (Iwabuchi et al., 2007, 2010; Tsuboi et al., 2007). The nuclear avoidance response is dependent on actin filaments in both mesophyll and pavement cells of Arabidopsis (Iwabuchi et al., 2010). Recently, it was shown that the nuclear avoidance response relies on cp-actin-dependent movement of pavement cell plastids, where nuclei are associated with pavement cell plastids of Arabidopsis (Higa et al., 2014a). In mesophyll cells, nuclear avoidance response is likely dependent on cp-actin filament-mediated chloroplast movement, because the mutants deficient in chloroplast movement were also defective in nuclear avoidance response (Higa et al., 2014a). Thus, phots mediate both chloroplast (and pavement cell plastid) and nuclear photorelocation movement by regulating cp-actin filaments.

Molecular genetic analyses of Arabidopsis mutants deficient in chloroplast photorelocation movement have identified many molecular factors involved in signal transduction and/or motility systems as well as those involved in the photoreceptor system for chloroplast photorelocation movement (and thus, nuclear photorelocation movement; for review, see Suetsugu and Wada, 2012; Wada and Suetsugu, 2013; Kong and Wada, 2014). CHLOROPLAST UNUSUAL POSITIONING1 (CHUP1; Oikawa et al., 2003) and KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT (KAC; Suetsugu et al., 2010b) are key factors for generating and/or maintaining cp-actin filaments. Both proteins are highly conserved in land plants and essential for the movement and attachment of chloroplasts to the plasma membrane in Arabidopsis (Oikawa et al., 2003, 2008; Suetsugu et al., 2010b), A. capillus-veneris (Suetsugu et al., 2012), and P. patens (Suetsugu et al., 2012; Usami et al., 2012). CHUP1 is localized on the chloroplast outer membrane and binds to globular and filamentous actins and profilin in vitro (Oikawa et al., 2003, 2008; Schmidt von Braun and Schleiff, 2008). Although KAC is a kinesin-like protein, it lacks microtubule-dependent motor activity but has filamentous actin binding activity (Suetsugu et al., 2010b). An actin-bundling protein THRUMIN1 (THRUM1) is required for efficient chloroplast photorelocation movement (Whippo et al., 2011) and interacts with cp-actin filaments (Kong et al., 2013a). chup1 and kac mutant plants were shown to lack detectable cp-actin filaments (Kadota et al., 2009; Suetsugu et al., 2010b; Ichikawa et al., 2011; Kong et al., 2013a). Similarly, cp-actin filaments were rarely detected in thrum1 mutant plants (Kong et al., 2013a), indicating that THRUM1 also plays an important role in maintaining cp-actin filaments.

Other proteins J-DOMAIN PROTEIN REQUIRED FOR CHLOROPLAST ACCUMULATION RESPONSE1 (JAC1; Suetsugu et al., 2005), WEAK CHLOROPLAST MOVEMENT UNDER BLUE LIGHT1 (WEB1; Kodama et al., 2010), and PLASTID MOVEMENT IMPAIRED2 (PMI2; Luesse et al., 2006; Kodama et al., 2010) are involved in the light regulation of cp-actin filaments and chloroplast photorelocation movement. JAC1 is an auxilin-like J-domain protein that mediates the chloroplast accumulation response through its J-domain function (Suetsugu et al., 2005; Takano et al., 2010). WEB1 and PMI2 are coiled-coil proteins that interact with each other (Kodama et al., 2010). Although web1 and pmi2 were partially defective in the avoidance response, the jac1 mutation completely suppressed the phenotype of web1 and pmi2, suggesting that the WEB1/PMI2 complex suppresses JAC1 function (i.e. the accumulation response) under strong light conditions (Kodama et al., 2010). Both web1 and pmi2 showed impaired disappearance of cp-actin filaments in response to strong blue light (Kodama et al., 2010). However, the exact molecular functions of these proteins are unknown.

In this study, we characterized mutant plants deficient in the PMI1 gene and two homologous genes PLASTID MOVEMENT IMPAIRED1-RELATED1 (PMIR1) and PMIR2. PMI1 was identified through molecular genetic analyses of pmi1 mutants that showed severe defects in chloroplast accumulation and avoidance responses (DeBlasio et al., 2005). PMI1 is a plant-specific C2-domain protein (DeBlasio et al., 2005; Zhang and Aravind, 2010), but its roles and those of PMIRs in cp-actin-mediated chloroplast and nuclear photorelocation movements remained unclear. Thus, we analyzed chloroplast and nuclear photorelocation movements in the single, double, and triple mutants of pmi1, pmir1, and pmir2.

RESULTS

PMI1 Is Essential for Chloroplast Photorelocation Movement in Mesophyll Cells

We screened mutants using a band assay to identify those deficient in chloroplast photorelocation movement (Kagawa et al., 2001; Oikawa et al., 2003; Suetsugu et al., 2005; Kodama et al., 2010). We isolated a mutant with severe defects in chloroplast movement, and rough mapping and sequencing of candidate genes revealed a mutation in its PMI1 gene (Fig. 1). The defect in chloroplast movement was complemented by PMI1pro::PMI1-GFP (see below). This mutant allele was named pmi1-5, because pmi1-1, pmi1-2, pmi1-3, and pmi1-4 alleles have already been reported (DeBlasio et al., 2005; Rojas-Pierce et al., 2014). A 37-bp deletion (G172–T208 from start codon) was found in the PMI1 exon 1 of pmi1-5 (Fig. 1A). The pmi1-5 mutation is presumed to produce a premature stop codon. pmi1-5 was characterized in detail in this study.

Figure 1.

Gene structure of PMI1, PMIR1, and PMIR2 and chloroplast photorelocation movement in mesophyll cells of pmi1 and pmir1 pmir2 mutants. A, Gene structure and mutation sites of PMI1, PMIR1, and PMIR2 genes. Rectangles indicate exons (gray rectangles indicate 5′ or 3′ untranslated region), and intervening bars indicate introns. The gray bar in PMI1 shows the promoter region used in PMI1pro::PMI1-GFP. LB, Left border of T-DNA. B, Changes in leaf transmittance caused by chloroplast photorelocation movement. After transmittance measurement started, dark-adapted samples were kept in darkness for an additional 10 min. Then, samples were sequentially irradiated with continuous blue light at 3, 20, and 50 µmol m⁻² s⁻¹ for 60, 40, and 40 min indicated by white, sky blue, and blue arrows, respectively. Light was turned off at 150 min (black arrow). Mean values from three independent experiments are shown. Error bars indicate ses. C, Changes in leaf transmittance rates from 2 to 6 min after changes in light fluence rate (3, 20, and 50 µmol m⁻² s⁻¹) are indicated as percentage transmittance change over 1 min. Mean values from three independent experiments are shown. Error bars indicate ses. WT, Wild type.

Chloroplast photorelocation movement in the wild type, pmi1-5, and pmi1-2 (a transfer DNA [T-DNA] insertion mutant described in DeBlasio et al. [2005]; Fig. 1A) was analyzed by measuring changes in leaf transmittance. Both chloroplast accumulation and avoidance responses (a weak light-induced decrease and a strong light-induced increase in leaf transmittance, respectively) were severely impaired in pmi1-5 (Fig. 1, B and C; Supplemental Table S1). These impaired responses were similar to those described previously for pmi1-1, a strong pmi1 allele (DeBlasio et al., 2005; Fig. 1A). Compared with pmi1-5, pmi1-2 showed weaker defects in chloroplast photorelocation movement (Fig. 1, B and C; Supplemental Table S1), similar to the previous report that pmi1-2 was weaker than pmi1-1 (DeBlasio et al., 2005). Although pmi1-1 and pmi1-5 were severely impaired in chloroplast photorelocation movement, they retained partial chloroplast movement. Because there are two PMI1-like genes in the Arabidopsis genome (At5g20610 and At5g26160 designated as PMIR1 and PMIR2, respectively; DeBlasio et al., 2005), we assumed a possibility that the subtle chloroplast photorelocation movement in pmi1 could be caused by PMIR1 and PMIR2. We obtained T-DNA insertion lines for each gene (Fig. 1A) and generated double and triple mutants of pmi1 and pmir mutants. Contrary to our expectations, the pmir1-1pmir2-1 double mutant exhibited stronger chloroplast photorelocation movement compared with the wild type. The pmi1pmir1pmir2 triple mutants showed similar chloroplast photorelocation movement to that of pmi1 single mutants (both pmi1-2 and pmi1-5; Fig. 1, B and C; Supplemental Table S1). Between wild-type and pmi1 mutant plants, we did not observe any clear difference in leaf morphology, leaf color, and chloroplast distribution pattern in dark-adapted cells as described previously (DeBlasio et al., 2005). Indeed, initial transmittance in dark-adapted leaves was similar, and the slight differences in the initial transmittance did not correlate with the differences in the transmittance changes among genotypes (Supplemental Fig. S1). These results indicated that PMI1 plays the major role in chloroplast movement compared with PMIR1 and PMIR2. Hereafter, all experiments were performed using pmi1-5, pmir1-1, and pmir2-1 alleles.

Genetic Interaction between pmi1 and Other Mutants Partially Defective in Chloroplast Photorelocation Movement in Mesophyll Cells

To elucidate the function of PMI1 in chloroplast photorelocation movement, we analyzed the genetic interaction between PMI1 and PHOT1, PHOT2, JAC1, WEB1, and PMI2 (and its homolog PMI15; Luesse et al., 2006; Fig. 2). For each gene, pmi1-5, phot1-5, phot2-1, jac1-2, web1-2, pmi2-2, and pmi15-1 alleles were used (Huala et al., 1997; Kagawa et al., 2001; Suetsugu et al., 2005; Luesse et al., 2006; Kodama et al., 2010). Although phot1 was partially defective in the accumulation response (Fig. 2A; Sakai et al., 2001), the avoidance response in phot1 was enhanced under certain conditions (Fig. 2A; Ichikawa et al., 2011). phot2 was severely defective in the avoidance response but not the accumulation response (Fig. 2A; Jarillo et al., 2001; Kagawa et al., 2001). pmi1phot2 showed a weak accumulation response similar to that of pmi1 and an impaired avoidance response similar to that of phot2 (Fig. 2A; Supplemental Table S1). However, there was a synergistic genetic interaction between the pmi1 and phot1 mutations. pmi1phot1 showed a very weak avoidance response (Fig. 2A; Supplemental Table S1). This result indicated that PMI1 is necessary for phot2-mediated chloroplast movements, especially the avoidance response, in the absence of phot1. jac1 was shown to be severely defective in the accumulation response and partially defective in the avoidance response (Suetsugu et al., 2005; Kodama et al., 2010). Like phot1pmi1, the pmi1jac1 double mutant was severely impaired in both the accumulation and avoidance responses, similar to the phot2jac1 double mutant (Suetsugu et al., 2005; Fig. 2B). Thus, PMI1 has an important role in the phot2 signaling pathway that regulates the avoidance response.

Figure 2.

Changes in leaf transmittance rates in mesophyll cells of mutants crossed between pmi1 and phot, jac1, web1, or pmi2. Changes in leaf transmittance rates from 2 to 6 min after changes in light fluence rate (3, 20, and 50 µmol m⁻² s⁻¹). A, Genetic interaction between PMI1 and PHOT genes. B, Genetic interaction between PMI1 and JAC1, WEB1, and PMI2 (and PMI15) genes. C, Genetic interaction between PMI1, JAC1, and WEB1 genes. D, Genetic interaction between PMI1, JAC1, and PMI2 (and PMI15) genes. For details, see Figure 1C. Mean values from three independent experiments are shown. Error bars indicate ses. WT, Wild type.

We evaluated the genetic interaction between PMI1 and WEB1/PMI2 by analyzing pmi1web1 and pmi1pmi2pmi15. PMI15 is homologous to PMI2. The defect in chloroplast movement was slightly stronger in pmi2pmi15 than in the pmi2 single mutant (Luesse et al., 2006; Fig. 2B). Interestingly, the defect in the accumulation response of pmi1 was partially suppressed by web1 and pmi2pmi15 mutations. Thus, the accumulation responses were greater in pmi1web1 and pmi1pmi2pmi15 than in pmi1 (Fig. 2B; Supplemental Table S1). However, the avoidance response was greatly impaired in pmi1web1 and pmi1pmi2pmi15, especially at 50 µmol m⁻² s⁻¹ (Fig. 2B; Supplemental Table S1). Superficially, the phenotypes of pmi1web1 and pmi1pmi2pmi15 were similar to that of phot2. The enhanced accumulation response in pmi1web1 and pmi1pmi2pmi15 was suppressed by jac1 mutation. pmi1web1jac1 and pmi1pmi2pmi15jac1 exhibited similar phenotypes to that of pmi1jac1 (that is, the severe attenuation of both the accumulation and avoidance responses; Fig. 2, C and D; Supplemental Table S1). These findings indicated that the suppression of the weak accumulation response in pmi1 by the web1 or pmi2pmi15 mutations depends on JAC1 activity.

PMI1 Is Localized Mainly in the Cytoplasm in Both Mesophyll and Pavement Cells

The previous results (DeBlasio et al., 2005) and analyses of large-scale transcriptome (Zimmermann et al., 2004; Winter et al., 2007) and translatome data (Mustroph et al., 2009) indicated that PMI1 was preferentially expressed in leaf tissues (Supplemental Fig. S2, A and B). PMIR1 was ubiquitously expressed in various tissues, although the expression level of PMIR1 was lower than that of PMI1 in leaf tissues. No expression data were available for PMIR2, because there was no microarray probe set for PMIR2. The proteome data (Joshi et al., 2011) indicated that PMI1 protein was expressed in various organs. Compared with the PMI1 peptide, a much smaller amount of PMIR1 peptide was detected in leaves, and no PMIR2 was detected in leaves (Supplemental Fig. S2C).

To investigate the subcellular localization of PMI1, we generated transgenic pmi1 lines expressing the PMI1-GFP fusion protein under the control of the putative PMI1 promoter (Fig. 3). Transgenic lines with approximately three-quarters gentamycin resistance were selected from the T2 generation; these lines contained a single copy of the transgene. Chloroplast photorelocation movement was examined in T3 homozygous siblings. Most of the transgenic lines examined were complemented by PMI1pro::PMI1-GFP, indicating that PMI1-GFP was a functional protein (Supplemental Fig. S3). When confocal microscopic analysis was performed using the fully rescued PMI1pro::PMI1-GFP transgenic lines, PMI1-GFP fluorescence was consistently detected in the cytosol of mesophyll cells and the thin layer of cytoplasm in the pavement cells without specific localization on the membrane or organelles (Fig. 3A).

Figure 3.

Subcellular localization of PMI1 and fractionation of protein factors regulating chloroplast movement in pmi1. A, Subcellular localization of PMI1-GFP. Transverse sections of pavement cells and mesophyll cells were observed under a confocal laser-scanning microscope. Image is false colored to indicate fluorescence of GFP (green) and chlorophyll (red). Arrows indicate PMI1-GFP fluorescence in the cytoplasm. B, Immunoblot analysis of PHOT1, PHOT2, JAC1, CHUP1, and KAC proteins in various mutants. Total protein extracts (T) were fractionated into soluble (S) and microsomal (M) fractions by ultracentrifugation (100,000g for 30 min at 4°C). Immunoblotting was performed using indicated antisera (Suetsugu et al., 2010b). Numbers on the left indicate the molecular weight of protein markers in the far left lanes. Arrows indicate deduced full-length bands of indicated proteins. The small arrow indicates the phot1 protein band recognized by phot2 antisera. WT, Wild type.

To determine the possible effects of the pmi1 mutation on the abundance and fractionation profiles of phot1, phot2, JAC1, KAC, and CHUP1, we performed immunoblot analyses on fractionated proteins from wild-type and pmi1 rosette leaves (Fig. 3B). phot1, phot2, and CHUP1 were enriched in the microsomal fraction, and KAC was detected mainly in the soluble fraction as described previously (Suetsugu et al., 2010b). JAC1 was detected exclusively in the microsomal fraction, although a previous transient expression analysis of GFP-JAC1 suggested that JAC is a soluble protein (Suetsugu et al., 2005). The protein levels and fractionation patterns of these proteins in pmi1 were the same as those in wild-type plants. Thus, the defects in the chloroplast photorelocation movement of pmi1 were not caused by impaired protein expression or altered localization of these proteins that regulate chloroplast photorelocation movement.

PMI1 Is Involved in Regulating Cp-Actin Filaments in Mesophyll Cells

To examine the role of PMI1 on the regulation of cp-actin filaments, we observed the dynamics of actin filaments visualized with GFP-talin using confocal laser-scanning microscopy (for details, see “Materials and Methods”; Kong et al., 2013a). In wild-type cells (Fig. 4; Supplemental Movie S1), a small amount of cp-actin filaments was detectable around the entire rims of chloroplasts before blue light irradiation (Fig. 4A, white arrows). After irradiation with strong blue light, cp-actin filaments rapidly disappeared from the irradiated area (Fig. 4A, white arrows at 2 min 4 s [02:04]). Thereafter, an asymmetric distribution of cp-actin filaments was established with the accumulation of cp-actin filaments at the front regions of moving chloroplasts (Fig. 4A, yellow arrows), and the chloroplasts moved to the nonirradiated area. However, in pmi1 mutant cells, chloroplasts did not move away from the strong light-irradiated area (Fig. 4B; Supplemental Movie S1). Also, cp-actin filaments were not detectable on the chloroplasts (Fig. 4B).

Figure 4.

Observation of cp-actin filaments on moving chloroplasts in mesophyll cells of wild-type and pmi1 cells. A and B, Time-lapse images of reorganization of cp-actin filaments in wild-type (A) and pmi1 (B) cells during chloroplast movement in response to strong blue light (BL). Actin filaments were probed with GFP-mouse talin fusion protein (green). Blue broken lines indicate the BL-irradiated area. Irradiation times (minutes-seconds) are shown at the top left corner. Note that cp-actin filaments rapidly reorganized on the rims of moving chloroplasts (numbers 1–6). White arrows indicate rapid disappearance of cp-actin filaments from the rear region of moving chloroplasts; yellow arrows indicate reappearance of cp-actin filaments in the front region of moving chloroplasts. For the full time-lapse series, see Supplemental Movie S1. Bar = 10 µm.

However, when the pmi1 mutant cells were incubated in the dark for 4 min (4 min) after a 30-s irradiation with blue light (30 s), cp-actin filaments were detected in these cells as in wild-type cells, although there was a smaller amount of cp-actin filaments in pmi1 mutant cells than in wild-type cells (Fig. 5). After irradiation with strong blue light, cp-actin filaments disappeared more rapidly from pmi1 cells than from wild-type cells but reappeared after an additional 4-min dark incubation (4 min; Fig. 5, A and B). It should be noted here that any significant difference was not detected in the cortical actin filament patterns in wild-type and pmi1 mutant cells (Figs. 4 and 5A), indicating that the defect of pmi1 was not the cause of any possibility, such as differential photobleach of the fluorescent protein. These findings suggested that the cp-actin filaments were unstable in the pmi1 mutant cells. We, therefore, speculated that the imaging blue laser (488 nm) used to detect GFP likely caused the disappearance of cp-actin filaments in pmi1 cells. To address this possibility, we examined the chloroplast avoidance response with an imaging laser of 516 nm, which is out of the absorption spectra of phots (Sakai et al., 2001). The chloroplast avoidance response was effectively induced in the pmi1 mutant cells by the 458-nm stimulating laser when the 516-nm laser was set for imaging (Fig. 5, C and D; Supplemental Movie S2). This result was consistent with the partial chloroplast photorelocation movement detected by measuring the change in leaf transmittance, in which red light was used to read transmittance (Fig. 1, B and C). Collectively, these findings indicated that the defects in chloroplast photorelocation movement in pmi1 result from the impaired regulation of cp-actin filaments.

Figure 5.

Reorganizations of cp-actin filaments in mesophyll cells under different light conditions. A, Light-dependent reorganization of cp-actin filaments. Cells of wild-type (WT) and pmi1 leaves were irradiated with serial scans of a 458-nm laser for 30 s (blue light [BL] 30 s) and then incubated in the dark for 4 min (dark [D] 4 min). Next, 3-min serial scans with 458- and 488-nm lasers (BL 3 min) were carried out to induce disappearance of cp-actin filaments. Finally, cells were incubated in the dark for 4 min (D 4 min). Images are false colored to show GFP (green) and chlorophyll (red) fluorescence. Note that cp-actin filaments disappeared after BL irradiation and reappeared after 4 min of adaptation in the dark in both the wild type and pmi1. Bar = 5 µm. B, BL-induced disappearance of cp-actin filaments in wild-type and pmi1 mutant cells. Fluorescence intensities of cp-actin filaments were measured at chloroplast edges in wild-type and pmi1 mutant cells, representing changes in the amount of cp-actin filaments during BL irradiation for 3 min after the 4-min dark adaption. Values are mean ± sd (n = 5 squares) in arbitrary units. C and D, Effect of 488- (C) and 516-nm (D) imaging lasers on avoidance response in pmi1 mutant cells. Time-lapse images were collected at approximately 30-s intervals with two different imaging lasers (488 and 516 nm) for 15 min and 8 s. The blue rectangular regions (region of interest [roi], 10 × 20 µm) were irradiated with a stimulating laser (458 nm) during intervals between the image acquisitions of chlorophyll fluorescence images with the imaging lasers. Chlorophyll fluorescence is false colored in red. Right shows moving paths of individual chloroplasts (a–d). For the full time-lapse series, see Supplemental Movie S2. Bars = 10 µm.

PMI1 Alone Is Essential for the Nuclear Avoidance Response in Mesophyll Cells

We recently showed that cp-actin-dependent photorelocation movement of pavement cell plastids attached to nuclei is required for the motive force generation for nuclear photorelocation movement in Arabidopsis pavement cells and also, mesophyll cells (Higa et al., 2014a). We guessed that pmi1 single mutants but not pmir1pmir2 might be severely defective in the nuclear avoidance response in mesophyll cells, because pmi1 but not pmir1pmir2 exhibited severe defects in chloroplast photorelocation movement (Fig. 1). In both wild-type and pmir1pmir2 plants, approximately 25% of nuclei in dark-adapted plants were in the light position (i.e. approximately 75% of nuclei in the dark position; Fig. 6). Strong blue light induced the nuclear avoidance response, and the response was saturated after 6 h (about 60%–70% of nuclei were light positioned; Fig. 6). However, pmi1 and pmi1pmir1pmir2 mutant plants showed almost no nuclear avoidance response in mesophyll cells, and approximately 25% of nuclei were in the light position over the light irradiation period (Fig. 6). These results showed that PMI1 is necessary for nuclear avoidance response as well as chloroplast photorelocation movement in mesophyll cells.

Figure 6.

Distinct roles of PMI1 and PMIRs on nuclear photorelocation movement in mesophyll cells. Time-course analysis of nuclear avoidance response in mesophyll cells of wild-type (WT), pmi1, pmir1pmir2 double-mutant, and their triple-mutant plants. Nuclear avoidance response was induced by strong blue light (50 µmol m⁻² s⁻¹). The percentage of cells in which the nucleus was in the light position is depicted in mean ± sd. Each data point was obtained from five leaves; 100 cells were observed in each leaf.

PMI1 and PMIR1 Are Essential for the Nuclear Avoidance Response in Pavement Cells

In pavement cells of wild-type plants, most of nuclei were positioned on the cell bottom in darkness (dark position; Fig. 7A, dark) and moved to the anticlinal walls in response to strong blue light (light position; Fig. 7A, blue light; Iwabuchi et al., 2007, 2010; Higa et al., 2014a). We measured the percentage of pavement cells in which the nucleus was in the light position during the irradiation with strong blue light (Fig. 7, B–D). In wild-type plants, approximately 30% of nuclei in dark-adapted plants were in the light position (Fig. 7B), and thus, approximately 70% of nuclei were in the dark position. Strong blue light induced the movement of nuclei from the cell bottom to the anticlinal cell wall. This response was saturated after 9 h (about 70% of nuclei were light positioned; Fig. 7B), reproducing the results reported previously (Higa et al., 2014a). pmir1 and pmir1pmir2 double mutant but not pmir2 similarly showed a slight impairment in strong light-induced nuclear movement. Although the population of nuclei in the light position sharply increased at 3 h after strong blue light irradiation in pmir1 and pmir1pmir2 like in the wild type, the light positioning was almost saturated around 60% at 6 h and even at 12 h after light irradiation, which was slightly less than that of the wild type (approximately 70%; Fig. 7B; Supplemental Table S1), indicating that PMIR1 but not PMIR2 is involved in nuclear photorelocation movement in pavement cells. This result is consistent with the fact that PMIR2 is not expressed in green parts (only very weak expression in roots; Supplemental Fig. S2). In pmi1, nuclear photorelocation movement in pavement cells was greatly impaired; even after 12 h, only 57% of nuclei were in the light position (Fig. 7, C and D; Supplemental Table S1). Notably, pmi1pmir1 double- and pmi1pmir1pmir2 triple-mutant plants lacked light-induced nuclear movement, and approximately 40% to 50% of nuclei were in the light position, regardless of the light conditions (Fig. 7, C and D). The defective light-induced nuclear movement in the pmi1pmir2 double- and pmi1pmir1pmir2 triple-mutant plants was similar to that in the pmi1 single- and pmi1pmir1 double-mutant plants (Fig. 7D; Supplemental Table S1). When light-adapted plants were transferred to dark conditions, the nuclei moved from the anticlinal walls to the cell bottom, and it took approximately 20 h to complete the dark positioning (Supplemental Fig. S4). Although dark positioning occurred in pmi1, pmir1pmir2, and pmi1pmir2, there was no detectable dark positioning in pmi1pmir1 and pmi1pmir1pmir2, mirroring the defective light-induced nuclear movement in these mutants (Supplemental Fig. S4). Importantly, clear blue light-induced avoidance movement of pavement cell plastids occurred in the wild type (8 of 11 examined plastids) and pmi1 (5 of 13 examined plastids) but not in pmi1pmir1pmir2 (0 of 7 examined plastids; Supplemental Movie S3). These results indicated that, in pavement cells, PMI1 and PMIR1 redundantly mediate the avoidance responses of nuclei and pavement cell plastids.

Figure 7.

Distinct roles of PMI1 and PMIRs on nuclear photorelocation movement in pavement cells. A, Representative images showing dark position (left) and light position (right) of nuclei under the strong blue light (BL) in pavement cells of wild-type Arabidopsis. Bar = 25 µm. B to D, Time-course analysis of nuclear avoidance response in pavement cells of wild-type (WT), pmi1, pmir1, pmir2 single, and their double- and triple-mutant plants. The other details are the same as in Figure 6.

DISCUSSION

Although PMI1 was identified through the analysis of a mutant deficient in chloroplast photorelocation movement a decade ago (DeBlasio et al., 2005), the roles of PMI1 and its homologous proteins PMIR1 and PMIR2 in not only chloroplast photorelocation movement but also nuclear photorelocation movement remained to be determined. Therefore, we aimed to analyze the physiological and cellular functions of PMI1 and homologous PMIR proteins in Arabidopsis. Our findings showed that the pmi1 mutant plants are defective in both chloroplast accumulation and the avoidance response and that the defective chloroplast movement resulted from the impaired regulation of cp-actin filaments in pmi1 mutant cells. Furthermore, our results revealed that PMI1 and PMIR1 are essential for the nuclear avoidance response.

PMI1 is a plant-specific protein in the C2-domain superfamily (DeBlasio et al., 2005; Zhang and Aravind, 2010). The typical C2 domain of protein kinase C binds lipid in a calcium-dependent manner and thus, is involved in membrane targeting (Rizo and Südhof, 1998; Zhang and Aravind, 2010). PMI1 contains a C2 domain at the N terminus and a C-terminal conserved region that is found in plant PMI1 and PMIR proteins (DeBlasio et al., 2005). PMI1 is further classified into the N-terminal C2 (NT-C2) family within the C2 superfamily (Zhang and Aravind, 2010). As its name suggests, the NT-C2 family contains the C2 domain at the N terminus; this family was recently identified as one of the four new C2 subfamilies (Zhang and Aravind, 2010). Although the exact function of the C2 domain in NT-C2 family proteins has yet to be determined, the N-terminal conserved region including the C2 domain of PMI1 might be essential for PMI1 function. pmi1-2 carries a T-DNA insertion that might result in a truncated PMI1 consisting of the entire N-terminal region, including the C2 domain. The phenotype of pmi1-2 is weaker than that of pmi1-5. The sequence of pmi1-5 carries a premature stop codon that might result in a PMI1 N-terminal fragment lacking the intact conserved N-terminal region, suggesting that the N-terminal region including the C2 domain retains some function of PMI1 if it is expressed.

Several NT-C2-domain family proteins contain a domain at the C terminus that is involved in regulating actin filaments (for example, the Dilute- and Calponin-homologous domains; Zhang and Aravind, 2010), suggesting that NT-C2 family proteins might function in regulating actin filaments. A previous study reported that the pmi1 mutant showed a normal pattern of cortical actin filaments (DeBlasio et al., 2005). However, we found that the pmi1 mutant was defective in the regulation of cp-actin filaments, which are essential for photorelocation movement and the attachment of chloroplasts to the plasma membrane (Kadota et al., 2009; Kong et al., 2013a). These observations indicated that PMI1 mediates chloroplast photorelocation movement by the regulation of cp-actin filaments. Although our genetic analyses suggested that PMI1 functions primarily in the phot2 signaling pathway, the defects in cp-actin filaments differed between phot2 and pmi1. cp-actin filament dynamics in the phot2 mutant cells were defective specifically in the process of depolymerization in response to strong blue light (Kadota et al., 2009; Kong et al., 2013a). Although the fundamental processes of cp-actin filament dynamics, including actin polymerization and depolymerization, were normal in pmi1 cells, they were much more sensitive to blue light-dependent depolymerization than wild-type cells. Consequently, the asymmetric distribution of cp-actin filaments was poorly established in pmi1 cells, in which the 488-nm imaging laser may have been sufficient to activate the phot signal. These results suggested that PMI1 is a downstream signaling factor that functions in the signaling pathway from light perception to actin-based movement, including the regulation of cp-actin filaments.

Because the interface between chloroplasts and the plasma membrane is the important site for generation of cp-actin filaments and thus, the motive force for chloroplast movement (Kadota et al., 2009; Suetsugu et al., 2010a; Kong et al., 2013a), factors for chloroplast photorelocation movement must be present in this area. CHUP1 and some phots (especially phot2) are localized on the chloroplast outer envelope (Oikawa et al., 2008; Schmidt von Braun and Schleiff, 2008; Kong et al., 2013b), although most phots are localized on the plasma membrane (Sakamoto and Briggs, 2002; Kong et al., 2006). KAC proteins were present in both the soluble and microsomal fractions, suggesting that some portion of KAC proteins is localized on the plasma membrane (Suetsugu et al., 2010b). JAC1 was detected in the microsomal fraction (Fig. 3B). PMI1-GFP fluorescence was detected mainly in the cytoplasm of mesophyll cells (Fig. 3A). Although PMI1 proteins were identified in the proteome data for the plasma membrane protein (Nühse et al., 2003, 2004; Zhang and Peck, 2011), we could not detect a specific association of PMI1-GFP with the plasma membrane and/or organelles in the microscopic analysis.

A previous study identified PMI1 homologs in monocot (rice [Oryza sativa] and corn [Zea mays]) and legume species (soybean [Glycine max] and Medicago trunculata; DeBlasio et al., 2005). Two Arabidopsis proteins (PMIR1 and PMIR2) distantly similar to PMI1 (DeBlasio et al., 2005) were also identified. Detailed database searches and phylogenetic analyses revealed that PMI1/PMIR proteins are present in most land plants and the green alga Klebsormidium flaccidum (Supplemental Fig. S5). However, PMI1-clade proteins are found only in seed plants, indicating that the separation between PMI1 and PMIR clades occurred before the separation between gymnosperms and angiosperms. Thus, it is plausible that ancestral PMI1/PMIR proteins (i.e. nonseed plant PMI1/PMIR proteins) have the ability to regulate chloroplast photorelocation movement and that the functional divergence between PMI1 and PMIR clades in seed plants occurred during the seed plant evolution in such a way of tissue-specific expression.

Although the involvement of PMIR1 and PMIR2 in chloroplast photorelocation movement is unclear in mesophyll cells, PMIR1 together with PMI1 are essential for the nuclear avoidance response in pavement cells (Supplemental Fig. S6). The nuclear avoidance response is mediated by nucleus-attached pavement cell plastids in a cp-actin filament-dependent manner (Higa et al., 2014a). The pmi1pmir1pmir2 plants were defective in the blue light-induced avoidance response of pavement cell plastids, although pmi1 retained the avoidance response of pavement cell plastids (Supplemental Movie S3), indicating that PMI1 and PMIR1 redundantly mediate the blue light-induced avoidance response of pavement cell plastids. A tissue-specific translatome analysis showed that PMIR1 was expressed preferentially in leaf pavement cells (Mustroph et al., 2009; Supplemental Fig. S2C), supporting the specific function of PMIR1 in pavement cells.

Although both PMI1 and PMIR1 were required for the avoidance responses of pavement cell plastids and nuclei in pavement cells, PMI1 alone was essential for chloroplast and nuclear avoidance responses in mesophyll cells (Supplemental Fig. S6). Thus, defects in the photorelocation movements of pavement plastids and chloroplasts were strongly correlated with the defective nuclear avoidance response in both pavement and mesophyll cells, respectively. The chup1 mutant showed impaired chloroplast and nuclear avoidance responses in mesophyll cells (Higa et al., 2014a). Furthermore, in the jac1 mutant, chloroplasts and nuclei were localized constitutively on the anticlinal walls (Suetsugu et al., 2005; Higa et al., 2014a). Therefore, it is plausible that light-induced movement of chloroplasts is essential for the nuclear avoidance response in mesophyll cells. However, there is no direct evidence for the chloroplast-mediated nuclear movement, because it is too difficult to analyze the nuclear movement independent of chloroplasts in mesophyll cells in which the nucleus is always surrounded with many chloroplasts.

In conclusion, our results showed that PMI1 plays an important role in cp-actin-mediated chloroplast photorelocation movement in mesophyll cells and that PMIR1 together with PMI1 are essential for cp-actin-mediated photorelocation movement of pavement cell plastids (Supplemental Fig. S6). Our results also showed that PMI1- and PMI1/PMIR1-dependent photorelocation movements of chloroplasts and pavement cell plastids are required for the motive force generation for nuclear photorelocation movement in mesophyll and pavement cells, respectively. Because cryptogamic land plants, such as bryophytes and lycophytes, have PMI1-like genes, it is plausible that PMI1 like is necessary for chloroplast and nuclear photorelocation movements in these plants as well. Detailed analyses of PMI1/PMIR1 in Arabidopsis and PMI1 orthologs in cryptogamic land plants are required to unravel the molecular mechanism of these responses.

MATERIALS AND METHODS

Plant Materials, Plant Growth, and Mutant Screening

Arabidopsis (Arabidopsis thaliana) seeds (Columbia-0, glabrous1) were sown on one-third-strength Murashige and Skoog culture medium containing 1% (w/v) Suc and 0.8% (w/v) agar. After incubation for 2 d at 4°C, the seedlings were cultured under white light at approximately 100 µmol m⁻² s⁻¹ under a 16-h/8-h light-dark cycle at 23°C in a growth chamber. Approximately 2-week-old seedlings were used for mutant screening and analyses of chloroplast and nuclear photorelocation movements. The band assay used to screen mutants and isolate those deficient in chloroplast photorelocation movement has been described previously (Kagawa et al., 2001; Oikawa et al., 2003; Suetsugu et al., 2005; Kodama et al., 2010). The SALK T-DNA insertion lines (set of SALK T-DNA lines [CS27943]; pmi1-2 [SALK_141795; DeBlasio et al., 2005]; pmir1-1 [SALK_098762]; and pmir2-1 [SALK_055706]) and the N7 nuclear marker line (Cutler et al., 2000) were provided by the Arabidopsis Biological Stock Center. According to previous reports (DeBlasio et al., 2005; Rojas-Pierce et al., 2014), our pmi1 mutant line was named pmi1-5. Double- and triple-mutant plants were generated by genetic crossing. Mutant lines containing the N7 nuclear marker and GFP-mouse-talin (Kadota et al., 2009; Kong et al., 2013a) were generated by genetic crossing.

Generation of Transgenic Plants

To construct the PMI1pro::PMI1-GFP vector, GFP complementary DNA was cloned into the pPZP221/35S-nopaline synthase terminator (nosT) binary vector (Hajdukiewicz et al., 1994) using the KpnI and SalI restriction sites, yielding pPZP221/35S::GFP-nosT. A PMI1 gene fragment including the 2,817-bp 5′ sequence (before the start codon) and the gene body region including the open reading frame but lacking the stop codon were cloned into the KpnI site of pPZP221/35S-GFP-nosT. The pmi1-5 mutants were transformed with pPZP221/PMI1pro::PMI1-GFP-nosT by the floral-dipping method using Agrobacterium tumefaciens (GV3101::pMP90).

Analyses of Chloroplast Photorelocation Movement

Chloroplast photorelocation movement was analyzed by measuring changes in leaf transmittance as described previously (Kodama et al., 2010; Wada and Kong, 2011). The third leaves were detached from 16-d-old seedlings and placed on 1% (w/v) gellan gum in a 96-well plate. Samples were dark adapted at least for 1 h before transmittance measurements.

Analyses of Nuclear Photorelocation Movement

Time-course experiments for nuclear photorelocation movement were performed as described previously (Higa et al., 2014a). For strong light-induced nuclear movement, 2-week-old plants were dark adapted for 24 h and irradiated with 50-µmol m⁻² s⁻¹ blue light for 12 h. The leaves were collected and fixed at 0, 3, 6, 9, and 12 h after light irradiation as described previously (Higa et al., 2014a). To analyze dark-induced nuclear movement, 2-week-old plants were irradiated with 50-µmol m⁻² s⁻¹ blue light for 12 h and then dark adapted. The leaves were collected and fixed after 12, 16, 20, and 24 h of dark adaptation.

Immunoblot Analyses

Crude protein extracts were prepared from 2-week-old rosette leaves and fractionated as described previously. Immunoblot analysis was performed as previously described (Suetsugu et al., 2010b).

Confocal Laser-Scanning Microscopy

The subcellular localization of PMI1-GFP and cp-actin filaments and nuclear photorelocation movement were observed under a confocal microscope (SP5; Leica Microsystems) as described previously (Kong et al., 2013a; Higa et al., 2014a). The multi-Ar laser was used at 488 nm for GFP and 458 nm (the output laser power of 2.8 µW) for the chloroplast and nuclear avoidance responses. The fluorescent signals were captured through the narrow bands of 500 to 550 nm for GFP and 650 to 710 nm for chlorophyll autofluorescence.

Phylogenetic Analysis of PMI1 and PMIR Proteins

Multiple alignment, alignment curation, phylogenetic tree construction, and tree visualization were performed using MUSCLE (Edgar, 2004), Gblocks (Castresana, 2000), PhyML (Guindon and Gascuel, 2003), and TreeDyn (Chevenet et al., 2006) outputs, respectively, according to a predefined pipeline at the Phylogeny.fr server (Dereeper et al., 2008).

Sequence data from this article can be found in The Arabidopsis Information Resource (TAIR10) database under accession numbers PMI1 (At1g42550), PMIR1 (At5g20610), and PMIR2 (At5g26160). Accession numbers and gene identifiers for genes used in phylogenetic analyses are provided in Supplemental Figure S5.

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Initial transmittance in leaves of dark-adapted wild-type and pmi1/pmir mutant plants.

• Supplemental Figure S2. Transcript and protein expression data of PMI1, PMIR1, and PMIR2 from Arabidopsis genome-wide transcriptome, translatome, and proteome database.

• Supplemental Figure S3. Leaf transmittance changes indicative of chloroplast photorelocation movement in mesophyll cells in PMI1pro::PMI1-GFP lines.

• Supplemental Figure S4. PMI1 and PMIR1, but not PMIR2, are essential for nuclear dark positioning in pavement cells.

• Supplemental Figure S5. Phylogenetic tree of PMI1/PMIR proteins.

• Supplemental Figure S6. Roles of PMI1/PMIR proteins.

• Supplemental Table S1. Statistical tests for the data mentioned in the text.

• Supplemental Movie S1. Reorganization of cp-actin filaments in wild-type and pmi1 cells during strong blue light-induced chloroplast avoidance response.

• Supplemental Movie S2. Strong blue light-induced chloroplast avoidance response in pmi1 mutant cells.

• Supplemental Movie S3. Observation of pavement cell plastid irradiated with strong blue light in pmi1 and pmi1pmir1pmir2 pavement cells.

Glossary

cp-actin

chloroplast-actin

T-DNA

transfer DNA