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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Plastid Movement Impaired 2, a New Gene Involved in Normal Blue-Light-Induced Chloroplast Movements in Arabidopsis^1,[W]

Department of Biology, Indiana University, Bloomington, Indiana 47405

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Chloroplasts move in a light-dependent manner that can modulate the photosynthetic potential of plant cells. Identification of genes required for light-induced chloroplast movement is beginning to define the molecular machinery that controls these movements. In this work, we describe plastid movement impaired 2 (pmi2), a mutant in Arabidopsis (Arabidopsis thaliana) that displays attenuated chloroplast movements under intermediate and high light intensities while maintaining a normal movement response under low light intensities. In wild-type plants, fluence rates below 20 µmol m^–2 s^–1 of blue light lead to chloroplast accumulation on the periclinal cell walls, whereas light intensities over 20 µmol m^–2 s^–1 caused chloroplasts to move toward the anticlinal cell walls (avoidance response). However, at light intensities below 75 µmol m^–2 s^–1, chloroplasts in pmi2 leaves move to the periclinal walls; 100 µmol m^–2 s^–1 of blue light is required for chloroplasts in pmi2 to move to the anticlinal cell walls, indicating a shift in the light threshold for the avoidance response in the mutant. The pmi2 mutation has been mapped to a gene that encodes a protein of unknown function with a large coiled-coil domain in the N terminus and a putative P loop. PMI2 shares sequence and structural similarity with PMI15, another unknown protein in Arabidopsis that, when mutated, causes a defect in chloroplast avoidance under high-light intensities.

In Arabidopsis and other angiosperms studied, light-induced chloroplast movement is initiated by blue light via members of the phototropin family of photoreceptors, whereas in algae, moss, and ferns, red light can also initiate movement (Kagawa and Wada, 1994, 1996, Kagawa and Wada, 2000; Jarillo et al., 2001; Kagawa et al., 2001, 2003; Sakai et al., 2001). Previous studies have shown that phototropin1 (phot1) and phototropin2 (phot2) function redundantly under low-intensity blue light to mediate the accumulation response, whereas phot2 appears to drive the avoidance response to high-intensity blue light. Specifically, mutations in phot1 show a slight attenuation in accumulation under low-intensity blue light, but behave normally under high-intensity blue light (Kagawa and Wada, 2000). Mutants in phot2 show chloroplast accumulation on the periclinal cell walls in response to all blue-light intensities tested, even when exposed to high-light conditions that would initiate an avoidance response in wild-type and phot1 plants (Kagawa et al., 2001; Sakai et al., 2001). Plants with mutations in both phototropins lack light-induced chloroplast movement under all tested light conditions (Sakai et al., 2001).

In Lemna triscula, the actin-depolymerizing agent cytochalasin B eliminated chloroplast movement, whereas microtubule-depolymerizing drugs had no impact on movement (Tlalka and Gabrs, 1993). In Arabidopsis mesophyll cells, actin is associated with the outer chloroplast envelope and forms a network of thick and thin filaments, which, when disrupted by lantrunculin B, resulted in abnormal chloroplast aggregation (Kandasamy and Meagher, 1999). Chloroplasts in the chloroplast unusual positioning 1 (chup1) mutant aggregate along abaxial walls in mesophyll cells and fail to move in response to any light treatment (Oikawa et al., 2003). CHUP1 localizes to the chloroplast outer envelope and glutathione S-transferase fusion proteins containing the predicted actin-binding domain of CHUP1 coprecipitated with F-actin, consistent with a possible role in mediating interactions between the actin cytoskeleton and chloroplasts.

Here we present the identification and characterization of a new chloroplast movement mutant, plastid movement impaired 2 (pmi2). Under low-light conditions, pmi2 shows normal accumulation of chloroplasts along periclinal cell walls. However, under high-light conditions, movement to the anticlinal cell walls is severely attenuated compared to wild type. The PMI2 gene is predicted to encode a protein with long coiled-coil domains, a putative P loop, and putative nuclear-localization signals. A T-DNA mutant in a homologous gene, pmi15, also displayed attenuated chloroplast movements under high blue light. PMI2 and PMI15 appear to represent newly identified proteins involved in light-induced chloroplast movements.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Chloroplast Movements in pmi2 Leaves

The pmi2 mutant was isolated in a screen designed to identify chloroplast movement mutants by measuring changes in red-light transmittance through leaves (Walczak and Gabrs, 1980; DeBlasio et al., 2005). In Arabidopsis, red light does not initiate chloroplast movement, but it is absorbed efficiently by chlorophyll. As chloroplasts relocate in leaf cells, the amount of red light transmitted through the leaf changes, leading to decreases in light transmittance as chloroplasts accumulate along the periclinal cell walls and increases in transmittance when chloroplasts gather on anticlinal walls (Trojan and Gabrys, 1996; Kagawa and Wada, 2000).

Wild-type leaves exposed to sequential 60-min treatments of low (0.2 µmol m^–2 s^–1), intermediate (20 µmol m^–2 s^–1), and high (60 µmol m^–2 s^–1) blue light responded with distinct chloroplast movement responses, as indicated by changes in red-light transmittance (Fig. 1A ). Exposure to 0.2 µmol m^–2 s^–1 resulted in chloroplast accumulation along periclinal cell walls and a corresponding drop in red-light transmittance. Subsequent treatments of 20 and 60 µmol m^–2 s^–1 led to an avoidance response as seen by increased red-light transmittance as the chloroplasts moved to anticlinal sides of cells. Leaves from pmi2 displayed a normal accumulation response under 0.2 µmol m^–2 s^–1, but failed to achieve an avoidance response under higher light intensities and instead showed accumulation on the periclinal walls (Fig. 1A). There were, however, slight transient increases in red-light transmittance immediately following initiation of the high blue-light treatments, indicating that pmi2 can perceive this signal but is unable to develop normal chloroplast movements to anticlinal walls.

View larger version (23K): [in this window] [in a new window]   Figure 1. Chloroplast movements in the pmi2 mutant. A, Chloroplast movements in wild-type and two pmi2 mutants in response to sequential treatments of increasing fluence rates of blue light. Red-light transmittance was measured for 90 min in dark-acclimated leaves to establish a baseline, followed by sequential, 60-min treatments of 0.2, 20, and 60 µmol m–2 s–1 blue light. Blue-light intensity changes occurred at 90, 150, and 210 min (indicated by arrows) for low-, intermediate-, and high-light treatments. Red-light transmittance was recorded every 5 min. For wild type, pmi2-1, and pmi2-2, n = 17, 7, and 7, respectively. B, The fluence-rate response of wild type and pmi2 after 90-min exposures to single fluence rates of blue light. Data points represent the final time points from C and D. C and D, Time course of red-light transmittance changes through leaves in response to different fluence rates of blue light. Red-light transmittance was measured for 60 min in dark-acclimated leaves to establish a baseline before the blue-light treatments were initiated. Red-light transmittance was recorded every 3 min. Data at 5 µmol m–2 s–1 were similar to data at 1 µmol m–2 s–1 and were omitted to reduce crowding on the graph. The data are the average ± SE from leaves of five to 15 plants per light treatment.

The pmi2 phenotype is most pronounced under high fluence rates of blue light. In wild type, 100 µmol m^–2 s^–1of blue light caused robust avoidance movement of chloroplasts to the anticlinal cell walls, which increased red-light transmittance by about 3% (Fig. 1, B and C). At that intensity of blue light, the response in pmi2 leaves was strongly attenuated, reaching a change in light transmittance slightly above the initial dark value (Fig. 1, B and D). At light intensities between 20 and 100 µmol m^–2 s^–1, which induce a robust avoidance response in wild type, pmi2 showed biphasic transmittance changes similar to those displayed by wild type under 10 µmol m^–2 s^–1 of blue light. As the intensity of blue light increased above about 20 µmol m^–2 s^–1, the magnitude of the accumulation phase decreased and the longevity of the avoidance phase of the biphasic response increased in pmi2. These data show that, although pmi2 can perceive high-light treatments, it is defective in some aspect of chloroplast movement following perception of intermediate and high-light intensities because it fails to develop a normal movement response at all fluence rates above 10 µmol m^–2 s^–1 (Fig. 1B).

To confirm that the observed changes in red-light transmittance reflected changes in chloroplast movement in pmi2, chloroplast movements were recorded by time-lapse microscopy of dark-acclimated leaves during exposure to sequential treatments of low- and high-intensity white light (Supplemental Movie 1). Under low-intensity white light, chloroplasts of both wild type and pmi2 were found to accumulate along the periclinal walls of palisade cells. Subsequent irradiation with high-intensity white light initiated movement to the anticlinal walls. However, the high-light-induced movements in pmi2 showed a decrease in the rate of movement as well as differences in chloroplast positioning.

Analysis of cross sections of leaves that had been irradiated for 60 min with low (5 µmol m^–2 s^–1) or high (60 µmol m^–2 s^–1) blue light allowed visualization of the final chloroplast position in all cell layers. After dark treatment, both wild type and pmi2 had about 57% of their chloroplasts located on the periclinal cell walls and 43% on the anticlinal walls (Fig. 2A ). After exposure to low-fluence-rate blue light, 70% of chloroplasts in both wild-type and pmi2 cells were located along periclinal cell walls. However, under high fluence rates, only 40% of wild-type chloroplasts remained along periclinal cell walls, whereas pmi2 had 53% residing there. Although the pmi2 defect is seen throughout the leaf, the defect is most pronounced in the palisade layer, which shows a 17% difference in chloroplast location between wild type and pmi2 compared to a 10% difference in both lower mesophyll layers (Fig. 2B). Consistent with the light transmittance results, pmi2 chloroplasts have roughly the same distribution after dark and high-light treatments.

View larger version (24K): [in this window] [in a new window]   Figure 2. Chloroplast distribution in mesophyll cells. A, Average distribution of chloroplasts along anticlinal and periclinal walls of all cell layers. B, Distribution of chloroplasts in mesophyll cell layers after high blue-light treatment. Data in A and B represent the average percentage (±SD) from 15 cross sections.

Lines expressing a green fluorescent protein (GFP) reporter fused to the actin-binding domain of mouse talin (GFP:mTalin) were crossed with pmi2 to allow the actin cytoskeleton of living pmi2 cells to be examined (Kost et al., 1998). As previously reported, Arabidopsis mesophyll cells have thick and thin filaments of actin, as well as shells of polymerized actin surrounding wild-type and pmi2 chloroplasts (Kost et al., 1998; Kandasamy and Meagher, 1999). No noticeable differences in the arrangement of the actin network in wild-type (Fig. 3, A and B ) and pmi2 (Fig. 3, C and D) leaf cells were observed by confocal microscopy. It should be noted, however, that although GFP:mTalin is often used to study actin-based processes in living cells, in our experiments it attenuated light-induced chloroplast movements to roughly one-half the magnitude of that seen in wild-type leaves (Fig. 3E). The cause of this inhibition is unknown, but raises the possibility that the GFP:mTalin may alter the actin cytoskeleton sufficiently to obscure subtle differences in mutant plants.

View larger version (69K): [in this window] [in a new window]   Figure 3. Actin cytoskeleton of wild-type and pmi2 mesophyll cells. Fluorescence images of wild-type (A and B) and pmi2 (C and D) leaves expressing GFP:mTalin. A and C, Composite image of GFP and chlorophyll autofluorescence. B and D, Only GFP. Bar = 10 µm. E, Chloroplast movement in wild type (n = 17) and in plants expressing GFP:mTalin (n = 7) as measured in Figure 1A.

Genetic polymorphisms and recombination frequencies were analyzed in F₂ plants obtained from a cross between pmi2 Columbia (Col) and Landsberg erecta (Ler) to map pmi2 to a 134-kb region on chromosome I, which was covered by one complete and two partial bacterial artificial chromosomes (BACs). The largest of these, F4N21, spanning 97 kb, was partially digested and transformed into Escherichia coli. A contiguous group of clones was used to transform pmi2-1 plants. The mutant phenotype was repeatedly rescued by two clones, analysis of which identified a 13-kb interval that contained one partial and four complete open reading frames. Sequencing of this region in pmi2 revealed a cytosine to Tyr mutation resulting in a premature stop codon at amino acid 323 of At1g66840 (Fig. 4A ). A Salk insertion line (SALK_088187 designated pmi2-2), with a T-DNA insert in the second exon of At1g66840 (Alonso et al., 2003), also displayed the pmi2 phenotype (Fig. 1A) and failed to complement pmi2-1 (data not shown). Taken together, these results indicated that PMI2 is At1g66840.

View larger version (19K): [in this window] [in a new window]   Figure 4. Location of pmi2 mutations and predicted protein structure. A, Diagram of PMI2 exons (boxes) and introns (lines) as well as the location of the mutations in pmi2-1 and pmi2-2. B, Diagram of PMI2 predicted protein structure.

A search of the PROSITE database (Gattiker et al., 2002) indicated a putative ATP-/GTP-binding motif A (P loop) from amino acids 560 to 567 (Saraste et al., 1990) and a putative nuclear-localization signal starting at amino acid 584. Further analysis with PredictNLS identified an additional potential nuclear-localization signal in PMI2 in addition to the one identified by PROSITE (Cokol et al., 2000). The first is located at the C terminus of the protein, spanning amino acids 584 to 603. The other, located in the coiled-coil region and extending from amino acids 81 to 88, is also predicted by PredictNLS to bind DNA.

Localization of PMI2

A GFP:PMI2 fusion containing the putative P loop and the C-terminal nuclear-localization domain was created by inserting the carboxy terminus of PMI2 (Glu-509 to Gln-607) into the vector pEGAD (creating construct pP2CTPGD). Wild-type and pmi2-1 plants expressing pP2CTPGD and the control pEGAD showed strong GFP fluorescence in the cytoplasm, but no noticeable localization to the nucleus or any organelle was observed for pP2CTPGD (data not shown). Furthermore, analysis of chloroplast movement in pmi2 plants transformed with pP2CTPGD showed that this construct is not sufficient to rescue the pmi2 mutant phenotype (Fig. 5 ). Leaves from wild-type plants transformed with pEGAD or pP2CTPGD and visibly expressing GFP showed normal chloroplast movements upon exposure to 60 µmol m^–2 s^–1 blue light (Fig. 5C). However, leaves from wild-type plants transformed with pP2CTPGD, but with no visible GFP, displayed only about 0.8% change in red-light transmittance. Although RNA and protein levels in these plants were not examined, activity of the GFP, pP2CTPGD, and native PMI2 were most likely limited by gene silencing, providing further evidence that the mutation found in At1g66840 is responsible for the pmi2 phenotype.

View larger version (13K): [in this window] [in a new window]   Figure 5. Expression of pP2CTPGD. Chloroplast movements in plants transformed with pP2CTPGD and exposed to 75 µmol m–2 s–1 of blue light. Transformed wild-type plants not expressing GFP (presumably due to gene silencing) showed an attenuated response, whereas plants expressing GFP showed a robust movement response.

To determine where PMI2 is transcribed, reverse transcription (RT)-PCR was performed on RNA collected from different parts of wild-type and pmi2 plants (Fig. 6 ). Results indicate that PMI2 is expressed in rosette and cauline leaves, stems, flowers, and roots of wild-type plants as well as in leaves of pmi2-1 and pmi2-2. The presence of PMI2 RNA in pmi2 mutant leaves indicates that transcription is not inhibited by the mutations in these lines. The presence of PMI2 RNA in nonphotosynthetic tissue suggests that PMI2 may have functions in addition to the regulation of chloroplast movement. RT-PCR using primers downstream of the T-DNA insert in pmi2-2 resulted in cDNA amplification, indicating that transcription in this mutant continues through the insert (data not shown).

View larger version (38K): [in this window] [in a new window]   Figure 6. PMI2 gene expression. RT-PCR was performed on RNA extracted from rosette leaves of pmi2-1 (2-1) and pmi2-1 (2-2) and from various parts of wild-type plants: rosette leaves (RL), cauline leaves (CL), flowers (F), roots (R), and stems (S). Bands represent a 162-bp product. DNA extract was used as a negative control and primers designed to ubiquitin-conjugating enzymes represent a positive control.

PMI2-Related Genes

Analysis of the Arabidopsis genome revealed that At5g38150 (GenBank accession no. NM_123175) has 55% identity and 65% similarity to PMI2. Structurally, At5g38150 is also predicted to contain a long coiled-coil domain in the N terminus, but lacks the consensus sequences for a P-loop or nuclear localization. To determine whether At5g38150 functions in chloroplast movement, lines with T-DNA insertions in the predicted At5g38150 open reading frame were obtained from the SALK collection and tested for light-induced chloroplast movements. Leaves from SALK_047862 plants displayed attenuated chloroplast movement in response to a high blue-light treatment (Fig. 7 ). The change in red-light transmittance was 2%, compared to 3% in wild type, after 60 min of 60 µmol m^–2 s^–1 of blue light. Due to the pmi phenotype, we have designated this T-DNA mutant as pmi15.

View larger version (27K): [in this window] [in a new window]   Figure 7. Chloroplast movements in pmi15 and pmi2 pmi15. Chloroplast movements in wild type, pmi2, pmi15, and pmi2 pmi15 double mutants measured as described in Figure 1A. For wild type, pmi2-1, pmi15, and pmi2 pmi15, n = 17, 7, 11, and 8 plants, respectively.

Analysis of available genomes has revealed a protein in rice (Oryza sativa; BAD82493, GenBank accession no. NP_947041) that is predicted to encode a 1,051-amino acid protein that shares 31% identity and 45% similarity with PMI2 in its carboxy terminus. Like PMI2, it has a long coiled-coil segment (amino acids 480–980) followed by a region with no known motifs. The N-terminal region of BAD82493 does not appear to share significant homology with other known proteins. The consensus sequences for nuclear localization and the putative P loop found in PMI2 are not present in BAD82493; however, PredictNLS indicates three other putative nuclear-localization signals located at positions 271, 326, and 340. Alignments of PMI2, PMI15, and BAD82493 revealed the presence of two highly conserved regions between amino acids 166 to 191 and 448 to 487 in PMI2 (Fig. 8 ). However, these conserved regions do not appear to share homology with any known functional domains.

View larger version (62K): [in this window] [in a new window]   Figure 8. Alignment of PMI2 (top) and PMI15 (bottom). Black shading indicates identical amino acid sequence in both proteins. Gray shading indicates similar amino acid composition. Dashes indicate gaps inserted to facilitate alignment.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

We have identified PMI2 and PMI15 as genes that are required for proper light-induced chloroplast movements. Under low blue light, pmi2 mutants displayed normal chloroplast movement, whereas under high blue light, pmi2 had markedly attenuated chloroplast movements. Whereas wild-type leaves displayed an avoidance response when exposed to light intensities >20 µmol m^–2 s^–1, pmi2 leaves required at least 75 to 100 µmol m^–2 s^–1 to produce avoidance movement (Fig. 1). Analysis of leaf cross sections confirmed that the blue-light-induced changes in red-light transmittance measured in pmi2 were a result of fewer chloroplasts moving from the periclinal to the anticlinal cell walls in all cell layers of leaves (Fig. 2).

The two alleles of pmi2 we have identified displayed slightly different phenotypes, with pmi2-2 (T-DNA) showing a less severe reduction in movement under intermediate and high-light conditions than pmi2-1 (ethyl methanesulfonate nonsense; Fig. 1A). The cause of this is unknown. It is possible that pmi2-2 contains a full-length protein, including an insert, which reduces its functionality but does not eliminate it. Conversely, it is possible that the early stop codon in pmi2-1 produces a truncated protein that can have a dominant negative effect on chloroplast movement. However, this is less likely because pmi2-1 appears to be fully recessive. Antibodies to PMI2 will be required to determine whether there is a stable protein product in either allele. In any case, the phenotypes of both pmi2-1 and pmi2-2 support the requirement of pmi2 for normal light-induced chloroplast movement under intermediate and high-light intensities.

The phenotype of pmi2 is distinct from that of other known chloroplast movement mutants. For example, the pmi1 and chup1 mutants display no light-induced chloroplast movement (Oikawa et al., 2003; DeBlasio et al., 2005), the phot1 mutant has a slightly attenuated low-light response (Kagawa and Wada, 2000), phot2 has an accumulation response under any fluence rate of light (Kagawa et al., 2001; Sakai et al., 2001), and the jac1 mutant lacks the low-light accumulation response while retaining the high-light avoidance response (Suetsugu et al., 2005). To some degree, the phenotype of pmi2 resembles that of phot2 because it shows an accumulation response under high light. However, pmi2 displays unique responses when exposed to increasing fluence rates of light, whereas phot2 develops and maintains the same magnitude of chloroplast accumulation under all tested fluence rates between 1 and 120 µmol m^–2 s^–1 (Jarillo et al., 2001; Kagawa et al., 2001). Based on the upward trajectory in our fluence-rate response data for pmi2 (Fig. 1B), we expect that increasing the blue-light intensity above the 100 µmol m^–2 s^–1 we have tested would lead to a more robust avoidance response in pmi2.

Analysis of the predicted structure of PMI2 has provided few clues to its possible function. Publicly available motif databases suggest three possible functional domains within PMI2: a long coiled-coil domain, two putative nuclear-localization signals, and a putative P loop. BLAST analysis of the Arabidopsis genome with PMI2 revealed one homologous gene, PMI15, for which the predicted amino acid sequences have 55% identity and 65% similarity. Analysis of chloroplast movement in T-DNA mutant pmi15 indicates that it has an attenuated response under high blue light, although the phenotype is not as severe as in the pmi2 mutant (Fig. 7). The pmi2 pmi15 double mutant had a reduced chloroplast movement response very similar to pmi2 single mutants. Although the double mutant had a slightly greater attenuation under intermediate light conditions, it is apparent that PMI2 is the major player for chloroplast movement.

Structural comparison of PMI2 and PMI15 may provide some insight into the potential functional domains in PMI2. Analysis of PMI15 sequence indicates that the putative P-loop and nuclear-localization motifs predicted in PMI2 are absent in PMI15. Furthermore, the program PredictNLS did not identify any nuclear-localization sequences in PMI15. The only predicted functional domain that is shared between the two proteins is the long coiled-coil span that makes up the N terminus. Interestingly, the coiled-coil segments share a 50% identity, whereas the C-terminal portions of the protein that are not predicted to form coils share a 68% identity.

Analysis of other available plant genomes revealed the presence of BAD82493, a similar protein in rice. The most conserved regions of PMI2, PMI15, and BAD82493 are present in two domains of unknown function, one within the coiled-coil region and one immediately after. Whereas sequence comparisons have given no hints as to the functions of these domains, it is likely that they play an important role in the function of these proteins.

The predicted domain that is most conserved in both the Arabidopsis homolog and the related rice genes is the long coiled-coil region that covers most of the N-terminal two-thirds of the protein. Proteins with long coiled-coil domains have been shown to have diverse functions from structural support to DNA binding. Many coiled-coil proteins are involved in cytoskeletal functions, including roles in microtubule nucleation, spindle organization, and structural support (Gergely et al., 2000; Strilkov et al., 2003). Because PMI2 has no apparent actin-binding domain, it seems unlikely that it directly interacts with actin filaments. Golgins, a family of proteins with long coiled-coil domains that form long rod-like structures, interact with the Golgi apparatus to form a three-dimensional matrix around the Golgi, lending structural support and aiding in vesicle docking (Barr and Short, 2003). The nuclear-localized lamins have long coiled-coil domains and have been shown to comprise part of the nuclear lamina, bind chromatin, and play a role in DNA synthesis (Goldman et al., 2002). Structural maintenance of chromosomes 1 (SMC1) contains two long coiled-coil domains separated by a short hinge region and an ATP-/GTP-binding domain (Strunnikov and Jessberger, 1999). These proteins have been shown to bind DNA and likely play a role in sister chromatid cohesion and DNA recombination (Akhmedov et al., 1998).

The program PredictNLS identified two potential nuclear-localization signals in PMI2. In nonplant systems, proteins containing these sequences were found to be nuclear localized 98% and 99% of the time, and 98% of proteins containing the interior-localization signal have been shown to bind DNA (Cokol et al., 2000). However, to our knowledge, neither sequence has been shown to cause nuclear localization in plants. Furthermore, no identifiable nuclear-localization signals are present in PMI15, suggesting that that PMI2 may not be localized to the nucleus.

We have attempted to examine the localization of PMI2 using a GFP:PMI2-C-terminal fusion protein that contained both the putative P loop and the C-terminal nuclear-localization domain. Our analysis of plants expressing this construct indicated that it was localized in the cytoplasm (Fig. 5). These results suggest that the carboxy-terminal putative nuclear-localization domain does not function in PMI2 to direct the protein to the nucleus. However, until a functional full-length GFP fusion or antibodies can be made, the localization of PMI2 will remain unconfirmed. Ongoing efforts to create a full-length PMI2::GFP fusion have been unsuccessful because constructs containing the coiled-coil portion of the protein have been lethal to E. coli. This problem has hampered efforts to determine localization, overexpress PMI2, produce effective antibodies, test the putative P loop in vitro, and rescue the mutant phenotype.

Although we are unable to assign a specific function to PMI2 at this time, it seems unlikely that PMI2 is required for basic cytoskeletal functions such as actin nucleation, filament stability, or actin basket breakdown because these types of defects would probably cause alterations of chloroplast movements under both low- and high-light conditions. Also, defects in basic cytoskeletal function may disrupt aspects of cell biology, such as mitochondrial movement, cell size, and overall structure of the actin cytoskeleton, which were not seen in pmi2 (Fig. 3; Supplemental Fig. 2). The ability of pmi2 mutant leaves to develop an avoidance response when exposed to high light (Fig. 1, B and D) may indicate that the movement machinery is functional and suggests that the defect lies in the signal for the response rather than its execution. However, it remains possible that the movement mechanism for each response is distinct and uses a unique set of cytoskeletal components to achieve chloroplast relocation.

Based on the physiological analyses of light-induced chloroplast movements in the pmi2 mutant, it seems likely that PMI2 is involved in some aspect of signal transduction for the avoidance response. It has been suggested that phot1 and phot2 operate through separate, yet intertwined, signaling pathways when mediating high- and low-light phototropism (Sakai et al., 2000). It is possible that they function in a similar fashion when controlling chloroplast movement and that PMI2 acts in the phot2 portion of the pathway. The similarity between the phenotypes of pmi2 and phot2 certainly suggests this. It is also possible that PMI2 is not involved in the signaling cascade, but rather has a structural role providing support for phot2 or other components of the pathway. For example, given the function of other coiled-coil proteins, PMI2 could function to orient or hold phot2 to the plasma membrane where it is known to locate (Sakamoto and Briggs, 2002).

In conclusion, we have identified two new genes that are required for a normal chloroplast avoidance response to high fluence rates of blue light. Plants with mutations in the PMI2 gene have normal chloroplast movement under low light intensities, but display aberrant chloroplast orientation under both intermediate and high blue light. PMI2 encodes a protein of unknown function composed mostly of a long coiled-coil motif. A mutation in PMI15, a gene related to PMI2 in Arabidopsis, also affects chloroplast avoidance under high light intensities. Although mutations in phot2 eliminate the avoidance response, we are not aware of other mutants that specifically attenuate the high-light chloroplast movement. These two proteins add to the growing list of proteins of unknown biochemical function that are associated with light-induced chloroplast movements (Oikawa et al., 2003; DeBlasio et al., 2005; Suetsugu et al., 2005). Because several of the proteins required for chloroplast motility, including phot1, phot2, CHUP1, JAK1, and PMI1, appear to be unique to plants, the mechanism of chloroplast motility may be different from other organelles.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotype Col gl1 was used as the wild type in this study. The pmi2-1 mutant is in the Col background and was isolated from a population mutagenized with ethyl methanesulfonate (Sigma-Aldrich). The pmi2-1 seeds used for these studies were backcrossed three times. The pmi2-2 T-DNA insertion line was isolated from the SALK collection (http://signal.salk.edu). Phototropin mutants (Col ecotype) were obtained from Emmanuel Liscum (University of Missouri, Columbia; phot1-5) and Takatoshi Kagawa (University of Tsukuba, Japan; phot2-1). GFP:mTalin-expressing lines (Ler ecotype) were provided by Zenbiao Yang. Seeds were sown in damp Scott's plug mix (Scotts-Sierra) and incubated at 4°C for 48 to 72 h. Plants were germinated and grown at 23°C in a growth room with a 12-h photoperiod under 60 to 70 µmol m^–2 s^–1 of light produced by mixture of cool-white and warm-white fluorescent bulbs (General Electric). Plants were fertilized with K-Grow all-purpose plant food (Kmart) every 2 weeks after germination.

Mutant Isolation and Light Transmittance Measurements

Details of the procedure for identifying chloroplast movement mutants have been previously described (DeBlasio et al., 2005). For characterization of chloroplast movements in mutant and wild-type plants, leaves were excised from plants after 11 h of darkness near the end of their subjective 12-h night period. The excised leaves were placed in a dark humid chamber with their petioles held in water-filled microfuge tubes for 15 min to 6 h. To prepare for measurement, a leaf blade was sandwiched between two glass microscope slides (VWR International) with its petiole protruding from the slides. The petiole was wrapped with wet paper toweling to ensure hydration. The assembly was then arranged on a stage so the leaf covered a 5-mm diameter red Plexiglas window (Rohm and Haas no. 2423; Dayton Plastics). The sensor from a LI-COR 1800 spectroradiometer (Li-COR) was placed directly below the red Plexiglas window and a red light-emitting diode (660 nm; Radio Shack) was mounted directly above the leaf to provide 20 to 30 µmol m^–2 s^–1 red light for measurements of leaf transmittance. Blue light to induce movement was generated by passing light from a halogen fiber-optic light microscope illuminator (Cole Palmer) through a blue interference filter 450 ± 25 nm (03FIB304; Melles Griot). The light from the fiber-optic assembly illuminated the leaf at an angle of 60° relative to the leaf surface. Changes in blue-light intensity were achieved using neutral density filters. Red-light transmittance through leaves was measured with a LI-COR 1800 spectroradiometer by integrating the quantum flux between 650 to 670 nm for each time point. For each leaf, the change in percent of red-light transmittance was calculated as (I_t/I_o) x 100/I_A, where I_t and I_o are the incident and transmitted red-light fluence rate, respectively, and I_A is the average red-light transmittance value measured prior to the blue-light treatments. Results are presented as the average change in percent of red-light transmittance ± SE.

Mapping and Identification of PMI2

Crosses were made between pmi2 (Col ecotype) and the Ler ecotype. Analysis of polymorphisms in 194 F₂ plants homozygous for pmi2 placed the mutation in a 135-kb region between markers at 35,308 bp on BAC T12I7 and 35,116 bp on BAC T4024.

Plasmids containing BAC F4N21 (Arabidopsis Biological Resource Center [ABRC]) were partially digested by Sau3AI (New England Biolabs) for 30 min at 37°C to obtain 15- to 25-kb fragments. These fragments were ligated (T4 DNA ligase; New England Biolabs) at 4°C overnight to binary vector pCLD04541 (ABRC) that had been digested with BamHI (New England Biolabs). Plasmid DNA was introduced into Escherichia coli (DH5) using a Gigapack III XL cosmid packaging kit (Stratagene). E. coli colonies were screened with PCR markers located every 10 kb along the length of BAC F4N21. Colonies containing 10- to 30-kb fragments were identified that formed a contiguous sequence across the entire BAC. These were mated into Agrobacterium tumefaciens (GV3101) using E. coli helper strain pRK2013 and used to transform pmi2-1 plants by floral dipping (Clough and Bent, 1998). Transformants were isolated as described in DeBlasio et al. (2005).

Microscopy

Cross sections of leaves were made to measure chloroplast positions in leaf cells. Leaves that had been exposed to darkness, low-intensity (1 µmol m^–2 s^–1), or high-intensity (60 µmol m^–2 s^–1) blue light were cut into small pieces (1 x 0.5 mm), dehydrated in acetone, fixed in a solution of 4% formaldehyde and 5% gluteraldehyde (Electron Microscopy Sciences), and embedded in soft Spurr's resin (Electron Microscopy Sciences). The embedded leaf pieces were sectioned using an automated ultramicrotome and a glass knife. Chloroplasts and cell walls were stained using bromphenol blue (Sigma-Aldrich). Micrographs obtained from the sections were analyzed by counting chloroplasts positioned along the top, bottom, left, and right cell wall. The data from each micrograph were considered one data point.

To analyze chloroplast movement in palisade mesophyll cells with time-lapse microscopy, 1-mm pieces of live, dark-acclimated leaves, excluding the midvein, were cut with a razor and mounted on a slide in water. Images of the adaxial surface of palisade mesophyll cells were captured every 30 s with MetaMorph software (Universal Imaging) and a Hamamatsu ORCA-ER CCD camera mounted on a Nikon E800 microscope. While imaging, the leaf sections were given 1-h sequential irradiations of low and high light from below using the built-in bright-field lamp. To measure cell and chloroplast area, leaf pieces were prepared as described above. The diameter of palisade mesophyll cells and the chloroplasts within were determined using ImageJ (http://rsb.info.nih.gov/ij).

Mitochondria were observed in live tissue by vacuum infiltrating 1-mm pieces of freshly cut leaves with the green fluorescent dye MitoTracker Green (Invitrogen). Images of cells with stained mitochondria were captured every second to detect mitochondria movements with the same equipment used for visualization of chloroplast movement.

To view PMI2 localization, a gene construct was made by fusing the carboxy terminus of PMI2 with GFP. A 300-bp C-terminal region of PMI2 was amplified with primers (forward primer 5'-GGATCCATGGAAGAGAGGGAATCGTTTAGGATG-3' and reverse primer 5'-GGATCCAACTTACACTCACTTCAACAAGATAAC-3') containing BamHI restriction sites. The amplified fragment was ligated into the vector pGEM-T (Invitrogen). The insert was then excised from pGEM-T by digestion with BamHI and ligated into vector pEGAD (ABRC) that had been cut with BamHI to create plasmid pP2CTPGD. The transformation vector pEGAD contains a polylinker cloning region downstream of GFP. Using the floral-dip method, pP2CTPGD was transformed into wild-type and pmi2-1 plants. Leaves were prepared for microscopy as described above. GFP fluorescence from leaves expressing GFP:mTalin, pP2CTPGD, and pEGAD was recorded using a spinning disc confocal microscope (Perkin-Elmer). The images shown are projected views from between 10 and 40 optical sections taken every 0.5 to 0.1 nm.

Tropisms

The phototropic response of pmi2 was determined by germinating seeds on vertical square petri dishes containing 0.5x Murashige and Skoog medium (Sigma-Aldrich). After 60 h of etiolated growth, seedlings were exposed to 6 h of 1 µmol m^–2 s^–1 unilateral blue light. Gravitriopism was tested by a 90° reorientation of 60-h-old etiolated seedlings grown on vertical petri dishes containing 0.5x Murashige and Skoog and 1% Suc. Curvature was measured using ImageJ (http://rsb.info.nih.gov/ij).

Confirmation of T-DNA Inserts from the SALK Database

The position of the T-DNA insert in pmi2-2 (SALK_088187) was confirmed through PCR amplification with primer LBa-1 (located on the T-DNA insert: 5'-TGGTTCACGTAGTGGGCCATCG-3') and primers flanking the predicted insert (5'-AACCAATCATCATCATCTCCTTC-3' and 5'-CTTCATTCGCAACATCAACTTC-3'). The T-DNA insert in AT5g38150 in plants homozygous for SALK_047862 was confirmed by PCR using primers LBa-1 and primers flanking the predicted insertion site (5'-GTTGGAAGTGGTATCTATCGCC-3' and 5'-TCAACATTTTCAGACAACTATAACC-3').

RT-PCR

Isolation of total RNA was performed using the RNeasy plant mini kit (Qiagen). Rosette leaf and root pieces were collected from 6-week-old plants. Cauline leaves, stem segments, and flowers were collected form 10-week-old plants. RT-PCR was performed on a 0.5-µg sample of RNA from each sample using the Titan One Tube RT-PCR system (Roche Diagnostics). Primers (5'-GGAATTTAGATGGAACTGTATCAG-3' and 5'-CCTTAGCAGACTCAGCAACTC-3') were designed to flank the intron in PMI2 to differentiate RNA amplification from amplification of contaminating DNA. For a loading control, amplification of the Arabidopsis ubiquitin-conjugating enzyme (At5g25760) was used (5'-TCAAATGGACCGCTCTTATCAAAGG-3' and 5'-TCCAACCAGGACTCCAAGCATTC-3'), designed around an intron.

Sequence Alignments

Sequence alignments of PMI2, At5G38150, and BAD82493 were produced by the publicly available program TCOFFEE. Outputs were produced by BOXSHADE (Swiss EMBnet node server, Swiss Institute of Bioinformatics; http://www.ch.embnet.org/index.html).

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers NM_105355 (pmi1), NM_123175 (pmi15), and NP_947041 (BAD82493).

Received March 13, 2006; returned for revision June 1, 2006; accepted June 1, 2006.

	FOOTNOTES

 
¹ This work was supported by a grant from the National Science Foundation (IOB–0416741).

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: Roger P. Hangarter (rhangart{at}indiana.edu).

^[W] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.080333.

^* Corresponding author; e-mail rhangart{at}indiana.edu; fax 812–855–6082.

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