RESULTS PSII Activity Is Reduced in lpa2-1 The lpa2-1 mutant was obtained by screening the Scheible and Somerville T-DNA Arabidopsis lines ( Weigel et al., 2000) for high chlorophyll fluorescence phenotypes ( Meurer et al., 1996; Peng et al., 2006). Chlorophyll fluorescence induction experiments revealed that the ratio of variable fluorescence to maximum fluorescence ( Fv/Fm), which reflects the maximum potential of photochemical reactions of PSII, was significantly lower in the lpa2-1 mutant (0.54 ± 0.03) than in wild-type plants (0.83 ± 0.02) ( Figure 1A). The decreased Fv/Fm ratios suggest that the mutants have defects in electron transfer within PSII or a partial loss of PSII capacity. The redox kinetics of P700 showed that P700 can be oxidized in the lpa2-1 mutant, indicating that PSI is functional in it ( Figure 1B). The amplitude of changes in A820 induced by far-red light was lower in the mutant than in wild-type plants. These spectroscopic analyses showed that the photosynthetic deviations of lpa2-1 are similar to those of lpa1 and primarily reflect reductions in PSII activity ( Peng et al., 2006). ![Figure 1. Figure 1.](picrender.fcgi?artid=1955735&blobname=tpc1901980f01.gif) | Figure 1. Spectroscopic Analysis of Wild-Type and lpa2-1 Plants. |
The lpa2-1 mutant displayed a slightly pale green phenotype, and its growth was greatly reduced ( Figure 2A). The leaf areas of mutant plants were ~70% smaller than those of wild-type plants at 26 d after germination ( Figure 2B). ![Figure 2. Figure 2.](picrender.fcgi?artid=1955735&blobname=tpc1901980f02.gif) | Figure 2. Phenotypes of lpa2-1, lpa2-2, Wild-Type, and lpa2-1 Transformant Plants Complemented with the Open Reading Frame of the At5g51545 Gene. |
Molecular Cloning of the LPA2 Gene The genetic defect in lpa2-1 segregated as a single recessive mutation. Cosegregation of the mutant phenotype and the phosphinotricin resistance conferred by the T-DNA confirmed that the mutation was induced by the T-DNA insertion (data not shown). To determine the genetic basis of the lpa2-1 phenotype, thermal asymmetric interlaced (TAIL) PCR was performed and the genomic region flanking the left border sequences of the T-DNA was isolated. Sequence analysis revealed that the T-DNA was inserted in the 5′ untranslated region of At5g51545, at position −12 relative to the ATG codon ( Figure 3A), and the gene was expressed at levels that were barely detectable by RT-PCR in the lpa2-1 mutant ( Figure 3B). However, two annotated genes close to the insertion site ( At5g51540 and At5g51550) were expressed at similar levels to those in wild-type plants, as shown by RT-PCR analysis ( Figure 3B). ![Figure 3. Figure 3.](picrender.fcgi?artid=1955735&blobname=tpc1901980f03.gif) | Figure 3. Identification of the lpa2 Mutation. |
To confirm that the mutated gene is responsible for the observed phenotype, we analyzed an independent T-DNA insertion line, SAIL_293_G05, in the sequence-indexed Arabidopsis T-DNA insertion mutant stock. This homologous line, lpa2-2, carries a T-DNA insertion at nucleotide position 452 in the second exon of the At5g51545 gene ( Figure 3A). RT-PCR analysis showed that the expression of At5g51545 was also suppressed in this mutant ( Figure 3B), and its chlorophyll fluorescence induction kinetics were similar to those of lpa2-1 (data not shown). The lpa2-2 mutant also had a pale-green phenotype ( Figure 2A) and reduced growth rate (data not shown). To obtain direct evidence that disruption of the LPA2 gene was responsible for the lpa2 mutant phenotype, a complementation experiment was performed with the isolated At5g51545 full-length cDNA. The resulting clone containing the cDNA sequence under the control of the cauliflower mosaic virus 35S promoter was introduced into a homozygous lpa2-1 mutant using the floral dip method ( Clough and Bent, 1998). Sixteen successfully complemented transgenic plants had similar growth rates and chlorophyll fluorescence induction kinetics to wild-type plants ( Figures 1 and 2). Thus, it can be concluded that the lpa2 phenotype is due to the inactivation of At5g51545. PSII Proteins Are Severely Reduced in lpa2-1 We hypothesized that the defect in the electron transfer of PSII could be associated with altered levels of proteins in the PSII complex. To examine steady state levels of thylakoid proteins in wild-type and lpa2-1 plants, thylakoid membranes were isolated from 5-week-old wild-type and lpa2-1 leaves and immunoblot analysis was performed using antibodies raised against specific subunits of the photosynthetic thylakoid membrane protein complexes. The results showed that the levels of plastid-encoded PSII subunits D1, D2, CP47, and CP43 were all decreased to ~30% of wild-type levels ( Figure 4A). By contrast, levels of the nucleus-encoded 33-kD protein of the oxygen-evolving complex and LHCII were only slightly reduced in the mutant ( Figure 4A). The amounts of PSI reaction center proteins PsaA/B were also reduced to ~75% of wild-type levels. The contents of cytochrome f of the cytochrome b 6/f complex and the β subunit of the ATP synthase were slightly increased per unit of chlorophyll ( Figure 4A). The contents of these proteins in the lpa2-1 mutant, compared with those in the wild type, were further decreased slightly when they were normalized to levels of cytochrome f of the cytochrome b 6/f complex or the β subunit of the ATP synthase ( Figure 4B). To examine whether the accumulation of PSII proteins is dependent on the developmental stage, immunoblot analysis was performed to examine the content of PSII proteins from 12-d-old young wild-type and lpa2-1 seedlings. The results showed that the levels of D1 and CP43 proteins in the mutant were decreased to ~30% of wild-type levels per unit of chlorophyll ( Figure 4C). ![Figure 4. Figure 4.](picrender.fcgi?artid=1955735&blobname=tpc1901980f04.gif) | Figure 4. Immunoblot Analysis of Chloroplast Proteins in lpa2-1 and Wild-Type Plants. |
To explore putative structural alterations of the thylakoid membrane protein complexes in the mutant, thylakoid membranes from mutant and wild-type plants were solubilized with dodecyl-β- d-maltopyranoside (DM) and the chlorophyll protein complexes (with equal amounts of chlorophyll) were separated by blue-native (BN) PAGE ( Figure 5A). Six major bands labeled I to VI were resolved after the first-dimensional separation ( Figure 5A), apparently representing PSII supercomplexes (band I), monomeric PSI and dimeric PSII (band II), monomeric PSII (band III), CP43-free PSII (band IV), trimeric LHCII/PSII reaction center (band V), and monomeric LHCII (band VI) ( Guo et al., 2005). As clearly shown in Figure 5A, the largest chlorophyll-containing protein complexes (labeled I) were absent, and there was a nearly complete loss of band IV, in the mutant. Since the separated proteins were detected by Coomassie blue staining ( Figure 5B), it did not show PSII reaction center composition but only the presence of CP43 together with some LHC and (probably) some minor antenna complexes (for additional information, see Figure 7E below). Analyses of the two-dimensional SDS-urea-PAGE gels after Coomassie blue staining showed that the relative levels of PSII core proteins D1, D2, CP47, and CP43 were reduced in the mutant, especially the PSII supercomplexes, which were barely detectable ( Figure 5B). It is interesting that the CP43 doublet becomes a single band in the mutant ( Figure 5B). In addition, the amount of PSI was decreased slightly and the contents of cytochrome b 6f and ATP synthase were increased slightly per unit of chlorophyll in the mutant ( Figure 5B). ![Figure 5. Figure 5.](picrender.fcgi?artid=1955735&blobname=tpc1901980f05.gif) | Figure 5. Accumulation of Chloroplast Proteins, Phosphorylation of CP43, and Binding of Chlorophyll to CP43 in Wild-Type and lpa2-1 Plants. |
![Figure 7. Figure 7.](picrender.fcgi?artid=1955735&blobname=tpc1901980f07.gif) | Figure 7. In Vivo Synthesis and Assembly of Chloroplast Proteins. |
CP43 is known to undergo two cotranslational or early posttranslational changes: chlorophyll binding and phosphorylation. It has been observed previously that changes or defects in CP43 phosphorylation accompany changes in antenna-PSII core formation ( de Vitry et al., 1989), with a spectacular loss of PSII-LHCII supercomplexes ( Swiatek et al., 2001). To examine the phosphorylation of CP43, we performed immunoblot analysis with anti-phosphothreonine antibodies. There were no apparent changes in the phosphorylation level of CP43 between the wild-type and mutant plants when normalized to the protein level of CP43 ( Figure 5C). To examine the chlorophyll binding to CP43, we detected the presence of the chlorophyll binding form of CP43 by monitoring the abnormal migration of holo-CP43 versus apo-CP43 on two-dimensional gels ( de Vitry et al., 1989). In the wild-type plants, CP47 and CP43 were detected as spots off the diagonal by two-dimensional gel electrophoresis ( Figure 5D, arrows). In the lpa2-1 mutant, the content of CP43 was reduced and CP43 was detected as spots off the diagonal in two-dimensional gel electrophoresis ( Figure 5D, arrows). CP47 and CP43 were not detectable after heat treatment before the first-dimensional electrophoresis in the wild-type and mutant plants (data not shown). Thus, these two events may not account for the presence of the CP43 doublet observed in the wild type and the single CP43 band in the lpa2-1 mutant. Steady State mRNA Levels and Polysome Association Are Unaffected in lpa2-1 The reduced PSII contents in the lpa2-1 mutant could be due to impaired accumulation of transcripts that encode one or more PSII proteins. To assess this possibility, we performed RNA gel blot hybridization to examine levels of the plastid-encoded PSII transcripts. Our results indicate that identical amounts of psbA and psbC (encoding the D1 protein and CP43 of PSII, respectively) accumulated in 5-week-old lpa2-1 and wild-type plants ( Figure 6A). The abundance and pattern of transcripts of other PSII operons (such as the psbD/C, psbEFLJ, and psbKI operons) and transcripts of psaA and petA (which encode the PSI reaction center protein PsaA and the cytochrome b 6f subunit cytochrome f, respectively) were also unaltered in the mutant ( Figure 6A). ![Figure 6. Figure 6.](picrender.fcgi?artid=1955735&blobname=tpc1901980f06.gif) | Figure 6. mRNA Expression and Polysome Accumulation in Chloroplasts. |
The effects of the lpa2-1 mutation on the protein synthesis capacity of chloroplasts were investigated by analyzing changes in the polysome association of psbA, psbB, psbC, and psbD transcripts following sucrose gradient fractionation. The results showed that the association of these transcripts with polysomes was largely unaffected in 5-week-old mutant plants ( Figure 6B). Synthesis of Thylakoid Membrane Proteins in lpa2-1 The decreased accumulation of PSII may be due to either reduced protein synthesis or increased degradation of its protein components. Therefore, we studied the synthesis of plastid-encoded thylakoid membrane proteins by pulse-chase labeling of mutant leaves in the presence of cycloheximide, which inhibits the translation of nucleus-encoded proteins. As shown in Figures 7A and 7B, after pulse labeling for 20 min, the rates of synthesis of the PSII proteins CP47, D1, and D2, the PSI reaction center proteins PsaA/B, and the α and β subunits of ATP synthase (CF1α/β) in 12-d-old young mutant seedlings and 5-week-old plants were comparable to those in their wild-type counterparts. However, labeling of the PSII subunit CP43 in the lpa2-1 mutant was dramatically reduced, to <10% of wild-type levels in both young seedlings and 5-week-old plants ( Figures 7A and 7B). When the time of pulse labeling was shortened to 10 min, the amount of radioactivity incorporated into CP43 in the lpa2 mutant was >25% of wild-type levels in young seedlings ( Figure 7C). Pulse labeling for 20 min was followed by a chase with unlabeled Met to monitor the turnover rates of plastid-encoded proteins in young seedlings. The results showed that the turnover rates of CP47 and the weakly synthesized CP43 are relatively unaffected in the mutant, and the turnover rate of D1 is much more strongly affected than that of D2 ( Figure 7D). The Assembly of PSII Is Impaired in lpa2-1 To study the assembly of photosynthetic protein complexes, thylakoid membrane proteins were separated by BN gel electrophoresis and the different PSII assembly intermediates of free PSII protein, PSII reaction center, CP43-free PSII monomers, intact PSII monomers, PSII dimers, and PSII supercomplexes were visualized autoradiographically. After a 20-min pulse, most of the radiolabeling was found in protein complexes of ~100 kD (PSII reaction centers), 220 kD (CP43-free PSII monomers), and 250 kD (PSII monomers) ( Figure 7E). Protein complexes of 220, 250, and 500 kD (PSII dimers) were the most strongly labeled complexes after a 15-min chase, and larger protein complexes (>500 kD) became clearly visible in the autoradiogram obtained after a 30-min chase in the wild-type plants. During the chase period, the incorporation of radioactivity into PSII monomers, dimers, and supercomplexes gradually increased, with concomitant losses of label in PSII reaction centers and CP43-free PSII complexes. There was no evidence of the further assembly of PSII after 60 min of chasing ( Figure 7E). However, in the lpa2-1 mutant, most of the radioactivity in the thylakoid membrane preparations was found to be associated with PSII reaction centers and CP43-free PSII after 20 min of pulse labeling ( Figure 7E). The radioactivity in PSII monomers increased after a 15-min chase, and the PSII dimer was clearly labeled after a 30-min chase. The accumulation of radioactivity in PSII supercomplexes was barely detectable even after a 60-min chase ( Figure 7E). The pulse–chase samples were also subjected to BN-SDS-PAGE analysis to follow the assembly of the major PSII core proteins, CP47, CP43, D1, and D2, into PSII complexes ( Figure 7F). The PSII reaction center protein D1 was the most heavily labeled protein, even though very young leaves were used for the pulse–chase experiments. After a 20-min pulse labeling, ~20% of the labeled D1 protein was found in PSII reaction centers, ~35% was found in CP43-free PSII monomers, and ~25% was found in PSII monomers ( Figure 7F). As the chase time increased, the radiolabeled D1 protein appeared in larger PSII complexes, such as PSII dimers and PSII supercomplexes. After a 30-min chase, most of the radiolabeled D1 protein was incorporated into PSII monomers (~30%), dimers (~25%), and supercomplexes (~25%) and only ~20% was found in CP43-free PSII monomers ( Figure 7F). However, the assembly from CP43-free PSII monomers to PSII monomers and the formation of PSII supercomplexes were distinctively slower in the lpa2-1 mutant than in wild-type plants. In the mutant, ~70% of the radiolabeled D1 protein was incorporated into CP43-free PSII monomers after a 20-min pulse, which is consistent with the results of the one-dimensional BN gel analysis described above ( Figures 7E and 7F). Even after a 60-min chase, ~40% of the labeled D1 protein was still in CP43-free PSII monomers, indicating that CP43 assembly was severely hampered in the mutant. Only a small amount of radiolabeled D1 protein (<5%) had accumulated in PSII supercomplexes after a 60-min chase ( Figure 7F). LPA2 Is an Intrinsic Thylakoid Membrane Protein The open reading frame of LPA2 encodes a polypeptide of 185 amino acids with a calculated molecular mass of 20 kD. Database searches did not reveal the presence of any recognizable motif or domain in LPA2 ( Figure 8A). However, the N-terminal sequence of LPA2 is rich in positive and hydroxylated amino acid residues, which is characteristic of chloroplast transit peptides. Analysis by the TMHMM program showed that LPA2 has two transmembrane domains, in the regions 112 to 135 and 150 to 173, suggesting that LPA2 is probably a membrane protein ( Figure 8A). BLAST searches of the complete Arabidopsis sequence indicated that the LPA2 gene is present in a single copy in the nuclear genome. Database searches and protein sequence alignments revealed that LPA2 shares significant sequence identity with an unknown rice ( Oryza sativa) protein, XP_463951 (68% identity, 80% similarity) ( Figure 8A). No homologous protein to LPA2 was found in cyanobacteria or Chlamydomonas. The homologous protein in rice also had a chloroplast transit peptide, suggesting that they may have similar functions. ![Figure 8. Figure 8.](picrender.fcgi?artid=1955735&blobname=tpc1901980f08.gif) | Figure 8. Amino Acid Sequence Alignment and Immunolocalization of LPA2. |
To determine the localization of LPA2, polyclonal antiserum was raised against recombinant LPA2 protein (amino acids 7 to 114). An ~20-kD protein was detected in thylakoid membrane proteins and total proteins prepared from the wild-type plants, but no signal was detected in total protein preparations from either the lpa2-1 or lpa2-2 plants ( Figure 8B). The level of LPA2 in total protein preparations of complemented plants was comparable to that in wild-type plants ( Figure 8B). These findings indicate that the T-DNA insertion leads to the loss of LPA2 protein accumulation. To further investigate whether LPA2 is a transmembrane protein, thylakoid membrane fractions of wild-type plants were sonicated and treated with different salts and the proteins were then subjected to immunoblot analysis. LPA2 was still retained in the membranes, even when membrane preparations were sonicated in the presence of 250 mM NaCl, 1 M CaCl 2, or 6 M urea ( Figure 8C). During these treatments, PsbO, the 33-kD luminal protein, and CP47, the PSII core protein, were used as controls. LPA2 Interacts with the PSII Core Protein CP43 Since LPA2 appears to be required for the efficient assembly of PSII, we tested the possibility that LPA2 is an integral subunit of PSII or part of a multiprotein complex. For this purpose, isolated thylakoid membranes of wild-type plants were solubilized by DM, separated by sucrose gradient sedimentation, and subjected to immunoblot analysis ( Zhang et al., 1999; Peng et al., 2006). After centrifugation, 20 fractions of equal volume were collected from the top to the bottom of the gradients and the proteins in each fraction were immunodetected using specific antibodies. The immunoblots indicated that LPA2 was present in fractions at ~100 kD, based on the migration of molecular standards, and that it did not comigrate with PSII proteins. Thus, it appears that LPA2 is not a component of PSII ( Figure 9). ![Figure 9. Figure 9.](picrender.fcgi?artid=1955735&blobname=tpc1901980f09.gif) | Figure 9. Sucrose Gradient Fractionation Analysis of Thylakoid Proteins. |
However, if LPA2 is involved in the assembly of PSII, it may interact with one or more subunits of the PSII complex. To test this possibility, we examined the interaction of LPA2 with the subunits of PSII in vivo using a modified split-ubiquitin system designed to assess interactions of membrane proteins ( Pasch et al., 2005; Peng et al., 2006). A prey construct plasmid, in which the NubG moiety was fused to the N terminus of LPA2, was prepared and subsequently transformed into the NWY32 strain in which bait proteins were expressed. The bait plasmids were constructed to generate the fusion proteins X-Cub-LexA-VP16 (where X represents D1, D2, or CP43). The resulting transformants were analyzed for growth on plates lacking His, Leu, and Trp (SD-His-Leu-Trp), and their β-galactosidase activities were assayed. As shown in Figure 10, coexpression of CP43-Cub-LexA-VP16 with NubG-LPA2 resulted in positive β-galactosidase activity and growth on SD-His-Leu-Trp plates ( Figure 10). However, coexpression of D1-Cub-LexA-VP16 or D2-Cub-LexA-VP16 with NubG-LPA2 produced transformants that showed no β-galactosidase activity or growth on SD-His-Leu-Trp plates ( Figure 10). These results suggest that LPA2 interacts with CP43 but not with D1 or D2. CP43-LPA2 complexes may not be sufficiently stable to accumulate in the membranes, based on immunoblot analysis of the distribution of proteins over the sucrose gradient fractions ( Figure 9). LPA2 Interacts with Alb3 Recent studies have revealed that Arabidopsis Alb3 interacts with the PSII proteins D1, D2, and CP43 ( Pasch et al., 2005). To examine whether LPA2 interacts with Alb3, the fractions obtained from sucrose gradient fractionation were immunoblotted with anti-Alb3 antibodies. Alb3 was found to be present in two distinct complexes: one of ~60 to 140 kD, and one >600 kD ( Figure 9). The interaction between LPA2 and Alb3 was further investigated by yeast two-hybrid analysis. Coexpression of Alb3-Cub-LexA-VP16 with NubG-LPA2 resulted in positive β-galactosidase activity and growth on SD-His-Leu-Trp plates ( Figure 10). In previous studies, we have shown that LPA1 is required for efficient PSII assembly ( Peng et al., 2006). The interaction between LPA1 and LPA2 was also investigated. Coexpression of LPA1-Cub-LexA-VP16 with NubG-LPA2 produced transformants that showed no β-galactosidase activity or growth on SD-His-Leu-Trp plates ( Figure 10). Thus, the above results suggest the interaction of LPA2 with Alb3, but not with LPA1. |
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