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First published online February 7, 2008; 10.1104/pp.107.113423 Plant Physiology 146:1469-1481 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Deregulation of Maize C4 Photosynthetic Development in a Mesophyll Cell-Defective Mutant1,[C],[W],[OA]Department of Plant Biology (S.C., W.M., K.J.v.W.), and Boyce Thompson Institute for Plant Research (J.M.K., T.P.B.), Cornell University, Ithaca, New York 14853; and Department of Statistics, Iowa State University, Ames, Iowa 50011 (P.L.)
During maize (Zea mays) C4 differentiation, mesophyll (M) and bundle sheath (BS) cells accumulate distinct sets of photosynthetic enzymes, with very low photosystem II (PSII) content in BS chloroplasts. Consequently, there is little linear electron transport in the BS and ATP is generated by cyclic electron flow. In contrast, M thylakoids are very similar to those of C3 plants and produce the ATP and NADPH that drive metabolic activities. Regulation of this differentiation process is poorly understood, but involves expression and coordination of nuclear and plastid genomes. Here, we identify a recessive allele of the maize high chlorophyll fluorescence (Hcf136) homolog that in Arabidopsis (Arabidopsis thaliana) functions as a PSII stability or assembly factor located in the thylakoid lumen. Proteome analysis of the thylakoids and electron microscopy reveal that Zmhcf136 lacks PSII complexes and grana thylakoids in M chloroplasts, consistent with the previously defined Arabidopsis function. Interestingly, hcf136 is also defective in processing the full-length psbB-psbT-psbH-petB-petD polycistron specifically in M chloroplasts. To determine whether the loss of PSII in M cells affects C4 differentiation, we performed cell-type-specific transcript analysis of hcf136 and wild-type seedlings. The results indicate that M and BS cells respond uniquely to the loss of PSII, with little overlap in gene expression changes between data sets. These results are discussed in the context of signals that may drive differential gene expression in C4 photosynthesis.
In maize (Zea mays), photosynthetic activities are partitioned between two morphologically and biochemically distinct cell types, mesophyll (M) and bundle sheath (BS; Edwards and Walker, 1983  ). M and BS cells are organized as concentric files around the vasculature in a classical Kranz anatomy. Functionally, these two cell types cooperate in photosynthesis, carbon fixation (Edwards et al., 2001a; Majeran et al., 2005), nitrogen metabolism (Rathnam and Edwards, 1975, 1976; Harel et al., 1977; Becker et al., 1993), and sulfur assimilation (Burgener et al., 1998). Notably, M plastids contain grana thylakoids, perform linear electron transport, and photoreduce NADP+ (Andersen et al., 1972). In contrast, BS chloroplasts are agranal (Andersen et al., 1972; Kirchanski, 1975; Miller et al., 1977), PSII depleted (Schuster et al., 1985), and perform most of the reactions of the Calvin cycle (Chollet, 1973; Kagawa and Hatch, 1974). Partitioning of photosynthetic activities between M and BS cell types is mediated by cell-specific localization of multiple transcripts (Furumoto et al., 2000; Sawers et al., 2007) and proteins (Majeran et al., 2005). However, little is known about the transcriptional program regulating C4 differentiation.
Previous studies have suggested that a small number of regulatory changes are sufficient to establish the C4 syndrome (Ku et al., 1996
Another factor that may influence the differentiation process is redox poise. In the leaf blade, M cells contain both PSII and PSI activities and perform linear photosynthetic electron transport (PET). In contrast, BS cells lack detectable levels of functional PSII and are believed to be restricted to cyclic electron transport (Gregory et al., 1979
The characterization of mutants that are selectively disrupted in either M or BS cell photosynthetic differentiation may prove useful in understanding the networks that drive this process. For instance, bundle sheath defective2 (bsd2) seedlings do not accumulate Rubisco (Roth et al., 1996
Several maize mutants have been reported with defects in PSII function, including high chlorophyll fluorescence3 (hcf3), hcf19G, and hcf19YG (Leto and Miles, 1980
Ac-Tagged Zmhcf136 Is Seedling Lethal
The Zmhcf136 mutant was first identified in sand bench screens of an Ac-mutagenized population as a recessive hcf seedling-lethal mutant (see "Materials and Methods"). DNA-blot analysis identified a 2.5-kb EcoRI fragment containing an Ac insertion that cosegregated with the mutant phenotype. Inverse PCR with primers designed to Ac (Kolkman et al., 2005
To identify full-length maize coding sequences for Hcf136, the 265 bp flanking the Ac was used to search available genomic and EST databases and a nearly full-length pseudomolecule of Hcf136 transcript was assembled. To confirm the cloning of Hcf136 and recover noncoding sequences associated with the Hcf136 gene, we exploited the somatic instability of an active Ac allele to selectively amplify sequences flanking the Ac insertion in ZmHcf136. By utilizing a genome-walking technique known as Ac casting (Singh et al., 2003
HCF136 Proteins Are Highly Conserved
As shown in Figure 1B, HCF136 homologs are highly similar across monocots, dicots, algae, and cyanobacterial species. TargetP predicts that ZmHCF136 is chloroplast localized with a 25-amino-acid transit peptide (Emanuelsson et al., 2000
Using in vivo fluorescence induction curves, we examined the functional status of PSII in hcf136 leaves (see "Materials and Methods"). Mutant seedlings displayed hcf, but no variable fluorescence, consistent with the absence of PSII activity (hcf136 Fv/Fm = 0; wild type = 0.8). Light microscopy of cross sections of wild-type and mutant leaf tissue revealed smaller chloroplasts in both M and BS cells of hcf136 (Supplemental Fig. S1B). Plastid ultrastructure was examined in greater detail using transmission electron microscopy (Fig. 2 ). In the hcf136 mutant, grana are absent or display aberrant ultrastructure in M plastids (Fig. 2B). In contrast, plastid ultrastructure in hcf136 BS cells appears normal (Fig. 2D). These results are consistent with the prediction that the primary defect in Zmhcf136 is a disruption in PSII assembly and accumulation.
ZmHcf136 Transcripts Accumulate Preferentially in M Cells
To determine whether ZmHcf136 transcript accumulation is M cell specific, RNA-blot analysis of several cell types and tissues was performed using an Hcf136-specific probe (Fig. 3
). RNA was isolated from light-grown wild-type M cell protoplasts, BS strands, total leaf tissue, total hcf136 mutant leaf tissue, and total leaf tissue from wild-type dark-grown plants. To control for changes in gene expression due to M cell protoplast isolation, RNA was also extracted from total wild-type light-grown tissue that was stress-treated by a mock protoplast digestion (see "Materials and Methods"). The RNA samples were hybridized with probes derived from the cell-specific markers Pepc and Rbcs, which accumulate preferentially in M and BS cell types, respectively (Sheen and Bogorad, 1987
The psbB-psbH-psbT-petB-petD Polycistron Is Misprocessed in M Cells
When analyzing the mutant for changes in PSII transcript regulation, we unexpectedly observed a defect in the processing/stability of the psbB-psbH-psbT-petB-petD polycistron (Fig. 4A
). Components of PSII (psbB, psbH, psbN, and psbT) and Cyt b6f (petB and petD) are encoded by this polycistron, which is processed into many overlapping RNAs that are capable of directing protein synthesis (Barkan, 1988
Zmhcf136 Lacks HCF136 and PSII Proteins To examine the accumulation and localization of ZmHCF136, the profiles of wild-type and hcf136 stroma-enriched and thylakoid peripheral and lumenal proteins were compared by two-dimensional (2D) gel electrophoresis with immobilized pH gradient (IPG) strips in the first dimension and SDS-PAGE in the second dimension. A single spot was identified in Sypro Ruby-stained 2D gels as a spot that is present in plastid protein extracts of wild-type plants, but absent in hcf136 mutants (Supplemental Fig. S3). This spot was excised, trypsin digested, and analyzed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and identified as HCF136, confirming the identity of the Ac-tagged gene (TC296744; http://ppdb.tc.cornell.edu; Supplemental Table S1. To identify plastid-localized proteins that differentially accumulate in the hcf136 mutant, thylakoid membranes were isolated, subfractionated into membrane and soluble components, and separated by one-dimensional (1D) SDS-PAGE (Fig. 5 ). Strong differential accumulation was observed for a number of bands in the membrane fractions, but not in the soluble fraction. Eight major bands showing differential accumulation were identified by peptide mass finger printing (PMF) using a matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) mass spectrometer (Supplemental Table S2) as FtsH1 (band 1, TC292243), CP47 (band 2, NP_043049.1), OEC33-like (band 3, TC279249), PSII-D2 (band 4, NP_043009.1), light-harvesting complex (LHCII-1; band 5, TC286614), pyruvate, orthophosphate dikinase (PPDK; band 6, TC286559), cpHSP70 (band 7, TC293193), and small subunit of Rubisco (RBCS; band 8, TC286731). These identifications likely represent the most abundant protein in the band. In hcf136, FtsH1 metalloprotease accumulation is reduced, and the CP47, OEC33-like, and D2 subunits of PSII are absent or dramatically reduced. A slight reduction in the accumulation of the major LHCII-1 band is observed likely due to the absence of accumulation of its interacting PSII complex. PPDK, cpHSP70, and RBCS proteins have increased accumulation in the hcf136 membrane fraction, but no differential accumulation in the soluble fraction, suggesting that these proteins interact more strongly with thylakoid membranes in plastids that lack PSII or grana. It is unlikely that a treatment effect from thylakoid preparation accounts for this result because other abundant chloroplast-soluble components are not found in the membrane fraction.
To improve resolution of the thylakoid proteome analysis and to determine the assembly state of the major photosynthetic complexes in wild type and hcf136, thylakoids were solubilized with the nonionic detergent n-dodecyl β-D-maltoside and analyzed by Blue Native (BN) gel electrophoresis followed by SDS-PAGE (2D BN SDS-PAGE; Fig. 6 ). Major photosynthetic complexes in wild-type and mutant tissues were identified by PMF analysis (Supplemental Table S3). In hcf136, PSII reaction center and core subunits are absent from thylakoid membranes, but there is no dramatic effect on the accumulation of PSI. The hcf136 mutant also has a different oligomeric assembly state of the major LHCII, which is present in a monomeric form rather than the trimeric form typical of wild-type thylakoids (Dekker and Boekema, 2005 ). This may be a consequence of the loss of its interaction partner, PSII core complex. Alternatively, the absence of membrane stacking may lead to increased accessibility to detergent and destabilization of LHC oligomers during membrane preparation. Other protein complexes, including ATP synthase and Cyt b6f, accumulate to similar levels in both wild-type and mutant plastids.
Changes in Protein Accumulation Do Not Correlate with RNA Levels
To determine the extent of transcriptional control on the observed changes in protein accumulation, the relative abundance of several transcripts was measured by quantitative real-time PCR (qPCR) in M and BS cell preparations (see "Materials and Methods"). Relative transcript levels were assayed using primer pairs specific to the following nuclear-encoded genes (Supplemental Table S4): Rbcs (TC286731), Ppdk (TC286559), FtsH1 (TC292243), cpHsp70 (TC293193), PsbO OEC33 (TC279249), Lhcb1 (TC286614), Lhca3 (TC286618), PsaD (TC293201, TC293200), PsaE (TC279867), PsaF (TC299208, TC299217, TC299206), and AtpC (TC287102). The plastid-encoded genes psbB (NP_043049), psbD (NP_043009), and psbE (TC279867) were also assayed (see Supplemental Table S2). A comparison of transcript levels in M and BS cells of hcf136 and wild type is shown in Figure 7
. A value >1 indicates transcripts were more abundant in the mutant than in the wild type, and a value <1 indicates transcript abundance was greater in the wild type. These data show that the observed disruption in plastid protein accumulation does not correspond to a general reduction in corresponding transcript accumulation. Examination of the qPCR data shown in Figure 7 indicated that, in general, transcripts accumulated to higher levels in mutant M cells and lower levels in BS. This finding suggests that disrupting PSII activity can enhance the differential expression of M-enriched transcripts (e.g. psbB; Kubicki et al., 1994
Loss of PSII Leads to Changes in C4 Spatial Regulation
To further explore the disruption of PSII activity on gene expression, transcript profiles from separated M and BS cells were examined using two-label microarray analysis (see "Materials and Methods"). To avoid confounding treatment effects associated with direct comparisons of M and BS transcriptomes (Sawers et al., 2007
Using a false discovery rate (FDR) of 5%, we identified 2,568 differentially expressed features between hcf136 and wild type in the M cell data set. When a more stringent 1% FDR cutoff is applied, 1,078 features are differentially expressed of which 162 have at least a 2-fold change in expression and 773 are more abundant in the mutant relative to wild type. In the BS experiment, 1,669 features are differentially expressed between hcf136 and wild type at a 5% FDR and 586 at a 1% FDR. In the 1% FDR BS data set, 195 features change by at least 2-fold relative to wild type and 306 are more abundant in the mutant relative to wild type. When the differentially expressed genes are compared at a 5% FDR between M and BS data sets, 573 features are identified that are common to both cell types (Fig. 8B). This overlap is reduced to 147 features when significance is controlled at a 1% FDR (Fig. 8C). Because only 14% or 9% (high and low FDR, respectively) of differentially expressed features are shared, these data suggest there is a cell-specific transcriptional response to the loss of PSII.
A comparison of these data to a previous study (Sawers et al., 2007 To verify the altered transcriptional profiles determined by microarray analysis, RNA blots were performed (Fig. 9 ). Probes were designed to a number of plastid- and nuclear-encoded genes with highly abundant transcripts involved in photosynthesis that are differentially expressed between hcf136 and wild type at a 5% FDR in at least one cell type. From the M cell data, chloroplast-encoded psaAB, rbcL, psbH, matK and nuclear-encoded Lhcb were chosen for verification. From the BS data, chloroplast-encoded rbcL and matK and nuclear-encoded PsbS, Lhcb, and Rbcs were chosen for confirmation. As shown in Figure 9, RNA-blot analysis confirmed the differential accumulation of these genes between wild-type and mutant plants. The expression change of PsbS in BS cells was at the limit of detection (Fig. 9), but these data were confirmed using qPCR (Supplemental Fig. S4). Collectively, these data validate a subset of the microarray results indicating differential responses of M and BS cells to a loss of HCF136 function.
HCF136 Function in Maize
Using the transposable element Ac as a molecular tag, the ZmHcf136 gene was cloned and characterized. The pale green, seedling-lethal Zmhcf136 mutant displays reduced thylakoid stacking in M plastids, an absence of PSII complexes, and no detectable PSII reaction center functionality (Fv/Fm = 0). These data are consistent with the previously assigned function of HCF136 as a PSII reaction center assembly or stability factor (Meurer et al., 1998
Although PSII reaction center and core proteins fail to accumulate to detectable levels in hcf136, the corresponding transcripts of both nuclear and chloroplast genes accumulate to near wild-type levels. This lack of correlation between proteome and transcriptome profiles is likely a consequence of protein degradation of unassembled PSII reaction center and core proteins in the chloroplast. In contrast, nuclear-encoded protein components of PSI (PsaD, E, F) and ATP synthase (CF1
The microarray data revealed that, whereas many features are detectable in both M and BS cells of hcf136 (5670), only 573 features are differentially expressed between wild type and mutant at a 5% FDR and 147 at a 1% FDR. These data suggest that M and BS cells are responding differently to a perturbation in HCF136 function. Striking examples of these differences in regulation can be observed in transcripts encoded by the plastid genome (5% FDR). For instance, different sets of genes encoding PSII components are misregulated in hcf136 M and BS plastids. In mutant M cells, psbH, J, M, and N are differentially expressed relative to wild type, whereas in BS cells, psbD, E, J, and K show altered accumulation profiles. Also, three components of the ATP synthase (atpA, B, E) and four components of the NADH dehydrogenase (ndhE, F, G, I) are differentially expressed in M cells, but not in the BS. Additionally, significant changes in psaB, petA, petD, rpoA, rpoB, and infA expression are only detected in M cell comparisons. In contrast, transcripts for psaJ, rpoC2, atpI, and ndhJ are differentially expressed solely in the BS. Another striking trend in the M cell expression data is that nearly twice as many features are differentially expressed in M cells (2,568) relative to the BS (1,669) at a 5% FDR. This trend is most evident for plastid-encoded transcripts, where 57 genes are differentially expressed between wild-type and mutant in M chloroplasts and only 18 genes are differentially expressed in BS plastids. For example, of 21 rpl and rps genes detected in both cell types, all 21 are differentially expressed in the M cells, but only two of those 21 are differentially expressed in BS strands. In general, transcripts encoded by the plastid genome are more abundant in the mutant relative to wild type when differentially expressed. Specifically, only psbH, psaB, ndhJ, atpI, rps14, and rbcL are less abundant in hcf136 than in wild type. Consequently, these data suggest that pools of plastid mRNA, particularly in the M, are responding in concert and are either more stable or more highly expressed in the mutant. It is possible that the smaller global response in the BS may reflect its naturally PSII-depleted state. A comparison of M and BS cell data sets shows that a greater percentage of differentially expressed features change by more than 2-fold in BS relative to M cells at a 1% FDR (33% versus 15%). This indicates that BS features are capable of a strong transcriptional response to the loss of PSII. For example, putative maize homologs of Phosphatidylcholine acyltransferase (MZ00018920), Peroxidase (MZ00015594), U2 snRNP auxiliary factor (MZ00006052), H2B histone (MZ00013518), and BTH-induced ERF transcriptional factor1 (MZ00017004) are increased in accumulation by more than 2-fold only in the BS. Similarly, Phosphenolpyruvate carboxykinase (MZ00013533) and a putative Inositol 1,3,4-trisphosphate 5/6-kinase (MZ00029181) decrease by more than 2-fold in hcf136 BS cells. In addition, some features are differentially expressed in both cell types, but the magnitude of the response is greater in the BS. For example, Thylakoid formation1 (MZ00043318) increases 2.6-fold in the BS and only 1.9-fold in M cells. Similarly, Cytochrome c (MZ00013468) increases 2.3-fold in BS and 1.5-fold in M hcf136 cells. Thus, M and BS cells are capable of independently regulating gene expression in response to a disruption of PSII.
We identified 296 features that were previously shown to differentially accumulate in BS and M cells (Sawers et al., 2007
Current models propose that the evolution of C4 biology from the basal C3 state requires the recruitment of cis- and trans-acting regulatory elements to alter gene expression (Sage, 2004
An example of misexpression due to a change in cellular environment may be the aberrant processing of the psbB-psbH-psbT-petB-petD polycistron detailed in Figure 4. This defect is likely due to a change in the environment of M cell plastids that is associated with the loss of PSII (e.g. pH change, redox poise, thylakoid membrane structure). Although we have not ruled out a direct role for HCF136 in RNA metabolism, the psbB polycistron is aberrantly processed in several nonallelic hcf mutants, including hcf2, hcf38, and hcf43 (Barkan et al., 1986
Many nuclear genes respond to plastid-derived signals that are integrated through a common pathway in the chloroplast (Koussevitzky et al., 2007 In summary, the hcf136 mutant has provided an opportunity to examine the effects of altered M and BS cellular environments on C4 differentiation. The loss of PSII impacts M and BS protein composition, PET, redox poise, energy, and sugar metabolite gradients. As a result, there is a general increase in RNA transcript accumulation in the M cell, and M- and BS-enriched genes become more and less differentially expressed, respectively. Additionally, altering the BS cellular environment results in decreased transcript accumulation for a number of features and this may reflect a shift to a more basal C3 state in this cell type.
Identification of ZmHcf136
The maize (Zea mays) homolog of Arabidopsis (Arabidopsis thaliana) Hcf136 was identified as part of a regional mutagenesis screen using Ac/Dissociation transposition in the W22 inbred line of maize. The mutant family JK03-77.24 was created by selecting transposition events from bti00228::Ac and subsequent screening of self-pollinated populations (Kolkman et al., 2005
DNA-blot analysis was performed using an Ac-specific fragment (Ac900; Kolkman et al., 2005
Ac casting was used to obtain genomic DNA sequence for the full ZmHcf136 gene (Singh et al., 2003
Plants were grown in 16-h days and constant 28°C under low-light conditions of 80 µmol m–2 s–1 for fluorescence, electron microscopy, and protein analyses and 40 µmol m–2 s–1 for all other experiments. Etiolated seedlings were grown in darkness at 28°C until their light-grown siblings were at the third leaf-emerging stage. Mutants were identified from segregating families, and near-isogenic comparisons made with phenotypically wild-type siblings.
In vivo fluorescence induction curves for Fv/Fm were obtained at room temperature from the second leaf tip of seedlings at the third leaf-emerging stage of development using an actinic light source and bright saturating pulse as previously described (Maxwell and Johnson, 2000
Electron microscopy was performed on wild-type and mutant plants at the third leaf-emerging stage of development. Tips of the second leaves of 10-d-old wild-type and mutant seedlings were harvested in the morning to deplete overnight starch reserves. Samples were fixed for 0.5 h at room temperature and 1.5 h at 4°C in 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 6.8. The samples were rinsed at 4°C in 0.1 M sodium cacodylate buffer, pH 6.8, fixed in 1% osmium tetroxide, and rinsed in 0.1 M sodium cacodylate buffer, pH 6.8. Samples were dehydrated in a graded ethanol series, and then infiltrated with Spurr's resin. Sections were cut on a Reichert OmU2 Ultramicrotome and contrasted with uranyl acetate and lead citrate. The sections were viewed on a Tecnai 12 Biotwin transmission electron microscope (FEI Corporation). Digital images were acquired using a Gatan Multiscan Camera (model 791).
M protoplasts, BS strands, and the control stressed total tissue were prepared from second leaf blades as previously described (Markelz et al., 2003
Total RNA was isolated and analyzed by RNA blot as previously described (Sheehan et al., 2004
DNA probes used in RNA-blot analysis include Hcf136, Pepc, Rbcs, Me, Lhcb-m7, rbcL, PsbS, matK, psaAB, psbH, petD, psbB, psbD, petB, psbA, psbC, and atpF/H. The Hcf136 probe was a 648-bp fragment amplified from the 3'-end of the gene using the forward primer Hcf136 Common Exon (5'-GAGAGCGGACGGTGGACTTT) and reverse primer Hcf136 3'-UTR Common (5'-GGTTTTCAAGTTCCTAAGCAAGCAG). The rbcL probe was amplified from genomic maize DNA using the primers 5'-GCAGTAGCTGCGGAATCTTCTACT and 5'-GGTGAATGTGAAGAAGTAGGCCGT. PsbS was amplified using 5'-TCTCCATCATCGGCGAGATCATCA and 5'-TACAAGCAGACAACCCAACG. Other fragments were as previously described (Roth et al., 1996
Plants were grown as described above and tissue harvested for 2D IPG SDS-PAGE when the third leaf was emerging and for 2D BN SDS-PAGE and 1D SDS-PAGE when the fifth leaf was emerging. Proteins were extracted from whole seedlings for 2D IPG SDS-PAGE and from apical regions about 4 cm from the tips of third and fourth leaves for other PAGE experiments. The total leaf microsomal fraction was isolated in grinding buffer (350 mM sorbitol, 50 mM HEPES-KOH, pH 8, 2 mM EDTA, 5 mM ascorbic acid, 5 mM L-Cys) in a blender at half speed, followed by Miracloth filtration and low-speed centrifugation (1,000g). The thylakoid membrane fraction was purified from the microsomal pellet on discontinuous Percoll gradients as previously described (Friso et al., 2004
For 1D SDS-PAGE separation, proteins were equilibrated with SDS (0.2%), Na2CO3 (100 mM), dithiothreitol (100 mM), and Suc (10%) and separated on 12% Tricine gels (Schägger and von Jagow, 1987
For protein identification, Coomassie Brilliant Blue or Sypro Ruby stained spots were picked manually. Spots were automatically washed and digested with modified trypsin (Promega) as previously described (Shevchenko et al., 1996
Total RNA was isolated from the second leaf of plants as described above. Six biological replicates were used to compare wild-type and mutant transcript profiles in separate M and BS experiments. To maximize biological replication, different seedling pools were used for each of the 12 hybridizations. Microarray experiments and analyses were performed according to Sawers et al. (2007)
Three biological replicates were used for qPCR, with two internal technical replicates for each reaction. Total RNA (8 µg) was treated with 3 units of DNase I amplification grade enzyme (Invitrogen) at 37°C for 30 min to remove contaminating DNA in the presence of 80 units of RNaseOUT (Invitrogen). Enzymes and salts were removed from the RNA with TRIzol reagent (Invitrogen). One microgram of purified RNA was incubated at 70°C for 10 min with 50-ng random hexamers and the reaction cooled on ice. Additional reagents were added to a final concentration of 5 mM MgCl2, 0.01 M dithiothreitol, 0.5 mM dNTP, 40 units of RNaseOUT, and 200 units of SuperScript III reverse transcriptase (Invitrogen). Water was substituted for enzyme in the negative control. cDNA synthesis was performed by incubation at 25°C for 10 min, 50°C for 50 min, 80°C for 5 min, and a 4°C soak. Upon completion, the RNA template was destroyed with 2 units Escherichia coli RNase H, and cDNA was diluted with 60 µL of water. For qPCR reactions, the template was further diluted with three parts water, and the SYBR Green JumpStart Taq ReadyMix without MgCl2 kit (Sigma) was used with final concentrations of 2.3 mM MgCl2 and 24 ng/µL forward and reverse primers. Primer sequences are available in Supplemental Table S4. An internal reference dye was used to measure data quality. Samples were run at 95°C for 2 min, cycled 47 times between 95°C for 15 s and 60°C for 1 min, followed by a dissociation stage of 95°C for 15 s, 60°C for 15 s, and 95°C 15 s on an ABI Prism 7900HT sequence detection system (Applied Biosystems). Data were analyzed using ABI Prism SDS 2.1 software. Results were normalized using 18S rRNA reactions as a control. Sequence data for the maize homolog of Hcf136 can be found in the GenBank library under accession number EF587243. The data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) and are accessible through GEO Series accession number GSE9698. For Figure 1B, HCF136 homologs were aligned using the following accessions: Z. mays (ABQ53629), Oryza sativa (BAD62115.1), Arabidopsis (O82660), G. theta (NP_113453.1), and Synechocystis sp. PCC 6803 (NP_440411). S. bicolor protein information was assembled from CN132236, CN142773, CN142842, CN145337, CN150433, CN150507, and CN148500.
The following materials are available in the online version of this article.
We gratefully thank Dr. Thomas Owens, Dr. Robert Turgeon, Dr. Tesfamichael Kebrom, Ms. Phyllis Farmer, and Dr. Qi Sun for technical advice and helpful discussions, Shannon Caldwell and Anita Aluisio at the Cornell Integrated Microscopy Center (Veterinary Medical Center, Cornell University) for the electron micrographs, and Dr. Katia Wostrikoff for helpful discussions. Received November 18, 2007; accepted February 5, 2008; published February 7, 2008.
1 This work was supported by the National Science Foundation (grant no. DBI–0211935 to T.P.B. and K.J.v.W.) and the Natural Sciences and Engineering Research Council of Canada (to S.C.). 
2 Present address: Department of Plant Pathology, Cornell University, Ithaca, NY 14853. 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: Thomas P. Brutnell (tpb8{at}cornell.edu).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.113423 * Corresponding author; e-mail tpb8{at}cornell.edu.
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(atpA, TC303520); (9) CP47 (psbB, TC283413); (10) CF1
(AtpC, TC287102); (11) D1 (psbA, TC290677); (12) Cyt b6f Rieske iron-sulfur (TC286511); (13) unknown; (14) LHCII-1 (TC299123); (15) LHCII-1 (TC286602); (16) LHCII-1 (TC299123); (17) LHCII-3 (TC286603); (18) LHCI-4 (TC279557). Protein marker positions in kilodaltons are indicated between the two gel images. [See online article for color version of this figure.]
