PIF1 directly and indirectly regulates chlorophyll biosynthesis to optimize the greening process in Arabidopsis
Abstract
Plants depend on light signals to modulate many aspects of their development and optimize their photosynthetic capacity. Phytochromes (phys), a family of photoreceptors, initiate a signal transduction pathway that alters expression of a large number of genes to induce these responses. Recently, phyA and phyB were shown to bind members of a basic helix–loop–helix family of transcription factors called phy-interacting factors (PIFs). PIF1 negatively regulates chlorophyll biosynthesis and seed germination in the dark, and light-induced degradation of PIF1 relieves this negative regulation to promote photomorphogenesis. Here, we report that PIF1 regulates expression of a discrete set of genes in the dark, including protochlorophyllide oxidoreductase (POR), ferrochelatase (FeChII), and heme oxygenase (HO3), which are involved in controlling the chlorophyll biosynthetic pathway. Using ChIP and DNA gel-shift assays, we demonstrate that PIF1 directly binds to a G-box (CACGTG) DNA sequence element present in the PORC promoter. Moreover, in transient assays, PIF1 activates transcription of PORC in a G-box-dependent manner. These data strongly suggest that PIF1 directly and indirectly regulates key genes involved in chlorophyll biosynthesis to optimize the greening process in Arabidopsis.
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Light has a profound effect on plant growth and development. Plants not only rely on light signals to regulate developmental phases, but also to provide spatial and temporal information about their environment. Within plant cells, an array of photoreceptors detects several light characteristics such as wavelength, direction, duration, and intensity. Photoreceptors such as cryptochromes, phototropins, and an unidentified UV-B receptor perceive and respond to blue light, whereas phytochromes (phys) respond to the red (R) and far-red (FR) region of the spectrum (1, 2).
Phys exist in two spectral forms: a R light-absorbing Pr form and a FR light-absorbing Pfr form. R light induces conformation of phys to the Pfr, or “active” form; FR light coverts phys to the Pr, or “inactive” form. In Arabidopsis, phys are encoded by a small multigene family (PHYA-PHYE). All phys are active in R light; however, phyA is light labile and activated by both R and FR light. Both phyA and phyB are predominantly in the cytosol in the Pr form. The Pfr form is induced to translocate into nucleus upon light activation either by unmasking of Nuclear Localization Signal (NLS) present in the C-terminal domain (for phyB) (3) or through associated proteins (for phyA) (4). Activation of phys by light initiates a signaling cascade, which results in changes in gene expression that drive photomorphogenesis (2, 5, 6).
phyA and phyB interact in a conformer-specific manner with basic helix–loop–helix (bHLH) transcription factors called phy-interacting factors (PIFs) (7, 8). PIFs preferentially bind a G-box (CACGTG) DNA sequence element, which is a subclass of an E-box element (CANNTG) present in many light-regulated promoters (9, 10). Interactions between the Pfr form of phyB with PIF3 bound to a G-box promoter motif are hypothesized to directly regulate transcription of light-responsive genes involved in photomorphogenesis (10, 11). However, recent results show that PIFs are stable in the dark and are degraded in response to R and FR light in a phy-dependent manner (8, 12–17), suggesting that activated phys induce degradation of PIFs to promote photomorphogenesis.
Genetic analysis of PIF1 and PIF3-PIF5 suggests that these proteins function as negative regulators of distinct phy-signaling pathways (7, 8). For example, PIF3–PIF5 predominantly control hypocotyl length under R light (9, 18, 19, 20), whereas PIF1 functions as a negative regulator of chlorophyll biosynthesis in the dark and seed germination in FR light (13, 14, 21). PIF1 directly and indirectly regulates gibberellic acid biosynthesis and sensitivity to control seed germination (22). Compared with WT seedlings in the dark, pif1 seedlings accumulate higher amounts of free protochlorophyllide (Pchlide), a phototoxic intermediate in the chlorophyll biosynthetic pathway. Subsequent light exposure causes photooxidative damage and bleaching of pif1 seedlings (13, 21). PIF1 shows transcriptional activation activity in the dark, which is reduced by light-induced degradation of PIF1 to promote chlorophyll biosynthesis and seed germination in light (13, 14). However, the direct target genes by which PIF1 controls chlorophyll biosynthesis have not been identified. Here, we present evidence that PIF1 directly and indirectly regulates key genes in the chlorophyll biosynthetic pathway in the dark to optimize the greening process in Arabidopsis.
Results
PIF1 Regulates Expression of Tetrapyrrole Pathway Genes in the Dark.
Previously, we have shown that pif1 seedlings have higher levels of Pchlide than WT in the dark (21). Because PIF1 shows strong transcription activation activity in the dark (13, 21), we reasoned that identifying the genes differentially expressed in dark-grown pif1 and WT seedlings may provide further insight into the pif1 phenotype. To this end, we performed whole-genome expression profiling by using Affymatrix Microarray chips on RNA isolated from 4-day-old dark-grown WT and pif1 null mutant seedlings. Using P ≤ 0.05, the Bioconductor microarray analysis software identified only three genes (2.81X, PIF1; 1.96X, At4g17600; 1.91X, At5g44580) differentially expressed between WT and pif1 mutants. One of the three genes is PIF1, which shows a 2.8-fold reduction in expression between WT and the mutant, confirming the validity of our analysis method.
Because the Bioconductor software might be too stringent to detect small expression changes in pif1 seedlings, we used an alternative approach for data analyses as described (23). Using this approach, we identified additional differentially expressed genes (data not shown). Because of PIF1's role in chlorophyll biosynthesis, we focused our analyses on genes involved in the tetrapyrrole pathway [supporting information (SI) Table S1] (24). Interestingly, a few key genes encoding enzymes involved in tetrapyrrole pathway showed expression changes of at least 1.5-fold between the dark grown WT and pif1 samples (Fig. 1 and Table S1). To independently verify our microarray results, a semiquantitative RT-PCR assay was performed. The RT-PCR results largely support the microarray data (Fig. 1A and Table S1). Microarray analysis for ferrochelataseI (FeChI) (At5g26030) and ferrochelataseII (FeChII) (At2g30390), both of which are involved in the conversion of protoporphyrin IX (PPIX) to heme (25, 26), did not show a significant difference between the WT and pif1 samples (Table S1). However, semiquantitative and quantitative RT-PCR (qRT-PCR) analyses showed that FeChII is down-regulated in pif1 seedlings compared with WT (Fig. 1A and Table S1). Taken together, these results suggest that PIF1 is a subtle regulator that controls a small set of key genes involved in chlorophyll biosynthesis.
Fig. 1.
PIF1 Directly Regulates PORC in the Chlorophyll Biosynthesis Pathway.
Because PIFs bind the E/G-box DNA sequence element (CANNTG) (10, 21), we analyzed the upstream promoter region of the differentially expressed genes for the presence of these elements by using the PLACE web site (www.dna.affrc.go.jp/PLACE/signalscan.html). Results show that most of the differentially expressed genes have promoters with two or more E/G boxes (Table S2).
To determine whether these genes are directly regulated by PIF1, we transformed pif1 plants with a construct expressing PIF1 fused to a tandem affinity purification (TAP) tag, 35S:TAP-PIF1 (Fig. S1A) (27). As a control, we expressed a 35S:TAP-GFP construct in the WT background. After confirming that the 35S:TAP-PIF1 transgene complemented pif1 phenotypes (Fig. S1 B–G), we used both transgenic lines in a ChIP assay. After immunoprecipitation of protein–DNA complexes using antibody to the MYC tag, enriched DNA sequences were amplified by using primers to the promoter regions of the candidate genes. ChIP assay results show that the PORC promoter region was amplified from the immunoprecipitation (IP) fraction of 35S:TAP-PIF1 seedlings, but not in the 35S:TAP-GFP or without antibody samples (Fig. 2). Under these conditions, we observed no amplification of the promoter regions of the PORA, DVR, HO3, and FeChII genes. To determine whether these genes were targeted by PIF1 in slightly younger or older seedlings, the ChIP assay was performed on tissue from a range of developmental stages; however, no amplification of these promoters was observed (data not shown). These data suggest that PORC is a direct target of PIF1, whereas PORA, PORB, HO3, and FeChII genes are indirect targets of PIF1.
Fig. 2.
PIF1 Binds G-Box Motifs Within the PORC and FeChII Promoters.
Previously, we have shown that PIF1 binds a synthetic G-box motif by using a gel-shift assay (10, 21). To determine whether PIF1 directly binds the G/E boxes within PORC, a gel-shift assay was performed as described (9, 21). Results show that PIF1 binds the labeled PORC G-box fragment (Fig. 3A). The PORC promoter fragment containing a mutated G-box element did not compete with the WT G-box fragment for PIF1 binding. Because the FeChII promoter has an identical G-box as in the PORC promoter and FeChII expression is regulated by PIF1, we also examined whether PIF1 directly binds to the G-box present in the FeChII promoter. Cold FeChII promoter probe successfully competed with labeled PORC fragments for PIF1 binding (Fig. 3B). Further, mutated G-box FeChII probe did not compete for PIF1 binding with PORC. Control proteins, in vitro expressed-LUC and PIF3, did not bind the PORC G-box sequence in this assay (Fig. 3B). However, a similar PIF3 preparation bound a synthetic G-box originally identified as the PIF3 binding site (data not shown) (10). PIF1 did not bind to PORA and PORB E-box sequences under these experimental conditions (data not shown). These results suggest that PIF1 directly binds to the G-box present in both PORC and FeChII promoters in vitro in a sequence-specific manner.
Fig. 3.
PIF1 Regulates PORC and FeChII Expression in Vivo.
Given that PIF1 is a transcription factor, we wanted to determine whether PIF1 can activate transcription from a native promoter in vivo. As a control, we transiently expressed a non-PIF1 target promoter driving β-glucuronidase (GUS) (pACT2:GUS) in WT, pif1, and PIF1 overexpression (35S:LUC-PIF1) seedlings using the transient assay that we developed (21). GUS assay results show that all three genotypes express the same level of pACT2:GUS, suggesting that PIF1 does not control expression from this promoter (Fig. 4). To determine whether PIF1 can activate transcription from a native promoter, we transiently expressed the native PORC or FeChII promoters driving GUS expression in WT, pif1, and 35S:LUC-PIF1 seedlings (Fig. 4A). Results show that pPORC:GUS activity is significantly higher in 35S:LUC-PIF1 seedlings than in the WT or pif1 seedlings (Fig. 4B). To confirm our results, we measured endogenous PORC expression in these lines by using qRT-PCR assays and found a similar expression pattern as observed for the reporter GUS assays (Fig. 4B Inset). Strikingly, the increased GUS activity in 35S:LUC-PIF1 seedlings expressing pPORC:GUS is eliminated when the G-box within the PORC promoter is mutated (Fig. 4B). These results strongly suggest that PIF1 directly regulates PORC expression in a G-box-dependent manner.
Fig. 4.
GUS activity in pif1 lines expressing pFeChII:GUS was significantly reduced compared with GUS activity in pFeChII:GUS-expressing WT seedlings (Fig. 4C). Moreover, 35S:LUC-PIF1 lines in the pif1 background showed WT levels of FeChII expression, demonstrating rescue of the pif1 phenotypes in the dark (13). However, the 35S:LUC-PIF1 seedlings did not show overexpression of FeChII. Using qRT-PCR, we found that endogenous FeChII expression levels in WT, pif1, and 35S:LUC-PIF1 seedlings reflect the expression patterns found in the pFeChII:GUS assays (Fig. 4C Inset). In contrast to what was observed in the pPORCGm:GUS assays, the pFeChGm:GUS lines showed no significant change in GUS activity in the WT, pif1, and 35S:LUC-PIF1 backgrounds (Fig. 4C). These results suggest that PIF1 is necessary for activation of FeChII expression in a G-box-independent manner.
Because both PORC and FeChII are modestly induced in light (24), we investigated whether PIF1 plays a role in light regulation of these genes by using the qRT-PCR assays. Results show that the expression of PORC is modestly, but significantly, reduced in pif1 seedlings compared with WT seedlings (Fig. S2). However, pif1 seedlings display a WT FeChII expression level under these light conditions. Because PIF1 is rapidly degraded under light (13), and PORC and FeChII levels are reduced in the dark in the pif1 seedlings compared with WT seedlings (Figs. 1A, 4 B and C, and Fig. S2), these results suggest that PIF1 does not play a significant role in the light-induced expression of these genes.
pif1 Seedlings Have Reduced POR Enzyme Activity.
Microarray and RT-PCR data show that POR genes are down-regulated in pif1 seedlings compared with WT seedlings in the dark (Fig. 1A). To determine whether the transition from Pchlide to chlorophyllide (Chlide) was aberrant in pif1 seedlings, we performed spectrofluorometric analyses on acetone extracts of 4-day-old dark-grown pif1 and WT seedlings with or without a 5-min white light treatment. The results show that although dark-grown pif1 seedlings have a higher relative fluorescence peak at 632 nm, indicative of Pchlide, the relative fluorescence peak at 670 nm, indicative of Chlide, is lower in pif1 seedlings than in WT seedlings after the light treatment (Fig. 5). These in vivo enzyme assay results suggest that pif1 seedlings have reduced levels of POR enzyme activity and, consistent with our microarray data, support our hypothesis that PIF1 regulates expression of the POR genes in the dark (Fig. 1).
Fig. 5.
PIF1 Regulates Genes Involved in Heme Biosynthesis.
One of the major points of regulation in the chlorophyll pathway is the conversion of PPIX to either Mg-PP, which leads to chlorophyll production, or heme, which leads to phytochromobilin production (Fig. 1B) (25). Heme negatively regulates the chlorophyll pathway by down-regulating δ-aminolevulinic acid (ALA) production (Fig. 1B) (25, 28). Because pif1 seedlings show a reduced level of FeChII and an increased level of HO3 expression in the dark (Table S1 and Figs. 1A and 4C), it is possible that pif1 seedlings have reduced levels of heme compared with WT seedlings. Lower heme levels would result in less feedback inhibition of ALA production and a higher level of Pchlide production (25). Because direct measurement of heme in etiolated Arabidopsis seedlings poses significant technical challenges, we took an indirect approach as described (28). Exogenous application of the iron chelator 2′-2′-bipyridyl (BP) prevents conversion of PPIX to heme and allows accumulation of Mg-PP to detectable levels in seedlings. We measured Mg-PP levels in dark-grown WT and pif1 seedlings incubated with or without BP. Our results show that after BP treatment pif1 seedlings accumulate significantly higher amounts of Mg-PP than WT seedlings (Fig. S3). These data suggest that pif1 seedlings have a reduced amount of heme, possibly resulting from reduced expression of FeChII and an increased expression of HO3 (Figs. 1A and 4C). Alternatively, the higher levels of Mg-PP observed in the pif1 background may be a result of defects in the conversion of ALA to PPIX (Fig. 1B).
To address this notion, we measured PPIX levels in dark-grown seedlings treated with or without 10 mM ALA. Because Pchlide and PPIX fluorescence emission spectra overlap, and given that Pchlide levels are higher in the pif1 background (Fig. 5) (21), absorbance at 503 nm was measured. The results show that pif1 seedlings contain a WT level of PPIX (Fig. S4), suggesting that the elevated levels in Mg-PP found in the pif1 seedlings are a consequence of reduced levels of heme compared with WT seedlings.
Because heme is a negative feedback regulator of the early rate-limiting step in the pathway, reduced levels of heme are expected to increase the rate of ALA biosynthesis (Fig. 1B) (25). We measured the rate of ALA biosynthesis by using a protocol as described (29). The rate of ALA synthesis in pif1 seedlings is ≈2-fold higher than that in WT seedlings (Fig. 6). The modest increase in the rate of ALA synthesis is consistent with the modest increase in Pchlide levels in pif1 seedlings compared with WT seedlings (Fig. 5A). Taken together, these data suggest that PIF1 subtly regulates the level of heme in the dark to fine-tune the tetrapyrrole pathway in Arabidopsis.
Fig. 6.
Discussion
Exquisite regulation of the tetrapyyrole pathway in the dark is required to avoid photooxidative damage of seedlings upon illumination. This study provides genetic, molecular, and biochemical evidence that PIF1 directly and indirectly regulates key genes to fine-tune the tetrapyrrole pathway. Several lines of evidence suggest that PORC is a direct target of PIF1. First, microarray and RT-PCR/qRT-PCR assays established that PORC expression is reduced in dark-grown pif1 seedlings compared with WT seedlings (Fig. 1A and Table S1). Second, the ChIP assay shows that PIF1 binds to the promoter of PORC in vivo (Fig. 2). Third, PIF1 directly binds to the G-box element in the PORC promoter (Fig. 3A). Fourth, in transient expression assays PIF1 activates transcription of PORC in a G-box-dependent manner (Fig. 4 A and B). Fifth, regulation of PORC is consistent with our physiological data showing that after initial light exposure Chlide levels in pif1 seedlings are reduced compared with Chlide levels in WT seedlings (Fig. 5). Taken together, these results strongly suggest that PIF1 is a direct regulator of PORC expression.
Expression analyses data suggest that PIF1 regulates all three POR genes, with PORA and PORB displaying the most significant changes in expression (Table S1). However, direct interaction studies show that PORC is the only direct target of PIF1. One distinction between PORA, PORB, and PORC is the cis-elements present in their respective promoters. PORA and PORB promoters have E-boxes, whereas the PORC promoter contains a G-box motif (Table S2). The PIF1 homodimer binds only G-boxes and not E-boxes in in vitro gel-shift assays (Fig. 3 and data not shown). It is probable that PIF1 regulates PORA and PORB expression indirectly and PORC expression directly. Further, POR gene expression is developmentally regulated. PORA and PORB function in young seedlings during the transition from dark to light, and PORC functions in light-grown plants (25). Therefore, PIF1 might control chlorophyll biosynthesis not only during the initial dark-to-light transition, but also during daily light–dark cycles.
The tetrapyrrole pathway is primarily regulated by metabolic intermediates and transcriptional regulation of metabolic enzymes (25). Higher Pchlide content in dark-grown pif1 seedlings suggests that PIF1 either represses genes involved in Pchlide production or activates a repressor that down-regulates Pchlide production. Two well established repressors of the chlorophyll pathway are FLORESCENT (FLU) and heme (25). Both FLU and heme are negative feedback regulators targeting early steps in the chlorophyll pathway to repress production of downstream intermediates (25, 29) (Fig. 1B). Expression analyses confirm that PIF1 does not regulate FLU expression or the expression of other genes involved in conversion of ALA to Pchlide (Fig. 1A, Fig. S4, Table S1, and data not shown). Conversely, PIF1 indirectly activates the expression of FeChII and indirectly represses the expression of HO3 in the dark. FeChII encodes a ferrochelatase enzyme that converts PPIX to heme, and HO3 encodes a heme oxygenase enzyme that converts heme to biliverdin IXα (Table S1 and Figs. 1 and 4C). Although PIF1 regulation of FeChII is subtle (Fig. 1A), the net effect of FeChII and HO3 expression may lead to lower heme content in pif1 seedlings compared with WT seedlings. Reduced heme content relieves the feedback inhibition of ALA synthesis and results in a higher level of Pchlide in pif1 seedlings compared with WT seedlings (Fig. 5A) (21). Increased levels of Mg-PP in pif1 seedlings compared with WT seedlings after BP treatment (Fig. S3A) and the comparable level of PPIX after ALA treatment (Fig. S4A) suggest that pif1 seedlings have less endogenous heme than WT seedlings. Moreover, pif1 seedlings have a modest increase (≈2-fold) in the rate of ALA synthesis compared with WT seedlings (Fig. 6). Interestingly, a reduction in plastidic FeCh in tobacco resulted in an increased rate of ALA synthesis and higher chlorophyll production (25, 30), similar to our results. Combined, our data strongly suggest that PIF1 controls heme levels to optimize Pchlide production in the dark.
Previous work shows that PIF1 functions as a negative regulator of chlorophyll biosynthesis under prolonged light conditions (13, 21). Initially, this finding appears to contradict our conclusion that pif1 seedlings have reduced POR enzyme activity. However, because POR expression is reduced but not eliminated in the pif1 background (Fig. 1A), it is possible that the amount of Pchlide, not the POR enzyme levels, is a limiting factor for chlorophyll biosynthesis under prolonged light conditions. pif1 seedlings have an increased rate of ALA synthesis caused by reduced heme content compared with WT seedlings (Fig. S3 and Fig. 6), resulting in increased Pchlide synthesis in pif1 seedlings (Fig. 5). Therefore, the higher Pchlide level will result in higher chlorophyll synthesis in pif1 seedlings compared with WT seedlings upon prolonged light exposure. Further experiments are necessary to determine whether the POR enzymes or their substrate (Pchlide) is the rate-limiting factor under prolonged light conditions.
PIF1, PIF3, and PIF4 bind a G-box DNA sequence element present in light-regulated promoters, raising questions about how PIFs specify gene targets (Figs. 3 and 4) (9, 10, 21, 31). Our results show that PIF3 does not bind to the G-box present in the PORC and FeChII promoters (Fig. 3B). Both PORC and FeChII promoters contain the G-box sequence, A[CACGTG]T, flanked with an adenine (A) at the 5′ end and a thymine (T) at the 3′ end. Indeed, random DNA binding site selection studies for PIF3 did not isolate any G-box sequence flanked by a 3′ T (10). These results suggest that PIF binding is specified by the sequence flanking the G-box motif in gene promoters, as has been shown for animal bHLH DNA binding (32).
PIFs interact with differential affinities to phys, and PIFs function in distinct phy signaling pathways (8). However, how these interactions result in light regulation of gene expression is still unclear. Our data show that PIF1 constitutively activates gene expression in the dark and does not play a major role in light regulation of these genes (Figs. 1, 4, and S2), which is consistent with the light-induced degradation of PIF1. These results are also consistent with recent reports that both PIF1 and PIF3 constitutively activate gene expression in the dark (22, 33). Therefore, how phys regulate gene expression in response to light remains to be determined.
Although PIF1 regulates key genes in the tetrapyrrole pathway, the effects are subtle. Other bHLH proteins in addition to PIF1 may regulate the expression of PIF1 target genes. The promoters of most of these genes have multiple E/G-boxes within the 500 bp upstream of ATG (Table S2). It is possible that PIF1 binds E-box motifs as heterodimers with other bHLH proteins. The Arabidopsis genome encodes ≈162 bHLH proteins (32), and many of these factors regulate photomorphogenesis (8). It is likely that combinatorial control by multiple factors is necessary to optimize the greening process.
In conclusion, our data show that PIF1 directly and indirectly regulates key genes in the tetrapyrrole pathway in the dark to prepare young etiolated seedlings to respond to light. PIF1 appears to act both positively and negatively to fine-tune the chlorophyll biosynthetic pathway (Fig. 1). Because PIF1 is degraded in light and reaccumulates in the dark (13), PIF1 might provide plants an adaptive advantage under natural light–dark cycles by reducing the daily photooxidative damage at dawn, and thereby ensures robustness and fitness of plants under an ambient light environment.
Materials and Methods
Plant Material and Growth Conditions.
Microarray Analyses.
Total RNA was isolated from 4-day-old WT and pif1 dark-grown seedlings. Microarray hybridizations and probe synthesis were performed as in ref. 23 on RNA from three independent biological samples. To identify genes that are regulated by PIF1, the data files were also analyzed by using Microsoft Excel as described (23).
RNA Isolation, RT-PCR, and qRT-PCR.
Total RNA was isolated from 4-day-old dark-grown WT, pif1, and 35S:LUC-PIF1 transgenic seedlings by using the RNase Plant Mini Kit (Qiagen) and reverse-transcribed by using SuperScript II (Invitrogen) per the manufacturer's protocol. The qRT-PCR assays used the Power SYBR Green RT-PCR Reagents Kit (Applied Biosystems). Primer sequences used for RT-PCR and qRT-PCR can be found in Table S3, and additional details are available in SI Text.
ChIP Assay.
ChIP assays were performed as in ref. 34, except 3-day-old dark-grown 35S:TAP-PIF1 and 35S:TAP-GFP seedlings were vacuum-infiltrated with 1% formaldehyde for 1 h at 4°C, and cross-linking was quenched by vacuum infiltration with 0.125 M glycine for 3 min. mAb against MYC tag (Calbiochem) was used for IP.
DNA Gel-Shift Assay.
DNA gel-shift assays were performed as described (9, 10). PIF1, PIF3, and LUC were synthesized by using the Rabbit Reticulocyte TNT system (Promega) as described (9). A 70-bp PORC promoter fragment containing a G-box motif was labeled with 32P-dCTP. Cold competitor probe was generated from dimerized oligos of the PORC or FeChII promoter region containing the G-box promoter motif. Probe sequences are shown in Table S3.
Transient Transfection of Promoter-GUS Fusions.
To construct pPORC:GUS, a 1.6-kb promoter region of the PORC gene was cloned into the pENTR vector (Invitrogen), sequenced, and recombined into pBGWFS7 destination vector (35). The G-box element in the PORC promoter was mutated by using a site-directed mutagenesis kit (Stratagene) to produce pPORCGm:GUS. A 1.0-kb promoter region of the FeChII gene was used to construct pFeChII:GUS and pFeChIIGm:GUS as described above. A 1.4-kb promoter region of the ACT2 gene (At3g18780) was used to construct pACT2:GUS as described above. The DNA-coated beads were bombarded into 3.5-day-old WT, pif1, or 35S:LUC-PIF1 transgenic seedlings under dim light as described (21). Seedlings were grown vertically in individually wrapped plates in darkness and opened just before bombardment. Immediately after bombardment, the seedlings were exposed to 15 min of FR light (34 μmol·m−2·s−1) before growing in the dark for 16 h. Total protein was extracted in the darkroom under safe green light, and the protein concentration, Renilla Luciferase, and GUS activity were determined as described (13, 21).
Analysis of Chlorophyll Pathway Intermediates.
Pchlide and Chlide were extracted as in ref. 28 except 4-day-old dark-grown WT and pif1 seedlings were used. Spectrofluorometery (TimeMaster Pro; Photon Technologies International) was performed at an excitation wavelength of 440 nm and an emission wavelength of 600–700 nm, and data were curve-fitted by using PeakFit, version 4.11 (Systat Software). The ALA feeding experiment was carried out as described (28), except ALA or buffer control was vacuum-infiltrated for 5 min at 25 Hg into 4-day-old WT and pif1 seedlings. Measurement of ALA synthesis rate was carried out as in ref. 29 on 3-day-old seedlings grown in 8-h light/16-h dark cycles, and samples were harvested at the end of the dark period.
Additional details are provided in SI Text.
Data Availability
Data deposition: The sequences reported in this paper has been deposited in the GenBank database (accession no. GSE11594).
Acknowledgments.
We thank Joshua Russell for assistance with the spectrofluorometer, Sharyn Perry for help with ChIP assay, and Phi Luong and Julie Sottilo for technical assistance. This work was supported by National Science Foundation Grant IBN-0418653 and a set-up fund from the University of Texas at Austin (to E.H.).
Supporting Information
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© 2008 by The National Academy of Sciences of the USA.
Data Availability
Data deposition: The sequences reported in this paper has been deposited in the GenBank database (accession no. GSE11594).
Submission history
Received: December 11, 2007
Published online: July 8, 2008
Published in issue: July 8, 2008
Keywords
Acknowledgments
We thank Joshua Russell for assistance with the spectrofluorometer, Sharyn Perry for help with ChIP assay, and Phi Luong and Julie Sottilo for technical assistance. This work was supported by National Science Foundation Grant IBN-0418653 and a set-up fund from the University of Texas at Austin (to E.H.).
Notes
This article contains supporting information online at www.pnas.org/cgi/content/full/0803611105/DCSupplemental.
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Competing Interests
The authors declare no conflict of interest.
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PIF1 directly and indirectly regulates chlorophyll biosynthesis to optimize the greening process in Arabidopsis, Proc. Natl. Acad. Sci. U.S.A.
105 (27) 9433-9438,
https://doi.org/10.1073/pnas.0803611105
(2008).
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