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Originally published In Press as doi:10.1074/jbc.M601172200 on April 25, 2006

J. Biol. Chem., Vol. 281, Issue 31, 22190-22199, August 4, 2006
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A Basic Leucine Zipper Transcription Factor, G-box-binding Factor 1, Regulates Blue Light-mediated Photomorphogenic Growth in Arabidopsis*

Chandrashekara Mallappa1, Vandana Yadav1, Prem Negi, and Sudip Chattopadhyay2

From the National Centre for Plant Genome Research, Laboratory 101, Aruna Asaf Ali Marg, New Delhi 110067, India

Received for publication, February 7, 2006 , and in revised form, April 24, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Several transcriptional regulators have been identified and demonstrated to play either positive or negative regulatory roles in seedling development. However, the regulatory coordination between hypocotyl elongation and cotyledon expansion during early seedling development in plants remains unknown. We report the identification of a Z-box binding factor (ZBF2) and its functional characterization in cryptochrome-mediated blue light signaling. ZBF2 encodes a G-box binding factor (GBF1), which is a basic leucine zipper transcription factor. Our DNA-protein interaction studies reveal that ZBF2/GBF1 also interacts with the Z-box light-responsive element of light-regulated promoters. Genetic analyses of gbf1 mutants and overexpression studies suggest that GBF1 acts as a repressor of blue light-mediated inhibition in hypocotyl elongation, however, it acts as a positive regulator of cotyledon expansion during photomorphogenic growth. Furthermore, whereas GBF1 acts as a positive regulator of lateral root formation, it differentially regulates the expression of light-inducible genes. Taken together, these results demonstrate that GBF1 is a unique transcriptional regulator of photomorphogenesis in blue light.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Arabidopsis seedlings are genetically defined to follow two distinct developmental pathways: skotomorphogenesis or etiolation in the dark and photomorphogenesis or de-etiolation in the light (1-3). In the dark, seedlings grow with elongated hypocotyls, small and closed cotyledons, and the light-inducible genes are expressed either at low or below detectable levels. The presence of light inhibits hypocotyl elongation, promotes cotyledon opening and expansion, and thus results in photomorphogenesis. The light-inducible genes are expressed at high levels during photomorphogenic growth.

Plants are able to perceive various wavelengths of light through photoreceptors. Far-red and red light are perceived by phytochromes (phyA to phyE)3; whereas cryptochromes (cry1 and cry2) are involved in the perception of blue and UV-A light (4-7). Recent studies have made significant progress in the identification and functional characterization of downstream components in phytochrome signaling (1, 2, 6-8). HYH, AtPP7, and ZBF1/MYC2 have been reported as blue light (BL)-specific regulators of photomorphogenic growth in Arabidopsis (9-11). However, the connection of photoperception to transcription in BL still remains largely unclear (11).

Several transcription factors have been reported that are involved in early seedling development in Arabidopsis (11-22). COP1, a repressor of photomorphogenesis in the dark, acts as an ubiquitin ligase, and it interacts with and mediates the degradation of photomorphogenesis promoting factors such as HY5, HYH, LAF1, and HFR1 in the dark (9, 23-26). Recent studies have shown that COP1 interacts with SPA1, a negative regulator acting in far-red light, and this interaction is critical for proteasome-mediated degradation of HY5 and LAF1 (25, 27, 28).

Analyses of the promoter sequences of light-inducible genes, including CAB, RBCS, and CHS, have led to identification of at least four commonly found light-responsive elements (LREs): G, GATA, GT1, and Z-box, which have been demonstrated to be essential for light-mediated transcriptional activity (29-35). Several LRE-specific transacting factors have been identified earlier, and in some cases the genes that encode such factors have been cloned and their functions have been investigated (31, 36). A four-member gene family encoding proteins containing basic leucine zipper DNA binding domains (GBFs) have been reported (37, 38). Extensive DNA-protein interaction studies were carried out with GBFs. The light-regulated modification and subcellular localization of GBFs have also been investigated. It has been proposed from these studies that the limited nuclear access may be an important controller of the activities of GBFs (39). However, the in vivo functions of these genes are yet to be defined.

A Z-DNA-forming sequence (ATACGTGT) is present in CAB1 minimal promoter that is essential for light-dependent developmental expression of CAB1 gene (29). Recent studies have revealed that the Z-box containing synthetic and native promoters are responsive to phyA, phyB, and cry1 photoreceptors and are under the control of downstream regulatory components such as COP1 and HY5 (33, 35). To identify and clone ZBFs (Z-box binding factors), we have carried out DNA-ligand binding screening to screen an Arabidopsis cDNA expression library and have identified several such factors. One of these ZBFs, ZBF1/MYC2, has very recently been shown to be a negative regulator of blue light-mediated photomorphogenic growth. Furthermore, it has been demonstrated that ZBF1/MYC2 acts as a point of cross-talk among light, abscisic acid, and jasmonic acid signaling pathways (11, 40, 41). We have investigated the functional relevance to light-regulated gene expression and photomorphogenic growth of another ZBF (ZBF2/GBF1) in this study.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Materials and Transformations—Surface-sterilized seeds were sown on Murashige and Skoog plates, kept at 4 °C in darkness for 3-5 days, and transferred to light at 22 °C. The intensities of continuous light sources used in this study are: white light (100, 60, 30, 15, 5, and 1 µmol m-2 s-1); blue light (40, 30, 15, 5, and 1 µmol m-2 s-1); red light (95, 30, 15, and 5 µmol m-2 s-1); and far-red light (90, 30, 15, and 5 µmol m-2 s-1). Unless otherwise mentioned, the highest light intensities were used for the experiments.

The T-DNA-tagged mutant lines heterozygous or homozygous for the zbf2/gbf1 mutations were identified by genomic PCR analyses. Individual plants of T2 generation, obtained from a self-fertilized heterozygous plant, were examined by genomic PCR using the left border-specific primer LBP: 5'-GCGTGGACCGCTTGCTGCACCT-3' and GBF1-specific primers LP13: 5'-GTGCCATAAGGCGGCATCATA-3' and RP13: 5'-TGCAAACAAACACCTTTGCATGT-3' (for gbf1-1 mutants); and LP14: 5'-GCACCGAACCTTGGATTTCAC-3' and RP14: 5'-TTCCCATCCCCAGTTGGATCT-3' (for gbf1-2 mutants). A segregated wild-type (Col) line was used to compare the phenotypic and molecular differences with the gbf1 mutants.

For the generation of GBF1/ZBF2 over-expresser transgenic lines, a 1.15-kb fragment of GBF1 cDNA was PCR-amplified using primers 5'-GAAGATCTTGAGTAACACAAGTAAGTAGTAAGC-3' and 5'-GACTAGTAATCGTAGCTTTTGCAGCTT-3'. The PCR product was digested and cloned into BglII and SpeI site of pCAMBIA1303, a binary vector carrying the cauliflower mosaic virus (CaMV) 35 S promoter. For the complementation test, a genomic fragment containing full-length GBF1 and ~1.3 kb upstream DNA sequence was cloned into the SmaI site of pBI101.2 vector. The Agrobacterium strain GV3101 was transformed individually with each recombinant construct. The Arabidopsis wild-type (Ws) plants (for overexpression) or gbf1-1 mutant plants (for complementation) were transformed using Agrobacterium-mediated vacuum infiltration method. Transgenic plants (T1) were screened on 15 µg/ml hygromycin or 20 µg/ml kanamycin containing Murashige and Skoog plates. Several individual lines with a single T-DNA locus, as determined by the segregation of hygromycin- or kanamycin-resistant versus-sensitive ratios (3:1), were selected, and homozygous transgenic plants were generated for further studies.

For the generation of gbf1 cry1, gbf1 cry2, and gbf1 phyA double mutants, homozygous gbf1-1 mutant plants (Col) were crossed individually with hy4-2.23N (Ler (4)), cry2-1 (Col (42)), and phyA-101 (RLD (43)) homozygous mutant lines. F2 seedlings were grown in white light (WL, 60 µmol m-2 s-1) or far-red light (FR, 30 µmol m-2 s-1) for the identification of cry1, cry2, or phyA homozygous lines, respectively, and elongated seedlings were transferred to soil. To determine the genotype at GBF1 locus, ~40 seedlings from each line were tested by genomic PCR. F3 progeny that are homozygous for gbf1-1 mutant plants were further tested and designated as gbf1 cry1, gbf1 cry2, and gbf1 phyA double mutants. Because gbf1, cry1, cry2, and phyA were of different ecotype backgrounds, F2 seedlings, which were heterozygous for cry1, cry2, or phyA mutations but homozygous wild type for GBF1 were used as control (WT).

Transgenic Lines with Promoter-GUS Constructs and GUS Assays—The promoter-reporter constructs used in this study have been described (33, 35). GUS staining (using 20-30 seedlings in each sample) and GUS activity measurements (40-50 seedlings) has been described previously (34). Wild-type and gbf1 mutant plants containing the same transgene were stained for the same length of time.

Measurements of Epidermal Cell Length and Expansion—Measurements of epidermal cell length and expansion of 6-day-old seedlings were essentially carried out as described (44, 45).

Electrophoretic Mobility Shift (Gel Shift) Assays—The full-length GBF1 cDNA was cloned in pGEX4T-2 vector, and GST-GBF1 was induced using 1 mM isopropyl 1-thio-beta-D-galactopyranoside and overexpressed in Escherichia coli. The overexpressed GST-GBF1 was affinity-purified following the manufacturer's protocol (Amersham Biosciences). The DNA binding assays were performed as described (13). The 189-bp DNA fragment of CAB1 minimal promoter was cloned into pBluescript vector after PCR with primers: forward, 5'-CGGAATTCATAAGGATAGAGAGATCTATTC-3' and reverse, 5'-CGGGATCCTGAGGTTGCTATTGGCTAGTCAT-3' using genomic DNA as template. The 189-bp fragment was digested with EcoRI plus BamHI, purified, and 3'-end-labeled for using as probe for the DNA binding assays. One nanogram of labeled DNA was used for each binding reactions.

Northern and Immunoblot Analyses—Total RNA was extracted using the RNeasy plant minikit (Qiagen) following the manufacturer's instruction. We used a 1.15-kb full-length cDNA fragment of GBF1 for probe preparation using random priming kit (MegaprimeTM, Amersham Biosciences) following the manufacturer's instructions. The DNA fragments of CAB, RBCS, and CHS genes were used for probes as described (11). To quantify the Northern blot data, the intensity of each band was quantified by Fluor-S-MultiImager (Bio-Rad) and ratios of the genes GBF1, CAB, RBCS, or CHS versus its corresponding 18 S rRNA band were determined and plotted.

Protein extracts were prepared from wild-type or gbf1 mutant seedlings. 20 µg of total protein was used for immunoblot analysis. Proteins were separated by 8% SDS-PAGE. Pre-stained protein markers (Amersham Biosciences) were used for molecular mass determination. The gel was stained with Coomassie Brilliant Blue R-250 for visualization. For immunoblot analysis, the proteins were transferred to Hybond C-Extra (Amersham Biosciences), blocked with 5% bovine serum albumin in phosphate-buffered saline (10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 2.7 mM KCl) and probed with affinity-purified GBF1 polyclonal antibodies.

Chlorophyll and Anthocyanin Measurements—Chlorophyll and anthocyanin contents were measured following essentially the same protocols as described in (9). Sequence data (GBF1) from this article have been deposited with the EMBL/Gen-BankTM data libraries under accession number AJ843257 [GenBank] .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Molecular Cloning of GBF1—We screened a cDNA expression library by DNA-ligand binding screening for the identification of ZBFs. Several genes were identified and cloned from this screen, the products of which showed specific interactions with the Z-box (11, 35). We chose one of these genes, ZBF2 (Z-box binding factor 2), which was represented by three independent cDNA clones, for this study. One of the cDNAs of ZBF2 isolated from the ligand binding screen appeared to be a full-length cDNA (At1g36730). It codes for a protein of 315 amino acids with a basic leucine zipper (bZIP) DNA binding domain. The same protein was earlier shown to be interacting with the G-box and designated as GBF1 (37). Therefore, hence onwards we designate this gene as GBF1.

GBF1 Interacts with the Z- and G-box LREs of Light-regulated Promoters—To further examine whether GBF1 was able to specifically interact with the Z-box, we purified GST-GBF1 fusion protein from E. coli and performed electrophoretic mobility shift (gel shift) assays using the Z-box DNA as probe. A high affinity DNA-protein complex was detected along with the free probe as shown in Fig. 1A (lane 3). This DNA binding activity of GBF1 was efficiently competed by 50 or 100 molar excess unlabeled Z-box DNA (Fig. 1A, lanes 4 and 5). Because it was earlier reported that GBF1 could interact with the G-box (Schindler et al. (37)), we also competed this binding activity with a consensus G-box (13). As shown in Fig. 1A (lanes 6 and 7), the G-box was able to compete more efficiently this binding activity. Whereas 50 molar excess of Z-box was unable to complete the interaction completely, the unlabeled G-box was able to do so at the same molar excess. However, GT1 failed to compete for the GBF1 binding activity even at 100 M excess (Fig. 1A, lane 8).

To further test the relative affinity of GBF1 for the G- and Z-box LREs, we carried out similar experiments using the tetrameric G-box as probe. As shown in Fig. 1B, whereas 50 molar excess of unlabeled Z-box was unable to compete the binding activity completely, the G-box was able to efficiently compete the interaction at the same molar excess (lanes 4-7). In fact, further experiments using various amounts of unlabeled G- or Z-box revealed that, whereas 40 molar excess of unlabeled G-box was able to compete the binding activity, ~70 molar excess of Z-box was required to compete the binding activity of GBF1 completely (data not shown). Taken together, these results suggest that GBF1 interacts with the Z- and G-box LREs, and the protein may have slightly more affinity toward the G-box as compare with the Z-box.


Figure 1
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  		FIGURE 1.
GBF1 interacts with the Z- and G-box of light-responsive promoters. A, electrophoretic mobility shift assays (gel shift) using GST-GBF1 (GBF1) and the consensus dimeric Z-box LRE (35) as probe. Approximately 200 ng of recombinant protein was added (lanes 3-8) to the radioactively labeled Z-box. No protein was added in lane 1, and 500 ng of GST protein was added in lane 2. The protein-DNA complexes were resolved on 7% native polyacrylamide gel. The triangle indicates increasing concentrations of the competitors (Comp) and the plus and minus signs indicate the presence or absence of competitors, respectively. B, gel-shift assays using GST-GBF1 and the consensus tetrameric G-box (13) as probe. Experimental details are as in A. C, gel-shift assays using GST-GBF1 and the native CAB1 minimal promoter as probe. Approximately 200 ng of recombinant protein was added (lanes 3-7) to radioactively labeled CAB1 DNA fragment. Experimental details are as in A.

 
To further substantiate the interaction of GBF1 with the Z-box, we tested the ability of GBF1 to interact with the Z-box present in native CAB1 minimal promoter. The Z-box present within the minimal promoter region of CAB1 has been shown to be critical for light regulated expression of this gene (29). The 189-bp DNA fragment of CAB1 was used for gel shift assays. As shown in Fig. 1C, whereas GST alone did not show any binding activity, a strong low mobility DNA-protein complex was formed with GST-GBF1 fusion protein (lanes 2 and 3). This interaction was efficiently competed out with 80 or 120 molar excess of unlabeled Z-box (Fig. 1C, lanes 4 and 5) but not with 120 molar excess of GT1 or Zm, a mutated version of the Z-box (Fig. 1C, lanes 6 and 7). Taken together, these results demonstrate that GBF1 specifically binds to the Z-box of native CAB1 minimal promoter.

Isolation and Characterization of Null Mutations in GBF1—To investigate the in vivo function of GBF1, we searched for mutants in T-DNA knock-out collections (46). Two independent mutant lines with T-DNA insertion were identified, and the corresponding alleles were designated as gbf1-1 (zbf2-1) and gbf1-2 (zbf2-2). We performed PCR genotyping analyses to determine plants that are homozygous or heterozygous for gbf1-1 or gbf1-2 mutations. We monitored the segregation of self-fertilized plants heterozygous for gbf1-1 or gbf1-2. The segregation ratios determined by the analyses of genotyping PCR in T2 progeny suggested that a single T-DNA locus was present in each of gbf1-1 or gbf1-2 mutant lines. The junctions of T-DNA and GBF1 were amplified by PCR, and the DNA sequence analyses revealed that the T-DNA was inserted in nucleotide position 80 bp upstream to the start codon of GBF1 in gbf1-1, and in nucleotide position 660 bp downstream to the start codon of GBF1 in gbf1-2 mutants (Fig. 2A). Northern and immunoblot analyses were unable to detect any GBF1 mRNA or protein in gbf1-1 or gbf1-2 mutant backgrounds suggesting that gbf1-1 and gbf1-2 are likely to be null mutants (Fig. 2, B-D).


Figure 2
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  		FIGURE 2.
The identification of gbf1 mutants and the expression of GBF1 in wild-type seedlings. A, the schematic diagram of the T-DNA insertion sites in GBF1. The inverted triangles show the T-DNA insertion sites. The exons and introns are shown as boxes and arrowheads, respectively. B, RNA gel blot analysis of GBF1 in segregated wild-type (Col) and gbf1-1 mutant (Col) seedlings. 20 µg of total RNA was loaded onto each lane. The 1.15-kb full-length cDNA fragment of GBF1 was used as probe. The 18 S rRNA is shown as loading control. C, immunoblot of 20 µg of total protein prepared from wild-type (Col) and gbf1-1 mutants. Affinity-purified GBF1 polyclonal antibodies were used as primary antibody for detection of GBF1. Coomassie-stained protein gel (Total protein) is shown as loading control. D, immunoblot of 20 µg of total protein prepared from wild-type (Col) and gbf1-2 mutants (Col). Affinity-purified GBF1 polyclonal antibodies were used as primary antibody for detection of GBF1. The asterisk marks a cross-reacting protein band in the same blot indicating the loading control. E, time course of GBF1 transcript or protein accumulation. Upper panel: 5-day-old seedlings (Col) grown in constant darkness (D) or white light (WL) were transferred to WL or D, respectively, for 0.5, 1, 2, or 4 h. GBF1 transcript of 5-day-old seedlings grown in constant D or WL are shown as 0 h. Experimental details are as in B. Lower panel: time-course experiment of GBF1 protein accumulation. Experimental details are the same as in B (upper panel) and D. F, light-regulated expression of GBF1. 6-day-old wild-type (Col) seedlings grown in constant darkness (D), WL, FR, RL, or BL were used for RNA gel blot analyses. Experimental details are the same as in B. G, expression of GBF1 in different light qualities after WL pretreatment. 5-day-old seedlings grown in constant WL were transferred to RL, BL, or FR for 0.5, 1, 2, or 4 h, and the steady-state mRNA levels of GBF1 were determined. Experimental details are the same as in B. rRNA has been shown as loading control. H, tissue-specific expression of GBF1. Total RNA was isolated from root (R), stem (S), leaf (L), or flower (F) of 30-day-old wild-type Arabidopsis plants grown in WL (16-h light and 8-h dark cycle). Experimental details are the same as in B. I-K, quantification of the Northern blot data in E (upper panel), F, and H, respectively, by Fluor-S-MultiImager (Bio-Rad)

 
Previous studies revealed that GBF1 mRNA was present in both light and dark grown cotyledons of 5-day-old wild-type seedlings (37). To quantify and expand our understanding about the pattern of expression of GBF1 in wild-type background, we carried out time-course experiments. For these experiments, 5-day-old seedlings grown in constant darkness or WL were transferred to WL or darkness, respectively, for 0.5, 1, 2, or 4 h, and the steady-state mRNA levels were measured. About 3-fold reduction in the expression of GBF1 was detected after 4 h of exposure to WL as compared with dark grown seedlings (Fig. 2E, upper panel and I). In agreement with this observation, the expression of GBF1 was increased to ~4-fold after 4 h of exposure to darkness as compared with WL grown seedlings (Fig. 2E, upper panel and I). We performed similar time-course experiments to determine whether the level of GBF1 protein was also higher in dark grown seedlings. However, we could not detect any significant change at the protein level during dark to WL transitions or vice versa (Fig. 2E, lower panel). These results suggest that, although the transcript level of GBF1 varies depending on the presence or absence of WL, the protein level remains largely unaltered.

Because GBF1 is expressed in WL, we asked whether it was expressed under various wavelengths of light, including far-red light (FR), red light (RL), and blue light (BL). As shown in Fig. 2 (F and J), GBF1 was expressed in all light conditions tested with maximum level of expression in FR. To further examine the light-dependent expression of GBF1, we carried out time-course experiments. For these experiments, 5-day-old seedlings grown in constant WL were transferred to various wavelengths of light for 0.5, 1, 2, or 4 h, and the steady-state mRNA levels of GBF1 were determined. As shown in Fig. 2G, the expression of GBF1 slightly decreased or increased in RL or BL, respectively, however the expression of GBF1 was significantly elevated after 4 h of exposure to FR. The examination of tissue specific expression of GBF1 in adult plants revealed that the gene was expressed in root, stem, and flower at similar levels, however ~2-fold less expression was detected in the leaf tissues (Fig. 2, H and K).

gbf1 Mutants Exhibit Blue Light-specific Morphological Defects in Seedling Development—We monitored the growth of 6-day-old gbf1 mutant seedlings in constant darkness or WL conditions. As shown in Figs. 3A and 4 (A and B), no morphological difference was detected between wild-type and gbf1 mutants grown in constant darkness. However, gbf1 mutants displayed increased sensitivity to WL irradiation under various fluences and therefore, resulted in strikingly shorter hypocotyls as compared with the wild-type seedlings (Figs. 3B, 3C, 3J, and 4A). The effects appeared to be more pronounced within 5-30 µmol m-2 s-1 fluence rates of WL. We asked whether the hypersensitive phenotype of gbf1 was specific to a particular wavelength of light. To address this question, the growth of 6-day-old seedlings under various wavelengths of light was tested. The enhanced inhibition in hypocotyl elongation in gbf1 was observed in constant BL, however, no significant change in hypocotyl length was observed in constant RL or FR under various fluences (Figs. 3D, 3E, 3F, 3K, and 4B). Furthermore, although the hypocotyls of gbf1 displayed hypersensitivity to WL and BL, the cotyledons were found to be less sensitive to WL and BL. Thereby, the cotyledons of gbf1 mutants were found to be significantly smaller as compared with wild-type seedlings under WL and BL grown conditions (Figs. 3G, 3H, 3L, 4C, and 4D). Taken together, these results suggest that GBF1 acts as a negative regulator of inhibition of hypocotyl elongation, however, it functions as a positive regulator of cotyledon expansion in BL. The examination of the leaf size of wild-type and gbf1 mutant plants, however, revealed no significant differences (Fig. 3R). A genomic fragment containing GBF1 and its upstream sequence of ~1.3 kb was introduced into the gbf1-1 mutants plants for complementation test. The transgenic seedlings were unable to display BL-specific phenotypes suggesting that the observed phenotypes of gbf1 mutants are due to the loss of GBF1 functions (data not shown).


Figure 3
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  		FIGURE 3.
Mutation in GBF1 results in various effects. Segregated wild-type (Col) and gbf1-1 (Col) mutants (A-I) or gbf1-2 (Col) mutants (J-L) are shown on the left and right, respectively. A-F, 6-day-old seedlings were grown in constant darkness, WL (5 µmol m-2 s-1), WL (30 µmol m-2 s-1), BL (30 µmol m-2 s-1), RL (95 µmol m-2 s-1), or FR (90 µmol m-2 s-1), respectively. G and H, cotyledons of 6-day-old seedlings grown in constant WL (30 µmol m-2 s-1), or BL (30 µmol m-2 s-1), respectively. I, 16-day-old plants grown in constant WL (100 µmol m-2 s-1). J-K, 6-day-old seedlings were grown in constant WL (5 µmol m-2 s-1), or BL (30 µmol m-2 s-1), respectively. L, cotyledons of 6-day-old seedlings grown in constant BL (30 µmol m-2 s-1). M-O, hypocotyl epidermal cells of 6-day-old wild-type, gbf1-1, and gbf1-2 seedlings, respectively, grown in constant BL (30 µmol m-2 s-1). P and Q, imprints of cotyledon epidermal cells of 6-day-old wild-type and gbf1-1 seedlings, respectively, grown in constant BL (30 µmol m-2 s-1). R, 16-day-old wild-type, gbf1-1, and gbf1-2 (from left to right) plants grown in WL (30 µmol m-2 s-1).

 
To determine whether the enhanced inhibition in hypocotyl elongation or smaller cotyledon size of gbf1mutants is due to the altered cell elongation or expansion, we examined the size of epidermal cells of gbf1 mutants and compared with the wild-type 6-day-old seedlings grown in BL. As shown in Figs. 3 (M-O) and 6B, the epidermal cells of hypocotyls were significantly shorter in gbf1 mutants as compared with the wild type. Similarly, the epidermal cells of cotyledons were found to be significantly less expanded in gbf1 mutants as compared with 6-day-old wild-type seedlings (Fig. 3, P and Q). These results indicate that GBF1 acts as a regulator of growth that promotes cell elongation and expansion and thus the loss of GBF1 function mutants result in shorter hypocotyls and less expanded cotyledons during seedling development.

To investigate whether gbf1 mutants have any additional morphological defects, we examined and compared the root growth of gbf1 mutants with wild-type plants. The gbf1 mutant plants produced significantly less number of lateral roots as compared with wild-type plants suggesting that GBF1 is essential for optimum lateral root formation (Figs. 3I and 6D).

While propagating gbf1 mutant plants, we observed that the gbf1 mutation caused early flowering. Whereas long-day-grown (16-h light/8-h dark cycles) wild-type plants start flowering after the formation of ~10-11 rosette leaves, gbf1 mutants flower after producing ~7-8 rosette leaves (Fig. 6, A and C). However, such effect was not detected in short-day-grown (8-h light/16-h dark cycles) gbf1 mutant plants (data not shown).

Mutations in GBF1 Result in Altered Chlorophyll Accumulation—Chlorophyll and anthocyanin syntheses are two important physiological responses regulated by light. To examine whether gbf1 mutants have altered chlorophyll or anthocyanin accumulation, we measured the chlorophyll and anthocyanin contents in gbf1 mutant seedlings. We measured the chlorophyll content of cotyledons and normalized the chlorophyll content by cotyledon size. The chlorophyll content was found to be significantly lower in gbf1 mutants as compared with wild-type seedlings (Fig. 4E). No difference in accumulation of anthocyanin was detected between wild-type and gbf1 mutant seedlings (data not shown). gbf1 Mutants Are Epistatic to cry1 and cry2—To determine the involvement of photoreceptors such as cry1, cry2, and phyA in BL-specific functions of GBF1, we performed epistasis analyses. We generated gbf1 cry1, gbf1 cry2, and gbf1 phyA double mutants and examined the hypocotyl length in comparison to cry1 (Ler), cry2 (Col), phyA (RLD), and gbf1 (Col) mutants. Measurements of hypocotyl length revealed that double mutants such as gbf1 cry1 and gbf1 cry2 displayed similar hypocotyl lengths as gbf1 mutants in BL (Fig. 4, F and G). However, gbf1 phyA double mutants exhibited hypocotyl length similar to phyA mutants in BL (Fig. 4H). These results suggest that GBF1 likely acts downstream to both cry1 and cry2 photoreceptors and the increased sensitivity to BL caused by the gbf1 mutation requires blue light perception by phyA.


Figure 4
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  		FIGURE 4.
Characterization of gbf1 mutants. ~25-30 seedlings were used for the measurement of hypocotyl length, cotyledon area, or chlorophyll accumulation. The error bars indicate standard deviations. The gbf1 mutants are in Columbia background. A and B, quantification of hypocotyl length of 6-day-old wild-type (Col) and gbf1 mutant seedlings grown at various fluence rates in constant WL or BL, respectively. C and D, quantification of cotyledon area of 6-day-old wild-type (Col) and gbf1 mutant seedlings grown in constant WL (30 µmol m-2 s-1) or BL(30 µmol m-2 s-1), respectively. E, accumulation of chlorophyll a and b in the cotyledons after normalized by cotyledon size in 6-day-old wild-type (Col) and gbf1-1 mutant seedlings. F-H, hypocotyl lengths of 6-day-old wild-type (W T: see "Experimental Procedures"), gbf1, cry1 (Ler), gbf1 cry1, cry2 (Col), gbf1 cry2, phyA (RLD), and gbf1 phyA seedlings grown at various fluence rates of BL.

 
GBF1 Over-expressers Display BL-specific Regulation of Hypocotyl and Cotyledon Growth in Opposite Manner—Because the loss of GBF1 function resulted in shorter hypocotyls and less expanded cotyledons, we asked whether higher levels of GBF1 cause opposite effects. Several independent transgenic lines expressing GBF1 cDNA driven by CaMV 35 S promoter were generated for this study. We selected multiple transgenic lines segregating for a single T-DNA locus, determined by hygromycin resistance, for the production of homozygous lines and further analysis. Examination of photoresponsiveness revealed that the transgenic lines displayed significant reduction of inhibition in hypocotyl elongation in WL and BL with no visible effect in RL or FR (Fig. 5, A-D, I-J, and data not shown). Furthermore, cotyledons of the overexpresser transgenic lines were strikingly more expanded as compared with wild-type seedlings (Fig. 5, E, F, and K). Determination of GBF1 transcript and protein levels showed dramatically elevated levels of expression of this gene in over-expresser lines relative to wild-type background (Fig. 5, G and H). These results indicate that the altered phenotypes of the over-expresser lines observed were likely to be caused due to the elevated levels of GBF1.


Figure 5
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  		FIGURE 5.
Regulation of blue light-mediated photomorphogenic growth in GBF1 over-expresser lines. In each panel from A to F, wild-type (Ws) and GBF1 over-expresser seedlings (OE1 or OE2 in Ws background) are shown on the left and right, respectively. A, 6-day-old wild-type and OE1 seedlings grown in constant WL (5 µmol m-2 s-1); B, 6-day-old wild-type and OE2 seedlings grown in constant WL (5 µmol m-2 s-1); C, 6-day-old wild-type and OE1 seedlings grown in constant BL (30 µmol m-2 s-1); D, 6-day-old wild-type and OE2 seedlings grown in constant BL (30 µmol m-2 s-1); E, cotyledons of 6-day-old wild-type and OE1 seedlings grown in constant BL (30 µmol m-2 s-1); F, cotyledons of 6-day-old wild-type and OE2 seedlings grown in constant BL (30 µmol m-2 s-1). G, RNA blot analysis of GBF1 in wild-type Ws (WT), OE1, and OE2 seedlings. 20 µg of total RNA was loaded onto each lane. The 1.15-kb full-length cDNA fragment of GBF1 was used as probe. rRNA is shown as loading control. H, immunoblot of 20 µg of total protein prepared from wild-type Ws (WT), OE1, and OE2 seedlings. Affinity-purified GBF1 polyclonal antibodies were used for the detection of GBF1. Coomassiestained protein gel (Total protein) is shown as the loading control. I and J, quantification of hypocotyl length of 6-day-old wild-type Ws (WT), OE1, and OE2 seedlings grown in constant WL (5 µmol m-2 s-1) or BL(30 µmol m-2 s-1), respectively. About 25 seedlings were used for the measurement of hypocotyl length. The error bars indicate standard deviations. K, quantification of cotyledon area of 6-day-old wild-type Ws (WT), OE1, and OE2 seedlings grown in BL (30 µmol m-2 s-1). Experimental details are the same as in I-J. L, formation of lateral roots in 16-day-old wild-type Ws, OE1, and OE2 plants (from left to right) grown in constant WL (80 µmol m-2 s-1). M-O, hypocotyl epidermal cells of 6-day-old wild-type, OE1, or OE2 seedlings, respectively, grown in constant BL (30 µmol m-2 s-1). P and Q, imprints of cotyledon epidermal cells of 6-day-old wild-type or OE1 seedlings, respectively, grown in constant BL (30 µmol m-2 s-1).

 
We examined the length and size of epidermal cells of 6-day-old over-expresser and wild-type seedlings grown in BL. The epidermal cells of hypocotyls were detected to be significantly longer in GBF1 over-expresser lines as compared with wild-type seedlings (Figs. 5 (M-O) and 6B). Similarly, the epidermal cells of cotyledons were significantly more expanded in over-expresser lines as compared with wild-type seedlings (Fig. 5P-Q). Taken together, these results firmly demonstrate that the bZIP protein, GBF1, is a transcriptional regulator of photomorphogenic growth that promotes cell elongation and expansion during early seedling development in Arabidopsis.

Because the loss of function mutant GBF1 displayed less lateral roots as compared with wild-type plants, we examined whether overexpression of GBF1 caused more lateral root formation in Arabidopsis plants. As shown in Figs. 5L and 6D, the over-expresser transgenic lines indeed formed more lateral roots as compared with wild-type plants suggesting that GBF1 acts as a positive regulator of lateral root formation. Examination of flowering time of the over-expresser lines revealed that GBF1 transgenic overexpresser lines flower significantly late after formation of 15-18 rosettes as compared with wild-type plants (Fig. 6, A and C).

GBF1 Differentially Regulates the Expression of Light-inducible Genes—The up-regulation of light-inducible genes such as CAB and RBCS is one of the important phenomena in photomorphogenic growth. Because GBF1 regulates the growth of hypocotyls and cotyledons in response to BL, we asked whether the bZIP transcription factor GBF1 also plays a role in light-regulated gene expression. For this study, we used 6-day-old wild-type and gbf1-1 mutant seedlings grown in constant dark or various light conditions and measured the relative steady-state mRNA levels of light-inducible genes. Whereas no difference in the expression of CHS was detected between wild-type and gbf1 mutants, the expression of RBCS was found to be significantly higher in gbf1 as compared with wild-type seedlings grown in BL or WL (Fig. 7A). No alteration in the expression of CAB was detected in WL, however, the expression of the gene was significantly reduced in BL grown seedlings (Fig. 7A). To further examine the BL-mediated regulation of CAB gene expression in gbf1-1 mutant background, 4-day-old seedlings grown in darkness were transferred to BL for 12, 24, and 48 h, and the transcript levels were measured. Whereas >7-fold induction in CAB gene expression was found at 24 h in wild-type, <4-fold induction was detected in gbf1-1 mutant background (Fig. 7, B and C), suggesting that the induction of CAB gene expression was significantly compromised in gbf1-1 mutants. These results suggest that, although GBF1 negatively regulates the expression of RBCS, it acts as a positive regulator of CAB gene expression (Fig. 7H).


Figure 6
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  		FIGURE 6.
Characterization of GBF1 over-expresser lines. A, 30-day-old wild-type (Col), gbf1-1, gbf1-2, GBF1 over-expresser 1 (OE1) and over-expresser 2 (OE2) plants (from left to right) grown in WL (80 µmol m-2 s-1) under 16-h light/8-h dark cycles. B, quantification of epidermal cell lengths of hypocotyls of 6-day-old wild-type (Col), gbf1-1, gbf1-2, wild-type (Ws), OE1, and OE2 seedlings grown in constant BL (30µmol m-2 s-1). C, number of rosette leaves formed at the time of bolting in wild-type (Col), gbf1-1, gbf1-2, wild-type (Ws), OE1, and OE2 plants grown under long-day conditions of 16-h WL (80 µmol m-2 s-1) and 8-h dark cycles. D, quantification of the number of lateral roots formed in wild-type (Col), gbf1-1, gbf1-2, OE1, and OE2 plants grown in constant WL (80 µmol m-2 s-1) at various days (from day 10 to 15).

 
To further investigate the above observation, we used two stable transgenic lines: Z/NOS101-GUS and CAB1-GUS (33, 34). Both these promoter-reporter constructs were individually introduced into gbf1-1 mutants by genetic crosses with wild-type transgenic lines (35). Mutant lines homozygous for each transgene were then generated for further studies. The Z/NOS101-GUS transgene was expressed in all the tissues in gbf1 mutants similar to wild-type seedlings in BL (Fig. 7D). Quantitative GUS activity measurements revealed that there was ~50% reduction in the activity of this promoter in gbf1 mutants as compared with wild-type background (Fig. 7F). The expression of CAB1-GUS transgene has been shown to be confined to the cotyledons in wild-type background (35). As shown in Fig. 7E, very little expression was detected (if any) of CAB1-GUS transgene in the gbf1 mutants, and the quantification GUS activity measurements revealed that the activity of the CAB1 promoter was reduced to ~4-fold in gbf1 mutants as compared with the wild-type background (Fig. 7G). Taken together, these results strongly suggest that GBF1 is required for the proper activation of the Z-box-containing promoters in BL.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although the GBF family of transcription factors has been known for more than a decade, the physiological functions of these genes remain elusive (37-39). Several transcription factors have been reported in light signaling that play either positive or negative regulatory roles in seedling development (9, 11, 14, 41). This study establishes GBF1 as a unique transcription factor in light signaling that plays both positive and negative regulatory roles in photomorphogenic growth and gene expression.

GBF1 Interacts with Both the G- and Z-box LREs—The DNA-protein interaction data in this study provide several lines of evidence that GBF1 interacts with both the Z- and G-box LREs of light regulated promoters. The competitive gel shift assays using several LREs, including the Z- and G-box, demonstrate that, although GBF1 interacts with both Z- and G-box, the protein may have higher affinity for the G-box as compared with the Z-box LRE. The recognition of the G- and Z-box LREs by GBF1 possibly indicates that these two LREs are functionally equivalent (11) with the context to GBF1 transcription factor.

Mutations in GBF1 Result in Multiple Effects—The analysis of seedling morphology of gbf1 mutants demonstrates that the shorter hypocotyl phenotype is restricted to BL. Therefore, although GBF1 is expressed at various wavelengths of light, it specifically acts as a negative regulator of BL-mediated inhibition of hypocotyl elongation. Our results further demonstrate that gbf1 mutants have smaller cotyledons as compared with wild-type seedlings in blue light, thus demonstrating a positive regulatory function of GBF1 in cotyledon expansion in a blue light-specific manner. The results of epistasis analyses indicate that GBF1 acts downstream to both cry1 and cry2 photoreceptors, and the increased sensitivity to BL caused by gbf1 mutation also requires light perception by phyA. Thus GBF1-mediated inhibition is likely to play an important role in negative or positive feedback control of cryptochrome signaling, although the function of phyA is likely to be independent of GBF1. Thus, GBF1 plays a dual but opposite regulatory role in early seedling development acting downstream to both cry1 and cry2 photoreceptors (Fig. 7H).

Overexpression of GBF1 has resulted in elongated hypocotyls but more expanded cotyledons in blue light, thereby confirming the differential regulatory role of GBF1 in cotyledon and hypocotyl growth. This finding further indicates that GBF1 transcripts may not be present at sufficiently high levels in wild-type seedlings and thus may be a rate-limiting factor for cotyledon expansion in blue light signaling. However, because higher levels of GBF1 result in elongated hypocotyls, a fine controlled level of GBF1 is likely to be essential for plants to obtain blue light-mediated optimum photomorphogenic growth. This notion is further supported by the fact that GBF1 promotes cell elongation and expansion in hypocotyl and cotyledon, respectively.


Figure 7
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  		FIGURE 7.
The light-regulated gene expression in gbf1 mutants. A, RNA gel-blot analysis of RBCS, CAB, and CHS genes in segregated wild-type (Col) and gbf1-1 mutant (Col) seedlings (M) grown in constant darkness (D), WL, or BL conditions. 10 µg of total RNA was loaded onto each lane. 18 S rRNA has been shown as loading control. B, RNA gel-blot analysis of CAB in segregated wild-type (Col) and gbf1-1 mutant seedlings. 4-day-old dark grown seedlings were transferred to BL for 12, 24, or 48 h, and total RNA was extracted from each sample for RNA gel-blot analysis. 10 µg of total RNA was loaded onto each lane. 6-day-old seedlings grown in darkness are shown as 0 h. rRNA was used as loading control. C, quantification of the data in B by Fluor-S-MultiImager (Bio-Rad). D and E, 6-day-old seedlings carrying Z/NOS101-GUS or 12-day-old plants carrying CAB1-GUS transgene, respectively, were grown in BL (30 µmol m-2 s-1) and used for GUS activity staining. In each panel wild-type (Col) and gbf1-1 mutants (Col) are shown on the left and right side, respectively. F and G, GUS activities of 6-day-old constant BL (30 µmol m-2 s-1) grown seedlings carrying Z/NOS101-GUS or CAB1-GUS transgenes, respectively. The error bars indicate standard deviations. The promoter-reporter constructs are diagrammed at the top of each panel. H, a working model shows BL-specific regulatory role of GBF1 during early seedling development in Arabidopsis.

 
HYH, AtPP7, SUB1, and MYC2/ZBF1 have been reported as downstream components in blue light signaling. Whereas SUB1, a Ca2+-binding protein, functions as a negative regulator in blue and far-red light signaling, AtPP7, an Ser/Thr protein phosphatase, acts as a positive regulator of blue light-mediated photomorphogenic growth (10, 42). HYH, a transcription factor and a close homolog of HY5, acts as a positive regulator in BL signaling (9). MYC2 is a Z-box-binding transcription factor, which acts as a negative regulator in BL-mediated photomorphogenic growth and is a point of cross-talk among light, abscisic acid, and jasmonic acid signaling (11).

GBF1 Differentially Regulates the Expression of Light-inducible Genes—It has been shown that PIF3 exhibits opposite regulatory effects on seedling morphology and light-regulated gene expression in an RL- and FR-specific manner (22). Furthermore, the presence of parallel and branched pathways of light-regulated gene expression has already been suggested (17, 47, 48). Analyses of light-regulated gene expression in gbf1 mutants have revealed that, although GBF1 is required for the proper activation of CAB gene expression, it acts as a negative regulator for RBCS gene expression (Fig. 7H). Transgenic studies with synthetic and native promoter-reporter constructs further indicate that GBF1 is required for the proper activation of the Z-box-containing promoters, including CAB1. Extensive heterodimerization of bZIP proteins has been reported (37). Thus, heterodimerization of GBF1 with other bZIP proteins could be a potential mechanism in vivo to generate positive and negative regulators, which in turn may play opposite roles for light-regulated gene expression and seedling development.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) AJ843257 [GenBank] .

* This work was supported by a grant from Department of Science and Technology, the Government of India, and an internal grant from National Centre for Plant Genome Research (to S. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipients of Council of Scientific and Industrial Research fellowships from the Government of India. Back

2 To whom correspondence should be addressed. Tel.: 91-11-2617-8614; Fax: 91-11-2671-6658; E-mail: sudipchatto{at}yahoo.com.

3 The abbreviations used are: phyA, -E, phytochromes A to E; cry1, -2, cryptochromes 1 and 2; GBF1, G-box binding factor 1; ZBF2, Z-box binding factor 2; bZIP, basic leucine zipper; LRE, light-responsive elements; GST, glutathione S-transferase; WL, white light; D, dark/darkness; FR, far-red light; RL, red light; BL, blue light; OE1, -2, over-expressers 1 and 2; Ler, Landsberg erecta; Col, Columbia; RLD, Reschiev; GUS, beta-glucuronidase. Back


   ACKNOWLEDGMENTS
 
We thank Prof. Sushil Kumar, Dr. Shikha Bhatia, and Sreeramaiah N. Gangappa for critically reading and commenting on the manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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