Arabidopsis bZIP16 transcription factor integrates light and hormone signaling pathways to regulate early seedling development

Abstract

Transcriptomic adjustment plays an important role in Arabidopsis thaliana seed germination and deetiolation in response to environmental light signals. The G-box cis-element is commonly present in promoters of genes that respond positively or negatively to the light signal. In pursuing additional transcriptional regulators that modulate light-mediated transcriptome changes, we identified bZIP16, a basic region/Leu zipper motif transcription factor, by G-box DNA affinity chromatography. We confirmed that bZIP16 has G-box-specific binding activity. Analysis of bzip16 mutants revealed that bZIP16 is a negative regulator in light-mediated inhibition of cell elongation but a positive regulator in light-regulated seed germination. Transcriptome analysis supported that bZIP16 is primarily a transcriptional repressor regulating light-, gibberellic acid (GA)-, and abscisic acid (ABA)-responsive genes. Chromatin immunoprecipitation analysis revealed that bZIP16 could directly target ABA-responsive genes and RGA-like2, a DELLA gene in the GA signaling pathway. bZIP16 could also indirectly repress the expression of phytochrome interacting factoR3-like5, which encodes a basic helix-loop-helix protein coordinating hormone responses during seed germination. By repressing the expression of these genes, bZIP16 functions to promote seed germination and hypocotyl elongation during the early stages of Arabidopsis seedling development.

Figure 1.

bZIP16 Is a G-Box Binding Protein. (A) A flowchart showing the procedure for identifying bZIP16 by combining DNA (G-box) affinity chromatography and liquid chromatography (LC) MS/MS. (B) EMSAs performed with recombinant bZIP16 protein and the 4×G-box used as a probe. HY5, a known G-box binding protein, was used as a positive control. G-box probe, 10 ng biotin 3′-end labeled 4×G-box; HY5, 70 ng HY5 recombinant protein; G-box competitor, unlabeled G-box tetramer at fivefold to 20-fold excess to the G-box probe as indicated. The free probe and protein–G-box complexes are marked.

Figure 2.

Molecular Characterization of bzip16 Mutants. (A) Diagram of bZIP16 gene and the location of T-DNA insertion sites for bzip16-1 (SALK_044834) in Col-0 and bzip16-2 (GT9934) in Ler background. (B) RNA gel blot and immunoblot analyses of levels of mRNA (top) and protein (bottom) of bZIP16 in wild-type and bzip16 mutants in 12-d-old seedlings. Ethidium bromide–stained rRNA and α-tubulin (Tub) protein were loading controls.

Figure 3.

bZIP16 Negatively Regulates RL-Mediated Photomorphogenesis in Arabidopsis. Hypocotyl lengths of 4-d-old seedlings grown under various fluences of RL under LD conditions. bzip16 mutants and transgenic lines in a Col-0 background (A) and Ler background (B). Representative photographs of seedlings are shown. Asterisk indicates hypocotyl length of bzip16 mutants or bZIP16ox lines is significantly different from that of Col-0 or Ler (P < 0.01, Student’s t test; n = 20 to 30). Bars = 5 mm. [See online article for color version of this figure.]

Figure 4.

bZIP16 Is a Transcriptional Repressor in the Dark. Hierarchical clustering of bZIP16-regulated genes differentially expressed in Ler and bzip16-2 under dark or RL. Expression data for all three biological repeats of each treatment are shown as relative expression of each gene to the median of 12 samples. The median expression of each gene is shown in yellow. Relative up- or downregulation from median expression is yellow and blue respectively. The k-means clustering (left panel) categorized the bZIP16-regulated genes into five clusters (I to V) according to their expression patterns illustrated in the right panel. Gray spots in the right panel indicate the levels of expression for each bZIP16-regulated gene in each expression cluster. Red lines illustrate the expression averages for Ler or bzip16 in each cluster.

Figure 5.

bZIP16 Is a Positive Regulator of Seed Germination. phyA-dependent ([A] and [B]) and phyB-dependent (C) assays of the germination rate (percentage) of Col-0, bzip16-1, bzip16-1/bZIP16-GFP, bZIP16ox, Ler, bzip16-2, phyA, bzip16-2 phyA, phyB, and bzip16-2 phyB. The light treatment schemes are shown above. FRp, 0.5 W/m²; Rp, 5 μE; FR, 3 W/m².

Figure 6.

bZIP16 Represses the Expression of RGL2 and PIL5 during Seed Germination. Real-time quantitative PCR of the mRNA expression of RGL2 and PIL5 in Ler, bzip16-2, and bzip16-2/bZIP16 seeds imbibed for the times indicated. phyA-dependent ([A] and [B]) and phyB-dependent (C) light treatment schemes are shown above. The expression of RGL2 and PIL5 at each time point was normalized to that of UBQ10. The expression at time zero was set to 1. The means and standard deviations were calculated from three technical replicates. Three independent biological experiments were performed with similar results. FRp, 0.5 W/m²; Rp, 5 μE; FR, 3 W/m².

Figure 7.

rgl2 and pil5 Are Epistatic to bzip16 in Seed Germination. The germination rate (percentage) of Col-0, Ler, F1 of Ler and Col-0 (Ler Col-0), bzip16-2, pil5-1, rgl2-5, bzip16-2 pil5-1 and bzip16-2 rg12-5 with FRp (A), prolonged FR light (B), and R light (C). The light treatment schemes are shown above. FRp, 0.5 W/m²; Rp, 5 μE; FR, 3 W/m².

Figure 8.

bZIP16 Directly Binds to the Promoter Regions of GA- and ABA-Responsive Genes. (A) Gene structures for the candidate target genes of bZIP16. Transcriptional start and gene unit are marked with an arrow and shaded arrow bar. Black line indicates intergenic region. Cis-elements are indicated with ovals in different colors. Target fragments assayed by ChIP-qPCR are indicated with horizontal black bars as fragment A, B, or C. (B) and (C) ChIP assays were performed in extracts isolated from germinating seeds (B) or 4-d-old etiolated seedlings (C). Immunoprecipitated DNA was quantified by qPCR with specific primer pairs for candidate fragments. Amplicons in UBQ10 were used as an internal control. Results from Col-0 (wild type [WT]) and bzip16-1/bZIP16-GFP (bZIP16) were normalized as percentage of the input DNA. Data are means ± sd (technical repeats, n = 3). Three independent experiments were performed with similar results.

Figure 9.

A Working Model Illustrating Molecular Actions of bZIP16 in Arabidopsis Early Seedling Development. bZIP16, primarily a transcriptional repressor, positively regulates seed germination and hypocotyl cell elongation by repressing RGL2, PIL5, and ABA-responsive genes. The model also highlights the roles of bZIP16 in coordinating light, GA, and ABA signaling pathways.