A pivotal role of the basic leucine zipper transcription factor bZIP53 in the regulation of Arabidopsis seed maturation gene expression based on heterodimerization and protein complex formation

Abstract

Transcription of Arabidopsis thaliana seed maturation (MAT) genes is controlled by members of several transcription factor families, such as basic leucine zippers (bZIPs), B3s, MYBs, and DOFs. In this work, we identify Arabidopsis bZIP53 as a novel transcriptional regulator of MAT genes. bZIP53 expression in developing seeds precedes and overlaps that of its target genes. Gain- and loss-of-function approaches indicate a correlation between the amount of bZIP53 protein and MAT gene expression. Specific in vivo and in vitro binding of bZIP53 protein to a G-box element in the albumin 2S2 promoter is demonstrated. Importantly, heterodimerization with bZIP10 or bZIP25, previously described bZIP regulators of MAT gene expression, significantly enhances DNA binding activity and produces a synergistic increase in target gene activation. Full-level target gene activation is strongly correlated with the ratio of the correspondent bZIP heterodimerization partners. Whereas bZIP53 does not interact with ABI3, a crucial transcriptional regulator in Arabidopsis seeds, ternary complex formation between the bZIP heterodimers and ABI3 increases the expression of MAT genes in planta. We therefore propose that heterodimers containing bZIP53 participate in enhanceosome formation to produce a dramatic increase in MAT gene transcription.

Figure 1.

Expression of bZIP53 in Seeds. (A) In situ mRNA hybridization for bZIP53 at different stages of seed development. Longitudinal sections of siliques with seeds at heart (I and I′), early torpedo (II and II′), and green cotyledon (III and III′) stages of development are shown. Sections were hybridized with a bZIP53 antisense (I, II, and III) or a sense probe (I′, II′, and III′). (B) Histochemical analysis of Columbia (Col-0) seeds harboring a bZIP53 promoter fused to GUS (ProbZIP53:GUS). GUS staining of late torpedo stage (II) and green cotyledon stage (III) embryo development are shown. The arrow indicates the developing endosperm; to facilitate viewing, the seed has been pressed to push out the embryo.

Figure 2.

Plants with Constitutive Expression of bZIP53 Have Growth Defects and Increased Levels of Seed Maturation Transcripts. (A) Plants overexpressing bZIP53 (Pro35S:bZIP53) have a stunted and late flowering phenotype compared with Col wild-type (Col-0 wt) plants. (B) mRNA samples from 2-week-old wild-type and two Pro35S:bZIP53 lines (#1 and #2) were analyzed by qRT-PCR to quantify the transcript levels of bZIP53, LEA76, CRU3, and ProDH. Expression levels are given relative to a UBIQUITIN gene for normalization. Given are mean values and standard deviation of two to three replicates.

Figure 3.

In Vivo and in Vitro Binding of the bZIP53 Protein to the Albumin 2S2 Promoter and Interaction with bZIP10 and bZIP25. (A) Chromatin extracts from wild-type plants and plants overexpressing a HA-tagged bZIP53 protein (Pro35S:HA-bZIP53) were subjected to qRT-PCR analysis with 2S2 promoter-specific primers before (input) and after immunoprecipitation with an anti-HA antibody (α-HA). Ct values for Pro35S:HA-bZIP53 samples were subtracted from the Ct values of the equivalent wild type, and the differentials are shown on top of the right bars in the graph. A value of 1 was assigned to the Col-0 samples. For normalization, an actin (ACT7) gene was used (see Methods). (B) Schematic view of the 2S2 promoter fused to GUS used as reporter in transient expression analysis. Depicted are RY (black) and G-box (white) elements. The sequence of the wild type and mutated G-box (mt) is shown. (C) Arabidopsis leaves were transformed with the reporter constructs containing sequences described in (B) and effector constructs containing specific bZIP genes under control of the Pro35S in cobombardment experiments as described by Lara et al. (2003). Three microliters of control plasmid Pro35S:NAN was included in all the experiments to normalize GUS expression values for differences in bombardment efficiencies (Kirby and Kavanagh, 2002). The x axis values are expressed as GUS activity relative to NAN. Given are mean values and standard deviation of three independent experiments. (D) In vitro binding of bZIP53 to the G-box sequence from the 2S2 promoter. A biotinylated oligonucleotide containing the G-box sequence was bound to streptavidin-coated wells and incubated with increasing amounts of a T7-tagged bZIP53 protein (1 to 1:90). Nonbound proteins were removed from the reaction wells, and the amount of T7-bZIP53 protein was quantified by immunodetection with an anti-T7 antibody (α-T7; left panel). The binding specificity of bZIP53 to the 2S2 G-box was analyzed in competition experiments where increasing amounts of unlabeled oligonucleotides (as indicated) containing a wild-type (2S2 wt; black bars) or a mutated 2S2 mt version (gray bars) were incubated with a fixed amount of the T7-bZIP53 protein and the biotinylated oligonucleotide containing the wild-type G-box sequence (right panel). (E) Effect of heterodimerization on bZIP53 and bZIP10 binding affinity. A biotinylated oligonucleotide containing the G-box sequence was bound to streptavidin-coated wells and incubated with increasing amounts (1 to 1:30) of a T7-bZIP53 protein in the absence (filled squares) or presence of a fixed amount of bZIP10 protein (open squares). Nonbound proteins were removed from the reaction wells, and the amount of T7-bZIP53 protein was quantified by immunodetection with an α-T7 antibody (left panel). The reciprocal experiment was performed with a T7-bZIP10 protein and a nontagged bZIP53 protein (right panel). Given are mean values and standard deviation of three repetitions.

Figure 4.

Interaction of bZIP53, bZIP10, and bZIP25 Homo- or Heterodimers with ABI3. (A) Protein interactions in yeast two- and three-hybrid systems. ABI3, ABI5, bZIP10, and bZIP25 proteins were fused to the GAL4 DNA BD or AD and introduced separately into a yeast strain containing the AD-bZIP53 (HF7c) or BD-bZIP53 (SFY526) protein, respectively. Activation of the reporter genes HIS3 (growth in a His-depleted medium; left panel) and LacZ (blue colored colonies; right panel) indicates positive protein–protein interactions. (B) Yeast strains (SFY526) expressing different combinations of BD-bZIP53, AD-ABI3, and bZIP10 or bZIP25 were assayed for β-galactosidase activity. The latter were provided in the three-hybrid vector pTFT (Egea-Cortines et al., 1999). Average values (Miller units) and standard errors from six replicates and two independent experiments are shown. (C) Schematic overview of a three-hybrid assay in Arabidopsis protoplasts. Constructs are shown on the left, and a model of reporter gene activation is shown on the right. (D) Activation of the GAL4-UAS₄:GUS reporter after cotransfection with the constructs indicated in (C). The x axis values are expressed as GUS activity relative to NAN (Ehlert et al., 2006). Average values and standard errors from four transfections are shown. Numbers along the y axis represent fold induction values relative to nontransfected control cells. The experiments were repeated three times with similar results.

Figure 5.

bZIP53, bZIP10, and ABI3 Protein–Protein Interaction Studied by BiFC (Walter et al., 2004). Onion epidermis cells have been transiently transformed by particle bombardment. Fusion proteins of the C-terminal part of YFP (CtYFP) and ABI3 and the N-terminal part of YFP (NtYFP) and bZIP53 have been coexpressed with HA-tagged bZIP10 (HA-bZIP10) (a and a′) or an empty vector control (b and b′). Bright-field images (top panels) and epifluorescence images taken by confocal microscopy (bottom panels) are shown.

Figure 6.

Effect of ABI3 on the Transcriptional Activation Mediated by the bZIP53/bZIP10 Heterodimers. (A) GUS reporter activity under the control of the 2S2 or CRU3 promoter was measured in transiently transformed Arabidopsis protoplasts after cotransfection of the effector constructs indicated. bZIP10, bZIP53, and ABI3 are expressed under the control of Pro35S. For immunodetection, 3xHA-epitope-tagged derivatives were used (see Supplemental Figure 3 online). The importance of bZIP heterodimerization was demonstrated by including bZIP10pp, which is impaired in zipper-mediated dimerization (Weltmeier et al., 2006). Given are mean values and standard deviation of four independent transfections. (B) Immunoblot analysis of transiently transformed protoplasts confirms that expression of HA-bZIP10 is comparable to HA-bZIP10pp-untransformed control protoplasts. HA epitope-tagged proteins were detected using an α-HA antiserum. The arrow indicates HA-bZIP10 and bZIP10pp proteins, and the asterisk shows degradation products. (C) Transient expression using a GUS reporter gene under the control of the 2S2 promoter. Combinations of bZIP53 and bZIP10 at different ratios (indicated are ratios of input DNA) in the presence (black bars) or absence (white bars) of ABI3 were used as effectors. The x axis values are expressed as GUS activity relative to NAN as described by Ehlert et al. (2006). Average values and standard errors from four transfections are shown.

Figure 7.

Effect of a bZIP53 T-DNA Insertion Mutant on Seed-Specific Gene Regulation. (A) mRNA levels detected of the seed maturation genes indicated by qRT-PCR in Col wild-type and bzip53 siliques at different stages of development. Lane 1, 0 d after flowering (DAF); lane 2, 2 DAF; lane 3, 4 DAF; lane 4, 6 DAF; lane 5, 9 DAF; lane 6, 12 DAF; lane 7, 15 DAF; lane 8, ≥18 DAF. Data are normalized using UBIQUITIN expression values. Average values and standard errors from at least two technical replicates are shown. (B) Transient expression by microparticle bombardment of Arabidopsis leaves from the wild type (white bars) and plants with increased (Pro35S:bZIP53; gray bars) or decreased (bzip53; black bars) expression of bZIP53. Effector constructs containing bZIP10 or ABI3 under the control of a 35S promoter (Pro35S) and a GUS reporter gene under the control of the 2S2 promoter were used. The x axis values are expressed as GUS activity relative to NAN (Kirby and Kavanagh, 2002). Average values and standard errors from four replicates and two different experiments are shown.

Figure 8.

Model for bZIP53 Regulation of Seed Maturation Gene Expression. Yellow and blue backgrounds represent expression in the seed and leaf, respectively. Positions of the indicated TFs were represented in a coordinate system according to relative expression values in seeds (y axis) and leaves (x axis) (data derived from AtGenExpress). Structure of a typical SSP promoter-like albumin (2S2) or cruciferin (CRU3) is depicted, indicating regulatory elements in their promoters (RY, RY-box; G, G-box; TATA, TATA-box). Continuous and dotted lines indicate DNA–protein interactions and protein–protein interactions, respectively. A displacement arrow (open line) indicates overexpression of bZIP53 in Pro35S:bZIP53 plants. bZIP53 increased expression in leaves favors heterodimer formation (hd) with group C bZIP10 and bZIP25 and triggers induction of seed gene expression in this organ.