Transactivation of the Brassica napus napin promoter by ABI3 requires interaction of the conserved B2 and B3 domains of ABI3 with different cis‐elements: B2 mediates activation through an ABRE, whereas B3 interacts with an RY/G‐box

Abstract

SummaryThe transcriptional activator ABI3 is a key regulator of gene expression during embryo maturation in crucifers. In monocots, the related VP1 protein regulates the Em promoter synergistically with abscisic acid (ABA). We identi®ed cis-elements in the Brassica napus napin napA promoter mediating regulation by ABI3 and ABA, by analyzing substitution mutation constructs of napA in transgenic tobacco plantlets ectopically expressing ABI3. In transient analysis using particle bombardment of tobacco leaf sections, a tetramer of the distB ABRE (abscisic acid-responsive element) mediated transactivation by ABI3 and ABI3-dependent response to ABA, whereas a tetramer of the composite RY/G complex, containing RY repeats and a G-box, mediated only ABA-independent transactivation by ABI3. Deletion of the conserved B2 and B3 domains of ABI3 abolished transactivation of napA by ABI3. The two domains of ABI3 interact with different cis-elements: B2 is necessary for ABA-independent and ABA-dependent activations through the distB ABRE, whereas B3 interacts with the RY/G complex. Thus B2 mediates the interaction of ABI3 with the protein complex at the ABRE. The regulation of napA by ABI3 differs from Em regulation by VP1, in that the B3 domain of ABI3 is essential for the ABA-dependent regulation of napA.

Genes directly regulated by LEAFY COTYLEDON2 provide insight into the control of embryo maturation and somatic embryogenesis

“…Consistent with this observation, LEC2, ABI3, and FUS3 share identical or conserved amino acid residues at positions in the B3 domain implicated as being responsible for DNA-binding specificity based on the solution structure of the B3 domain protein RAV1 (41). Thus, all three transcription factors bind RY motifs through their B3 domains and activate maturation-specific genes, although we note that ABI3 activity is influenced by an additional domain that binds ABA response elements (40).…”

“…1) (33,36,37). Our demonstration that LEC2 binds with the same DNA element bound by FUS3 and ABI3 (30,(38)(39)(40), the RY motif, provides a partial explanation for similarities in the gain-of-function phenotypes. Consistent with this observation, LEC2, ABI3, and FUS3 share identical or conserved amino acid residues at positions in the B3 domain implicated as being responsible for DNA-binding specificity based on the solution structure of the B3 domain protein RAV1 (41).…”

A Pivotal Role of the Basic Leucine Zipper Transcription Factor bZIP53 in the Regulation ofArabidopsisSeed Maturation Gene Expression Based on Heterodimerization and Protein Complex Formation

“…Three-hybrid and BiFC analyses confirmed that bZIP heterodimerization of bZIP10 or bZIP25 with bZIP53 does not interfere with ABI3 interaction and supports the existence of a ternary complex. ABI3 has been shown to bind to the RY element (Mö nke et al, 2004), and a functional interplay between G-box and RY-element in MAT gene regulation has been previously described (Ezcurra et al, 2000;Lara et al, 2003). In this report, we clearly demonstrate functional interplay between bZIP heterodimers and ABI3, presumably by ternary complex formation.…”

“…A nativesequence oligonucleotide (wild type) containing a functional bZIP (G-box) binding site (Ezcurra et al, 2000) derived from the 2S2 promoter and a mutated version were used ( Figure 3B). Doublestranded oligonucleotides were covalently attached to 96-well ELISA plates before doing the binding assays as described in Methods.…”

“…The corresponding genes clearly differ in expression and function despite being partially redundant. Although binding of ABI3, FUS3, and LEC2 to the RY element, mediated by the B3 DNA binding domain, has been shown in vitro and in yeast systems (Kroj et al, 2003;Mö nke et al, 2004;Braybrook et al, 2006), RY motifs are not sufficient to confer seed-specific expression patterns to a target promoter (Ezcurra et al, 2000;Nakashima et al, 2006). Cooperation with neighboring cis-elements and the cognate TFs has been demonstrated to be important to establish seed-specific transcriptional patterns.…”