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.
During seed maturation, the transcriptional activity of napin genes is regulated by developmental signals involving the transcriptional activator ABI3 and abscisic acid (ABA). To localize cis elements involved in the seed-specific activity of the napin napA promoter, a systematic analysis was performed focusing on two major element complexes, the B-box and RY/G. Substitution mutation analysis using promoter-reporter gene fusions in stable transgenic tobacco showed synergistic interactions between elements within these complexes. The distal part of the B-box shows similarities to abscisic acid response elements and the proximal portion contains a CA-rich element. In vitro studies involving Exonuclease III protection and electrophoretic mobility shift assays revealed binding by nuclear proteins to elements within the B-box. The distal and proximal parts of the B-box were found to bind distinct nuclear protein complexes. By gain-of-function analysis with a tetramer of the B-box fused to a truncated (-46) cauliflower mosaic virus (CaMV) 35S minimal promoter, it was demonstrated that the B-box mediates strong activity in seeds. Further, it was shown that the elements in the B-box constitute an ABA-responsive complex, since the B-box tetramer mediates ABA-responsiveness in vegetative tissues to a construct containing the CaMV virus 35S enhancer (-343 to -90). Thus, the seed-specific activity of the napA promoter relies on the combinatorial interaction between the RY/G complex and the B-box ABA-responsive complex during the ABA response in seed development.
SummaryBrassica napus complementary deoxyribonucleic acid (cDNA) clones encoding a DNA-binding protein, BnPEND, were isolated by Southwestern screening. A distinctive feature of the protein was a bZIP-like sequence in the amino-terminal portion, which, after expression in Escherichia coli, bound DNA. BnPEND transcripts were present in B. napus roots and flower buds, and to a lesser extent in stems, flowers and young leaves. Treatment in the dark for 72 h markedly increased the amount of BnPEND transcript in leaves of all ages. Sequence comparison showed that BnPEND was similar to a presumed transcription factor from B. napus, GSBF1, a protein deduced from an Arabidopsis thaliana cDNA (BX825084) and the PEND protein from Pisum sativum, believed to anchor the plastid DNA to the envelope early during plastid development. Homology to expressed sequence tag (EST) sequences from additional species suggested that BnPEND homologues are widespread among the angiosperms. Transient expression of BnPEND fused with green fluorescent protein (GFP) in Nicotiana benthamiana epidermal cells showed that BnPEND is a plastid protein, and that the 15 amino acids at the amino-terminal contain information about plastid targeting. Expression of BnPEND in Nicotiana tabacum from the Cauliflower Mosaic Virus 35S promoter gave stable transformants with different extents of white to light-green areas in the leaves, and even albino plants. In the white areas, but not in adjacent green tissue, the development of palisade cells and chloroplasts was disrupted. Our data demonstrate that the BnPEND protein, when over-expressed at an inappropriate stage, functionally blocks the development of plastids and leads to altered leaf anatomy, possibly by preventing the release of plastid DNA from the envelope.
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