SummaryA cDNA encoding a DNA-binding protein of the DOF class of transcription factors was isolated from a barley endosperm library. The deduced amino acid sequence for the corresponding protein is 94% identical through the DOF domain to the prolamin-box (P-box) binding factor PBF from maize. The gene encoding the barley PBF (BPBF) maps to chromosome 7H, and its expression is restricted to the endosperm where it precedes that of the hordein genes. The BPBF expressed in bacteria as a GST-fusion binds a P-box 5Ј-TGTAAAG-3Ј containing oligonucleotide derived from the promoter region of an Hor2 gene. Binding was prevented when the P-box motif was mutated to 5Ј-TGTAgAc-3Ј. A P-box binding activity, present in barley and wheat endosperm nuclei, interacted similarly to BPBF with this synthetic oligonucleotide, and the binding was abolished by 1,10-phenanthroline. Transient expression experiments in developing barley endosperms demonstrate that BPBF transactivates transcription from the Pbox element of a native Hor2 promoter and that direct binding of BPBF to its target site is essential for transactivation since mutations in the DOF DNA-binding domain or in the P-box motif of this promoter abolished both binding and transactivation. Evidence was also obtained for the presence in wheat of a Pbf homologue having similar DNA-binding properties to that of BPBF. These results strongly implicate this endosperm-specific DOF protein from barley as an important activator of hordein gene expression and suggest the evolutionary conservation of the Pbf gene function among small grain cereals.

SummaryHvGAMYB, a MYB transcription factor previously shown to be expressed in barley aleurone cells in response to gibberellin during germination, also has an important role in gene regulation during endosperm development. The mRNA was detected early (10 DAF) in the seeds where it accumulates, not only in the aleurone layer, starchy endosperm, nucellar projection and vascular tissue, but also in the immature embryo as shown by in situ hybridization analysis. The HvGAMYB protein, expressed in bacteria, binds to oligonucleotides containing the 5¢-TAACAAC-3¢ or 5¢-CAACTAAC-3¢ sequences, derived from the promoter regions of the endosperm-speci®c genes Hor2 and Itr1, encoding a B-hordein and trypsin-inhibitor BTI-CMe, respectively. Binding is prevented when these motifs are mutated to 5¢-TgACAAg-3¢ and 5¢-CgACTgAC-3¢. Transient expression experiments in co-bombarded developing endosperms demonstrate that HvGAMYB trans-activates transcription from native Hor2 and Itr1 promoters through binding to the intact motifs described above. Trans-activation of the Hor2 promoter also requires an intact prolamine box (PB). This suggests that HvGAMYB interacts in developing barley endosperms with the PB-binding factor BPBF, an endosperm-speci®c DOF transcriptional activator of the Hor2 gene. The in vivo interaction experiment between HvGAMYB and BPBF was done in the yeast twohybrid system, where HvGAMYB potentiates the BPBF trans-activation capacity through interaction with its C-terminal domain.

The expression of many seed storage protein genes in cereals relies on transcription factors of the bZIP class, belonging to the maize OPAQUE2 family. Here, we describe a survey of such factors in the genome of Arabidopsis thaliana, and the characterization of two of them, AtbZIP10 and AtbZIP25. Expression analysis by in situ hybridization shows that the occurrence of their mRNAs in the seed starts from early stages of development, peaks at maturation, and declines later in seed development, matching temporally and spatially those of the seed storage protein genes encoding 2S albumins and cruciferins. Gel mobility shift assays showed that AtbZIP10 and AtbZIP25 bind the ACGT boxes present in At2S and CRU3 promoters. Moreover, using the yeast two-hybrid system we show that AtbZIP10 and AtbZIP25 can interact in vivo with ABI3, an important regulator of gene expression in the seed of Arabidopsis. Transient expression analyses of a reporter gene under the control of the At2S1 promoter in transgenic plants overexpressing ectopically AtbZIP10, AtbZIP25, and ABI3 reveal that none of these factors could activate significantly the reporter gene when expressed individually. However, co-expression of AtbZIP10/25 with ABI3 resulted in a remarkable increase in the activation capacity over the At2S1 promoter, suggesting that they are part of a regulatory complex involved in seed-specific expression. This study shows a common mechanism of ABI3 in regulating different seed-specific genes through combinatorial interactions with particular bZIP proteins and a conserved role of O2-like bZIPs in monocot and dicot species.During seed development, storage reserves accumulate mostly in the form of carbohydrates and proteins, whose degradation upon germination will provide nutrients to the growing seedling before the photosynthetic capacity is fully acquired. Seed storage proteins (SSP) 1 are specifically synthesized in developing seeds, both in the endosperm and in the embryo. In the seed of monocotyledoneous species, the former is the predominant reserve tissue, whereas in dicotyledoneous plants the endosperm is commonly re-absorbed as maturation proceeds, and storage proteins are preferentially accumulated in the embryo. Because SSP-encoding genes are specifically induced and tightly regulated during seed development, they represent an interesting model system for studying the mechanisms of temporal and tissue-specific gene regulation.In the developing seed, different programs of gene expression have been defined that comprise distinct classes of genes that are coordinately regulated (1, 2). The MAT (maturation) class includes major SSP genes (like 2S albumins and 12S globulins) expressed at early and mid-maturation phases, whereas the LEA (late embryogenesis abundant) class includes primarily genes involved in the acquisition of desiccation tolerance expressed at later stages of maturation (3, 4). Unraveling the molecular basis of seed-specific gene expression has been mainly focused on the identification of cis-acting promoter el...

Background: Dof proteins are a family of plant-specific transcription factors that contain a particular class of zinc-finger DNA-binding domain. Members of this family have been found to play diverse roles in gene regulation of processes restricted to the plants. The completed genome sequences of rice and Arabidopsis constitute a valuable resource for comparative genomic analyses, since they are representatives of the two major evolutionary lineages within the angiosperms. In this framework, the identification of phylogenetic relationships among Dof proteins in these species is a fundamental step to unravel functionality of new and yet uncharacterised genes belonging to this group.

Despite evolutionary conserved mechanisms to silence transposable element activity, there are drastic differences in the abundance of transposable elements even among closely related plant species. We conducted a de novo assembly for the 375 Mb genome of the perennial model plant, Arabis alpina. Analysing this genome revealed long-lasting and recent transposable element activity predominately driven by Gypsy long terminal repeat retrotransposons, which extended the low-recombining pericentromeres and transformed large formerly euchromatic regions into repeat-rich pericentromeric regions. This reduced capacity for long terminal repeat retrotransposon silencing and removal in A. alpina co-occurs with unexpectedly low levels of DNA methylation. Most remarkably, the striking reduction of symmetrical CG and CHG methylation suggests weakened DNA methylation maintenance in A. alpina compared with Arabidopsis thaliana. Phylogenetic analyses indicate a highly dynamic evolution of some components of methylation maintenance machinery that might be related to the unique methylation in A. alpina.