EFFECTOR OF TRANSCRIPTION factors are novel plant-specific regulators associated with genomic DNA methylation in Arabidopsis.

Plant-specific EFFECTORS OF TRANSCRIPTION (ET) are characterised by a variable number of highly conserved ET repeats, which are involved in zinc and DNA binding. In addition, ETs share a GIY-YIG domain, involved in DNA nicking activity. It was hypothesised that ETs might act as epigenetic regulators. Here, methylome, transcriptome and phenotypic analyses were performed to investigate the role of ET factors and their involvement in DNA methylation in Arabidopsis thaliana. Comparative DNA methylation and transcriptome analyses in flowers and seedlings of et mutants revealed ET-specific differentially expressed genes and mostly independently characteristic, ET-specific differentially methylated regions. Loss of ET function results in pleiotropic developmental defects. The accumulation of cyclobutane pyrimidine dimers after ultraviolet stress in et mutants suggests an ET function in DNA repair.

Transcriptional regulator AtET2 is required for the induction of dormancy during late seed development.

During the late stages of seed development, the embryo patterning program is completed and maturation is initiated. One of the main events during the maturation phase is the acquisition of dormancy, characterized by the failure of a normally developed seed to germinate precociously. Dormancy is controlled by a complex regulatory mechanism that involves the phytohormone gibberellin (GA) and the transcription factor FUSCA3 (FUS3). Here, we demonstrate the importance of the previously characterized GA regulator EFFECTOR OF TRANSCRIPTION2 (AtET2) for correct seed development. We show that entering the maturation phase, seeds of the et2-1 mutant, which contain a non-functional AtET2 gene, fail to induce dormancy. This correlates well with the observed activity pattern of the AtET2 promoter, which is active in the maturing embryo. AtET2 action during seed development is dependent on a complex interaction with GA and the FUS3 gene, the latter evidenced by the phenotypes of the et2-1 fus3-T double mutant. We show that in vitro expressed AtET2 protein can bind to both linear and supercoiled DNA without any obvious sequence preference. This suggests that, within a larger protein complex, AtET2 might be required for the correct positioning upon the DNA.

Members of the RKD transcription factor family induce an egg cell-like gene expression program.

In contrast to animals, the life cycle of higher plants alternates between a gamete-producing (gametophyte) and a spore-producing (sporophyte) generation. The female gametophyte of angiosperms consists of four distinct cell types, including two gametes, the egg and the central cell, which give rise to embryo and endosperm, respectively. Based on a combined subtractive hybridization and virtual subtraction approach in wheat (Triticum aestivum L.), we have isolated a class of transcription factors not found in animal genomes, the RKD (RWP-RK domain-containing) factors, which share a highly conserved RWP-RK domain. Single-cell RT-PCR revealed that the genes TaRKD1 and TaRKD2 are preferentially expressed in the egg cell of wheat. The Arabidopsis genome contains five RKD genes, at least two of them, AtRKD1 and AtRKD2, are preferentially expressed in the egg cell of Arabidopsis. Ectopic expression of the AtRKD1 and AtRKD2 genes induces cell proliferation and the expression of an egg cell marker. Analyses of RKD-induced proliferating cells exhibit a shift of gene expression towards an egg cell-like transcriptome. Promoters of selected RKD-induced genes were shown to be predominantly active in the egg cell and can be activated by RKD in a transient protoplast expression assay. The data show that egg cell-specific RKD factors control a transcriptional program, which is characteristic for plant egg cells.

EFFECTOR OF TRANSCRIPTION2 is involved in xylem differentiation and includes a functional DNA single strand cutting domain.

EFFECTORS OF TRANSCRIPTION2 (ET) are plant-specific regulatory proteins characterized by the presence of two to five C-terminal DNA- and Zn-binding repeats, and a highly conserved cysteine pattern. We describe the structural characterization of the three member Arabidopsis thaliana ET gene family and reveal some allelic sequence polymorphisms. A mutation analysis showed that AtET2 affects the expression of various KNAT genes involved in the maintenance of the undifferentiated state of cambial meristem cells. It also plays a role in the regulation of GA5 (gibberellin 3-beta-dioxygenase) and the cell-cycle-related GASA4. A correlation was established between AtET2 expression and the cellular differentiation state. AtET-GFP fusion proteins shuttle between the cytoplasm and nucleus, with the AtET2 product prevented from entering the nucleus in non-differentiating cells. Within the nucleus, AtET2 probably acts via a single strand cutting domain. A more general regulatory role for ET factors is proposed, governing cell differentiation in cambial meristems, a crucial process for the development of plant vascular tissues.

Phylogenetic footprints in fern spore- and seed-specific gene promoters.

Spermatophyte seed-storage proteins have descended from a group of proteins involved in cellular desiccation/hydration processes. Conserved protein structures are found across all plant phyla and in the fungi and Archaea. We investigated whether conservation in the coding region sequence is paralleled by common gene regulatory processes. Seed- and spore-specific gene promoters of three phylogenetically diverse plants were analysed by transient and transgenic expression in Arabidopsis thaliana and tobacco. The transcription factors FUS3 and ABI3, which are central regulators of seed maturation processes, interact with cis-motifs of seed-specific promoters from distantly related plants. The promoter of a fern spore-specific gene encoding a seed-storage globulin-like protein exhibits strong seed-specific activity in both Arabidopsis and tobacco. The existence of phylogenetic footprints indicates good conservation of regulatory pathways controlling gene expression in fern spores and in gymnosperm and angiosperm seeds, reflecting the concerted evolution of coding and regulatory regions.