SummaryThe Arabidopsis mutants fus3 and abi3 show pleiotropic effects during embryogenesis including reduced levels of transcripts encoding embryo-speci®c seed proteins. To investigate the interaction between the B3-domain-containing transcription factors FUS3 and ABI3 with the RY cis-motif, conserved in many seed-speci®c promoters, a promoter analysis as well as band-shift experiments were performed. The analysis of promoter mutants revealed the structural requirements for the function of the RY ciselement. It is shown that both the nucleotide sequence and the alternation of purin and pyrimidin nucleotides (RY character) are essential for the activity of the motif. Further, it was shown that FUS3 and ABI3 can act independently of each other in controlling promoter activity and that the RY cis-motif is a target for both transcription factors. For FUS3, which is so far the smallest known member of the B3-domain family, a physical interaction with the RY motif was established. The functional and biochemical data demonstrate that the regulators FUS3 and ABI3 are essential components of a regulatory network acting in concert through the RY-promoter element to control gene expression during late embryogenesis and seed development.

In Arabidopsis thaliana (L.) Heynh. the seed-specific transcription factors ABI3 and FUS3 have key regulatory functions during the development of mature seeds. The highly conserved RY motif [DNA motif CATGCA(TG)], present in many seed-specific promoters, is an essential target of both regulators. Here we show that, in vitro, the full-length ABI3 protein, as well as FUS3 protein, is able to bind to RY-DNA and that the B3 domains of both transcription factors are necessary and sufficient for the specific interaction with the RY element. Flanking sequences of the RY motif modulate the binding, but the presence of an RY sequence alone allows the specific interaction of ABI3 and FUS3 with the target in vitro. Transcriptional activity of ABI3 and FUS3, measured by transient promoter activation, requires the B3 DNA-binding domain and an activation domain. In addition to the known N-terminal-located activation domain, a second transcription activation domain was found in the B1 region of ABI3.

SummaryFull length cDNAs encoding three amino acid permeases were isolated from seed-speci®c libraries of Vicia faba. The predicted proteins VfAAP1, VfAAP3 and VfAAP4 share up to 66% identity among themselves. Functional characterization of VfAAP1 and VfAAP3 in a yeast mutant showed that these permeases transport a broad range of amino acids. However, VfAAP1 had a preference for cysteine and VfAAP3 for lysine and arginine. VfAAP1 was highly expressed in cotyledons at early developmental stages and moderately in other sink tissues. Its peak of expression in cotyledons corresponded to the appearance of storage protein transcripts, suggesting that this transporter ful®lls an important role in providing amino acids for storage protein biosynthesis. VfAAP3 was expressed most abundantly in maternal tissues, that is in roots, stems, gynoecia, pods and seed coats at different developmental stages. VfAAP4 transcripts could not be detected by northern hybridization. In situ hybridization showed that VfAAP1 mRNA is distributed throughout cotyledon storage parenchyma cells, but could not be detected in the abaxial epidermal cell layer. It also accumulate in the chlorenchyma and thin-walled parenchyma cells of seed coats. VfAAP1 mRNA levels were lower in cotyledons cultured in the presence of glutamine, whereas expression of a vicilin storage protein gene was up-regulated under similar conditions. Cysteine repressed the expression of the GUS reporter gene under control of the VfAAP1 promoter, suggesting that this transporter is modulated at the transcriptional level. Regulation of amino acid transport in relation to storage protein accumulation is discussed.

The tissue-specific expression pattern and the intracellular distribution of the Ca(2+)-binding protein calreticulin at the mRNA and protein levels have been studied during somatic and zygotic embryogenesis of Nicotiana plumbaginifolia Viv. A full-length cDNA sequence encoding calreticulin was isolated from a lembda Zap cDNA library from early developmental stages of somatic embryogenesis. The deduced amino acid sequence of the calreticulin from N. plumbaginifolia shows high homology to the corresponding proteins of tobacco (98.2% identity), maize (80%) and barley (76.5%), and more than 55% homology to animal calreticulins, and the sequence motifs with established functions found in calreticulins of other species were quite conserved. Northern experiments revealed a developmental regulation of the calreticulin transcript with a maximum during the early stages of somatic embryogenesis and an auxin dependence during in-vitro cell culture. alpha-Naphthaleneacetic acid stimulated calreticulin expression whereas 2,4-dichlorophenoxyacetic acid reduced it. Immunohistological analysis of calreticulin distribution in the ovaries during zygotic embryogenesis showed that calreticulin biosynthesis started tissue specifically, with a high abundance in the endothelium of the integument in the ovules, followed by calreticulin accumulation in the embryo proper and in the associated endosperm at the late globular stage of embryogenesis. Using immunogold labeling, calreticulin was intracellularly localized with a high abundance to the Golgi compartment and to patches on the surface of dividing protoplasts. Smaller amounts were found in the endoplasmic reticulum and plasma membranes. The functional role of calreticulin in posttranslational processing and translocation processes, apart from its postulated function in cellular Ca2+ homeostasis, is discussed.

SUMMARYThe enzymological properties of AtAurora1, a kinase responsible for the cell cycle-dependent phosphorylation of histone H3 at S10, and its cross-talk with other post-translational histone modifications, were determined. In vitro phosphorylation of H3S10 by AtAurora1 is strongly increased by K9 acetylation, and decreased by K14 acetylation and T11 phosphorylation. However, S10 phosphorylation activity is unaltered by mono-, di-or trimethylation of K9. An interference of H3K9 dimethylation by SUVR4 occurs by a pre-existing phosphorylation at S10. Hence, cross-talk in plants exists between phosphorylation of H3S10 and methylation, acetylation or phosphorylation of neighbouring amino acid residues. AtAurora1 undergoes autophosphorylation in vivo regardless of the presence of substrate, and forms dimers in planta. Of the three ATP-competitive Aurora inhibitors tested, Hesperadin was most effective in reducing the in vivo kinase activity of AtAurora1. Hesperadin consistently inhibited histone H3S10 phosphorylation during mitosis in Arabidopsis cells, but did not affect other H3 post-translational modifications, suggesting a specific inhibition of AtAurora in vivo. Inactivation of AtAurora also caused lagging chromosomes in a number of anaphase cells, but, unlike the situation in mammalian cells, Hesperadin did not influence the microtubule dynamics in dividing cells.