Annegret Tewes

Seed-specific transcription factors ABI3 and FUS3: molecular interaction with DNA

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.

Calreticulin expression in plant cells: developmental regulation, tissue specificity and intracellular distribution.

Abstract. The tissue-specific expression pattern and the intracellular distribution of the Ca2+-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 λ 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. α-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.

Molecular characterization of cDNAs encoding G protein α and β subunits and study of their temporal and spatial expression patterns in Nicotiana plumbaginifolia Viv.

We have isolated cDNA sequences encoding α and β subunits of potential G proteins from a cDNA library prepared from somatic embryos of Nicotiana plumbaginifolia Viv. at early developmental stages. The predicted NPGPA1 and NPGPB1 gene products are 75–98% identical to the known respective plant α and β subunits. Southern hybridizations indicate that NPGPA1 is probably a single-copy gene, whereas at least two copies of NPGPB1 exist in the N. plumbaginifolia genome. Northern analyses reveal that both NPGPA1 and NPGPB1 mRNA are expressed in all embryogenic stages and plant tissues examined and their expression is obviously regulated by the plant hormone auxin. Immunohistological localization of NPGPα1 and NPGPβ1 preferentially on plasma and endoplasmic reticulum membranes and their immunochemical detection exclusively in microsomal cell fractions implicate membrane association of both proteins. The temporal and spatial expression patterns of NPGPA1 and NPGPB1 show conformity as well as differences. This could account for not only cooperative, but also individual activities of both subunits during embryogenesis and plant development.

Induction by methyl jasmonate of embryogenesis-related proteins and mRNAs in Nicotinia plumbaginifolia

Abstract Upon treatment with jasmonic acid methyl ester (methyl jasmonate, JaMe) detached leaf segments of in vitro-regenerated plants of Nicotiana plumbaginifolia accumulate novel abundant proteins and mRNAs. Among them are late embryogenesis abundant (Lea) transcripts, which appear in a temporally defined manner during somatic embryogenesis and normally do not occur in mature plant organs such as leaves or roots. To pursue this observation, the induction by methyl jasmonate of embryogenesis-related proteins and mRNAs in leaf and root tissues was investigated by comparing the two-dimensional patterns of in vivo- and in vitro-translated polypeptides. We found a selective reinduction by methyl jasmonate of several embryo-specific proteins and mRNAs, whose expression is associated with the formation of early and mature globular stages. Some of the jasmonate-induced embryo-specific proteins were also expressed in leaves, but not in roots, in response to abscisic acid (ABA) or osmotic stress (sorbitol) treatment, suggesting their role in osmoprotection of leaf and embryo tissues as discussed for LEA proteins. Western blot analyses with polyclonal antibodies raised against jasmonate-induced proteins (JIPs) of Mr 23 000 from barley highlighted the tissue-specific reappearance of a group of closely related, evolutionarily conserved embryo-specific proteins in N. plumbaginifolia leaves after JaMe, ABA and sorbitol treatment. Actin and tubulin, for which the transcript contents were estimated, increased in amount in the course of somatic embryogenesis, but did not change in abundance under the various conditions of treatment. Our results imply a specific role of JaMe in controlling gene expression during somatic embryogenesis in N. plumbaginifolia.

Ectopic Expression of EFFECTOR OF TRANSCRIPTION Perturbs Gibberellin-Mediated Plant Developmental Processes

The plant hormone gibberellin (GA) is known to modulate various aspects of plant cell differentiation and development. The current model of GA-mediated regulation is based on a de-repressible system and includes specific protein modification and degradation. HRT, a zinc finger protein from barley has been shown to have GA-dependent transcriptional repressing activity on the seed-specific α-amylase promoter [Raventos, D., Skriver, K., Schlein, M., Karnahl, K., Rogers, S.W., Rogers, J.C. and Mundy, J. 1998. J.␣Biol. Chem. 273: 23313–23320]. Here we report the characterization of a dicot homologue from Brassica napus (BnET) and provide evidence for its role in GA response modulation suggesting that this could be a conserved feature of this gene family. When BnET is ectopically expressed in either Arabidopsis or tobacco the phenotypes include dwarfism due to shorter internodes and late flowering, reduced germination rate, increased anthocyanin content and reduced xylem lignification as a marker for terminal cell differentiation. Transient expression in protoplasts supports the notion that this most likely is due to a transcriptional repression of GA controlled genes. Finally, histological analysis showed that in contrast to other GA deficient mutants the shorter internodes were due to fewer but not smaller cells, suggesting a function of BnET in GA-mediated cell division control.

Long-term cultures of barley synthesize and correctly deposit seed storage proteins

SummaryLong-term cultures of four different cultivars of barley (Hordeum vulgare L.) have been established. Both callus and suspension cultures formed embryogenic structures at high frequency even after more than 18 months of culture. These compact proembryogenic cell clusters synthesize seed storage globulins whereas loose cell aggregates in callus culture and suspension cultures of fine dispersed consistency were free of globulins. Globulin synthesis was especially intense in compact structures of callus cultures established from suspension culture-derived protoplasts. Within the cells storage globulins are deposited in the vacuolar compartment as in zygotic embryos. The molecular data provided recommend the system for studies on factors determining seed protein gene expression and intracellular protein transport.