Judith Müller

The organization of living cells is based on networks of interacting molecules. Systematic analysis of protein interactions of 3-aa loop extension (TALE) homeodomain proteins, fundamental regulators of plant meristem function and leaf development, revealed a highly connected, complex network. The network includes nine members of Arabidopsis thaliana ovate family proteins (AtOFPs), a plant-specific protein family, indicating a close functional connection to TALE homeodomain proteins. Evidence is provided that AtOFP1 is an essential pleiotropic developmental regulator. AtOFP1 and AtOFP5 are shown to associate with the cytoskeleton and to regulate subcellular localization of TALE homeodomain proteins, suggesting a previously unrecognized control mechanism in plant development.

This paper describes two-hybrid interactions amongst barley homeodomain proteins encoded by the Three Amino acid Loop Extension (TALE) superfamily. The class I KNOX protein BKN3 is shown to homodimerise and to associate with proteins encoded by the class I and II Knox genes BKn-1 and BKn-7. Furthermore, JUBEL1 and JUBEL2, two BELL1 homologous proteins, are identified and characterised as interacting partners of BKN3. Differences in the requirements of BKN3 derivatives for interactions with KNOX and JUBEL proteins imply the involvement of overlapping but slightly different domains. This set of results is an example for interactions amongst different classes of plant TALE homeodomain proteins, as previously described for related animal proteins. Apparently identical spatial and temporal expression patterns of BKn-1, BKn-3, BKn-7, JuBel1 and JuBel2, as determined by in situ hybridisation, are compatible with possible interactions of their protein products in planta. Contradictory to the common model, that the transcriptional down-regulation of certain class 1 Knox-genes is the prerequisite for organ differentiation, transcripts of all five genes were, similar to Tkn1 and Tkn2/LeT6 of tomato, detected in incipient and immature leaves as well as in meristematic tissues. A characteristic phenotype is induced by the overexpression of JuBel2 in transgenic tobacco plants.

The endoplasmic reticulum (ER) of eukaryotic cells serves as a checkpoint tightly monitoring protein integrity and channeling malformed proteins into different rescue and degradation routes. The degradation of several ER lumenal and membrane-localized proteins is mediated by ER-associated protein degradation (ERAD) in yeast (Saccharomyces cerevisiae) and mammalian cells. To date, evidence for the existence of ERAD-like mechanisms in plants is indirect and based on heterologous or artificial substrate proteins. Here, we show that an allelic series of single amino acid substitution mutants of the plant-specific barley (Hordeum vulgare) seven-transmembrane domain mildew resistance o (MLO) protein generates substrates for a postinsertional quality control process in plant, yeast, and human cells, suggesting conservation of the underlying mechanism across kingdoms. Specific stabilization of mutant MLO proteins in yeast strains carrying defined defects in protein quality control demonstrates that MLO degradation is mediated by HRD pathway-dependent ERAD. In plants, individual aberrant MLO proteins exhibit markedly reduced half-lives, are polyubiquitinated, and can be stabilized through inhibition of proteasome activity. This and a dependence on homologs of the AAA ATPase CDC48/p97 to eliminate the aberrant variants strongly suggest that MLO proteins are endogenous substrates of an ERAD-related plant quality control mechanism.

In the dominant mutant Hooded (K), the barley gene BKn3 is overexpressed as a result of a duplication of 305 bp in intron IV. When fused to a cauliflower mosaic virus 35S minimal promoter, the 305 bp element activates gene expression in tobacco, as does a 655 bp BKn3 promoter sequence. Both DNA fragments contain a (GA)8 repeat (GA/TC)8. A one-hybrid screen using the 305 bp element as the DNA target led to the cloning of the barley b recombinant (BBR) protein, which binds specifically to the (GA/TC)8 repeat. BBR is nuclear targeted and is a characterized nuclear localization signal (NLS) sequence, a DNA-binding domain extended up to 90 aa at the C-terminus and a putative N-terminal activation domain. The corresponding gene has no introns and is ubiquitously expressed in barley tissues. In co-transfection experiments, BBR activates (GA/TC)8-containing promoters, and its overexpression in tobacco leads to a pronounced leaf shape modification. BBR has properties of a GAGA-binding factor, but the corresponding gene has no sequence homology to Trl and Psq of Drosophila, which encode functionally analogous proteins. In Arabidopsis, (GA/TC)8 repeats occur particularly within 1500 bp upstream of gene start codons included in some homeodomain genes of different classes. The data presented suggest that expression of the barley BKn3 is regulated, at least in part, by the binding of the transcription factor BBR to GA/TC repeats.