Background: WRKY proteins belong to the WRKY-GCM1 superfamily of zinc finger transcription factors that have been subject to a large plant-specific diversification. For the cereal crop barley (Hordeum vulgare), three different WRKY proteins have been characterized so far as regulators in sucrose signaling, pathogen defense, and in response to cold and drought. However, their phylogenetic relationship remained unresolved. Results: In this study, we used available sequence information to identify a minimum number of 45 barley WRKY transcription factor (HvWRKY) genes. According to their structural features, the HvWRKY factors were classified into the previously defined polyphyletic WRKY subgroups 1 to 3. Furthermore, we could assign putative orthologs of the HvWRKY proteins in Arabidopsis and rice. While in most cases clades of orthologous proteins were formed within each group or subgroup, other clades were composed of paralogous proteins for the grasses and Arabidopsis only, which is indicative of specific gene radiation events. To gain insight into their putative functions, we examined expression profiles of WRKY genes from publicly available microarray data resources and found group specific expression patterns. While putative orthologs of the HvWRKY transcription factors have been inferred from phylogenetic sequence analysis, we performed a comparative expression analysis of WRKY genes in Arabidopsis and barley. Indeed, highly correlative expression profiles were found between some of the putative orthologs. Conclusion: HvWRKY genes have not only undergone radiation in monocot or dicot species, but exhibit evolutionary traits specific to grasses. HvWRKY proteins exhibited not only sequence similarities between orthologs with Arabidopsis, but also relatedness in their expression patterns. This correlative expression is indicative for a putative conserved function of related WRKY proteins in monocot and dicot species.

A discussion of the technological limitations and advantages of the most commonly used techniques for detecting in vivo protein-protein interactions is presente

One of the most important regulatory small molecules in plants is indole-3-acetic acid, also known as auxin. Its dynamic redistribution has an essential role in almost every aspect of plant life, ranging from cell shape and division to organogenesis and responses to light and gravity^1,2. So far, it has not been possible to directly determine the spatial and temporal distribution of auxin at a cellular resolution. Instead it is inferred from the visualization of irreversible processes that involve the endogenous auxin-response machinery^3–7; however, such a system cannot detect transient changes. Here we report a genetically encoded biosensor for the quantitative in vivo visualization of auxin distribution. The sensor is based on the Escherichia coli tryptophan repressor⁸, the binding pocket of which is engineered to be specific to auxin. Coupling of the auxin-binding moiety with selected fluorescent proteins enables the use of a fluorescence resonance energy transfer signal as a readout. Unlike previous systems, this sensor enables direct monitoring of the rapid uptake and clearance of auxin by individual cells and within cell compartments in planta. By responding to the graded spatial distribution along the root axis and its perturbation by transport inhibitors—as well as the rapid and reversible redistribution of endogenous auxin in response to changes in gravity vectors—our sensor enables real-time monitoring of auxin concentrations at a (sub)cellular resolution and their spatial and temporal changes during the lifespan of a plant.

Summary: The paralogous maize (Zea mays)LBD(Lateral Organ Boundaries Domain) genesrtcs(rootless concerning crown and seminal roots) andrtcl(rtcs‐like) emerged from an ancient whole‐genome duplication.RTCSis a key regulator of crown root initiation. The diversity of expression, molecular interaction and phenotype ofrtcsandrtclwere investigated. Thertcsandrtclgenes display highly correlated spatio‐temporal expression patterns in roots, despite the significantly higher expression ofrtcs. BothRTCSandRTCLproteins bind toLBDdownstream promoters and act as transcription factors. In line with its auxin inducibility and binding to auxin response elements ofrtcsandrtclpromoters,ARF34 (AUXIN RESPONSE FACTOR34) acts as transcriptional activator. Yeast two‐hybrid screening combined with bimolecular fluorescence complementation (BiFC) experiments revealed conserved and unique interaction partners ofRTCSandRTCL. Thertclmutation leads to defective shoot‐borne root elongation early in development. Cooperative action ofRTCSandRTCLduring shoot‐borne root formation was demonstrated byrtcs‐dependent repression ofrtcltranscription in coleoptilar nodes. AlthoughRTCSis instrumental in shoot‐borne root initiation,RTCLcontrols shoot‐borne root elongation early in development. Their conserved role in auxin signaling, but diverse function in shoot‐borne root formation, is underscored by their conserved and unique interaction partners.

Summary: 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.