Kenneth W. Berendzen

Phylogenetic and comparative gene expression analysis of barley (Hordeum vulgare) WRKY transcription factor family reveals putatively retained functions between monocots and dicots

BackgroundWRKY 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.ResultsIn 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.ConclusionHvWRKY 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.

The Arabidopsis thaliana response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds

BackgroundThe Arabidopsis response regulator 22 (ARR22) is one of two members of a recently defined novel group of two-component system (TCS) elements. TCSs are stimulus perception and response modules of prokaryotic origin, which signal by a His-to-Asp phosphorelay mechanism. In plants, TCS regulators are involved in hormone response pathways, such as those for cytokinin and ethylene. While the functions of the other TCS elements in Arabidopsis, such as histidine kinases (AHKs), histidine-containing phosphotransfer proteins (AHPs) and A-type and B-type ARRs are becoming evident, the role of ARR22 is poorly understood.ResultsWe present evidence that ARR22 is a preferentially cytoplasmic protein, exclusively expressed in the chalaza of developing seeds. ARR22 specifically interacts with AHP2, AHP3 and AHP5 in yeast and living plant cells. Two new loss-of-function alleles, arr22-2 and arr22-3, were isolated and characterized. With respect to their morphology and metabolite status, no significant difference in the developing seeds of the arr22 mutants was observed compared to wild type. The genetic complementation of the arr22 mutants with a genomic ARR22 fragment resulted in plants (arr22/gARR22) with a pleiotropic phenotype of different penetrance. This phenotype was not observed when the phosphorylatable Asp74 of ARR22 was changed to either a dominant-active Glu or a dominant-inactive Asn. The phenotype of the arr22/gARR22 plants was comparable to that of multiple ahk, ahp and B-type arr mutants.ConclusionOur results favor the model that ARR22 acts as a phospho-histidine phosphatase on specific AHPs in the cytoplasm of Arabidopsis chalaza cells. The lack of any aberrant morphological and metabolite phenotype in the seeds of the arr22 mutants indicates that ARR22 is probably primarily responsible for the fine tuning of specific branches of chalaza-based TCS signalling. Even when slightly mis-expressed, ARR22 interferes with hormone homeostasis in non-chalaza tissues. Our data indicate that the chromatin status might play a crucial role in maintaining the chalaza-restricted expression of ARR22.

A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta.

BackgroundMany established PCR-based approaches in plant molecular biology rely on lengthy and expensive methods for isolation of nucleic acids. Although several rapid DNA isolation protocols are available, they have not been tested for simultaneous RNA isolation for RT-PCR applications. In addition, traditional map-based cloning technologies often use ill-proportioned marker regions even when working with the model plant Arabidopsis thaliana, where the availability of the full genome sequence can now be exploited for the creation of a high-density marker systems.ResultsWe designed a high-density polymorphic marker set between two frequently used ecotypes. This new polymorphic marker set allows size separation of PCR products on agarose gels and provides an initial resolution of 10 cM in linkage mapping experiments, facilitated by a rapid plant nucleic acid extraction protocol using minimal amounts of A. thaliana tissue. Using this extraction protocol, we have also characterized segregating T-DNA insertion mutations. In addition, we have shown that our rapid nucleic acid extraction protocol can also be used for monitoring transcript levels by RT-PCR amplification. Finally we have demonstrated that our nucleic acid isolation method is also suitable for other plant species, such as tobacco and barley.ConclusionTo facilitate high-throughput linkage mapping and other genomic applications, our nucleic acid isolation protocol yields sufficient quality of DNA and RNA templates for PCR and RT-PCR reactions, respectively. This new technique requires considerably less time compared to other purification methods, and in combination with a new polymorphic PCR marker set dramatically reduces the workload required for linkage mapping of mutations in A. thaliana utilizing crosses between Col-0 and Landsberg erecta (Ler) ecotypes.

Volatiles of two growth-inhibiting rhizobacteria commonly engage AtWRKY18 function

Interactions with the (a)biotic environment play key roles in a plants fitness and vitality. In addition to direct surface-to-surface contact, volatile chemicals can also affect the physiology of organism. Volatiles of Serratia plymuthica and Stenotrophomonas maltophilia significantly inhibited growth and induced H(2) O(2) production in Arabidopsis in dual culture. Within 1 day, transcriptional changes were observed by promoter-GUS assays using a stress-inducible W-box-containing 4xGST1 construct. Expression studies performed at 6, 12 and 24 h revealed altered transcript levels for 889 genes and 655 genes in response to Se. plymuthica or St. maltophilia volatiles, respectively. Expression of 162 genes was altered in both treatments. Meta-analysis revealed that specifically volatile-responsive genes were significantly overlapping with those affected by abiotic stress. We use the term mVAMP (microbial volatile-associated molecular pattern) to describe these volatile-specific responses. Genes responsive to both treatments were enriched for W-box motifs in their promoters, and were significantly enriched for transcription factors (ERF2, ZAT10, MYB73 and WRKY18). The susceptibility of wrky18 mutant lines to volatiles was significantly delayed, suggesting an indispensable role for WRKY18 in bacterial volatile responses.

Cis-motifs upstream of the transcription and translation initiation sites are effectively revealed by their positional disequilibrium in eukaryote genomes using frequency distribution curves

BackgroundThe discovery of cis-regulatory motifs still remains a challenging task even though the number of sequenced genomes is constantly growing. Computational analyses using pattern search algorithms have been valuable in phylogenetic footprinting approaches as have expression profile experiments to predict co-occurring motifs. Surprisingly little is known about the nature of cis-regulatory element (CRE) distribution in promoters.ResultsIn this paper we used the Motif Mapper open-source collection of visual basic scripts for the analysis of motifs in any aligned set of DNA sequences. We focused on promoter motif distribution curves to identify positional over-representation of DNA motifs. Using differentially aligned datasets from the model species Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster and Saccharomyces cerevisiae, we convincingly demonstrated the importance of the position and orientation for motif discovery. Analysis with known CREs and all possible hexanucleotides showed that some functional elements gather close to the transcription and translation initiation sites and that elements other than the TATA-box motif are conserved between eukaryote promoters. While a high background frequency usually decreases the effectiveness of such an enumerative investigation, we improved our analysis by conducting motif distribution maps using large datasets.ConclusionThis is the first study to reveal positional over-representation of CREs and promoter motifs in a cross-species approach. CREs and motifs shared between eukaryotic promoters support the observation that an eukaryotic promoter structure has been conserved throughout evolutionary time. Furthermore, with the information on positional enrichment of a motif or a known functional CRE, it is possible to get a more detailed insight into where an element appears to function. This in turn might accelerate the in depth examination of known and yet unknown cis-regulatory sequences in the laboratory.

Techniques for the Analysis of Protein-Protein Interactions in Vivo

A discussion of the technological limitations and advantages of the most commonly used techniques for detecting in vivo protein-protein interactions is presented, emphasizing their application to plant research. Identifying key players and their interactions is fundamental for understanding biochemical mechanisms at the molecular level. The ever-increasing number of alternative ways to detect protein-protein interactions (PPIs) speaks volumes about the creativity of scientists in hunting for the optimal technique. PPIs derived from single experiments or high-throughput screens enable the decoding of binary interactions, the building of large-scale interaction maps of single organisms, and the establishment of cross-species networks. This review provides a historical view of the development of PPI technology over the past three decades, particularly focusing on in vivo PPI techniques that are inexpensive to perform and/or easy to implement in a state-of-the-art molecular biology laboratory. Special emphasis is given to their feasibility and application for plant biology as well as recent improvements or additions to these established techniques. The biology behind each method and its advantages and disadvantages are discussed in detail, as are the design, execution, and evaluation of PPI analysis. We also aim to raise awareness about the technological considerations and the inherent flaws of these methods, which may have an impact on the biological interpretation of PPIs. Ultimately, we hope this review serves as a useful reference when choosing the most suitable PPI technique.

Bioinformatic cis-element analyses performed in Arabidopsis and rice disclose bZIP- and MYB-related binding sites as potential AuxRE-coupling elements in auxin-mediated transcription

BackgroundIn higher plants, a diverse array of developmental and growth-related processes is regulated by the plant hormone auxin. Recent publications have proposed that besides the well-characterized Auxin Response Factors (ARFs) that bind Auxin Response Elements (AuxREs), also members of the bZIP- and MYB-transcription factor (TF) families participate in transcriptional control of auxin-regulated genes via bZIP Response Elements (ZREs) or Myb Response Elements (MREs), respectively.ResultsApplying a novel bioinformatic algorithm, we demonstrate on a genome-wide scale that singular motifs or composite modules of AuxREs, ZREs, MREs but also of MYC2 related elements are significantly enriched in promoters of auxin-inducible genes. Despite considerable, species-specific differences in the genome structure in terms of the GC content, this enrichment is generally conserved in dicot (Arabidopsis thaliana) and monocot (Oryza sativa) model plants. Moreover, an enrichment of defined composite modules has been observed in selected auxin-related gene families. Consistently, a bipartite module, which encompasses a bZIP-associated G-box Related Element (GRE) and an AuxRE motif, has been found to be highly enriched. Making use of transient reporter studies in protoplasts, these findings were experimentally confirmed, demonstrating that GREs functionally interact with AuxREs in regulating auxin-mediated transcription.ConclusionsUsing genome-wide bioinformatic analyses, evolutionary conserved motifs have been defined which potentially function as AuxRE-dependent coupling elements to establish auxin-specific expression patterns. Based on these findings, experimental approaches can be designed to broaden our understanding of combinatorial, auxin-controlled gene regulation.

Molecular interactions of ROOTLESS CONCERNING CROWN AND SEMINAL ROOTS, a LOB domain protein regulating shoot-borne root initiation in maize (Zea mays L.)

Rootless concerning crown and seminal roots (Rtcs) encodes a LATERAL ORGAN BOUNDARIES domain (LBD) protein that regulates shoot-borne root initiation in maize (Zea mays L.). GREEN FLUORESCENT PROTEIN (GFP)-fusions revealed RTCS localization in the nucleus while its paralogue RTCS-LIKE (RTCL) was detected in the nucleus and cytoplasm probably owing to an amino acid exchange in a nuclear localization signal. Moreover, enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that RTCS primarily binds to LBD DNA motifs. RTCS binding to an LBD motif in the promoter of the auxin response factor (ARF) ZmArf34 and reciprocally, reciprocal ZmARF34 binding to an auxin responsive element motif in the promoter of Rtcs was shown by electrophoretic mobility shift assay experiments. In addition, comparative qRT-PCR of wild-type versus rtcs coleoptilar nodes suggested RTCS-dependent activation of ZmArf34 expression. Consistently, luciferase reporter assays illustrated the capacity of RTCS, RTCL and ZmARF34 to activate downstream gene expression. Finally, RTCL homo- and RTCS/RTCL hetero-interaction were demonstrated in yeast-two-hybrid and bimolecular fluorescence complementation experiments, suggesting a role of these complexes in downstream gene regulation. In summary, the data provide novel insights into the molecular interactions resulting in crown root initiation in maize.

Detection of in vivo interactions between Arabidopsis class A-HSFs, using a novel BiFC fragment, and identification of novel class B-HSF interacting proteins.

Class A heat shock factors (Hsfs) of Arabidopsis are known to function as transcriptional activators of stress genes. Genetic and functional analysis suggests that HsfA1a and HsfA1b are central regulators required in the early phase of the heat shock response, which have the capacity to functionally replace each other. In order to examine Hsf interaction in vivo, we conducted interaction assays using bimolecular fluorescence complementation (BiFC) on Arabidopsis protoplasts co-transformed with suitable Hsf-YFP fusion genes. BiFC assays were quantified with confocal laser scanning microscopy and flow cytometry, and confirmed with immunoprecipitation assays. For each Hsf we could not only demonstrate homomeric interactions but also detect heteromeric interaction between HsfA1a and HsfA1b. Truncated versions of these of Hsfs, containing deletions of the oligomerization domains (ODs), provided clear evidence that the ODs are required and sufficient for the HSF interaction in vivo. By contrast there was only homomeric but no heteromeric interaction detected between two different class B Hsf transcription factors (HsfB1 and HsfB2b) in a yeast two-hybrid assay. HsfB1/HsfB2b functions are not directly linked with the expression of conventional heat shock genes; class B Hsfs are devoid of the activation domain motif conserved in class A Hsfs. In order to identify other proteins interacting with HsfB1 and HsfB2b we performed yeast two-hybrid screenings of cDNA libraries. Three of the identified proteins were common to both screenings. This suggests that HsfB1 and HsfB2b may be involved in complex regulatory networks, which are linked to other stress responses and signaling processes.

Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry.

Understanding protein and gene function requires identifying interaction partners using biochemical, molecular or genetic tools. In plants, searching for novel protein-protein interactions is limited to protein purification assays, heterologous in vivo systems such as the yeast-two-hybrid or mutant screens. Ideally one would be able to search for novel protein partners in living plant cells. We demonstrate that it is possible to screen for novel protein-protein interactions from a random library in protoplasted Arabidopsis plant cells and recover some of the interacting partners. Our screen is based on capturing the bi-molecular complementation of mYFP between an YN-bait fusion partner and a completely random prey YC-cDNA library with FACS. The candidate interactions were confirmed using in planta BiFC assays and in planta FRET-FLIM assays. From this work, we show that the well characterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3, HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This is one of the first randomin planta screens to be successfully employed.