David Chevalier

The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis

Proteins containing the forkhead-associated domain (FHA) are known to act in biological processes such as DNA damage repair, protein degradation, and signal transduction. Here we report that DAWDLE (DDL), an FHA domain-containing protein in Arabidopsis, acts in the biogenesis of miRNAs and endogenous siRNAs. Unlike mutants of genes known to participate in the processing of miRNA precursors, such as dcl1, hyponastic leaves1, and serrate, ddl mutants show reduced levels of pri-miRNAs as well as mature miRNAs. Promoter activity of MIR genes, however, is not affected by ddl mutations. DDL is an RNA binding protein and is able to interact with DCL1. In addition, we found that SNIP1, the human homolog of DDL, is involved in miRNA biogenesis and interacts with Drosha. Therefore, we uncovered an evolutionarily conserved factor in miRNA biogenesis. We propose that DDL participates in miRNA biogenesis by facilitating DCL1 to access or recognize pri-miRNAs.

Regulation of floral organ abscission in Arabidopsis thaliana

Abscission is a developmental program that results in the active shedding of infected or nonfunctional organs from a plant body. Here, we establish a signaling pathway that controls abscission in Arabidopsis thaliana from ligand, to receptors, to downstream effectors. Loss of function mutations in Inflorescence Deficient in Abscission (IDA), which encodes a predicted secreted small protein, the receptor-like protein kinases HAESA (HAE) and HAESA-like 2 (HSL2), the Mitogen-Activated Protein Kinase Kinase 4 (MKK4) and MKK5, and a dominant-negative form of Mitogen-Activated Protein Kinase 6 (MPK6) in a mpk3 mutant background all have abscission-defective phenotypes. Conversely, expression of constitutively active MKKs rescues the abscission-defective phenotype of hae hsl2 and ida plants. Additionally, in hae hsl2 and ida plants, MAP kinase activity is reduced in the receptacle, the part of the stem that holds the floral organs. Plants overexpressing IDA in a hae hsl2 background have abscission defects, indicating HAE and HSL2 are epistatic to IDA. Taken together, these results suggest that the sequential action of IDA, HAE and HSL2, and a MAP kinase cascade regulates the programmed separation of cells in the abscission zone.

14-3-3 and FHA Domains Mediate Phosphoprotein Interactions

Many aspects of plant growth and development require specific protein interactions to carry out biochemical and cellular functions. Several proteins mediate these interactions, two of which specifically recognize phosphoproteins: 14-3-3 proteins and proteins with FHA domains. These are the only phosphobinding domains identified in plants. Both domains are present in animals and plants, and are used by plant proteins to regulate metabolic, developmental, and signaling pathways. 14-3-3s regulate sugar metabolism, proton gradients, and control transcription factor localization. FHA domains are modular domains often found in multidomain proteins that are involved in signal transduction and plant development.

Molecular characterisation of the STRUBBELIG-RECEPTOR FAMILY of genes encoding putative leucine-rich repeat receptor-like kinases in Arabidopsis thaliana

BackgroundReceptor-like kinases are a prominent class of surface receptors that regulate many aspects of the plant life cycle. Despite recent advances the function of most receptor-like kinases remains elusive. Therefore, it is paramount to investigate these receptors. The task is complicated by the fact that receptor-like kinases belong to a large monophyletic family with many sub-clades. In general, functional analysis of gene family members by reverse genetics is often obscured by several issues, such as redundancy, subtle or difficult to detect phenotypes in mutants, or by decision problems regarding suitable biological and biochemical assays. Therefore, in many cases additional strategies have to be employed to allow inference of hypotheses regarding gene function.ResultsWe approached the function of genes encoding the nine-member STRUBBELIG-RECEPTOR FAMILY (SRF) class of putative leucine-rich repeat receptor-like kinases. Sequence comparisons show overall conservation but also divergence in predicted functional domains among SRF proteins. Interestingly, SRF1 undergoes differential splicing. As a result, SRF1 is predicted to exist in a standard receptor configuration and in a membrane-anchored receptor-like version that lacks most of the intracellular domain. Furthermore, SRF1 is characterised by a high degree of polymorphism between the Ler and Col accessions. Two independent T-DNA-based srf4 mutants showed smaller leaves while 35S::SRF4 plants displayed enlarged leaves. This is in addition to the strubbelig phenotype which has been described before. Additional single and several key double mutant combinations did not reveal obvious mutant phenotypes. Ectopic expression of several SRF genes, using the 35S promoter, resulted in male sterility. To gain possible insights into SRF gene function we employed a computational analysis of publicly available microarray data. We performed global expression profiling, coexpression analysis, and an analysis of the enrichment of gene ontology terms among coexpressed genes. The bioinformatic analyses raise the possibility that some SRF genes affect different aspects of cell wall biology. The results also indicate that redundancy is a minor aspect of the SRF family.ConclusionThe results provide evidence that SRF4 is a positive regulator of leaf size. In addition, they suggest that the SRF family is characterised by functional diversity and that some SRF genes may function in cell wall biology. They also indicate that complementing reverse genetics with bioinformatical data mining of genome-wide expression data aids in inferring hypotheses on possible functions for members of a gene family.

The Protein Phosphatases and Protein Kinases of Arabidopsis thaliana

Protein kinases and protein phosphatases are major post-translational regulators of numerous cellular processes. These enzymes regulate metabolic pathways and are intimately involved in cellular signaling networks. There are over 1000 genes (Wang et al., 2003) in Arabidopsis that encode protein kinases and another 112 genes (Kerk et al., 2002) that encode protein phosphatase catalytic subunits (Table 1)​1).. While Arabidopsis contains orthologs of many of the protein kinases found in other eukaryotes, Arabidopsis, and most likely plants in general, also has an unique set of protein kinases. These include the receptor-like protein kinases and related cytoplasmic protein kinases, the calcium-dependent protein kinases and several members of the putative mitogen-activated protein kinase kinase kinases (Wang et al., 2003). The Arabidopsis protein phos-phatase catalytic subunits encompass orthologs of the majority of the protein phosphatases found in other eukaryotes. However, the type 2C protein phosphatase family is notably large in number in Arabidopsis (Kerk et al., 2002). The distinct representation of genes encoding protein kinases and phosphatases in the Arabidopsis genome, relative to other eukaryotes, is a reflection of the evolutionary history of plants. The understanding that plants have developed cellular communication systems and basic developmental mechanisms independently from other multicellular eukaryotes (Meyerowitz, 2002) explains why plants have evolved a unique collection of enzymes that regulate protein phopshorylation. Indeed, we pointed out over a decade ago (Stone and Walker, 1995), before the exceptionality of the Arabidopsis kinome was fully appreciated, that plants have an unique repertoire of protein kinases that control the early steps in signaling pathways which is reflective of the unique developmental and environmental responses that govern plant growth and development. Table 1. Overview of protein kinases and phosphatases in Arabidopsis. Table 1 (continued). Overview of protein kinases and phosphatases in Arabidopsis. Progress in understanding the role of protein phosphorylation in plant development and environmental responses has made some significant steps in the past few years. While much of the research is focused on Arabidopsis, important insights are also being made in other plant species. Indeed, as genomic and functional data becomes more complete for other plant species, we should be better equipped to answer questions about the fundamental mechanisms plants employ to control their growth, development and responses to environmental stimuli and the role that protein phosphorylation plays in these processes. This chapter on the protein phosphatases and protein kinases of Arabidopsis takes a gene-centric approach to summarize our current understanding of the functional roles of these important mediators of cellular processes. We have tried to focus on the unique aspects of protein kinases and phosphatases.

DAWDLE, a forkhead-associated domain gene, regulates multiple aspects of plant development.

Phosphoprotein-binding domains are found in many different proteins and specify protein-protein interactions critical for signal transduction pathways. Forkhead-associated (FHA) domains bind phosphothreonine and control many aspects of cell proliferation in yeast (Saccharomyces cerevisiae) and animal cells. The Arabidopsis (Arabidopsis thaliana) protein kinase-associated protein phosphatase includes a FHA domain that mediates interactions with receptor-like kinases, which in turn regulate a variety of signaling pathways involved in plant growth and pathogen responses. Screens for insertional mutations in other Arabidopsis FHA domain-containing genes identified a mutant with pleiotropic defects. dawdle (ddl) plants are developmentally delayed, produce defective roots, shoots, and flowers, and have reduced seed set. DDL is expressed in the root and shoot meristems and the reduced size of the root apical meristem in ddl plants suggests a role early in organ development.

Fiber loop ringdown DNA and bacteria sensors

We report a new type of refractive index-based biosensor using a fiber loop ringdown evanescent field (FLRD-EF) sensing scheme, in which the sensing signal is a time constant and detection sensitivity is enhanced by the multipass nature of the ringdown technique. Bulk index-based detections of three different single strand DNAs and one type of bacteria are demonstrated for the FLRD-EF sensors that utilize a partially-etched single mode fiber as the sensor head. Stepwise coating of the sensor head with poly-L-lysine and a probe DNA has enabled surface index-based label-free target DNA sensing. We expect an array of FLRD-EF biosensors to be created, which are superior to counterparts in terms of simplicity, low cost, and high sensitivity.

DVL genes play a role in the coordination of socket cell recruitment and differentiation

Specialized plant cells arise from undifferentiated cells through a series of developmental steps. The decision to enter into a certain differentiation pathway depends in many cases on signals from neighbouring cells. The ability of cells to engage in short-range intercellular communication permits the coordination of cell actions necessary in many developmental processes. Overexpression of genes from the DEVIL/ROTUNDIFOLIA (DVL/ROT) family results in severe developmental alterations, but very little is known about their mechanism of action. This work presents evidence that suggests a role for these genes in local signalling, specifically in the coordination of socket cell recruitment and differentiation. Overexpression of different DVL genes results in protuberances at the base of the trichomes surrounded by several rows of elongated epidermal cells, morphologically similar to socket cells. Localized overexpression of DVL4 in trichomes and socket cells during early developmental stages activates expression of socket cell markers in additional cells, farther away from the trichome. The same phenomenon is observed in an activation tagged line of DVL1, which also shows an increase in the number of socket cells in contact with the trichome. The roles of individual DVL genes have been difficult to discover since their overexpression phenotypes are quite similar. In gl1 leaves that lack trichomes and socket cells DVL1 expression shows a 69% reduction, suggesting that this gene could be involved in the coordination of socket cell development in wild-type plants.