The Plant Transcription Factor Database (PlnTFDB; http://plntfdb.bio.uni-potsdam.de/v3.0/) is an integrative database that provides putatively complete sets of transcription factors (TFs) and other transcriptional regulators (TRs) in plant species (sensu lato) whose genomes have been completely sequenced and annotated. The complete sets of 84 families of TFs and TRs from 19 species ranging from unicellular red and green algae to angiosperms are included in PlnTFDB, representing >1.6 billion years of evolution of gene regulatory networks. For each gene family, a basic description is provided that is complemented by literature references, and multiple sequence alignments of protein domains. TF or TR gene entries include information of expressed sequence tags, 3D protein structures of homologous proteins, domain architecture and cross-links to other computational resources online. Moreover, the different species in PlnTFDB are linked to each other by means of orthologous genes facilitating cross-species comparisons.

The PhosPhAt database of Arabidopsis phosphorylation sites was initially launched in August 2007. Since then, along with 10-fold increase in database entries, functionality of PhosPhAt (phosphat.mpimp-golm.mpg.de) has been considerably upgraded and re-designed. PhosPhAt is now more of a web application with the inclusion of advanced search functions allowing combinatorial searches by Boolean terms. The results output now includes interactive visualization of annotated fragmentation spectra and the ability to export spectra and peptide sequences as text files for use in other applications. We have also implemented dynamic links to other web resources thus augmenting PhosPhAt-specific information with external protein-related data. For experimental phosphorylation sites with information about dynamic behavior in response to external stimuli, we display simple time-resolved diagrams. We have included predictions for pT and pY sites and updated pS predictions. Access to prediction algorithm now allows ‘on-the-fly’ prediction of phosphorylation of any user-uploaded protein sequence. Protein Pfam domain structures are now mapped onto the protein sequence display next to experimental and predicted phosphorylation sites. Finally, we have implemented functional annotation of proteins using MAPMAN ontology. These new developments make the PhosPhAt resource a useful and powerful tool for the scientific community as a whole beyond the plant sciences.

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Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogenactivated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance.

The oomycete Phytophthora infestans causes late blight, the most relevant disease of potato (Solanum tuberosum) worldwide. Field resistance to late blight is a complex trait. When potatoes are cultivated under long day conditions in temperate climates, this resistance is correlated with late plant maturity, an undesirable characteristic. Identification of natural gene variation underlying late blight resistance not compromised by late maturity will facilitate the selection of resistant cultivars and give new insight in the mechanisms controlling quantitative pathogen resistance. We tested 24 candidate loci for association with field resistance to late blight and plant maturity in a population of 184 tetraploid potato individuals. The individuals were genotyped for 230 single nucleotide polymorphisms (SNPs) and 166 microsatellite alleles. For association analysis we used a mixed model, taking into account population structure, kinship, allele substitution and interaction effects of the marker alleles at a locus with four allele doses. Nine SNPs were associated with maturity corrected resistance (P , 0.001), which collectively explained 50% of the genetic variance of this trait. A major association was found at the StAOS2 locus encoding allene oxide synthase 2, a key enzyme in the biosynthesis of jasmonates, plant hormones that function in defense signaling. This finding supports StAOS2 as being one of the factors controlling natural variation of pathogen resistance.

Phosphoproteomics involves identification of phosphoproteins, precise mapping, and quantification of phosphorylation sites, and eventually, revealing their biological function. In plants, several systematic phosphoproteomic analyses have recently been performed to optimize in vitro and in vivo technologies to reveal components of the phosphoproteome. The discovery of novel substrates for specific protein kinases is also an important issue. Development of a new tool has enabled rapid identification of potential kinase substrates such as kinase assays using plant protein microarrays. Progress has also been made in quantitative and dynamic analysis of mapped phosphorylation sites. Increased quantity of experimentally verified phosphorylation sites in plants has prompted the creation of dedicated web-resources for plant-specific phosphoproteomics data. This resulted in development of computational prediction methods yielding significantly improved sensitivity and specificity for the detection of phosphorylation sites in plants when compared to methods trained on less plant-specific data. In this review, we present an update on phosphoproteomic studies in plants and summarize the recent progress in the computational prediction of plant phosphorylation sites. The application of the experimental and computed results in understanding the phosphoproteomic networks of cellular and metabolic processes in plants is discussed. This is a continuation of our comprehensive review series on plant phosphoproteomics.

Phosphoproteomics can be defined as the comprehensive study of protein phosphorylation by identification of the phosphoproteins, exact mapping of the phosphorylation sites, quantification of phosphorylation, and eventually, revealing their biological function. Its place in today's research is vitally important to address the most fundamental question - how the phosphorylation events control most, if not all, of the cellular processes in a given organism? Despite the immense importance of phosphorylation, the analysis of phosphoproteins on a proteome-wide scale remains a formidable challenge. Nevertheless, several technologies have been developed, mostly in yeast and mammals, to conduct a large-scale phosphoproteomic study. Some of these technologies have been successfully applied to plants with a few modifications, resulting in documentation of phosphoproteins, phosphorylation site mapping, identification of protein kinase substrates, etc. at the global level. In this review, we summarize in vitro and in vivo approaches for detection and analysis of phosphoproteins including protein kinases and we discuss the importance of phosphoproteomics in understanding plant biology. These approaches along with bioinformatics will help plant researchers to design and apply suitable phosphoproteomic strategies in helping to find answers to their biological questions.

In the dioecious genus Populus, sex determination has been located to chromosome 19. However, despite a high degree of genome collinearity, various Populus species seem to differ with regard to the location of the sex-determining region on the respective chromosome and the apparent heterogametic sex. In this study, the boundaries of the recombination-suppressed, sex-linked region of the male P. tremuloides clone Turesson 141 were localised by genetic mapping using new SNP and InDel markers. The respective region seems to be located in a pericentromeric position. The corresponding P. trichocarpa genome region spans about two million bp and comprises 65 gene loci, which were bioinformatically evaluated for their potential as candidate genes for sex determination. Three putative transcription factor genes and four genes that are potentially involved in flower development processes, e.g. meristem transition from the vegetative to the reproductive phase, were identified. Populus tremuloides sequence data of the sex-linked region is required for a final search for candidate genes.