Activation of AtMPK9 through autophosphorylation that makes it independent of the canonical MAPK cascades.

Mitogen activated protein kinases (MAPKs) are part of conserved signal transduction modules in eukaryotes that are typically organised into three-tiered kinase cascades. The activation of MAPKs in these pathways is fully dependent on the bisphosphorylation of the TXY motif in the T-loop by the pertinent dual-specificity MAPK kinases (MAPKKs). The plant AtMPK9 is a member of an atypical class of MAPKs. Representatives of this MAPK family have TDY phosphoacceptor site, a long C-terminal extension, and lack the common MAPKK binding docking motif. Here, we present multiple in vitro and in vivo data that AtMPK9 is activated independently of any upstream MAPKKs but it is activated through autophosphorylation. We mapped the autophosphorylation sites by mass spectrometry to the TDY motif and to the C-terminal regulatory extension. We mutated the phosphoacceptor sites on the TDY, which confirmed the requirement for bisphorylation at this site for full kinase activity. Next, we demonstrated that the kinase inactive mutant form of AtMPK9 is not transphosphorylated on the TDY site when mixed with an active AtMPK9, implying that the mechanism of the autocatalytic phosphorylation is intramolecular. Furthermore, we show that in vivo AtMPK9 is activated by salt and is regulated by okadaic acid-sensitive phosphatases. We conclude that the plant AtMPK9 shows similarities to the mammalian atypical MAPKs, ERK7/8 in terms of MAPKK-independent activation mechanism

Genomic organization and evolutionary conservation of plant D-type cyclins

Plants contain more genes encoding core cell cycle regulators than other organisms but it is unclear whether these represent distinct functions. D-type cyclins (CYCD) play key roles in the G1-to-S-phase transition, and Arabidopsis (Arabidopsis thaliana) contains 10 CYCD genes in seven defined subgroups, six of which are conserved in rice (Oryza sativa). Here, we identify 22 CYCD genes in the poplar (Populus trichocarpa) genome and confirm that these six CYCD subgroups are conserved across higher plants, suggesting subgroup-specific functions. Different subgroups show gene number increases, with CYCD3 having three members in Arabidopsis, six in poplar, and a single representative in rice. All three species contain a single CYCD7 gene. Despite low overall sequence homology, we find remarkable conservation of intron/exon boundaries, because in most CYCD genes of plants and mammals, the first exon ends in the conserved cyclin signature. Only CYCD3 genes contain the complete cyclin box in a single exon, and this structure is conserved across angiosperms, again suggesting an early origin for the subgroup. The single CYCD gene of moss has a gene structure closely related to those of higher plants, sharing an identical exon/intron structure with several higher plant subgroups. However, green algae have CYCD genes structurally unrelated to higher plants. Conservation is also observed in the location of potential cyclin-dependent kinase phosphorylation sites within CYCD proteins. Subgroup structure is supported by conserved regulatory elements, particularly in the eudicot species, including conserved E2F regulatory sites within CYCD3 promoters. Global expression correlation analysis further supports distinct expression patterns for CYCD subgroups

Expression of a nondegradable cyclin B1 affects plant development and leads to endomitosis by inhibiting the formation of a phragmoplast

In plants after the disassembly of mitotic spindle, a specific cytokinetic structure called the phragmoplast is built, and after cytokinesis, microtubules populate the cell cortex in an organized orientation that determines cell elongation and shape. Here, we show that impaired cyclin B1 degradation, resulting from a mutation within its destruction box, leads to an isodiametric shape of epidermal cells in leaves, stems, and roots and retarded growth of seedlings. Microtubules in these misshaped cells are grossly disorganized, focused around the nucleus, whereas they were entirely missing or abnormally organized along the cell cortex. A high percentage of cells expressing nondestructible cyclin B1 had doubled DNA content as a result of undergoing endomitosis. During anaphase the cytokinesis-specific syntaxin KNOLLE could still localize to the midplane of cell division, whereas NPK1-activating kinesin-like protein 1, a cytokinetic kinesin-related protein, was unable to do so, and instead of the formation of a phragmoplast, the midzone microtubules persisted between the separated nuclei, which eventually fused. In summary, our results show that the timely degradation of mitotic cyclins in plants is required for the reorganization of mitotic microtubules to the phragmoplast and for proper cytokinesis. Subsequently, the presence of nondegradable cyclin B1 leads to a failure in organizing properly the cortical microtubules that determine cell elongation and shape

The kinome of Phytophthora infestans reveals oomycete-specific innovations and links to other taxonomic groups

<p>Abstract</p> <p>Background</p> <p>Oomycetes are a large group of economically and ecologically important species. Its most notorious member is <it>Phytophthora infestans</it>, the cause of the devastating potato late blight disease. The life cycle of <it>P. infestans </it>involves hyphae which differentiate into spores used for dispersal and host infection. Protein phosphorylation likely plays crucial roles in these stages, and to help understand this we present here a genome-wide analysis of the protein kinases of <it>P. infestans </it>and several relatives. The study also provides new insight into kinase evolution since oomycetes are taxonomically distant from organisms with well-characterized kinomes.</p> <p>Results</p> <p>Bioinformatic searches of the genomes of <it>P. infestans</it>, <it>P. ramorum</it>, and <it>P. sojae </it>reveal they have similar kinomes, which for <it>P. infestans </it>contains 354 eukaryotic protein kinases (ePKs) and 18 atypical kinases (aPKs), equaling 2% of total genes. After refining gene models, most were classifiable into families seen in other eukaryotes. Some ePK families are nevertheless unusual, especially the tyrosine kinase-like (TKL) group which includes large oomycete-specific subfamilies. Also identified were two tyrosine kinases, which are rare in non-metazoans. Several ePKs bear accessory domains not identified previously on kinases, such as cyclin-dependent kinases with integral cyclin domains. Most ePKs lack accessory domains, implying that many are regulated transcriptionally. This was confirmed by mRNA expression-profiling studies that showed that two-thirds vary significantly between hyphae, sporangia, and zoospores. Comparisons to neighboring taxa (apicomplexans, ciliates, diatoms) revealed both clade-specific and conserved features, and multiple connections to plant kinases were observed. The kinome of <it>Hyaloperonospora arabidopsidis</it>, an oomycete with a simpler life cycle than <it>P. infestans</it>, was found to be one-third smaller. Some differences may be attributable to gene clustering, which facilitates subfamily expansion (or loss) through unequal crossing-over.</p> <p>Conclusion</p> <p>The large sizes of the <it>Phytophthora </it>kinomes imply that phosphorylation plays major roles in their life cycles. Their kinomes also include many novel ePKs, some specific to oomycetes or shared with neighboring groups. Little experimentation to date has addressed the biological functions of oomycete kinases, but this should be stimulated by the structural, evolutionary, and expression data presented here. This may lead to targets for disease control.</p

Learning the lipid language of plant signalling.

Plant cells respond to different biotic and abiotic stresses by producing various uncommon phospholipids that are believed to play key roles in cell signalling. We can predict how they work because animal and yeast proteins have been shown to have specific lipid-binding domains, which act as docking sites. When such proteins are recruited to the membrane locations where these phospholipids are synthesized, the phospholipids activate them directly, by inducing a conformational change, or indirectly, by juxtaposing them with an activator protein. The same lipid-binding domains are present in Arabidopsis proteins. We believe that they represent an untapped well of information about plant lipid signalling

The role of the Arabidopsis E2FB transcription factor in regulating auxin-dependent cell division

The molecular mechanisms by which the phytohormone auxin coordinates cell division with cell growth and differentiation are largely unknown. Here, we show that in Arabidopsis thaliana E2FB, accumulation and stability are positively regulated by auxin. Coexpression of E2FB, but not of E2FA, with its dimerization partner A, stimulated cell proliferation in the absence of auxin in tobacco (Nicotiana tabacum) Bright Yellow-2 cells. E2FB regulated the entry into both S- and M-phases, the latter corresponding to the activation of a plant-specific mitotic regulator, CDKB1;1. Increased E2FB levels led to shortened cell cycle duration, elevated cell numbers, and extremely small cell sizes. In the absence of auxin, cells elongated with concomitant increase in their ploidy level, but both were strongly inhibited by E2FB. We conclude that E2FB is one of the key targets for auxin to determine whether cells proliferate or whether they exit the cell cycle, enlarge, and endoreduplicate their DNA

Gamma-tubulin is essential for acentrosomal microtubule nucleation and coordination of late mitotic events in Arabidopsis

Gamma-tubulin is required for microtubule (MT) nucleation at MT organizing centers such as centrosomes or spindle pole bodies, but little is known about its noncentrosomal functions. We conditionally downregulated gamma-tubulin by inducible expression of RNA interference (RNAi) constructs in Arabidopsis thaliana. Almost complete RNAi depletion of gamma-tubulin led to the absence of MTs and was lethal at the cotyledon stage. After induction of RNAi expression, gamma-tubulin was gradually depleted from both cytoplasmic and microsomal fractions. In RNAi plants with partial loss of gamma-tubulin, MT recovery after drug-induced depolymerization was impaired. Similarly, immunodepletion of gamma-tubulin from Arabidopsis extracts severely compromised in vitro polymerization of MTs. Reduction of gamma-tubulin protein levels led to randomization and bundling of cortical MTs. This finding indicates that MT-bound gamma-tubulin is part of a cortical template guiding the microtubular network and is essential for MT nucleation. Furthermore, we found that cells with decreased levels of gamma-tubulin could progress through mitosis, but cytokinesis was strongly affected. Stepwise diminution of gamma-tubulin allowed us to reveal roles for MT nucleation in plant development, such as organization of cell files, anisotropic and polar tip growth, and stomatal patterning. Some of these functions of gamma-tubulin might be independent of MT nucleation