Specificity and functional analysis of a PP2C protein phosphatase gene subfamily

Protein phosphatases and kinases are key regulatory enzymes in all eukaryotic cells. These enzymes have been characterised in yeast and animal cells as important components of signal transduction pathways involved in growth control and stress responses. Previously we have identified a Medicago PP2C-type protein phosphatase, (Medicago P2C) MP2C, as a negative regulator of a stress induced MAPK (mitogen activated protein kinase) pathway. We identified a specific substrate for this PP2C as being a MAPK, namely SIMK. SIMK, but not other MAPKs that we tested is dephosphorylated and inactivated by MP2C. To better understand the role of this phosphatase, we cloned a putative MP2C homolog from the model plant Arabidopsis and identified its substrate AtMPK6 (a homolog of SIMK). AP2C and MP2C contains a "kinase interaction motif "(KIM) at their N-terminus. KIM domain containing proteins interact with MAPKs in different organisms. We showed by domain swapping and site-directed mutagenesis that this domain is required for MAPK binding and inactivation. In Arabidopsis there are four genes closely resembling MP2C and thus forming a subfamily within the large number of PP2C genes. All of these PP2C proteins contain a KIM domain. We assume that these PP2Cs are involved in MAPK inactivation. The aims of the present grant proposal are to investigate the physiological role of these Arabidopsis PP2C genes using reverse genetics, to identify which of the MAPK pathways might be targeted by various PP2C proteins. To answer this questions, we suggest comparing the interaction and inactivation of MAPKs with these four KIM- domain-containing PP2Cs; to analyse the phenotype, and MAPK activities in PP2C insertional knock out mutants, and to search for further PP2C interacting partners, which might play important roles in determining specificity. An essential feature of signaling pathways is their transient activation, which is also important for their specificity. Therefore understanding the inactivation mechanism of MAPKs is a timely goal.

Stress in plants is communicated by signalling pathways that involve protein phosphorylation. Protein phosphatases can regulate this process but their functions are largely unstudied. Our work was focused on novel phosphatases (AP2Cs) from the model plant Arabidopsis seeking to uncover their functions. This task was successfully accomplished leading to important discoveries and making a substantial impact to the field of signal transduction research. Several essential biological processes were found to be regulated by two PP2C-type phosphatases: the plant response to pathogen attack or wounding; production of plant stress hormone jasmonate and resistance to spider mites being regulated by the first one, a stress-induced phosphatase; whereas the initiation of specific cells, called stomata that are localised on plant surfaces and are responsible for plant gas exchange, by the second one. Using bioinformatics analysis, molecular, cell biology, biochemical and approaches involving yeast cells we characterized the wound- and pathogen-induced PP2C-type phosphatase, AP2C1 as a key stress signalling component, which controls activation of stress-kinases (the proteins performing protein phosphorylation) in plants. We uncovered the pathway and build up the model, where AP2C1 interacts with/inactivates these kinases and that results in control of wound-produced gas ethylene in leaves and plant innate immunity against the necrotrophic fungus Botrytis. Moreover, we discovered that AP2C1 regulates the amounts of the important stress defence hormone jasmonate and controls plant herbivore-related defence. Thereby plants without the function of AP2C1 gene had higher jasmonate levels after wounding and were more resistant to herbivores. However, these plants had a wild type plant appearance, underlining that AP2C1 regulates processes that are induced only upon stress application and are not affecting plant development in normal growth conditions. This is very interesting finding that can be applied for study of plant protection against the herbivores. Another phosphatase was found to control different signalling pathways that are related to cell developmental decisions. Taken together, we uncovered a key role for the phosphatase AP2C1 as a novel regulator of stress/defence responses in plants and described a new link between wound-induced phosphorylation and jasmonate production in plants. Our findings highlight conserved mechanism and components in regulation of stress signal transduction and cell development decisions in plants and animals and prop up for more studies focused on regulation of signalling cascades by PP2Cs.