The MKK7-MPK6 MAP Kinase Module Is a Regulator of Meristem Quiescence or Active Growth in Arabidopsis.

Plant growth flexibly adapts to environmental conditions. Growth initiation itself may be conditional to a suitable environment, while the most common response of plants to adverse conditions is growth inhibition. Most of our understanding about environmental growth inhibition comes from studies on various plant hormones, while less is known about the signaling mechanisms involved. The mitogen-activated protein kinase (MAPK) cascades are central signal transduction pathways in all eukaryotes and their roles in plant stress responses is well-established, while increasing evidence points to their involvement in hormonal and developmental processes. Here we show that the MKK7-MPK6 module is a suppressor of meristem activity using genetic approaches. Shoot apical meristem activation during light-induced de-etiolation is accelerated in mpk6 and mkk7 seedlings, whereas constitutive or induced overexpression of MKK7 results in meristem defects or collapse, both in the shoot and the root apical meristems. These results underscore the role of stress-activated MAPK signaling in regulating growth responses at the whole plant level, which may be an important regulatory mechanism underlying the environmental plasticity of plant development.

Coevolving MAPK and PID phosphosites indicate an ancient environmental control of PIN auxin transporters in land plants.

Plant growth flexibly adapts to environmental conditions, implying cross-talk between environmental signalling and developmental regulation. Here, we show that the PIN auxin efflux carrier family possesses three highly conserved putative mitogen-activated protein kinase (MAPK) sites adjacent to the phosphorylation sites of the well-characterised AGC kinase PINOID, which regulates the polar localisation of PINs and directional auxin transport, thereby underpinning organ growth. The conserved sites of PIN1 are phosphorylated in vitro by two environmentally activated MAPKs, MPK4 and MPK6. In contrast to AGC kinases, MAPK-mediated phosphorylation of PIN1 at adjacent sites leads to a partial loss of the plasma membrane localisation of PIN1. MAPK-mediated modulation of PIN trafficking may participate in environmental adjustment of plant growth.

The DSPII splice variant is crucial for desmosome-mediated adhesion in HaCaT keratinocytes.

Desmosomes are intercellular junctions specialised for strong adhesion that are prominent in the epidermis and heart muscle. Defective desmosomal function due to inherited mutations in the constitutive desmosomal gene desmoplakin (DSP) causes skin or heart disorders and in some instances both. Different mutations have different disease-causing molecular mechanisms as evidenced by the varying phenotypes resulting from mutations affecting different domains of the same protein, but the majority of these mechanisms remain to be determined. Here, we studied two mutations in DSP that lead to different dosages of the two major DSP splice variants, DSPI and DSPII, and compared their molecular mechanisms. One of the mutations results in total DSP haploinsufficiency and is associated with autosomal dominant striate palmoplantar keratoderma (PPK). The other leads to complete absence of DSPI and the minor isoform DSPIa but normal levels of DSPII, and is associated with autosomal recessive epidermolytic PPK, woolly hair and severe arrhythmogenic dilated cardiomyopathy. Using siRNA treatments to mimic these two mutations and additionally a DSPII-specific siRNA, we found striking differences between DSP isoforms with respect to keratinocyte adhesion upon cellular stress with DSPII being the key component in intermediate filament (IF) stability and desmosome-mediated adhesion. In addition, reduction in DSP expression reduced the amount of plakophilin 1, desmocollin (DSC) 2 and DSC3 with DSPI having a greater influence than DSPII on the expression levels of DSC3. These results suggest that the two major DSP splice variants are not completely redundant in function and that DSPII dosage is particularly important for desmosomal adhesion in the skin.

Mitogen-activated protein kinase activity and reporter gene assays in plants.

Mitogen-activated protein (MAP) kinase pathways are conserved in eukaryotes and transmit a plethora of stimuli. MAP kinases (MAPKs) are part of signalling modules that consist of three to four tiers of protein kinases in a phosphorylation cascade. MAPKs are known to phosphorylate specific substrates at specific sites at a -threonine or serine residue followed by proline, but the surrounding amino acids of the phosphorylation site and docking interactions are also important for substrate recognition. MAPK activity can be assayed by detecting their phosphotransferase activity, their activation state, or detecting the switching on or off reaction of specific genes, or cellular responses. Prior to the kinase assay, specific MAPK proteins can be immunoprecipitated either by MAPK-specific antibodies or by the introduction of C-terminal epitope tags and expression of the fusion proteins in planta or transiently in protoplasts. Protoplasts derived from Arabidopsis thaliana cell cultures or leaves provide a valuable tool to co-express multiple gene constructs, thus in this system MAPKs can be co-expressed with upstream regulatory components or downstream targets. In protoplasts, the signalling activity through MAPK pathways can also be monitored by -co-transforming reporter genes fused to target promoters. Furthermore, components of the MAPK -signalling pathways can be silenced by co-transformation of RNAi or amiRNA constructs, and the impact of silencing on MAPK activation or gene expression can thus be determined.

Primary cilium-dependent and -independent Hedgehog signaling inhibits p16(INK4A).

In a genome-wide siRNA analysis of p16(INK4a) (p16) modulators, we identify the Hedgehog (Hh) pathway component SUFU and formally demonstrate that Hh signaling promotes mitogenesis by suppression of p16. A fragment of the Hh-responsive GLI2 transcription factor directly binds and inhibits the p16 promoter and senescence is associated with the loss of nuclear GLI2. Hh components partially reside in the primary cilium (PC), and the small fraction of cells in mass culture that elaborate a PC have the lowest expression of p16. Suppression of p16 is effected by both PC-dependent and -independent routes, and ablation of p16 renders cells insensitive to an Hh inhibitor and increases PC formation. These results directly link a well-established developmental mitogenic pathway with a key tumor suppressor and contribute to the molecular understanding of replicative senescence, Hh-mediated oncogenesis, and potentially the role of p16 in aging.

The Arabidopsis MAP kinase kinase MKK1 participates in defence responses to the bacterial elicitor flagellin.

Plants sense pathogens through both pathogen-associated molecular patterns and recognition of race-specific virulence factors, which induce basal defence or an accelerated defence (often manifest in the form of local cell death), respectively. A mitogen-activated protein kinase (MAPK) module in Arabidopsis was previously proposed to signal from perception of the bacterial elicitor flagellin to the activation of basal defence-related genes. Here, we present evidence for a parallel MAPK-signalling pathway involved in the response to flg22, a peptide corresponding to the most conserved domain of flagellin. The endogenous Arabidopsis MAP kinase kinase MKK1 is activated in cells treated with flg22, phosphorylates the MAPK MPK4 in vitro, and activates it in vivo in protoplasts. In mkk1 mutant plants, the activation by flg22 of MPK4 and two other flg22-induced MAPKs (MPK3 and MPK6) is impaired. In the mkk1 mutant, a battery of both flg22-induced and flg22-repressed genes show altered expression, indicating that MKK1 negatively regulates the activity of flagellin-responsive genes. Intriguingly, in contrast to the mpk4 mutant, mkk1 shows no morphological anomalies and is compromised in resistance to both virulent and avirulent Pseudomonas syringae strains. Thus, the MKK1 signalling pathway modulates the expression of genes responding to elicitors and plays an important role in pathogen defence.