Plant stress signalling via two MYB transcription factors

Mitogen-activated protein kinase (MAPK) cascades are signalling modules that are highly conserved in eukaryotic organisms. They minimally consist of a MAPK kinase kinase (MAPKKK), a MAPKK and a MAPK, which via a phosphorelay mechanism, amplify developmentally regulated or environmental signals and pass them on to MAPK-phosphorylated proteins, ultimating in an appropriate response. Several studies on MAPK(KK)-deficient mutant plants have emphasised the importance of MAPK cascades in the response to biotic and abiotic stresses. However, little is known about the molecular mechanisms and components through which stress-activated MAPK cascades translate incoming stress signals into appropriate defence and adaptation responses. Basis: We have recently isolated VIP1, a plant-specific bZIP protein, as an interacting protein of the Arabiodpsis thaliana MAPK MPK3 (Djamei, Pitzschke et al., 2007). Upon phosphorylation by MPK3, which is activated by a number of biotic and abiotic stresses, VIP1 translocates from the cytoplasm to the nucleus. Using a random DNA selection assay, a novel DNA motif bound by VIP1 was isolated. This motif is overrepresented in the promoter regions of early stress-responsive genes. Preliminary data strongly suggests two highly homologous transcription factors, MYB44 and MYB77, to be directly transcriptionally regulated by VIP1. In addition, MYB44 was isolated from a Yeast-Two-Hybrid screen for interaction partners of MKK4, the upstream regulating MAPKK of MPK3/MPK6; and MYB44 and MYB77 were found to be phosphorylated MPK3/MPK6 in vitro. mpk3 mutant plants are impaired in the stress-induced expression of MYB44/MYB77. Project: In the anticipated project the - apparently sophisticated - feedback regulation of MYB44/MYB77 in MAPK- mediated stress signalling shall be studied. To identify the genes and processes potentially regulated by these MYB transcription factors, microarray studies will be performed. Rapid accumulation of MYB44/MYB77 in the nucleus will be triggered by dexamethasone- treatment of transgenic plants constitutively expressing MYB44/MYB77-Glucocorticoid fusion proteins. It will be resolved whether MYB44/MYB77 target genes are stress-responsive, and whether their transcriptional control is regulated through a MAPK cascade involving MPK3/MPK6. The MAPK-regulated phosphorylation sites of MYB44/MYB77 will be mapped by mass spectrometry analysis of transgenic plants. Using a transient protoplast expression system and transgenic plants the potential impact of phosphorylation on the localisation and/or transcriptional regulatory capacity of MYB44/MYB77 will be assessed. Moreover, plants overexpressing MYB44/MYB77 derivatives mutated at the identified phosphosite(s) will be subjected to stress tolerance tests. Yeast-three-hybrid experiments shall resolve a potential role of MYB44/MYB77 as scaffolding protein in a ternary [MKK4-MPK3/MPK6-MYB44/MYB77] complex. A potential feedback regulation of MYB44/77 on MPK3/MPK6 activity will be investigated.

In this project we studied mechanisms that enable plants to withstand environmental stresses. Stress, such as drought and high salinity, activates MPK3 within minutes. MPK3 in turn modifies MYB44. MYB44 can then activate stress response genes. Plants in which this flow of information is disturbed are more susceptible to stress. In contrast, more rapid signaling enhances stress tolerance.In any living organism, gene expression is regulated by transcription factors. These proteins determine which gene is active in which cell and under which condition. If all genes in all cells were equally active, distinct organs such as roots and leaves would never be formed. The same applies to humans. Our kidney and skin cells contain the same DNA, but differential gene activation allows specificationAnimals and plants respond to environmental signals by altering protein activities and by activating or repressing appropriate genes. Often, stress responses have to happen rapidly to build an effective protection. For instance, a sudden cold snap has to be dealt with immediately, otherwise it will be fatal. In the model plant Arabidopsis, a protein called MPK3 is activated early upon stress. It is pivotal to numerous stress adaptation responses. These include e.g. the closure of stomates (leaf pores through which plants breathe) under drought stress and the production of antimicrobial substances upon bacterial attack. MPK3 is a kinase, not a transcription factor. How can it yet control gene expression?We have identified a transcription factor MYB44, which is a direct target of MPK3. Stress-activated MPK3 transfers a phosphate group onto MYB44. MYB44 subsequently induces genes required for stress tolerance. The modification of MYB44 by MPK3 is highly specific, since only one out of five theoretically possible positions accepts the phosphate group. Manipulation of this position renders MYB44 inactive. High-salinity stress is not an artificial stress condition. In fact, it reflects the effects of climate change. Drought and heat are the main reason for the increasing desertification and salinization of land.MPK3 and MYB44 deficiency severely compromises the performance of plants under high-salinity stress. In contrast, plants that overproduce MYB44 are more tolerant. This elevated tolerance strictly relies on MPK-mediated activation.MYB44 and MPK3 are interaction partners and form complexes in the nucleus, which is where gene activation occurs. If we turn the transcriptional activator into a transcriptional repressor (by adding a small motif at the end of the protein), MYB44 fails to induce appropriate stress responses. Plants containing such manipulated MYB44 protein perform very poorly under drought and salt stress. From this observation we learnt that MYB44 is a transcriptional activator, not a repressor.