Final Report Summary - MAPKINDROUGHT (MAPK signalling network to adapt leaf growth to drought conditions)
Summary description of the MAPKinDROUGHT project objectives
Drought stress signal is sensed either directly, e.g. in root cells, or indirectly through systemic spread of the signal by hormones and other messenger molecules, and the information is processed through complex signaling pathways to elicit growth adaptation responses. MAPK signalling pathways, a fundamental circuitry of stress signalling, constitute a hierarchical three-tiers system of protein kinases: MEKK, MEK and the MAPK, which phosphorylate specific substrate proteins, thereby changing cellular behavior. In an activation screening for stress-regulating processes, we found MKK7 and MKK9 and two downstream MAPKs, MPK3 and MPK6 to lead to stress adaptation and to regulate plant growth. The overall aim of the MAPKinDROUGHT project was to identify the targets of the MKK7/MKK9 – MPK3/MPK6 drought-responsive MAPK signaling pathway, and link these to the growth adaptation of plants under drought stress conditions. To do this we performed: (i) unbiased phosphoproteomics screen for MAPK targets using the inducible MKK7/MKK9 lines, (ii) computational predictions of MAPK substrates involved in drought responses, (iii) targeted approaches to test MAPK phosphorylation of key regulators in plant growth regulation, the RBR transcriptional repressor complex, stress responsive transcription factors including HSFA4A and RAP2s as well as the auxin efflux protein, PIN1 as well as the microtubule-associated proteins, γ-tubulin, EB1 and a 14-3-3 adaptor protein. Detailed phenotypic analysis of gain and loss of function MKK7, MKK9 and MPK3, MPK6 lines under control and drought stress conditions provided links of drought growth adaptation to MAPK signalling pathway. In the course of the project the MC fellow obtained training in interdisciplinary research; proteomics, computational biology, and gained transferable skills to become an all rounded scientist.
Description of the work performed in the MAPKinDROUGHT project
The work of the MAPKinDROUGHT project was organized around 5 tasks. In task 1 we utilized the lines we have developed whereby the MKK7 and MKK9 were placed under the β-estradiol-inducible promoters and collaborated with a mass spectrometry group expert in phosphoproteomics to conduct an unbiased phosphoproteomics screen for MAPK targets. In Task 2 we followed up on our work of computational predictions of MAPK substrates involved in drought response. We predicted 4 classes of transcription factors as MAPK substrates, and selected 2-3 TFs from each classes for experimental verifications. In task 3 we designed experiments to verify the MAPK substrates by in vitro as well as in vivo phosphorylation experiments. These involved in vitro translation of putative MAPK substrates in wheat germ translation system and phosphorylation bt MKK7/9-activated MPK3/6. We also did co-transformation experiments of MKKs, MPKs and substrates into protoplasts as well as their expression in planta. In Task 4 we mapped the phosphorylation sites on selected MAPK substrates, HSFA4A, PIN1 and S6. In Task 5 we studies the role of this phosphorylation by mutating the sites to non-phosphorylatable alanine or phosphomimic glutamic acid on HSFA4A, PIN1 and S6.
Description of the main results achieved in the MAPKinDROUGHT project
In Task 1 we used N14/15 labelling and tandem MOAC phosphopeptide enrichment in collaboration Dr. Gerold J.M. Beckers (RWTH-Aachen University, Aachen, Germany) to perform quantitative phosphoproteomics with MKK7 and MKK9 inducible overexpression lines. We identified 282 phosphopeptides belonging to 89 phosphoproteins. Among these 109 phopshorylation events were dependent on the β-estradiol-induced MKK7 and MKK9 expression; 48 were MKK7-, and 50 were MKK9-specific, while 11 were common to both MKK7 and MKK9. These phosphoproteins belonged to 41 differentially phosphorylated proteins, of which 25 were MKK7-, 12 were MKK9-dependent while the phosphorylation of 4 depended both on MKK7 and MKK9. Some of the identified phosphoproteins were known MPK6 and MPK3 substartes, such as the PHOS32 or the MAP65-1, showing that our activation-lines and phosphoproteomics protocols are well working. Within this work we also identified some novel leads, such as the MKK7-dependent phosphorylation of S6, which indicate a cross-talk between the MAPK pathway and TOR-S6K pathway in plants.
In task 2 we followed up on our work of computational predictions of MAPK substrates. In collaboration with the Paccanaro lab we extracted the MAPK-core modules (MEK, MAPK) from plant interactome databases (e.g. BioGrid) and constructed the Arabidopsis MAPK signaling network, which revealed to be highly connected and complex compared to yeast and animals. Based on sequence information on the docking interactions we identified a comprehensive list of substrates in Arabidopsis. Not all experimentally identified MAPK substrates however, have recognizable docking sequences. The Paccanaro group developed a machine learning approach that exploited all the experimentally known MAPK substrates in Arabidopsis (around 600) and created classifiers (e.g. accessibility, conservation score), that can be used by a machine learning approach to discriminate areas of the substrate sequences containing docking sites. Applying this classifier we extracted four sequence motifs that are potentially new docking sites. We selected 2-4 proteins from each of the categories of predicted MAPK targets with annotations in drought and abiotic stresses, cell cycle regulation, leaf growth and development for experimental verifications
In Task 3 we experimentally verified MAPK substrates. For proteins (~10) where we predicted new docking motives, we cloned the ORFs to a vector suitable for in vitro translation, produced the substrates as well as the activated MPK3, MPK4 and MPK6 MAPKs in wheat germ extract and carried out in vitro phosphorylations. These included the TSO1-like, CIR1, LHY-like, MYB, Blind-like, ERF-B3, DRE/CRT-binding TF, WKY75, EB1, γ-tubulin, PIN1. We found all of these proteins to get phosphorylated by MPK3, MPK4, MPK6, there were no preference among the MAPKs. For in vivo experiments, we focused on PIN1, HSFA4A, EB1, γ-tubulin. We tagged these proteins and transiently expressed them in protoplasts as well as in transformed Arabidopsis plants. We applied IEF in a capillary column (cIEF) to detect phosphorylation changes. With this technique we could show PIN1 phosphorylation by MPK6. For HSFA4A, EB1, γ-tubulin we looked for band shifts on SDS-PAGE and mapped the phosphorylation by mass spectrometry.
In Task 4 we mapped the phosphorylation sites on selected MAPK substrates, PIN1, HSFA4A, EB1. On PIN1 there are 4 predicted MAPK phosphorylation sites, one of which was experimentally detected in a phosphoproteomics experiment. We mutated all 4 sites in combinations, and proved that only one of this site contributes to MAPK phosphorylation. For HSFA4A and EB1 we mapped the phosphorylation sites by mass spectrometry.
In Task 5 we mutated the MAPK phosphorylation sites on PIN1 and HSFA4A. When these sites were mutated to alanine, we conformed the compromised or lack of phosphorylation in vitro and in vivo. The abiotic-stress-induced expression of HEAT SHOCK PROTEIN17.6A was strongly compromised by the serine-309 to alanine HSFA4A mutant form. For PIN1 we showed that the Ala mutation of the MAPK phosphorylation site (PIN1-A) is less abundant then wild type (opposite stability than with the phosphomimic glutamic acid mutation PIN1-E) and is resistant to MKK7 (neither stabilisation nor aggregate formation). The PIN1-A mutant is cytoplasmic and does not co-localise with the Golgi marker, and the ER co-localisation is also not prominent. We also had a weak, partly cytoplasmic signal in roots upon U0126 treatment. So it seems that phosphorylation plays a role in the polar membrane localisation and protein stability of PIN1.
Final results in the MAPKinDROUGHT project and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far)
Better understanding how plant growth is in tune with limiting environment is fundamental in efforts to improve stress tolerance. Suboptimal environmental conditions, among which water availability is in no doubt already the largest factor, limit crop yields to only 30% of the intrinsic capability of the plant, even under modern Western agricultural conditions. The overall aim of the proposed research is to identify the targets of the MKK7/MKK9 – MPK3/MPK6 drought-responsive MAPK signaling pathway, and link these to the growth adaptation of plants under drought stress conditions. To do this we performed: (i) unbiased phosphoproteomics screen for MAPK targets using the inducible MKK7/MKK9 lines, (ii) computational predictions of MAPK substrates involved in drought responses, (iii) targeted approaches to test MAPK phosphorylation of key regulators in plant growth regulation, including the RBR transcriptional repressor complex, the transcriptional regulators; HSFA4A and RAP2s, the auxin efflux protein, PIN1 as well as the microtubule-associated proteins, γ-tubulin, EB1 and a 14-3-3 adaptor protein. In summary, the MAPKinDROUGHT project identified novel targets of the MKK7 and MKK9 pathways that regulate meristematic growth and cell proliferation. Modulating these signalling pathways could help to obtain crops that better adapt their growth to limiting environmental conditions.
Drought stress signal is sensed either directly, e.g. in root cells, or indirectly through systemic spread of the signal by hormones and other messenger molecules, and the information is processed through complex signaling pathways to elicit growth adaptation responses. MAPK signalling pathways, a fundamental circuitry of stress signalling, constitute a hierarchical three-tiers system of protein kinases: MEKK, MEK and the MAPK, which phosphorylate specific substrate proteins, thereby changing cellular behavior. In an activation screening for stress-regulating processes, we found MKK7 and MKK9 and two downstream MAPKs, MPK3 and MPK6 to lead to stress adaptation and to regulate plant growth. The overall aim of the MAPKinDROUGHT project was to identify the targets of the MKK7/MKK9 – MPK3/MPK6 drought-responsive MAPK signaling pathway, and link these to the growth adaptation of plants under drought stress conditions. To do this we performed: (i) unbiased phosphoproteomics screen for MAPK targets using the inducible MKK7/MKK9 lines, (ii) computational predictions of MAPK substrates involved in drought responses, (iii) targeted approaches to test MAPK phosphorylation of key regulators in plant growth regulation, the RBR transcriptional repressor complex, stress responsive transcription factors including HSFA4A and RAP2s as well as the auxin efflux protein, PIN1 as well as the microtubule-associated proteins, γ-tubulin, EB1 and a 14-3-3 adaptor protein. Detailed phenotypic analysis of gain and loss of function MKK7, MKK9 and MPK3, MPK6 lines under control and drought stress conditions provided links of drought growth adaptation to MAPK signalling pathway. In the course of the project the MC fellow obtained training in interdisciplinary research; proteomics, computational biology, and gained transferable skills to become an all rounded scientist.
Description of the work performed in the MAPKinDROUGHT project
The work of the MAPKinDROUGHT project was organized around 5 tasks. In task 1 we utilized the lines we have developed whereby the MKK7 and MKK9 were placed under the β-estradiol-inducible promoters and collaborated with a mass spectrometry group expert in phosphoproteomics to conduct an unbiased phosphoproteomics screen for MAPK targets. In Task 2 we followed up on our work of computational predictions of MAPK substrates involved in drought response. We predicted 4 classes of transcription factors as MAPK substrates, and selected 2-3 TFs from each classes for experimental verifications. In task 3 we designed experiments to verify the MAPK substrates by in vitro as well as in vivo phosphorylation experiments. These involved in vitro translation of putative MAPK substrates in wheat germ translation system and phosphorylation bt MKK7/9-activated MPK3/6. We also did co-transformation experiments of MKKs, MPKs and substrates into protoplasts as well as their expression in planta. In Task 4 we mapped the phosphorylation sites on selected MAPK substrates, HSFA4A, PIN1 and S6. In Task 5 we studies the role of this phosphorylation by mutating the sites to non-phosphorylatable alanine or phosphomimic glutamic acid on HSFA4A, PIN1 and S6.
Description of the main results achieved in the MAPKinDROUGHT project
In Task 1 we used N14/15 labelling and tandem MOAC phosphopeptide enrichment in collaboration Dr. Gerold J.M. Beckers (RWTH-Aachen University, Aachen, Germany) to perform quantitative phosphoproteomics with MKK7 and MKK9 inducible overexpression lines. We identified 282 phosphopeptides belonging to 89 phosphoproteins. Among these 109 phopshorylation events were dependent on the β-estradiol-induced MKK7 and MKK9 expression; 48 were MKK7-, and 50 were MKK9-specific, while 11 were common to both MKK7 and MKK9. These phosphoproteins belonged to 41 differentially phosphorylated proteins, of which 25 were MKK7-, 12 were MKK9-dependent while the phosphorylation of 4 depended both on MKK7 and MKK9. Some of the identified phosphoproteins were known MPK6 and MPK3 substartes, such as the PHOS32 or the MAP65-1, showing that our activation-lines and phosphoproteomics protocols are well working. Within this work we also identified some novel leads, such as the MKK7-dependent phosphorylation of S6, which indicate a cross-talk between the MAPK pathway and TOR-S6K pathway in plants.
In task 2 we followed up on our work of computational predictions of MAPK substrates. In collaboration with the Paccanaro lab we extracted the MAPK-core modules (MEK, MAPK) from plant interactome databases (e.g. BioGrid) and constructed the Arabidopsis MAPK signaling network, which revealed to be highly connected and complex compared to yeast and animals. Based on sequence information on the docking interactions we identified a comprehensive list of substrates in Arabidopsis. Not all experimentally identified MAPK substrates however, have recognizable docking sequences. The Paccanaro group developed a machine learning approach that exploited all the experimentally known MAPK substrates in Arabidopsis (around 600) and created classifiers (e.g. accessibility, conservation score), that can be used by a machine learning approach to discriminate areas of the substrate sequences containing docking sites. Applying this classifier we extracted four sequence motifs that are potentially new docking sites. We selected 2-4 proteins from each of the categories of predicted MAPK targets with annotations in drought and abiotic stresses, cell cycle regulation, leaf growth and development for experimental verifications
In Task 3 we experimentally verified MAPK substrates. For proteins (~10) where we predicted new docking motives, we cloned the ORFs to a vector suitable for in vitro translation, produced the substrates as well as the activated MPK3, MPK4 and MPK6 MAPKs in wheat germ extract and carried out in vitro phosphorylations. These included the TSO1-like, CIR1, LHY-like, MYB, Blind-like, ERF-B3, DRE/CRT-binding TF, WKY75, EB1, γ-tubulin, PIN1. We found all of these proteins to get phosphorylated by MPK3, MPK4, MPK6, there were no preference among the MAPKs. For in vivo experiments, we focused on PIN1, HSFA4A, EB1, γ-tubulin. We tagged these proteins and transiently expressed them in protoplasts as well as in transformed Arabidopsis plants. We applied IEF in a capillary column (cIEF) to detect phosphorylation changes. With this technique we could show PIN1 phosphorylation by MPK6. For HSFA4A, EB1, γ-tubulin we looked for band shifts on SDS-PAGE and mapped the phosphorylation by mass spectrometry.
In Task 4 we mapped the phosphorylation sites on selected MAPK substrates, PIN1, HSFA4A, EB1. On PIN1 there are 4 predicted MAPK phosphorylation sites, one of which was experimentally detected in a phosphoproteomics experiment. We mutated all 4 sites in combinations, and proved that only one of this site contributes to MAPK phosphorylation. For HSFA4A and EB1 we mapped the phosphorylation sites by mass spectrometry.
In Task 5 we mutated the MAPK phosphorylation sites on PIN1 and HSFA4A. When these sites were mutated to alanine, we conformed the compromised or lack of phosphorylation in vitro and in vivo. The abiotic-stress-induced expression of HEAT SHOCK PROTEIN17.6A was strongly compromised by the serine-309 to alanine HSFA4A mutant form. For PIN1 we showed that the Ala mutation of the MAPK phosphorylation site (PIN1-A) is less abundant then wild type (opposite stability than with the phosphomimic glutamic acid mutation PIN1-E) and is resistant to MKK7 (neither stabilisation nor aggregate formation). The PIN1-A mutant is cytoplasmic and does not co-localise with the Golgi marker, and the ER co-localisation is also not prominent. We also had a weak, partly cytoplasmic signal in roots upon U0126 treatment. So it seems that phosphorylation plays a role in the polar membrane localisation and protein stability of PIN1.
Final results in the MAPKinDROUGHT project and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far)
Better understanding how plant growth is in tune with limiting environment is fundamental in efforts to improve stress tolerance. Suboptimal environmental conditions, among which water availability is in no doubt already the largest factor, limit crop yields to only 30% of the intrinsic capability of the plant, even under modern Western agricultural conditions. The overall aim of the proposed research is to identify the targets of the MKK7/MKK9 – MPK3/MPK6 drought-responsive MAPK signaling pathway, and link these to the growth adaptation of plants under drought stress conditions. To do this we performed: (i) unbiased phosphoproteomics screen for MAPK targets using the inducible MKK7/MKK9 lines, (ii) computational predictions of MAPK substrates involved in drought responses, (iii) targeted approaches to test MAPK phosphorylation of key regulators in plant growth regulation, including the RBR transcriptional repressor complex, the transcriptional regulators; HSFA4A and RAP2s, the auxin efflux protein, PIN1 as well as the microtubule-associated proteins, γ-tubulin, EB1 and a 14-3-3 adaptor protein. In summary, the MAPKinDROUGHT project identified novel targets of the MKK7 and MKK9 pathways that regulate meristematic growth and cell proliferation. Modulating these signalling pathways could help to obtain crops that better adapt their growth to limiting environmental conditions.