progress report

EUROPEAN COMMISSIONRESEARCH AND INNOVATION DGHUMAN RESOURCES ANDMOBILITY

Progress Report

Project No: 264474

Project Acronym: MERIT

Project Full Name: Metabolic Reprogramming by Induction ofTranscription

Progress Report

Period covered: from 01/01/2011 to 31/12/2012 Date of preparation: 15/06/2011

Period number: 1st Date of submission (SESAM): 25/01/2012

Project coordinator name:Dr. Johannes Hanson

Project coordinator organisation name:UNIVERSITEIT UTRECHT

Version: 1

Progress Report

PROJECT PROGRESS REPORTGrant Agreement number: 264474

Project acronym: MERIT

Project title: Metabolic Reprogramming by Induction ofTranscription

Funding Scheme: FP7-MC-ITN

Period covered - start date: 01/01/2011

Period covered - end date: 31/12/2012

Project co-ordinator:

Organisation PIC: 999985805

Organisation legal name: UNIVERSITEIT UTRECHT

Person in charge of scientific aspects:

Title: Dr.

First name: Johannes

Name: Hanson

Tel: +31 30 2533132

Fax: +31 30 2532837

E-mail: [email protected]

Project website address: http://theory.bio.uu.nl/MERIT/html/index.html

Project No.: 264474Period number: 1stRef: 264474_Progress_Report11_20120125_135630_CET.pdf

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OVERALL PROGRESS OF THE PROJECT:

Comments:

Please find the 1.Progress Report attached as a pdf.Summary:In January 2011 MERIT (MEtabolic Reprogramming by Induction of Transcription) started as acooperation of 7 beneficiaries from 6 countries (NL, DE, AT, SP, PT, BE) and 1 associated partner(Nunhems, NL). Within the project 10 ESR positions and 2 ER positions are offered. At thebeginning of the second year all positions are filled or at least suitable candidates are selected. Thepositions were announced widespread at several sites, e.g the project website at Utrecht University,EURAXESS, mailing lists (e.g. of the German Society of Botany or Experimental Plant Science),TAIR (Arabidopsis Google Group), Nature jobs, Academic Transfer and also by personal contacts.Most of the ESR´s have started within the last months. They are now used to the facilities and basicexperimental methods and initiated first experiments, which are running quite successful. The firsttransgenic plants have been generated, established and tested. The ER´s are starting in January andearly spring 2012.Two month after the official project start of MERIT the kickoff meeting was organized by JohannesHanson, Utrecht University. During the meeting each beneficiary gave a short overview about the labactivities, followed by organizational information by the coordinator. The meeting was combinedwith talks and individual job interviews for candidates for the open ESR positions. With 26participants it was a successful start with fruitful discussions about the specific ESR projects, theexchange of students between the project partners and further details regarding the MERIT project.In November 2011 the 1. Annual Meeting was organized in Madrid by Jesus Vicente-Carbajosa,UPM, Madrid. The meeting was scheduled for 2 days and associated with the first networkskill-training course about how to give scientific oral presentations. During the meeting each ESR /ER introduced himself and gave a short presentation about former projects.Associated with the 1. Annual Meeting all hired / selected ESR´s and ER´s followed a 1-day courseabout how to give efficient oral presentations of scientific data (CST 1). This course was open toexternal researchers, thus 5 students from outside the network also attend. The course was organizedby Jesus Vicente-Carbajosa, UPM, Madrid, and held by Carmen Sancho Guinda, UniversidadPolitécnica de Madrid. The fellows learned the essential basics of giving scientific presentations. Inthe afternoon a selected group of volunteers gave their presentations, which were prepared for themeeting of the next day. The presentations were discussed and improved by input from both thefellows and the professional trainer with the focus on constructive result-oriented criticism. Specialcare was taken to give clear and understandable oral presentation. During the meeting the next twodays the fellows than presented them selves and their research to the other participants of the projecton an impressive high level. Afterwards the course was evaluated by the fellows and got aremarkable good review. The results can be found on the MERIT webpage.Beside this all fellows are involved in internal meetings, (literature) seminars, workshops ordiscussion groups of their department.For each ESR a Career Development Plan was made and discussed during the 1. Annual Meetingwith the ESR, the supervisor and the co-supervisor. This plan will be updated by the ESR themselvesand after each MERIT meeting in accordance with the supervisors. Aim of the CDP is to giveorientation and guidance during the promotion period.Individual progress summaryESR 1: The fellow is establishing the necessary transgenic lines to be used for the planed studies, andhas also started with optimizing the protoplast system for further metabolome analysis. First resultsare promising.ESR 2:The fellow just started and is now getting familiar with the facilities. The training on analysisof micro-array data has been initiated.ESR 3: Within the first 4 months the needed transgenic plants were established and tested. Thefellow confirmed phosphorylation sites defined by M. Teige (University of Vienna) and studiescurrently the regulation of bZIP function by phosphorylation. A transgenic knock-down approach hasbeen initiated.ESR 4: The fellow generated transgenic plants (multiple CDPK-ko lines) and tested them. Theinteraction between CDKPs and bZIP63 has been confirmed and methods for further investigationhave been established. A collaboration with E. Baena-Gonzalez has been started to analyse


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sumoylation sites using MS.ESR 5: A douple enrichment method for phosphoproteome screen is validated and established by thefellow. Material for akin10 ko, ox and control lines screening is available. For 2012 two labexchanges are planned.ESR 6: Due to difficulties in finding a suitable candidate the fellow will start 01.01.2012.ESR 7: Due to visa administration problems the fellow started only two month ago. During this timehe gets familiar with the facilities and started the examination and verification of mutant lines.Currently, treatment conditions, which are than used for material preparation, are tested.ESR 8: The fellow is familiar with several methods and instruments (e.g. FACS, confocalmicroscopy or ELISA). The interaction between GBF1 and bZIP was analysed and will be extendedin collaboration with W. Dröge-Laser (University of Würzburg). First phenotyping experiments areinitiated and studied now in detail.ESR 9: Due to general space limitation for the lab institutes the recruitment was significantlydelayed. Initiative parts of the project were started already by the group, which enables the fellow togo into more detail with his experiments. Currently the fellow works on the establishment of assaysto evaluate interaction reactions and to consolidate the first data. The first publication is expectedbefore the Mid-term Meeting.ESR 10: The fellow is currently performing several cloning experiments involving KIN10 andKIN11. New approaches were developed, tested and optimization is ongoing. Beside this a qPCRplatform to detect KIN10 marker genes is being optimized.ER 1: The fellow will start 01.01.2012.ER 2: The fellow will start after defending his thesis, probably in spring 2012.


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CONTRACT DELIVERABLES UPDATEM - Months

RESR - Researcher

FAC B - Fixed amount contract B (%)

RECRUITMENT

Participants

Early Stage Researchers (ESR) Experienced Researchers (ER) Visiting scientists (VS 10)

Foreseen Implemented Difference Foreseen Implemented Difference Foreseen Implemented Difference Foreseen Implemented Difference

M RESR FACB M RESRFAC

B M RESRFAC

B M RESRFAC

B M RESRFAC

B M RESRFAC

B M RESRFAC

B M RESRFAC

B M RESRFAC

B M RESRFAC

B M RESRFAC

B M RESRFAC

B

UU 72 2 10 2 62 0 24 1 0 24 1 0 0 0 0

UWUERZ 36 1 4.25 1 31.75 0 0 0 0 0 0 0

UNIVIE 72 2 11 2 61 0 24 1 0 24 1 0 0 0 0

UPM 36 1 0 36 1 0 0 0 0 0 0

UTU 72 2 10.5 2 61.5 0 0 0 0 0 0 0

FCG-IGC 36 1 2.5 1 33.5 0 0 0 0 0 0 0

BBS 36 1 8 1 28 0 0 0 0 0 0 0

Total 360 10 46.25 9 313.75 1 48 2 0 48 2 0.00 0 0 0.00 0 0


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Comments:

Some delay during recruitement due to some problems with visa administration.


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INTERNATIONAL CONFERENCES / EVENTS OPEN TO EXTERNAL RESEARCHERS

EventNumber

Participant hosting theevent Type of Event

Month whenthe event took

place

Start date ofthe event

End date of theevent

Total number ofresearchers outside thenetwork attending the

event

Total number ofresearcher days forresearchers from

outside the networkattending the event

Website of the event

1 UNIVERSITEITUTRECHT

kick off meeting 3 01/03/2011 03/03/2011 8 24

2 UNIVERSIDADPOLITECNICA DE

MADRID

CST 1 - How to give anoral presentation?

11 21/11/2011 21/11/2011 5 5

3 UNIVERSIDADPOLITECNICA DE

MADRID

1. annual meeting 11 22/11/2011 23/11/2011 5 10

Total number of researchers outside the network attending the event Total number of researcher days for researchers from outside the networkattending the event

18 39

Planned number of researcher days for researchers from outside the network attending the event: 0

Remaining number of researcher days for researchers from outside the network attending the event: -39


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Comments:


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Attachments 1. progress report.pdf

Name

Date

Signature


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1. Progress Report

Grant Agreement Number 264474

MERIT Metabolic REprogramming by

Induction of Transcription Table of content

General summary .............................................................................................................................................. 3 Scientific progress ............................................................................................................................................. 3 Network meetings .............................................................................................................................................. 3 Training program ............................................................................................................................................... 3 Career Development Plan ................................................................................................................................. 4 Summary individual project progress ................................................................................................................ 4

ESR 1: Monika Tomar, Differential impact of bZIP heterodimers on metabolic reprogramming ........... 4 ESR 2: Alessia Peviani, Bioinformatic investigation of the regulation of low energy syndrome ............ 4 ESR 3: Lorenzo Pedrotti, Function of the C/S1 bZIP transcription factor network in low energy

signaling .................................................................................................................................... 4 ESR 4: Andrea Simeunovic, Regulation of C/S1 bZIP transcription factors by phosphorylation ........... 4 ESR 5: Ella Nukarinen, A mass western approach for the bZIP - SnRK1 signaling network ................ 4 ESR 6: Desiree Caudullo, C/S1 bZIP networks in the seed: integration of developmental, metabolic

and environmental responses ................................................................................................... 4 ESR 7: Abhroop Garg, Analysis of the combinatorial control of bZIP10-dependent stress responses . 4 ESR 8: Carles Llorca, Role of GBF1 in the crosstalk between LES and natural senescence ............... 4 ESR 9: Mattia Adiamo, Novel regulatory mechanisms of the energy sensor SnRK1 protein kinases ... 5 ESR 10: Filipa Tomé, The role of NAD metabolism in SnRK1 dependent regulation during stress ....... 5 ER 1: Magdalena Gamm, Identification of proteins interacting with the regulatory Sucrose-control

peptide of bZIP11 ...................................................................................................................... 5 ER 2: Thomas Nägele, Metabolic reprogramming in response to stress - analysis and regulation .... 5

Attended meetings and conferences by the fellows .......................................................................................... 5 Presentations and publications by the fellows ................................................................................................... 5

Oral presentations ...................................................................................................................................... 5 Poster presentations .................................................................................................................................. 6

Individual work package reports ........................................................................................................................ 7 Work package 1: Regulatory mechanisms of the SnRK1 kinases

(WP leader Elena Baena-Gonzalez) .......................................................................................................... 7 Work package 2: bZIP transcription factors executing low energy signals

(WP leader Wolfgang Dröge-Laser) ........................................................................................................... 7 Work package 3: Metabolic reprogramming, regulation and consequences

(WP leader Wolfram Weckwerth) ............................................................................................................... 8 Work package 4: Growth and physiological phenotyping (WP leader Matthew Hannah) .......................... 9 Work package 5: Coordinated European-wide training (WP leader M. Teige) ........................................ 10

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Work package 6: Transparent Network Management (WP leader J. Hanson) ........................................ 10 Individual progress reports .............................................................................................................................. 11

ESR 1: Differential impact on bZIp heterodimers on metabolic reprogramming ...................................... 11 ESR 2: Bioinformatic investigation of the regulation of low energy syndrome ......................................... 14 ESR 3: Function of the C/S1 bZIP transcription factor network in low energy signaling .......................... 16 ESR 4: Regulation of C/S1 bZIP transcription factors by phosphorylation .............................................. 18 ESR 6: C/S1 bZIP networks in the seed: integration of development, metabolism and environmental responses ................................................................................................................................................. 22

ESR 7: Analysis of the combinatorial control of bZIP10-dependent stress responses ............................ 24 ESR 8: Role of GBF1 in the crosstalk between LES and natural senescence ........................................ 27 ESR 9: Identification and characterization of the SnRK1 phosphatase(s) ............................................... 30 ESR 10: The role of NAD metabolism in SnRK1 dependent regulation during stress ............................. 32

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General summary

In January 2011 MERIT (MEtabolic Reprogramming by Induction of Transcription) started as a cooperation of 7 beneficiaries from 6 countries (NL, DE, AT, SP, PT, BE) and 1 associated partner (Nunhems, NL). Within the project 10 ESR positions and 2 ER positions are offered. At the beginning of the second year all positions are filled or at least suitable candidates are selected. The positions were announced widespread at several sites, e.g the project website at Utrecht University, EURAXESS, mailing lists (e.g. of the German Society of Botany or Experimental Plant Science), TAIR (Arabidopsis Google Group), Nature jobs, Academic Transfer and also by personal contacts. Most of the ESR´s have started within the last months. They are now used to the basic experimental methods and to initiate first experiments, which are running quite successful. The ER´s are starting in January and early spring 2012.

Scientific progress

Most fellows started during the last months of the first year. They started with getting familiar with the facilities and instruments and are now in the state to start experiments. The first transgenic plants have been generated, established and tested. Several lab exchanges for collaboration are planned for 2012.

Network meetings

Kickoff Meeting Two month after the official project start of MERIT the kickoff meeting was organized by Johannes Hanson, Utrecht University. During the meeting each beneficiary gave a short overview about the lab activities, followed by organizational information by the coordinator. The meeting was combined with talks and individual job interviews for candidates for the open ESR positions. With 26 participants it was a successful start with fruitful discussions about the specific ESR projects, the exchange of students between the project partners and further details regarding the MERIT project. 1. Annual Meeting In November 2011 the 1. Annual Meeting was organized in Madrid by Jesus Vicente-Carbajosa, UPM, Madrid. The meeting was scheduled for 2 days and associated with the first network skill-training course about how to give scientific oral presentations. During the meeting each ESR / ER introduced himself and gave a short presentation about former projects.

Training program

Associated with the 1. Annual Meeting all hired / selected ESR´s and ER´s followed a 1-day course about how to give efficient oral presentations of scientific data (CST 1). This course was open to external researchers, thus 5 students from outside the network also attend. The course was organized by Jesus Vicente-Carbajosa, UPM, Madrid, and held by Carmen Sancho Guinda, Universidad Politécnica de Madrid. The fellows learned the essential basics of giving scientific presentations. In the afternoon a selected group of volunteers gave their presentations, which were prepared for the meeting of the next day. The presentations were discussed and improved by input from both the fellows and the professional trainer with the focus on constructive result-oriented criticism. Special care was taken to give clear and understandable oral presentation. During the meeting the next two days the fellows than presented them selves and their research to the other participants of the project on an impressive high level. Afterwards the course was evaluated by the fellows and got a remarkable good review. The results can be found on the MERIT webpage. Beside this all fellows are involved in internal meetings, (literature) seminars, workshops or discussion groups of their department.

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Career Development Plan

For each ESR a Career Development Plan was made and discussed during the 1. Annual Meeting, together with the ESR, the supervisor and the co-supervisor. This plan will be updated by the ESR themselves and after each MERIT meeting in accordance with the supervisors. Aim of the CDP is to give orientation and guidance during the promotion period.

Summary individual project progress

ESR 1: Monika Tomar, Differential impact of bZIP heterodimers on metabolic reprogramming Supervisor J. Hanson, Utrecht University, start 01.04.2011 The fellow is establishing the necessary transgenic lines to be used for the planed studies, and has also started with optimizing the protoplast system for further metabolome analysis. First results are promising.

ESR 2: Alessia Peviani, Bioinformatic investigation of the regulation of low energy syndrome Supervisor B. Snel, Utrecht University, start 01.12.2011 The fellow just started and is now getting familiar with the facilities. The training on analysis of micro-array data has been initiated.

ESR 3: Lorenzo Pedrotti, Function of the C/S1 bZIP transcription factor network in low energy signaling Supervisor W. Dröge-Laser, University of Würzburg, start 22.08.2011 Within the first 4 months the needed transgenic plants were established and tested. The fellow confirmed phosphorylation sites defined by M. Teige (University of Vienna) and studies currently the regulation of bZIP function by phosphorylation. A transgenic knock-down approach has been initiated.

ESR 4: Andrea Simeunovic, Regulation of C/S1 bZIP transcription factors by phosphorylation Supervisor M. Teige, University of Vienna, start 01.07.2011 The fellow generated transgenic plants (multiple CDPK-ko lines) and tested them. The interaction between CDKPs and bZIP63 has been confirmed and methods for further investigation have been established. A collaboration with E. Baena-Gonzalez has been started to analyze sumoylation sites using MS.

ESR 5: Ella Nukarinen, A mass western approach for the bZIP - SnRK1 signaling network Supervisor W. Weckwerth, University of Vienna, start 01.08.2011

A douple enrichment method for phosphoproteome screen is validated and established by the fellow. Material for akin10 ko, ox and control lines screening is available. For 2012 two lab exchanges are planned.

ESR 6: Desiree Caudullo, C/S1 bZIP networks in the seed: integration of developmental, metabolic and environmental responses Supervisor J. Vicente-Carbajosa, University of Madrid, start 01.01.2012 Due to difficulties in finding a suitable candidate the fellow has been started 01.01.2012.

ESR 7: Abhroop Garg, Analysis of the combinatorial control of bZIP10-dependent stress responses Supervisor C. Chaban, start 01.10.2011 Due to visa administration problems the fellow started only two month ago. During this time he gets familiar with the facilities and started the examination and verification of mutant lines. Currently, treatment conditions, which are than used for material preparation, are tested.

ESR 8: Carles Llorca, Role of GBF1 in the crosstalk between LES and natural senescence Supervisor U. Zentgraf, start 15.05.2011 The fellow is familiar with several methods and instruments (e.g. FACS, confocal microscopy or ELISA). The interaction between GBF1 and bZIP was analyzed and will be extended in collaboration with W. Dröge-Laser (University of Würzburg). First phenotyping experiments are initiated and studied now in detail.

5

ESR 9: Mattia Adiamo, Novel regulatory mechanisms of the energy sensor SnRK1 protein kinases Supervisor E. Baena-Gonzalez, start 14.10.2011 Due to general space limitation for the lab institutes the recruitment was significantly delayed. Initiative parts of the project were started already by the group, which enables the fellow to go into more detail with his experiments. Currently the fellow works on the establishment of assays to evaluate interaction reactions and to consolidate the first data. The first publication is expected before the Mid-term Meeting.

ESR 10: Filipa Tomé, The role of NAD metabolism in SnRK1 dependent regulation during stress Supervisor M. Hannah, start 02.05.2011, The fellow is currently performing several cloning experiments involving KIN10 and KIN11. New approaches were developed, tested and optimization is ongoing. Beside this a qPCR platform to detect KIN10 marker genes is being optimized.

ER 1: Magdalena Gamm, Identification of proteins interacting with the regulatory Sucrose-control peptide of bZIP11 Supervisor J. Hanson, start 01.01.2012 The fellow has currently defended her thesis successfully and is just started officially. She already participated into the 1. Annual Meeting in Madrid and gave a presentation about her PhD project.

ER 2: Thomas Nägele, Metabolic reprogramming in response to stress - analysis and regulation Supervisor W. Weckwerth, start probably spring 2012 The fellow will start after defending his thesis, probably in spring 2012. He already participated into the 1. Annual Meeting in Madrid and gave a presentation about his PhD project.

Attended meetings and conferences by the fellows

o Annual Meeting, 19.-23. November 2011, Madrid, Spain (9 fellows + 2 ER candidates)

o Meeting of the German Botanical Society, September 2011, Berlin, Germany (1 fellow) o COSI conference on Plant Organellar signaling, 31.08-2.09.2011, Primosten, Croatia (1 fellow)

o 12th Symposium on Plant protein phosphorylation, 14.-16.09.2011, Tübingen, Germany (2 fellows)

o 5th Regio Plant Science Meeting; 5.11.11, Ulm, Germany (1 fellow)

o Umea Plant Science Centre Conference, 29-30 August 2011, Umea, Sweden (2 fellows) o EPS PhD day, 20 May 2011 Wageningen University, The Netherlands (1 fellow)

o Lunteren meetings, April, 2011 Lunteren, The Netherlands (1 fellow)

o PhD retreats, 5-8 July 2011,Orsay University (1 fellow)

o PhD summerschool,22/8-24/8-2011,Utrecht University, The Netherlands (1 fellow)

Presentations and publications by the fellows

Oral presentations Adamo, M. “Presentation on former research experience and introduction to current research (aimed at the Identification and characterization of the SnRK1 phosphatase(s))”, 1. Annual Meeting, November 2011, Madrid.

Garg, A. and Chaban, C. “Introduction to: Analysis of the combinatorial control of bZIP10-dependent stress responses”, 1. Annual Meeting, November 2011, Madrid, Spain.

Llorca, C.M. and Zentgraf, U. “The role of GBF1in developmental senescence and the crosstalk to LES” 1. Annual meeting, November 2011, Madrid.

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Nukarinen, E. “Phosphoproteome and proteome analyses of SnRK1/bZIP signalling network ”, 1. Annual meeting, November 2011, Madrid. Pedrotti, L. “General project overview”, 1. Annual Meeting, November 2011, Madrid, Spain.

Peviani, A. “Integration of protein-protein interaction networks and gene expression data in cancer outcome prediction”, 1. Annual meeting, November 2011, Madrid, Spain.

Simeunovic, A. “Regulation of plant metabolic adaptation to stress by calcium dependent phosphorylation” , public presentation of PhD reserch project (Expose`)in front of the PhD committee, 16.12.2011 at MFPL, Vienna, Austria.

Simeunovic, A. “Regulation of plant metabolic adaptation to stress by calcium dependent phosphorylation”, 1. Annual Meeting, November 2011, Madrid, Spain.

Tomar, M. Genetic Dissection of Metabolic reprogramming, 1. Annual meeting, November 2011, Madrid.

Tome, F. Progress Report, Bio-032 Meeting, December 2011, Gent

Tome, F. Introduction to the Merit Project, 1. Annual meeting, November 2011, Madrid.

Tome, F. Progress Report, Bio-032 Meeting, October 2011, Gent

Tome, F. Progress Report, Bio-032 Meeting, August 2011, Gent

Poster presentations Llorca, C.M., Jäschke, A., Smykowski, A., Zentgraf, U. “Posttranscriptional regulation of the bZIP transcription factor GBF1 during leaf senescence in Arabidopsis thaliana”, 12th International Symposium on Plant Protein Phosphorylation, Tubingen 14.-16.09.2011. Mair, A., Wurzinger, B., Simeunović, A., Anrather, D., Dietrich, K., Fragner, L., Vicente Carbajosa, J., Hanson, J., Baena-Gonzales, E., Chaban, C., Harter, K., Weckwerth, W., Dröge-Laser, W., Teige, M. “Multisite phosphorylation of a bZIP transcription factor in metabolic reprogramming“, COSI Conference, Primosten 31.08-2.09.2011. NOTE: this poster was also shown at 12th Symposyum on Plant Protein Phosphorylation, Tubingen 14.-16.09.2011.

Selvanayagam, J., Tomar, M., Eck-Stouten, E., Hansen, M., Ehlert, A., Dröge-Laser, W., Smeekens, S., Hanson, J., “Cracking the code of bZIP Dimerizatiob in Arabidopsis”, 2nd PhD retreat, Orsay University France, 05.07. – 08.07.2011. NOTE: this poster was also shown at UPSC day 29-30.08.2011, Umea Univeristy, Sweden and at PhD Summerschool 22-28.08.2011, Utrecht University, The Netherlands.

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Individual work package reports

Work package 1: Regulatory mechanisms of the SnRK1 kinases (WP leader Elena Baena-Gonzalez) The objective of WP1 is to understand the role of SnRK1 kinases in low energy signaling with a particular emphasis on how the activity of SnRK1 is regulated. For this purpose, major aims of WP1 are the identification of SnRK1 interacting factors and the characterization of SnRK1 activity in relation to its phosphorylation targets, bZIPs and possibly other transcription factors. This has been analyzed through four main lines of research, focusing on:

o The posttranscriptional modification of SnRK1 and its effect on SnRK1 activity and subcellular localization (EBG)

o The regulation of SnRK1 by upstream phosphatases (EBG)

o The regulation of SnRK1 by NAD metabolism (MH) o The co-regulation of bZIP63 by SnRK1 and CDPK kinases (MT).

The proposed deliverables and the corresponding progress for the first year are the following: o Provide materials for metabolic analysis in WP3. The material will be samples in which the

activity or level of kinases are manipulated to determine the effect on metabolism of individual kinases. Progress:

o bZIP63 knockout mutant material has been analysed (MT, WW) and material from knock-in lines is being prepared (MT, WW)

o KIN10 (SnRK1.1) knockout and overexpressor lines are being analyzed in parallel (EBG, MT, WW)

o Mass-western protocol established. Progress:

o Since the regulatory SnRK1 phosphatases are one major line of research (EBG), priority will be given at this point to phosphoproteomics analysis of SnRK1 phosphorylation in different Arabidopsis genotypes (wild type and transgenics with altered PP2C levels) rather than to mass-western analyses. This is expected to help elucidate the mechanism through which these phosphatases regulate SnRK1.

Work package 2: bZIP transcription factors executing low energy signals (WP leader Wolfgang Dröge-Laser) WP2 will provide insight how low energy signals control bZIP activity by post-transcriptional mechanism, in particular via SnRK1 mediated phosphorylation (MT, WDL), uORF regulation (JH) or protein-protein interaction (CC). Heterodimerisation of bZIP factors provides an additional important regulatory level in low energy signaling. Heterodimer formation, their impact on DNA binding and target gene regulation will be studied on a genome-wide scale using transcriptomic and ChIP-seq techniques (JH, WDL, JVC, BS, CC) and with respect to particular functional aspects such as seed maturation (JVC), senescence (UZ), stress responses (JVC, WDL, CC) and primary metabolism (JH, WDL). Data sets recorded by the various approaches will be integrated using bioinformatic tools (BS). The progress of the first year:

o Most of the ESRs have been working on their project for 6 month or less, which allowed them to get used to the basic experimental methods and to initiate the generation of time consuming tools. In this respect, several transgenic approaches have been initiated or are in progress. However, the generation of homozygous plant lines will last until late 2012 (e. g. ESR2, 3, 4 or 8).

o As several of the WP2 projects are tightly linked between partners, the collaborative time-lines have been established and plants and other tools have been exchanged to get started. Here selected examples are described:

o Partner 3 (MT) has defined bZIP63 as a KIN10 target, which has been confirmed in protoplasts by partner 2 (WDL). Metabolomic studies on bzip63 and overexpressors have already been performed in co-operation with partner 3 (WW). Planed transcriptomic analyses done by partner

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1 (JH, BS) and studies in protoplast (partner 2) will assist in defining target genes and mechanism of gene regulation.

o In general, bZIP proteins bind and regulate transcription as dimmers. Heterodimerisation within the C/S1 network has been studied by microarray analysis of transiently transformed protoplast (partner 1, JH; partner 2, WDL). Co-transformation of several bZIP combinations allows to define the impact of bZIP heterodimers. In order to identify putative cis-binding sites, bioinformatic analyses of these data will be performed by partner 1 (BS). Nevertheless, these putative cis-elements need to be confirmed experimentally by a novel DNA binding method developed in the lab of partner 5 (CC) and/or by ChIP-seq techniques (CC).

o As demonstrated by partner 5 (UZ), the group C member bZIP63 heterodimerizes not only within the group C/S1 network, but also with the senescence–related group G member GBF1. Hence, this work of partner 5 (UZ) links low energy signaling with natural senescence. This result opens up the important question how these functions are connected. Consequently, a more sophisticated heterodimerisation pattern has to be taken into account. The bZIP heterodimerisation properties of GBF1 and bZIP63 will be studied in co-operation with partner 2 (WDL) using a high-throughput protoplast two-hybrid assay.

Work package 3: Metabolic reprogramming, regulation and consequences (WP leader Wolfram Weckwerth) Dynamic SnRK1-bZIP signaling networks and protein complex formations are correlated to changes in metabolite levels. Central in WP3 is efficient profiling of metabolites. Metabolite profiles will be determined from protoplasts, whole plants as well as from seeds. The progress since June 2011: In collaboration with MT, WDL and EBG we have analysed metabolomic profiles of several Arabidopsis mutants in the bZIP-signaling pathway: bZIP1, bZIP53, bZIP1/53, bZIP63 and AKIN10. Mutants were analysed in normal day/night rhythm and under extended night conditions driving the plant into the low-energy-syndrome (LES). The metabolome profiles of the mutants were analysed with our standard gas-chromatography-mass spectrometry methods (Weckwerth et al. (2004) Proteomics 4, 78-83; Morgenthal et al. (2007) Methods Mol Biol 358, 57-75). Intriguingly, all mutants showed distinct metabolite profiles as a response to dark/light and LES conditions (figure 1 A and B).

-6 -5 -4 -3 -2 -1 0 1 2 3-3

-2

-1

0

1

2

3

PC 1

PC 2

1/53KO D

1OEX D

1OEX L

153KO L

35S::bZIP63-GFP 6h dark

35S::bZIP63-GFP 6h extended night

35S::bZIP63-GFP 6light

53OEX D

53OEX L

WT D

WT L

akin10 k.o. 6h dark

akin10 k.o. 6h extended night

akin10 k.o. 6light

bzip63 k.d. 6h dark

bzip63 k.d. 6h extended night

bzip63 k.d. 6light

wt 6h dark

wt 6h extended night

wt 6light

53OX Dark/Light

light

dark

63 KD Dark/LES

63 OX Light

1 OX Dark

1 OX Light

A B Figure 1: A Initial Principal components analysis of all mutants showing distinct behaviour to dark/light/LES conditions; B Detailed analysis of metabolomic profiles for bZIP 63 mutants (Mair et al. manuscript in preparation). Initial results from metabolomic comparisons of different bZIP mutants demonstrate their specific metabolic behaviour in a typical diurnal rhythm as well as under LES conditions such as extended night. These individual metabolic signatures will be further investigated with respect to a functional characterisation of the distinct bZIP transcription factors. Further studies on the dynamics of phosphoproteins will be linked to these metabolic changes.

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Work package 4: Growth and physiological phenotyping (WP leader Matthew Hannah) The objective of this WP is to robustly evaluate the physiological consequences for developmental senescence, abiotic stress and pathogen responses, of modulating target genes relevant to the pathways involved in LES (low energy syndrome). Biotic and abiotic stress and developmental senescence have a large, and likely partially overlapping, impact on metabolic reprogramming. This WP will therefore use 3 complementary platforms in order to phenotype plant material and to evaluate gene involvement in these responses.

o Developmental senescence phenotypes will be analyzed by detailed analysis of development rate (including flowering time) and leaf senescence by partner 5 (UZ) supported by physiological (leaf number, chlorophyll fluorescence) and molecular (expression of early and late senescence associated genes) measurements.

o Abiotic stress phenotypes will be robustly evaluated for multiple plant lines by utilizing the battery of standardized abiotic stress phenotypic assays available to the industrial partner 7 (MH) using field relevant (e.g. high-light, temperature, salt) and model (osmotic stress, oxidative stress) conditions and recovery from extended darkness. Non-destructive image analysis and statistical evaluation tools are in place to allow efficient and accurate assessments of plant growth phenotypes.

o Pathogen responses will be analyzed with respect to altered susceptibility to various biotrophic and necrotrophic pathogens, which are well established in the laboratory of partner 3 (WDL). Pathogen propagation and induction of typical marker genes will be analyzed.

Within WP4 the following items were planned to be delivered after 12 months:

o Decision on which lines to phenotype in established assays. Progress: o At our 1. Annual Meeting, held in Madrid, the projects of all ESRs and ER were

discussed among the relevant PIs/ERs/ESRs and updated. This planning included an overview of all the genetic material that will be considered for phenotyping during the project and which exchanges will occur to allow this phenotyping to occur. The relevant genetic material and exchanges are detailed within the individual CDPs. Further optimization of both will occur during the project based on the results obtained in the partner labs.

o Methods for senescence, growth response to extended darkness and pathogens, etc established. Progress:

o The following methods with respect to the necessary phenotypic assays have been established or are in development:

§ Abiotic Stress: Digital imaging of growth established (in vitro, in soil)

§ Darkness response: Digital imaging of growth in optimization phase (in vitro) and in planning (in soil)

§ Pathogen Stress

• Pseudomonas syringae (biotrophic bacterium): Quantification by bacterial growth assays

• Botrytis cinerea (necrotrophic fungus): Quantification by PCR amplification of fungal DNA

§ Senescence

• Natural senescence of soil grown plants: Quantification by temporal analysis of leaf number, biomass, number and height of the bolts, chlorophyll analysis and expression of SAGs

• Dark-induced Senescence on soil grown-plants: Quantification by Chlorophyll measurement

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Work package 5: Coordinated European-wide training (WP leader M. Teige) Within the WP 5 the project ensures a state-of-the-art interdisciplinary research training to maximize the research potential and employability of the fellows. They will develop complementary skills needed for a successful career in industry or in the academic institutions. A network of expertise for consultancy, reviewing and support will be provided to the MERIT researchers and will include both scientific and industry related skill training and complementary skill training, such as presentation and writing skills. WP5 has so far been carried out as outlined in the proposal.

o The selection of ESRs/ERs was coordinated by JH and MT in close collaboration with all involved group leaders. Starting with their first working days, the local training started under the responsibility of the direct supervisor, and a personal career development plan (CDP) was developed for each employed researcher.

o The first network-wide training activity was the skill training course (CST1) on presentation skills, organized by Jesus Vicente-Carbajosa at the University of Madrid. Researchers arrived at the weekend before the course and had social activities during the weekend before, and received lectures and practical training in presentation skills and subsequently presented their projects during the meeting to the entire consortium and received feedback from all group leaders.

o The next training on complementary skills in the context with the network meetings training is scheduled for the midterm meeting in Lisbon, End 2012. Additionally, exchanges between the partner labs were adjusted to the progress of the individual projects.

o The date for the first skill training course on Statistics and Bio-Informatics in Utrecht is scheduled for 23.01. – 03.02.2012. The information letter containing further instructions/directions on the venue and schedule of the course was distributed via email and is also available on the website.

Work package 6: Transparent Network Management (WP leader J. Hanson) The project management serves as a joint connection between the project partners, the fellows and the EU/REA. It will ensure an efficient communication channel and financial management. The coordinator is supported by a project manager, who started 01.03.2012 (part time, 16 hours / week).

Main objectives are the reporting to the commission, establishment of Internet based infrastructure and the coordination of the training and research activities within MERIT, as well as the financial management. The deliverables for the first year were the recruitment of the ESRs and ERs, as well as the establishment of a communication infrastructure.

o The recruitment is finished, within the first year all positions were filled or at least the candidates are selected. ESR 6, ER1 and ER2 will start 01.01.2012 and spring 2012, respectively, depending on the date of his thesis defense. The positions were announced widespread at several sites, e.g. the first project website at Utrecht University, EURAXESS, mailing lists (e.g. of the German Society of Botany), TAIR (Arabidopsis Google Group), Nature jobs and also by personal contacts. The project management received almost 100 applications directly, beside this the project leaders were contacted individually. Lots of the candidates were not eligible regarding the EU criteria or educated in too different fields. This lack of suitable candidates caused some delay in the recruitment process, as well as some problems with visa administration.

o The communication infrastructure is realized as a website, new constructed by the project manager. Here the project related information are placed and available, not only for the MERIT members but also visible for the public. The website contains one password protected area restricted to the MERIT members on which notes of meetings, contractual details or other internal information is available.

The site can be found at http://theory.bio.uu.nl/MERIT/html/index.html

Beside this the project management was involved in the organization of the 1. Annual Meeting in Madrid and supported the local organizer. A newsletter is introduced and will be published twice a year. The newsletter can also be found on the website of the project.

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Individual progress reports

Name Monika Tomar Project title

ESR 1: Differential impact on bZIp heterodimers on metabolic reprogramming

ESR number 1 Supervisor Johannes Hanson Co-supervisor Ulrike Zentgraf Official Starting date 1 April 2011 Recruitment process The position was advertised In EURAXESS and at Nature jobs, Academic

transfer, Utrecht university and the MERIT website. It was also sent to various mailing lists including “Experimental plant science”, and Dutch and Swedish lists. It was also personally communicated by Johannes Hanson. In total more than 50 persons responded of which a majority were formally not eligible or had CVs not matching the expectations. Three persons were interviewed over Skype and finally the decision was made to hire Monika Tomar.

Project introduction

Transcription factors regulate the activity of genes. The factors studied in this project can only regulate genes in pairs. Interestingly the pairs consisting of different factors regulate different genes. The project will determine how the different factors in cooperation with other factors regulate genes in plants.

Deliverables 4 Provide material for metabolic analysis in WP3 Initiated

6 Methods for senescence, growth response to extended darkness and pathogens, etc. established

Assays for darkness induced starvation responses established 8 Provide at least three sets of transcriptomic data to identify putative bZIP target genes.

Dataset of Protoplast, with overexpressed bZIP factors performed 10 Establishment of unbiased metabolic profiling protocol for Arabidopsis protoplasts and seeds.

Sugar free protoplast method establishment initiated 15 Provide at least 5 transgenic lines to study bZIP function in low energy signaling

Two bZIP11 EGFP fusion lines established, amiRNA as well as overexpressor lines are initiated.

Progress / Results

Generally progress is good. Several deliverables are achieved earlier that expected. Details progress: 1) Metabolome effect of bZIPs in protplasts Optimization of protoplast system for metabolome analysis initiated, First results are promising but at present no strategic decision can be made and further experimentation are required. 2) Further confirmation of mirco-array results in protoplasts Construct using the ProDH, PPD5 and PPD6 promoters have been used to confirm the results form the microarray analysis. These constructs has as well been used for characterization of the response on the expression of these genes in response to all possible combinations of S1 and C bZIP transcription factors.

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3) Characterization of plants in which group S1 genes have been inactivated Constructs for transgenic lines have been constructed and transformed to wild-type Arabidopsis. Stable lines have not yet been established or characterized. This awaits later generations of the lines.

Future plans / expected results

Emphasis will be on generating material and optimizing protoplast procedures for metabolic analysis as well as further characterizing the transgenic lines.

Local training The fellow has followed the standard program of seminars in the department as well as the activities stipulated by the graduate school of the university.

Network training / activities

The fellow has participated in all network activities as yet offered by the MERIT network including the Kickoff meeting, first annual meeting as well as firs complementary skill training course.

Collaborations No new collaboration are established during this period Planned lab exchange For the Metabolomic analysis ESR1 will go to laboratory of Prof. Dr. Wolfram

Weckwerth and Dr. Markus Teige, Universitat Wein,Veinna, Austria in the coming months,2012.

Published Poster, Articles, Presentations, Books, Others

Oral presentation: Tomar, M. Genetic Dissection of Metabolic reprogramming, 1. Annual meeting, November 2011, Madrid.

Selvanayagam, J., Tomar, M., Eck-Stouten, E., Hansen, M., Ehlert, A., Droge laser, Wolfgang., Smeekens, S and Hanson, J. (2011) Cracking the code of bZIP Dimerizatiob in Arabidopsis. Poster at 2nd PhD retreat 5-8 July,Orsay University France. Selvanayagam, J., Tomar, M., Eck-Stouten, E., Hansen, M., Ehlert, A., Droge laser, Wolfgang., Smeekens, S and Hanson, J. (2011) Cracking the code of bZIP Dimerizatiob in Arabidopsis. Poster at UPSC day 29-30 Aug, Umea Univeristy. Selvanayagam, J., Tomar, M., Eck-Stouten, E., Hansen, M., Ehlert, A., Droge laser, Wolfgang., Smeekens, S and Hanson, J. (2011) Cracking the code of bZIP Dimerizatiob in Arabidopsis. Poster at PhD Summerschool 22-28 Aug, Utrecht University.

Attended Conferences, meetings, summer schools,

1. EPS PhD day, 20 May 2011 Wageningen University 2. Lunteren meetings,April,2011 Lunteren

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ect. (incl. contribution) 3. PhD retreats, 5-8 July 2011,Orsay university, Poster Presentation on the topic Cracking the code of bZIP dimerization in Arabidopsis.

4. UPSC day, 29-30 August, 2011,Umea University, Poster Presentation on Cracking the code of bZIP dimerization in Arabidopsis.

5. PhD summerschool,22/8-24/8-2011,Utrecht University, Poster Presentation Cracking the code of bZIP dimerization in Arabidopsis.

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Name Alessia Peviani Project title

ESR 2: Bioinformatic investigation of the regulation of low energy syndrome

ESR number ESR2 Supervisor Berend Snel Co-supervisor Wolfram Weckwerth Official Starting date 01/12/11 Recruitment process The position was announced on nature jobs for three seperate periodes

throughout 2011, and for most of the year on the web-page of the department of theoretical biology and bioinformatics, the web-page of the Snel group as well as the webpage of the MERIT consortium. A number of people were contacted via phone and skype. Four people were invited to give a presentation and for an interview.


The general goal of MERIT is to elucidate the regulation of the low energy syndrome. In order to achieve this, massive amounts of data will be generated in the project. Also there are likely important clues present (but sofar undetected) in publically available data and in the evolutionary history of the components of the system as retrievable from the genome sequences of plants and algae. We will perform advanced bioinformatic analyses to integrate these three sources of information to find new hypotheses on the biological organsiation of the regulation of the low energy syndrome as well as on its evolution as a system.

Deliverables The project has started 2 weeks ago. Hence no deliverables can be reported yet.

Progress / results

The project just started. The training of the PhD student has been iniated by performing under close supervision the analysis of micro-array data from MERIT partner Hanson. These data are relevant because they are from bZIP mutants and over-experssion lines which are transcription factors that are likely essential for the regulatio nof the low energy syndrome. In addition the close supervision of the analyses should provide a very productive learning environment for the PhD student.


We expect to obtain biologically relevant results from the analyses of micro-array data from the consortium as well as from their comparison to publically available data. Such results could include but are not limted to predicted motifs specific for bZIP subtypes, shared and unique targets of bZIPs, pathways targetted by bZIP transcription factors that potentially provide feedback to SNRK kinase signalling. Furthermore we expect to have started on the phylogenomic analyses of a few key proteins from our network, such as the KIN10/KIN11 kinases, the C and S type bZIPs and the casein kinases. Finally we hope to be able to adopt genome alignments / phylogenetic footprinting in our pipeline in order to differentiate spurious bZIP motifs from evolutionary constrained motifs with the aim of finding more functional relevant targets.

Local training No local course have been followed yet. Network training / activities

The PhD student attended the first MERIT annual meeting, Madrid November 2011. The PhD student participated in the MERIT complementary skill training on “scientific presentations”, Madrid November 2011, 2 ECTS.

Collaborations

We obviously iniated the local collaboration with the group of Johannes Hansson.

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One potential collaboration is planned with the ESR from the Chaban group who will visiting our lab to perform in depth phylogenetic analyses of C subtype bZIPs in order to determine the ancestral sequences. Another potential collaboration is planned with the Teige group who are cloning known components of the low energy syndrome regulatory pathway in primitive plants and algae in order to be able to study this network in a less redundant system. We are planning to perform the phylogenetic analyses to provide a comparative genomic framework to interprete and guide the experiments on these components in primitive plants and algae.

Planned lab exchange

The ESR from Chaban will visit our lab in february (after the MERIT course) to be trained in phylogenetic analyses. Alessia will visit the lab of Wolfram Weckwerth in 2012 to learn about the bioinformatic analysis of other omics data types such as proteomics or metabolomics and to discuss the progress of her research.


oral presentation: Peviani, A. “Integration of protein-protein interaction networks and gene expression data in cancer outcome prediction”. Annual meeting November 2011, Madrid.

Attended Conferences, meetings, summer schools, ect. (incl. contribution)

So far no time was found yet for attendance.

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Name Lorenzo Pedrotti Project title

ESR 3: Function of the C/S1 bZIP transcription factor network in low energy signaling

ESR number 3 Supervisor Prof. Dr. Wolfgang Dröge-Laser Co-supervisor Dr. Christina Chaban Official Starting date August 22nd, 2011 Recruitment process The position has been internationally advertised at different portals and

candidates were invited for selection on the MERIT kick-off meeting in Utrecht. As no suitable candidate was found, a second round of selection was initiated. Lorenzo Pedrotti was contacted directly by the PI, as this candidate was well recommended by colleagues.


Plants are able to convert solar energy into organic compounds. In nature, there are many factors, which can affect photosynthetic activity such as the normal day-night rhythms, pathogen infection and salt stress. Due to their phototrophic life style, plants have to adjust their metabolism to withstand transient energy deprivation. Previous work has shown that SnRK1 kinases, together with group C and S1 bZIP transcription factors play a crucial role in reprogramming the metabolism in response to low energy stress (LES). The goal of this research is to understand the regulatory function of the C/S1 network as an integral node in SnRK1 mediated low energy signaling.

Deliverables Deliverable 1, WP 6, Lead beneficiary 1: Recruitment of ESR3 Deliverable 7, WP 6, Lead beneficiary 3: Personal CDP of Lorenzo Pedrotti approved. Deliverable 15, WP 2, 4, Lead beneficiary 2: homozygous KIN10 and KIN11 amiRNA plants have been generated. Deliverable 15, WP 2, 4, Lead beneficiary 2: bzip53 /bzip1 knock-down plants have been transformed with a amiRNAbZIP2/11/44 construct. Selection of homozygous plants is in progress.

Progress / results

1. Impact of KIN10/11 in transcriptional and metabolic reprogramming during low energy stress. Transgenic constitutive KIN10 and KIN11 amiRNA plants have been established and confirmed. Estradiol inducible lines and double knock-down approaches have been initiated and will be available in summer 2012. In co-operation with partner 1 (JH, BS) and partner 3 (WW), these plants will be analyzed for the impact of KIN10/KIN11 in low energy stress. 2. Impact of bZIP transcription factors in mediating low energy stress responses. Partner 3 has identified bZIP63 as a target of KIN10. Phosphorylation sites defined by partner 3 (MT) were confirmed by analyzing bZIP63 transcription factor function in protoplasts using bZIP63 mutations at these phosphorylation sites. Currently, protoplast assays are used to study how phosphorylation is regulating bZIP63 function. A transgenic knock-down approach for all group S1 members has been initiated and will probably be available in summer 2012.


1. Generation of homozygous plants (inducible amiRNA KIN10/11 plants, group S1 knock-down).

2. Establishment of conditions for LES and estradiol treatments. 3. Harvest plant material which will be analyzed in co-operation with

partner 1 (transcriptomics) and partner 3 (metabolomics). 4. Use of protoplast systems to define the function of bZIP63

phosphorylation. 5. 5. Analyze bZIP heterodimerisation with partner 1 and 5.

6. Optimize infection conditions with biotrophic and necrotrophic pathogens.

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Local training - Journal Club each Friday 9.00 - 10.00 (during the semester) - Progress Report: twice a month, Wednesday 9.00 – 11.00


- MERIT, 1st Annual meeting, November, 22nd- 23rd 2011; - CST1, efficient oral presentation of scientific data.

Collaborations

- With partner 1, 5 (JH) bZIP heterodimerisation - With partner 3 (MT): bZIP63 phosphorylation - With partner 3 (WW): metabolic analyses


The following secondments are planed for Lorenzo Pedrotti: - partner 1: microarray analysis (end of 2012) - partner 3: metabolome anaylsis (end of 2012)


Oral presentation:

- Pedrotti, Introduction about the project,

1. Annual meeting, November 2011, Madrid.


Attended conferences: - Meeeting of the German Botanical Society, September 2011, Berlin.

(no poster as the work has started 1 month before) - 1st Annual MERIT meeting, November 2011, Madrid

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Name Andrea Simeunovic Project title

ESR 4: Regulation of C/S1 bZIP transcription factors by phosphorylation

ESR number ESR4 Supervisor Markus Teige Co-supervisor Elena Baena-González Official Starting date 01.03.2011 Recruitment process The position has been internationally advertised at different portals (e.g.

Euraxess) and in the international call for the Vienna PhD programme. In a faculty wide selection process candidates have been selected and interviewed and in this highly competitive process Andrea Simeunovic has been selected and started on March 1, 2011.


The main goal of this research is to define the regulatory inputs on bZIP transcription factors by phosphorylation. In this context bZIP63 and its potential interaction partners are the main targets and we study the interplay of different protein kinases like SnRK1 type kinases and CDPKs.

Deliverables 1.2 Identification of SnRK1 kinase interaction partners. We performed yeast-two hybrid, BiFC and direct phosphorylation assays with bZIP63 and AKIN10 and 11 and could establish a specific interaction with bZIP63. Regulatory phosphatases will be analysed in collaboration with EBG. 1.3 Generation of multiple CDPK knockout lines has been initiated. Cpk3,6,11 triple ko is available. 1.4 bzip ko mutant material has been analysed together with WW and material from knock-in lines is being prepared. 2.3 bzip63 mutant lines have been grown for transcriptomic analysis together with JH. 5.1 PCD plans have been approved within the MERIT network and also within the faculty in december 2011.

Progress / results

Multiple cdpk ko lines have been generated and constructs for complementation have been tested. Material for transcriptome analysis has been generated. Interaction between CDPKs and bZIP63 has been confirmed. Methods to address importance of sub-cellular localization of CDPKs established. In collaboration with EBG several sumoylation sites have been analysed by MS.


Analysis of different bzip63 mutant lines under control and extended night conditions will reveal important tartget genes to be focused on in future studies. Phenotyped of cdpk multiple ko lines will be done under stress conditions and RNAi of SnRK1 will reveal redundancy of CDPKs and SnRKs1.

Local training - Attending VBC Plant Molecular Biology Seminars offered by University of Vienna - Max F.Perutz Laboratories (MFPL) Seminars - Regular presentation of work in progress in a joined group seminar and literature seminars organized within the groups working in the field of plant molecular biology at MFPL, Vienna - Two Introductory Lectures to BioOptics for the use of the confocal microscope offered by Microscopy facility at MFPL , followed by hand-on sessions


- Attendance at the kick-off meeting in Utrecht - Merit Scientific oral presentation course organized in Madrid from 19.11-23.11.2011

Collaborations On SnRK1 sumoylation with EBG, on metabolomic profiles with WW, ontranscriptomics with JH, on functional assays in protoplasts with WDL

Planned lab exchange With JH for transcriptomics early next year, more to be defined, for example

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to WDL later next year to study impact of CDPKs on bZIP63 mediated gene expression


Simeunovic, A. “Regulation of plant metabolic adaptation to stress by calcium dependent phosphorylation” , public presentation of PhD reserch project (Expose`)in front of the PhD committee, 16.12.2011 at MFPL ,Vienna

Simeunovic, A. “Regulation of plant metabolic adaptation to stress by calcium dependent phosphorylation” ,presentation of research project within Merit program at the first annual meeting in Madrid 19.-23.11.2011

Andrea Mair, Bernhard Wurzinger, Andrea Simeunović, Dorothea Anrather, Katrin Dietrich, Lena Fragner, Jesús Vicente Carbajosa, Johannes Hanson, Elena Baena-Gonzales, Christina Chaban, Klaus Harter, Wolfram Weckwerth, Wolfgang Dröge-Laser, Markus Teige „Multisite phosphorylation of a bZIP transcription factor in metabolic reprogramming“, poster presentation at COSI Conference, Primosten 31.08-2.09.2011

Andrea Mair, Bernhard Wurzinger, Andrea Simeunović, Dorothea Anrather, Katrin Dietrich, Lena Fragner, Jesús Vicente Carbajosa, Johannes Hanson, Elena Baena-Gonzales, Christina Chaban, Klaus Harter, Wolfram Weckwerth, Wolfgang Dröge-Laser, Markus Teige „Multisite phosphorylation of a bZIP transcription factor in metabolic reprogramming“, poster presentation at 12th Symposyum on Plant Protein Phosphorylation, Tubingen 14.-16.09.2011


CST1 and 1st Annual meeting, 19.-23.11.2011 2011, Madrid -COSI conference on Plant organellar signalin in Primosten , 31.08-2.09.2011 (participating in the group’s poster presentation) - 12th Symposium on Plant protein phosphorylation , Tubingen 14.-16.09.2011 (participating in the group’s poster presentation)

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Name Ella Nukarinen Project title ESR 5: A mass western approach for the bZIP - SnRK1 signaling

network ESR number ESR5 Supervisor Wolfram Weckwerth Co-supervisor Johannes Hanson Official Starting date 01.08.2011 Recruitment process The position has been internationally adviertised at different portals and in

the international call for the Vienna PhD programme. In a faculty wide selection process candidates have been selected and interviewed and in this highly competitive process Ella Nukarinen has been selected and started on August, 2011.


The purpose of this research project is to identify potential phosphorylation substrates and upstream regulators of SnRK1/bZIP network and to accurately quantitate their abundance in different conditions and tissues and cell compartments in response to the energy state of the plant. Further aims are to characterize the dynamic metabolome in response to this complex regulatory network and energy state of the plant.

Deliverables 5.1 Screening of SnRK1 substrates. We are studying influence of extended night to phosphoproteome of akin10 ko and ox lines together with control lines. Other energy restricted conditions will also be included in the study. 5.2 Mass Western technology will be used to analyse the quantity of proteins and their phosphorylation sites involved into SnRK1/bZIPs regulatory network. Also ratio between phosphorylated and non-phosphorylated SnRK1 substrate proteins will be quantitated by Mass Western. 5.3 The Mass Western approach is established for various tissues. 5.4 Correlative analysis of mRNA and metabolite levels will be done.

Progress / results

Douple enrichment method for phosphoproteome screen is validated and established. We have material for akin10 ko, ox and control lines screening.


By analysis of proteomes and phosphoproteomes of SnRK1 and bZIP mutant lines we will obtain information about the low energy syndrome (LES) signalling network at protein level. It is planned to correlate this to metabolomic responses.

Local training Attendance to courses of Department of Molecular Systems Biology: Proteomics, metabolomics and bioinformatics.


Merit Scientific oral presentation course organized 19.11-23.11.2011 in Madrid.

Collaborations Analysis of proteins that putatively participate in regulation of SnRK1/bZIPs network together with EBG and WDL, and correlative analysis of metabolites, proteines and mRNA levels with JH.


Protoplast assays with WDL in spring 2012. Correlative analysis of metabolite, protein and mRNA levels with JH end of 2012.

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Oral presentation at the first annual meeting in Madrid.


CST1 and 1st Annual meeting, November 2011, Madrid.

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Name Desiree Lucy Anna Rita CAUDULLO (Nationality: Italian) Project title

ESR 6: C/S1 bZIP networks in the seed: integration of development, metabolism and environmental responses

ESR number ESR6 Supervisor Jesús Vicente Carbajosa. UPM Spain Co-supervisor Wolfgang Dröge-Laser, University of Würzburg, Germany Official Starting date 01.01.2012 Recruitment process Advertised at the MERIT webpage and linked sites. Recruitment involved

emailing, phone conversations and and web interview Project introduction

Our focus is on TFs involved in seed development. Most of these are involved in antagonistic metabolic processes under distinct hormonal regulation. We have analyzed functionality conserved cis-regulatory elements present in the promoters of maturation and germination specific genes (Vicente-Carbajosa et al. (2005) Int J Dev Biol 49, 645-651). In addition, we could establish regulatory networks based on protein-protein interactions and functional analyses in transient expression assays. In collaboration with partner 2 (WDL) we have shown that AtbZIP10, 25 and 53 are key players in the regulation of seed-maturation genes in conjunction with ABI3, an important regulator of seed development (Alonso et al. (2009) Plant Cell 21, 1747-1761). Within this project we will generate both constitutive and inducible mutants with gain or loss-of-function in different C/S1 bZIP members and study their participation during seed development, and in nutrient and abiotic stress responses.

Deliverables The UPM group has so far contributed the following deliverables: • Del.1.2 and 1.3: Plant materials with altered components in the

C/S1 system that will be further analysed in other WPs (2 and 3) in collaboration with other members of the network

• Del. 2.2: experimental data on specific binding of heterodimers of C/S1 bZIPs.

• Del. 5.3: Organisation of Complementary Skill Training worskshops, CST1 (Madrid, November 2011).

• Del. 6.2 and 6.3: Recruitment of ESRs Progress / results

The project has been developed so far by other member of the UPM laboratory. Different mutant lines have been generated in C/S1 members that will be studied according to the proposed plan by ESR6.


Inmediate plans following the incorporation of ERS6 include: • Transcriptomic analyses of developing seeds of C/S1 mutant lines to

uncover the particular contribution of different bZIPs in the control of seed gene expression

• Phenotipic analyses of C/S1 altered lines under abiotic and nutrient stress conditions. Preliminary results indicate that these factors have an important role in plant responses to these factors and will serve to determine experimental cnditions to further characterised the molecular mechanisms involved.

Local training The ESR will follow courses within the Master Programme of the Biotechnology Department UPM (www.bit.etsia.upm.es/doctorado_i.htm). Due to the incorporation date, ESR6 missed the CST1 activity during the Madrid Meeting (Nov, 2011). ESR6 will followed this activity in the next months, since it is regularly offered by the Institute of Education at UPM.


Since the incorporation is January 2012, network activities have not been started by the ESR (see above).

Collaborations

During the reported period, our laboratory has been connected to WDL´s group for the generation of plant materialas that will be further analysed by ESR6 after her incorporation.

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The inmediate analyses include transcriptomic analyses of C/S1 bZIP loss-of-function combinations during seed development, and phenotypic characterisation under abiotic stress conditions.


During this period a temporary stay of one member from our group in WDL’s laboratory has helped in the establisment of new techniques and protocols in our lab. Assitance of ERS6 to the Utrecht groups is expected in the next term to attend a Skill Training Workshop in Statistics and to perform microarray data analyses as part of a planned secondment within the network activities.



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Name Abhroop Garg Project title

ESR 7: Analysis of the combinatorial control of bZIP10-dependent stress responses

ESR number 7 Supervisor Christina Chaban Co-supervisor Jesus Vicente-Carbajosa Official Starting date 1.10.2011 Recruitment process The position was announced at the official MERIT website. More than 15

applications were obtained, mainly from the young researchers from China, India, Ukraine, Russia and the North African states. The applications had been thoroughly examined and the best applications were then selected for the discussions between the group members. At the Kick-off Meeting in Utrecht, several applicants have been interviewed. Unfortunately, none of them corresponded to the desired requirements. Therefore, a second round of interviews was organized by CC and UZ at the UTU, to which 5 applicants were invited. Due to the lengthy procedure for obtaining visa, only 4 could actually participate in the interviewing process, which took place on 4th-5th April, 2011. In order to familiarize the potential researcher with the living and working conditions, the tours through the Institute facilities, as well as through the city were organized. Abhroop Garg was among the interviewed candidates and has been selected for the actual position. Again, due to the delay in obtaining the visa, he could not start before 1st October, 2011.


The member of C-group bZIP transcription factors bZIP10 is under the main focus of the project. It forms functional dimers with several bZIP factors, which belong to the S1-group and are in the focus of other MERIT participants. It was shown that interaction of bZIP10 and its closest homolog bZIP25, with the S1-group member bZIP53 is necessary to activate both, the Proline Dehydrogenase (ProDH) and the seed storage albumin 2S2 (Alonso et al., 2009; Weltmeier et al., 2006). bZIP10 also interacts with Lesion Simulating Disease1 (LSD1) that functions as a negative regulator of the plant cell death (Dietrich et al., 1997). Since plants with enhanced bZIP10 expression level demonstrate higher rate of cell death only in the lsd1 mutant background, it is believed that bZIP10 function is in some way inhibited by LSD1 (Kaminaka et all., 2006). However, the mechanisms of this inhibition, the molecular pathways involving bZIP10 function, the involvement of S1-group bZIPs in LSD1-dependent regulation of bZIP10 as well as the direct target genes of bZIP10 are yet unknown. Therefore, the main objectives of the project are as follows: - Identification of the direct bZIP10 target genes; - The effect of LSD1 on bZIP10-S1 heterodimer formation, localization and

activity; - Influence of the external factors (different stress signals) and internal

signals (SnRK1-dependent phosphorylation) on bZIP10 intracellular distribution and expression of bZIP10 target genes;

- Analysis of the redox-dependent bZIP10 regulation and identification of the bZIP10/LSD1-interacting proteins;

- Analysis of the bZIP10 mutant lines (in different backgrounds) upon various treatments (for example, cold, salt, osmotic stress etc.)

Deliverables Deliverable 5: Provide material for metabolic analysis in WP3. - Delivery date was settled for 12-th project month. However, the position has been occupied only from October 1-st, i.e. for around three months. Therefore, the actual delivery date should be postponed. Currently, ERS7 has started with examination of mutant lines and testing treatment conditions, which will be then used for material preparation.

Progress / results

Major task: The effect of LSD1 on bZIP10-S1 heterodimer formation, localization and activity.

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- cloning of LSD1 versions containing Nuclear Export Signal (NES): the NES has been added to the LSD1 coding sequence either at the N-, C-, or both termini by PCR amplification. The resulting fragments have been cloned in the pENTR/D-TOPO vector from Invitrogen. The generated clones are currently under verification. - cloning of different fragments of ProDH promoter: two promoter fragments have been amplified by PCR, (a) containing only ACGT-core sequences; (b) containing both ACGT-core and ACTCAT-cis-element. The GUS reporter gene construct containing only ACTCAT-cis-element (bZIP53 binding site, Weltmeier at al., 2006) has been provided by WDL. Major task: Analysis of the bZIP10 mutant lines (in different backgrounds) upon various treatments (for example, cold, salt, osmotic stress etc.). - cold treatment experiment: seeds of the following lines have been sown and individually picked in pots filled with soil: Col-0, bzip10-ko, bzip25-ko, bzip10-ko/ bzip25-ko, bZIP10-HA#21, bZIP25-GFP. Seedlings will be kept at the ambient temperature (control) or at 4-6°C (cold treated).


Major task: The effect of LSD1 on bZIP10-S1 heterodimer formation, localization and activity. - Generate binary constructs of the translational fusions of LSD1 versions

containing NES with RFP or mCherry. Use these constructs for the transient expression in tobacco leaves and Arabidopsis protoplasts.

- Follow the localization of bZIP10 and LSD1 when expressed alone or co-expressed. Analyze the influence of oxidative signal on the localization of wt and cysteine mutants of bZIP10.

- Perform yeast two hybrid (Y2H) and FRET-FLIM analysis of the interaction between bZIP10 (wt and Cys-mutants), S1-group bZIPs and LSD1; test the influence of oxidative signal on the interaction.

- Initiate the reporter gene activation studies. Major task: Identification of the direct bZIP10 target genes. - Continue generating the transgenic lines expressing GFP-tagged versions

of bZIP10. Major task: Analysis of the mutant lines (in different backgrounds) upon various treatments. - Monitor growth responses of different mutant lines to cold treatment; select

the lines with similar phenotypic responses for further metabolomic analysis.

Local training A Personal Career Development Plan (PCDP) has been formulated by the supervisor in cooperation with the ESR7 and has been approved by the co-supervisor. At the host institution, ESR7 followed the following courses / activities: - Retreat of Plant Physiology Department; 5.10.11 – 7.10.11. ESR7 gave a

short presentation on his Master’s research project results and got acquainted with the main research projects carried out at the department.

- One-day workshop on CLC Genomics Workbench; 29.09.11. - Weekly seminars on “Progress in Plant Physiology”; no own presentation

has been made yet. - Weekly literature seminar in plant physiology and plant biochemistry; no

own presentation has been made yet. Network training / activities

Starting from his appointment at the position, ESR7 has followed following network training / activities: - 1st MERIT Annual Meeting M1, 19.11.11 – 23.11.11, Madrid - CST1, workshop on efficient oral presentation of scientific data; 21.11.11, Madrid.

Collaborations No new collaboration was built up during reported period.

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No exchanges have been done yet. However, the visiting of the lab of BS has been already discussed and planned for the beginning of February 2012 (duration up to one week). During this time, ERS7 will learn how to perform phylogenetic analysis of protein families and will analyze phylogeny of bZIP factors with especial accent on the evolutionary events involved in the formation of the highly conserved DNA-binding domain of these factors.


Oral presentations:

Garg, A. and Chaban, C. Introduction to: Analysis of the combinatorial control of bZIP10-dependent stress responses, 1. Annual meeting, November 2011, Madrid.


ESR7 attended following conferences, meetings, summer schools: - 5th RegioPlantScience Meeting; 5.11.11, Ulm, Germany. - 1st MERIT Annual Meeting M1, 19.11.11 – 23.11.11, Madrid (oral presentation) - CST1, workshop on efficient oral presentation of scientific data; 21.11.11, Madrid.

27

Name Carles Marco Llorca Project title

ESR 8: Role of GBF1 in the crosstalk between LES and natural senescence

ESR number ESR 8 Supervisor Prof. Dr. Ulrike Zentgraf Co-supervisor Dr. Matthew Hannah (MERIT), Prof Dr. Harter (local) Official Starting date May 15, 2011 Recruitment process The position was announced on the EURAXESS web page of the European

Commission and by the German Society of Botany mailing list. Two mini symposia for the selection of suitable candidates were performed, one in combination with the Kick-off meeting in Utrecht and one in Tübingen on 4/5th of April, 2011. Among the five candidates invited to Tübingen, Carles Marco Llorca was selected as the best candidate for ESR8 because of his two master degrees, one in biology and one in biochemistry which ideally combine the necessary basic knowledge demanded to perform the project.


Developmental as well as dark-induced leaf senescence is accompanied by a massive change in the transcriptome clearly implying an important role of transcription factors in these overlapping processes. We could show that G-box binding factor1 (GBF1), belonging to the G-group bZIPs, is involved in the induction of developmental leaf senescence since gbf1 mutant plants exhibit a delayed senescence phenotype. Further characterization of gbf1 mutant plants revealed that GBF1 is a negative regulator of CAT2 and RUBISCO expression. Senescence-specific CAT2 downregulation and increasing levels of hydrogen peroxide, which can be observed in wildtype plant, are lost in gbf1 mutants. This increasing level of hydrogen peroxide is most likely a signal to induce the expression of senescence-associated transcription factors and genes. However, GBF1 expression is not transcriptionally up-regulated during leaf senescence; therefore, other mechanisms have to be responsible for activation of GBF1 as negative regulator of leaf senescence. This could be achieved either by the formation of specific heterodimers with other bZIP factors or by modification of the protein e.g. by specific kinases or regulation of translation or by changing of the intracellular localization. The aim of this project will be to characterize this senescence-specific activation of GBF1 and the crosstalk to low energy stress in more detail. Therefore, the interaction with several pre-selected candidate bZIP factors will be tested using bimolecular fluorescence complementation assays (BiFC) and FACS analyses of transiently transformed protoplasts. The intracellular localization of the GBF1 heterodimers and their impact on the specific target gene selection will be characterized. Furthermore, the role of casein kinase II and sugar non-fermenting related protein kinase1 (SnRK1) in leaf senescence and cross talk between leaf senescence and low energy syndrome will be addressed.

Deliverables Del. No.1, WP6, Lead beneficiary 1: Recruitment of ESR8 Del. No. 7, WP6 Lead beneficiary 3: personal career plan of Carles Llorca approved Del. No.15: WP2, 4, Lead beneficiary 2: ChII homozygous knock-out line lines to study bZIP function in low energy signaling and senescence. Del. No.6, WP4, Lead beneficiary 7: Methods for senescence phenotyping is established Del. No.22, WP4, Lead beneficiary 7: Identification of pZIP63 overexpressor with statistically significant phenotype of altered senescence. Still has to be confirmed by repeating the evaluation.

Progress / results

• The interaction of GBF1 with bZIP factors potentially related to the LES, namely, bZIP1, bZIP2, bZIP53 and bZIP63 were analyzed through BiFC using protoplast transformation with subsequent FACS analyses and confocal microscopy. An interaction matrix between

28

these factors was established. A significant interaction of GBF1 was only determined with bZIP63 and GBF1 itself. However, all tested bZIPs could interact with bZIP63. bZIP2 can form homodimers whereas bZIP1 and bZIP53 cannot or show only very weak interaction. These interaction analyses will be extended in the secondment in Würzburg, which has been postponed to spring 2012 since more constructs for P2H will be available at that time for a high throughput analyze of interaction. Using an ELISA based DNA-binding assay it could be shown that the formation of the heterodimer with bZIP63 had a negative effect on the DNA binding of GBF1 to the CAT2 and RUBISCO promoter sequences.

• Three conserved Serine residues in the GBF1 basic domain, which

are computationally predicted to be phosphorylation sites, were mutated to ASP to mimic phosphorylation or to ALA. The effect of these mutations on the formation of the GBF1 homodimer or the heterodimer with bZIP63 was evaluated. The mutations in the DNA-binding region appear to have no influence on the interaction but these experiments need to be repeated to get conclusive results.

• The phenotype of A. thaliana transgenic plants overexpressing

bZIP63 and the bZIP63-knockout mutants have been evaluated for their senescence phenotype. In the first experiment bZIP63 overexpressors showed a delayed senescence phenotype whereas the mutants seemed to be not affected. However, leaves appeared to be dark green and chlorophyll contents were higher in early stages of development. The phenotyping is currently repeated in more detail.


• CAT2 and RUBISCO promoter-GUS assays in protoplasts are planned to analyze the effect of heterodimer formation on the expression of GBF1 target genes in vivo. Co-transformation with CKII and KIN10 will be performed to characterize the effect of these kinases on target gene expression.

• In vitro kinase assays with CKII and KIN10 will be performed to characterize the phosphorylation sites of the different kinases. Different versions of bZIP63 mutated in phosphorylation sites for KIN10 will be use to analyze their influence on heterodimer formation, intracellular localization and DNA binding of GBF1.

• A collection of bZIPs will be tested for interaction. If there are new candidates for GBF1 regulation by interaction, overexpressing plants will be created and knock-out lines will be characterized and phenotyped. The effect of interaction on DNA-binding and intracellular localization will be evaluated.

• Additional putative phosphorylation sites will be mutated outside the DNA binding domain of GBF1 and the effect of the mutations on DNA-binding, interaction and intracellular localization will be addressed.

Local training Introduction to the use of confocal microscopes. Half-day course given by Dr. York-Dieter Stierhof on August 1st 2011. German course Mondays and Wednesdays from 18:00 to 20:00 since the 17th October at the Language Center of the University of Tuebingen. PhD seminar each Monday during the semester term from 17.00-18.00 (talks on relevant literature or PhD work were given) Progress report every Thursday 9.00 to 10.00


CST1 - Efficient oral presentation of scientific data (2 ECTS) ahead of the 1st annual Meeting of the MERIT Network in Madrid between the 19 and 23 of November.

29

Collaborations

Seeds of KIN10 overexpressors and knock-out plants have been provided by Elena Baena-Gonzales to evaluate the developmental senescence phenotype of these plants. These plants will be transformed with inducible GBF1-GFP overexpressing constructs to evaluate the influence of KIN10 phosphorylation on GBF1 localization during development. Mutated versions of bZIP63 have been provided by Andrea Maier in Vienna to test the effect of phosphorylation on GBF1-bZIP63 interaction


No lab exchanges have been done yet. The first secondment in Würzburg has been postponed until more constructs for P2H are available to test in the high throughput protoplast transformation system. Two secondments are planned now for the next period. One in Wuerzburg at the end of February for one week to find new interaction partners of GBF1 using a high throughput protoplast transformation system. The second is planned in Vienna end of March for one week in Markus Teiges lab to analyze the phosphorylation of GBF1 by KIN10 and KIN11.


Poster: Llorca, C.M., Jäschke, A., Smykowski, A. and Zentgraf, U. Posttranscriptional regulation of the bZIP transcription factor GBF1 during leaf sene

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