Melbourne-Potsdam PhD Programme Joint projects Application round 2019/2020 Participating Institutions University of Melbourne, Australia Max Planck Institute of Molecular Plant Physiology, Germany University of Potsdam, Germany Table of Contents Page TABLE: Overview of joint projects 2 Descriptions of the joint projects 3 – 20 Project code Project title P1_AFL+FK_1219 Transgenerational memory: will plants learn to adapt to phosphorus starvation? 3 P2_BE+JH+JL+MS_1219 Understanding how plants mobilize carbon to build trees 5 P3_BE+JH+UR+JK_1219 Untangling the sugar code: How do plants glycosylate metabolites? 7 P4_JG_ML_1219 Elucidating the mechanism of KLU action in growth control 9 P5_MH+RZ_1219 Circadian-regulated dynamics of translation in plants 11 P6_SP+GK+ZN_1219 Environmental constraints of root growth; how does a root change its ability to grow through different layers of soil? 13 P7_SP+JH+ZN_1219 Getting in shape: Understanding principles of how cells obtain their shapes 15 P8_UR+JK+RB_1219 Metabolic adjustments of novel synthetic tobacco species: Allopolyploidy induced metabolome evolution 17 P9_VM+UR+RH_1219 Understanding plant adaptations to abiotic stress via regulation of purine metabolism 19 File compiled by: Dr. Ina Talke Scientific Coordinator PhD Program IMPRS "Primary Metabolism and Plant Growth" Max Planck Institute of Molecular Plant Physiology [email protected]Version: 06 December 2019
P9_VM+UR+RH_1219 Understanding plant adaptations to abiotic stress via regulation of purine metabolism
19
File compiled by: Dr. Ina Talke Scientific Coordinator PhD Program IMPRS "Primary Metabolism and Plant Growth" Max Planck Institute of Molecular Plant Physiology [email protected] Version: 06 December 2019
TABLE: Overview of joint projects | Melbourne-Potsdam PhD Programme | project start: 2020 Legend: UoM, University of Melbourne; MPI, Max Planck Institute of Molecular Plant Physiology; UP, University of Potsdam; | in a year indicates that the first half of the year is spent in one institution, the second in the other (e.g. UoM | MPI – first half at UoM, second half at MPI-MP). * The columns “Year 1, 2, 3 spent at” indicate approximate timing; exact times may need to be adjusted depending on the project needs.
Project code ID Project title PIs / Supervisors MELBOURNE
PIs / Supervisors POTSDAM
Home base
Year 1 spent at *
Year 2 spent at *
Year 3 spent at *
Methods
P1_AFL+FK_1219 Transgenerational memory: will plants learn to adapt to phosphorus starvation?
Alex Fournier-Level Fritz Kragler MPI MPI UoM MPI CRISPR/Cas9 genome editing, long non-coding and small RNAs analysis, Assay for Transposase-Accessible Chromatin (ATAC) sequencing, metabolite analysis, bioinformatic data analysis
P2_BE+JH+JL+MS_1219 Understanding how plants mobilize carbon to build trees
P3_BE+JH+UR+JK_1219 Untangling the sugar code: How do plants glycosylate metabolites?
Berit Ebert, Joshua Heazlewood, Ute Roessner
Joachim Kopka UoM UoM MPI UoM Heterologous expression of plant GTs in yeast or bacteria, synthetic biology of plant pathways in yeast, GC- and LC-MS, reverse genetics and expression analysis (Arabidopsis)
P4_JG_ML_1219 Elucidating the mechanism of KLU action in growth control
John Golz Michael Lenhard UoM UoM UP UoM Transcriptomics (RNA seq), comparative metabolite profiling, molecular biology, in silico promoter analysis, plant work with Arabidopsis, barley and Marchantia
P5_MH+RZ_1219 Circadian-regulated dynamics of translation in plants
Mike Haydon Reimo Zoschke UoM UoM MPI UoM Ribosome profiling, bioinformatics data analysis, molecular biology, plant phenotype analysis
P6_SP+GK+ZN_1219 Environmental constraints of root growth; how does a root change its ability to grow through different layers of soil?
Staffan Persson, Ghazanfar Khan
Zoran Nikoloski UoM UoM MPI /UP
UoM Plant root growth analyses, electron microscopy, X-ray computed tomography, in silico model generation, finite element modelling
P7_SP+JH+ZN_1219 Getting in shape: Understanding principles of how cells obtain their shapes
Staffan Persson, Joshua Heazlewood
Zoran Nikoloski UoM UoM MPI /UP
UoM Combination of cell biology and computational biology: plant mutant screen, microscopy, molecular biology, computational analysis and modelling
MPI MPI UoM MPI GC- and LC-MS metabolite profiling, LC-MS/MS and spatial metabolomics, 15N isotope feeding experiments, abiotic and biotic stress experiments with plants
P9_VM+UR+RH_1219 Understanding plant adaptations to abiotic stress via regulation of purine metabolism
Vanessa Melino, Ute Roessner
Rainer Höfgen MPI MPI UoM MPI In silico promoter analysis, yeast-1-hybrid screen, metabolic analyses (allantoin, hormones), molecular biology, phenotypic analyses in response to N and S deficiency + osmotic stress in rice and Arabidopsis
Page 2
P1_AFL+FK_1219
Transgenerational memory: will plants learn to adapt to phosphorus starvation? Keywords: Plant memory, epigenetics, transgenerational plasticity, molecular imprinting, cellular signalling, nutrient starvation. THE TEAM The Kragler lab at the Max Planck Institute of Molecular Plant Physiology focuses on the function of macromolecular signals transported to distant tissues. Mobile RNAs are transported across tissues, acting as signals and potentially imposing epigenetic modifications. In particular, the male gametophyte producing tissue has the potential to epigenetically alter traits in the next generation. By combining single-cell sequencing with stress response we aim to show that a limited number of cells in distinct flowers receive RNA signals potentially leading to adaptation to stresses in the offspring formed in these flowers. Thus, adaptation to stresses may occur only in a fraction of offspring obscuring the analysis of adaptation rates measured within seed populations. The Adaptive Evolution lab at the University of Melbourne (PI: Alex Fournier-Level) utilises natural diversity in Arabidopsis thaliana grown in multiple ecologically realistic environmental conditions to decipher the genetic basis of adaptation. We use genomic analysis tools to understand how specific genotypes “learn” to adapt. We combine experimental capacity with bioinformatics and integrative genomics skills and this allows us to use genome-wide association and whole transcriptome analysis to identify the genes responsible for subtle variation in phenotype. The two labs are combining molecular and experimental expertise with systems-level epigenomic analysis to propel our understanding of the extent of transgenerational memory and reveal its potential impact on growth of offspring challenged by nutrient stress. THE PROJECT Problem: Earth's non-renewable phosphorus is being depleted at an alarming rate increasing the costs of fertilisers and, thus, crop production Nearly 90% of phosphorus is used in the global food supply chain, most of it in crop fertilisers. At current consumption levels, we will run out of phosphor deposits that can be mined with reasonable costs in around 80 years. Thus, there is an urgent need to dramatically reduce phosphorus requirements in crops and one way of achieving it would be to prime the plant adaptive memory to enhance their response to nutrient starvation. Phenotypic alterations over generations necessarily start with the sensing of environmental stress that is signalled to the offspring through epigenetic modifications. But beyond sensing the nutrient availability, how can plants “memorise” and readily respond in the next generation? The immediate response to phosphorus starvation is starting to emerge, with the involvement of transcription factors, micro/si RNAs and transporters creating a dramatic change in metabolic flow in phosphorus-starved plants. During plant embryogenesis, this flow of metabolite circulates through alternate layers of maternal and offspring tissues putatively modulating the epigenetic imprinting of the embryo. The plant epigenome regulates transcription in response to nutrient variation, but we ignore how some epigenetic marks are carried over to the next generation when others are lost. These effects have been occasionally described at the phenotypic level, but neither the molecular bases nor the genes
Conceptual framework for the epigenomic analysis of the transgenerational response using high-throughput sequencing.
Page 3
P1_AFL+FK_1219
regulating the memory process are known. A plant model enables to track the epigenomic modification over time and across tissues. We would gain unprecedented mechanistic insights on how epigenome contributes to adaptation to environmental stress in diverse genetic backgrounds. Hundreds of different natural accessions of the model plant Arabidopsis thaliana have been screened in Melbourne for their response to phosphorus starvation, leading to the identification of extremely responsive genotypes. Single-cell transcriptomic and epigenomic analysis has been established at the Max Planck Institute to gain a cellular understanding of the epigenetic signalling mechanism. This project aims to explore the mechanisms whereby the flow of macromolecules between the maternal tissue and embryo results in the imprinting of a transgenerational signal allowing plant to readily respond to phosphorus starvation in the next generation. We will genetically manipulate the epigenetic response pathways in contrasted genetic backgrounds using CRISPR/Cas9 genome editing. The mutant lines will be tested for the transfer of epigenetic imprinting molecules (long non-coding and small RNAs) between the different maternal and offspring tissues at embryognesis when exposed to phosphorus starvation. The flow of metabolites will be measured using mass-spectrometry and the chromatin state of the embryo tissue will be monitored using Assay for Transposase-Accessible Chromatin (ATAC) sequencing. The team combines distinguished expertise in genomics and molecular transport physiology. We will train the applicant with the quantitative skills to analyse multi-omics data across multiple genotypes; the Kragler group has a unique experience with molecular transport experiments to achieve the testing. Strong functional hypotheses were generated about the signaling of the phosphorus starvation response and as a team, we gather the best experimental and analytical platform. This combination of adaptive evolution and molecular physiology is optimal for the challenge of identifying the signal controlling transgenerational memory. We will push the boundaries of our current expertise by using novel techniques to test the hypothesis of cell-to-cell imprinting of the transgenerational memory using a combination of wet lab and computational techniques. We will monitor changes in epigenetic state of the meristem over time, from the onset of flowering until embryogenesis, and determine the key epigenetic changes specific to the “learning” plants.
PROJECT PLAN and TIMELINE
Year 1: the project starts at the Max Planck Institute with the generation of the CRISPR mutants and the dissection and tissue preparation for sequencing.
Year 2: at the University of Melbourne, the single cell RNA library will be prepared and sequenced. The analysis of the genomic data will be conducted and the most promising molecular mechanisms identified
Year 3: at the Max Planck Institute, the mechanisms identified will be tested in planta through a novel set of CRISPR mutant and using quantitative complementation across genotypes.
SUPERVISORS WEBSITES Fritz Kragler: https://www.mpimp-golm.mpg.de/6650/3kragler Alex Fournier-Level: adaptive-evolution.org
Confocal Laser Scanning Microscopy images of GFP-tagged early embryos. Loss of GFP fluorescence indicates spread of epigenetic silencing to the offspring (arrows).
Page 4
P2_BE+JH+JL+MS_1219
Project: Understanding how plants mobilize carbon to build trees Supervisors: Dr John Lunn and Prof Mark Stitt - Max-Planck Institute of Molecular Plant Physiology Dr Berit Ebert and A/Prof Joshua Heazlewood - University of Melbourne
Links to supervisor websites: Joshua Heazlewood - http://www.heazleome.org/ Berit Ebert - https://blogs.unimelb.edu.au/ebert-lab/ Mark Stitt - https://www.mpimp-golm.mpg.de/9333/Mark_Stitt
Background: The plant cell wall constitutes the most abundant renewable bioresource on the planet with about 160 billion tonnes produced each year. While representing a major sink for global CO2, cell walls are a vital source of materials for construction, textiles and feedstocks for the chemical industry, as well as a globally important renewable fuel source. Cell wall biosynthesis is a highly intricate process that involves many hundreds of enzymes and an array of metabolic reactions, intracellular transport of proteins and wall precursors, to assemble the different cell wall polymers. Growing cells are surrounded by the primary cell wall, which provides mechanical strength but can also expand. The much thicker and stronger secondary wall is deposited after the cell has stopped growing, and accounts for most of the carbohydrate in plant biomass. This transition from a flexible primary cell wall to a rigid secondary cell wall represents a major developmental change requiring extensive metabolic re-programming. This includes changes in the patterns of cellulose deposition, a reduction in pectin biosynthesis, changes to hemicellulose composition (increased xylan) and the inclusion of lignin (Fig. 1). Such a transition is also likely to have major impacts on photosynthesis, sucrose and starch synthesis, amino acid biosynthesis, glycolysis and the TCA cycle. While studying this transition in plants has been difficult, in recent years a simple experimental system has been developed that enables the controlled induction of secondary cell wall development through a transcription factor (VND7). Thus, VND7 inducible plants provide a unique system to examine the controlled transition from primary to secondary cell walls.
This project will involve carbon isotope labelling studies to provide information about the rates of synthesis of cell wall components and primary metabolism during the transition from primary to secondary cell walls. The project will employ photosynthetic fixation of 13CO2 using stable (13C) carbon isotopes (Fig. 2) followed by liquid and gas chromatography coupled to mass spectrometry to determine 13C changes in both nucleotide sugars (LC-MS/MS), the composition of assembled cell wall polymers using HPAEC-PAD / GC-MS and a range of primary metabolites by mass spectrometry.
The project will also address the role of sugar signalling, especially the sucrose-signal trehalose 6-phosphate (Tre6P), during the transition to secondary cell walls. Our unpublished work indicates that TPS1 – the main Tre6P-synthesizing enzyme – interacts with the primary cell wall biosynthetic apparatus. The project will exploit the VND induction system to perform co-immunoprecipitation (co-IP) experiments with TPS1 during the induced transition to secondary cell wall synthesis, to test whether TPS1 also interacts with the secondary cell wall machinery, and to investigate levels of Tre6P and other central metabolites during the transition to secondary cell walls. Depending on initial results, we will also
Fig 1. Primary (left) and Secondary (right) Cell Walls
Page 5
P2_BE+JH+JL+MS_1219 cross VND expressing lines into mutants disturbed in Tre6P synthesis and signalling to learn if Tre6P has an important role in coordinating metabolic reconfiguration during secondary cell wall induction. A major component of secondary cell walls is the hemicellulose xylan, which contains a xylose backbone derived from UDP-xylose. UDP-xylose biosynthetic and transport mutants will be used in combination with 13C labelling to elucidate the pathway and subcellular
compartmentation of xylan biosynthesis. Xylan appears to originate from a cytosolic source of UDP-xylose (UDP-Xyl) that is transported into the Golgi. Curiously, plants also biosynthesise a Golgi source of UDP-Xyl but this seems to be a precursor for UDP-arabinose biosynthesis within the Golgi. We have generated mutants that are unable to biosynthesize the cytosolic or lumenal forms of UDP-Xyl and these will be employed to understand how these metabolic pools are used during the transition to secondary cell walls.
This project will examine how the transition to secondary cell walls impacts carbon metabolism and cell wall biosynthesis. Outcomes should provide significant insight into how carbon is being mobilized to form the secondary cell wall –one of the most important renewable resources on the
planet.
Proposed timeline: First year: UoM – (i) generate inducible-VND7 mutants; (ii) optimize induction conditions and perform time course induction experiment; (iii) prepare soluble extracts from samples to measure nucleotide sugars (LC-MS/MS), and send samples to MPIMP for Tre6P analysis; (iv) hydrolyse insoluble (cell wall) material and send samples to MPIMP for sugar analysis (LC-MS/MS); (v) depending on the result, initiate crossing of inducible-VND7 lines with Tre6P mutants; (vi) selection and validation of cell wall (xylan biosynthesis) mutants. Second year: MPIMP – (i) 13CO2 labelling of Arabidopsis wildtype, inducible-VND7 and UDP-Xyl mutants; (ii) isotopomer analysis of soluble metabolites (nucleotide sugar analysis at UoM) and sugar monomers from cell wall hydrolysis using LC-MS/MS; (iii) co-IP experiments on induced and non-induced VND7 lines; (iv) immunoblotting and proteomic analysis of co-IP samples; (v) if (iv) indicates interaction of TPS1 with secondary cell wall synthesis machinery, cross established GFP-tagged TPS1 lines with RFP-tagged CesA4/CesA7/CesA8 lines to investigate interaction in vivo. Third year: UoM – (i) 13C data analysis; (ii) confocal microscopy of GFP/RFP-tagged lines; (iii) write thesis.
Petzold CJ, Pauly M, Willats WGT, Heazlewood JL, Scheller HV (2015) Identification and characterization of a Golgi localized UDP-xylose transporter family from Arabidopsis. Plant Cell 27: 1218-1227
2. Verbančič J, Lunn JE, Stitt M and Persson S (2018) Carbon Supply and the Regulation of Cell Wall Synthesis. Molecular Plant 11: 75-94
3. Figueroa CM, Lunn JE (2016) A tale of two sugars - trehalose 6-phosphate and sucrose. Plant Physiology 172; 7–27
Fig 2. Schematic of 13CO2 labelling experiments modified from Verbančič et al., 2017
Page 6
P3_BE+JH+UR+JK_1219
Project: Untangling the sugar code: How do plants glycosylate metabolites?
Supervisors: Dr Joachim Kopka - Max-Planck Institute of Molecular Plant Physiology A/Prof Joshua Heazlewood, Dr Berit Ebert and Prof Ute Roessner - University of Melbourne
Links to supervisors websites: Kopka lab - http://www.mpimp-golm.mpg.de/5909/3kopka Heazlewood lab - http://www.heazleome.org/ Ebert lab - https://blogs.unimelb.edu.au/ebert-lab/ Roessner lab - http://roessnerlab.biosciences.uom.org.au/ Background: When we think of sugar, it’s of the white crystals that we use to make sweets. However simple sugar molecules can be fused to create powerful structures that are linked to all aspects of life including health and foods. Long sugar chains are called glycans, and the attachment of sugars onto proteins, lipids or glycan structures, is essential to the life of each cell. However, unlike, other building blocks of life, such as DNA, complex sugar molecules are built without an apparent template. The decoration of metabolites, proteins and lipids with sugar residues allows creating a huge diversity among them since a vast number of combinations are possible due to the different starting molecules and the countless ways these can be linked together. It has been hypothesised that these sugar attachments create sugar-based ‘words’ that can be ‘read,’ and their message subsequently be translated into cellular effects. For example, plants produce a multitude of natural products (metabolites) and many of those are decorated with a range of different sugar residues or even oligosaccharide chains. Interestingly, studies involving similar metabolites decorated with different sugar residues suggest that the sugar can have a major effect on the bioactivity of the metabolite.
Problem: We know that the biosynthesis of sugar structures is carefully controlled by all organisms on earth, as often serious problems occur when they go wrong. Yet, the functions of individual sugar structures and most components that make them remain poorly understood.
The attachment of sugar residues onto such compounds is facilitated by a huge class of enzymes called glycosyltransferases (GTs); they catalyze the transfer of a glycosyl moiety from UDP sugars to a wide range of acceptor molecules. Bioinformatic approaches have predicted that more than 500 GTs are represented in plants, which have been classified into families based on amino acid sequence similarities in the carbohydrate active enzyme (CAZy) database. The CAZy database is continually increasing, adding new GTs as they are discovered, and currently contains ~91 GT families overall and 45 in plants.
A) Schematic of GT function B) Example compounds decorated with sugar residues C) Number of GTs present in each of the 45 GT families present in plants. The GT1 family is by far the largest family found in plants.
Page 7
P3_BE+JH+UR+JK_1219
To address this problem, we aim to study members of the GT family of Arabidopsis. We have previously generated a clone collection containing nearly all members of the GT family. Leveraging this clone collection, we will express GTs in yeast or bacteria and screen for changes of the glycosylation patterns of metabolites using mass spectrometry. Given the diversity of plant sugars involved in glycosylating metabolites, the project will employ synthetic biology techniques to generate yeast with a plant sugar profile. Candidate GTs involved in glycosylation will then in the second part of the PhD be analysed in detail using reverse genetics. We will also look at the expression of these GTs and investigate whether they might be linked to specific stresses, signalling paths or organs.
Proposed project plan and timeline: UoM (first year) Synthesis of preliminary data and set-up of the experimental design, generation of destination clones, expression in bacteria/yeast, synthetic biology of plant pathways in yeast MPIMP (second year) Analysis of generated bacteria/yeast lines using GC and LC methods UoM (third year) Plant mutant characterisation, finalise experiments, data analysis and interpretation, thesis and manuscript preparation Selected publications:
1. Gal A, Wirth R, Kopka J, Fratzl P, Faivre D, Scheffel A (2016) Macromolecular recognition directs calcium ions to coccolith mineralization sites. Science 353(6299): 590-593.
2. Dethloff F, Orf I, Kopka J (2017) Rapid in situ 13C tracing of sucrose utilization in Arabidopsis sink and source leaves. Plant Methods 140: 96-109.
3. Lao, J., Oikawa, A., Bromley, J. R., McInerney, P., Suttangkakul, A., Smith-Moritz, A. M., Plahar, H., Chiu, T. Y., Gonzalez Fernandez-Nino, S. M., Ebert, B. et al. & Heazlewood, J. L. (2014) The plant glycosyltransferase clone collection for functional genomics, Plant Journal 79, 517-29.
4. Ebert, B. … Heazlewood, J. L., Bacic, A., Clausen, M. H., Willats, W. G. T. & Scheller, H. V. (2018) The Three Members of the Arabidopsis Glycosyltransferase Family 92 are Functional beta-1,4-Galactan Synthases, Plant Cell Physiology 59, 2624-2636.
5. Ebert B, Rautengarten C, McFarlane HE, Rupasinghe T, Zeng W, Ford K, Scheller HV, Bacic A, Roessner U, Persson S and Heazlewood JL. (2018) A Golgi UDP-GlcNAc transporter delivers substrates for N-linked glycans and sphingolipids. Nature Plants. 4, 792-801.
6. Roessner U and Beckles DM. (2012) Metabolomics for salinity research. Methods Mol Biol. 913, 203-215.
Page 8
P4_JG+ML_1219
Elucidating the mechanism of KLU action in growth control
Supervisors: John Golz (University of Melbourne, UoM) and Michael Lenhard (University of Potsdam, UP)
The cytochrome P450 CYP78A5/KLUH (KLU) promotes growth of shoot organs and seeds in a non-cell autonomous manner by contributing the synthesis of a hitherto unknown mobile signal. Loss-of-function mutants form smaller organs, while overexpression in the endogenous expression domain leads to larger organs than in wild type. The KLU signal appears to be widely conserved throughout angiosperms, as mutants in orthologues from dicotyledonous (Arabidopsis, tomato) and monocotyledonous species (rice, barley) all show very similar phenotypes with reduced leaf and floral organ size, and a faster rate of organ initiation. Sector analysis in Arabidopsis thaliana has indicated that the KLU-dependent signal can travel over long distances, including between different flower buds in the same inflorescence. However, neither its nature, its dynamics, downstream signaling events nor target genes are known.
The proposed project will address these questions by focusing on the following objectives:
1. The student will establish a Dexamethasone-inducible overexpression line of KLU in a homozygous klu mutant background and use this to identify downstream target genes via time-course RNA-seq after induction.
2. Based on the target genes, the student will identify enriched sequence elements in the promoters of KLU-induced or –repressed genes and use these elements to create a synthetic, KLU-dependent transcriptional reporter akin to the DR5-reporter for auxin.
3. This reporter will be used to retrace the signal spread and dynamics following locally restricted KLU induction.
4. To identify candidate metabolites for the KLU-dependent signal, the inducible overexpression line will be combined with klu mutants in A. thaliana and in barley (multinoded dwarf6) to perform comparative metabolite profiling.
5. Candidate compounds will be further characterized for their ability to induce the synthetic transcriptional reporter established above and to promote organ growth when applied to klu mutant seedlings.
6. Determine whether KLU functions are conserved in the basal land plant Marchantia
Together, work on these objectives will provide important insights into the activity and molecular nature of a highly conserved growth-promoting signal in flowering plants.
Timeline:
UoM (Home base, year 1 and 3)
· Generating an inducible KLU over-expression line and performing RNA-seq · Identifying KLU-response elements in target genes and synthesis of a KLU-responsive
reporter · Identifying and characterizing KLU orthologs from Marchantia
Page 9
P4_JG+ML_1219
UP (year 2)
· Comparative metabolomic analysis of klu and multinoded dwarf6. · Testing of the KLU-dependent compound for growth changes in wild type and klu
mutants.
Links to supervisor’s website:
Golz lab – blogs.unimelb.edu.au/golzlab/
Lenhard lab – https://lenhardlab.wordpress.com/
Dr John Golz – School of BioSciences, University of Melbourne
· Expertise in developmental genetics, particularly understanding the function of the Groucho/TUP1-like co-repressors of Arabidopsis
· Extensive experience with gene expression analyses including RNA-seq, RNA in situ hybridization, qRT-PCR and protein-protein techniques including yeast two-hybrid and bimolecular fluorescence complementation
Prof Michael Lenhard – Institute of Biochemistry and Biology, University of Potsdam
· Expertise in developmental genetics, with an emphasis on size control mechanisms in plant lateral organs and seeds
· Extensive experience with techniques associated with the analysis of plant development including cutting edge confocal and scanning electron microscopy
Page 10
P5_MH+RZ_1219 Joint PhD project in Haydon and Zoschke lab
1 of 2
Circadian-Regulated Dynamics of Translation in Plants Mike Haydon, Plant Cell Signalling University of Melbourne (UoM), Melbourne, Australia (Primary Institution) https://blogs.unimelb.edu.au/haydonlab/ Reimo Zoschke, Translational Regulation in Plants Max Planck Institute of Molecular Plant Physiology (MPI-MP), Potsdam, Germany (Secondary Institution) https://www.mpimp-golm.mpg.de/2070554/translational-regulation-in-plants
Research in the Haydon and Zoschke Labs
The Haydon lab is interested in plant cell signalling, with a particular interest in integration of external and internal signals and their impact on plant physiology. For example, our research has revealed a role for daily rhythms of sugars produced from photosynthesis in setting the pace of the circadian clock in Arabidopsis. This discovery presents an intriguing paradigm for how external (e.g. light) and internal (e.g. metabolism) cues converge to affect plant behaviour. Ongoing research in the lab aims to identify the signalling pathways by which sugars feed into the circadian clock and other regulatory networks. We use genetic and small molecule screens, transcriptomics and molecular genetic tools to better under-stand the integration of sugar and light signals in plant cells.
In the Zoschke lab we have a research focus on translational regulation in plants. We are fascinated by translation as the interface between RNA and protein metabolism. Our research projects aim at an un-derstanding of the molecular mechanisms of translational regulation in response to in- and external trig-gers. We use molecular biology, biochemical and genetic approaches to analyse translational regulation, identify the regulatory cis-elements and trans-factors involved and unravel their molecular mode of ac-tion.
Relevant publications from our labs can be found at our webpages.
Project
Background and Project Goals
Circadian clocks evolved in all kingdoms of life to adjust diverse cellular processes to the predictable daily oscillations of external triggers (e.g. light and temperature). These rhythms are driven by a complex regulatory network with multiple layers of transcriptional, translational and post-translational control of gene expression. This complexity enhances robustness of these rhythms, buffering the core oscillator in fluctuating environmental conditions. Two decades of intensive research has defined a core, circadian oscillator in Arabidopsis comprised of multiple, interlocking, so called transcription-translation feedback loops. The primary focus of this research has been on the transcriptional regulation of these compo-nents, and to some extent on post-translational control. A critical gap in our knowledge of the circadian network in plants is that of translational control.
Ribosome profiling is a cutting-edge technique that uses next-generation sequencing to identify the posi-tion and density of ribosomes on mRNAs to calculate the translational efficiency of the transcriptome. Through an established collaboration between the Haydon and Zoschke labs, we have used this tech-nique to measure daily rhythms of translation in Arabidopsis and determine the extent of control by the
Page 11
P5_MH+RZ_1219 Joint PhD project in Haydon and Zoschke lab
2 of 2
circadian clock. This provides a rich dataset to uncover novel mechanisms of circadian regulation and to deeply understand global dynamics of translation in plants.
This joint PhD project will exploit this data set and integrate this with further ribosome profiling to identify and experimentally verify potential elements of dynamic translational regulation in plants with a particular focus on carbon metabolism and chloroplast function.
Aim 1. Identify elements of translational regulation from circadian ribosome profiling data.
Aim 2. Explore dynamic regulation of translation by photosynthetic metabolites by ribosome profiling of carbon-starved plants.
Aim 3. Genetic, biochemical, and physiological characterisation of novel elements of translational control.
Major Approaches and Timeline
The Haydon and Zoschke labs have generated a high-resolution ribosome profiling data set to capture daily and circadian translation dynamics in Arabidopsis seedlings. The planned PhD project will com-mence at the University of Melbourne with deep analysis of this dataset to identify features and potential regulatory elements (cis-elements and trans-factors) that interconnect the translational control of daily and circadian rhythms, metabolism and chloroplast functions. From these analyses, candidate genes and/or putative translational regulatory elements in nuclear and chloroplast genes will be identified for experimental investigation. To this end, the student will utilise site-directed mutagenesis of plant expres-sion constructs and isolate mutants in candidate nuclear genes putatively involved in translational regula-tion. The second phase of the project will be conducted at MPI-MP and includes ribosome profiling of a carbon starvation time course, the bioinformatic analysis of these data and their comparison to the daily and circadian ribosome profiling data. In parallel, the generation of mutants will continue, including mu-tants of elements that control the rhythmic translational behaviour of chloroplast genes. During the final phase of the project at the University of Melbourne, transient and stable transformation in Arabidopsis will be used to characterise circadian and metabolic phenotypes in mutant lines.
Overall, this project aims to systematically and comprehensively investigate the daily rhythms of transla-tional regulation and define their interconnection with the circadian clock, carbon metabolism and chloro-plast functions. A timeline of the project is shown in Table 1.
Table 1 Timeline of planned work in the PhD project (for details see Major Approaches and Timeline). Project aims 1st year 2nd year 3rd year 1. Ribosome profiling analysis Deep analysis of circadian ribosome profiling data Mutagenesis of identified elements controlling circadian rhythms 2. Ribosome profiling of carbon starvation and mutant isolation Ribosome footprint preparation and data analyses Mutation of potential regulatory elements of chloroplast genes 3. Functional characterisation of mutants from 1. and 2. Phenotyping mutants using a transient transformation system Characterisation of stable transgenic lines
UoM
MPI-MP
Page 12
Environmental constraints of root growth; how does a root change its ability to grow through
different layers of soil?
Supervisors:
Prof Zoran Nikoloski, University of Potsdam, Germany
Getting in shape: Understanding principles of how cells obtain their shapes Supervisors: Prof Zoran Nikoloski, University of Potsdam, Germany https://www.uni-potsdam.de/ibb-bioinformatik/index.html Prof Staffan Persson, University of Melbourne, Australia https://blogs.unimelb.edu.au/persson-lab/ A/Prof Joshua Heazlewood, University of Melbourne, Australia http://www.heazleome.org/ Cells come in many different shapes and sizes. These characteristics reflect and influence their biological functions and underpin the features of organisms. Plants contain a wide variety of different cell types (approx. 40 depending on species) that have a range of different shapes. For example, trichomes are protruding hair-like structures involved in plant defence whereas leaf epidermal cells typically have jigsaw-like shapes (see Figure). Understanding what components that contribute to cell shape will allow us to resolve certain design principles in biology. However, a major obstacle to achieve this goal is the lack of tools that precisely describe shape.
Figure: Arabidopsis epidermal cells. Two types of cells are to be observed, guard cells that form a circle-shaped stomata and epidermal cells which have jigsaw-like shapes. The protrusions of one cell are indentations of the neighbouring cell, referred to as lobes and necks, respectively. Identification of lobes and necks is part of the cell shape characterization. Note that there is variability in shape complexity in the same cell type.
We have recently developed a new tool to characterize complex shapes and we aim to further develop and use this tool to identify genetic components that drive cell shape. We further aim to extend this to measure 3D shapes and connect such shapes with the ability of cells to scatter light. Hence, this project comprises an exciting mix of computational, plant physiology, and molecular & cell biology to understand cell shape. In brief, we developed a tool to measure shapes based on the concept of visibility graphs. This tool is based on centrality measures on visibility graphs to identify positions on the contour of an object that coincide with necks or lobes. However, the visibility graph has another representative feature – the matrix of Euclidean distances between the nodes comprising the graph. This provides a tractable means to compare shapes, which we will do by firstly devise and refine a tool for cell shape differential analysis; in essence, by finding a projection of matrices that maximizes their similarity.
• The tool will be applied on epidermal leaf images of an Arabidopsis homozygous T-DNA mutant library. The epidermal cells of Arabidopsis have complex puzzle-piece like shapes, which we will use as an input to estimate shape variations. This approach will allow us to identify genes that are associated with cell shape variations. The corresponding gene products will subsequently be functionally characterized to understand how they work in context of leaf cell shape determination.
• The tool will be extended to also measure 3D shapes. We aim to obtain 3D approximations of spongy mesophyll cells inside of leaves. The complex shapes of these cells might be associated with light-scattering properties to maximize light uptake in photosynthesis. We therefore aim to put the 3D shapes in context to “random walks” of light through the leaf to see how the shapes can reflect light. This may subsequently be linked through actual light path measurements and combined with mutant analyses that lack complex spongy mesophyll cell shapes that we have at hand in the lab.
Together, these projects will provide an exciting mix of computational biology, plant physiology, and molecular & cell biology to address how cell shape is regulated and how it contributes to key processes in plant biology. Time line:
Year 1: University of Melbourne: Screen mutant collection for pavement cell defects; identify genes associated with shape changes; proceed to develop tools to analyse the genes. Image spongy mesophyll cell volumes in different genotypes. Year 2: University of Potsdam/MPI-MP: Improve computational tools to extend capabilities and implement random walks of light; proceed with cell biology-based experiments to analyse genes from year 1 (via collaborator Dr Arun Sampathkumar). Year 3: University of Melbourne: Finalize the characterization of genes from years 1 & 2, measure how light goes through leaves in different genotypes, implement new tools on existing images (from year 1) to detect for example changes in stomata patterns and other cell-related patterning and shape changes. Selected references: CytoSeg 2.0: automated extraction of actin filaments (Conditionally accepted) Nowak J, Gennerman K, Persson S, Nikoloski Z. Bioinformatics
A molecular mechanism for salt stress-induced microtubule array formation in Arabidopsis (2019) Kesten C, Wallmann A, et al., Oschkinat H, Persson S. Nature Comm. System-wide quantification of the actin cytoskeleton captures design principles that underpin organelle transport in plant cells (2017) Breuer D, Ivakov A, Nowak J, Persson S, Nikoloski Z. Proc Natl Acad Sci U S A. A Mechanism for Sustained Cellulose Synthesis during Salt Stress. (2015) Endler A, Kesten C, Schneider R, Zhang Y,
Ivakov A, Froehlich A, Funke N, Persson S. Cell.
Page 16
talke
Typewritten Text
P7_SP+JH+ZN_1219
P8_UR+JK+RB_1219 Metabolic Adjustments of Novel Synthetic Tobacco Species: Allopolyploidy Induced Metabolome Evolution Supervisors: Dr. Joachim Kopka and Prof. Ralph Bock - Max-Planck Institute of Molecular Plant Physiology (MPIMP) Prof. Ute Roessner - University of Melbourne (UoM)
Links to supervisors websites: Ute Roessner: http://roessnerlab.science.unimelb.edu.au/ Joachim Kopka: https://www.mpimp-golm.mpg.de/10802/Joachim_Kopka Ralph Bock: https://www.mpimp-golm.mpg.de/10981/Ralph_Bock We recently discovered grafting as a non-transgenic technique to induce asexual speciation events [1]. Grafting creates synthetic, allopolyploid species and allows the merging of plant genomes that are naturally isolated due to barriers of sexual propagation (Fig. 1).
Potential advantages of such new species include superior growth properties due to heterosis-like hybrid vigor (Fig. 1) [1], and new chemotypes that arise from the combination of the differentially specialized metabolomes of the parent species. New chemotypes alter stress tolerance and may enhance tolerance especially to biotic stresses. Further expected advantages are superior properties of the new species as production hosts of heterologous, synthetic pathways for the production of small pharmaceutical molecules, for example, artemisinic acid that is generated by joined modification of the plastid and nuclear genomes of N. tabacum, one of the parent species of N. tabauca (Fig. 1; [2]). The heterosis-like hybrid vigor of N. tabauca led us to expect that the chemotypes of the new allopolyploid species comprise non-additive or even novel metabolic effects, especially related to the synthesis of molecules in specialized metabolism with bioactive properties. An initial metabolic screen of two independent speciation events of N. tabauca revealed non-additive effects at the level of typical alkaloids of the genus Nicotiana. N. tabauca lost the ability to accumulate nicotine in leaves (Fig. 2). This biosynthetic property should originate from the N. tabacum parent and is not replaced by anabasine production, a pathway that is active in N. glauca and should be transmitted by the genome fusion. Given these first observations of non-additive behaviour, we expect that the chemotypes of new allopolyploid species will reveal novel biosynthetic competences, especially under stress conditions that induce specialized metabolic pathways, and in tissues and cell types that accumulate specialized metabolites. To address non-additive and new metabolic competence and discover novel synthetic bioactive compounds, we will combine the metabolome profiling and discovery technologies that are available and developed at the MPIMP and UoM, e.g. [3-8], and use especially the spatial metabolomics technologies developed by Prof. Roessner´s lab at UoM [6-8]. The joined technology platforms and expertise will allow us to comprehensively explore allopolyploidy-induced changes in metabolism. Based on this knowledge we will initiate functional studies with the aim to reveal molecular mechanisms that allow two newly combined genomes to merge successfully and exploit the metabolic benefit of genome fusions. The synthetic nature of the allopolyploidization events enables insights not possible by natural speciation events that have occurred a long time ago and are constrained by sexual barriers.
Fig. 1. Synthetic plant species by inter-species grafting. The new species, Nicotiana tabauca with intermediate morphology originates from grafting of the natural species N. tabacum and N. glauca. (Figure modified after Fuentes et al., 2014)
Page 17
P8_UR+JK+RB_1219 Our first insights into alkaloid synthesis in N. tabauca open a line of functional research that we will target - in parallel to our screening approaches - at understanding the molecular mechanism of the lack of nicotine accumulation in N. tabauca leaves and its consequences for this new species (considering, e.g., silencing of the nicotine biosynthesis pathway or of the transport mechanisms that transfer nicotine from the root system into leave tissue of the N. tabacum parent). The latter we will address by stable isotope feeding experiments. In summary, this project will reveal new insights into the molecular mechanisms that contribute to the evolution of the highly diverse universe of plant
specialized metabolism.
This project will explore how metabolism adjusts after two plant genomes merged to form synthetic, allopolyploid species. We expect the discovery of novel metabolites and/or mechanisms of suppression of the competence to synthesize specific metabolites that are present in the progenitor species. Functional studies will reveal molecular mechanisms that act during speciation events to shape the metabolism of new species and are not observable in natural allopolyploid species. Project outline: MPIMP (first year: Kopka/Bock) – Screen synthetic tobacco species [1] for differential accumulation of metabolites in diverse root, shoot and flower materials using multiplexed targeted and non-targeted GC- and LC-MS metabolite profiling technologies at MPIMP, e.g. [3-5]. Build a knowledgebase of differentially accumulating tobacco metabolites and potentially novel metabolites. Analyse nicotine/ alkaloid transport processes by stable 15N isotope feeding experiments and initiate functional molecular studies, e.g. [3].
UoM (second year: Roessner) – Extend the knowledgebase by applying diverse abiotic and biotic stresses. Focus on specific tissue types using LC-MS/MS and spatial metabolomics [6] to characterize tissues types that produce relevant differentially accumulating metabolites. Classify and identify relevant differentially accumulating metabolites using specialized metabolomics technologies [6-8].
MPIMP (third year: Bock/Kopka) – Select metabolic pathways and continue work on transport processes that are likely subject to induced metabolic evolution, and perform functional studies towards understanding mechanisms of induced metabolic evolution following allopolyploidization.
Selected publications: 1. Fuentes I, Stegemann S, Golczyk H, Karcher D, Bock R (2014) Horizontal genome transfer as an asexual path to the
formation of new species. Nature 511: 232-235 (doi:10.1038/nature13291) 2. Fuentes P, Zhou F, Erban A, Karcher D, Kopka J, Bock R (2016) High-level production of the antimalarial drug precursor
artemisinic acid in tobacco plants. eLife 5: e13664 (doi: 10.7554/eLife.13664.001) 3. Dethloff F, Orf I, Kopka J (2017) Rapid in situ 13C tracing of sucrose utilization in Arabidopsis sink and source leaves.
Plant Methods 140: 96-109 (doi: 10.1186/s13007-017-0239-6) 4. Dunn WB, Erban A, Weber RJM, Creek DJ, Brown M, Breitling R, Hankemeier T, Goodacre R, Neumann S, Kopka J,
Viant MR (2013) Mass appeal: metabolite identification in mass spectrometry-focused untargeted metabolomics. Metabolomics 9: S44–S66 (doi: 10.1007/s11306-012-0434-4)
5. Kopka J, Schauer N, Krueger S, Birkemeyer C, Usadel B, Bergmueller E, Doermann P, Weckwerth W, Gibon Y, Stitt M, Willmitzer L, Fernie AR, Steinhauser D (2005) [email protected]: the Golm Metabolome Database. Bioinformatics 21: 1635-1638 (doi: 10.1093/bioinformatics/bti236)
6. Boughton BA, Thinagaran D, Sarabia D, Bacic A, Roessner U (2016) Mass spectrometry imaging for plant biology: a review. Phytochem Rev 3: 445-488 (doi: 10.1007/s11101-015-9440-2)
7. Sarabia LD, Boughton, BA, Rupasinghe T, van de Meene, AML, Callahan DL, Hill CB, Roessner U (2018) High-mass-resolution MALDI mass spectrometry imaging reveals detailed spatial distribution of metabolites and lipids in roots of barley seedlings in response to salinity stress. Metabolomics 14:63 (doi: 10.1007/s11306-018-1359-3)
8. Gupta S, Rupasinghe T, Callahan DL, Natera SHA, Smith PMC, Hill CB, Roessner U, Boughton BA (2019) Spatio-temporal metabolite and elemental profiling of salt stressed barley seeds during initial stages of germination by MALDI-MSI and μ-XRF spectrometry. Frontiers in Plant Sci 10: 1139- (doi: 10.3389/fpls.2019.01139)
Acknowledgement: We thank Itay Maoz, PhD, and Paulina Fuentes, PhD, for contributing to the development of this project proposal.
Fig 2. The synthetic species N. tabauca has new and unexpected metabolic properties. N. tabauca (green) fails to accumulate nicotine in leaves but does not gain competence for enhanced anabasine production (unpublished).
Page 18
P9_VM+UR+RH_1219
Understanding plant adaptations to abiotic stress via regulation of purine metabolism
There are numerous plant responses to deficiencies of macronutrients leading to metabolic reprogramming 1-3 and inducing cellular and whole-plant-physiological changes. Macronutrient uptake is also dependent on water availability. Plants respond to changing concentrations of macronutrients by regulating their transport, activating hormones, reactive oxygen species and related signaling cascades. Plants under nutritional stress also have reduced content of RNA, amino acids and proteins 1, 4. Nitrogen-rich purine nucleotides, derived from the catabolism of RNA, were found to represent 3-4% of the total nitrogen pool of young plants, and their catabolism enabled plants to maintain photosynthesis and growth under stress 4. Specifically, the purine catabolite allantoin was found to be reduced in plants in response to nitrogen deprivation in wheat 4 and rice 5, which was coordinated with up-regulation of purine degradation genes 4, 6. In contrast, allantoin was found to accumulate under drought, salinity, osmotic stress, and in sulphur-deprived Arabidopsis plants 1. Arabidopsis insertion mutants, with impaired allantoinase function, accumulate allantoin and perform better under dehydration, salt and osmotic stress. Furthermore, these mutants have enhanced absicic acid (ABA) and jasmonic acid (JA) metabolism. Allantoinase is transcriptionally regulated, in opposite directions, by nitrogen availability and sulphate availability as well as drought stress (see Figure 1) 6. We have identified a number of highly conserved motifs in the promoter region of aln from cereal plant species as possible regulators of aln expression. However, the mechanism of regulation of allantoinase, which serves as a sensitive switch in response to nitrogen pool sizes, and thereby of purine catabolism is unknown.
Figure 1. Leaf, wheat plants growing under high nitrogen (HN) and low nitrogen (LN). Right, current concept of a plant adapting to drought and salinity stress and regulation of purine and ABA metabolism.
How will we address these unknowns?
Firstly, we will establish the metabolic and phenotypic response of rice and Arabidopsis insertion mutants and inducible overexpression lines in aln background to N and S deficiency, and osmotic stress (drought and salinity). Most of the lines are available at project start. Secondly, an Arabidopsis transcription factor (TF) library will be screened using a yeast-1-hybrid (Y1H) screening technique in order to identify transcription factor candidates, which bind to elements of the aln promotor. Knockout and overexpresser lines will be analysed in comparison to ALN mutant lines.
Page 19
P9_VM+UR+RH_1219 Tasks and timeline MPIMP, Germany; Home institution (Year 1)
• In silico analysis of ALN promoter (support by UoM) based on previous studies • Y1H screen ALN promoter to identify TFs • Producing forward and reverse lines of selected candidate TFs • Start of characterization of candidate lines
UoA, Australia (Year 2) · Establishing growth conditions and · Characterizing mutant and transgenic ALN and TF Arabidopsis and rice lines · Determination of allantoin and hormone levels, especially ABA and JA · Refined in silico promotor analysis based on previous studies of purine degradation pathway
genes (Arabidopsis and rice)
MPIMP, Germany (Year 3) • Continuing analysis of TF candidate lines using the conditions established at UoM • Integrating data of molecular analysis of mutant ALN and TF lines • Conclusions w/r to molecular mechanisms, physiology • Manuscript preparation • Thesis preparation
The candidate will be offered support from leaders in salinity stress tolerance (Roessner)7 and nutrient stress tolerance (Hoefgen and Melino). There will be further support by MPI-MP and UoM PIs. Selected Publications from the PI/CI
1. Nikiforova, V.J., Kopka, J., Tolstikov, V., Fiehn, O., Hopkins, L., Hawkesford, M.J., Hesse, H. & Hoefgen, R. Systems Rebalancing of Metabolism in Response to Sulfur Deprivation, as Revealed by Metabolome Analysis of Arabidopsis Plants. Plant Physiology 138, 304-318 (2005).
2. Heyneke, E., Watanabe, M., Erban, A., Duan, G., Buchner, P., Walther, D., Kopka, J., Hawkesford, M.J. & Hoefgen, R. Characterization of the Wheat Leaf Metabolome during Grain Filling and under Varied N-Supply. Frontiers in Plant Science 8 (2017).
3. Bielecka, M., Watanabe, M., Morcuende, R., Scheible, W.-R., Hawkesford, M.J., Hesse, H. & Hoefgen, R. Transcriptome and metabolome analysis of plant sulfate starvation and resupply provides novel information on transcriptional regulation of metabolism associated with sulfur, nitrogen and phosphorus nutritional responses in Arabidopsis. Frontiers in Plant Science 5 (2015).
4. Melino, V.J., Casartelli, A., George, J., Rupasinghe, T., Roessner, U., Okamoto, M. & Heuer, S. RNA Catabolites Contribute to the Nitrogen Pool and Support Growth Recovery of Wheat. Frontiers in Plant Science 9 (2018).
5. Casartelli, A., Riewe, D., Hubberten, H.M., Altmann, T., Hoefgen, R. & Heuer, S. Exploring traditional aus-type rice for metabolites conferring drought tolerance. Rice 11, 9 (2018).
6. Casartelli, A., Melino, V.J., Baumann, U., Riboni, M., Suchecki, R., Jayasinghe, N.S., Mendis, H., Watanabe, M., Erban, A., Zuther, E., Hoefgen, R., Roessner, U., Okamoto, M. & Heuer, S. Opposite fates of the purine metabolite allantoin under water and nitrogen limitations in bread wheat. Plant Molecular Biology (2019).
7. Hill, C.B., Cassin, A., Keeble-Gagnère, G., Doblin, M.S., Bacic, A. & Roessner, U. De novo transcriptome assembly and analysis of differentially expressed genes of two barley genotypes reveal root-zone-specific responses to salt exposure. Sci Rep-Uk 6, 31558 (2016).
