Download PDF with detailed project information

Melbourne-Potsdam PhD Programme
Joint projects
Application round 2016/2017
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
Descriptions of the joint projects
Project code
2–3
4 – 21
Project title
P1_DB+SG_1016
Dismantling the evolutionary forces underlying
maternal organelle inheritance
4
6
P2_MH+AS+LW_1016
Defining small molecule-protein interactions to reveal
novel components of sugar signalling in plant growth
and metabolism
P3_MH+RZ_1016
Circadian-regulated dynamics of translation in plants
8
P4_JH+AF_1016
Metabolic transport into plant mitochondria:
promiscuous or specific?
10
P5_JH+AS_1016
BUILDING A WALL – Developing small molecule
biosensors to visualize cell wall biosynthesis
12
14
P6_MM+ZN_1016
Modelling host-pathogen interactions: dissecting the
metabolic interaction between Leishmania parasites
and their macrophage host cells
P7_SP+MG_1016
How are microtubule behaviour and cell plate
progression coordinated during plant cell division?
16
P8_SP+UR+RH_1016
Improving plant root performance
18
20
P9_UR+JK_1016
The effect of sub-optimal temperatures on the cellular
and membrane physiology of developing Arabidopsis
or wheat leaves
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: 07 November 2016
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 2 of 21
TABLE: Overview of joint projects | Melbourne-Potsdam PhD Programme
Legend: | 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); UoM, University
of Melbourne; MPI, Max Planck Institute of Molecular Plant Physiology; UP, University of Potsdam.
* 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
PI /
Supervisor 1
PI /
Supervisor 2
P1_DB+SG_1016
Dismantling the evolutionary
forces underlying maternal
organelle inheritance
D. Balding
(UoM)
S. Greiner
(MPI)
P2_MH+AS+LW_1016
Defining small moleculeprotein interactions to reveal
novel components of sugar
signalling in plant growth
and metabolism
M. Haydon
(UoM)
A. Skyricz
(MPI)
P3_MH+RZ_1016
Circadian-regulated
dynamics of translation in
plants
M. Haydon
(UoM)
P4_JH+AF_1016
Metabolic transport into
plant mitochondria:
promiscuous or specific?
P5_JH+AS_1016
P6_MM+ZN_1016
Home
base
Year 1
spent
at *
Year 2
spent
at *
Year 3
spent
at *
Methods
MPI
MPI
UoM
UoM or
MPI
UoM
UoM
MPI
UoM
R. Zoschke
(MPI)
UoM
UoM
MPI
UoM
J.
Heazlewood
(UoM)
A. Fernie
(MPI)
UoM
UoM
UoM |
MPI
MPI
BUILDING A WALL –
Developing small molecule
biosensors to visualize cell
wall biosynthesis
J.
Heazlewood
(UoM)
A. Sampathkumar (MPI)
UoM
UoM
MPI
UoM
mathematical modelling
and experimental
verification; NGS sequence
data generation and
analysis
transcriptomics, size
exclusion chromatography
(SEC) + affinity
chromatography,
physiological assays,
molecular genetics
ribosome profiling,
luciferase reporter assays,
molecular biology
techniques
metabolite measurements,
transporter assays,
biochemical and molecular
biology techniques
FRET sensors, molecular
techniques, cell biology,
cutting edge microscopy,
biochemistry
Modelling host-pathogen
interactions: dissecting the
metabolic interaction
between Leishmania
parasites and their
macrophage host cells
M.
McConville
(UoM)
Z. Nikoloski
(MPI)
MPI
MPI
UoM
MPI
Table continues on next page.
PI /
Supervisor 3
L. Willmitzer
(MPI)
mathematical modelling
and experimental
approaches (13C-labelling
studies and 13C-flux
analysis)
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 3 of 21
Contd. TABLE: Overview of joint projects | Melbourne-Potsdam PhD Programme
Project code ID
Project title
PI /
Supervisor 1
PI /
Supervisor 2
P7_SP+MG_1016
How are microtubule
behaviour and cell plate
progression coordinated
during plant cell division?
S. Persson
(UoM)
M. Grebe (UP)
P8_SP+UR+RH_1016
Improving plant root
performance
S. Persson
(UoM)
U. Roessner
(UoM)
P9_UR+JK_1016
The effect of sub-optimal
temperatures on the cellular
and membrane physiology
of developing Arabidopsis or
wheat leaves
U. Roessner
(UoM)
J. Kopka (MPI)
PI /
Supervisor 3
R. Höfgen
(MPI)
Home
base
Year 1
spent
at *
Year 2
spent
at *
Year 3
spent
at *
Methods
UoM
UoM
UP
UoM
UoM
UoM
UoM
MPI
MPI
MPI
UoM
MPI
cutting-edge cell biology
including live cell imaging
and electron microscopy,
functional biochemical in
vitro assays, genetic
analyses, computational
biology
transcriptomics,
metabolomics, lipidomics,
ionomics, cell biology
techniques
metabolomics, lipidomics,
root-imaging mass
spectrometry
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 4 of 21
Project code: P1_DB+SG_1016
Dismantling the evolutionary forces underlying maternal organelle inheritance
Research groups involved and Area of Expertise
UoM:
David Balding: Mathematical Modeling
http://www.ms.unimelb.edu.au/Personnel/profile.php?PC_id=1520
MIP-MP:
Stephan Greiner: Cytoplasm Genetics, NGS Sequencing
http://www.mpimp-golm.mpg.de/6946/3greiner
Project’s home base:
MPI-MP, Potsdam/Golm, Germany
Summary
In all eukaryotes organelle genomes are transmitted preferentially by the mother, but the underlying reasons for
this fundamental biological principal are far from being understood. The project proposed here aims to elucidate
this question by mathematical modeling and experimental verification of the obtained theoretical predictions.
Project Description
Background and addressed Questions
Why the DNA-containing organelles, chloroplasts and mitochondria, are inherited maternally is a long standing
and unsolved question. Uniparental inheritance excludes organelles from sexual recombination. However,
recombination is believed to be necessary to allow genomes to escape mutational meltdown (the accumulation of
deleterious mutations, a process known as Muller’s ratchet). Uniparental (maternal) organelle transmission
should therefore be an evolutionary dead end. However, accumulating evidence for at least occasional biparental
transmission (or paternal leakage) provides opportunities for sporadic sexual recombination events between
organellar genomes. Those could significantly slow down Muller’s ratchet, but the validity of this assumption for
organelles remains to be assessed. Their genomes fundamentally differ in genome size, organization and
recombination rates from the nucleus. If the low recombination frequencies assumed for organelle genomes are
sufficient to stop the ratchet from clicking is unclear.
Although uniparental inheritance might not be absolute, there must be a selection pressure towards its
predominance. This can be the avoidance of spread of selfish cytoplasmic elements. Such elements can be fast
replicating mutant or incompatible organellar genomes which are maladaptive to the organism. Hence, organelle
inheritance could be an evolutionarily unstable trait, as a trade-off between the evolution of strict uniparental
inheritance (to avoid the spread of selfish cytoplasmic elements) and occurrences of biparental transmission or
paternal leakage (that allows organelle genomes to escape from mutation meltdown by sexual recombination;
see above). The nature of this trade-off is unclear.
In particular, the hypothesis that selfish cytoplasmic elements are the driving forces for uniparental
inheritance has been modeled extensively. From this work, several theoretical problems arose. In brief, the
available models confirm that selfish cytoplasmic elements can drive the fixation of uniparental inheritance in a
population. However, the models fail to explain why (i) the paternal mode of organelle inheritance is rare and (ii)
why “killing one’s own paternal cytoplasm” occurs; i.e. the control of uniparental inheritance by the paternal
gamete, in that it kills its own organelle. Strikingly, this mechanism is frequent in nature. That previous modeling
is insufficient here is mainly due to the fact that non-recombining organelle genomes are assumed. Under this
presumption, the mechanistic problem arises that an inheritance modifier that kills its own (paternal) cytoplasm
cannot be genetically linked with the fittest alleles of the organelle. Hence, the current theoretical problem
connected with uniparental organelle inheritance is not its strong sex linkage per se, but the unclear contribution
of occasional sexual organelle DNA recombination, the dominance of the maternal over the paternal mode, and
the frequent observation that the paternal gamete kills its own organelle.
Modeling and experimental Verification
The proposed project aims to clarify these issues by mathematical modeling and experimental analyses. Under
the assumption of a higher mutational load of the paternal gamete and the possibility for sexual recombination of
oDNA, modeling could yield a compelling explanation for the organelle inheritance pattern observed in nature:
1
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 5 of 21
First, a higher mutational load of the paternal gamete, if confirmed, could explain the predominance of
the maternal inheritance mode. Second, in the occurrence of occasional sexual organelle DNA recombination, the
fittest alleles of the paternal organelle might be able to escape a uniparental inheritance modifier. This could
explain why “killing one’s own paternal cytoplasm” is frequently observed. Third, allowance of sexual
recombination in modeling approaches will clarify whether this is sufficient to overcome Muller’s ratchet.
The key question then will be whether the theoretical values that can be inferred from refined modeling
approaches are in agreement with experimental data on observed paternal leakage frequencies, oDNA
recombination rates, and the mutational load of paternal and maternal gamete.
First work package
We will use modeling to investigate the recombination frequencies needed to overcome Muller’s ratchet in a
small, effectively-haploid organelle genome, an approach previously not undertaken. We will elucidate the tradeoff between biparental inheritance (allowing sexual recombination of organelle DNAs) and the spread of selfish
cytoplasmic elements.
For experimental verification, we will assess the recombination frequencies of the chloroplast genome in
natural populations of Oenothera biennis. This organism displays a biparental chloroplast inheritance, evidence
for sexual recombination of chloroplast DNA is available, and selfish cytoplasmic elements in the form of
incompatible faster-replicating chloroplast genomes are present. A huge collection of accessions is available in the
germplasm collection of Stephan Greiner. Further, data for the strength of biparental chloroplast inheritance of
this organism is already available from the literature and our own data. Plant lines which allow measuring the
fitness effects of selfish cytoplasmic elements are available as well.
Second work package
We will assess whether a higher mutational load of the paternal gamete is able to explain the maternal
dominance of uniparental organelle inheritance. Mutational load and selection coefficients from the modeling will
be verified in an experimental set up, again taking advantage of the biparental chloroplast transmission of O.
biennis. We will measure the mutational load of maternally and paternally inherited chloroplast DNA using NGS
sequencing to search for minor allele variation as indicator for mutational load.
Time Plan
1st year:
2nd year:
3nd year:
Setting up the conceptual framework of the modeling, NGS sequence data generation and initial
analysis (MPI-MP)
With the expertise of Stephan Greiner on cytoplasm genetics the most promising modelling
approaches will be identified and experimental data generated
Modeling (UoM)
Theoretical and data modeling under the supervision of David Balding
Refine modelling and incorporation of sequence data, writing paper and thesis (either MPI-MP or
UoM)
At this stage the student should be largely independent, location of the student according to
his/her preference
Further Reading
Birky C.W. (1995). Uniparental inheritance of mitochondrial and chloroplast genes: Mechanisms and
evolution. Proceedings of the National Academy of Sciences 92: 11331-11338.
Greiner S, Sobanski J, and Bock R (2015). Why are most organelle genomes transmitted maternally?
BioEssays 37: 80-94.
Hoekstra R.F. (2011). Nucleo-cytoplasmic conflict and the evolution of gamete dimorphism. In The Evolution of
Anisogamy, T. Togashi and P.A. Cox, eds (Cambridge: Cambridge University Press), pp. 111-130.
2
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 6 of 21
Project code: P2_MH+AS+LW_1016
Defining small molecule-protein interactions to reveal novel components of sugar
signalling in plant growth and metabolism.
PI 1 Dr Mike Haydon, School of BioSciences, University of Melbourne (Primary Institution)
http://blogs.unimelb.edu.au/haydonlab/
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, Dr Haydon’s
research has shown a role for daily rhythms of sugars produced from photosynthesis in
setting the pace of the circadian clock in Arabidopsis (Haydon et al., Nature, 2013).
Presently, the primary research focus in the lab is to identify novel components of sugar
signalling that act independently of light input. We use genetic and small molecule screens,
transcriptomics and molecular genetic tools to better understand the integration of sugar and
light signals in plant cells.
PI 2 Dr Aleksandra Skirycz/Prof Lothar Willmitzer, MPIMP
http://www.mpimp-golm.mpg.de/2031673/Protein-metabolite_interactome_to_unravel_smallmolecule_signaling
The Willmitzer group is interested in small molecule signalling and regulation. We aim to
understand how diverse metabolites regulate plant growth and development by interacting
with their protein receptors. We have developed a methodological pipeline to study proteinmetabolite interactions in a cell-wide manner. We exploit classical biochemical methods,
state of the art metabolomics and proteomics platforms and bio-physical tools to assess the
binding. Importantly, our experimental approach is suitable not only for endogenous small
molecules, but also for the identification of protein partners of exogenous compounds, such
as drugs and herbicides. At present, the small molecule signalling group (established in
February 2015) comprises two doctoral students and six post-doctoral researchers from
Germany, Poland, Brazil and Columbia.
Proposed project
Carbohydrate metabolism is fundamental to plant growth and biomass production. Sugars
provide the building blocks and energy for life, but also act as modulators of key
developmental and physiological processes. Presently, we have a limited understanding of
the precise molecular pathways that underpin these signalling processes in plant cells. To
define key components of plant sugar signalling, a sensitive luciferase-based assay has
been developed to report dynamic transcriptional responses to sugars in living Arabidopsis
seedlings. A high-throughput small molecule screen based on this assay identified ~80 druglike compounds with significant, reproducible effects on the transcriptional output. These
compounds are predicted to target signalling proteins including kinases, phosphatases and
receptor proteins, but the specific identity of many of the targets in plant cells is unknown.
The aim of this project is to define the specificity of a subset of these compounds on
transcriptional networks using a combination of luciferase-reporter assays, qRT-PCR and
RNA-Seq to investigate their impact on transcripts associated with the circadian clock,
known sugar signalling pathways and novel markers defined by in-house transcriptomics
datasets. The project will also investigate the effects of these compounds on growth (preand post-germination, developmental transitions, circadian rhythms) and metabolism (sugar
quantification, chlorophyll content, chlorophyll fluorescence). A combined strategy of size
exclusion chromatography (SEC) and affinity chromatography (Veyel et al. in revision) will
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 7 of 21
be used to detect in vivo small molecule-protein interactions and identify the specific target
of effector compounds. Molecular genetic tools will then be applied to confirm the role and
mechanism of the target protein in sugar signalling towards defining the contribution to
physiology and development.
Timeline
Project aims
1. Define transcriptional and physiological effects of selected compounds
- Luciferase reporter assays, qRT-PCR, RNA-Seq
- Germination assays, plant growth assays, circadian clock experiments
- Photosynthesis measurements by chlorophyll fluorescence
2. Identify and validate compound-protein interactions
- SEC, Affinity chromatography
- Recombinant protein production for validation of interaction
3. Molecular genetic characterization of target protein
- characterization of mutant or knock-down Arabidopsis plants
- generate transgenic Arabidopsis lines for in vivo protein characterization
University of Melbourne
MPIMP
Year 1
Year 2
Year 3
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 8 of 21
Project code: P3_MH+RZ_1016
Circadian-Regulated Dynamics of Translation in Plants
Mike Haydon, Plant Cell Signalling
University of Melbourne (UoM), Melbourne, Australia (Primary Institution)
http://blogs.unimelb.edu.au/haydonlab/
Reimo Zoschke, Translational Regulation in Plants
Max Planck Institute of Molecular Plant Physiology (MPI-MP), Potsdam, Germany (Secondary Institution)
http://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 understand 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 understanding of the molecular mechanisms of translational regulation in response to in- and external triggers. 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 action.
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 components, 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 (NGS) to identify
the position and density of ribosomes on mRNAs to calculate the translational efficiency of the transcriptome. This technique has revealed the extent of translational regulation in eukaryotes. For example,
1 of 2
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 9 of 21
there is a pronounced change in translational efficiencies of transcripts in maize and Arabidopsis grown
in stress conditions, which is often correlated with differential expression of upstream ORFs (uORFs).
We aim to use ribosome profiling to analyse translational dynamics in circadian regulation at a genomewide scale.
Our goal is (i) to identify transcripts, which are translationally regulated by the circadian clock (i.e., oscillate in translational activity). Additionally, following the circadian dynamics of translational activity will
allow us to (ii) define a comprehensive plant translatome and is anticipated to (iii) lead to the discovery of
numerous novel ORFs (including upstream and short ORFs), which are only temporally expressed in a
limited time window. uORFs are involved in translational regulation of diverse cellular processes. However, only a few uORFs have been assigned to circadian regulation so far. For selected ORFs/uORFs,
which exhibit outstanding clock-driven translational regulation, we aim at a molecular understanding of
the underlying regulatory mechanisms and the downstream consequences and function of their circadian-regulated translation.
Major Approaches and Timeline
Gene expression has been studied at a transcriptome-wide scale. However, transcription is only the first
step of gene expression and much regulation occurs at translational level. Recently, ribosome profiling
revolutionized translation analyses by enabling the genome-wide examination of translational regulation
at high resolution. Ribosome profiling determines the in vivo positions and abundances of ribosomes on
mRNAs by deep sequencing RNase-resistant footprints that translating ribosomes leave on their mRNA
template. Ribosome profiling has been extensively used to study translational regulation and ribosome
behaviour and it currently provides the most comprehensive way to identify translated regions and define
ORFs in an unprecedented depth. However, so far its application in plants has been limited. Altogether,
ribosome profiling provides an excellent novel entry point into the systematic exploration of translational
regulation in circadian oscillations.
Luciferase reporters have proved to be an invaluable tool for the circadian clock field to infer in vivo transcriptional activity in Arabidopsis seedlings. The luciferase reporter can also be used as a translational
reporter by fusing 5’ regulatory sequences in frame with the luciferase gene and/or the target gene coding sequence. By modifying putative elements in the regulatory sequences, such as uORFs, in these
constructs by site-directed mutagenesis and transforming into wild-type and mutant seedlings, we can
determine the specific contribution of selected regulatory elements for translational regulation and gene
function.
Table 1 Timeline of planned work in the PhD project.
Project aims
1. Perform time-course, generate reporter constructs
circadian time-course experiment, sample preparation
preparation of translational reporter constructs
2. Ribosome profiling (RP) and subsequent analyses
ribosome footprint preparation, cDNA library production, NGS
analyses of RP results, identification of novel uORFs
3. Functional analyses of circadian translation
definition of the circadian translatome
uORF analyses: mutation, complementation of reporter constructs
2 of 2
st
1 year
nd
2
year
rd
3 year
UoM
MPI-MP
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 10 of 21
Project code: P4_JH+AF_1016
Metabolic transport into plant mitochondria: promiscuous or specific?
Supervisors
A/Prof Joshua Heazlewood (UoM) - http://www.heazleome.org/
Dr. Alisdair Fernie (MPI) - http://www.mpimp-golm.mpg.de/9205/Alisdair_Fernie
Mitochondria carry out a variety of biochemical processes within the plant cell, however their
primary role is the oxidation of organic acids via the tricarboxylic acid (TCA) cycle and the
synthesis of ATP via the electron transport chain. While many of these reactions are
partitioned from the rest of the cell, there is active transport of many TCA cycle intermediates
between the mitochondria and the cytosol. The inner mitochondrial membrane is relatively
impermeable and consequently transport of metabolites and other solutes across the inner
membrane is catalysed by a series of specific carriers that operate as exchangers/cotransporters. The reference plant Arabidopsis encodes around 50 carrier-type transporters,
with most localizing to the plastid, mitochondria and peroxisome. While a range of
biochemical analyses on Arabidopsis members has occurred over the past 15 years, the in
vitro conclusions of a broad substrate specificity of some members to TCA intermediates has
been surprising, for example, the transport of substrates not considered to occur outside
mitochondria.
Recently we have developed an elegant and robust transporter assay for Golgi localized
nucleotide sugar transporters. The approach, which is coupled to LC-MS/MS detection of
metabolites, enables the assessment of unlabelled substrates and can be readily applied to
‘screen’ substrate specificities of a particular transporter. The assay is especially suited to
antiport transporters since the co-substrate can be readily pre-loaded into liposomes
containing the transporter of interest, all potential substrates are added and then transported
metabolites detected and quantified by mass spectrometry.
The aim with this project is to adapt a transporter assay tailored for nucleotide sugar
transporters to the carrier-type transporter family and then assess substrate specifies of
candidates. Candidate transporters found to have more constrained (specific) substrate
specificities will be further characterized in planta.
How promiscuous are the
mitochondrial transporters in
plants?
The student will initially optimize LC-MS/MS separation and detection approaches (HILIC)
to detect and quantify TCA intermediates and related metabolites. Candidate mitochondrial
carriers from Arabidopsis implicated to have roles in the transport of TCA intermediates will
be cloned and expressed in yeast to conduct proteo-liposome assays with various
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 11 of 21
combinations of proposed metabolic substrates. Concurrently, homozygous knock-out lines
will be obtained for candidate mitochondrial carriers and double knock-out lines created for
paralogues. These mutant lines will be analysed for changes in metabolic pools to provide
clues as to their substrate specificities or to support findings from the transporter assays.
Isolated mitochondria from mutant plant lines will be subjected to biochemical
characterizations using an oxygen electrode, while the physiological analysis of plant growth
and development will be undertaken to examine the effect of inhibiting the exchange of TCA
intermediates.
The findings will substantially increase our understanding of how metabolism is partitioned
between mitochondria and the rest of the cell. The information will be essential for the
rational engineering of plants (crops) with increased energy production capabilities.
Timeline:
UoM – Home Institution (first and second year)
- Optimize detection of metabolites by LC-MS/MS (first year)
- Select candidate transporters, clone and express in yeast (first year)
- Order knock-out lines for candidate transporters (first year)
- Conduct transporter assays (first and second year)
MPI (second and third year)
- Biochemical analysis of isolated mitochondria from mutant plants (second year)
- Physiological and biochemical analysis of mutants plants (second and third year)
Selected publications of the PIs/CIs:
Ebert B, Rautengarten C, Guo X, Xiong G, Solomon PS, Smith-Moritz AM, Herter T, Chan
LJG, Adams PD, 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
Palmieri L, Santoro A, Carrari F, Blanco E, Nunes-Nesi A, Arrigoni R, Genchi F, Fernie
AR, Palmieri F (2008). Identification and characterization of ADNT1, a novel
mitochondrial adenine nucleotide transporter from Arabidopsis. Plant Physiol.
148:1797-808
Palmieri F, Rieder B, Ventrella A, Blanco E, Do PT, Nunes-Nesi A, Trauth AU, Fiermonte
G, Tjaden J, Agrimi G, Kirchberger S, Paradies E, Fernie AR, Neuhaus HE (2009).
Molecular identification and functional characterization of Arabidopsis thaliana
mitochondrial and chloroplastic NAD+ carrier proteins. J Biol Chem, 284: 31249-59
Palmieri F, Pierri CL, De Grassi A, Nunes-Nesi A, Fernie AR. (2011). Evolution, structure
and function of mitochondrial carriers: a review with new insights. Plant J 66: 161181
Rautengarten C, Ebert B, Liu L, Stonebloom S, Smith-Moritz AM, Pauly M, Orellana A,
Scheller HV, Heazlewood JL (2016) The Arabidopsis Golgi-localized GDP-L-fucose
transporter is required for plant development. Nat Commun 7
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 12 of 21
Project code: P5_JH+AS_1016
BUILDING A WALL – Developing small molecule biosensors to visualize
cell wall biosynthesis
Supervisors:
A/Prof Joshua Heazlewood (UoM) - http://www.heazleome.org/
Dr Arun Sampathkumar (MPI-MP) - http://www.mpimp-golm.mpg.de/8353/4sampathkumar
The plant cell wall is an intriguing structure; not only for its critical importance for plant
growth and development, but also as an essential source of renewable biomass and industrial
applications. However, our understanding for how cell walls are put together is still very
limited. Nucleotide sugar transporters (NSTs) are key regulators of cell wall synthesis as they
provide the substrates to make cell wall polymers. Recently, we developed an assay to detect
transport activities and substrate preferences of NSTs (Rautengarten et al., 2014) using this
assay we identified novel transporters for all major nucleotide sugar substrates in plants.
NSTs transport activated sugars, which are predominantly made in the cytosol, into the Golgi
lumen where they serve as substrates for enzymes that make polysaccharides. It is therefore
likely that the NST activity determines the rate with which polysaccharides are made, and
thus that such activity may be used as a proxy for cell wall polymer biosynthesis. Genetically
encoded Foerster resonance energy transfer (FRET) sensors have been proven innovative and
powerful tools that allow monitoring of ion and metabolite levels. The FRET nanosensor
technology relies on the activity of a FRET sensor protein which recognizes and binds a
specific substrate. Substrate binding leads to substrate-dependent conformational
rearrangements of the binding (sensory) domain of the protein. NSTs represent ideal
candidate proteins for FRET-based analysis since the transport of cell wall precursors from
the cytosol into the endomembrane system is based on direct binding of the nucleotide sugars
and a conformational change of the NST to mediate the transport.
We aim to establish FRET-based nanosensors for selected NSTs and measure the rate of
transport at different developmental stages and in response to changes in the
environment.
A precise control of cell wall biosynthesis at different developmental stages is
necessary for formation of complex cell (A) and tissue (B) forms in plants.
A
B
The student will generate gene constructs combining biochemically characterized NSTs with
FRET nanosensors (with Prof. Frommer, Stanford University). We will initially test the
sensor constructs in yeast to assure rapid progression of the project. Sensors that work in the
yeast system will be introduced into Arabidopsis plants. Both the UBI10 promoter and the
endogenous promoter of respective NSTs will be employed to generate FRET constructs for
in planta studies. The constructs will be transformed into the respective NST knock-out lines
to confirm the functionality of the constructs by rescue of the mutant phenotype. Ultimately,
the plant lines will be used to quantitatively measure real-time NST activity and thus monitor
real-time cell wall biosynthesis. Those plants will also be used to unravel regulatory aspects
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 13 of 21
of cell wall biosynthesis during different developmental stages and/or due to environmental
changes and hence allow the correlation of NST activity with subsequent changes in the wall.
The student will be exposed to a number of complementary approaches including molecular
techniques, cell biology, cutting edge microscopy and biochemistry.
The results from this project will greatly improve our understanding of plant cell wall
biosynthesis and potential provide a tool box to selectively improve cell wall synthesis for
industrial applications.
Timeline:
UoM (first year and part of second and third years)
- Generation of constructs and testing in yeast (first year)
- Introduce sensor constructs into Arabidopsis mutant and wild type plants and imaging
of them in different tissues and under varying external conditions (second year)
- Analyse cell wall epitopes in cells where FRET sensors have been used and in
response to corresponding external treatments (third year)
MPIMP (second, and part of third year)
- Co-relative image analysis of FRET sensors with gene expression patterns of cell wall
biosynthetic genes (second and third years)
Selected publications from PI/CIs:
The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis.
Rautengarten C, Ebert B, et al., Heazlewood JL, Scheller HV, Orellana A. PNAS. 111: 11563-8
Identification and Characterization of a Golgi-Localized UDP-Xylose Transporter Family from
Arabidopsis. (2015) Ebert B, Rautengarten C et al Heazlewood JL, Scheller HV Plant Cell. 27:
1218-1227.
The Arabidopsis Golgi-localized GDP-fucose transporter is required for plant development. (2016)
Rautengarten C, Ebert B, Liu L, Smith-Moritz AM, Pauly M, Orellana A, Scheller HV, Heazlewood
JL. prov. accepted Nature Comm. 7.
Visualization of cellulose synthases in Arabidopsis secondary cell walls. (2015) Watanabe Y,
Meents MJ, McDonnell LM, Barkwill S, Sampathkumar A, Cartwright HN,
Demura T, Ehrhardt DW, Samuels AL, Mansfield SD. Science 350: 198–203
Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis
cotyledon pavement cells. (2014) Sampathkumar A, Krupinski P, Wightman R, Milani P, Berquand
A, Boudaoud A, Hamant O, Jönsson H, Meyerowitz EM. Elife. 3: e01967.
Physical Forces Regulate Plant Development and Morphogenesis. (2014) Sampathkumar A, Yan A,
Krupinski P. Meyerowitz EM. Curr. Biol. 24, 475-483
Patterning and life-time of plasma membrane localized cellulose synthase is controlled by actin
organization in Arabidopsis interphase cells. Sampathkumar A, Gutierrez R, McFarlane HE,
Bringmann M, Lindeboom J, Emons AE, Samuels L, Ketelaar T, Ehrhardt D, Persson S (2013). Plant
Physiol 162(2):675-88
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 14 of 21
Project code: P6_MM+ZN_1016
Modelling host-pathogen interactions: dissecting the metabolic interaction between
Leishmania parasites and their macrophage host cells.
Supervisors
Dr. Zoran Nikoloski (MPIMP) is interested in the development of computational methods for
integration and analysis of data from high-throughput technologies in the context of genomescale metabolic networks. The methods are based on a combination of techniques from convex
optimisation, graph theory, mathematical statistics, and dynamical systems.
Website:
http://www.mpimp-golm.mpg.de/13175/6nikoloski
Prof. Malcolm McConville (UoM) is interested in studying the metabolism of medically
important eukaryotic (protist) pathogens that cause diseases such as malaria, toxoplasmosis
and leishmaniasis, with the view of identifying new drug targets. His group exploits
metabolomic, genetic, cell and structural biology approaches to identify and characterize
parasite metabolic pathways essential for virulence.
Website:
http://biomedicalsciences.unimelb.edu.au/research2/biochemistry-andmolecular-biology-research/molecular-parasitology
Project Description
Leishmania spp are insect-transmitted protozoa parasites that cause life-threatening infection
in more than 12 million people world-wide. These parasites invade macrophages, an important
class of host immune cells, and proliferate within the lysosome compartment. The major goal
of this project is to develop detailed genome-scale models of Leishmania and macrophage
metabolism and to integrate them in a combined metabolic model for the infected
macrophages. The combined model will incorporate metabolic interchange that occurs
between host and pathogen. This study will be used to understand how these parasites survive
within the macrophage and identify potential drug targets in both the parasite and the host.
The project will build on the existing genome-scale metabolic reconstructions of Leishmania
and human macrophages and will also consider the incorporation of signaling networks. The
model refinement and validation will utilize data from comprehensive 13C-labeling studies on
Leishmania amastigotes recently undertaken in the McConville lab (Saunders et al. (2014) PloS
Pathogens; Kloehn et al. (2015) PloS Pathogens).
The following approaches will be used to address this goal:
Aim 1. Species-specific stoichiometric models of Leishmania amastigote metabolism will be
developed by further refining existing genome-wide reconstructions. The models will be
validated by comparing the predicted fluxes with flux estimates from (in)stationary 13Cmetabolic flux analyses with simplified model variants (see Szecowka et al. (2013) Plant Cell).
Flux estimates from 13C-labeling experiments with genetically defined Leishmania metabolic
mutants will be used as another point of model validation. Altogether, these models will
provide the basis for experimental as well as in silico comparative analysis of metabolic
rerouting capabilities of Leishmania metabolic mutants.
Aim 2. Models of human THP-1 macrophage metabolism will be developed with additional
incorporation of publicly available transcriptomics and proteomics data sets based on methods
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 15 of 21
proposed in the Nikoloski lab (see Robaina-Estevez et al. PLoS ONE (2015)). McConville’s lab
has recently undertaken a genome-wide siRNA knock-down analysis of THP-1 macrophages to
identify genes that either inhibit or promote intracellular growth (Dagley et al (2015) ASSAY
and Drug Development Technologies). The results of the screen will be used to refine and
calibrate the model. 13C-labeling studies will be undertaken on THP-1 macrophages in which
selected genes have been repressed to determine the impact of knock-down on the host cell
metabolism.
Aim 3. Models developed in Aims 1 and 2 will be combined to understand how parasite and
host cell metabolisms are coupled. These models will be initially investigated in silico by
predicting gene essentiality and effects on growth, as well as to implement flux-coupling driven
prediction of intervention targets. The best ranked targets will be tested genetically (by
deleting genes in Leishmania or siRNA knock-down of genes in the macrophage), to validate
the predictions about effect on growth as well as metabolic rewiring.
Timeline:
MPI-MP (first year and third year)
- development and refinement of genome-wide models of Leishmania and TPH1 macrophage
metabolism (first year)
- Training in (in)stationary 13C-MFA and constraint-based modeling (first year)
- Analysis of 13C-data and refinement of the model variants (third year)
- Thesis write-up (third year)
UoM (second year)
- 13C-labeling studies and development of 13C-flux analysis for axenic amastigotes, macrophages
and infected macrophages
Selected publications of CIs:
Saunders EC, Ng WW, Kloehn J, Chambers JM, Ng M, McConville MJ (2014) Induction of a
stringent metabolic response in intracellular stages of Leishmania mexicana leads to increased
dependence on mitochondrial metabolism. PloS Pathogens 10(1) e1003888
Kloehn J, Saunders EC, O'Callahan S, McConville MJ (2015) Characterization of metabolically
quiescent Leishmania parasites in granulomatous murine lesions using heavy water labeling.
PloS Pathogens 11(2) e1004683
Dagley MJ, Saunders EC, Simpson KJ, McConville MJ (2015) High-content assay for measuring
intracellular growth of Leishmania spp. in human macrophages. ASSAY and Drug Development
Technologies 13(7), 389-401
Szecowka M, Heise R, Tohge T., Nunes-Nesi A, Vosloh D, Huege J, Nikoloski Z, Fernie A R, Stitt
M, Arrivault S (2013) Metabolic fluxes of an illuminated Arabidopsis thaliana rosette. The Plant
Cell 25(2), 694-714.
Robaina-Estevez S, Nikoloski Z (2015) Context-specific metabolic model extraction based on
regularized least squares optimization. PLoS ONE DOI: 10.1371/journal.pone.0131875
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 16 of 21
Project code: P7_SP+MG_1016
How are microtubule behaviour and cell plate progression coordinated during plant
cell division?
Supervisors:
Prof. Markus Grebe (UP): http://www.uni-potsdam.de/index.php?id=13521
Prof. Staffan Persson (UoM): http://blogs.unimelb.edu.au/persson-lab/
Plant growth is underpinned by plant cell division and expansion. A key difference between plant
and animal cells is the presence of a strong but dynamic cell wall that surrounds each plant cell. The
strength of this structure controls the morphology of plant cells and tissues, but has also led to
differences in how plant and animal cells divide. Plant cells divide by building a cell plate that is
supported by a unique microtubule structure called phragmoplast (Fig. 1A). Several proteins
directly binding to, or closely associated with, microtubules, including CLASP, CMU1 and 2 (1) or
SABRE (SAB) (2) are important for microtubule dynamics and impact the progression of the cell
plate and the behaviour of the phragmoplast.
The aim of this project is to elucidate, how the interplay of the CMUs, SAB and CLASP genes
and proteins impact phragmoplast dynamics and cell plate progression.
To this end, the project will employ a range of experimental approaches, such as cutting-edge cell
biology including live cell imaging and electron microscopy, functional biochemical in vitro assays,
genetic analyses and computational biology (cf. refs. 1,4), to resolve the role of these players in
plant cell division, which is of outstanding importance to plant growth and development.
Figure 1. A Schematic view of plant cell division (3); PPB; PreProphase Band, PM; Plasma membrane. B GFP-CMU1
localizes to the cell plate in between the phragmoplast outlined by the microtubule marker mCherry-TUA5 and remains
there until the plate is fully developed (1). C SAB-3xYpet (green) co-localizes with the cell plate marker KNOLLE
(magenta) (2). D Cell division plane orientation is disturbed in the sab-5 mutant (E) compared to the wild type (wt) in
D. Microtubules are marked by mCherry-TUA5 (magenta) and chromatin by H2B-YFP (green) in D, E. Note the tilted
division plane in E sab-5 vs. wt in D (2).
1
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 17 of 21
CLASP can engage with microtubules by moving with their plus ends, where it affects the
dynamics of the microtubules as well as their ability to bend and to interact with the cell cortex. In
addition, we have recently shown that the CMU and the SAB proteins associate or co-localize with
microtubules, respectively, and that the corresponding genes genetically interact with CLASP to
affect microtubule-dependent processes (1,2). Furthermore, SAB localizes to the cell plate and
mutations in SAB affect cell plate orientation (Fig. 1C-E; ref. 2). Similarly, the microtubuleinteracting CMU proteins localize to the cell plate rather than to microtubules during cytokinesis
(Fig. 1B; ref. 1). Taken together, these results lay the foundation to determine the coordinated
action of these proteins during cell plate progression and, thus, cell division.
The student will resolve genetic interactions between the mutants of the above-mentioned
components, in terms of seedling growth, cell plate formation and microtubule behaviour. Using
compatible fluorescently tagged versions of the components the student will investigate, how the
proteins behave during cell division and how their behaviours are coordinated with the microtubules
and the cell plate. These data will be complemented by transmission electron microscopy analyses
(cf. ref. 5). These cell biology endeavours will be qualitatively and quantitatively assessed using
custom-made scripts in imaging software. The student will also express full-length or parts of the
proteins to investigate how they can interact with microtubules in vitro and determine how these
interactions occur using TIRF microscopy.
Expected outcome:
The findings will substantially increase our understanding of how microtubules are coordinated
with the growing cell plate during plant cell division. As cell division drives organism growth, the
project will provide crucial information to understand how plants grow.
Timeline
UoM – Home Institution (first and third years)
- Multiple mutant and reporter line generation (year 1)
- Seedling growth analyses (year 1)
- Expression of components and in vitro characterization (year 1 and/or year 3)
- Advanced microtubule live cell imaging (TIRF microscopy) and image analyses (year 3)
UP (second year)
- Genetic interaction studies during cell plate formation
- Live cell imaging and immunolabelling analyses during cell plate formation
- Transmission electron microscopy analyses
Selected publications
1. Liu Z, Schneider R, Kesten C, Zhang Y, Somssich M, Zhang Y, Fernie AR, Persson S (2016).
Cellulose-Microtubule Uncoupling proteins prevent lateral displacement of microtubules during
cellulose synthesis in Arabidopsis. (2016) Developmental Cell 38: 305-315.
2. Pietra S, Gustavsson A, Kiefer C, Kalmbach L, Hörstedt P, Ikeda Y, Stepanova AN, Alonso JM,
Grebe M (2013). Arabidopsis SABRE and CLASP interact to stabilize cell division plane
orientation and planar polarity. Nature Communications 4: 2779.
3. Barr FA, Gruneberg U (2007). Cytokinesis: placing and making the final cut. Cell 131: 847-860.
4. Endler A, Kesten C, Schneider R, Zhang Y, Ivakov A, Froehlich A, Funke N, Persson S (2015).
A Mechanism for Sustained Cellulose Synthesis during Salt Stress. Cell 162: 1353-64.
5. Boutté Y, Frescatada-Rosa M, Men S, Chow CM, Ebine K, Gustavsson A, Johansson L, Ueda T,
Moore I, Jürgens G, Grebe M (2010). Endocytosis restricts Arabidopsis KNOLLE syntaxin to
the cell division plane during late cytokinesis. EMBO Journal 29: 546-558.
2
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 18 of 21
Project code: P8_SP+UR+RH_1016
Improving plant root performance
Staffan Persson, UoM
Ute Roessner, UoM
Rainer Hoefgen, MPI-MP
Nutrient starvation is one of the major growth-limiting factors for plants and causes substantial
losses to agriculture. Nutrient uptake mainly occurs via roots at the soil – root interface. Plants
can change their root architecture in the soil to explore soil zones for nutrients. These changes
are specific to different nutrients (Fig. 1). For example, phosphate deficiency leads to stunted
primary root and increased lateral root induction, while nitrate starvation leads to elongated
lateral roots. Since all plants cells are surrounded by cell walls that steer cell and tissue
morphology, the changes in root architecture is likely linked to changes in cell wall structures.
Our main question is therefore; How do plant roots change their cell wall structures during
nutrient starvation and re-supply, and how do these changes affect the architecture of the
root?
Fig 1. Nutrient concentration-dependent change in the root architecture of Arabidopsis
seedlings. Arabidopsis seedlings were grown on nutrient rich media with low or high
concentrations of indicated nutrients (Lopez-Bucio et al. 2003).
As mentioned above, changes in cell growth and shape are required for changes in the root
architecture. Cell division and cell elongation are the major factors that drive growth and shape.
These processes are dependent on cell wall synthesis and re-modelling, and modifications of the
cell walls are therefore also essential for changes of root architecture. This notion is further
supported by substantial changes in the gene expression of cell wall related genes in response to
changes in nutrient levels. Yet, the mechanisms of how cell wall biosynthesis and
composition is regulated in response to nutrient availability are unknown.
How will we address these questions?
We will analyse cell wall contents of roots exposed to different nutrients and monitor such
changes by a battery of cell wall specific antibodies. We will use live cell imaging to look at
incorporation of cell wall components and the behaviour of enzymes that produce cell walls. We
will also measure how transcripts and metabolites change in response to the same nutrient
changes. And, finally we will use known mutants that have defects in nutrient sensing or
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 19 of 21
transport and see how cell wall changes occur in these plants. Together, these experiments
should give us much new insight into how plant roots respond to changes in their environment,
which should lead to insights into how to tailor crop plants for growth on nutrient deficient soil.
The three groups of Persson, Roessner, and Hoefgen contribute the expertise to address the
above questions. More than pure academic interest, this investigation might provide a basis for
improving plant growth under conditions otherwise reducing yields.
Timeline:
UoM – Home Institution (first and second years)
- Make suitable crosses for subsequent analyses (first year)
- Analyze crossed material using cutting edge microscopy (second year)
- Metabolite, and transcript analyses (first year)
MPI (third year)
- Mutant performance and omics analyses
- Computational modelling (in collaboration with Dr Zoran Nikoloski’s group)
Webpages:
Staffan Persson:
Rainer Hoefgen:
Ute Roessner:
http://blogs.unimelb.edu.au/persson-lab/
http://www.mpimp-golm.mpg.de/10698/Rainer_Hoefgen
http://roessnerlab.biosciences.uom.org.au/
Selected publications of the PIs/CIs:
1. BRASSINOSTEROID INSENSITIVE2 Down-Regulates Cellulose Synthesis in Arabidopsis by Phosphorylating
Cellulose Synthase1. (Accepted) Sanchez-Rodriguez C, Ketelaar KJ, Schneider R, Somerville CR, Persson S*,
Wallace IS*. Proc Natl Acad Sci U S A.
2. Cellulose-Microtubule Uncoupling Proteins Prevent Lateral Displacement of Microtubules during Cellulose
Synthesis in Arabidopsis. (2016) Liu Z, Schneider R, Kesten C, Zhang Y, Somssich M, Zhang Y, Fernie AR,
Persson S. Developmental Cell. 38(3):305-15
3. 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. 162: 1353-64.
4. Sulfur deficiency-induced repressor proteins optimize glucosinolate biosynthesis in plants. Aarabi F, Kusajima M,
Tohge T, Konishi T, Gigolashvili T, Takamune M, Sasazaki Y, Watanabe M, Nakashita H, Fernie AR, Saito K,
Takahashi H, Hubberten HM, Hoefgen R, Maruyama-Nakashita A. (2016) Sci Adv. 2(10):e1601087.
5. Local and systemic regulation of sulfur homeostasis in roots of Arabidopsis thaliana. (2012) Hubberten HM,
Drozd A, Tran BV, Hesse H, Hoefgen R. Plant J. 72(4):625-35.
6. Nitrogen assimilation system in maize is regulated by developmental and tissue-specific mechanisms. (2016) Plett
D, Holtham L, Baumann U, Kalashyan E, Francis K, Enju A, Toubia J, Roessner U, Bacic A, Rafalski A, Dhugga
KS, Tester M, Garnett T, Kaiser BN. Plant Mol Biol. 92(3):293-312
7. The response of the maize nitrate transport system to nitrogen demand and supply across the lifecycle. (2013)
Garnett T, Conn V, Plett D, Conn S, Zanghellini J, Mackenzie N, Enju A, Francis K, Holtham L, Roessner U,
Boughton B, Bacic A, Shirley N, Rafalski A, Dhugga K, Tester M, Kaiser BN. New Phytol. 198(1):82-94
8. Metabolite profiling reveals distinct changes in carbon and nitrogen metabolism in phosphate-deficient barley
plants (Hordeum vulgare L.). (2008) Huang CY, Roessner U, Eickmeier I, Genc Y, Callahan DL, Shirley N,
Langridge P, Bacic A. Plant Cell Physiol. 49(5):691-703
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 20 of 21
Project code: P9_UR+JK_1016
The effect of sub-optimal temperatures on the cellular and membrane physiology of
developing Arabidopsis or wheat leaves
Supervisors:
Joachim Kopka (MPI); website: http://www.mpimp-golm.mpg.de/5909/3kopka
Ute Roessner (UoM); website: http://roessnerlab.biosciences.uom.org.au/
Introduction
Securing food production under challenging environments is one of the priority aims in
basic and applied plant sciences. The ability to grow at suboptimal temperature is essential for
the cultivation and yield.
Young leaves are the first to be exposed to challenging chilling conditions. Thus, cold stress
responses of plants will be the focus of this project. The analysis of small young leaves is
challenging due to the small sample size. Enhanced sensitivity of metabolite profiling and
spatial metabolomics imaging techniques now allow investigations of these small samples.
This project aims to gain new molecular understanding of early leaf development under
sub-optimal temperatures taking both a comparative integrative systems analytical
approach and a targeted functional analysis approach using both Arabidopsis thaliana, a
dicot model plant, and wheat, a monocot crop that is economically important both for
German and Australian agronomy.
We will use Arabidopsis ribosome biogenesis mutants (reil mutants) recently discovered by
the group of Joachim Kopka. These mutants are conditionally cold (10°C) sensitive when
they arrest leaf development but maintain development at optimal conditions (20°C). Not
much is known on specific adaptations of young leaves at low temperatures.
In order to analyze the effects of the REIL ribosome biogenesis factors on ribosome
abundance, we established sucrose sedimentation profiling of Arabidopsis ribosome subunits
and mono- to oligosomes. This profiling method of ribosome composition, which separates
eukaryotic from plastid ribosome components, will enable new insights into temperature
effects on ribosome subunit composition.
Also, young leaves respond to low temperatures by adjusting the availability of
carbohydrates, amino acids, and osmo-protectants. Cellular membranes are also highly
affected by cold temperatures. The composition of cellular membranes varies under suboptimal temperatures to maintain fluidity, integrity and function. The membranes are lipid
bilayers built from a vast array of lipid species, which allow the required fluidity, integrity
and functionality for a given environment. The biophysical structure of lipid bilayers is
known to be temperature responsive. Mammalians maintain cellular membrane structure and
functions through homeostasis of body temperatures however much less is known about how
plants cope with non-homeostatic sub-optimal temperatures.
The conditional growth of the reil mutants and the current lack of information on the
involvement of ribosome biogenesis / translation compared to the transcriptional control of
metabolism driving growth at suboptimal temperature in Arabidopsis make the mutant and
wildtype a highly promising model for the investigation of underlying functions that allow
winter annuals to grow under unfavorable temperature conditions.
The groups of Ute Roessner and Joachim Kopka are well established to perform a
functional systems analysis of leaf growth at suboptimal temperature. The highly localized
Melbourne-Potsdam PhD Programme: Application round 2016/2017 | Joint Projects | page 21 of 21
expression of the reil gene indicates that one mechanism may be restricted to meiosis and
respective cell division zones in the leaf. For this purpose, the group of Ute Roessner
provides the essential opportunity of metabolite and lipid imaging of leaf tissues (MALDIIMS). A translation of this work from Arabidopsis to commercially relevant crops such as
wheat will provide novel insights in how wheat adjusts and adapts to sub-optimal
temperature.
With the joined potential for systems wide molecular and biochemical analysis technologies
of Ute Roessner´s and Joachim Kopka´s research groups we propose to investigate the
involvement of ribosomal biogenesis in the control of leaf growth together with aspects of
metabolic and transcriptional control. This will be studied by complementation of the reil
mutants and with wildtype controls. Experimental designs will use temperature shifts and
explore responses of developing leaves from wild type and mutants during cold acclimation
and after re-acclimation to optimal temperature.
The hypothesis of this project is:
Central and lipid metabolism as well as ribosome biogenesis are altered under sub-optimal
temperatures providing sustainability of physiological and biochemical processes. We
hypothesize, under chilling or cold, developing leaves adjust ribosome biogenesis, primary
metabolism and membrane functions (fluidity and integrity) by membrane remodeling in
order to maintain leaf growth at suboptimal temperatures.
Suggested project plan:
Year 1 (MPI):
ARA WORK - growth (mutant and WT), temperature regimes, metabolomics, ribosome
analysis (work at MPI)
WHEAT WORK – establishment of wheat cultivation under standardized temperature
regimes, metabolomics, ribosome analysis (work at MPI)
Year 2 (UoM):
ARA WORK – lipidomics, imaging mass spectrometry of developing dicot leaves
WHEAT WORK – lipidomics, imaging mass spectrometry of developing monocot leaves
Year 3 (MPI):
Data analysis, interpretation, manuscript preparation
Selected publications:
Schmidt S, Dethloff F, Beine-Golovchuk O, Kopka J (2013) The REIL1 and REIL2 proteins
of Arabidopsis thaliana are required for leaf growth in the cold. Plant Physiology 163: 1623–
1639
Schmidt S, Dethloff F, Beine-Golovchuk O, Kopka J (2014) REIL proteins of Arabidopsis
thaliana interact in yeast-2-hybrid assays with homologs of the yeast Rlp24, Rpl24A,
Rlp24B, Arx1 and Jjj1 proteins. Plant Signaling and Behavior 9: e28224