urc 2016 poster - nathan jayne
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Investigating Cell Plate Formation Using the Cytokinesis
Inhibitor Endosidin 7
Nathan Jayne, Destiny Davis, Steve McDowell, Eunsook Park, Georgia DrakakakiUniversity of California at Davis | Department of Plant Sciences | Drakakaki Lab
Introduction
Cell plate formation is a complex process,
involving the fusion of vesicles and their
associated cargo. The mechanisms of vesicle
trafficking to the cell plate has yet to be
elucidated. Genetic approaches to disrupt cell
plate formation lead to lethal mutations, therefore
we use a small molecule, Endosidin 7 (ES7) to
study the formation of the cell plate. ES7 disrupts
callose deposition at the cell plate in a non-lethal
concentration dependent manner. However, the
mechanism by which ES7 inhibits cell plate
formation is not known. To uncover this
mechanism we will take the following steps:
1. Identify tolerant mutants in an ES7 tolerance
screen.
2. Characterize subcellular phenotypes, namely
callose deposition at the cell plate.
3. Sequence ES7 tolerant mutants and use Next
Generation Sequencing to identify the target of
ES7.
Identifying the target of ES7 may identity novel
proteins involved in cell plate maturation and
callose deposition.
Materials and Methods
Conclusion
Future Work
Acknowledgements
I will continue by backcrossing and outcrossing
the mutant lines and selecting for ES7 tolerant
progeny, in order to remove extraneous EMS
mutations (Figure 4). These tolerant seedlings will
be screened via root length assay and cell file
organization by confocal microscopy. After
confirming tolerance, I will create a mapping
population to use for sequencing and identification
of the target of ES7.
The molecular mechanisms of vesicle
trafficking at the cell plate will be elucidated by
analyzing the target of ES7. Understanding this
key step in plant cell division will be vital as the
bioenergy industry surges forward, pushing the
boundaries of plant growth.
Both mutants showed striking tolerance to ES7 in
root length assays (Figure 3), yet the callose
phenotype tells a different story. The most
promising mutant by root length, mutant 327.2
does not have restored callose at the cell plate as
expected. This suggests that there might be a
secondary mutation unrelated to cell plate callose
that causes the root length tolerance. Further
characterization is needed to determine the cause
of the root length phenotype and how/if it is
connected to the cell plate callose deposition
pathway.
ES7 Tolerance Screen
A population of A. thaliana seeds, with a YFP-
RABA2A cell plate marker, were mutagenized with
EMS. Seeds were grown on ¼ MS media
containing varying concentrations of ES7 and
screened for tolerance via root length assay
(Figure 1). A secondary screen was done with
DMSO as a control and both 5µM and 10µM ES7
(Figure 3).ImageJ was used to quantify tolerance
in comparison with the parent YFP-RABA2A line.
Subcellular Characterization
-Short Term Treatment-
o Callose deposition at the cell plate was also
analyzed in 3 day old mutant seedlings after a 2
hour treatment of 50μM ES7 compared to the
RABA2A line (Figure 2).
RABA2A Line 288.1
DMSO
5µM
10µM
YFP-RABA2A Callose Merge
Mutant Line 327.2
DM
SO
ES
7
20μm
Following root length analysis, three mutants were selected for further characterization based on ES7
tolerant growth phenotypes. Subcellular analysis was then conducted to check for callose at the cell plate.
Subcellular analysis of the most promising mutant by root length assay, mutant 327.2, is shown below (Figure
2). Cell plates labeled with callose (by aniline blue fluorochrome) are seen in the control (DMSO treated)
sample. However, following ES7 treatment no callose is detected at the cell plates of mutant 327.2, suggesting
that either the amount of callose is too low for aniline blue fluorochrome dye detection or the root length
tolerance is not due to a restoration of cell plate callose in this EMS mutant.
Results
Figure 3
Root Length Analysis of Mutant Lines Top: Line 288.1 vs RABA2A (red lines), the mutant line shows
only a 12.33% root length reduction when treated with 5µM ES7 compared to RABA2A with 65.24%
reduction. This trend continues across increasing concentrations. Bottom: Line 327.1 shows steady
tolerance across increasing ES7 concentrations, yet has less significant tolerance when exposed to
5µM ES7 causing a 29.94% reduction in root length comparted to 70.16% reduction in RABA2A.
Figure 1
Root length analysis of RABA2A parent line and mutant line 288.1 seedlings after 5
days of growth. Seedlings were grown on DMSO (control), 5μM and 10μM ES7, where
the mutant line shows tolerance to ES7 treatment. All three mutant lines showed ES7
tolerance via root length analysis
Contact Information
Figure 2
Subcellular analysis of mutant 327.2 by
confocal microscopy of the root tip. YFP-
RABA2A (green) is used to visualize the cell
plate and aniline blue fluorochrome (blue;
Biosupplies) enables fluorescent callose
labeling in live cells. Under ES7 treatment,
cell plate maturation is disrupted, visible in
the upper cell plate of the bottom row root
tip. DMSO treated samples exhibit normal
cell plate maturation with callose present at
late stages. Following ES7 treatment,
callose is no longer detected and cell plate
defects are visible.
Figure 4
Several A. thaliana plants grown for crosses. The three mutant lines were crossed to the parent
RABA2A line and wild type to observe segregation patterns and prepare for the creation of a
mapping population. The mutants were also crossed with each other to check for allelism.
Destiny Davis – Graduate Student and Mentor
Georgia Drakakaki – Principle Investigator
Funding for this work is provided by NSF Grant
10S 1258135 and the UC Davis 2015 Provost
Undergraduate Fellowship
Nathan Jayne
University of California at Davis
Department of Plant Sciences
Drakakaki Lab
Biotechnology Major
Senior
(510) 565-2576
Plants in Preparation for Crosses
0
0.2
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DMSO 5µM 10µM
Ro
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Len
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(cm
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Line 288.1
RABA2A
288.1
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Line 327.2
RABA2A
327.2