Page 181 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

*[O151] SNAKE CHARMMING: UNDERSTANDING COBRA THROUGH BIOINFORMATICS AND
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MUTATIONAL ANALYSIS. Kamryn Diehl and Geoffrey Wasteneys . Department of Botany, University
of British Columbia
Correspondence to: kamryn.diehl@botany.ubc.ca; geoffery.wasteneys@ubc.ca

Cellulose, the most abundant biopolymer on the planet, is the main component of plant cell walls and
plays a pivotal role in plant development and adaptation to their dynamic environments. Despite its
importance, the precise mechanisms facilitating cellulose biosynthesis and its regulation remain
disjointed. COBRA, a glycosylphosphatidylinositol (GPI)-anchored protein on the outer leaflet of the
plasma membrane, has emerged as a critical player in cellulose biosynthesis with the potential to bridge
some of our knowledge gaps related to the deposition of cellulose in the cell wall. The null mutant, cob-4,
is unable to produce sufficient cellulose and is seedling lethal, cementing COBRA’s critical role in plant
survival. The cob-1 allele, caused by a point mutation, has a conditional phenotype resulting in reduced
cellulose production and root-tip swelling when seedlings are grown on high-sucrose media. It is unclear
why cob-1 misregulates cellulose production, specifically when supplied with high sucrose. Theoretically,
there should be an ample supply of UDP-glucose for building cellulose polymers.

My research aims to utilize molecular dynamic simulations such as CHARMM to dissect COBRA’s
functional domains and post-translational modifications in combination with the analysis of unexplained
mutant phenotypes. COBRA contains an N-terminal cellulose binding domain (CBD), a central cysteine-
rich domain, a C-terminal GPI anchor, and is N-glycosylated at 9 locations. Prediction software on protein
structure suggests the CBD is distal to the plasma membrane, with three highly conserved aromatic
residues. Substitution of these amino acids result in seedling lethality, consistent with COBRA’s function
requiring interaction with cellulose. Furthermore, the cob-1 and hulk-1 conditional mutants have point
mutations between the cysteine-rich domain and the CBD. These mutations alter the mutants' surface
electrostatic charge, potentially affecting cellulose binding.

To further understand why cellulose loss is triggered in cob-1 by high levels of exogenous sucrose,
mutants were plated on defined media with varying amounts of sucrose. Whereas radial swelling of root
tips was induced on Hoagland’s medium with 4.5% sucrose, swelling could be also be induced on ½
Murashige-Skoog (MS) medium with just 1% sucrose. The presence of ammonium in the MS formulation
could account for the enhanced phenotype, potentially by inhibiting uptake of potassium, which along with
nitrate, is known to alleviate ammonium toxicity partially through the expression of cellulose synthase
genes. This finding opens up new avenues to explore the complex relationship between COBRA,
cellulose biosynthesis, and plant adaptation to environmental stress.

[O152] FROM SINGLE CELLS TO COMPLEX TISSUES - THE MOLECULAR DECODING OF PLANT
SEXUAL REPRODUCTION AT SINGLE CELL RESOLUTION. Katharina Bräutigam . Cell and
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Systems Biology, University of Toronto, Toronto, ON, Canada; and Department of Biology, University of
Toronto Mississauga, Mississauga, ON, Canada
Correspondence to: katharina.braeutigam@utoronto.ca

Plant organs are complex structures that are composed of a variety of cell types. At the molecular level,
organ formation reflects precisely coordinated cell differentiation programs and an integrated network for
gene regulation. Here, molecular complexity can be informative. For example, epigenetic factors such as
chromatin organization into accessible or inaccessible regions or DNA methylation patterns set the stage
for the regulation of gene expression.

In our work, we chart for the first time, the epigenome and transcriptome landscape of reproductive
organs in Populus at single cell resolution. We created cell type inventories and characterize molecular
cell differentiation paths. We further detect waves of chromatin re-organization that precede changes in
the transcriptome, and we characterize early divergent meristem identities that mark early stages in sex
determination. This wealth of data allows us now to integrate the different types of information to create
models for the separate male and female reproductive development.
We selected sexual reproduction for our study as it represents one of the most central processes in life.
The focus on the dioecious Populus allowed us to study reproductive development in flowers with

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