Page 155 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

hydroxy-fatty acids requires terminal hydroxylation of fatty acid substrates, while production of
dicarboxylic acids requires sequential oxidation of fatty acid substrates through hydroxy- and oxo-fatty
acids. Whether one or two enzymes are involved is unknown. Previous study demonstrated a positive
correlation between expression levels of CYP86A37 and CYP86A38 and the deposition of 18-
hydroxyoleic acid in soybean hairy roots. However, definitive proof for substrate specificities of the
respective enzymes is lacking. Additionally, the enzyme(s) responsible for catalysis of 18-dicarboxylic
acid in soybean remains unknown. My research focuses on employing biochemical techniques and
genome editing to characterize the molecular and functional roles of CYP86A37 and CYP86A38 in
aliphatic suberin biosynthesis in soybean. Screening recombinant protein using in vitro enzyme assays, I
surveyed the substrate specificity of CYP86A37 and CYP86A38. Among the two recombinant enzymes,
CYP86A38 was non-functional for all the substrates used in the study, while recombinant CYP86A37
hydroxylated 16:0, 18:0, 18:1, 20:0, 22:0 and 24:0 fatty acids, oleic acid (18:1) was the preferred
substrate. In planta, both 18-hydroxy oleic acid and 1,18-dicarboxylic oleic acid were reduced in
cyp86a37/cyp86a38 CRISPR soybean lines. These results are novel as they confirm the role of
CYP86A37 as a functional fatty acid ω-hydroxylase responsible for the production of soybean aliphatic
suberin monomers 18-hydroxyoleic acid directly and 1,18-dicarboxylic oleic acid indirectly. Further
understanding of key enzymes involved in aliphatic suberin biosynthesis is important as it establishes the
foundational research towards the protection and improvement of one of Canada’s most important crops.

*[O106] BUILDING OF SUBERIN - THE IMPORTANCE OF TIMING AND A STRONG FOUNDATION.

Jessica L. Sinka and Mark A. Bernards . Department of Biology, Western University, London, ON,
1 1
1
Canada, N6A 5B7
Correspondence to: jsinka2@uwo.ca

Suberin is a cell wall-associated biopolymer that has both poly(phenolic) and poly(aliphatic) elements
assembled into chemically and spatially distinct domains. Domain-specific monomers are formed via a
branched pathway between phenolic and aliphatic metabolisms. I previously conducted stable isotope
13
labeling experiments in which [ C]-glucose was administered to wound-healing potato tuber (Solanum
tuberosum) discs at different times post-wounding. This revealed highly coordinated, temporal changes in
the regulation of the phenolic and aliphatic metabolic ‘branches’. Notably, during early stages of wound-
healing, carbon from glucose was rapidly incorporated into phenolic-destined metabolites, while at later
stages it was shared between phenolic- and aliphatic-destined metabolites. This data supported
previously published transcript accumulation data (RNAseq). But, what is the importance of these
dynamic changes in suberin monomer biosynthesis, and more specifically how does the preferential
synthesis of phenolics affect suberin assembly and ultrastructure? To assess this, RNAi-mediated
silencing of an uncharacterized StHCT (hydroxycinnamoyl transferase) was employed to disrupt phenolic
biosynthesis upstream of ferulic acid (a key component of the phenolic domain and esterified phenolics of
the aliphatic domain). This work is premised on the idea that the phenolic domain acts as an anchor
within the primary cell wall to facilitate attachment of the aliphatic domain and the corollary that a
disrupted phenolic domain will compromise suberin function. Here I present chemical analyses to assess
composition, permeability measurements to assess the functionality, and electron microscopy to evaluate
the ultrastructure of suberin collected from StHCT-RNAi tubers. Suberin is an attractive target for crop
enhancement as it acts an innate physical barrier that confers resistance to drought, pathogens, and
desiccation during crop storage. Better understanding of its temporal regulation and ultrastructure can
help inform strategies for crop enhancement through genetic engineering and/or marker-assisted
breeding.

*[O107] SUBERIN PRODUCTION IN SOYBEAN IS MICROBIOME-RESPONSIVE. Alicia Halhed , Isabel
1
2
1 1
Molina , and Owen Rowland . Department of Biology and Institute of Biochemistry, Carleton University,
2
1125 Colonel By Dr, Ottawa, ON K1S 5B6; and Department of Biology, Algoma University, 1520 Queen
St E, Sault Ste. Marie, ON P6A 2G4
Correspondence to: aliciahalhed@cmail.carleton.ca

Plant stress response mechanisms allow crop species to be resilient and productive in the face of
environmental stress. Plant-associated microorganisms (i.e., the microbiome) contribute to this stress
tolerance, including outcompeting pathogens. To further tolerate stress, plants naturally reinforce the cell

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