Page 241 - PC2019 Program & Proceedings

PLANT CANADA 2019

P55. Interactions between starch biosynthetic enzymes and 14-3-3 adaptor proteins in maize
endosperm
*
Carswell, M. ; I. Tetlow; M. Emes
University of Guelph

Storage starch deposited within the endosperm of cereal grains supports the majority of human caloric
intake and serves several industrial applications. The process of synthesizing starch in storage tissues
occurs within amyloplasts and is a highly regulated process involving the activities of various starch
synthases (SSs), starch branching enzymes (SBEs) and starch debranching enzymes (DBEs). In Zea mays,
amyloplasts display a phosphorylation-dependent trimeric protein complex formed between two starch
synthases and a starch branching enzyme, SSI-SSIIa-SBEIIb. However, the precise nature of these
interactions remains elusive. A class of regulatory proteins called 14-3-3 have been hypothesized to
facilitate protein-protein interactions associated with a phosphorylation-dependent complex. Ubiquitous
within all eukaryotes, 14-3-3 proteins are known to bind numerous client proteins at specified
phosphorylated motifs. Such interactions can elicit altered enzyme activities, localization or provide
scaffolding for protein interactions of client proteins. Large proteomic analyses from maize and barley
whole cell extracts have shown 14-3-3 to interact in vitro with enzymes related to various biological
processes including primary carbohydrate metabolism. To establish a regulatory role for 14-3-3 proteins
in starch biosynthesis the subcellular localization of an amyloplast specific isoform was determined.
Expression of a recombinant tagged 14-3-3 was used in combination with pull-down assays to identify in
vitro putative interactors from amyloplast lysate.

Matthew Carswell (mcarswel@uoguelph.ca)

P56. Functional characterization of Arabidopsis thaliana HXXXD-motif (BAHD) acyltransferases
involved in suberin metabolism
*1
2
1
Queralta Castillo, I. ; M. Bernards ; I. Molina
1 The University of Western Ontario
2 Algoma University

Plants synthesize suberin, a lipophilic extracellular barrier that controls water and nutrient loss and
protects plants against pathogen infection. In Arabidopsis thaliana, suberin is deposited in the cell walls
of seed coats, endodermis, and periderm of roots. Suberin is a complex polymer formed by an aliphatic
polyester and an aromatic polymer, co-deposited with soluble waxes. The aliphatic polyester is composed
of ω-hydroxy fatty acids, α,ω-dicarboxylic acids, fatty alcohols, glycerol, and ferulate. Alkyl ferulates,
alkyl coumarates and alkyl caffeates are major components of suberin-associated root waxes, which also
contain alkanes, fatty acids and fatty alcohols. Two enzymes of the HXXXD-motif family of
acyltransferases, ASFT (AT5G41040) and FACT (AT5G63560), function as feruloyl- and caffeoyl-CoA
transferases, respectively. However, the enzyme responsible for transferring coumarate remains unknown.
Individual mutants of these enzymes are not affected in either alkyl coumarates or suberin-bound
coumarate, but in vitro both enzymes can transfer coumaroyl-CoA to fatty alcohols, suggesting that these
enzymes may have partially redundant function in suberin and wax biosynthesis. To investigate redundant
functionality in vivo, we generated double mutants (asft x fact). In addition, co-expression analysis with
suberin-specific genes identified two BAHD candidate genes. We will report our progress in the
characterization of asft x fact double mutants, and the new candidate genes.

Indira Queralta Castillo (yqueralt@uwo.ca)

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