Page 237 - PC2019 Program & Proceedings

PLANT CANADA 2019

P47. Post-translational modification in the regulation of starch branching enzyme 2.2 from
Arabidopsis thaliana
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MacNeill, G. ; I. Tetlow; M. Emes
University of Guelph

Starch, an insoluble carbon store comprised of glucose chains found in higher plants, is a major
component of the human diet. Unlike storage starch which is produced for long term or cross-generational
energy storage, transient starch is produced and degraded over the diurnal cycle. This temporary store of
carbon in chloroplasts provides a source of energy and fixed carbon while plants are not
photosynthesizing. Biosynthesis occurs through the coordinated activity of multiple classes of enzymes.
Starch synthases polymerize ADP-glucose into linear α-glucan chains, while starch branching enzymes
(SBE) introduce branch points to the growing glucan. SBEs form phosphorylation dependent complexes
with other starch biosynthetic enzymes important for normal function. Two SBE isoforms exist in
Arabidopsis (SBE2.1 and SBE2.2), of which SBE2.2 accounts for most of the measurable activity.
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Recombinant SBE2.2 was phosphorylated by chloroplast extracts on residues Ser and Ser . Site-
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directed mutagenesis was used to alter a conserved putative protein-protein interaction domain,
phosphorylatable serines, and Cys residues to investigate their importance in catalysis and the formation
of heteromeric complexes in vitro. The in vivo relevance of these post-translational modifications is being
investigated by functional complementation of a sbe2.1/sbe2.2 knockout Arabidopsis line with wildtype
and mutated SBE2.2 sequences. Effects on starch biosynthesis and granule structure will be determined.
This research is significant for its applications to crop production and targeted manipulation of starch
structure.

Gregory MacNeill (macneilg@uoguelph.ca)

P48. Natural variation in glucosinolate profiles in Camelina sativa and its wild relative
Amyot, L.; A. Hannoufa; T. McDowell; J. Renaud
Agriculture and Agri-Food Canada

Glucosinolates (GSs) are the major limiting factor for the use of Camelina sativa seed meal in animal
feed. In addition to their bitter taste, GSs can be toxic at high levels. The goal of our work was to evaluate
GS composition in C. sativa and its wild relatives to determine whether there is potential for introducing
wild germplasm into breeding programs.

Three major glucosinolates were identified in our study using LC-MS/MS and were shown to have
species-specific profiles. Principal component analysis revealed similar clustering at the metabolome
level suggesting that GSs are chemotaxonomic markers for Camelina species.

Quantification by HPLC showed that C. microcarpa cytotypes 4x and 6x had the lowest total GS content.
Interestingly, these same species had relatively higher amounts of GS nitrile (GSn) and GS isothiocyanate
(GSi) degradation products indicating that there are species-specific differences in GS catabolism. C. laxa
and the two C. hispida subspecies, which also had relatively low total GS content, had the lowest GS
catabolite levels.

Based on differences in GS composition and metabolism, some wild relatives could be incorporated into
breeding programs to reduce GS levels in C. sativa.

Lisa Amyot (Lisa.Amyot@canada.ca)

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