Page 200 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

is also the strongest nutrient activator of TOR, although many amino acids and sugars also have the
ability to activate TOR to a lesser degree. By contrast, in both the roots and shoots of Arabidopsis
seedlings exogenous Gln does not appreciably activate TOR under non-stressed conditions.
Furthermore, in developing pea seeds Gln activation of TOR is independent of light, whereas in mature
leaves and seedlings TOR signalling is strongly dependent on light. The effect of TOR regulation on
certain aspects of leaf metabolism is also light dependent. Using specific inhibitors we observe that,
besides Gln, TOR activation in developing pea seeds is completely dependent on auxin signalling and
respiratory ATP generation. Therefore, as in other tissues and organisms, TOR activity in developing
seeds serves to integrate information from multiple signals in order to direct seed growth and metabolic
processes. Phosphoproteomic analysis of TOR signalling in developing pea seeds identified established
and novel downstream TOR phosphorylation substrates and revealed a clear categorical enrichment of
proteins involved translation regulation among the differentially phosphorylated targets. Subsequent
experiments demonstrated that bulk protein accumulation in developing pea cotyledons is dependent on
TOR signalling. Therefore, Gln fed to developing seeds via the phloem promotes protein synthesis both
as a substrate and as a signal via TOR. Manipulation of Gln-TOR signalling in seeds, once better
understood, could be used to maximize seed yield and quality traits, like protein content.

*[O192] EXPLORING THE ALKENE BIOSYNTHETIC PATHWAY IN POPULUS TRICHOCARPA.
Jessica Hu , Jeff Chen , Bianca Ortiz , and Eliana Gonzales-Vigil . Department of Biological
1
1,2 1
1,2
1,2
Sciences, University of Toronto - Scarborough, Scarborough, ON, Canada, M1C A14; and Department of
2
Cell and Systems Biology, University of Toronto, Toronto, ON, Canada, M5S 3G5
Correspondence to: e.gonzalesvigil@utoronto.ca

Plants use Very-Long-Chain Fatty Acids (VLCFAs, more than 18-carbons long), for a myriad of purposes,
including building membranes, storing energy and sealing surfaces. VLCFAs are elongated two carbons
at a time from long-chain fatty acids at the ER surface by the Fatty Acid Elongation (FAE) complex. The
first enzyme of the complex, 3-ketoacyl-CoA Synthase (KCS), catalyzes the rate-limiting step and selects
the substrate that are elongated in each cycle of the complex. In Populus trichocarpa, PtKCS1 has been
shown to be the gene responsible for alkene biosynthesis in leaves, elongating cis-ω9 VLCFAs that
subsequently get modified into alkenes deposited on the cuticle. The adjacent gene PtKCS2, which is
73% similar at the protein level, shows preference towards saturated VLCFAs. Surprisingly, rationally
engineered chimeric proteins combining domains of the two genes show promiscuous and high activity
with larger accumulation of VLCFAs products than PtKCS1 and PtKCS2, even when supplemented with
exogenous substrates that are not present in native yeast such as polyunsaturated and odd-chain fatty
acids. We further explored the elongation of cis-ω9 and cis-ω7 substrates and observed possible product
competition among the two unsaturated VLCFAs series. These results serve as a starting point to extend
our knowledge on the alkene biosynthetic pathway in poplar and provide potential candidates for
engineering specific unsaturated VLCFAs in plants at higher levels.

[O193] POPLAR LEAF BUD RESIN BIOCHEMISTRY: SEASONAL PATTERNS AND ENZYMES FOR
RESIN SYNTHESIS IN BLACK COTTONWOOD (POPULUS TRICHOCARPA). C. Peter Constabel,
David Ma, and Eerik-Mikael Piirtola
Correspondence to: cpc@uvic.ca

The synthesis and copious accumulation of resin is a mechanism by which temperate trees protect
dormant leaf buds against frost, herbivory, and other stresses. Such resins contain a diverse array of
secondary metabolites including terpenoids, benzenoids, and phenolics. Populus trichocarpa and P.
balsamifera leaf bud resin is distinguished from resin of other poplars by a high O-methylated
dihydrochalcone content. The biosynthesis of leaf bud resin is poorly understood, and to date no
enzymes involved in leaf bud resin synthesis have been characterized. We used transcriptomics and
differential gene expression analysis to identify a gene encoding a dihydrochalcone-specific O-
methyltransferase, which we named PtDOMT1. This enzyme is a highly selective and regiospecific O-
methyltransferase which methylates only the 4- and 4’-positions of dihydrochalcones. Similar to other
plant O-methyltransferases, it uses S-adenosyl-L-methionine as a methyl donor. PtDOMT1 was not active
with any other flavonoid or phenolic substrate tested, and thus represents a unique molecular tool for
investigating resin-associated gene expression. A seasonal time series in P. trichocarpa indicated that in
lateral leaf buds, resin biosynthesis and accumulation occurs primarily in late summer synchronous with

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