Page 199 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

compounds, which could be leveraged for agricultural or medicinal applications. The challenge is to
identify unique compounds and their corresponding bioactivity by producing them at scale. Pterocarpan
biosynthesis is tightly regulated and requires elicitation in narrow lineages and tissues. To this end, we are
attempting to unravel pterocarpan biosynthesis by drawing on several legumes for “parts” (genes) and
reassembling the pathway in Nicotiana benthamiana. We have introduced enzymes to transform
flavanone substrates, first to an isoflavone scaffold and further downstream, culminating in the formation
of a pterocarpan, (+/-)-medicarpin. The final steps in pterocarpan biosynthesis are catalyzed by a unique
class of enzymes named dirigent proteins (DPs). Our results indicate novel subcellular localization of
uncharacterized DPs in two subclasses: isoflav-3-ene synthase (I3S) and pterocarpan synthase (PTS).
We conducted Agrobacterium-mediated transient expression in N. benthamiana, expressing legume DP
orthologs fused translationally at both the N and C-terminus with yellow fluorescent tags (YFP). Further,
co-infiltration was performed with organelle markers tagged with a cyan fluorescent protein (CFP) to
confirm accurate localization. The appropriate targeting of enzymes in such a multi-protein complex could
ultimately dictate the titers and output in an engineered bioproduction platform. In addition, our
heterologous pterocarpan pathway will serve as a synthetic biology tool to characterize other unknown
features and find optimal isoforms across the legume world. This bioproduction system can enable a
larger-scale yield of pterocarpans for rigorous testing of the suggested agricultural (e.g., pesticides) and
biomedical activities (e.g., antimicrobial, phytoestrogenic, and anticancer drugs).

*[O190] CHARACTERIZATION OF A CYSTEINE PROTEASE FROM PHYTOLACCA AMERICANA
1
AND ITS ASSOCIATION WITH POKEWEED ANTIVIRAL PROTEIN. Annabelle Audet and Katalin A.
1 1
Hudak . Department of Biology, York University, 4700 Keele Street, Toronto, ON, Canada, M3J 1P3
Correspondence to: abellea@yorku.ca

The plant apoplast is an essential extracellular space that harbors a diverse array of proteins crucial for
plant defense. Papain-like cysteine proteases (PLCPs) and ribosome inactivating proteins (RIPs) are
prominent constituents of the Phytolacca americana (American pokeweed) apoplast; however, their
functions remain largely unexplored. We recently identified that the RIP Pokeweed Antiviral Protein (PAP)
binds a putative cysteine protease in pokeweed that we refer to as Phytolacca americana cysteine
protease 1 (PaCP1) and this interaction may be involved in the plant stress response. We hypothesize
that binding inhibits either protein’s activity or that PaCP1 may proteolytically process PAP. Through
bioinformatic predictions, PaCP1 was identified as a papain-like cysteine protease exhibiting conserved
structural features specific to these proteins. Enzymatic activity assays confirmed PaCP1's functionality
and its classification as a cysteine protease. Furthermore, yeast-two hybrid assays validated the PAP-
PaCP1 interaction, while localization studies indicated their extracellular co-localization in the apoplast.
Subsequent enzymatic assays demonstrated that PaCP1proteolytically cleaves PAP, suggesting a
potential role for the distinct degradation products. Future investigations will focus on elucidating the
biological significance of the PAP-PaCP1 interaction under various stress conditions. Differential
expression studies under drought, salicylic acid treatment, and bacterial infection will shed light on the
adaptive responses of PAP and PaCP1. Moreover, analysis of PAP cleavage products during these
stresses will provide insights into their potential role in the plant’s stress response. Since plants are
constantly being challenged by a multitude of abiotic and biotic stresses, this work will contribute to
identifying how different components of the plant’s defense system may work together in the extracellular
space. This ultimately improves our understanding of plant defense strategies, emphasizing the role of
the extracellular space as the plant's primary line of defense.

[O191] GLUTAMINE ACTIVATION OF TOR REGULATES PROTEIN SYNTHESIS IN DEVELOPING
PEAS. Brendan O`Leary, Vinti Kumari, and Christoph Rampitsch
Correspondence to: brendan.oleary@agr.gc.ca

TOR kinase is the hub of an important signalling network in eukaryotes that integrates both nutritional and
hormonal signals to regulate cellular activities. Most studies on upstream regulation of TOR in plants have
been conducted on Arabidopsis seedlings, particularly meristems, where TOR is most strongly responsive
to light and sucrose, and little information is available on TOR function in other plant tissues. Here we
observe clear differences in the nutritional regulation of TOR in different tissues, highlighted by Gln being
the sole observable nutrient activator of TOR in developing pea seeds. In mature Arabidopsis leaves, Gln
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