Page 271 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

aggressiveness in the other. Two F. avenaceum isolates recovered from pea (AET-070 and AET-093)
and one from wheat (FaLH29), all with moderate levels of aggressiveness in durum wheat, pea and lentil,
were selected and an hygromycin expression cassette was inserted into these isolates using CRISPR.
One hygromycin resistant strain from each isolate with growth and disease phenotype similar to their
respective wild-type was used to inoculate pea, CDC Meadow, by a soil inoculation method. Root rot
disease severity, shoot length and emergence were assessed 14 days post inoculation. The inoculated
strains were re-isolated from infected pea roots on potato dextrose agar medium with hygromycin to
select for the strains and streptomycin sulfate and Penicillin G potassium salt to prevent bacterial growth.
Single spore isolates were obtained from the isolated strains. The inoculation and re-isolation were
repeated two more times in CDC Meadow, and each time root rot disease severity, shoot length and
emergence were recorded. For the first and second passage, there were no statistically significant
differences for disease severity, shoot length or emergence between the strains that were passed through
pea roots and their respective hygromycin resistant strains. The third passage is underway. The strains
re-isolated from the third passage will be inoculated onto durum wheat, lentil and pea to assess whether
the serial passage through pea roots alters aggressiveness in these hosts. Similarly, the hygromycin
strains will be passed through durum wheat spikes three times and the strains after the third passage will
be assessed for changes in aggressiveness in the aforementioned cultivars.

[P136] INVESTIGATING THE REGULATORY MECHANISMS OF TRICIN BIOSYNTHESIS IN RICE.
1 1
3
2
1
Yiming Gan , Andy CW Lui , Lydia PY Lam , and Clive Lo . School of Biological Sciences, The
University of Hong Kong, Pokfulam, Hong Kong, China; Plant Breeding and Genetics Section, School of
2
3
Integrative Plant Science, Cornell University, Ithaca,NY 14853, USA; and Center for Crossover
Education, Graduate School of Engineering Science, Akita University, Tegata Gakuen-machi 1-1, Akita
City, Akita 010-8502, Japan
Correspondence to: u3547344@connect.hku.hk

Tricin is a 3',5'-dimethoxyflavone belonging to a large family of plant secondary metabolites named
flavonoids which is characterized by a diphenyl propane (C6-C3-C6) backbone. Tricin has been studied
as O-conjugates for decades due to its widespread distribution in the grass family (Poaceae) and its
protective effects against pathogenic fungi and weeds. Tricin has also been reported to possess
pharmacological bioactivities including anti-inflammatory activity. Recently, tricin is identified as an
insoluble component in lignin polymer to form secondary cell wall in grasses.

In rice (Oryza sativa), the biosynthetic pathway of tricin has been elucidated in our laboratory. The
biosynthesis begins with the formation of naringenin chalcone by chalcone synthase which is then
isomerized by chalcone isomerase to form naringenin. To generate flavone, CYP93G1 functions as
flavone synthase II to convert naringenin into apigenin which is hydroxylated by flavonoid 3'-hydroxylase
(CYP75B4) to form luteolin. To generate tricin, luteolin goes through several methylation and
hydrolyzation reactions catalyzed by ROMT9 and CYP75B4, respectively.

The underlying regulatory mechanisms for tricin biosynthesis have remained unclear. In recent years, a
group of transcription factors (TFs) named Myeloblastosis (MYB) proteins has been reported to regulate
the biosynthesis of phenylpropanoid-derived compounds, including flavonoids, in many plant species.
Here, an MYB protein (provisionally named M14) with similar gene expression patterns to those of tricin
biosynthetic genes has been selected for investigations. To identify the potential physical interaction
between M14 and the tricin biosynthetic genes, yeast one-hybrid assay was conducted. M14 was found
to bind to the promoters of CYP75B4 and ROMT9. The results of DAP-seq also supported that M14
directly bound to the promoters of CYP75B4 and ROMT9. Meanwhile, electromobility shift assay showed
M14 directly bound to ACII motif on the promoters. Also, the results of dual-luciferase reporter assay
suggested that M14 has transcriptional activating activity by interacting with the promoters of CYP75B4
and ROMT9. To understand the role of M14 playing on tricin accumulation, CRISPR/Cas9 mutant lines
were generated. By LC-MS/MS analysis of the flavone profile, it was found that the mutation in M14 led to
a significant reduction in tricin content in the shoots during seedling and heading stages. Further analyses
will be done to characterize the regulatory role of M14 on tricin biosynthesis in rice.

In general, better understanding of the regulatory mechanisms of tricin biosynthesis will provide new
insights into tricin bioengineering for improvement of plant performance and biomass utilization.

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