Utilising CRISPR/Cas9 mutagenesis to understand stress-induced flavonoid biosynthesis and regulation in Marchantia polymorpha : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Palmerston North, New Zealand. EMBARGOED until 17th May 2025.

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Flavonoids are secondary metabolites that evolved during land plant colonisation to provide protection against the plethora of abiotic and biotic stresses present in a terrestrial environment. The bryophytes lineage is thought have arisen at the first branching event during evolution of the extant land plants, making them the sister group to all other land plants. Moreover, extant bryophytes retain features in common with early land plant ancestors. Together, these aspects make bryophytes particularly important for understanding land plant evolution. While the biosynthesis and regulation of flavonoids in angiosperms (flowering plants) is well understood, this knowledge is lacking for bryophytes. Thus, there is growing interest in using Marchantia polymorpha (hereafter, Marchantia), a model species for bryophytes, in comparative studies with angiosperms to investigate the conservation of the flavonoid biosynthetic pathway among land plants. Marchantia produces two main flavonoids under stress conditions: colourless flavones that function in UV-B light protection and a cell wall-bound red pigment called auronidin. The Marchantia Flavone synthase (MpFNSI) and BTB-TAZ Domain 2 (MpBT2) candidate genes were hypothesised to be involved in flavone biosynthesis and auronidin regulation, respectively. Gene function was assessed by using CRISPR/Cas9 mutagenesis and Agrobacterium tumefaciens-mediated Marchantia spore transformation to generate fnsI and bt2 loss-of-function mutants and characterising their ability to produce flavonoids under abiotic stress conditions. The fnsI mutants did not produce flavones, confirming that MpFNSI is required for flavone biosynthesis. Flavonoid analysis of bt2 mutants indicated that MpBT2 does not regulate auronidin production, as no significant differences in auronidin content were observed compared to wild-type control plants. Functional characterisation of MpFNSI and MpBT2 contributed to understanding of the conservation of the flavonoid biosynthetic pathway among land plants. Flavones can be produced by two types of FNS enzymes in flowering plants, so identifying that Marchantia uses FNSI established a conserved function of 2-oxoglutarate-dependent dioxygenase-type enzymes in flavone biosynthesis among land plants. The retention of FNS throughout land plant evolution also emphasises the importance of flavones in protecting plants against damaging UV-B light in a terrestrial environment. Identifying that MpBT2 does not regulate auronidin production in Marchantia established differences in the regulation of red pigmentation among land plants, because the production of anthocyanins is regulated by BT2 in flowering plants. This proposes that the regulation of flavonoids by BT2 proteins was acquired with the evolution of anthocyanins after the divergence from bryophytes.
Figure 1.3 is re-used with the publisher's permission. Figure 1.5 is re-used under a CC BY 4.0 Deed Attribution 4.0 International license. Embargoed until 17th May 2025.