Auronidin flavonoid pigments are a central component of the response of Marchantia polymorpha to carbon/nitrogen imbalance

The emergence of land plants around 450 million years ago transformed the terrestrial environment. The transition of plants to land required adaptations to a host of abiotic environmental challenges, such as drought, UV-B light, temperature fluctuations, and nutrient deficiency. Key for coping with these stresses was the evolution of specialist metabolite pathways. Of particular note for stress tolerance functions are the flavonoids, which are thought to have arisen with land plants. Recently, a new group of red flavonoid compounds named auronidins were identified in the liverwort model species Marchantia polymorpha (hereafter, Marchantia). The functions of auronidin in abiotic stress resistance are uncharacterised. To address this, we generated near-isogenic lines of Marchantia with either greatly increased or no auronidin production, by overexpression (35 S:MpMYB14) or CRISPR/Cas9 knockout (Mpmyb14), respectively, of MpMYB14, the key R2R3MYB transcription factor regulating auronidin biosynthesis. Among a range of abiotic stresses tested, auronidin production was specifically induced by carbon/nitrogen (C/N) imbalance and by light stress. The presence of auronidin was associated with delayed plant senescence, enhanced photo-protection, and improved plant survival rates under C/N imbalance stress. Generation and analysis of loss-of-function and over-expression Marchantia lines for the homologue to the angiosperm BT2 gene, which induces flavonoid production in response to C/N balance in Arabidopsis, showed that the MpBT gene is not involved in C/N signalling. Thus, the function of BT2 in the regulation of anthocyanin biosynthesis may have arisen through neofunctionalisation during angiosperm evolution. Our study furthers understanding of the function of auronidins in enhancing plant abiotic stress tolerance, revealing that they improve Marchantia tolerance to nutrient imbalance, potentially by serving as a carbon sink.