Plants are thought to have colonized the land around 500 million years ago. One of the major challenges the first pioneers faced was protection against UV radiation. UV has severe detrimental effects on plant cells
and was at particularly high levels during the period of land colonisation because the ozone layer was not fully developed. Seed plants use a mix of secondary metabolites as UV ‘sunscreens’, and of these the flavonoid
group of phenylpropanoids is of particular importance

Albert, N. W.

Clayton, William

Davies, K. M.

Deroles, S. C.

Jordan, Brian R.

Schwinn, K. E.

Lincoln University Research Archive

UV4Plants Bulletin, 2016, no. 1 Ongoing researchRegulation of UV-Induced Flavonoid Production inMarchantia polymorpha: a role in the evolution of plants forland colonisation?Brian Jordan1, ORCID: 0000-0003-0280-681XNick Albert2, ORCID: 0000-0002-8579-529XWilliam Clayton2, ORCID: 0000-0002-3042-4694Simon Deroles1,2, ORCID: 0000-0001-8154-1922Kathy Schwinn2, ORCID: 0000-0002-6337-3627Kevin Davies2, ORCID: 0000-0001-5652-50151Lincoln University, Christchurch, New Zealand2The New Zealand Institute for Plant & Food Research Limited, Palmerston North, New ZealandDOI: 10.19232/uv4pb.2016.1.13 © 2016 The Authors, licensed underPlants are thought to have colonized theland around 500 million years ago. One ofthe major challenges the ﬁrst pioneers facedwas protection against UV radiation. UVhas severe detrimental eﬀects on plant cellsand was at particularly high levels duringthe period of land colonisation because theozone layer was not fully developed. Seedplants use a mix of secondary metabolites asUV ‘sunscreens’, and of these the ﬂavonoidgroup of phenylpropanoids is of particularimportance. UV-B is perceived in seed plantsby the UVR8 photoreceptor, whose activationstimulates a cascade of transcription factorsthat up-regulate ﬂavonoid gene transcription.Of particular importance are the initial tar-get protein, the bZIP HY5, and the directactivators of the ﬂavonoid genes, which to-gether form the MYB-bHLH-WDR (MBW) tran-scriptional complex. There are no convin-cing reports of the identiﬁcation of ﬂavon-oids from algae and it is generally acceptedthat the phenylpropanoid pathway arose inland plants. However, it is an open questionwhether inducible ﬂavonoid production forUV-B tolerance is a universal system in plantsthat may have evolved during land coloniza-tion, as data is lacking from organisms out-side the angiosperms. Indeed, an alternativeproposal is that the ﬂavonoid pathway arosefor control of auxin action, as ﬂavonols aremodulators of auxin activity.As the closest living relatives of the ﬁrstland plants, bryophytes can help to inform usabout systems that have an early evolution-ary origin. With funding from The MarsdenFund of New Zealand, we are deﬁning theUV tolerance mechanisms of the liverwortMarchantia polymorpha (marchantia) andcomparing them with those of angiosperms,with a focus on the ﬂavonoids. Marchantiais an outstanding model system: it is a smallrapidly growing plant, it is easily transform-able, the main life stage is haploid, it canbe rapidly clonally propagated via gemmaeand it has a small, recently sequenced, gen-ome. Marchantia produces ﬂavones, whichare a key UV-induced ﬂavonoid of many an-giosperms and a red pigment thought to berelated to anthocyanins (Riccionidin A). Inother liverworts that have been studied, Ric-cionidin A appears to be bound to the cellwall, as opposed to the usual vacuolar loca-tion of anthocyanins in angiosperms. Liver-worts also produce a great variety of othersmall metabolites, including many that arethought to be derived from the phenylpro-panoid pathway. Of particular note are thebibenzyls, which are produced in abundanceand have great structural variety in liver-worts, including marchantia. Bibenzyls showanti-fungal activities and are also induced bygeneral stresses such as ABA treatment orUV-C exposure.©2016 by the authors 176 B. Jordan et al.: Regulation of UV-Induced Flavonoid ProductionFigure 6.1: Marchantia polymorpha plant withan archegonia (female sexual organ) and a closeup image of the clonal reproductive organ, thegemmae cup, stained to visualise ﬂavonoidsNothing is known on the biosynthesisof ﬂavonoids in marchantia. We haveused RNAseq analysis of UV-treated plantsand Blast interrogation of transcriptomeand genome resources to identify biosyn-thetic and regulatory gene candidates for themarchantia ﬂavonoid pathway. While theearly steps of the shikimate and phenylpro-panoid pathway can be identiﬁed with con-ﬁdence from sequence similarity to angio-sperm genes, the later steps of ﬂavonoid bio-synthesis will require functional studies forgene assignment. With regard to ﬁnding can-didate regulatory genes, these are even morechallenging to identify based on phylogen-etic analysis alone. Thus, we are character-izing our candidate biosynthetic and regulat-ory genes using transgenic over-expression,CRISPR mutagenesis and functional analysisin genetically deﬁned angiosperm mutants.Results to date include the identiﬁcationof a UV-induced transcription factor thatwhen over-expressed in marchantia confersconstitutive production of large amounts ofﬂavonoids. Strikingly, the over-expressiontransgenics are deep red in colour, from pro-duction of large amounts of Riccionidin A.When we generate CRISPR lines for this gene,the transgenics lose the ability to form redpigmentation.A great advantage of marchantia is the easeof transformation combined with its haploidgenome, as CRISPR events are obtained in theinitial transgenic generation. We are now gen-erating CRISPR knockouts for other genes ofinterest that may relate to the ﬂavonoid/UV-B system. UVR8 and HY5 have been re-ported as conserved across plants and thisis the case in marchantia, which has singlecopy candidate genes for each. To test thefunction of both of these we are currentlygenerating CRISPR mutant lines, and wouldbe interested to collaborate on the func-tional analysis of these with other research-ers. Moreover, studies on evolution are mostpowerful when data is available from a widerange of organisms, and we would be pleasedto hear from other groups that are study-ing ﬂavonoids and UV-B responses in speciesbeyond seed plants.Edited by: P. J. Aphalo, T. M. Robson and M.Jansen.18 ©2016 by the authorsUV4Plants Bulletin, 2016, no. 1The Team We are studying the mechanism of UV-B tolerance in some of our most basicplant species, the liverworts, so it can be compared to the well characterised ﬂavonoid-based systems of angiosperms. The project is based in the Plant Pigments Team of Plant& Food Research. The team of about a dozen staﬀ is led by Kathy Schwinn and locatedin Palmerston North in the North Island of New Zealand. The group carries out a widerange of both applied and fundamental research on plant pigments. The liverwort projectis led by Kevin Davies and Simon Deroles in collaboration with Brian Jordan. Brian waspreviously the leader of the Plant Pigments team, having joined the team from the Instituteof Horticulture Research, UK in 1994. Brian’s research expertise has focused on lightand plant development and since 1990 he has been recognized as an authority on plantcellular responses to UV-B. Brian is now based at Lincoln University, but has stayed a closefriend and colleague of the Palmerston North team. In 2011 the opportunity was taken todevelop a research collaboration on UV-B regulation of the ﬂavonoid pathway in the modelliverwort species Marchantia.In 2013 a prestigious New Zealand Marsden Grant was awarded and funded the appoint-ment of post-doctoral and PhD fellows. Enter Will and Nick! Will Clayton joined the teamafter completing his Master’s in biotechnology at Massey University, Palmerston North. Hestarted as a PhD student on the project in 2014, studying changes in ﬂavonoid production,plant physiology and gene expression following UV-B exposure of marchantia. Currentlybased at Lincoln University but working closely with the team in Palmerston North, Willis developing many new skills and enjoying the challenge of working towards unravel-ling this novel UV-B story. Nick Albert originally completed his PhD with Kathy and theteam, and after a few years working elsewhere returned for the Marsden post-doctoralappointment. He has developed molecular tools for marchantia, including CRISPR-basedmutagenesis and is identifying the genes that regulate ﬂavonoid production in liverworts.We are now two thirds through the project with many exciting results and concepts emer-ging. We very much hope that the UV4Plants community ﬁnds our research interestingand we look forward to future collaborations. Best Wishes from New Zealand.Figure 6.2: On the left, the Palmerston North team, from left to right Kevin Davies, Simon Deroles,Kathy Schwinn and Nick Albert, and on the right Brian and Will.©2016 by the authors 19

Regulation of UV-induced flavonoid production in Marchantia polymorpha: a role in the evolution of plants for land colonisation?

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