Genetic Analysis of the Liverwort Marchantia Polymorpha Reveals That
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Research Genetic analysis of the liverwort Marchantia polymorpha reveals that R2R3MYB activation of flavonoid production in response to abiotic stress is an ancient character in land plants Nick W. Albert1 , Amali H. Thrimawithana2 , Tony K. McGhie1 , William A. Clayton1,3, Simon C. Deroles1 , Kathy E. Schwinn1 , John L. Bowman4 , Brian R. Jordan3 and Kevin M. Davies1 1The New Zealand Institute for Plant & Food Research Limited, Private Bag 11600, Palmerston North, New Zealand; 2The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland Mail Centre, Auckland 1142, New Zealand; 3Faculty of Agriculture and Life Sciences, Lincoln University, Christchurch 7647, New Zealand; 4School of Biological Sciences, Monash University, Melbourne, Victoria 3800, Australia Summary Author for correspondence: The flavonoid pathway is hypothesized to have evolved during land colonization by plants Kevin M. Davies c. 450 Myr ago for protection against abiotic stresses. In angiosperms, R2R3MYB transcription Tel: +64 6 3556123 factors are key for environmental regulation of flavonoid production. However, angiosperm Email: [email protected] R2R3MYB gene families are larger than those of basal plants, and it is not known whether the Received: 29 October 2017 regulatory system is conserved across land plants. We examined whether R2R3MYBs regulate Accepted: 19 December 2017 the flavonoid pathway in liverworts, one of the earliest diverging land plant lineages. We characterized MpMyb14 from the liverwort Marchantia polymorpha using genetic New Phytologist (2018) 218: 554–566 mutagenesis, transgenic overexpression, gene promoter analysis, and transcriptomic and doi: 10.1111/nph.15002 chemical analysis. MpMyb14 is phylogenetically basal to characterized angiosperm R2R3MYB flavonoid regulators. Key words: CRISPR/Cas9, evolution, Mpmyb14 knockout lines lost all red pigmentation from the flavonoid riccionidin A, flavonoid, liverwort, MYB, stress. whereas overexpression conferred production of large amounts of flavones and riccionidin A, activation of associated biosynthetic genes, and constitutive red pigmentation. MpMyb14 expression and flavonoid pigmentation were induced by light- and nutrient-deprivation stress in M. polymorpha as for anthocyanins in angiosperms. MpMyb14 regulates stress-induced flavonoid production in M. polymorpha, and is essential for red pigmentation. This suggests that R2R3MYB regulated flavonoid production is a con- served character across land plants which arose early during land colonization. Introduction Many flowering plants (angiosperms) produce colourless UV- B absorbing flavone and flavonol glycosides in response to UV-B Flavonoid biosynthesis is a central component of plant secondary exposure, as well as following other abiotic stresses. They also metabolism that is thought to have evolved during land coloniza- produce red/purple anthocyanin flavonoid pigments in response tion, c. 450–500 Myr ago (Ma) (Vogt, 2010; Weng, 2013; de to nutrient deprivation, drought, wounding, cold and high light, Vries et al., 2017). Derived from the larger phenylpropanoid as well as for signalling to animals for pollination and seed disper- pathway, the acquisition of flavonoids is hypothesized to have sal (Allan et al., 2008; Agati & Tattini, 2010; Davies et al., been a key adaptation for coping with the additional abiotic 2012a; Cheynier et al., 2013). The flavones/flavonols and antho- stresses faced with the transition to a nonaquatic lifestyle. These cyanins can directly screen excess UV-B or white light energy, included drought, extreme temperature fluctuations and, in par- respectively, but also are thought to provide broad stress tolerance ticular, increased exposure to UV-B light (Bj€orn et al., 2002). through scavenging of reactive oxygen species (ROS; Agati & UV-B has severe detrimental effects on plant cells, and was at Tattini, 2010; Agati et al., 2012; Landi et al., 2015). particularly high intensities during the period of land coloniza- In all angiosperm species examined to date, R2R3MYB tran- tion because the ozone layer was not fully developed. Flavonoids scription factors are essential for environmental and developmen- are ubiquitous in extant land plants, including the liverworts, tal regulation of flavonoid biosynthesis (Albert et al., 2011; Feller which may be the closest living relatives of the first land plants. et al., 2011; Cheynier et al., 2013; Xu et al., 2015; Lloyd et al., However, they are absent from all algae, including Charophytes, 2017). For activation of the anthocyanin or proanthocyanidin which are thought to be most similar to the aquatic ancestors of pigment pathways, R2R3MYBs of subgroups (SGs) 5 or 6 act as land plants (de Vries et al., 2017). part of an R2R3MYB-bHLH-WDR (MBW) transcriptional 554 New Phytologist (2018) 218: 554–566 Ó 2018 Plant & Food Research www.newphytologist.com New Phytologist Ó 2018 New Phytologist Trust New Phytologist Research 555 complex. Additionally, members of SG4 can have a repressive acting redundantly in regulation of flavone biosynthesis. role on genes of the phenylpropanoid pathway as part of the Riccionidin A production is induced in liverworts by similar MBW complex and contain transcriptional repressor domains in abiotic stress factors as previously characterized for anthocyanin their C-terminal (Matsui et al., 2008; Albert et al., 2014; production in angiosperms. It is notable that structurally dis- Cavallini et al., 2015). For activation of flavonol biosynthesis tinct red flavonoid pigments are produced in marchantia and R2R3MYBs of SG7 act independently of the MBW complex. angiosperms, although some of the same flavonoid biosynthetic Although the MBW complex is often regarded as universal for genes are activated in each plant group. The results suggest that anthocyanin regulation, it is actually an open question as to what R2R3MYB proteins were co-opted for regulation of the aspects of the system are conserved across the major land plant flavonoid pathway from the early stages of land plant evolution, groups (Albert et al., 2014; Liu et al., 2015; Xu et al., 2015; Lloyd and supports the hypothesis that an inducible flavonoid path- et al., 2017). The recent finding of a conserved MBW complex way was an early evolutionary adaption to the abiotic stresses of able to activate flavonoid biosynthesis in a gymnosperm species life on land. (Nemesio-Gorriz et al., 2017) suggests an ancient origin in the plant lineage, as angiosperms and gymnosperms are thought to Materials and Methods have diverged c. 300 Ma (Lu et al., 2014). However, genomes of the basal plant groups liverworts (Bowman et al., 2017) and Plant lines and growth conditions mosses (Rensing et al., 2008) contain markedly fewer MYB and bHLH genes than those of angiosperms and gymnosperms, sug- Marchantia polymorpha L. accessions Sey-1 (male) and Aud-2 (fe- gesting a more limited range of regulatory activities. male) were grown on Jiffy-7 peat pellets (Egmont Seeds, New Liverworts together with mosses and hornworts form the three Plymouth, New Zealand), and were induced to produce sexual main lineages of the basal land plant group, the bryophytes. structures with supplemental far-red light (Chiyoda et al., 2008) Marchantia polymorpha (hereafter, marchantia) has been devel- and crossed to generate spores for transformations. oped as a model species for liverwort studies. Marchantia has a Plant lines were maintained asexually through the propagation dominant haploid gametophytic generation, rapid growth rate, is of gemmae, either plated directly on agar medium (0.5 9 Gam- dioecious and can reproduce asexually in large numbers through borg’s B5 medium (Duchefa Biochemie, Haarlem, the Nether- single-cell derived clonal gemmae (Bowman et al., 2016). The lands) 1% (w/v) sucrose, 1.2% (w/v) agar) or onto a sterile filter last common ancestor of liverworts and angiosperms is thought paper disc covering the medium. Standard culture conditions À À to have existed c. 450 Ma, so comparative studies of the two plant were 25°C, 16 h photoperiod and 30 lmol m 2 s 1 light inten- groups can help to inform us about systems that have an early sity provided by cool fluorescent tubes. evolutionary origin. Liverworts lack some of the typically For nutrient deprivation experiments, gemmae (three per tub/ flavonoid pigmented tissues of angiosperms, such as flowers, biological replicate; three biological replicates per treatment) were fruits and seed coats, and knowledge about flavonoid production plated onto filter papers on complete media (0.5 9 B5, 1% in liverworts is comparatively limited. They produce a range of sucrose, 1.2% agar) and grown for 4 wk. After this time, plants vacuolar-located flavone O-orC-glycosides similar to those of were transferred on the filter paper to fresh complete media or angiosperms (Asakawa et al., 2013). Additionally, although liver- onto minimal media (1% sucrose, 1.2% agar) and grown for a worts lack the diversity of anthocyanins reported for further 13 d. Tissue was collected and immediately frozen in liq- angiosperms, they do produce a cell wall-localized red flavonoid uid nitrogen; aliquots were used for RNA extraction or freeze- pigment that has been identified as the aglycone anthocyanidin dried for metabolite analysis. riccionidin A, including from marchantia (Kunz et al., 1993). For light induction experiments, gemmae (three to four per The analysis of Bowman et al. (2017) identified only 21 tub/biological replicate; three biological replicates per treatment) R2R3MYB genes in the marchantia genome, as compared for were plated onto complete media (0.5 9 B5, 1% sucrose, 1.2% example to the estimated 138 in Arabidopsis, 157–188 in agar) and grown for 4 wk under high or low light conditions. maize (Zea mays) and 244–288 in soybean (Glycine max) Illumination was provided by a combination of warm and cool À À (Stracke et al., 2001; Feller et al., 2011; Liu et al., 2015). Often white LEDs (high light, 200 lmol m 2 s 1). The low light treat- À À the marchantia genome contains one or two genes that are basal ment (50 lmol m 2 s 1) was performed using the same light to large clades containing multiple angiosperm subgroups with source, except that plants were shielded with shade cloth.