Simple and Efficient Transformation and Gene Editing of Marchantia polymorpha Spores

Marchantia polymorpha (Marchantia) has become a model species for liverwort studies, owing to its rapid growth in vitro, ease of propagation, simple genetics, small genome, haploid-dominant life cycle, and because it is amenable to genetic transformation. Efficient transformation provides a foundation for many molecular and genetic analyses. The protocol described here is a simple and robust procedure for transforming Marchantia for a variety of applications, including gene overexpression and CRISPR genome editing. This simplified Agrobacterium tumefaciens-mediated transformation protocol targets spores, using common Agrobacterium strains GV3101 or EHA105, and overcomes challenges experienced in other methods. Spores are sterilized and distributed over sterile filter papers, which effectively retain spores and regenerating spores (known as sporelings). This approach enables the sporelings to be transferred to different agar growth media at different stages of transformation. A critical feature is preculturing the spores with acetosyringone (AS) prior to co-cultivation with Agrobacterium. This treatment profoundly enhances the transformation rate, particularly for Agrobacterium strain GV3101. GV3101 is preferred for its rapid growth rate, simple transformation, and lack of a recombinase (recA), stabilizing plasmids. The protocol is suitable for transforming Marchantia with constructs for CRISPR gene editing. Editing efficiency can be increased by introducing a heat-shock treatment during the transformation procedure, which increases the proportion of plants with larger edited sectors, facilitating mutant identification and propagation. Constructs and strategies for both overexpression and multiplex genome editing with sgRNA arrays using new and existing vectors are described. Using this spore transformation protocol for CRISPR gene editing, we routinely achieve 60% to 70% mutation rates, significantly reducing the effort required to generate and isolate mutants for functional analyses.fals

Adaptation to life on land: how plants developed UV sunscreens

Terrestrial plant life evolved from an aquatic ancestor 400-6-- million years ago. This significant step from water to land involved a suite of genetic adaptations allowing for colonisation of new environments. One such adaptation was for tolerance of incident UV-B radiation. UV-B radiation is particularly damaging to plant life because of its absorption by molecules such as DNA and the production of reactive oxygen species. Therefore the development of UV-B tolerance by early land plants would have been particularly important. Flavonoid synthesis contributes significantly to UV-B tolerance in higher plants, yet it is not known whether the acquisition of flavonoid production allowed the first land plants to tolerate UV-B and colonise the land environment. Here we examine the model bryophyte Marchantia polymorpha, from one of the oldest extant land plant lineages , to determine the effect of flavonoid production in response to UV-B. Under set environmental conditions and UV-B irradiance M. polymorpha is shown to respond to UV-B with an increased production of flavonoid compounds (predominantly flavones). These preliminary results may lead into an understanding of the importance of flavonoid production in enabling the successful colonisation of land by plant life

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

The flavonoid pathway is hypothesized to have evolved during land colonization by plants c. 450 Myr ago for protection against abiotic stresses. In angiosperms, R2R3MYB transcription factors are key for environmental regulation of flavonoid production. However, angiosperm R2R3MYB gene families are larger than those of basal plants, and it is not known whether the regulatory system is conserved across land plants. We examined whether R2R3MYBs regulate the flavonoid pathway in liverworts, one of the earliest diverging land plant lineages. We characterized MpMyb14 from the liverwort Marchantia polymorpha using genetic mutagenesis, transgenic overexpression, gene promoter analysis, and transcriptomic and chemical analysis. MpMyb14 is phylogenetically basal to characterized angiosperm R2R3MYB flavonoid regulators. Mpmyb14 knockout lines lost all red pigmentation from the flavonoid riccionidin A, 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 conserved character across land plants which arose early during land colonization

Auronidins are a previously unreported class of flavonoid pigments that challenges when anthocyanin biosynthesis evolved in plants

Anthocyanins are key pigments of plants, providing color to flowers, fruit, and foliage and helping to counter the harmful effects of environmental stresses. It is generally assumed that anthocyanin biosynthesis arose during the evolutionary transition of plants from aquatic to land environments. Liverworts, which may be the closest living relatives to the first land plants, have been reported to produce red cell wall-bound riccionidin pigments in response to stresses such as UV-B light, drought, and nutrient deprivation, and these have been proposed to correspond to the first anthocyanidins present in early land plant ancestors. Taking advantage of the liverwort model species Marchantia polymorpha, we show that the red pigments of Marchantia are formed by a phenylpropanoid biosynthetic branch distinct from that leading to anthocyanins. They constitute a previously unreported flavonoid class, for which we propose the name “auronidin,” with similar colors as anthocyanin but different chemistry, including strong fluorescence. Auronidins might contribute to the remarkable ability of liverworts to survive in extreme environments on land, and their discovery calls into question the possible pigment status of the first land plants