A Polyphenol Oxidase Catalyzes Aurone Synthesis in Marchantia polymorpha.

Aurones constitute one of the major classes of flavonoids, with a characteristic furanone structure that acts as the C-ring of flavonoids. Members of various enzyme families are involved in aurone biosynthesis in different higher plants, suggesting that during evolution plants acquired the ability to biosynthesize aurones independently and convergently. Bryophytes also produce aurones, but the biosynthetic pathways and enzymes involved have not been determined. The present study describes the identification and characterization of a polyphenol oxidase (PPO) that acts as an aureusidin synthase (MpAS1) in the model liverwort, Marchantia polymorpha. Crude enzyme assays using an M. polymorpha line overexpressing MpMYB14 with high accumulation of aureusidin showed that aureusidin was biosynthesized from naringenin chalcone and converted to riccionidin A. This activity was inhibited by N-phenylthiourea, an inhibitor specific to enzymes of the PPO family. Of the six PPOs highly induced in the line overexpressing MpMyb14, one, MpAS1, was found to biosynthesize aureusidin from naringenin chalcone when expressed in Saccharomyces cerevisiae. MpAS1 also recognized eriodictyol chalcone, isoliquiritigenin and butein, showing the highest activity for eriodictyol chalcone. Members of the PPO family in M. polymorpha evolved independently from PPOs in higher plants, indicating that aureusidin synthases evolved in parallel in land plants.

Physiological function of photoreceptor UVR8 in UV-B tolerance in the liverwort Marchantia polymorpha.

MAIN CONCLUSION: The physiological importance of MpUVR8 in UV-B resistance and translocation in a UV-B-dependent manner from the cytosol into the nucleus is characterized in Marchantia polymorpha. UV RESISTANCE LOCUS 8 (UVR8) is an ultraviolet-B (UV-B) light receptor functioning for UV-B sensing and tolerance in Arabidopsis thaliana and other species. It is unclear whether UVR8 physiologically functions in UV-B-induced defense responses in Marchantia polymorpha, which belongs to the earliest diverging group of embryophyte lineages. Here, we demonstrate that UVR8 has a physiological function in UV-B tolerance and that there is a UVR8-dependent pathway involved. In addition, a UVR8-independent pathway is revealed. We examine the tissue-specific expression pattern of M. polymorpha UVR8 (MpUVR8), showing that it is highly expressed in the apical notch in thalli and gametangiophores, as well as in antheridial and archegonial heads. Furthermore, Mpuvr8KO plant transformants, in which the MpUVR8 locus was disrupted, were produced and analyzed to understand the physiological and molecular function of MpUVR8. Analysis using these plants indicates the important roles of MpUVR8 and MpUVR8-regulated genes, and of MpUVR8-independent pathways in UV-B tolerance. Subcellular localization of Citrine-fused MpUVR8 in M. polymorpha cells was also investigated. It was found to translocate from the cytosol into the nucleus in response to UV-B irradiation. Our findings indicate strong conservation of the physiological function of UVR8 and the molecular mechanisms for UVR8-dependent signal transduction through regulation of gene expression in embryophytes.

Biosynthesis of riccionidins and marchantins is regulated by R2R3-MYB transcription factors in Marchantia polymorpha.

R2R3-MYB transcription factors constitute the largest gene family among plant transcription factor families. They became largely divergent during the evolution of land plants and regulate various biological processes. The functions of R2R3-MYBs are mostly characterized in seed plants but are poorly understood in non-seed plants. Here, we examined the function of two R2R3-MYB genes of Marchantia polymorpha (Mapoly0073s0038 and Mapoly0006s0226) that are closely related to subgroup 4 of the R2R3-MYB family. We performed LC/MS/MS metabolomics, RNA-seq analysis and expression analysis in overexpressors and knockout mutants of MpMYB14 and MpMYB02. Overexpression of MpMYB14 remarkably increased the amount of riccionidins, which are specific anthocyanins in liverworts and a few flowering plants. In contrast, overexpression of MpMYB02 increased the amount of several marchantins, which are characteristic cyclic bis (bibenzyl ether) compounds in M. polymorpha and related liverworts. Knockouts of MpMYB14 and MpMYB02 abolished the accumulation of riccionidins and marchantins, respectively. The expression of MpMYB14 was up-regulated by UV-B irradiation, N deficiency, and NaCl treatment, whereas the expression of MpMYB02 was down-regulated by NaCl treatment. Our results suggest that the regulatory framework of phenolic metabolism by R2R3-MYB was already established in early land plants.

Cloning of a FLOWERING LOCUS T ortholog in Wasabia japonica (Matsum).

A FLOWERING LOCUS T ortholog (WjFT) was identified in Wasabia japonica. Heterologous expression of WjFT remarkably promoted the flowering of Arabidopsis. The expression of WjFT was examined in field-grown wasabi in October and November of 2009, and February of 2010 because the differentiation of flower buds occurs in autumn in field-grown wasabi. No expression of WjFT was detected in October, it was slightly increased in November, and highly increased in February. WjFT might be useful for examining the flowering response of wasabi.

GEMMA CUP-ASSOCIATED MYB1, an Ortholog of Axillary Meristem Regulators, Is Essential in Vegetative Reproduction in Marchantia polymorpha.

A variety of plants in diverse taxa can reproduce asexually via vegetative propagation, in which clonal propagules with a new meristem(s) are generated directly from vegetative organs. A basal land plant, Marchantia polymorpha, develops clonal propagules, gemmae, on the gametophyte thallus from the basal epidermis of a specialized receptacle, the gemma cup. Here we report an R2R3-MYB transcription factor, designated GEMMA CUP-ASSOCIATED MYB1 (GCAM1), which is an essential regulator of gemma cup development in M. polymorpha. Targeted disruption of GCAM1 conferred a complete loss of gemma cup formation and gemma generation. Ectopic overexpression of GCAM1 resulted in formation of cell clumps, suggesting a function of GCAM1 in suppression of cell differentiation. Although gemma cups are a characteristic gametophyte organ for vegetative reproduction in a taxonomically restricted group of liverwort species, phylogenetic and interspecific complementation analyses support the orthologous relationship of GCAM1 to regulatory factors of axillary meristem formation, e.g., Arabidopsis REGULATOR OF AXILLARY MERISTEMS and tomato Blind, in angiosperm sporophytes. The present findings in M. polymorpha suggest an ancient acquisition of a transcriptional regulator for production of asexual propagules in the gametophyte and the use of the regulatory factor for diverse developmental programs, including axillary meristem formation, during land plant evolution.

Characterization of root cells of anl2 mutant in Arabidopsis thaliana.

A mutation in the ANTHOCYANINLESS2 (ANL2) gene of Arabidopsis thaliana causes the formation of several extra cells called intervening cells (IV cells) between the cortical and epidermal layers of the primary root. The origin and character of IV cells were examined. Microscopic observation of serial sections of the root tissue showed that IV cells developed from epidermal cells, and the outer cells overlying the epidermal cells developed into epidermal-like cells. The IV cells expressed the marker gene for cortical cells, which indicated that the IV cells were cortical in nature. IV cells were primarily observed in a cleft between two cortical cells or between a cortical and an IV cell, which suggests the involvement of positional information between cortical and epidermal cells. Ectopic root hairs were formed, and the expression patterns of ß-glucuronidase driven by the GL2 and CPC promoters were irregular in the primary root of the anl2 mutant. These results indicate that ANL2 is required for the maintenance of epidermal cell lineage.

Anthocyaninless1 gene of Arabidopsis thaliana encodes a UDP-glucose:flavonoid-3-O-glucosyltransferase.

We isolated several mutants of Arabidopsis thaliana (L.) Heynh. that accumulated less anthocyanin in the plant tissues, but had seeds with a brown color similar to the wild-type. These mutants were allelic with the anthocyaninless1 (anl1) mutant that has been mapped at 15.0 cM of chromosome 5. We performed fine mapping of the anl1 locus and determined that ANL1 is located between the nga106 marker and a marker corresponding to the MKP11 clone. About 70 genes are located between these two markers, including three UDP-glucose:flavonoid-3-O-glucosyltransferase-like genes and a glutathione transferase gene (TT19). A mutant of one of the glucosyltransferase genes (At5g17050) was unable to complement the anl1 phenotype, showing that the ANL1 gene encodes UDP-glucose:flavonoid-3-O-glucosyltransferase. ANL1 was expressed in all tissues examined, including rosette leaves, stems, flower buds and roots. ANL1 was not regulated by TTG1.