Stomatal regulators are co-opted for seta development in the astomatous liverwort Marchantia polymorpha.

The evolution of special types of cells requires the acquisition of new gene regulatory networks controlled by transcription factors (TFs). In stomatous plants, a TF module formed by subfamilies Ia and IIIb basic helix-loop-helix TFs (Ia-IIIb bHLH) regulates stomatal formation; however, how this module evolved during land plant diversification remains unclear. Here we show that, in the astomatous liverwort Marchantia polymorpha, a Ia-IIIb bHLH module regulates the development of a unique sporophyte tissue, the seta, which is found in mosses and liverworts. The sole Ia bHLH gene, MpSETA, and a IIIb bHLH gene, MpICE2, regulate the cell division and/or differentiation of seta lineage cells. MpSETA can partially replace the stomatal function of Ia bHLH TFs in Arabidopsis thaliana, suggesting that a common regulatory mechanism underlies setal and stomatal formation. Our findings reveal the co-option of a Ia-IIIb bHLH TF module for regulating cell fate determination and/or cell division of distinct types of cells during land plant evolution.

Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha.

Marchantia polymorpha is one of the model species of basal land plants. Although CRISPR/Cas9-based genome editing has already been demonstrated for this plant, the efficiency was too low to apply to functional analysis. In this study, we show the establishment of CRISPR/Cas9 genome editing vectors with high efficiency for both construction and genome editing. Codon optimization of Cas9 to Arabidopsis achieved over 70% genome editing efficiency at two loci tested. Systematic assessment revealed that guide sequences of 17 nt or shorter dramatically decreased this efficiency. We also demonstrated that a combinatorial use of this system and a floxed complementation construct enabled conditional analysis of a nearly essential gene. This study reports that simple, rapid, and efficient genome editing is feasible with the series of developed vectors.

Phytochrome-mediated regulation of cell division and growth during regeneration and sporeling development in the liverwort Marchantia polymorpha.

Light regulates various aspects of development throughout the life cycle of sessile land plants. Photoreceptors, such as the red (R) and far-red (FR) light receptors phytochromes, play pivotal roles in modulating developmental programs. Reflecting high developmental plasticity, plants can regenerate tissues, organs, and whole bodies from varieties of cells. Among land plants, bryophytes exhibit extraordinary competency of regeneration under hormone-free conditions. As an environmental factor, light plays critical roles in regeneration of bryophytes. However, how light regulates regeneration remains unknown. Here we show that using the liverwort Marchantia polymorpha, which contains a single phytochrome gene, the phytochrome regulates re-entry into the cell cycle and cell shape in newly regenerating tissues. Our morphological and cytological observations revealed that S-phase entry of G1-arrested epidermal cells around the midrib on the ventral surface of thallus explants was greatly retarded in the dark or under phytochrome-inactive R/FR cycle irradiation conditions, where, nevertheless, small, laterally narrow regenerants were eventually formed. Thus, consistent with earlier descriptions published over a century ago, light is not essential for, but exerts profound effects on regeneration in M. polymorpha. Ventral cells in regenerants grown under R/FR cycle conditions were longer and narrower than those under R cycle. Expression of a constitutively active mutant of M. polymorpha phytochrome allowed regeneration of well grown, widely expanded thalli even in the dark when sugar was supplied, further demonstrating that the phytochrome signal promotes cell proliferation, which is rate-limited by sucrose availability. Similar effects of R and FR irradiation on cell division and elongation were observed in sporelings as well. Thus, besides activation of photosynthesis, major roles of R in regeneration of M. polymorpha are to facilitate proliferation of rounder cells through the phytochrome by mechanisms that are likely to operate in the sporeling.

CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L.

Targeted genome modification technologies are key tools for functional genomics. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 system (CRISPR/Cas9) is an emerging technology for targeted genome modification. The CRISPR/Cas9 system consists of a short guide RNA (gRNA), which specifies the target genome sequence, and the Cas9 protein, which has endonuclease activity. The CRISPR/Cas9 system has been applied to model animals and flowering plants, including rice, sorghum, wheat, tobacco and Arabidopsis. Here, we report the application of CRISPR/Cas9 to targeted mutagenesis in the liverwort Marchantia polymorpha L., which has emerged as a model species for studying land plant evolution. The U6 promoter of M. polymorpha was identified and cloned to express the gRNA. The target sequence of the gRNA was designed to disrupt the gene encoding auxin response factor 1 (ARF1) in M. polymorpha. Using Agrobacterium-mediated transformation, we isolated stable mutants in the gametophyte generation of M. polymorpha. CRISPR/Cas9-based site-directed mutagenesis in vivo was achieved using either the Cauliflower mosaic virus 35S or M. polymorpha EF1α promoter to express Cas9. Isolated mutant individuals showing an auxin-resistant phenotype were not chimeric. Moreover, stable mutants were produced by asexual reproduction of T1 plants. Multiple arf1 alleles were easily established using CRIPSR/Cas9-based targeted mutagenesis. Our results provide a rapid and simple approach for molecular genetics in M. polymorpha, and raise the possibility that CRISPR/Cas9 may be applied to a wide variety of plant species.

Development and validation of the Japanese version of organization-based self-esteem scale.

OBJECTIVES: The purpose of this study was to validate a Japanese version of the organization-based self-esteem (OBSE) scale, a positive self-concept that has been proven to play an important role in predicting employees' work attitudes and behaviors primarily in a Western organizational setting. METHODS: Two independent field studies, Study One and Study Two, involving 1,562 Japanese employees from a variety of organizations were conducted by employing a web-based questionnaire. RESULTS: In Study One, initial evidence for the factorial validity of an eight-item, single factor scale, with high internal consistency (0.93 and 0.88 for Sample 1 and Sample 2, respectively) and test-retest reliability (r=0.84) was provided. In Study Two, the eight-factor structure was replicated and multiple-group confirmatory factor analysis (CFA) demonstrated measurement invariance across two samples. In addition, a series of CFA demonstrated that the Japanese OBSE is a distinct construct from global self-esteem, job complexity and work engagement. Furthermore, the nomological validity evidence was demonstrated through structural equation modeling. CONCLUSIONS: The Japanese OBSE scale is presented as a psychometrically sound measure that can contribute to furthering substantive OBSE research.