Daisuke Takezawa

Dormancy is a crucial trait that allows land plants to withstand harsh terrestrial environments by pausing growth until favorable conditions return. The liverwort Marchantia polymorpha produces dormant propagules, gemmae, for asexual reproduction. The phytohormone ABA is known to regulate dormancy in the seeds of flowering plants and the gemmae of M. polymorpha. Here, we identify a basic helix–loop–helix protein, MpHYPNOS (MpHYP), as a central regulator of gemma dormancy. MpHYP expression induces germination arrest in gemmae. Transcriptome analysis revealed that MpHYP represses a D-type cyclin gene MpCYCD;1 and induces genes involved in ABA biosynthesis. Repression of MpCYCD;1 expression and germination arrest are both maintained even in an ABA-receptor mutant. By contrast, Mphyp and ABA-related mutants lack the desiccation tolerance observed in wild-type gemmae. We propose that MpHYP regulates gemma dormancy through two parallel pathways: an ABA-independent pathway that suppresses germination through cell cycle repression, and an ABA-dependent pathway that enhances desiccation tolerance via ABA biosynthesis. These findings highlight an integrated regulatory mechanism of dormancy via phytohormonal and cell cycle control in bryophytes.

ETHYLENE INSENSITIVE 2 (EIN2) is an Nramp family transmembrane protein recognized as an essential component of ethylene signaling in angiosperms. However, its functions in other plant systems are not fully understood. Here, we demonstrate that ppein2ab mutants of the moss Physcomitrium patens, in which both EIN2-like genes have been disrupted, do not show a typical ethylene-mediated escape response following submergence. Interestingly, ppein2ab mutants showed reduced sensitivity to abscisic acid (ABA), a phytohormone that mediates drought stress responses. The ppein2ab plants were sensitive to hyperosmosis and freezing stress and exhibited reduced late embryogenesis abundant protein accumulation. Furthermore, ppein2ab mutants showed reduced activation of both SNF1-related protein kinase2 (SnRK2), the central activator of ABA and osmostress signaling, and the B3-RAF kinase ARK/PpCTR1L, a positive regulator of SnRK2. These results indicate that EIN2 is a dual function signaling component mediating both submergence and drought signaling in bryophytes. The diminished ABA responses in ppein2ab were restored by introduction of Arabidopsis EIN2 and the EIN2 orthologs of the Charophyceaen alga Chara braunii, suggesting functional conservation of EIN2 orthologs in Phragmoplastophyta.

Cell-to-cell communication is essential for multicellular organisms. Plasmodesmata (PD) are plant-specific nanopore structures pivotal for cell-to-cell communication and plant survival. However, how PD form and their structure, regulation, and evolution remain largely unknown. Here, we demonstrate that the exogenous supply of abscisic acid (ABA), a well-conserved phytohormone in land plants, reduces primary PD density in the moss Physcomitrium patens . This regulation requires all core components of the ABA signaling pathway. Furthermore, we reveal that ABA-INSENSITIVE 5, a well-conserved transcription factor in the ABA signaling pathway of land plants, plays a pivotal role in PD density regulation, whereas ABA-INSENSITIVE 3 does not. Our findings show that the ABA-induced reduction in primary PD density is mediated by these ABA-responsive factors in P. patens . Considering previous reports on ABA-dependent PD regulation in both moss and angiosperms, we propose that the ABA-mediated control of PD biogenesis and permeability represents a conserved mechanism in land plants, with critical implications for cell-to-cell communication and stress adaptation.

Low‐molecular‐weight sugars serve as protectants for cellular membranes and macromolecules under the condition of dehydration caused by environmental stress such as desiccation and freezing. These sugars also affect plant growth and development by provoking internal signaling pathways. We previously showed that both sugars and the stress hormone abscisic acid (ABA) enhance desiccation tolerance of gemma, a dormant propagule of the liverwort Marchantia polymorpha. To determine the role of ABA in sugar responses in liverworts, we generated genome‐editing lines of M. polymorpha ABA DEFICIENT 1 (MpABA1) encoding zeaxanthin epoxidase, which catalyzes the initial reaction toward ABA biosynthesis. The generated Mpaba1 lines that accumulated only a trace amount of endogenous ABA showed reduced desiccation tolerance and reduced sugar responses. RNA‐seq analysis of sucrose‐treated gemmalings of M. polymorpha revealed that expression of a large part of sucrose‐induced genes was reduced in Mpaba1 compared to the wild‐type. Furthermore, Mpaba1 accumulated smaller amounts of low‐molecular‐weight sugars in tissues upon sucrose treatment than the wild‐type, with reduced expression of genes for sucrose synthesis, sugar transporters, and starch‐catabolizing enzymes. These results indicate that endogenous ABA plays a role in the regulation of the positive feedback loop for sugar‐induced sugar accumulation in liverworts, enabling the tissue to have desiccation tolerance.

Phytohormone abscisic acid (ABA) plays a key role in stomata closure, osmostress acclimation, and vegetative and embryonic dormancy. Group B3 Raf protein kinases (B3-Rafs) serve as positive regulators of ABA and osmostress signaling in the moss Physcomitrium patens and the angiosperm Arabidopsis thaliana. While P. patens has a single B3-Raf called ARK, specific members of B3-Rafs among six paralogs regulate ABA and osmostress signaling in A. thaliana, indicating functional diversification of B3-Rafs in angiosperms. However, we found that the liverwort Marchantia polymorpha, belonging to another class of bryophytes, has three paralogs of B3-Rafs, MpARK1, MpARK2, and MpARK3, with structural variations in the regulatory domains of the polypeptides. By reporter assays of the P. patens ark line and analysis of genome-editing lines of M. polymorpha, we found that these B3-Rafs are functionally redundant in ABA response, with respect to inhibition of growth, tolerance to desiccation and expression of stress-associated transcripts, the majority of which are under the control of the PYR/PYL/RCAR-like receptor MpPYL1. Interestingly, gemmae in gemma cups were germinating only in mutant lines associated with MpARK1, indicating that dormancy in the gametophyte is controlled by a specific B3-Raf paralog. These results indicated not only conservation of the role of B3-Rafs in ABA and osmostress response in liverworts but also functional diversification of B3-Rafs, which is likely to have occurred in the early stages of land plant evolution.

Significance: SNF1-related protein kinase 2s (SnRK2s) are central regulators in abscisic acid (ABA) signaling and stress responses in plants. However, SnRK2 substrates are largely unknown. Here, we identified Arabidopsis group C Raf-like kinases Raf36 and Raf22 as SnRK2 substrates. Reverse genetic analyses showed that Raf36 and Raf22 negatively regulate ABA signaling. Under optimal growth conditions, Raf36 accumulates in the cell and phosphorylates substrates to suppress ABA responses. Conversely, under abiotic stress conditions, ABA-activated SnRK2s phosphorylate Raf36 and promote Raf36 degradation, leading to stronger ABA responses. Taken together, this study provides insight into molecular and physiological functions of group C Raf kinases in ABA signaling.

The Raf-like protein kinase abscisic acid (ABA) and abiotic stress-responsive Raf-like kinase (ARK) previously identified in the moss Physcomitrium (Physcomitrella) patens acts as an upstream regulator of subgroup III SNF1-related protein kinase2 (SnRK2), the key regulator of ABA and abiotic stress responses. However, the mechanisms underlying activation of ARK by ABA and abiotic stress for the regulation of SnRK2, including the role of ABA receptor-associated group A PP2C (PP2C-A), are not understood. We identified Ser1029 as the phosphorylation site in the activation loop of ARK, which provided a possible mechanism for regulation of its activity. Analysis of transgenic P. patens ark lines expressing ARK-GFP with Ser1029-to-Ala mutation indicated that this replacement causes reductions in ABA-induced gene expression, stress tolerance, and SnRK2 activity. Immunoblot analysis using an anti-phosphopeptide antibody indicated that ABA treatments rapidly stimulate Ser1029 phosphorylation in the wild type (WT). The phosphorylation profile of Ser1029 in ABA-hypersensitive ppabi1 lacking protein phosphatase 2C-A (PP2C-A) was similar to that in the WT, whereas little Ser1029 phosphorylation was observed in ABA-insensitive ark missense mutant lines. Furthermore, newly isolated ppabi1 ark lines showed ABA-insensitive phenotypes similar to those of ark lines. Therefore, ARK is a primary activator of SnRK2, preceding negative regulation by PP2C-A in bryophytes, which provides a prototype mechanism for ABA and abiotic stress responses in plants.

The Raf-like protein kinase ARK previously identified in the moss Physcomitrella patens acts as an upstream regulator of subgroup III SnRK2, the key regulator of abscisic acid (ABA) and abiotic stress responses. However, the mechanisms underlying activation of ARK by ABA and abiotic stress for the regulation of SnRK2 including the role of ABA receptor-associated group A PP2C (PP2C-A) are not understood. We identified Ser1029 as the phosphorylation site in the activation loop of ARK, which provided a possible mechanism for regulation of its activity. Analysis of transgenic ark lines expressing ARK-GFP with Ser1029-to-Ala mutation indicated that this replacement causes reductions in ABA-induced gene expression, stress tolerance and SnRK2 activity. Immunoblot analysis using an anti-phosphopeptide antibody indicated that ABA treatments rapidly stimulate Ser1029 phosphorylation in wild type. The phosphorylation profile of Ser1029 in ABA-hypersensitive ppabi1 lacking PP2C-A was similar to that in wild type, whereas little Ser1029 phosphorylation was observed in ABA-insensitive ark missense lines. Furthermore, newly isolated ppabi1 ark lines showed ABA-insensitive phenotypes similar to those of ark lines. These results indicate that ARK is a primary activator of SnRK2, preceding negative regulation by PP2C-A in bryophytes, which provides a prototypal mechanism for ABA and abiotic stress-responses in embryophytes. One sentence summary: Physiological characterization of various moss mutants revealed a common mechanism for phytohormone responses under water deficit in all land plants.