Abscisic acid (ABA) controls seed dormancy and stomatal closure through binding to the intracellular receptor Pyrabactin resistance1 (Pyr1)/Pyr1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) in angiosperms. Genes encoding PYR/PYL/RCAR are thought to have arisen in the ancestor of embryophytes, but the roles of the genes in nonvascular plants have not been determined. In the liverwort Marchantia polymorpha, ABA reduces growth and enhances desiccation tolerance through increasing accumulation of intracellular sugars and various transcripts such as those of Late Embryogenesis Abundant (LEA)-like genes. In this study, we analyzed a gene designated MpPYL1, which is closely related to PYR/PYL/RCAR of angiosperms, in transgenic liverworts. Transgenic lines overexpressing MpPYL1-GFP showed ABA-hypersensitive growth with enhanced desiccation tolerance, whereas Mppyl1 generated by CRISPR-Cas9-mediated genome editing showed ABAinsensitive growth with reduced desiccation tolerance. Transcriptome analysis indicated that MpPYL1 is a major regulator of abiotic stress-associated genes, including all 35 ABA-induced LEA-like genes. Furthermore, these transgenic plants showed altered responses to extracellular Suc, suggesting that ABA and PYR/PYL/RCAR function in sugar responses. The results presented here reveal an important role of PYR/PYL/RCAR in the ABA response, which was likely acquired in the common ancestor of land plants. The results also indicate the archetypal role of ABA and its receptor in sugar response and accumulation processes for vegetative desiccation tolerance in bryophytes.

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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 summaryPhysiological characterization of various moss mutants revealed a common mechanism for phytohormone responses under water deficit in all land plants.