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.

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. 1 | INTRODUCTION Low molecular-weight sugars such as sucrose are fundamental compounds serving as energy and carbon sources to support plant growth Nobiza Khatun and Akihisa Shinozawa equally contributed to this study and share first authorship.