Abscisic acid-mediated sugar responses are essential for vegetative desiccation tolerance in the liverwort Marchantia polymorpha.

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.

Differential regulations of abscisic acid-induced desiccation tolerance and vegetative dormancy by group B3 Raf kinases in liverworts.

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.

Activation of SnRK2 by Raf-like kinase ARK represents a primary mechanism of ABA and abiotic stress responses.

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.

Archetypal Roles of an Abscisic Acid Receptor in Drought and Sugar Responses in Liverworts.

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 ABA-insensitive 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.