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Land plants exhibit various adaptation responses to unfavorable water environments, such as drought and flooding. The phytohormone abscisic acid (ABA) and ethylene play essential roles in plant adaptation to drought and flooding, respectively. It remains largely unknown how plants integrate environmental information for water availability. In the moss Physcomitrium patens, we recently reported that not only ethylene/flooding signaling but also ABA/osmostress signaling are mediated by ethylene receptor-related sensor histidine kinases (ETR-HKs). Subfamily I ETR-HKs of this moss were found to interact with a RAF kinase (ARK) and were required for ABA-dependent activation of SNF1-related protein kinase 2 (SnRK2) via ARK activation. To elucidate the mechanisms of ARK regulation by ETR-HKs, here we employed targeted in vivo mutagenesis of PpHK5, a member of subfamily I ETR-HKs. Analyses of ABA-insensitive Pphk5 mutants indicated that PpHK5 mutations affecting the interaction with ARK resulted in loss of PpHK5 function to activate ABA signaling. We also identified a PpHK5 mutation that does not affect ARK interaction but resulted in loss of PpHK5 function. These results suggest that physical interaction between ETR-HK and ARK is essential but not sufficient for the regulation of ARK activity, and the C-terminal response regulator domain is involved in regulating ARK activation.

Late embryogenesis abundant (LEA) proteins, which accumulate to high levels in seeds during late maturation, are associated with desiccation tolerance. A member of the LEA protein family was found in cultured cells of the liverwort Marchantia polymorpha; preculture treatment of these cells with 0.5M sucrose medium led to their acquisition of desiccation tolerance. We characterized this preculture-induced LEA protein, designated as MpLEA1. MpLEA1 is predominantly hydrophilic with a few hydrophobic residues that may represent its putative signal peptide. The protein also contains a putative endoplasmic reticulum (ER) retention sequence, HEEL, at the C-terminus. Microscopic observations indicated that GFP-fused MpLEA1 was mainly localized in the ER. The recombinant protein MpLEA1 is intrinsically disordered in solution. On drying, MpLEA1 shifted predominantly toward α-helices from random coils. Such changes in conformation are a typical feature of the group 3 LEA proteins. Recombinant MpLEA1 prevented the aggregation of α-casein during desiccation-rehydration events, suggesting that MpLEA1 exerts anti-aggregation activity against desiccation-sensitive proteins by functioning as a "molecular shield". Moreover, the anti-aggregation activity of MpLEA1 was ten times greater than that of BSA or insect LEA proteins, which are known to prevent aggregation on drying. Here, we show that an ER-localized LEA protein, MpLEA1, possesses biochemical and structural features specific to group 3 LEA proteins.

Plant responses to environmental stresses are mediated in part by signaling processes involving cytosolic Ca2+ and a Ca(2+)-binding protein, calmodulin. Screening with radiolabeled calmodulin of a cDNA library of the moss Physcomitrella patens resulted in identification of genes encoding novel membrane transporter-like proteins, MCamb1 and MCamb2. These proteins each had a central hydrophobic domain with two putative membrane spans and N- and C-terminal hydrophilic domains, and showed sequence similarity to mammalian inward rectifier potassium channels. Calmodulin binds to MCamb1 and MCamb2 via interaction with basic amphiphilic amino acids in the C-terminal domain. Levels of MCamb1 and MCamb2 transcripts increased dramatically following treatment with low temperature, hyperosmotic solutes, and the stress hormone abscisic acid, all of which were previously shown to increase cellular tolerance to freezing stress. These results suggest that calmodulin participates in cellular signaling events leading to enhancement of stress resistance through regulation of novel transporter-like proteins.