Daisuke Takezawa

Evolutionarily Conserved Regulatory Mechanisms of Abscisic Acid Signaling in Land Plants: Characterization of ABSCISIC ACID INSENSITIVE1-Like Type 2C Protein Phosphatase in the Liverwort Marchantia polymorpha

Abscisic acid (ABA) is postulated to be a ubiquitous hormone that plays a central role in seed development and responses to environmental stresses of vascular plants. However, in liverworts (Marchantiophyta), which represent the oldest extant lineage of land plants, the role of ABA has been least emphasized; thus, very little information is available on the molecular mechanisms underlying ABA responses. In this study, we isolated and characterized MpABI1, an ortholog of ABSCISIC ACID INSENSITIVE1 (ABI1), from the liverwort Marchantia polymorpha. The MpABI1 cDNA encoded a 568-amino acid protein consisting of the carboxy-terminal protein phosphatase 2C (PP2C) domain and a novel amino-terminal regulatory domain. The MpABI1 transcript was detected in the gametophyte, and its expression level was increased by exogenous ABA treatment in the gemma, whose growth was strongly inhibited by ABA. Experiments using green fluorescent protein fusion constructs indicated that MpABI1 was mainly localized in the nucleus and that its nuclear localization was directed by the amino-terminal domain. Transient overexpression of MpABI1 in M. polymorpha and Physcomitrella patens cells resulted in suppression of ABA-induced expression of the wheat Em promoter fused to the β -glucuronidase gene. Transgenic P. patens expressing MpABI1 and its mutant construct, MpABI1-d2, lacking the amino-terminal domain, had reduced freezing and osmotic stress tolerance, and associated with reduced accumulation of ABA-induced late embryogenesis abundant-like boiling-soluble proteins. Furthermore, ABA-induced morphological changes leading to brood cells were not prominent in these transgenic plants. These results suggest that MpABI1 is a negative regulator of ABA signaling, providing unequivocal molecular evidence of PP2C-mediated ABA response mechanisms functioning in liverworts.

ABA in bryophytes: how a universal growth regulator in life became a plant hormone?

Abscisic acid (ABA) is not a plant-specific compound but one found in organisms across kingdoms from bacteria to animals, suggesting that it is a ubiquitous and versatile substance that can modulate physiological functions of various organisms. Recent studies have shown that plants developed an elegant system for ABA sensing and early signal transduction mechanisms to modulate responses to environmental stresses for survival in terrestrial conditions. ABA-induced increase in stress tolerance has been reported not only in vascular plants but also in non-vascular bryophytes. Since bryophytes are the key group of organisms in the context of plant evolution, clarification of their ABA-dependent processes is important for understanding evolutionary adaptation of land plants. Molecular approaches using Physcomitrella patens have revealed that ABA plays a role in dehydration stress tolerance in mosses, which comprise a major group of bryophytes. Furthermore, we recently reported that signaling machinery for ABA responses is also conserved in liverworts, representing the most basal members of extant land plant lineage. Conservation of the mechanism for ABA sensing and responses in angiosperms and basal land plants suggests that acquisition of this mechanism for stress tolerance in vegetative tissues was one of the critical evolutionary events for adaptation to the land. This review describes the role of ABA in basal land plants as well as non-land plant organisms and further elaborates on recent progress in molecular studies of model bryophytes by comparative and functional genomic approaches.

Group A PP2Cs evolved in land plants as key regulators of intrinsic desiccation tolerance

Vegetative desiccation tolerance is common in bryophytes, although this character has been lost in most vascular plants. The moss Physcomitrella patens survives complete desiccation if treated with abscisic acid (ABA). Group A protein phosphatases type 2C (PP2C) are negative regulators of abscisic acid signalling. Here we show that the elimination of Group A PP2C is sufficient to ensure P. patens survival to full desiccation, without ABA treatment, although its growth is severely hindered. Microarray analysis shows that the Group A PP2C-regulated genes exclusively overlap with genes exhibiting a high level of ABA induction. Group A PP2C disruption weakly affects ABA-activated kinase activity, indicating Group A PP2C action downstream of these kinases in the moss. We propose that Group A PP2C emerged in land plants to repress desiccation tolerance mechanisms, possibly facilitating plants propagation on land, whereas ABA releases the intrinsic desiccation tolerance from Group A PP2C regulation.

Cold acclimation in the moss Physcomitrella patens involves abscisic acid-dependent signaling.

Overwintering plants develop tolerance to freezing stress through a cold acclimation process by which the cells provoke internal protective mechanisms against freezing. The stress hormone abscisic acid (ABA) is known to increase freezing tolerance of plant cells, but its role in cold acclimation has not been determined. In this study, we used ABA-insensitive lines of the moss Physcomitrella patens to determine whether cold acclimation in bryophytes involves an ABA-dependent process. Two ABA-insensitive lines, both impaired in ABA signaling without showing ABA-induced stress tolerance, were subjected to cold acclimation, and changes in freezing tolerance and accumulation of soluble sugars and proteins were compared to the wild type. The wild-type cells acquired freezing tolerance in response to cold acclimation treatment, but very little increase in freezing tolerance was observed in the ABA-insensitive lines. Analysis of low-molecular-weight soluble sugars indicated that the ABA-insensitive lines accumulated sucrose, a major compatible solute in bryophytes, to levels comparable with those of the wild type during cold acclimation. However, accumulation of the trisaccharide theanderose and of specific LEA-like boiling-soluble proteins was very limited in the ABA-insensitive lines. Furthermore, analysis of cold-induced expression of genes encoding LEA-like proteins revealed that the ABA-insensitive lines accumulate only small amounts of these transcripts during cold acclimation. Our results indicate that cold acclimation of bryophytes requires an ABA-dependent signaling process. The results also suggest that cold-induced sugar accumulation, depending on the sugar species, can either be dependent or independent of the ABA-signaling pathway.

Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2

Significance Plants can sense loss of water caused by drought and stimulate internal mechanisms for protecting cells from damage with the aid of the stress hormone abscisic acid (ABA). Analysis of a mutant of the basal land plant, the moss Physcomitrella patens, revealed that an impairment of a protooncogene Raf-like protein kinase, designated “ARK” (for “ABA and abiotic stress-responsive Raf-like kinase”), causes a loss of both ABA sensitivity and osmotic stress tolerance. We show evidence that ARK has a role in integrating ABA and osmotic signals upstream of the sucrose nonfermenting 1-related protein kinase2, known to be a central regulator of stress signaling in plants. Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated “ARK” (for “ABA and abiotic stress-responsive Raf-like kinase”) plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms.

Long- and short-term freezing induce different types of injury in Arabidopsis thaliana leaf cells

In nature, intact plant cells are subjected to freezing and can remain frozen for prolonged periods. We assayed the survival of Arabidopsis thaliana leaf cells following freezing and found that short- and long-term exposures produced different types of cellular injury. To identify the cause of these injuries, we examined the ultrastructure of the cell plasma membranes. Our results demonstrate that ultrastructural changes in the plasma membrane due to short-term freezing are associated with interbilayer events, including close apposition of the membranes. In both acclimated and non-acclimated leaf cells, these interbilayer events resulted in “fracture-jump lesions” in the plasma membrane. On the other hand, long-term freezing was associated with the development of extensive protein-free areas caused by the aggregation of intramembrane proteins with consequent vesiculation of the affected membrane regions; this effect was clearly different from the ultrastructural changes induced by interbilayer events. We also found that prolonged exposure of non-acclimated leaf cells to a concentrated electrolyte solution produced effects that were similar to those caused by long-term freezing, suggesting that the ultrastructural changes observed in the plasma membrane following long-term freezing are produced by exposure of the leaf cells to a concentrated electrolyte solution. This study illustrates multiple causes of freezing-induced injury in plant cells and may provide useful information regarding the functional role of the diverse changes that occur during cold acclimation.

Epoxycarotenoid-mediated synthesis of abscisic acid in Physcomitrella patens implicating conserved mechanisms for acclimation to hyperosmosis in embryophytes

Plants acclimate to environmental stress signals such as cold, drought and hypersalinity, and provoke internal protective mechanisms. Abscisic acid (ABA), a carotenoid-derived phytohormone, which increases in response to the stress signals above, has been suggested to play a key role in the acclimation process in angiosperms, but the role of ABA in basal land plants such as mosses, including its biosynthetic pathways, has not been clarified. Targeted gene disruption of PpABA1, encoding zeaxanthin epoxidase in the moss Physcomitrella patens was conducted to determine the role of endogenous ABA in acclimation processes in mosses. The generated ppaba1 plants were found to accumulate only a small amount of endogenous ABA. The ppaba1 plants showed reduced osmotic acclimation capacity in correlation with reduced dehydration tolerance and accumulation of late embryogenesis abundant proteins. By contrast, cold-induced freezing tolerance was less affected in ppaba1, indicating that endogenous ABA does not play a major role in the regulation of cold acclimation in the moss. Our results suggest that the mechanisms for osmotic acclimation mediated by carotenoid-derived synthesis of ABA are conserved in embryophytes and that acquisition of the mechanisms played a crucial role in terrestrial adaptation and colonization by land plant ancestors.

ABA as a Universal Plant Hormone

Abscisic acid (ABA) is a sesquiterpene known to regulate environmental stress responses in angiosperms, such as water-loss-induced stomatal closure, development of seed desiccation tolerance during maturation, and salt-, desiccation-, and freezing-stress tolerance of vegetative tissues. An ABA-induced increase in stress tolerance is also reported in other land plant lineages, including nonvascular bryophytes that diverged from vascular plants more than 420 million years ago. Thus, it is hypothesized that acquisition of sensing and response mechanisms for ABA by land plant ancestors was critical for invasion of and adaptation to land. Because bryophytes are key organisms in plant evolution, clarification of their ABA-dependent processes is important for understanding land plant evolutionary adaptation. Based on past and current studies on ABA in non-seed plants and phylogenetic analysis of genome information from various plant species, we discuss the evolution of ABA function and biosynthesis, transport, and signaling network pathways as well as calcium signaling because of its importance in ABA signaling in angiosperms. Future directions of ABA research in the evo-devo field are also discussed.

Abscisic acid-induced rearrangement of intracellular structures associated with freezing and desiccation stress tolerance in the liverwort Marchantia polymorpha.

The plant growth regulator abscisic acid (ABA) is known to be involved in triggering responses to various environmental stresses such as freezing and desiccation in angiosperms, but little is known about its role in basal land plants, especially in liverworts, representing the earliest land plant lineage. We show here that survival rate after freezing and desiccation of Marchantia polymorpha gemmalings was increased by pretreatment with ABA in the presence of increasing concentrations of sucrose. ABA treatment increased accumulation of soluble sugars in gemmalings, and sugar accumulation was further increased by addition of sucrose to the culture medium. ABA treatment of gemmalings also induced accumulation of transcripts for proteins with similarity to late embryogenesis abundant (LEA) proteins, which accumulate in association with acquisition of desiccation tolerance in maturing seeds. Observation by light and electron microscopy indicated that the ABA treatment caused fragmentation of vacuoles with increased cytosolic volume, which was more prominent in the presence of a high concentration of external sucrose. ABA treatment also increased the density of chloroplast distribution and remarkably enlarged their volume. These results demonstrate that ABA induces drastic physiological changes in liverwort cells for stress tolerance, accompanied by accumulation of protectants against dehydration and rearrangement and morphological alterations of cellular organelles.

Biochemical and structural characterization of an endoplasmic reticulum-localized late embryogenesis abundant (LEA) protein from the liverwort Marchantia polymorpha.

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.