Archetypal Roles of an Abscisic Acid Receptor in Droughtand Sugar Responses in Liverworts1

Akida Jahan,a,2 Kenji Komatsu,b,2 Mai Wakida-Sekiya,a Mayuka Hiraide,a Keisuke Tanaka,c Rumi Ohtake,c

Taishi Umezawa,d Tsukasa Toriyama,e Akihisa Shinozawa,e Izumi Yotsui,e Yoichi Sakata,e andDaisuke Takezawaa,f,3,4

aGraduate School of Science and Engineering, Saitama University, Saitama 338-8570, JapanbDepartment of Bioresource Development, Tokyo University of Agriculture, Kanagawa 243-0034, JapancThe NODAI Genome Research Center (NGRC), Tokyo University of Agriculture, Tokyo 156-8502, JapandGraduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture andTechnology, Tokyo 184-8588, JapaneDepartment of Bioscience, Tokyo University of Agriculture, Tokyo 156-8502, JapanfInstitute for Environmental Science and Technology, Saitama University, Saitama 338-8570, Japan

ORCID IDs: 0000-0003-3750-0503 (T.U.); 0000-0001-6751-7205 (D.T.).

Abscisic acid (ABA) controls seed dormancy and stomatal closure through binding to the intracellular receptor Pyrabactinresistance1 (Pyr1)/Pyr1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) in angiosperms. Genes encodingPYR/PYL/RCAR are thought to have arisen in the ancestor of embryophytes, but the roles of the genes in nonvascularplants have not been determined. In the liverwort Marchantia polymorpha, ABA reduces growth and enhances desiccationtolerance through increasing accumulation of intracellular sugars and various transcripts such as those of Late EmbryogenesisAbundant (LEA)-like genes. In this study, we analyzed a gene designated MpPYL1, which is closely related to PYR/PYL/RCARof angiosperms, in transgenic liverworts. Transgenic lines overexpressing MpPYL1-GFP showed ABA-hypersensitive growthwith 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 ofabiotic stress-associated genes, including all 35 ABA-induced LEA-like genes. Furthermore, these transgenic plants showedaltered responses to extracellular Suc, suggesting that ABA and PYR/PYL/RCAR function in sugar responses. The resultspresented here reveal an important role of PYR/PYL/RCAR in the ABA response, which was likely acquired in the commonancestor of land plants. The results also indicate the archetypal role of ABA and its receptor in sugar response and accumulationprocesses for vegetative desiccation tolerance in bryophytes.

Abscisic acid (ABA) is a phytohormone that mediatesdevelopmental and physiological processes in vegeta-tive and reproductive organs in plants. These pro-cesses include seed maturation and dormancy, buddormancy, closure of leaf stomata, and acquisition of

tolerance to various environmental stresses, such asdrought and desiccation (Milborrow, 1974; Vishwakarmaet al., 2017). ABA triggers the expression of stress-associated transcripts such as those encoding Late Em-bryogenesis Abundant (LEA)-like proteins (Cuming andLane, 1979; Chandler and Robertson, 1994). ABA elicitsthe plant responses mentioned through binding to in-tracellular receptors known as Pyrabactin resistance1(Pyr1)/Pyr1-like/regulatory components of ABA re-ceptors (PYR/PYL/RCAR). All of the 14 PYR/PYL/RCARs in Arabidopsis (Arabidopsis thaliana) have beenshown either by mutant analysis or transient gene ex-pression assays to function as ABA receptors (Maet al., 2009; Park et al., 2009; Fuchs et al., 2014). Thepyr1pyl1pyl2pyl4 quadruple mutant shows reducedsensitivity to ABA in seed germination and stomatalclosure (Park et al., 2009; Nishimura et al., 2010), and thepyr1pyl1pyl2pyl4pyl5pyl8 sextuple mutant shows strongABA insensitivity with little inhibition even by 50 mMABA (González-Guzmán et al., 2012). In the presence ofABA, the ABA-receptor complex binds to group Aprotein phosphatase 2C (PP2C), which is a negativeregulator of ABA signaling, and inhibits its phosphatase

1This work was supported by the Program for the Strategic Re-search Foundation at Private Universities (S1311017), JST PRESTO(13413773 to T.U.), Grant-in-Aid for Scientific Research (15H04383to T.U.), and MEXT of Japan (18H04774 to D.T.).

2These authors contributed equally to the article.3Author for contact: takezawa@mail.saitama-u.ac.jp.4Senior author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Daisuke Takezawa (takezawa@mail.saitama-u.ac.jp.).

A.J., K.K., and D.T. wrote and organized the manuscript. M.W.-S.produced transgenic liverwort plants. K.T. and R.O. conducted theRNA-Seq analysis. M.H. and I.Y. analyzed functions of gene paral-ogs. T.T. and A.S. conducted yeast two-hybrid assays. T.U. and Y.S.contributed to experimental design in the transgenic study and tran-sient assays.

www.plantphysiol.org/cgi/doi/10.1104/pp.18.00761

Plant Physiology�, January 2019, Vol. 179, pp. 317–328, www.plantphysiol.org � 2019 American Society of Plant Biologists. All Rights Reserved. 317 www.plantphysiol.orgon February 26, 2020 - Published by Downloaded from

Copyright © 2019 American Society of Plant Biologists. All rights reserved.

activity. Inhibition of the activity of PP2C causes acti-vation of subclass III Suc nonfermenting1 (SNF1)-relatedprotein kinase2 (SnRK2), a central regulatory kinase thatphosphorylates and activates various key cellular mol-ecules necessary for ABA responses (Umezawa et al.,2009; Vlad et al., 2009; Cutler et al., 2010). Among theSnRK2 substrates are the bZIP transcription factors thatrecognize the ABA-responsive elements (ABREs) con-served in promoters of ABA-induced genes (Marcotteet al., 1989; Uno et al., 2000). With other promoter ele-ments, such as CE3 and Sph/RY, ABREs facilitate atissue-specific ABA response (Hattori et al., 1992).

Functions of ABA as a phytohormone have beenshown not only in angiosperms but also in nonvas-cular basal land plants such as mosses. ABA treatmentincreases tolerance to rapid drying in the moss Funariahygrometrica (Werner et al., 1991) and freezing toler-ance in the moss Physcomitrella patens (Minami et al.,2003). In P. patens protonemata, treatment with ABAinduces accumulation of stress-associated transcripts,including those of LEA-like genes, and low-molecular-weight soluble sugars, such as Suc (Knight et al., 1995;Nagao et al., 2005). Completed sequencing of the P.patens genome has revealed four genes for putativePYR/PYL/RCAR, two genes for group A PP2C, andfour genes for subclass III SnRK2 (Rensing et al., 2008;Sakata et al., 2014). Disruption of both group A PP2Cgenes, PpABI1A and PpABI1B, results in a constitutiveABA response and desiccation tolerance (Komatsuet al., 2013). Furthermore, failure in SnRK2 activationin the P. patens mutant line AR7 causes loss of bothABA sensitivity and abiotic stress tolerance (Saruhashiet al., 2015). These results indicate that core signalingmechanisms for ABA signaling are common in em-bryophytes, although the physiological function ofPYR/PYL/RCAR receptors in basal land plants hasnot been demonstrated.

A role of ABA in stress signaling has also been dem-onstrated in liverworts, comprising another class of basalland plants. A rise in the level of endogenous ABA isassociatedwith the development of desiccation tolerancein thalli of the liverworts Exormotheca holstii and Ricciafluitans (Hellwege et al., 1994, 1996). We previouslyreported that gemma, a dormant form for asexual re-production in the liverwort Marchantia polymorpha,shows a sensitive response toABA (Tougane et al., 2010).ABA induces accumulation of LEA-like transcripts andsoluble sugars, mainly Suc, in M. polymorpha, with en-hancement of tolerance to freezing and desiccation(Akter et al., 2014). Exogenously applied Suc with ABAinhibited growth of gemmae and further enhanced thedesiccation tolerance, and therefore, Suc may affect theABA signaling pathway and accelerate the accumulationof LEA-like transcripts and further accumulation of Suc(Akter et al., 2014).

To understand the mechanisms underlying ABA re-sponses in liverworts, we conducted a search for genesfor the PYR/PYL/RCAR-like receptor inM. polymorpha.We analyzed the gene designated M. polymorpha PYR/PYL/RCAR-like1 (MpPYL1) by transient gene expression

assays and in transgenic liverworts to determine itsroles in ABA response, desiccation tolerance, and sugarresponse.

RESULTS

Identification of PYR/PYL/RCAR-Related Genes inM. polymorpha

By phylogenetic analysis based upon a sequenceanalysis of the M. polymorpha genome (Bowman et al.,2017; Sussmilch et al., 2017), we identified five genesas putative orthologs of PYR/PYL/RCAR of otherland plant groups (Fig. 1A). These genes are desig-nated as MpPYL1 (Mapoly0030s0080), MpPYL2(Mapoly0313s0001), MpPYL3 (Mapoly0508s0001), MpPYL4(Mapoly0030s0140), and MpPYL5 (Mapoly0145s0004). Ofthese, the polypeptide encoded by MpPYL1 had thehighest sequence similarity to the products of 14PYR/PYL/RCARs of Arabidopsis and four related genes of P. patens.Structural modeling of MpPYL1 indicated that three-dimensional features of the polypeptide resemble thoseof Arabidopsis PYR/PYL/RCAR with the gate and latchstructures (Supplemental Fig. S1). Furthermore, aminoacids for interaction with ABA and group A PP2C(Mosquna et al., 2011) are conserved in the MpPYL1-encoded polypeptide (Supplemental Fig. S2). To deter-mine the role of MpPYL1 in ABA response, its cDNAwasused in transient assays of P. patens with that of Arabi-dopsis PYL6 for comparison. Protonemal cells of P. patenshave been used for assays of ABA-induced gene expres-sionwith thewheat (Triticum aestivum)Empromoter fusedto the GUS reporter gene (proEm-GUS) in bryophytes(Knight et al., 1995; Marella et al., 2006). When we intro-duced MpPYL1 cDNA into the protonemal cells withproEm-GUS as a reporter, GUS activity significantly in-creased without ABA treatment (Fig. 1B), and the activitywas similar to that achieved by introduction ofArabidopsis PYL6.

To determine whether MpPYL1 activates ABA-induced gene expression via an ABA-specific pathway,we used promoters with mutations in conserved cis-regulatory elements for transient gene expressionassays. The wheat Em promoter contains RY (CATG-CATG) and ABRE (ABRE1a: GACACGTGGC) motifs.Disruption of either of thesemotifs results in reduction ofABA-induced gene expression in P. patens protonemalcells (Sakata et al., 2010). To determine the role of RY andABRE motifs in MpPYL1-induced activation of proEm-GUS, we conducted transient assays using mutant pro-moters. We introduced Em promoters with mutations inABRE (mABRE), RY element (mRY), and both elements(mABRE/mRY) into P. patens protonemal cells with orwithout the MpPYL1 construct to analyze GUS expres-sion. GUS activity was increased by MpPYL1 in cellscointroduced with nonmutated proEm-GUS and proEm(mRY)-GUS. But this increase effect was reduced in cellsin which proEm(mABRE)-GUS and proEm(mABRE/mRY)-GUSwere introduced, indicating that the effect of

318 Plant Physiol. Vol. 179, 2019

Abscisic Acid Receptor in Bryophytes

www.plantphysiol.orgon February 26, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.

MpPYL1 requires ABRE but not RY (Fig. 2A). To confirmthe roles of ABRE inMpPYL1-activated gene expression,we used the pro4xABRE-GUS construct with quadruplerepeats of ABRE fused for assays. Overexpression ofMpPYL1 increased pro4xABRE-GUS expression withoutABA (Fig. 2B).Similar assays conductedusingMarchantia callus cells

(Ghosh et al., 2016a) indicated that overexpression ofMpPYL1 cDNA enhanced proEm-GUS and pro4xABRE-GUS expression (Supplemental Fig. S3, A and B). Fur-thermore, disruption of ABRE resulted in reduction ofthe MpPYL1-enhanced gene expression (SupplementalFig. S3A).

Generation of Transgenic Liverwort Lines OverexpressingMpPYL1-GFP and Genome-Editing Lines withMutated MpPYL1

To analyze the ABA response in intact liverwort plants,we generated transgenicM. polymorpha carryingMpPYL1cDNA fused in frame to GFP (Niwa, 2003) driven by theM. polymorpha EF1 promoter (proMpEF1:MpPYL1-GFP)for overexpression. Among several transgenic lines gen-erated, we obtained two lines, MpPYL1ox8 and MpPY-L1ox9, expressing high levels of theMpPYL1-GFPprotein

as shown by immunoblot analysis (Fig. 3A). To test thesensitivity to ABA of the Takaragaike1 (TAK1) wild typeand transgenic lines, we grew gemmae of these plants ona medium containing ABA, which inhibits growth(Tougane et al., 2010). Growth of both MpPYL1ox8 andMpPYL1ox9 was more severely inhibited by ABA thanwas the growth of TAK1, indicating that the MpPYL1-GFP overexpression lines are hypersensitive to ABA(Fig. 3, B and C; Supplemental Fig. S4).We also generated MpPYL1-deficient lines (Mppyl1)

by CRISPR-Cas9-mediated genome editing (Suganoet al., 2018. We designed and expressed two target se-quences of 18 nucleotides (tg1 and tg2; Fig. 4A) withinthe MpPYL1 gene in transgenic M. polymorpha with theCas9 gene.We then analyzed the nucleotide sequence ofthe MpPYL1 locus of the generated transgenic lines foreach construct. All of the generated lines, Mppyl1ge1a,Mppyl1ge1b, Mppyl1ge1c, Mppyl1ge2a, Mppyl1ge2b, andMppyl1ge2c, had either addition or deletion of a singlenucleotide causing a frame shift within the tg1 and tg2regions (Fig. 4A). When we grew gemmalings of theselines on a medium containing ABA, the plants of all ofthe genome-edited lines tested grew similarly in thepresence or absence of ABA in the medium, indicatingthat they were insensitive to ABA (Fig. 4, B and C;Supplemental Fig. S5).

Figure 1. PYR/PYL/RCAR-Like Genes in March-antia polymorpha. A, Phylogenetic analysis of PYR/PYL/RCAR-related proteins in Arabidopsis, Ambor-ella trichopoda, Picea abies, Selaginella moellen-dorffii, Physcomitrella patens, and M. polymorpha.Amino acid sequences were aligned with MUSCLE.Phylogenetic relationship among PYR/PYL/RCAR-like proteins was inferred using the neighbor-joiningmethod. Numbers on the branches indicate boot-strap values (100 replicates). The bar represents thenumber of amino acid changes per branch length.Phylogenetic analyses were conducted in MEGA7.B, Effect of overexpression of MpPYL1 on ABA-induced gene expression. The cDNA of MpPYL1fused with the rice actin promoter was introducedinto the P. patens protonemata cells by particlebombardment. The ABA-inducible Em promoterfused with the GUS reporter gene was used as a re-porter construct. The tissues were incubated with orwithoutABA (10mM)andused for fluorometricGUSassays. GUS activity was normalized by expressionof the cointroduced luciferase (LUC) reporter gene.Results of PYL6 of Arabidopsis (AtPYL6, At2g40330)are shown for comparison. *P , 0.05 and ***P ,0.001 by the t test.

Plant Physiol. Vol. 179, 2019 319

Jahan et al.

www.plantphysiol.orgon February 26, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.

Transcriptome Analysis of Transgenic Lines withAltered MpPYL1

To determine changes in ABA-induced gene expres-sion by manipulation of MpPYL1, we subjected ABA-responsive transcriptomes in representative transgeniclines, MpPYL1ox8 and Mppyl1ge2b, and TAK1 to RNA-sequencing (seq) analysis. Analysis of 19,287 expressedgenes of gemmalings cultured with or without 1 mMABA for 6 h revealed that of 567 ABA-induced genes inTAK1, expression of 455 genes (80%) was decreased inMppyl1ge2b. Expression of 236 genes (42%) was increasedin MpPYL1ox8 (Fig. 5A; Supplemental Table S1). Fur-thermore, among 378 ABA-repressed genes in TAK1,expression of 235 genes (62%) was increased inMppyl1ge2b. Hierarchical clustering and heatmap analy-sis also indicated that ABA-responsive expression of themajority of transcripts was affected in Mppyl1ge2b(Fig. 5B). Gene ontology analysis using 455 genes (whichwere induced by ABA in TAK1 but reduced inMppyl1ge2b) revealed that transcripts of genes involvedin abiotic stress response and seed development(Supplemental Fig. S6), including genes encoding

various LEA-like proteins, were under the control ofMpPYL1. Of 35 LEA-like transcripts induced by ABA inTAK1, expression of all transcripts was reduced inMppyl1ge2b. Expression of 32 transcripts was increasedin MpPYL1ox8 (Fig. 6A; Supplemental Table S2). Wepreviously showed that expression of several MpLEALgenes encoding LEA-like proteins was strongly inducedbyABA (Ghosh et al., 2016b). Both RT-PCR (Fig. 6B) andRNA gel-blot analysis (Supplemental Fig. S7) indicatedthat accumulation of MpLEAL transcripts was enhancedin MpPYL1ox8 and reduced in Mppyl1ge2b, being con-sistent with the results of RNA-seq analysis.

Sugar Responses in Transgenic Lines

In our previous study, bothABA treatment and sugartreatment caused inhibition of gemma growth and en-hancement of desiccation tolerance with increased ex-pression of LEA-like transcripts (Akter et al., 2014).Here, we grew gemmae of TAK1, MpPYL1ox8, andMppyl1ge2b on media containing 0, 0.1 and 0.2 M Suc.Growth of MpPYL1ox8 gemmae was more severelyinhibited by 0.1 M Suc than was growth of TAK1 gem-mae (Fig. 7). Such Suc hypersensitivity was also ob-served in MpPYL1ox9 (Supplemental Fig. S8). Theseresults indicated that enhancement of ABA signals byoverexpression of MpPYL1-GFP might have increasedsensitivity to sugars in the gemmalings. Thus, we testedhow externally applied ABA and sugar coordinatelyaffect accumulation of representative MpLEAL tran-scripts inMpPYL1ox8 andMppyl1ge2b. Accumulation ofthe MpLEAL transcripts was induced by 0.1 M Suc onlyslightly after 6-h treatment in TAK1, whereas they weremore abundantly induced by Suc in MpPYL1ox8.When the medium contained both ABA and Suc, ex-pression of the MpLEAL genes were strongly inducedin TAK1, and the difference in abundance of the tran-scripts between TAK1 and MpPYL1ox8 was small. Incontrast, there was little accumulation of MpLEALtranscripts in Mppyl1ge2b with or without ABA and Suc(Fig. 8A; Supplemental Figs. S9 and S10).

In addition to MpLEAL transcripts, we analyzedABA- and Suc-increased accumulation of low-molecu-lar-weight soluble sugars associated with desiccationtolerance (Akter et al., 2014) in gemmalings. We de-termined levels of soluble sugars in tissues of TAK1,MpPYL1ox8, and Mppyl1ge2b with or without ABA and0.1 M Suc in the medium. Without external Suc or ABA,levels of soluble sugars in tissues were similar in allthree lines. Treatment with ABA but without Suc in-duced sugar accumulation in tissues of MpPYL1ox8and TAK1 but not in Mppyl1ge2b (Fig. 8B). In the pres-ence of 0.1 M Suc butwithoutABA in themedium, sugarcontents in tissues were similar in TAK1 and MpPY-L1ox8, but sugar content was significantly less thanTAK1 in Mppyl1ge2b. ABA treatment with 0.1 M Suc inthe medium increased sugar levels in tissues of all threelines, but the sugar levels were less in Mppyl1ge2b thanin TAK1 and MpPYL1ox8 (Fig. 8C). These results

Figure 2. MpPYL1-EnhancedGene Expression Is Mediated by ABRE. A,Transient assays of Physcomitrella patens protonemata cells using theGUS reporter gene fused to the nonmutated Em promoter and thepromoters with mutations in ABRE (mABRE), RY (mRY), or both(mABRE/mRY). B, Enhanced expression by MpPYL1 of GUS fused withthe 4xABRE promoter (pro4xABRE-GUS). The GUS construct withoutthe promoter (GUS) was used as a control. GUS expression was nor-malized by the cointroduced LUC gene. Error bars indicate SE (n = 3).*P , 0.05 by the t test.

320 Plant Physiol. Vol. 179, 2019

Abscisic Acid Receptor in Bryophytes

www.plantphysiol.orgon February 26, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.

indicated that MpPYL1 is required for ABA- and Suc-induced sugar accumulation. We also analyzed desic-cation tolerance in TAK1, MpPYL1ox8, andMppyl1ge2b.Gemmalings of these plants were incubated with orwithout ABA and Suc and then dried in a chambercontaining silica gel.We found that treatment with bothABA and Suc increased desiccation tolerance in TAK1.Treatment with ABA alone could induce desiccationtolerance in MpPYL1ox8. Either of these treatments(and the treatment with both ABA and Suc) did notincrease desiccation tolerance in Mppyl1ge2b (Fig. 9).

DISCUSSION

Important Role of PYR/PYL/RCAR Receptors in ABASignaling in Embryophytes

Among several candidates for the ABA receptors,PYR/PYL/RCARs identified in Arabidopsis have beenrecognized as bona fide ABA receptor family proteins.Overexpression of PYR/PYL/RCAR orthologs in to-mato (Solanum lycopersicum) and rice (Oryza sativa) re-sults in enhanced tolerance to drought, indicating thatPYR/PYL/RCAR-mediated stress signaling is commonin angiosperms (Kim et al., 2012; González-Guzmánet al., 2014). Phylogenetic analysis indicates that PYR/

PYL/RCAR-like genes are conserved in various em-bryophyte groups (Sakata et al., 2014; Sussmilch et al.,2017). However, there has also been controversy onwhether ABA receptors function in liverworts, repre-senting one of the earliest landplant lineages (Chen et al.,2017), and physiological roles of PYR/PYL/RCAR-likegenes in liverworts have not been demonstrated. Of fiveputative PYR/PYL/RCAR orthologs identified in theM.polymorpha genome, MpPYL1 appears to be the onlyPYR/PYL/RCAR-like gene strongly expressed in ga-metophytes (gemma and thalli), while four other paral-ogs (MpPYL2, MpPYL3, MpPYL4, and MpPYL5) arepreferentially expressed in sporophytes (Bowman et al.,2017). We showed that overexpression of MpPYL1-GFPin the pyr1/pyl1/pyl2/pyl4 quadruple mutant of Arabi-dopsis could partially complement ABA-insensitivity ofthemutant (Bowman et al., 2017). In this study, we showthat MpPYL1 plays a primary role in ABA signaling inthe liverwort gametophyte by analysis of transgenic M.polymorpha. Preferential expression in the sporophyte offour other PYR/PYL/RCAR-like genes of M. polymorphadoes not necessarily indicate these genes are nonfunctional.Transient gene expression assays indicated that over-expression of MpPYL2 and MpPYL3 can enhance expres-sion of proEm-GUS, indicating that these paralogs mightalso encode functional ABA receptors (SupplementalFig. S11). In contrast, overexpression of MpPYL4 with an

Figure 3. Enhanced ABA Sensitivity in MpPYL1-GFPOverexpression Lines of TransgenicMarchantiapolymorpha. A, Two transgenic lines, MpPYL1ox8and MpPYL1ox9, were subjected to immunoblotanalysis using an anti-GFP antibody. Molecular sizemarkers are shown in kilodalton (kDa). B and C,Growth of TAK1 (the wild type) and the transgeniclines on a medium containing 0 to 5 mM ABA areshown by area (B) and appearance (C) of the thalli.Error bars indicate SE (n = 3). *P , 0.05 comparedwith TAK1 of the same treatment by the t test. Long-term effect of ABA on growth of the thalli is shown inSupplemental Fig. S4.

Plant Physiol. Vol. 179, 2019 321

Jahan et al.

www.plantphysiol.orgon February 26, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.

extra N-terminal amino acid stretch and MpPYL5 lackingseveral conserved amino acids (Supplemental Fig. S1) didnot enhance the proEm-GUS expression.

Transcriptome analysis with gene ontology enrich-ment indicated that MpPYL1 controls the expression ofgenes involved in abiotic stress tolerance, including the

Figure 4. Generation and Growth Analysis ofMpPYL1 Genome-Edited Lines. A, Nucleotide se-quences of theMpPYL1 genome region of TAK1 (thewild type) and genome-edited lines Mppyl1ge1b,Mppyl1ge1c, Mppyl1ge2b, andMppyl1ge2c. A hyphenrepresents a deletion of a nucleotide in the genome-edited lines. B, Fresh weight of gemmalings culturedon medium containing 10 mM ABA for two weeks.Error bars indicate the SE (n=3). *P,0.05 comparedwith the minus ABA control by the t test. C, Ap-pearance of the representative gemmalings shown in(B). Effects of higher concentration of ABA on growthare shown in Supplemental Fig. S5.

Figure 5. Transcriptome Analysis ofMpPYL1-Dependent Genes. A, Venn dia-grams showing the number of MpPYL1-regulated genes revealed by RNA-seqanalysis. The upper diagram shows rela-tionships between 567 genes induced byABA in TAK1 (the wild type), 397 genes forwhich expression in ABA-treated MpPY-L1ox8wasmore than2-fold of that inABA-treated TAK1 and 1013 genes for whichexpression in ABA-treatedMppyl1ge2bwasless than 2-fold of that in ABA-treatedTAK1. The lower diagram shows relation-ships between378genes repressedbyABAin TAK1, 182 genes forwhich expression inABA-treated MpPYL1ox8 was less than2-fold of that in ABA-treated TAK1, and423 genes for which expression in ABA-treated Mppyl1ge2b was more than 2-foldof that in ABA-treated TAK1. B, A heatmapshows hierarchical clustering of transcripts.Gene expression values are represented asrelative to the mean of all samples; bluecolor represents lower expression, and redcolor represents higher expression.

322 Plant Physiol. Vol. 179, 2019

Abscisic Acid Receptor in Bryophytes

www.plantphysiol.orgon February 26, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.

majority of ABA-induced LEA-like transcripts (Fig. 6;Supplemental Fig. S6; Supplemental Table S2). Resultsalso indicated that MpPYL1 regulates the expression of

Mapoly0072s0050 encoding an ABA-responsive element-binding protein (AREB)-like transcription factor(Supplemental Table S1), which activates ABA-induced

Figure 6. ABA-Induced Expression of LEA-Like Genes in TAK1, MpPYL1ox8, and Mppyl1ge2b. A, Venn diagram showing ex-pression of LEA-like genes in TAK1 (the wild type) and expression of 38 genes inMpPYL1ox8 andMppyl1ge2b. The diagram showsthe relationship between 35 LEA-like genes for which expression was induced by ABA in TAK1, 33 genes for which expressionwas increased in MpPYL1ox8, and 38 genes for which expression was decreased in Mppyl1ge2b. B, RT-PCR analysis ofRNA isolated from gemmalings treated with 1 mM ABA for 6 h. Primers for MpLEAL1 (Mapoly0112s0030), MpLEAL2(Mapoly0119s0008), MpLEAL3 (Mapoly0035s0082), MpLEAL4 (Mapoly0016s0148), MpLEAL5 (Mapoly0087s0015), MpLEAL6(Mapoly0027s0114), and MpLEAL7 (Mapoly0025s0042) were used for amplification. Results for MpEF1 (Mapoly0024s0116)unchanged by ABA and ethidium bromide-stained rRNA bands are shown for comparison.

Figure 7. Sensitivity of Growth to Suc in Gemmal-ings of TAK1, MpPYL1ox8, andMppyl1ge2b lines. A,Growth appearance onmedia containing 0, 0.1, and0.2 M Suc after 2 weeks of culture. B, Fresh weightmeasurement of plants shown in (A). Error bars in-dicate SE (n = 3). *P, 0.05 by the t test.

Plant Physiol. Vol. 179, 2019 323

Jahan et al.

www.plantphysiol.orgon February 26, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.

genes by binding to an ABRE (Uno et al., 2000; Fujitaet al., 2005). We previously showed that ABA-inducedgene expression in M. polymorpha is dependent on anABRE as shown for both the wheat Em promoter andendogenous MpDHN1 promoter (Ghosh et al., 2016a).ABA-induced gene expression by both promoters wasinhibited by overexpression of M. polymorpha ABI1A(MpABI1A) encoding group A PP2C, a negative regu-lator of ABA signaling (Tougane et al., 2010; Ghosh et al.,2016a). Yeast two-hybrid assays indicated that MpPYL1interactedwithMpABI1A (Supplemental Fig. S12). WithMpPYL1-dependent gene activation mediated by ABRE(Fig. 2), results of our study indicate that the core sig-naling pathway leading to ABA-induced gene expres-sion is common in nonvascular plants, such asliverworts, and in vascular plants. Phylogenetic analysisof PYR/PYL/RCAR indicated that its orthologs are alsopresent in the moss P. patens but not in the CharophyteKlebsormidium flaccidum (Klebsormidium nitens), impli-cating that PYR/PYL/RCAR had emerged in the com-mon ancestor of land plants (Sakata et al., 2014). Thegenes for other elements required for the ABA response,namely, group A PP2C, SnRK2, and clade A bZIP, areidentified not only in embryophytes but also in Char-ophytes (Corrêa et al., 2008; Fuchs et al., 2013; Hori et al.,2014; Bowman et al., 2017). The results of our study

therefore suggest that acquisition of PYR/PYL/RCARby the common ancestor of embryophytes is associatedwith establishment of ABA as a phytohormone duringthe process of adaptation to a terrestrial environmentwith limited water availability (Ligrone et al., 2012;Sakata et al., 2014).

Possible Roles of ABA and MpPYL1 in Sugar Response forDesiccation Tolerance

We previously reported that ABA induces accumu-lation of low-molecular-weight soluble sugars, espe-cially Suc, and that ABA and Suc synergistically inhibitgrowth and enhance desiccation tolerance of gemmal-ings of M. polymorpha (Akter et al., 2014). Results ofexpression studies indicated that the Suc-induced geneexpression is mediated by PYR/PYL/RCAR (Fig. 8A;Supplemental Fig. S10), suggesting signal crosstalkbetween ABA and Suc in liverworts. Crosstalk betweenABA and sugar signaling processes is well documentedin angiosperms through identificationofABA-biosyntheticand ABA-insensitive mutants in sugar response mu-tant screens of Arabidopsis seeds (Arenas-Huerteroet al., 2000; Laby et al., 2000; Rolland et al., 2002).However, the physiological significance of the relationship

Figure 8. Effects of ABA and Suc on Gene Expres-sion and Sugar Accumulation. A, ABA- and Suc-induced gene expression in gemmalings of TAK1,MpPYL1ox8, and Mppyl1ge2b. Gemmalings werecultured for 6 h in a medium with or without 0.1 M

Suc, and 1 mM ABAwere used for RNA extraction.The extracted RNA was used for RT-PCR analysisusing primers of LEA-like genes, MpLEAL1 (Map-oly0112s0030), MpLEAL2 (Mapoly0119s0008), andMpLEAL3 (Mapoly0035s0082). Results for MpEF1(Mapoly0024s0116) and ethidium bromide-stainedrRNA bands are shown for comparison. B and C,ABA-induced sugar accumulation is shown ingemmalings of TAK1, MpPYL1ox8, andMppyl1ge2b.Gemmalings were cultured for one day with orwithout 1 mM ABA in a half-strength B5 mediumcontaining no sugar (B) or 0.1 M Suc (C). Theextracted sugars were used for quantitation by an-throne assays with Glc as a standard. Error bars in-dicate SE (n = 3). *P, 0.05 by the t test.

324 Plant Physiol. Vol. 179, 2019

Abscisic Acid Receptor in Bryophytes

www.plantphysiol.orgon February 26, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.

between ABA and sugar responses is not clearly un-derstood. Sugars are among the essential metaboliccomponents as an energy source, structural compo-nents, and osmolytes for maintenance of cell turgor(Koch, 2004). Sugars also play a role in desiccation tol-erance by protecting cellular membranes and proteinsfrom dehydration-induced damage (Hoekstra et al.,2001). It has been suggested that Suc serves as a sig-naling molecule that plays a distinct role in metabolicand developmental responses in plants by affectinggene expression (Chiou and Bush, 1998). Throughcrosstalk between other environmental signals such aschanges in light, water potential, and temperatures orlevels of phytohormones, it is thought that plants con-trol carbohydrate metabolism for growth, storage, orenvironmental stress tolerance (Smeekens, 1998;Tognetti et al., 2013).Signal transduction for sugars is known to be depen-

dent on the activity of Suc non-fermenting-related kinase1 (SnRK1), by which the cell senses low Suc levels undera stressful condition (Baena-González and Sheen, 2008).SnRK1 likely mediates ABA signaling, because trans-genic plants overexpressing SnRK1.1 show hypersensi-tivity to Glc and ABA (Jossier et al., 2009). Two group APP2Cs, ABSCISIC ACID INSENSITIVE1 and PP2CA,interact with SnRK1 and act as negative regulators(Rodrigues et al., 2013). It is thus likely that SnRK1 isunder the control of ABA and PYR/PYL/RCAR. Inaddition to SnRK1, SnRK2 might also participate inABA-mediated metabolic regulation of sugars, becauseoverexpression of SnRK2.6 increases Suc accumulationin Arabidopsis (Zheng et al., 2010). M. polymorpha hasgenes for proteins with high similarity to SnRK1 (Map-oly0160s0010) and SnRK2 (Mapoly0061s0075 and Map-oly0011s0096), although their functions in ABA andsugar responses have not been determined.

Our results also indicate that MpPYL1 is involved inaccumulation of soluble sugars in tissues (Fig. 8, B andC). This is consistent with the results of gene ontologyanalysis of the transcriptome showing that the expres-sion of genes involved in polysaccharide catabolism,possibly contributing to degradation of starch to pro-duce low-molecular-weight soluble sugars, is MpPYL1dependent (Supplemental Fig. S4). The analysis alsorevealed that the expression of genes for putative sugartransporters (Mapoly0009s0099, Mapoly0050s0068, andMapoly0175s0014) for sugar uptake can be controlled byMpPYL1 (Supplemental Table S1). We previouslyreported that accumulation of soluble sugars is in-creased by exogenous ABA, which enhances freezingand desiccation tolerance in P. patens (Nagao et al.,2005), indicating that there might be a common mech-anism for ABA-induced accumulation of sugars inbryophytes. It is likely that the accumulated sugarscontribute to protecting cells from damage caused bycellular dehydration during freezing and desiccationwith assistance of various species of LEA-like proteinsinduced by ABA.

Conclusion

PYR/PYL/RCAR plays a role in ABA-induced ac-cumulation of stress-associated transcripts throughconserved promoter elements in liverworts and angio-sperms. Our results not only clarify the role of the ABAreceptor in gene expression but also indicate that theABA signaling pathway mediates sugar response andaccumulation for vegetative desiccation tolerance.These responses represent the archetypal function ofABA, which was likely acquired before establishmentof the ABA-mediated regulation of stomata (Chen et al.,2017) or determination of sex (McAdam et al., 2016) invascular plants. Our future works will focus on iden-tification of key signaling molecules regulated by Sucand ABA through transcriptome and proteome analy-ses of transgenic plants, in which levels of MpPYL1have been manipulated, along with analysis of solublesugars and their intermediate metabolites.

MATERIALS AND METHODS

Chemicals and Plant Materials

Chemicals were purchased from Wako Jun-yaku (Osaka, Japan) unlessotherwise stated. Abscisic acid (ABA) was from Sigma (A4906, St. Louis, MO).Gametophytes ofMarchantia polymorphawere cultured at 22°C on half-strengthGamborg’s B5 agar medium containing 1% (w/v) Suc under continuous light(Kubota et al., 2013). Protonemata of Physcomitrella patens andMarchantia calluscells were cultured as described previously (Tougane et al., 2010).

Reporter Assays by Particle Bombardment

P. patens protonemata cultured on cellophane-overlaid BCDAT agar me-dium for 5 d were used for particle bombardment (Tougane et al., 2010).Marchantia callus cells were cultured in 1M51C liquid medium for 2 to 3 dand collected on a filter paper for bombardment (Ghosh et al., 2016a). Theplasmids with cDNA of Mapoly0030s0080 (MpPYL1), Mapoly0313s0001

Figure 9. Desiccation Tolerance of Gemmalings of TAK1, MpPYL1ox8,and Mppyl1ge2b. Gemmalings cultured with or without 10 mM ABA(+ABA) and 0.1 M Suc (+Suc) were desiccated in a container with silicagel for two days in the dark. The rehydrated gemmalings were trans-ferred onto agar medium and cultured for two weeks.

Plant Physiol. Vol. 179, 2019 325

Jahan et al.

www.plantphysiol.orgon February 26, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.

(MpPYL2), Mapoly0508s0001 (MpPYL3), Mapoly0030s0140 (MpPYL4), Map-oly0145s0004 (MpPYL5), or PYL6 (AT2G40330) fused to the rice (Oryza sativa)actin promoter were used as the effector constructs. The reporter constructproEm-GUS and its mutation constructs, proEm(mABRE)-GUS, proEm(mRY)-GUS, and proEm(mABRE/mRY)-GUS, were described in detail previously(Sakata et al., 2010). The 4xABRE-GUS construct has four ACGT-core repeats,AGCTCCACGTGTCGGCA, upstream of the minimal promoter of PpLEA1(Yotsui et al., 2013). The cells were bombarded with DNAs of the effector andreporter constructs and the Ubi-LUC reference constructs coated on 1-mM goldparticles using the PDS-1000He particle delivery system (Bio-Rad, Hercules,CA). The bombarded cells were incubated in a medium with or without ABAand were used for GUS and LUC assays (Jefferson et al., 1987; Tougane et al.,2010).

Generation of Transgenic M. polymorpha

The MpPYL1-GFP construct was fused with the MpEF1 promoter for over-expression (Althoff et al., 2014) using pCambia1300. For the MpPYL1 genome-editing construct, double-strand oligonucleotides containing the target guideRNA seq were fused downstream to the U6 promoter in the pMpGE010 vectorcontaining the Cas9 gene cassette. Agrobacterium tumefaciens (Rhizobium radio-bacter) C58 strain harboring the above constructs was used for infection of two-week-cultured gemmalings of M. polymorpha as described by Kubota et al.(2013). After selection on a medium containing 10 mg L21 hygromycin and100 mg L21 cefotaxime, the generated transgenic lines were individually cul-tured on a fresh selection medium for further propagation.

Protein Gel Electrophoresis and Immunoblot Analysis

Tissues of gemmalings were homogenized using 50 mM Tris-Cl (pH 7.5),2 mM EDTA, 100 mM NaCl, 2 mM DTT, and a 1:100 volume of the proteinaseinhibitor cocktail (Sigma, St. Louis, MO). After centrifugation, the supernatantwas subjected to SDS-PAGE. For immunoblot analysis, the electrophoresedproteins were transferred onto a polyvinylidene fluoride membrane. Afterblocking with 3% skim milk diluted in TBS-T (25 mM Tris-Cl, pH 7.5, 150 mM

NaCl, and 0.05% (w/v) Tween 20), the proteins were reacted with a 1:1000dilution of an anti-GFP antibody (MBL, Nagoya, Japan) and thenwith a 1:10000dilution of a horseradish peroxidase-conjugated secondary antibody (170-6515,Bio-Rad, Hercules, CA). The membrane was reacted with chemiluminescencereagent (Chemi-Lumi One, Nacalai Tesque, Kyoto, Japan) and exposed to x-rayfilm for signal detection.

Extraction of Total RNA, Reverse Transcription (RT)-PCR,and RNA Gel-Blot Analysis

Gemmalings of M. polymorpha cultured for four days in a liquid mediumwere used for ABA and sugar treatment. RNA extraction, first-strand cDNAsynthesis, and PCR reactions were conducted according to Ghosh et al. (2016b).For RNA gel-blot analysis, total RNA electrophoresed in a formaldehyde aga-rose gel was blotted on a nylon membrane and hybridized with 32P-labeledcDNA probes of M. polymorpha LEA-like (MpLEAL) genes (Akter et al., 2014).Hybridization was conducted using Ultrahyb hybridization buffer (Ambion,Austin, TX) at 42°C overnight, andwashingwas conducted at 65°C for one hourin a buffer containing 0.2 X SSC (30 mM NaCl and 3 mM Na citrate) and 0.2%(w/v) SDS. The membrane was then exposed to x-ray film to detect radioactivesignals.

RNA-Seq Library Construction and Sequencing

RNA-seq libraries were prepared using a TruSeq RNA sample prep kit v2(Illumina, San Diego, CA), and library quality control was performed withBioanalyzer 2100 (Agilent, Santa Clara, CA). The libraries had inserts with a sizerange of approximately 200–500 bp. Library quantification was conducted us-ing RT-quantitative PCR (RT-qPCR), and the concentration of each library wasadjusted to 10 nM. The prepared libraries were sequenced using the IlluminaHiSeq 2500 system. The raw reads for each library were deposited into DDBJand are available through Sequence Read Archive with the accession numberDRA005869.

RNA-Seq Alignment and Data Analysis

Single reads fromeach librarywereprocessedusingCASAVAversion1.8.2 inFASTQ format. The FASTQ files were imported to CLC Genomics Workbench(QIAGEN, USA) for subsequent analysis. The Trim sequences tool in the suitewas used to remove adapters and to filter out low-quality bases (,Q13), andonly the reads that showed a quality score of 13 or greater were retained.The filtered sequence reads were mapped onto the M. polymorpha genomev3.1 (Phytozome 12; https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Mpolymorpha) using the CLC assembler with default parameters.Expression values were reported as reads per kilobase per million (RPKM)values. Differentially expressed genes were detected by the Empirical Analysisof Digital Gene Expression algorithm in the CLC software. The results werefiltered on the basis of a False Discovery Rate (FDR), with P-values less than 0.05and a corrected fold change greater than 2. Hierarchical clustering and heatmapanalysis were performed on the basis of Euclidian distance using the CLCsoftware. RPKM values were used for the hierarchical clustering analysis. Geneontology enrichment analysis was performed using BiNGO (Biological Net-work Gene Ontology) software, a Cytoscape plug-in (available at www.cytoscape.org) for putative Arabidopsis (Arabidopsis thaliana) orthologs ofMpPYL1-dependent ABA-induced genes of M. polymorpha. These putativeArabidopsis orthologs were identified by a BLASTP search against the TAIR10protein database. The degree of functional enrichment for a given cluster wasquantitatively assessed by a hypergeometric test, and a multiple test correctionwas applied using the FDR algorithm implemented in the BiNGO plug-in.Overrepresented GO terms were generated after FDR correction, with a sig-nificance level of 0.05.

Sugar Extraction and Quantitation

Gemmalings were weighed, frozen, and crushed using amortar and a pestlein 80% (v/v) ethanol. After removal of insoluble materials by centrifugation at14,000 g for 10 min at 4°C, the supernatant was dried and suspended in water.After removal of water-insoluble material by centrifugation, the supernatantwas used as a sugar sample. The amounts of sugars were determined by theanthrone-sulfuric acid assay, with Glc as a standard (Yemm and Willis, 1954).

Tests for Desiccation Tolerance

Gemmaewere cultured for threedays in1/2B5 liquidmediumwithorwithout10 mMABA or 0.1 M Suc and transferred to a sheet of cellophane set on wet filterpaper in a petri dish. The dish was then placed in a container containing silica geland kept at 23°C for one day in the dark for desiccation treatment. The driedgemmae on the cellophane were rehydrated by adding 1/2 B5 liquid medium.The gemmae with the cellophane were then transferred to a fresh 1/2 B5 agarmedium and cultured for 2 weeks to determine survival.

Accession Numbers

Accession numbers of genes mentioned in this study are listed inSupplemental Table S3.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Structural Models of MpPYL1.

Supplemental Figure S2. Comparison of the Amino Acid Sequences ofPYR/PYL/RCAR-Like Proteins.

Supplemental Figure S3. Transient Assays of Marchantia callus Cells forAnalysis of MpPYL1 Functions.

Supplemental Figure S4. Long-Term Effect of ABA Treatment on TAK1,MpPYL1ox8, and Mppyl1ge2b.

Supplemental Figure S5. Growth Response to a 50 mM ABA in TAK1 andMppyl1ge2b.

Supplemental Figure S6. GO Term Network Analysis on 455 MpPYL1-Regulated Genes.

326 Plant Physiol. Vol. 179, 2019

Abscisic Acid Receptor in Bryophytes

www.plantphysiol.orgon February 26, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.

Supplemental Figure S7. RNA Gel-Blot Analysis of TAK1, MpPYL1ox8,and Mppyl1ge2b.

Supplemental Figure S8. Growth Response to Suc in TAK1, MpPYL1ox8,and MpPYL1ox9.

Supplemental Figure S9. Analysis of ABA- and Suc-Induced Expression ofLEA-Like Genes.

Supplemental Figure S10. Effects of Suc on MpLEAL1 Gene Expression inTAK1 and Mppyl1ge2b.

Supplemental Figure S11. Transient Assays of P. patens protonemata andMarchantia callus Cells for Analysis of PYR/PYL/RCAR Paralogs.

Supplemental Figure S12. Interaction of MpPYL1 with MpABI1A in YeastCells.

Supplemental Table S1. ABA-Responsive Genes and Those Altered inMpPYL1ox8 and Mppyl1ge2b.

Supplemental Table S2. ABA-Induced LEA-Like Genes in M. polymorpha.

Supplemental Table S3. List of Accession Numbers of Genes Mentioned inThis Study.

ACKNOWLEDGMENTS

We thank Ryuichi Nishihama (Kyoto University), Takayuki Kohchi (KyotoUniversity), and Kimitsune Ishizaki (Kobe University) for providing thepMpGE10 vector and for critical discussion. We also thank Yasuo Niwa forGFP(S65T), Ralph Quatrano for proEm-GUS, Tuan-hua David Ho for Ubi-LUCconstructs, and Totan Kumar Ghosh and Shuuhei Murai for technicalassistance.

Received June 25, 2018; accepted October 27, 2018; published November 15,2018.

LITERATURE CITED

Akter K, Kato M, Sato Y, Kaneko Y, Takezawa D (2014) Abscisic acid-induced rearrangement of intracellular structures associated withfreezing and desiccation stress tolerance in the liverwort Marchantiapolymorpha. J Plant Physiol 171: 1334–1343

Althoff F, Kopischke S, Zobell O, Ide K, Ishizaki K, Kohchi T, Zachgo S(2014) Comparison of the MpEF1a and CaMV35 promoters for appli-cation in Marchantia polymorpha overexpression studies. Transgenic Res23: 235–244

Arenas-Huertero F, Arroyo A, Zhou L, Sheen J, León P (2000) Analysis ofArabidopsis glucose insensitive mutants, gin5 and gin6, reveals a centralrole of the plant hormone ABA in the regulation of plant vegetativedevelopment by sugar. Genes Dev 14: 2085–2096

Baena-González E, Sheen J (2008) Convergent energy and stress signaling.Trends Plant Sci 13: 474–482

Bowman JL, Kohchi T, Yamato KT, Jenkins J, Shu S, Ishizaki K, YamaokaS, Nishihama R, Nakamura Y, Berger F, et al (2017) Insights into landplant evolution garnered from the Marchantia polymorpha genome. Cell171: 287–304.e15

Chandler PM, Robertson M (1994) Gene expression regulated by abscisicacid and its relation to stress tolerance. Annu Rev Plant Physiol 45:113–141

Chen ZH, Chen G, Dai F, Wang Y, Hills A, Ruan YL, Zhang G, Franks PJ,Nevo E, Blatt MR (2017) Molecular evolution of grass stomata. TrendsPlant Sci 22: 124–139

Chiou TJ, Bush DR (1998) Sucrose is a signal molecule in assimilate par-titioning. Proc Natl Acad Sci USA 95: 4784–4788

Corrêa LG, Riaño-Pachón DM, Schrago CG, dos Santos RV, Mueller-Roeber B, Vincentz M (2008) The role of bZIP transcription factors ingreen plant evolution: Adaptive features emerging from four foundergenes. PLoS One 3: e2944

Cuming AC, Lane BG (1979) Protein synthesis in imbibing wheat embryos.Eur J Biochem 99: 217–224

Cutler SR, Rodriguez PL, Finkelstein RR, Abrams SR (2010) Abscisic acid:Emergence of a core signaling network. Annu Rev Plant Biol 61: 651–679

Fuchs S, Grill E, Meskiene I, Schweighofer A (2013) Type 2C proteinphosphatases in plants. FEBS J 280: 681–693

Fuchs S, Tischer SV, Wunschel C, Christmann A, Grill E (2014) Abscisicacid sensor RCAR7/PYL13, specific regulator of protein phosphatasecoreceptors. Proc Natl Acad Sci USA 111: 5741–5746

Fujita Y, Fujita M, Satoh R, Maruyama K, Parvez MM, Seki M, Hiratsu K,Ohme-Takagi M, Shinozaki K, Yamaguchi-Shinozaki K (2005) AREB1is a transcription activator of novel ABRE-dependent ABA signalingthat enhances drought stress tolerance in Arabidopsis. Plant Cell 17:3470–3488

Ghosh TK, Kaneko M, Akter K, Murai S, Komatsu K, Ishizaki K, YamatoKT, Kohchi T, Takezawa D (2016a) Abscisic acid-induced gene ex-pression in the liverwort Marchantia polymorpha is mediated by evolu-tionarily conserved promoter elements. Physiol Plant 156: 407–420

Ghosh TK, Kaneko M, Takezawa D (2016b) Transient assays of gemmal-ings of the liverwort Marchantia polymorpha for studies of abscisic acid-induced gene expression. Japanese Society for Cryobiology and Cry-otechnology 62: 57–60

González-Guzmán M, Pizzio GA, Antoni R, Vera-Sirera F, Merilo E,Bassel GW, Fernández MA, Holdsworth MJ, Perez-Amador MA,Kollist H,et al (2012) Arabidopsis PYR/PYL/RCAR receptors play amajor role in quantitative regulation of stomatal aperture and tran-scriptional response to abscisic acid. Plant Cell 24: 2483–2496

González-Guzmán M, Rodríguez L, Lorenzo-Orts L, Pons C, Sarrión-Perdigones A, Fernández MA, Peirats-Llobet M, Forment J, Moreno-Alvero M, Cutler SR, et al (2014) Tomato PYR/PYL/RCAR abscisicacid receptors show high expression in root, differential sensitivity tothe abscisic acid agonist quinabactin, and the capability to enhance plantdrought resistance. J Exp Bot 65: 4451–4464

Hattori T, Vasil V, Rosenkrans L, Hannah LC, McCarty DR, Vasil IK(1992) The Viviparous-1 gene and abscisic acid activate the C1 regulatorygene for anthocyanin biosynthesis during seed maturation in maize.Genes Dev 6: 609–618

Hellwege EM, Dietz KJ, Volk OH, Hartung W (1994) Abscisic acid and theinduction of desiccation tolerance in the extremely xerophilic liverwortExormotheca holstii. Planta 194: 525–531

Hellwege EM, Dietz KJ, Hartung W (1996) Abscisic acid causes changes ingene expression involved in the induction of the landform of the liver-wort Riccia fluitans L. Planta 198: 423–432

Hoekstra FA, Golovina EA, Buitink J (2001) Mechanisms of plant desic-cation tolerance. Trends Plant Sci 6: 431–438

Hori K, Maruyama F, Fujisawa T, Togashi T, Yamamoto N, Seo M, Sato S,Yamada T, Mori H, Tajima N, et al (2014) Klebsormidium flaccidum ge-nome reveals primary factors for plant terrestrial adaptation. NatCommun 5: 3978

Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions:b-glucuronidase as a sensitive and versatile gene fusion marker inhigher plants. EMBO J 6: 3901–3907

Jossier M, Bouly JP, Meimoun P, Arjmand A, Lessard P, Hawley S,Grahame Hardie D, Thomas M (2009) SnRK1 (SNF1-related kinase 1)has a central role in sugar and ABA signalling in Arabidopsis thaliana.Plant J 59: 316–328

Kim H, Hwang H, Hong JW, Lee YN, Ahn IP, Yoon IS, Yoo SD, Lee S, LeeSC, Kim BG (2012) A rice orthologue of the ABA receptor, OsPYL/RCAR5, is a positive regulator of the ABA signal transduction pathwayin seed germination and early seedling growth. J Exp Bot 63: 1013–1024

Knight CD, Sehgal A, Atwal K, Wallace JC, Cove DJ, Coates D, QuatranoRS, Bahadur S, Stockley PG, Cuming AC (1995) Molecular responses toabscisic acid and stress are conserved between moss and cereals. PlantCell 7: 499–506

Koch K (2004) Sucrose metabolism: Regulatory mechanisms and pivotalroles in sugar sensing and plant development. Curr Opin Plant Biol 7:235–246

Komatsu K, Suzuki N, Kuwamura M, Nishikawa Y, Nakatani M, OhtawaH, Takezawa D, Seki M, Tanaka M, Taji T, et al (2013) Group A PP2Csevolved in land plants as key regulators of intrinsic desiccation toler-ance. Nat Commun 4: 2219

Kubota A, Ishizaki K, Hosaka M, Kohchi T (2013) EfficientAgrobacterium-mediated transformation of the liverwort Marchantiapolymorpha using regenerating thalli. Biosci Biotechnol Biochem 77:167–172

Plant Physiol. Vol. 179, 2019 327

Jahan et al.

www.plantphysiol.orgon February 26, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.

Laby RJ, Kincaid MS, Kim D, Gibson SI (2000) The Arabidopsis sugar-insensitive mutants sis4 and sis5 are defective in abscisic acid synthesisand response. Plant J 23: 587–596

Ligrone R, Duckett JG, Renzaglia KS (2012) Major transitions in the ev-olution of early land plants: A bryological perspective. Ann Bot 109:851–871

Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, Grill E(2009) Regulators of PP2C phosphatase activity function as abscisic acidsensors. Science 324: 1064–1068

Marcotte WR Jr., Russell SH, Quatrano RS (1989) Abscisic acid-responsivesequences from the em gene of wheat. Plant Cell 1: 969–976

Marella HH, Sakata Y, Quatrano RS (2006) Characterization and func-tional analysis of ABSCISIC ACID INSENSITIVE3-like genes fromPhyscomitrella patens. Plant J 46: 1032–1044

McAdam SAM, Brodribb TJ, Banks JA, Hedrich R, Atallah NM, Cai C,Geringer MA, Lind C, Nichols DS, Stachowski K, et al (2016) Abscisicacid controlled sex before transpiration in vascular plants. Proc NatlAcad Sci USA 113: 12862–12867

Milborrow BV (1974) The chemistry and physiology of abscisic acid. AnnuRev Plant Physiol 25: 259–307

Minami A, Nagao M, Arakawa K, Fujikawa S, Takezawa D (2003) Ab-scisic acid-induced freezing tolerance in the moss Physcomitrella patens isaccompanied by increased expression of stress-related genes. J PlantPhysiol 160: 475–483

Mosquna A, Peterson FC, Park S-Y, Lozano-Juste J, Volkman BF, CutlerSR (2011) Potent and selective activation of abscisic acid receptorsin vivo by mutational stabilization of their agonist-bound conformation.Proc Natl Acad Sci USA 108: 20838–20843

Nagao M, Minami A, Arakawa K, Fujikawa S, Takezawa D (2005) Rapiddegradation of starch in chloroplasts and concomitant accumulation ofsoluble sugars associated with ABA-induced freezing tolerance in themoss Physcomitrella patens. J Plant Physiol 162: 169–180

Nishimura N, Sarkeshik A, Nito K, Park SY, Wang A, Carvalho PC, Lee S,Caddell DF, Cutler SR, Chory J, et al (2010) PYR/PYL/RCAR familymembers are major in-vivo ABI1 protein phosphatase 2C-interactingproteins in Arabidopsis. Plant J 61: 290–299

Niwa Y (2003) A synthetic green fluorescent protein gene for plant bio-technology. Plant Biotechnology 20: 1–11

Park SY, Fung P, Nishimura N, Jensen DR, Fujii H, Zhao Y, Lumba S,Santiago J, Rodrigues A, Chow TF, et al (2009) Abscisic acid inhibitstype 2C protein phosphatases via the PYR/PYL family of START pro-teins. Science 324: 1068–1071

Rensing SA, Lang D, Zimmer AD, Terry A, Salamov A, Shapiro H,Nishiyama T, Perroud PF, Lindquist EA, Kamisugi Y, et al (2008) ThePhyscomitrella genome reveals evolutionary insights into the conquestof land by plants. Science 319: 64–69

Rodrigues A, Adamo M, Crozet P, Margalha L, Confraria A, Martinho C,Elias A, Rabissi A, Lumbreras V, González-Guzmán M, et al (2013)ABI1 and PP2CA phosphatases are negative regulators of Snf1-relatedprotein kinase1 signaling in Arabidopsis. Plant Cell 25: 3871–3884

Rolland F, Moore B, Sheen J (2002) Sugar sensing and signaling in plants.Plant Cell 14: S185–S205

Sakata Y, Nakamura I, Taji T, Tanaka S, Quatrano RS (2010) Regulation ofthe ABA-responsive Em promoter by ABI3 in the moss Physcomitrellapatens: Role of the ABA response element and the RY element. PlantSignal Behav 5: 1061–1066

Sakata Y, Komatsu K, Takezawa D (2014) ABA as a universal plant hor-mone. Prog Bot 75: 57–96

Saruhashi M, Kumar Ghosh T, Arai K, Ishizaki Y, Hagiwara K, KomatsuK, Shiwa Y, Izumikawa K, Yoshikawa H, Umezawa T, et al (2015)Plant Raf-like kinase integrates abscisic acid and hyperosmotic stresssignaling upstream of SNF1-related protein kinase2. Proc Natl Acad SciUSA 112: E6388–E6396

Smeekens S (1998) Sugar regulation of gene expression in plants. CurrOpin Plant Biol 1: 230–234

Sugano SS, Nishihama R, Shirakawa M, Takagi J, Matsuda Y, Ishida S,Shimada T, Hara-Nishimura I, Osakabe K, Kohchi T (2018) EfficientCRISPR/Cas9-based genome editing and its application to conditionalgenetic analysis in Marchantia polymorpha. PLoS One 13: e0205117

Sussmilch FC, Atallah NM, Brodribb TJ, Banks JA, McAdam SAM (2017)Abscisic acid (ABA) and key proteins in its perception and signalingpathways are ancient, but their roles have changed through time. PlantSignal Behav 12: e1365210

Tognetti JA, Pontis HG, Martínez-Noël GM (2013) Sucrose signaling inplants: A world yet to be explored. Plant Signal Behav 8: e23316

Tougane K, Komatsu K, Bhyan SB, Sakata Y, Ishizaki K, Yamato KT,Kohchi T, Takezawa D (2010) Evolutionarily conserved regulatorymechanisms of abscisic acid signaling in land plants: Characterization ofABSCISIC ACID INSENSITIVE1-like type 2C protein phosphatase in theliverwort Marchantia polymorpha. Plant Physiol 152: 1529–1543

Umezawa T, Sugiyama N, Mizoguchi M, Hayashi S, Myouga F,Yamaguchi-Shinozaki K, Ishihama Y, Hirayama T, Shinozaki K (2009)Type 2C protein phosphatases directly regulate abscisic acid-activatedprotein kinases in Arabidopsis. Proc Natl Acad Sci USA 106:17588–17593

Uno Y, Furihata T, Abe H, Yoshida R, Shinozaki K, Yamaguchi-Shinozaki K (2000) Arabidopsis basic leucine zipper transcription fac-tors involved in an abscisic acid-dependent signal transduction pathwayunder drought and high-salinity conditions. Proc Natl Acad Sci USA 97:11632–11637

Vishwakarma K, Upadhyay N, Kumar N, Yadav G, Singh J, Mishra RK,Kumar V, Verma R, Upadhyay RG, Pandey M, et al (2017) Abscisicacid signaling and abiotic stress tolerance in plants: A review on currentknowledge and future prospects. Front Plant Sci 8: 161

Vlad F, Rubio S, Rodrigues A, Sirichandra C, Belin C, Robert N, Leung J,Rodriguez PL, Laurière C, Merlot S (2009) Protein phosphatases 2Cregulate the activation of the Snf1-related kinase OST1 by abscisic acidin Arabidopsis. Plant Cell 21: 3170–3184

Werner O, Ros Espín RM, Bopp M, Atzorn R (1991) Abscisic-acid-induceddrought tolerance in Funaria hygrometrica Hedw. Planta 186: 99–103

Yemm EW, Willis AJ (1954) The estimation of carbohydrates in plant ex-tracts by anthrone. Biochem J 57: 508–514

Yotsui I, Saruhashi M, Kawato T, Taji T, Hayashi T, Quatrano RS,Sakata Y (2013) ABSCISIC ACID INSENSITIVE3 regulates abscisic acid-responsive gene expression with the nuclear factor Y complex throughthe ACTT-core element in Physcomitrella patens.New Phytol 199: 101–109

Zheng Z, Xu X, Crosley RA, Greenwalt SA, Sun Y, Blakeslee B, Wang L,Ni W, Sopko MS, Yao C, et al (2010) The protein kinase SnRK2.6 me-diates the regulation of Suc metabolism and plant growth in Arabi-dopsis. Plant Physiology 153: 99–113

328 Plant Physiol. Vol. 179, 2019

Abscisic Acid Receptor in Bryophytes

www.plantphysiol.orgon February 26, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.