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Plant Signaling & Behavior

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Abscisic acid (ABA) and key proteins in itsperception and signaling pathways are ancient,but their roles have changed through time

Frances C. Sussmilch, Nadia M. Atallah, Timothy J. Brodribb, Jo Ann Banks &Scott A. M. McAdam

To cite this article: Frances C. Sussmilch, Nadia M. Atallah, Timothy J. Brodribb, Jo Ann Banks &Scott A. M. McAdam (2017): Abscisic acid (ABA) and key proteins in its perception and signalingpathways are ancient, but their roles have changed through time, Plant Signaling & Behavior, DOI:10.1080/15592324.2017.1365210

To link to this article: http://dx.doi.org/10.1080/15592324.2017.1365210

Accepted author version posted online: 25Aug 2017.Published online: 25 Aug 2017.

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ARTICLE ADDENDUM

Abscisic acid (ABA) and key proteins in its perception and signaling pathways areancient, but their roles have changed through time

Frances C. Sussmilch a, Nadia M. Atallahb, Timothy J. Brodribb a, Jo Ann Banks b, and Scott A. M. McAdam a,b

aSchool of Biological Sciences, University of Tasmania, Hobart, TAS, Australia; bDepartment of Botany and Plant Pathology, Purdue University, WestLafayette, IN, USA

ARTICLE HISTORYReceived 20 July 2017Revised 4 August 2017Accepted 5 August 2017

ABSTRACTHomologs of the Arabidopsis core abscisic acid (ABA) signaling component OPEN STOMATA1 (OST1) arebest known for their role in closing stomata in angiosperm species. We recently characterized a fern OST1homolog, GAMETOPHYTES ABA INSENSITIVE ON ANTHERDIOGEN 1 (GAIA1), which is not required forstomatal closure in ferns, consistent with physiologic evidence that shows the stomata of these plantsrespond passively to changes in leaf water status. Instead, gaia1 mutants reveal a critical role in ABAsignaling for spore dormancy and sex determination, in a system regulated by antagonism betweenABA and the gibberellin (GA)-derived fern hormone antheridiogen (ACE). ABA and key proteins, includingABA receptors from the PYR/PYL/RCAR family and negative regulators of ABA-signaling from Group A ofthe type-2C protein phosphatases (PP2Cs), in addition to OST1 homologs, can be found in all terrestrialland plant lineages, ranging from liverworts that lack stomata, to angiosperms. As land plants haveevolved and diversified over the past 450 million years, so too have the roles of this important planthormone and the genes involved in its signaling and perception.

KEYWORDSAbscisic acid (ABA); OST1;fern; evolution; PYL/RCAR;PP2Cs; land plants; sexdetermination

We recently characterized several allelic mutants from the fernCeratopteris richardii that showed insensitivity to the phyto-hormone abscisic acid (ABA). We found these mutants to con-tain defects in the GAMETOPHYTES ABA INSENSITIVE ONANTHERDIOGEN 1 (GAIA1) gene, which encodes an SNF1-related kinase2 (SnRK2) protein homologous to the Arabidop-sis core ABA signaling component OPEN STOMATA1(OST1).1 OST1 is best known for its role in minimising waterloss in Arabidopsis, where OST1 mediates the active/metabolicpathway for stomatal closure in response to ABA synthesizedin leaves when water status declines.2,3 OST1 does this by inter-acting with S-type anion channels (SLACs) within the guardcell membrane to control guard cell turgor and thus the size ofthe stomatal pore.4 In accordance with this role, ost1 mutantshave a severe and characteristic wilted phenotype under lowhumidity or when desiccated.2 While an OST1-independentpathway for SLAC activation has been identified, involving cal-cium-dependent protein kinases (CPKs),5,6 the considerableseverity of the ost1mutant stomatal phenotype when comparedwith the weak/absent stomatal phenotypes of cpk loss of func-tion mutants indicates that OST1 plays a major role in the con-trol of stomatal aperture via SLAC activation.5,7

In contrast, we found that stomatal behavior in C. richardiigaia1 mutants was identical to wild-type plants,1 indicatingthat, unlike angiosperm OST1 kinases, GAIA1 does not play acritical role in ABA signaling for stomatal closure in the fern C.richardii. Indeed, our findings indicated that both the fern C.richardii, and the lycophyte Selaginella moellendorffii lack what

is considered an essential requirement for active ABA-mediatedstomatal control: functional, guard-cell specific SnRK2-SLACpairs.8 These findings are consistent with the results of physio-logic studies showing that, in contrast to the dramatic stomatalclosure elicited by ABA in seed plants, biologically relevant lev-els of ABA (within the same order of magnitude of the levelsthese plants are able to produce endogenously) fail to elicit ormaintain stomatal closure in basal vascular plants includinglycophytes and ferns. This finding is based on measurements ofboth stomatal conductance as a measure of leaf gasexchange,9,10 and stomatal aperture,11 which needs to be mea-sured carefully following the same stomata from open to closedto ensure measurements are from live stomata, and using singleblind methodology (without knowing the genotype) to avoidunconscious bias.12 Unnaturally high levels of ABA, approxi-mately 1000x higher than endogenous levels, have been foundto elicit a small reduction in stomatal aperture in some moss,13

hornwort,14 lycophyte,15 and fern species.16 However, the bio-logic relevance of these levels is debatable, especially given thesmaller scale of response these levels elicit in basal land plantswhen compared with the complete stomatal closure elicited bymuch lower, biologically relevant ABA levels in seedplants.17-19 Furthermore basal vascular plants do not show astrong hysteresis in the recovery of stomatal opening followinga period of water deficit, which is characteristic of ABA-medi-ated stomatal control, resulting from lingering ABA levels andslow rates of ABA catabolism. Instead the stomata of basal vas-cular plants show ‘passive’ stomatal responses that are directly

CONTACT Scott A. M. McAdam smcadam@purdue.edu School of Biological Sciences, University of Tasmania, Hobart, TAS 7001, Australia.Addendum to: McAdam SAM, Brodribb TJ, Banks JA, Hedrich R, Atallah NM, Cai C, Geringer MA, Lind C, Nichols DS, Stachowski K, Geiger D, Sussmilch FC. Abscisic acid con-trolled sex before transpiration in vascular plants. Proc Natl Acad Sci U S A. 2016;113:12862–7. doi:10.1073/pnas.1606614113.© 2017 Taylor & Francis Group, LLC

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Figure 1. The hypothesized GA signaling pathway in Arabidopsis and the sex determination pathway in the fern Ceratopteris richardii including the involvement of ABA.Arrows indicate activating events, while bars represent repressive events. Dashed lines indicate putative effects. Adapted from ref. 37.

Figure 2. The land plant PYR/PYL/RCAR receptor family. Themaximum likelihood phylogenetic tree of PYR/RCAR-like (PYRL) genes was generated using PhyML 3.0 with SmartModelSelection38 from a MAFFT alignment of full length predicted protein sequences for genes identified by reciprocal BLAST search in available resources for representative angiosperm(At, Arabidopsis thaliana, TAIR10; AmTr, Amborella trichopoda, v1.0), gymnosperm (Pa, Picea abies, v1.0, http://congenie.org/), fern (Cr, Ceratopteris richardii, unpublished transcrip-tome,1 Supplemental Text), lycophyte (Sm, Selaginella moellendorffii, v1.0), moss (Pp, Physcomitrella patens, v3.3), and liverwort (Mp/Mapoly, Marchantia polymorpha,v3.1) species at Phytozome (http://phytozome.jgi.doe.gov/) unless otherwise indicated. Vascular plant clades are indicated. Large, single-species sub-clades have been col-lapsed for figure clarity as follows: AtII (AtPYL4/RCAR10_AT2G38310, AtPYL5/RCAR8_AT5G05440, AtPYL6/RCAR9_AT2G40330, AtPYL13/RCAR7_AT4G18620, AtPYL11/RCAR5_AT5G45860, AtPYL12/RCAR6_AT5G45870); PaIIa (MA_7378814 g0010, MA_74427 g0010, MA_9600951 g0010, MA_10177719 g0010, MA_14451g0010, MA_52202 g0010, MA_10427386 g0010, MA_554564 g0010, MA_15750 g0010, MA_407237 g0010); PaIIb (MA_10117348 g0010, MA_9939588 g0010, MA_99662g0010, MA_181781 g0010, MA_18800 g0010, MA_10302927 g0010, MA_10355251 g0010, MA_10388164 g0010); Pp (PpPYRL1_Pp3c26_15240, PpPYRL2_Pp3c13_7110,PpPYRL3_Pp3c7_26290; PpPYRL4_Pp3c9_19760, PpPYRL5_Pp3c3_660, PpPYRL6_Pp3c7_4410, PpPYRL7_Pp3c15_19950, PpPYRL8_Pp3c20_7480). Bootstrap values from1000 replicates are shown as percentages for clades with>50% support. The scale bar indicates amino acid changes.

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controlled by leaf water status and plant hydraulics.20,21 Inter-mediate between the stomatal behaviors of basal vascular plantsand angiosperms, gymnosperm species have active ABA-medi-ated stomatal closure in response to drought, but not inresponse to more subtle daily changes in air humidity.20 Takentogether, these results support a gradualistic model for the evo-lution of ABA-mediated control of stomatal aperture, whichsuggests that the most basal vascular plant stomata respondedpassively to changes in leaf water status, and ‘active’, ABA-driven mechanisms for stomatal responses to water statusevolved after the divergence of seed plants, culminating in thecomplex, and highly sensitive ABA-mediated responsesobserved in modern angiosperms.22

Instead of a role in stomatal responses, we found the SnRK2-ABA signaling pathway involving GAIA1 in C. richardii playedimportant roles in spore dormancy and in sex determination infern gametophytes, in a system regulated by antagonismbetween ABA and the gibberellin (GA)-derived fern hormoneantheridiogen (ACE).

1 In C. richardii, the pathway for sex deter-mination has been elucidated from the epistatic interactions ofmore than 100 mutants.23-26 This pathway includes an indirectnegative feedback loop between 2 major classes of sex-regulat-ing loci: the TRANSFORMER loci (TRAs; necessary for femaletraits) and the FEMINIZATION1 locus (FEM1; required formale traits; named from mutant phenotype). ACE determineswhether FEM or TRAs are activated in the gametophyte, byactivating the TRA-repressing HERMAPHRODITIC loci(HERs). We can now update this model to include GAIA1,

which links the ABA and ACE signaling pathways, and repressesthe ACE response (Fig. 1).

1 In Arabidopsis, the OST1 subgroupSnRK2s act at multiple points within a comparable GA signal-ing pathway (Fig. 1), with GA biosynthesis, receptor and signal-ing genes upregulated in the ost1 snrk2–2 snrk2–3 triplemutant.27 Although the precise mechanism for GAIA1 regula-tion of sex determination in C. richardii is not yet clear, it ispossible that it acts at multiple points in this pathway, in a simi-lar manner.

Roles have also been identified for ABA and GAs/GA pre-cursors in spore dormancy in mosses, seed dormancy in seedplants, and sex determination in angiosperms.28-30 Further-more, there is clear evidence for an ancient role for ABA indesiccation tolerance, through the upregulation of proteinsthat function in osmoregulation,31 separate from the role ofABA described in modern seed plants in the prevention ofdesiccation via stomatal closure. Accordingly, and despite arecent report to the contrary,32 ABA receptors belonging tothe PYR/PYL/RCAR receptor family are found in all lineagesof land plants from bryophytes (including liverworts, whichlack stomata) to angiosperms (Fig. 2). Phylogenetic analysisshows that this family diverged into 2 major clades in thevascular plants, indicating a duplication event after separa-tion from the bryophyte lineages. Clade II includes 2 sub-clades of genes in fern, gymnosperm and angiosperm models(previously characterized as clades II and III in Arabidop-sis),33 indicating a second duplication occurred in this cladeafter lycophyte divergence.

Figure 3. Land plant Group A PP2C phosphatases. The maximum likelihood phylogenetic tree was generated using PhyML 3.0 with Smart Model Selection38 from aMAFFT alignment of full length predicted protein sequences for genes identified by reciprocal BLAST search in available resources for representative species for eachland plant lineage, as described for Figure 2. Vascular plant clades are indicated. Abbreviations as follows: At, Arabidopsis thaliana; AmTr, Amborella trichopoda; Cr, Cera-topteris richardii; Mp, Marchantia polymorpha; Pa, Picea abies; Pp, Physcomitrella patens; Sm, Selaginella moellendorffii (accessions indicated where full length sequencewas obtained from GenBank). Bootstrap values from 1000 replicates are shown as percentages for clades with >50% support. The scale bar indicates aminoacid changes.

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While the PYR/PYL/RCAR receptor family is land plant-spe-cific,34 other ABA-signaling components, including the SnRKs andtype-2C protein phosphatases (PP2Cs), belong to older proteinfamilies that were present in an early eukaryote ancestor. Geneswithin Group A of the PP2C family, which includes the key Arabi-dopsis ABA-signaling gene ABA-INSENSITIVE 1 (ABI1), arefound in all land plants (Fig. 3). While these genes evolved a role asnegative regulators of ABA-signaling at an early stage during plantevolution,35 the nature of this role has changed over time. Specifi-cally, while ABI1 phosphatases physically prevent SnRK2 functionin the absence of ABA in angiosperms, orthologous phosphatasesin bryophytes act downstream of SnRK2 kinases to control anancient desiccation tolerance pathway.36

In conclusion, the results from both physiologic and geneticstudies highlight the important role that the plant hormoneABA has played in plant development throughout land plantevolution. Importantly, these findings show that the roles ofABA itself, in addition to key components of the ABA percep-tion and signaling pathways, have also evolved and diversifiedthrough time.

Funding details

This work was supported by the Australian Research Council under GrantsDE140100946 (S.M.) and DP140100666 (T.B.); and the National ScienceFoundation under Grant IOS1258091 (J.B.).

ORCID

Frances C. Sussmilch http://orcid.org/0000-0002-8659-1125Timothy J. Brodribb http://orcid.org/0000-0002-4964-6107Jo Ann Banks http://orcid.org/0000-0002-7762-7261Scott A. M. McAdam http://orcid.org/0000-0002-9625-6750

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