The Analysis of Pinus Pinaster Snrks Reveals Clues of the Evolution of This Family and a New Set of Abiotic Stress Resistance Biomarkers

Article The Analysis of Pinus pinaster SnRKs Reveals Clues of the Evolution of This Family and a New Set of Abiotic Stress Resistance Biomarkers

Francisco Javier Colina * , María Carbó , Ana Álvarez , Luis Valledor * and María Jesús Cañal

Plant Physiology, Department of Organisms and Systems Biology and University Institute of Biotechnology (IUBA), University of Oviedo, 33006 Oviedo, Spain; [email protected] (M.C.); [email protected] (A.Á.); [email protected] (M.J.C.) * Correspondence: [email protected] (F.J.C.); [email protected] (L.V.)

Received: 15 January 2020; Accepted: 17 February 2020; Published: 19 February 2020

Abstract: Climate change is increasing the intensity and incidence of environmental stressors, reducing the biomass yields of forestry species as Pinus pinaster. Selection of new stress-tolerant varieties is thus required. Many genes related to plant stress signaling pathways have proven useful for this purpose with sucrose non-fermenting related kinases (SnRK), conserved across plant evolution and connected to different phosphorylation cascades within ABA- and Ca2+-mediated signaling pathways, as a good example. The modulation of SnRKs and/or the selection of specific SnRK alleles have proven successful strategies to increase plant stress resistance. Despite this, SnRKs have been barely studied in gymnosperms. In this work P. pinaster SnRK sequences (PpiSnRK) were identified through a homology- and domain-based sequence analysis using Arabidopsis SnRK sequences as query. Moreover, PpiSnRKs links to the gymnosperm stress response were modeled out of the known interactions of PpiSnRKs orthologs from other species with different signaling complexity. This approach successfully identified the pine SnRK family and predicted their central role into the gymnosperm stress response, linking them to ABA, Ca2+, sugar/energy and possibly ethylene signaling. These links made the gymnosperm kinases promising candidates into the search for new stress resistance-related biomarkers, which would be useful into future breeding strategies.

Keywords: stress; forestry; Pinus pinaster; SnRK

1. Introduction The ever-increasing intensity of environmental stressors and the changes into their seasonal incidence, both associated to climate change, have a negative and global impact in plant biomass yields [1–4]. Pinus pinaster is a gymnosperm species with a key environmental and forestry relevance into the northwestern Mediterranean area, where it is used for afforestation due to its fast growth, high phenotypic plasticity and stress tolerance, soil stabilization capacity and the quality of its timber and oleoresins [5,6]. Moreover, this species is also a good example of the climate-associated reductions in plant biomass yield related to regional climatic changes, increasing abiotic—heat and drought—and biotic stress pressure, which are also a main constraint for other Pinus species [7–10]. Multiple efforts have been started globally to avoid climate change effects over plant production directed to the use of better management strategies and the selection of more stress tolerant and productive plant varieties [3]. Despite new stress tolerant varieties are required to cope with the climate change effects and/or increase productivity, their generation faces the complexity of plant traits as stress resistance, growth speed and wood quality. Compared to crops, in tree species and particularly in gymnosperms as P. pinaster the complexity problem add up to the large and poorly characterized

Agronomy 2020, 10, 295; doi:10.3390/agronomy10020295 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 295 2 of 13 genomes. These large sizes complicate the characterization of forestry desirable traits since they are generally polygenic. Many pathways aimed at these stress- and biomass-associated traits, as the ones involving abscisic acid (ABA), reactive oxygen species (ROS), jasmonates/salicylates and Ca2+, have been largely characterized in herbaceous plant models as Arabidopsis thaliana, and the modulation of genes within these routes improves stress tolerance in many plant species [11–13], however the knowledge in pine is scarce. Therefore, the characterization of these pathways in gymnosperms and their role into the modulation of these species’ agronomical traits will contribute to better understand the species stress response and potentially identify new biomarkers for breeding. Sucrose non-fermenting related kinases (SnRK) are central and conserved elements in the phosphorylation cascades within ABA- and Ca2+-mediated plant stress signaling pathways [14]. This kinase family divides into three functionally divergent subfamilies, namely SnRK1, SnRK2 and SnRK3. The SnRK1 subfamily, conserved across eukaryotes, links central metabolism with stress response to modulate the cell responses to energy stress [15]. SnRK1 kinases are part of protein complexes involving different regulatory subunits, namely β, γ and βγ where β modulates the kinase substrate specificity and its cell location [16], and γ acts as a sensor of the ADP-AMP levels monitoring the cell energy status [17]. βγ is a plant specific subunit functionally equivalent to the animal γ subunit, as plant γ orthologs do not interact with SnRK1 [18]. SnRK2 and three subfamilies are also Plantae-specific elements, which share with SnRK1 a common Ser/Thr kinase domain. This kinase domain is followed in SnRK1 by a UBA and KA domains, which allow its interaction with their different regulatory subunits. Conversely, the kinase domain is followed by different regulatory domains in subfamilies 2 and 3. Regulatory domain I follows the kinase domain into all SnRK2 and is related to their activation by osmotic stress, while the kinase domain of all SnRK3 is followed by a FISL/NAF domain, which allows the interaction of SnRK3 kinases with Ca2+ signaling [14]. Some SnRK2 have a second regulatory domain (domain II) allowing their modulation by ABA. Arabidopsis SnRK2 sequences could be organized by their ABA sensitivity into the groups SnRK2 I (ABA insensitive), SnRK2 II (low ABA sensitivity) and SnRK2 III (high ABA sensitivity) where only group II and III sequences contain the ABA regulatory domain II [13]. SnRK kinases participate in the stress response both through the direct modulation of stress response effectors as ion channels, and the regulation of transcription factors—bZIP family—and epigenetic modulation mechanisms such as the SWI/SNF complex, which in turn control the expression of broad stress-related gene groups. The modulation of the expression of the SnRKs and/or their related elements as bZIP transcription factors through the use of genetic engineering or approaches exploiting their natural variation enhances stress resistance into both herbaceous species as A. thaliana and rice, and tree species as poplar [14,19,20]. Moreover, SnRKs are linked to the modulation of different agronomical traits under stress including wood quality. SnRK1, involved in sugar and energy signaling, modulates the flux of sucrose towards cellulose [21]. Furthermore, other SnRK related elements and signalers are also involved in xylogenesis. Poplar genes related to wood formation are enriched in bZIP binding motifs [22] and ABA abundance changes correlate with stress effects on wood structure [23]. Therefore, P. pinaster SnRK orthologs as probable ABA and bZIP intermediaries in the gymnosperm and possible links between the pine stress response and its wood quality are interesting targets in this species’ enhancement strategies. The description of the P. pinaster SnRKs, thereafter PpiSnRKs, will allow the identification of their orthologs in species with better characterized stress response systems as A. thaliana. These species would contribute first to predict and then to characterize the function of the pine kinases. Model species as A. thaliana, hereafter Arabidopsis, have already been used for the validation of candidate genes from tree species [24]. Moreover, the identification of the PpiSnRK orthologs into related species with close but simpler stress response systems would further ease the characterization of the pine kinases. Amborella trichopoda, an ancient angiosperm closer to gymnosperms than Arabidopsis, Selaginella moellendorffii, a non-seed plant, and Marchantia polymorpha, a non-vascular plant, (hereafter Amborella, Selaginella and Marchantia) are, along the less evolved chlorophyte microalgae Agronomy 2020, 10, 295 3 of 13 species, promising species for this purpose. The modulation of the PpiSnRK orthologs of these species and/or their substitution for their P. pinaster counterparts would allow the characterization of the P. pinaster PpiSnRKs, reducing the required time-consuming transformation and/or selection strategies in the tree species. Moreover, the identification of the orthology links with these plant and microalgae species, with different positions in relation to the last microalgae-plant common ancestor, would trace down the evolution of the complex plant SnRK families and evidence many of the specific features of the gymnosperms SnRK families. The known relevance of SnRKs for the enhancement of stress resistance and biomass production in plants, and the lack of knowledge of this kinase family in gymnosperms, has motivated the description of the SnRK family of the gymnosperm species P. pinaster. PpiSnRKs were described, and their interactions modeled through an approach based on sequence analysis and protein interaction prediction, comparing the pine SnRKs with their orthologs from different plant species. These approaches would give hints about the evolution of SnRK family in gymnosperms and predict the specific role of the identified PpiSnRKs, which would ultimately contribute to the selection of specific ones related to some traits desired in forestry. These selected PpiSnRKs and or their related proteins could be characterized in simpler plant systems and then possibly used as biomarkers to drive future P. pinaster breeding programs.

2. Materials and Methods

2.1. Description of the P. Pinaster PpiSnRK Family P. pinaster transcriptome and proteome [25] available at Plaza [26] were used for the identification of the gymnosperm SnRK orthologs to the Arabidopsis SnRK family [14]. All of the Arabidopsis sequences of the family were used as query into BLAST- and BLASTP-based searches against P. pinaster transcriptome and proteome using the default NCBI BLAST and BLASTP parameters. Only hits with e-values lower than e-25 were considered for further analyses. Non-SnRK sequences were filtered out from homology results through the analysis of their domain composition using the version 75.0 of the Inter Pro Scan database [27]. Only P. pinaster sequences with the Arabidopsis SnRKs canonical domain composition—including Ser/Thr kinase domains (PTHR24343, PTHR43895), UBA (IPR015940), KA1/ αCTD (IPR001772), Immunoglobulin E-set (IPR014756), AMPK glycogen binding subunit (IPR032640), ASC (IPR006828), Immunoglobulin E-set (IPR014756), CBS (IPR000644) and NAF/FISL (IPR018451) domains (Table S1)—were maintained for further analyses as previously specified [28]. Pinus pinaster PpiSNRK transcript sequences were used for the identification of possible PpiSNRK isoforms. PpiSNRK transcripts were aligned with MUSCLE alignment algorithm using default parameters, considering a group of aligned transcripts isoforms when sharing 3 and 50 UTR regions. Pinus pinaster PpiSNRK transcripts [25] and P. taeda [26] transcript orthologs to the Arabidopsis SnRK sequences were used for the curation of the incomplete and fragmented P. pinaster PpiSnRK protein models. The P. pinaster and P. taeda transcript sequences were translated, and the resulting protein sequences aligned using the M-Coffee consensus alignment method with default parameters to their respective PpiSNRK protein models for their curation.

2.2. Alignment of SnRK Sequences PpiSnRK sequences were aligned with other SnRK sequences belonging to Arabidopsis and different microalgae species [28] (Table S2) using the M-Coffee consensus alignment method with default parameters [29]. Alignment was filter-curated through the Transitive Consistency Score algorithm (TCS) using default parameters [30]. Curated alignment distances were used in the generation of a maximum likelihood (ML) tree whose consistency was evaluated through the Transfer Bootstrap Expectation score (TBE, 500 replicates) included in the FastTree workflow within the Booster platform [31] due to the high number of involved sequences. SnRK2 sequences from microalgae and different plant species including P. pinaster PpiSnRK2, Arabidopsis, Amborella and Selaginella (Table S2) were aligned using M-Coffee multiple alignment Agronomy 2020, 10, 295 4 of 13 method with default parameters [29]. Different alignments were made by changing the included plant species set to test the resulting trees topology consistency. TCS weight-curated alignment distances were used to build rooted ML trees whose consistency was evaluated through Felsestein’s bootstrap (FBP, 500 replicates) into the PhyML-SMS workflow within the booster platform [31].

2.3. Bioinformatic and Statistical Analyses All the procedures for the identification and classification of P. pinaster PpiSnRK were performed locally employing the bioinformatics suite Geneious v7 (Biomatters Inc.), with the exception of Inter Pro Scan [27] searches, M-Coffee alignments and TCS-based alignment curation, and ML trees construction that were respectively performed at the European Bioinformatics Institute (ebi.ac.uk), T-Coffee (tcoffee.crg.cat) and booster (booster.pasteur.fr) websites. Protein–protein functional interactions between the pine PpiSnRK2 sequences were inferred using the STRING v11 [32] database. As P. pinaster or other gymnosperms were not available at the interaction database, PpiSnRK2 orthologs from related STRING-available species as Amborella, Selaginella and Arabidopsis were separately used as query into the STRING website (string-db.org). Three networks were made (one for each species) showing high confidence (over 0.7 STRING interaction score), known and predicted associations between proteins.

3. Results

3.1. Identification of the P. pinaster SnRK Family Members The search of the Arabidopsis SnRKs [14] in P. pinaster transcriptome and proteome by using BLAST and BLASTP revealed a large protein set including SnRK homologs and non-SnRK sequences sharing the conserved Ser/Thr kinase domain. The analysis of the domain composition of these proteins allowed to unequivocally distinguish the PpiSnRK sequences attending to the exclusive SnRK domains surrounding the conserved kinase domain and/or the specific features of their kinase domain. The combination of BLAST-based search and domain analysis over P. pinaster available sequence data resulted in the confident identification of 7 PpiSnRK1-associated regulatory subunits and 45 PpiSnRK kinases belonging to the subfamilies 1, 2 and 3, including multiple isoforms (Table S1). The M-Coffee alignment tree of the identified P. pinaster kinases with their orthologs in Arabidopsis and different microalgae species (Table S2, Figure1) together with the description of the pine PpiSnRK domain structure were the bases for defining the three catalytic clusters of this family in pine, namely PpiSnRK1, PpiSnRK2 and PpiSnRK3 (Figure1, Table S1). A fourth cluster, described by the domain analysis, contained the PpiSnRK1-associated regulatory subunits (Table S1). PpiSnRK1 cluster included the P. pinaster SnRK1 ortholog (PpiSnRK1; Ser/Thr kinase (PTHR24343), UBA (IPR015940) and KA1/ αCTD (IPR001772) domains) and the SnRK1-like sequence (PpiSnRK1-L; Ser/Thr kinase (PTHR24346)) along their orthologs in other species (Figure1, Table S1). Arabidopsis SnRK1/AKIN10 was the closest sequence to P. pinaster PpiSnRK1 and PpiSnRK1L clustered along the Chlamydomonas reinhardtii and Volvox carteri SnRK1 like sequences within this group (Figure1). The fourth and PpiSnRK1-associated cluster included the non-catalytic regulatory subunits of the PpiSnRK1 complex: PpiSnRKβ1, β2 (Immunoglobulin E-set (IPR014756) AMPK glycogen binding subunit (IPR032640) and ASC (IPR006828) domains), PpiSnRKβγ1, βγ2 (Immunoglobulin E-set (IPR014756) and CBS (IPR000644) domains) and related PpiSnRKγ1, γ2, γ3 (CBS domains (IPR000644); Table S1). PpiSnRK3 cluster (Ser/Thr kinase (PTHR43895) and NAF/FISL (IPR018451) domains) was the biggest sequence group in the tree (Figure1, Table S1), mostly represented by the Arabidopsis and P. pinaster elements. SnRK3 sequences from less evolved microalgae species as Coccomyxa subellipsoidea CsCKIN3 and Chlorella variabilis CvCKIN3 group together (PpiSnRK3 cluster, Group 1), placed at the base of this cluster followed by four more groups containing exclusively Arabidopsis and P. pinaster sequences (PpiSnRK3 cluster, Groups 2-5; Figure1). The closest plant SnRK3 group to the microalgae specific Group 1 was Group 2 and contained Arabidopsis SnRK3.11 and SnRK3.13, and P. pinaster Agronomy 2020, 10, x FOR PEER REVIEW 5 of 13

The third cluster, PpiSnRK2 sequences (Ser/Thr kinase (PTHR24343) domain), was divided into AgronomySnRK2(A)2020, 10, 295 and (B) clusters as previously described [28] with all the P. pinaster PpiSnRK2 and5 of 13 Arabidopsis SnRK2 sequences falling into the SnRK2(A) cluster (Figure 1, Table S1). SnRK2(A) also included a small group of plant-like microalgae SnRK2s conforming the Group 2 of SnRK2(A) (Figure PpiSnRK3.31). P. pinaster and PpiSnRK3.4 sequences as (Figure PpiSnRK2.5,1). Arabidopsis and PpiSnRK2.6SnRK3.11 isoforms or SOS2 wereis confidently involved clustered into the SOSalong salt stress signalingArabidopsis pathway SnRK2.1,[ 2.4,33]. 2.5, 2.9 and 2.10 sequences, but the tree failed to cluster remaining ones with any of the Arabidopsis SnRK2 sequences (Figure 1).

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Figure 1.FigureUnrooted 1. Unrooted maximum maximum likelihood likelihood (ML) (ML) tree tree of ofPinus Pinus pinaster pinaster (PpiSnRK)(PpiSnRK) and and ArabidopsisArabidopsis thaliana thaliana (AthSnRK)(AthSnRK) SnRK sequencesSnRK sequences along theiralong orthologs their ortholog into dis ffintoerent different microalgae microalgae species—CKIN—including species—CKIN— Chlamydomonasincludingreinhardtii Chlamydomonas(CreCKIN), reinhardtiiVolvox (CreCKIN), carteri (VcaCKIN), Volvox carteriDunaliella (VcaCKIN), salina (DsaCKIN), Dunaliella Chlorellasalina variabilis(DsaCKIN),(CvaCKIN), ChlorellaCoccomyxa variabilis subellipsoidea (CvaCKIN),(CsuCKIN) Coccomyxa and subellipsoideaOstreococcus (CsuCKIN) lucimarinus and(OluCKIN) Ostreococcus SnRK sequences.lucimarinus Tree branches (OluCKIN) are SnRK colored sequences. according Tree branches to their are TBE colored bootstrap according values. to their Branches TBE bootstrap with TBE values. Branches with TBE values below 0.8 are black, while these above 0.8 TBE are colored from red values below 0.8 are black, while these above 0.8 TBE are colored from red (0.8) to light green (1). Tree (0.8) to light green (1). Tree divided the sequences into the tree SnRK families (SnRK1, 2, 3). SnRK1 divided the sequences into the tree SnRK families (SnRK1, 2, 3). SnRK1 subfamily was divided into subfamily was divided into SnRK1 like and SnRK1 sequences, SnRK3 sequences was divided into five SnRK1different like and subgroups SnRK1 sequences, and SnRK2 SnRK3 sequences sequences were divided was divided between intoSnRK2A five and diff erentSnRK2B. subgroups P. pinaster and SnRK2PpiSnRK2 sequences sequences were divided were all between included SnRK2A into the SnRK2A and SnRK2B. group P.along pinaster ArabidopsisPpiSnRK2 SnRK2s. sequences were all included into the SnRK2A group along Arabidopsis SnRK2s.

The third cluster, PpiSnRK2 sequences (Ser/Thr kinase (PTHR24343) domain), was divided into SnRK2(A) and (B) clusters as previously described [28] with all the P. pinaster PpiSnRK2 and Arabidopsis SnRK2 sequences falling into the SnRK2(A) cluster (Figure1, Table S1). SnRK2(A) also included a small group of plant-like microalgae SnRK2s conforming the Group 2 of SnRK2(A) (Figure1). P. pinaster sequences as PpiSnRK2.5, and PpiSnRK2.6 isoforms were conﬁdently clustered along Arabidopsis Agronomy 2020, 10, 295 6 of 13

SnRK2.1, 2.4, 2.5, 2.9 and 2.10 sequences, but the tree failed to cluster remaining ones with any of the Arabidopsis SnRK2 sequences (Figure1).

3.2. Description of the P. pinaster PpiKIN2 Subfamily Structure and Sequence Features M-Coffee-based trees of P. pinaster PpiSnRK2, and other plant and microalgae SnRK2 orthologs confidently separated plant SnRK2 and microalgae CKIN2/SnRK2 (Figure2) as the previous analysis (Figure1). Arabidopsis SnRK2 sequences were confidently grouped into the three previously described groups, namely I (ABA insensitive), II (Low ABA sensitivity) and III (high ABA sensitivity) [34] into all SnRK2 trees (Figure2). Pinus pinaster PpiSnRK2.5, 2.6.1 and 2.6.2, and PpiSnRK2.1, 2.2 and 2.3 fell within Groups I and III respectively into all tested tree topologies. Conversely, PpiSnRK2.4 isoforms did not cluster consistently with any of the previous groups (Figure2). Most P. pinaster and Amborella (phylogenetically closer to pine than Arabidopsis) SnRK2 were consistently clustered within I and III Groups along the Arabidopsis sequences. On the other hand, the consistency of Group II was lower across trees and Arabidopsis SnRK2(II) sequences—SnRK2.7 and SnRK2.8—were the only constantly present in this branch (Figure2). Amborella SnRK2(II) sequence only clustered with Arabidopsis SnRK2(II) sequences into one of the trees (Figure2b), and P. pinaster PpiSnRK2.4 isoforms were either equidistant to all groups (Figure2a,c) or to II and III groups (Figure2b). Selaginella sequences had a basal position in the plant SnRK2 branch of the SnRK2/CKIN2 tree and none of its sequences were clustered within the previously defined groups (Figure2c). Furthermore, built trees were unable to converge into a basal SnRK2 group (Figures1 and2). The ABA-sensitive cluster III of SnRK2 was highly similar between Arabidopsis and P. pinaster. PpiSnRK2.1, 2.2 and 2.3 had highly conserved regulatory domains I and II (ABA box) after the characteristic SnRK Ser/Thr kinase domain, supporting their involvement in the gymnosperm ABA signaling (Figure3c). P. pinaster PpiSnRK2.5, 2.6.1 and 2.6.2, within the Group I SnRK2, also conserved a regulatory domain I after the Ser/Thr kinase domain (Figure3a). PpiSnRK2.4 isoforms conserved the regulatory domain I, which was followed by a C-terminal sequence with features observed in the regulatory domain II of Group II and III SnRK2 sequences (Figure3b). Selaginella SnRK2 homolog sequences had a basal position in the SnRK2 tree and did not cluster with any of the deﬁned SnRK2 groups (Figure2c). In spite of this, these sequences showed analogy to Arabidopsis ABA sensitive II and III SnRK2. Selaginella SnRK2 had a conserved Domain I followed by an aspartate-rich domain II-like. These acidic aspartate residues are characteristic of the SnRK2s terminal domains of Groups II and III (Figures2 and3, Figure S1).

3.3. Arabidopsis, Amborella and Selaginella Orthologs to P. Pinaster PpiSnRK2 Are Connected to Stress Signaling and Metabolism Modulation The Arabidopsis, Amborella and Selaginella orthologs to P. pinaster PpiSnRK2 were used to build three different STRING-based protein interaction networks from which a network outline was created pointing to the potential function of the pine PpiSnRK2 sequences (Figure4, Figure S1). All the PpiSnRK2 orthologs across the different included species shared a connection to the SnRK1-related regulatory PpiSnRKβγ subunit and different starch modulatory enzymes, and to many PP2C and bZIP elements (Figure4, Figure S1). In Amborella and Arabidopsis PP2Cs were functionally connected to the ABA receptors PYL/PYR/RCAR, which were also directly associated to these species group III SnRK2 kinases (Figure4, Figure S1). The ABA receptor Mg chelatase (GUN5 or CHLH) and different ubiquitination-related elements were other conserved associations across the different species SnRK2 sequences. Despite the multiple connections conserved between the different species SnRK orthologs Selaginella and Amborella showed exclusive interactions. A protein phosphatase 2C and cyclic nucleotide-binding/kinase domain-containing protein (PP2C-L) was associated in the Selaginella and Amborella STRING networks (Figure4, Figure S2b,c) with the conserved cluster of SnRK1, its associated regulatory βγ subunits and starch regulatory enzymes, while Amborella SnRK2(III) and Selaginella OST2.1 were connected to ethylene- and protein glycosylation-related elements respectively (Figure4, Figure S1). Agronomy 2020, 10, 295 7 of 13 Agronomy 2020, 10, x FOR PEER REVIEW 6 of 13

(a) CvaCKIN2.6 CsuCKIN2.7 OluCKIN2.2 OluCKIN2.3 CreCKIN2.8 VcaCKIN2.8

CvaCKIN2.8 SnRK2

CsuCKIN2.8 Plant-like AthSnRK2.7 0.84 AthSnRK2.8 II PpiSnRK2.4.2 PpiSnRK2.4.1 ? PpiSnRK2.5 0.98 <0.5 PpiSnRK2.6.2 PpiSnRK2.6.1 1 AthSnRK2.4 AthSnRK2.10 I <0.5 AthSnRK2.9 AthSnRK2.1 AthSnRK2.5 PpiSnRK2.1 0.98 PpiSnRK2.2 PpiSnRK2.3 AthSnRK2.6 III AthSnRK2.2 (b) AthSnRK2.3 OluCKIN2.2 CsuCKIN2.7 CvaCKIN2.6 OluCKIN2.3 CreCKIN2.8 VcaCKIN2.8

CsuCKIN2.8 SnRK2

CvaCKIN2.8 Plant-like PpiSnRK2.5 PpiSnRK2.6.2 PpiSnRK2.6.1 1 AmSnRK2(I) AthSnRK2.4 I AthSnRK2.10 AthSnRK2.9 1 AthSnRK2.1 AthSnRK2.5 AmSnRK2(II) 0.87 AthSnRK2.7 II AthSnRK2.8 0.86 PpiSnRK2.4.2 PpiSnRK2.4.1 ? 0.49 PpiSnRK2.1 1 PpiSnRK2.2 PpiSnRK2.3 AmSnRK2(III) III AthSnRK2.6 AthSnRK2.2 (c) AthSnRK2.3 OluCKIN2.2 CsuCKIN2.7 CvaCKIN2.6 OluCKIN2.3 CreCKIN2.8 VcaCKIN2.8 lant-like CsuCKIN2.8 SnRK2 CvaCKIN2.8 P SeOST2.1 SeOST3.1 SeOST1.1 1 AmSnRK2(II) <0.5 AthSnRK2.7 0.84 0.9 AthSnRK2.8 II PpiSnRK2.6.2 PpiSnRK2.5 PpiSnRK2.6.1 0.77 1 AmSnRK2(I) AthSnRK2.4 I AthSnRK2.10 AthSnRK2.9 <0.5 AthSnRK2.1 AthSnRK2.5 PpiSnRK2.4.2 PpiSnRK2.4.1 ? <0.5 PpiSnRK2.1 1 PpiSnRK2.2 PpiSnRK2.3 AmSnRK2(III) III AthSnRK2.6 AthSnRK2.2 Tree scale: 0.1 AthSnRK2.3

Figure 2. Rooted SnRK2 ML trees including SnRK2 orthologs from different sets of species along Pinus pinaster PpiSnRK2s: (a) P. pinaster (PpiSnRK2) and Arabidopsis thaliana (AthSnRK2) SnRK2 sequences along the plant-like SnRK2 orthologs (CKIN2) of different microalgae species including Chlamydomonas reinhardtii (CreCKIN2), Volvox carteri (VcaCKIN2), Dunaliella salina (DsaCKIN2), Chlorella variabilis (CvaCKIN2), Coccomyxa subellipsoidea (CsuCKIN2) and Ostreococcus lucimarinus (OluCKIN2); (b) same species and sequence set including Amborella trichopoda (AmSnRK2) SnRK2 orthologs and (c) same previous species and sequence set including A. trichopoda (AmSnRK2) and Selaginella moellendorfii (SeOST) SnRK2 orthologs. Selaginella and Amborella SnRK2 sequences, plant species with simpler SnRK2 subfamilies, were added to this SnRK2 sequence trees to evaluate the orthology links between the P. pinaster and these species SnRK2 subfamilies and their possible use as models for the characterization of the gymnosperm PpiSnRK2. Tree branches were colored according to their FBP bootstrap value, thus, branches with FBP values below 0.5 are black while these above 0.5 FBP are colored from red (0.5) to light green (1). Agronomy 2020, 10, x FOR PEER REVIEW 7 of 13

3.2. Description of the P. pinaster PpiKIN2 Subfamily Structure and Sequence Features M-Coffee-based trees of P. pinaster PpiSnRK2, and other plant and microalgae SnRK2 orthologs Agronomyconfidently 2020, separated10, x FOR PEER plant REVIEW SnRK2 and microalgae CKIN2/SnRK2 (Figure 2) as the previous analysis 8 of 13 (Figure 1). Arabidopsis SnRK2 sequences were confidently grouped into the three previously conserveddescribed groups,a regulatory namely domain I (ABA insensitive),I after the Ser/ThrII (Low ABA kinase sensitivity) domain and (Figure III (high 3a). ABA PpiSnRK2.4 sensitivity) isoforms conserved[34] into all the SnRK2 regulatory trees (Figure domain 2). PinusI, which pinaster was PpiSnRK2.5, followed 2.6.1by a andC-terminal 2.6.2, and sequence PpiSnRK2.1, with 2.2 features observedand 2.3 infell the within regulatory Groups domain I and IIIII of respectively Group II and into III all SnRK2 tested sequences tree topologies. (Figure Conversely, 3b). PpiSnRK2.4Selaginella isoforms SnRK2 did homolog not cluster sequences consistently had wi ath basal any ofpositi the previouson in the groups SnRK2 (Figure tree and 2). Most did notP. cluster withpinaster any andof the Amborella defined (phylogenetically SnRK2 groups closer(Figure to 2c).pine In than spite Arabidopsis of this, these) SnRK2 sequences were consistently showed analogy toclustered Arabidopsis within ABA I and sensitive III Groups II and along III the SnRK2. Arabidopsis Selaginella sequences. SnRK2 On thehad other a conserved hand, the Domainconsistency I followed of Group II was lower across trees and Arabidopsis SnRK2(II) sequences—SnRK2.7 and SnRK2.8— by an aspartate-rich domain II-like. These acidic aspartate residues are characteristic of the SnRK2s were the only constantly present in this branch (Figure 2). Amborella SnRK2(II) sequence only terminal domains of Groups II and III (Figures 2 and 3, Figure S1). clustered with Arabidopsis SnRK2(II) sequences into one of the trees (Figure 2b), and P. pinaster PpiSnRK2.4 isoforms were either equidistant to all groups (Figure 2a,c) or to II and III groups (Figure 3.3.2b). Arabidopsis, Selaginella sequences Amborella had and a Selaginella basal position Ortholog in thes plantto P. PinasterSnRK2 branch PpiSnRK2 of the Are SnRK2/CKIN2 Connected totree Stress Signaling and Metabolism Modulation Agronomyand2020 none, 10 , 295of its sequences were clustered within the previously defined groups (Figure 2c).8 of 13 Furthermore,The Arabidopsis, built trees Amborella were unable and to Selaginella converge intoorthologs a basal toSnRK2 P. pinaster group (FiguresPpiSnRK2 1 and were 2). used to build three different STRING-based protein interaction networks from which a network outline was (a)

PpiSnRK2.5 createdPpiSnRK2.6.1 pointing to the potential function of the pine PpiSnRK2 sequences (Figure 4, Figure S1). All PpiSnRK2.6.2 AthSnRK2.9 AthSnRK2.10 theAthSnRK2.1 PpiSnRK2 orthologs across the different included species shared a connection to the SnRK1- AthSnRK2.4 AthSnRK2.5 γ related(b) regulatory PpiSnRKβ subunit and different starch modulatory enzymes, and to many PP2C

PpiSnRK2.4.1 andPpiSnRK2.4.2 bZIP elements (Figure 4, Figure S1). In Amborella and Arabidopsis PP2Cs were functionally AthSnRK2.7 AthSnRK2.8

connected(c) to the ABA receptors PYL/PYR/RCAR, which were also directly associated to these species

AthSnRK2.2 groupAthSnRK2.3 III SnRK2 kinases (Figure 4, Figure S1). The ABA receptor Mg chelatase (GUN5 or CHLH) and AthSnRK2.6 PpiSnRK2.1 PpiSnRK2.2 differentPpiSnRK2.3 ubiquitination-related elements were other conserved associations across the different speciesFigure 3.SnRK2M-Co ﬀsequences.ee alignments Despite of P. pinaster the multiplePpiSnRK2 connections and Arabidopsis conservedSnRK2 between sequences the (AthSnRK2) different species SnRKfocused Figureorthologs on 3. the M-Coffee C-terminalSelaginella alignments regionand ofAmborella ofP. pinaster thesekinases PpiSnRK2showed containing andexclusive Arabidopsis the interactions. regulatorySnRK2 sequences domainsA protein (AthSnRK2) I andphosphatase/or II: 2C and(a) Groupcyclicfocused Inucleotide-binding/kinase SnRK2;on the C-terminal (b) Group region II SnRK2 of these domain-containing and kinase (c)s Group containing III SnRK2.the proteinregulatory All (PP2C-L) pine domains PpiSnRK2 I and/orwas regulatory associatedII: (a) in the Group I SnRK2; (b) Group II SnRK2 and (c) Group III SnRK2. All pine PpiSnRK2 regulatory sequences Selaginellasequences showedand Amborella high resemblance STRING to networks the Arabidopsis (Figuresequences 4, Figure into theirS2b,c) respective with the groups conserved including cluster of showed high resemblance to the Arabidopsis sequences into their respective groups including SnRK1,PpiSnRK2.4 its associated isoform, with regulatory conserved βγ aspartate subunits residues and atstarch positions regulatory corresponding enzymes, with while those ofAmborella PpiSnRK2.4 isoform, with conserved aspartate residues at positions corresponding with those of SnRK2(III) and Selaginella OST2.1 were connected to ethylene- and protein glycosylation-related ArabidopsisArabidopsisSnRK2(II) SnRK2(II) sequences sequences regulatory regulatory domain domain II.II. elements respectively (Figure 4, Figure S1). The ABA-sensitive cluster III of SnRK2 was highly similar between Arabidopsis and P. pinaster. PpiSnRK2.1, 2.2 and 2.3 had highlyUbiquitination conserved regulatory domains I and II (ABA box) after the Interaction legend characteristic SnRK Ser/Thr kinase domain, supporting their involvementSelaginella in the gymnosperm ABA signaling (Figure 3c). P.bZIP pinaster PpiSnRK2.5,I 2.6.1 and 2.6.2, withinAmborella the Group I SnRK2, also Arabidopsis

Starch metabolism

PP2C II SnRK1

PP2C-like II/III-L Protein glycosilation PYL/PYR/ RCAR III Ethylene receptor

Mg Chelatase

FigureFigure 4. Network 4. Network outline outline summarizing summarizing the known the andknown predicted and associationspredicted associations in the STRING in networksthe STRING for thenetworksArabidopsis for theSnRK2s Arabidopsis and their SnRK2s orthologs and intheirAmborella orthologsand inSelaginella Amborella. Most and SnRK2Selaginella groups. Most across SnRK2 the includedgroups across species the are included related to species the same are processes related to and the nodes same highlighting processes and bZIP nodes transcription highlighting factors, bZIP ABA-relatedtranscription PP2C factors, signaling ABA-relate and sensingd PP2C and signaling carbon metabolism. and sensing Some and carbon speciﬁc metabolism. features also Some arose specific as the interactionfeatures also of arose the Amborella as the interactionSnRK2 III of ortholog the Amborella with an SnRK2 ethylene III ortholog sensor or with the an interaction ethylene ofsensor the or Selaginellathe interactionsequences of with the elements Selaginella related sequences to themodulation with elements of protein relate glycosylation.d to the modulation All connections of protein shownglycosylation. in this outline All areconnections possibly conservedshown in amongthis outline PpiSnRK2 are possibly sequences conserved taking into among account PpiSnRK2 the intermediatesequences pine taking position into betweenaccount Arabidopsisthe intermediate, Amborella pineand positionSelaginella between. Arabidopsis, Amborella and Selaginella. 4. Discussion Despite the great divergence between P. pinaster and Arabidopsis, the identiﬁed pine PpiSnRK and

Arabidopsis SnRK families had similar size, structure, and sequence features, and potentially also similar interactions and roles. These parallelisms could be extended to the SnRK2 subfamilies of Amborella—a sequenced embryophyte close to the last gymnosperm-angiosperm common ancestor—and Selaginella, an also sequenced protovascular plant more ancient than gymnosperms. The observed similarities between these distant species point to the early origin of the SnRK family configuration and function known in Arabidopsis and other land plants [14,28]. Furthermore, the conservation evidences and the simpler SnRK families of these ancient model species motivated their election for the characterization of the pine PpiSnRK elements. Pinus pinaster and Arabidopsis share large and similar SnRK3 subfamilies. A reduced group of these SnRK3 sequences could be considered founder as were close in the sequence tree to Agronomy 2020, 10, 295 9 of 13 microalgae SnRK3/CKIN3 (Figure1). Interestingly, between the plant sequences included in this group are Arabidopsis SnRK3.11 or SOS2, a salt stress responsive kinase within the SOS pathway [33], osmostress-sensitive SnRK3.13 [35] and P. pinaster PpiSnRK3.3 and PpiSnRK3.4. This closeness points at once to the ancient (microalgae) origin of this pathway and by analogy to the possible involvement of both microalgae and P. pinaster PpiSnRK3.3 and PpiSnRK3.4 into salt and/or osmotic stress responses. Many of the remaining Arabidopsis SnRK3 sequences, more distant from the microalgae ones, and clustering into the 3-5 SnRK3 clusters within the sequence tree (Figure1) are involved in ABA stress signaling and expressed under certain developmental times and in specific tissues [36]. Thus, the diversification of the plant SnRK3 subfamily from the founder elements might have been related to the increase in structure and life cycle complexity of land plants and/or to their adaption to the more stressing land environment. Same explanation and function thus could be applied to the P. pinaster PpiSnRK3 sequences clustering along with the Arabidopsis ones in the 3-5 groups (Figure1). As SnRK3 and PpiSnRK3 subfamilies, PpiSnRK2 subfamily had a group division highly similar to Arabidopsis SnRK2. The SnRK2 subfamily sequence and structure similarity could also be expanded to Amborella, at the base of angiosperm group. Interestingly, although Amborella has a small SnRK2 family (three members), its members represent the three Arabidopsis SnRK2 groups. Again, the shared SnRK complexity and features between these distantly related species points to ancient duplication/divergence events—before angiosperm and gymnosperm division—but also to the conservation of these elements giving origin to the observed family similarity and pointing to their shared relevance and function. Besides this, the small size of Amborella SnRK2 family also points to gymnosperm and angiosperm independent SnRK2 duplication events. These independent duplication events could explain the observed grouping issues between elements sharing group II SnRK2 features as Arabidopsis SnRK2.7, SnRK2.8, Amborella AmSnRK2(II) and P. pinaster PpiSnRK2.4 isoforms. On the other hand, Selaginella, a non-seed plant, has a simpler family structure exclusively composed by elements close to Arabidopsis SnRK2 II and III groups. The absence of ABA-insensitive group I SnRK2 sequences in Selaginella and their presence in Amborella, Arabidopsis and P. pinaster makes them the most recent SnRK2s. Group I SnRK2 would have originated at some point between vascular plant and seed plant origin. Moreover, the Selaginella SnRK2 subfamily points to the earlier origin of the ABA-sensitive SnRK2s and thus to the early involvement of ABA in SnRK2-based signaling pathways. This SnRK2(III) exclusive subfamilies have also been found in the moss Physcomitrella patens and charophyte algae supporting also the earlier origin of the ABA-sensitive elements [37]. Strikingly, C. reinhardtii CKIN2.8, a plant-like SnRK2, expression is not sensitive to exogenous ABA treatments [28]. Approaches including sequence and domain/motif analysis have been widely used along tree-based sequence clustering for the identification and description of many plant gene families, including the SnRK family. These strategies combined sequence-based methods with the evaluation of the identified sequences expression patterns in order to link them to particular stresses [28,38,39]. Other works [28,40] complemented this approach with the prediction of the proteins associated to the newly identified ones through databases such as STRING containing known relations between the orthologs to these elements. The involvement of apple bHLH transcription factors and C. reinhardtii CKIN/SnRK into their stress response system was predicted using this database. STRING predicted the link with ABA signaling of some of the identified apple bHLH genes and the link of C. reinhardtii CKIN/SnRKs to Ca2+ and PP2C signaling, and to the modulation of carbon and nitrogen metabolism [28,40]. STRING database was also chosen to predict the PpiSnRK2 sequences function and associations with the gymnosperm stress response system and metabolism using data from related plant and microalgae species with different positions within the Plantae species tree and different stress response system complexity. The overlap of the known and predicted STRING-based protein–protein interaction networks draw from the P. pinaster PpiSnRK2 orthologs in Arabidopsis, Amborella and Selaginella (Figure S2) outlined a network model of the possible associations of the P. pinaster PpiSnRKs with other stress related elements (Figure4). These networks showed the conserved SnRK2 connections Agronomy 2020, 10, 295 10 of 13 with PP2C, bZIP transcription factors, ABA sensing/signaling and carbon metabolism modulation in Arabidopsis, Amborella and Selaginella (Figure S2, Figure4) and microalgae [ 28]. This conservation makes highly probable the involvement of the P. pinaster kinases with these signaling elements. SnRK1-2 related bZIP transcription factors are involved in plant abiotic and energetic stress responses in different plant species [41,42]. The overexpression of the Arabidopsis bZIP ABF2 confers tolerance to multiple abiotic stresses [43]. Interestingly, the pine PpiSnRK2 could have conserved connections to ethylene signaling and protein glycosylation modulation not observed into the Arabidopsis family. The prediction of the PpiSnRK interactions using species more ancient than P. pinaster and Arabidopsis (Amborella, Selaginella and microalgae) have also allowed the identification of possible SnRK2 interactions to ethylene signaling, protein glycosylation and alternative central regulators as a PP2C-like protein. Despite these connections were not conserved in the Arabidopsis network, they would have been inherited by the P. pinaster sequences. To overcome pine experimental limitations, model species as Arabidopsis, but specially Selaginella and Amborella—with simpler but close SnRK families—would contribute to the characterization of PpiSnRKs. The knowledge of the specific role of these kinases within the gymnosperm stress response and the identification of their related elements would provide promising targets to drive new selection and breeding strategies. Between these targets would be the PpiSnRKs themselves, orthologs to the conserved bZIP transcription factors and their modulated genes but also novel elements, which are absent from the characterized angiosperms SnRK families. These elements would be interesting candidates for screening programs aimed at the identification of P. pinaster genotypes carrying specific sequence changes linked to higher endurance to environmental stress. Previous works based on the characterization of the genotype and secondary metabolism profile among Mediterranean P. pinaster populations adapted to different environmental conditions found differences related to the individuals tolerance to stress [5,6]. The candidates found in this work are also an interesting base to identify novel stress associated variations, which could be used as biomarkers of better stress endurance and contribute to breeding/selection programs for the generation of more stress tolerant varieties both in P. pinaster and other close gymnosperm species.

5. Conclusions This work successfully identified and described the SnRK family members of P. pinaster, a non-sequenced tree species with a large genome. The success of this approach was based on the search of the orthologs to the well described and characterized Arabidopsis SnRKs into the available transcriptome/proteome of P. pinaster and other close Pinus species. The identification of the gymnosperm SnRKs (PpiSnRK) search was complemented by the prediction of their associations with other stress response-related elements. This prediction required the comparison of the PpiSnRK elements with other known plant SnRK families including Arabidopsis and other plant species such as Amborella and Selaginella with simpler SnRK families. The pine PpiSnRK family showed a high resemblance to those of Arabidopsis but also to Amborella and Selaginella, giving hints about the pine family evolution, structure and function. SnRK2 subfamilies of Arabidopsis, Amborella and P. pinaster shared the same structure divided into ABA sensitive (Groups II, III PpiSnRK2.4 and PpiSnRK2.1, 2.2, 2.3 respectively) and insensitive (group I PpiSnRK2.5, 2.6) elements. This similarity was extended to the SnRK3 subfamily, PpiSnRK3.3 and 3.4 closeness to salt-sensitive Arabidopsis SOS2 pointed to the involvement of these PpiSnRK3 into the P. pinaster osmotic and/or salt stress response. Moreover, STRING protein interaction networks over related species suggested the conservation of the gymnosperm PpiSnRK2 interaction with known SnRK2 interactors as PP2C, bZIP and SnRK1 while highlighting possible novel interactions. These results supported the use of these model species especially the ones with simpler but close SnRK systems as Amborella for the characterization of the gymnosperm SnRKs, overcoming the species experimental difficulties. The knowledge of the specific role of these kinases within the gymnosperm stress response and the identification of their related elements would provide promising targets to drive new selection and breeding strategies. Between these targets would be the PpiSnRKs themselves, orthologs to the conserved bZIP transcription Agronomy 2020, 10, 295 11 of 13 factors and their modulated genes but also novel elements, which are absent from the characterized angiosperm SnRK families. These elements would be interesting candidates for screening programs aimed at the identification of P. pinaster genotypes carrying specific sequence changes linked to higher endurance to environmental stress. These candidates would be used as biomarkers of better stress endurance and contribute to breeding/selection programs for the generation of more stress tolerant varieties both in P. pinaster and other close gymnosperm species.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4395/10/2/295/s1, Figure S1: M-Coffee alignments of Selaginella and Arabidopsis SnRK2 orthologs, Figure S2: STRING based interaction networks of PpiSnRK orthologs, Table S1: P. pinaster PpiSnRK sequence names, clusters, protein and domain identifiers (ID), Table S2: sequences used for the design of the Figure1 and /or Figure2 sequence tree. Author Contributions: Conceptualization, L.V., M.J.C. and F.J.C.; Data curation, F.J.C., M.C.; Investigation, F.J.C., M.C.; Methodology, F.J.C., M.C.; Software, F.J.C., M.C.; Writing—original draft preparation, F.J.C., M.C.; Funding acquisition, M.J.C. and L.V.; Project administration, M.J.C. and L.V; Supervision, M.J.C. and L.V.; Writing—review and editing, L.V., F.J.C., M.C., A.Á. and M.J.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Spanish Ministry of Science, Innovation and University, project numbers AGL2016-77633-P and AGL2017-83988-R. F.J.C., M.C. and L.V. were respectively and generously granted by the BP14-138, BP19-137 (Programa de Ayudas Predoctorales Severo Ochoa, Autonomous Community of Asturias, Spain; to F.J.C. and M.C.) and RYC-2015-17871 (Ramón y Cajal Programme, Spanish Ministry of Economy and Competitiveness; to L.V.) fellowships. Conflicts of Interest: The authors declare no conflict of interest.

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