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Article Genome-Wide Identiﬁcation, Characterization, and Expression Analyses of P-Type ATPase Superfamily Genes in Soybean

Bingqian Zhao 1, Haicheng Wu 1, Wenjing Xu 2, Wei Zhang 2, Xi Chen 3, Yiyong Zhu 4 , Huatao Chen 2,* and Houqing Zeng 1,*

1 College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121, China; [email protected] (B.Z.); [email protected] (H.W.) 2 Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; [email protected] (W.X.); [email protected] (W.Z.) 3 Department of Biochemistry and Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing 210095, China; [email protected] 4 College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China; [email protected] * Correspondence: [email protected] (H.C.); [email protected] (H.Z.)

Abstract: P-type ATPases are transmembrane pumps of cations and phospholipids. They are energized by hydrolysis of ATP and play important roles in a wide range of fundamental cellular and physiological processes during plant growth and development. However, the P-type ATPase superfamily genes have not been characterized in soybean. Here, we performed genome-wide bioinformatic and expression analyses of the P-type ATPase superfamily genes in order to explore the potential functions of P-type ATPases in soybean. A total of 105 putative P-type ATPase genes were identified in the soybean genome. Phylogenetic relationship analysis of the P-type ATPase genes indicated that they can be divided into five subfamilies including P1B, P2A/B, P3A, P4 and P5. Proteins belonging to the same subfamily shared conserved domains. Forty-seven gene pairs were related to Citation: Zhao, B.; Wu, H.; Xu, W.; segmental duplication, which contributed to the expansion of the P-type ATPase genes during the Zhang, W.; Chen, X.; Zhu, Y.; Chen, H.; Zeng, H. Genome-Wide evolution of soybean. Most of the P-type ATPase genes contained hormonal- and/or stress-related Identification, Characterization, and cis-elements in their promoter regions. Expression analysis by retrieving RNA-sequencing datasets Expression Analyses of P-Type suggested that almost all of the P-type ATPase genes could be detected in soybean tissues, and ATPase Superfamily Genes in some genes showed tissue-specific expression patterns. Nearly half of the P-type ATPase genes Soybean. Agronomy 2021, 11, 71. were found to be significantly induced or repressed under stresses like salt, drought, cold, flooding, https://doi.org/10.3390/agronomy and/or phosphate starvation. Four genes were significantly affected by rhizobia inoculation in root 11010071 hairs. The induction of two P2B-ATPase genes, GmACA1 and GmACA2, by phosphate starvation was confirmed by quantitative RT-PCR. This study provides information for understanding the evolution Received: 13 November 2020 and biological functions of the P-type ATPase superfamily genes in soybean. Accepted: 29 December 2020 Published: 31 December 2020 Keywords: P-type ATPase; ion pump; phylogenetic analysis; expression analysis; tissue expression pattern; stress; duplication; evolution; cis-acting element Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- ms in published maps and institutional affiliations. 1. Introduction The P-type ATPases are cation and lipid pumps energized by hydrolysis of ATP. They were named P-type ATPases because they form a phosphorylated (hence P-type) reaction Copyright: © 2020 by the authors. Li- cycle intermediate during catalysis [1]. The ATP hydrolyzing machinery of P-type ATPases censee MDPI, Basel, Switzerland. consists of three interconnected cytoplasmic domains: the phosphorylation (P), nucleotide This article is an open access article binding (N), and actuator (A) domain. A conserved sequence motif, DKTGT, is contained distributed under the terms and con- in the P domain of P-type ATPases, where the Asp (D) residue is phosphorylated during ditions of the Creative Commons At- catalysis [1]. The P-type ATPases form a large superfamily that can be divided into five tribution (CC BY) license (https:// creativecommons.org/licenses/by/ distinct evolutionarily related subfamilies, P1–P5. Each of the subfamilies can be further 4.0/). divided into subgroups [2,3]. The major subgroups of P-type ATPases include P1A-ATPases

Agronomy 2021, 11, 71. https://doi.org/10.3390/agronomy11010071 https://www.mdpi.com/journal/agronomy Agronomy 2021, 11, 71 2 of 25

(part of bacterial K+ transport system), P1B-ATPases (heavy metal pumps), P2A-ATPases (Ca2+ pumps), P2B-ATPases (Ca2+ pumps), P2C-ATPases (Na+/K+ and H+/K+ pumps of animals), P2D-ATPases (Na+ pumps of fungi), P3A-ATPases (H+ pumps), P4-ATPases (phospholipid pumps), and P5-ATPases (the substrate of P5-ATPases is still unclear) [1,4,5]. The P-type ATPases play critical roles in a wide range of fundamental cellular processes, such as generating and maintaining the electrochemical gradient that is used as the driving force for secondary transporters (H+-ATPases in plants and fungi and Na+/K+-ATPases in animals), calcium signaling (Ca2+-ATPases), the transport of essential micronutrients (heavy metal-ATPases), and the generation of membrane phospholipid asymmetry [6–10]. The subfamilies of P1B-, P2A-, P2B-, P3A-, P4-, and P5-ATPases have been found to exist in higher plants, while the genes of P1A, P2C, and P2D subfamilies are absent [3,11,12]. In Arabidopsis (Arabidopsis thaliana L.), a total of 47 P-type ATPase genes have been identified, including eifht P1B, four P2A, eleven P2B, eleven P3A, twelve P4, and one P5 gene(s) [11–13]. In rice (Oryza sativa L.), a total of 45 P-type ATPase genes have been identified, including nine P1B, three P2A, twelve P2B, ten P3A, ten P4, and one P5 gene(s) [11,13]. P1B-ATPase is a subfamily of P-type ATPases. It is also named as heavy metal ATPase (HMA). HMAs are divided into two groups based on their substrate specificity: Cu/Ag transporters (Cu+-ATPases) and Zn/Cd/Co/Pb transporters (Zn2+-ATPases) [14,15]. In addition to the conserved domains of P-type ATPases, HMAs typically possess six to eight transmembrane domains, an HP locus, a CPx/SPC motif, and putative metal-binding domains in the N- and/or C-terminal regions [9]. HMAs mainly function in the transport of metal cations (e.g., Cu+, Cu2+, Ag+, Zn2+, Co2+, Cd2+, and Pb2+) across biological membranes in a wide range of organisms [9,16,17]. P1B-ATPases in Arabidopsis and rice have been divided into six groups [9,11]. Arabidopsis AtHMA1–4 have been suggested to be Zn2+-ATPases, while AtHMA5–8 have been suggested to be Cu+-ATPases [9,17,18]. Rice OsHMA1–3 belong to the Zn2+-ATPase group, while OsHMA4–9 have been indicated to belong to the Cu+-ATPase group [16,19,20]. P2-ATPases in plants, which are also named Ca2+ pumps, are divided into two subgroups: P2A (ER-type Ca2+-ATPases, ECAs) and P2B (autoinhibited Ca2+-ATPases, ACAs) [8,21]. Both ECAs and ACAs contain all the characteristic motifs of P-type ATPases and of the P2 subfamily ATPases, such as the highly conserved sequence DKTGT, the PEGL motif playing a critical role in energy transduction, and the KGAXE sequence involved in nucleotide binding [8,11,21]. ACAs contain an N-terminal autoinhibitory domain that binds to calmodulin and activates the Ca2+ pump [22,23]. Plant P-type ATPases are impli- cated in Ca2+ signaling and Ca2+ homeostasis by pumping Ca2+ from the cytoplasm into intracellular compartments or into the apoplast [8,21]. Plant P2-ATPases are involved in many cellular and physiological processes, such as pollen tube growth, and abiotic and biotic stress tolerance [21,24–27]. The P3A-ATPases are plasma membrane (PM) H+-ATPases, which are universal elec- trogenic H+ pumps hydrolyzing ATP to pump H+ out of the cytosol from fungi to vascular plants [28,29]. The PM H+-ATPase is a functional monomer with a molecular weight about 100 kDa that can oligomerize to form dimeric and hexameric complexes [30]. The PM H+-ATPases have N- and C-terminal segments protruding into the cytoplasm, and ten transmembrane segments [28]. Generally, the C-terminal region of the PM H+-ATPase consisting of approximately 100 amino acids is known as an autoinhibitory domain keeping the H+-ATPase in a low-activity state via an interaction with the catalytic domain under normal conditions [28]. The phosphorylation of the penultimate Thr and subsequent binding of the 14-3-3 protein to the phosphorylated penultimate Thr results in the activation of H+-ATPase [31,32]. All 12 PM H+-ATPase gene members in Arabidopsis (AHA1–AHA12), 10 members in rice (OSA1–OSA10), and 12 members in sorghum (Sorghum bicolor L.) have been classified into five subfamilies [11,29,33]. By generating an H+ electrochemical gradient, maintaining intracellular pH homeostasis, and providing driving force for the active influx and efflux of ions and metabolites across the PM, PM H+-ATPases have been indicated to be involved in many important physiological processes during plant growth Agronomy 2021, 11, 71 3 of 25

and development, such as nutrient uptake and transportation, stomatal movement, cell elongation, and environmental stress tolerance [6,7,28,30,34–36]. The P4-ATPases, also known as phospholipid flippases, are unique to eukaryotic organisms. They are present in the plasma membrane and membranes of the secretory pathway to generate phospholipid asymmetry, which is required for vesicle budding and fusion in the secretory pathway [4,37]. A characteristic feature of the P4-ATPase subfamily proteins is the high frequency of hydrophobic or aromatic residues within those transmembrane domains that are commonly involved in cation transport in other P-type ATPases [4]. Mutation of some of these residues causes changes in lipid specificity, but none of these residues seem to be involved in direct lipid binding [37,38]. There are 12 P4-ATPase subfamily proteins in Arabidopsis, designated as Aminophospholipid ATPase1 (ALA1) to ALA12 [12]. Ten ALA proteins were identified in rice [11]. Some Arabidopsis ALA proteins have been characterized to be phospholipid flippases and have diverse physiological functions [37]. For example, AtALA1 is involved in chilling tolerance [39]; AtALA3 is a broad-specificity phospholipid flippase localized in the Golgi apparatus, where it contributes to the formation of secretory vesicles [10]; AtALA10 transports up to six different phospholipids, including sphingomyelin, a ceramide-derived phospholipid [40]. In contrast to the well-known cation transporters of the P-type ATPases, the substrate of P5 ATPases is still unclear [5]. In both Arabidopsis and rice, there is only one P5 ATPase gene [11]. In addition to model plants Arabidopsis and rice, genes encoding P-type ATPase proteins have been identified at the whole-genome scale in some other plants [41,42]. Soybean is an important economic crop providing oil- and protein-rich food for humans around the world. The genome sequence of a cultivated soybean developed in the 1980s (Glycine max L. var. Williams 82) has been released ten years ago [43]. The reference genome has opened the door for soybean functional genomics [44,45]. The entire P-type ATPase superfamily in the whole genome of soybean has not been identified, and its roles in developmental regulation and environmental stress tolerance are largely unknown. In this study, we carried out a genome-wide search and characterized and analyzed all the putative P-type ATPase genes in the soybean genome. RNA-sequencing and microarray data were used to analyze their expression patterns in different tissues of soybean and their transcriptional responses to environmental stresses and rhizobia infection. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to validate the expression of two genes in response to phosphorus deficiency. These results provide useful information for further functional studies on P-type ATPase genes and their possible roles in stress tolerance, nutrient utilization, and symbiosis.

2. Materials and Methods 2.1. Identification and Annotation of P-Type ATPase Genes in the Soybean Genome Proteins encoding putative P-type ATPase proteins in the soybean genome were identified by searching the soybean genome database SoyBase (https://www.soybase.org/) by BLASTP with the representative P-type ATPase proteins (including all five subfamilies of P-type ATPases, P1B, P2A/B, P3A, P4, and P5) from Arabidopsis and rice. The default settings were used. The nucleotide and protein sequences were obtained from the SoyBase website (https://www.soybase.org/). Protein domains were analyzed by using HMMScan (https://www.ebi.ac.uk/Tools/hmmer/search/hmmscan)[46] and the InterPro database (http://www.ebi.ac.uk/interpro/) [47]. The TMHMM2.0 program (http://www.cbs.dtu. dk/services/TMHMM/) was used to predict transmembrane helices [48]. Proteins that do not contain the characteristic domains and motifs of the P-type ATPase subfamilies were eliminated. Putative genes encoding P-type ATPase proteins were named according to their orthologues in Arabidopsis and their positions in the phylogenetic trees.

2.2. Phylogenetic Tree Construction and Chromosome Distribution The full-length amino acid sequences of P-type ATPase proteins from soybean, Ara- bidopsis, and rice were retrieved from TAIR (http://www.arabidopsis.org/), RGAP Agronomy 2021, 11, 71 4 of 25

(http://rice.plantbiology.msu.edu/index.shtml), and SoyBase (https://www.soybase.org/), respectively. These sequences were aligned using ClustalW, and the neighbor-joining trees were constructed using the Molecular Genetics Analysis (MEGA) 6.0 with the “pairwise deletion” option and “Poisson correction” model [49]. Bootstrap replications were set to 1000 to evaluate the reliability of internal branches. Chromosomal locations of all the putative genes encoding P-type ATPase proteins were obtained from the SoyBase website (https://www.soybase.org/). The P-type ATPase genes were mapped on the 20 soybean chromosomes based on their physical positions on chromosomes.

2.3. Gene Duplication Analysis and Calculation of Ka/Ks Values The duplication of putative P-type ATPase genes on segmentally duplicated regions was determined according to the results of phylogenetic trees and through searching the plant genome duplication database (PGDD) [50]. If the distance between two neighboring paralogous genes were less than 100 kb and separated by ﬁve or less genes, they were considered to be tandemly duplicated genes [51]. The history of selection performed on coding sequences can be measured by the Ka (non-synonymous substitution rates)/Ks (synonymous substitution rates) ratio, and the Ka/Ks can be used to identify pairwise combinations of genes, where encoded proteins may have changed functions [52]. The direction and magnitude of natural selection enforcing on the different genes could be interpreted by the Ka/Ks ratio. A pair of sequences having Ka/Ks < 1 indicates purifying selection; Ka/Ks = 1 implies both sequences drift neutrally; and Ka/Ks > 1 indicates positive or Darwinian selection [53]. The Ks and Ka of each gene pair was calculated by using TBtools [54]. The divergence time (T) was calculated by T = Ks/(2 × 6.1 × 10−9) × 10−6 MYA (million years ago), in which 6.1 × 10−9 is divergence rate in millions of years translated from Ks value [53].

2.4. In Silico Expression Analysis of P-Type ATPase Genes in Soybean The expression patterns of P-type ATPase genes in different tissues of soybean were retrieved from two published soybean RNA-seq datasets [55,56]. The normalized Illumina- Solexa reads number (The Reads/Kb/Million, RPKM) was log2-transformed and visualized in the heatmap, which was drawn by the MultiExperiment Viewer program (http://mev. tm4.org/). Microarray and high-throughput sequencing datasets of short-term salt stress (1 h, 6 h, and 12 h) in soybean roots [57], 1 d cold stress in soybean shoots [58], 4 d dehydration stress in soybean shoots [58], 7 d ﬂooding stress in soybean leaves [59], 7 d drought stress in soybean leaves [59], and 7 d phosphate starvation stress in soybean roots [60], were acquired from published datasets. The published dataset of transcriptome response to symbiotic bacteria (Bradyrhizobium japonicum) in soybean root hairs was also retrieved [61].

2.5. Analysis of Cis-Acting Regulatory Elements Promoter sequences of 1.5 kb upstream from the transcription start sites of putative genes encoding P-type ATPase proteins were obtained from the SoyBase database (http: //www.soybase.org/). The location of cis-acting regulatory elements was analyzed using Regulatory Sequence Analysis Tools (RSAT) (http://rsat.eead.csic.es/plants/)[62].

2.6. Plant Growth and qRT-PCR Analysis Soybean seeds (Glycine max L. var. Williams 82) were germinated at room temper- ature in the dark between two layers of filter paper moistened with sterilized water. After four days, soybean seedlings were grown hydroponically in half-strength modified Hoagland nutrient solution, the pH of the nutrient solution was adjusted to 5.6, and the nutrient solution was changed every three days [63]. The seedlings were grown in a growth chamber with a photoperiod set at 16-h-light/8-h-dark at 26/22 ◦C and with a light intensity of 150 µmol m−2 s−1. After cultivating for two weeks, soybean seedlings were used for phosphorus deficiency treatment. Seedlings were transferred into nor- Agronomy 2021, 11, 71 5 of 25

mal nutrient solution (control) and phosphorus deficient solution (without KH2PO4). For the phosphorus deficient treatment, we replaced KH2PO4 with K2SO4 to keep the concentration of K+. Roots and leaves of soybean seedlings were separately sampled after treatment of phosphorus deficiency for one and seven days. Quantitative RT-PCR (qRT-PCR) was performed on a real-time PCR system (CFX96 system, Bio-Rad Company, Hercules, California, USA) as described previously [64]. Relative expression levels were normalized to that of an internal control GmACTIN11 (Glyma.18g290800). The primers used for amplification were as follows: 50-CGGTGGTTCTATCTTGGCATC-30 and 50- GTCTTTCGCTTCAATAACCCTA-30 for GmACTIN11; 50-TCGAGGCTTTCTTGGGTTGG-30 and 50-GCAGTTTGCCATGTCTCAGTC-30 for GmACA1; and 50-ACTACTTTTGGGGAAGT GGTAG-30 and 50- CATTTGGCCACTGTTACTATCG-30 for GmACA2. The calculated effi- ciency (E) of these primers was all around 2.0.

3. Results 3.1. Identification of Putative Genes Encoding P-Type ATPase Proteins in the Soybean Genome To investigate the P-type ATPase gene superfamily in the soybean genome, the BLAST algorithm was used to search the soybean genome database in the SoyBase website (https://www.soybase.org/) using Arabidopsis and rice P-type ATPase protein sequences as the query. In total, 105 genes were identified to putatively encode P-type ATPase proteins (Table S1). These genes can be classified into five subfamilies, e.g., P1B, P2A/B, P3A, P4, and P5, based on their homologues in Arabidopsis and rice (Figure1 and Table S1). There were nineteen P1B genes, seven P2A genes, twenty-six P2B genes, twenty-four P3A genes, twenty-seven P4 genes, and two P5 genes identified in the soybean genome (Figures1 and Agronomy 2021, 11, x FOR PEER REVIEW 6 of 25 2). The total number of P-type ATPase proteins in soybean is about 2.2 and 2.3 folds that in Arabidopsis and rice, respectively (Figure2).

Figure 1. Phylogenetic tree of all P-type ATPase superfamily genes in the soybean genome: genes in Figure 1. Phylogenetic tree of all P-type ATPase superfamily genes in the soybean genome: genes the same subfamily are shaded by the same color. in the same subfamily are shaded by the same color.

30 27 Ath Osa Gma 26 24 25

19 20

15 12 12 11 11 10 10 9 10 8 Numberofgenes 7 4 5 3 2 1 1 0 P1B P2A P2B P3A P4 P5

Figure 2. The number of genes encoding different P-type ATPase subfamilies in Arabidopsis (Ath), rice (Osa), and soybean (Gma).

3.2. P1B Subfamily Genes in Soybean Twenty P1B subfamily genes have been identified previously in soybean and they were sequentially named GmHMA1 to GmHMA20 according to their chromosomal positions [65]. Here, we used P1B proteins from Arabidopsis and rice as queries to BLAST against the soybean genome database (SoyBase, https://www.soybase.org/). These 20 genes were all covered in the BLAST result, but the gene or protein length of these GmHMAs in the latest version of soybean genome annotation (Wm82.a4.v1) is different from the previous report (Wm82.a1.v1) [65]. For example, the protein length of GmHMA16 is 278 amino acids, while the length was 793 in the previous report. Because of the lack of characteristic domains of P1B ATPases, GmHMA16 was removed in the P1B subfamily members. The gene and protein sequences of the other 19 GmHMAs in the latest version were then used for further analysis. The coding region of these soybean P1B Agronomy 2021, 11, x FOR PEER REVIEW 6 of 25

Agronomy 2021, 11, 71 6 of 25 Figure 1. Phylogenetic tree of all P-type ATPase superfamily genes in the soybean genome: genes in the same subfamily are shaded by the same color.

30 27 Ath Osa Gma 26 24 25

19 20

15 12 12 11 11 10 10 9 10 8 Numberofgenes 7 4 5 3 2 1 1 0 P1B P2A P2B P3A P4 P5

FigureFigure 2. 2. TheThe number number of of genes genes encoding encoding different different P-type P-type ATPase ATPase subfamilies subfamilies in in Arabidopsis Arabidopsis (Ath), (Ath),rice (Osa), rice (Osa), and soybean and soybean (Gma). (Gma). 3.2. P1B Subfamily Genes in Soybean 3.2. P1B Subfamily Genes in Soybean Twenty P1B subfamily genes have been identiﬁed previously in soybean and they Twenty P1B subfamily genes have been identified previously in soybean and they were sequentially named GmHMA1 to GmHMA20 according to their chromosomal po- were sequentially named GmHMA1 to GmHMA20 according to their chromosomal posi- sitions [65]. Here, we used P1B proteins from Arabidopsis and rice as queries to BLAST tions [65]. Here, we used P1B proteins from Arabidopsis and rice as queries to BLAST against the soybean genome database (SoyBase, https://www.soybase.org/). These 20 againstgenes were the allsoybean covered genome in the BLASTdatabase result, (Soy butBase, the https://www.soybase.org/). gene or protein length of these These GmH- 20 genesMAs inwere the latestall covered version in of the soybean BLAST genome result, annotationbut the gene (Wm82.a4.v1) or protein islength different of these from GmHMAsthe previous in reportthe latest (Wm82.a1.v1) version of soybean [65]. For genome example, annotation the protein (Wm82.a4.v1) length of GmHMA16 is different is from278 amino the previous acids, while report the length(Wm82.a1.v1) was 793 in[65]. the For previous example, report. the Because protein of thelength lack of of GmHMA16characteristic is domains278 amino of acids, P1B ATPases,while the GmHMA16length was 793 was in removed the previous in the report. P1B subfamily Because ofmembers. the lack of The characteristic gene and protein domains sequences of P1B ATPases, of the other GmHMA16 19 GmHMAs was removed in the latest in the version P1B subfamilywere then members. used for further The gene analysis. and protein The coding sequences region of of the these other soybean 19 GmHMAs P1B ATPase in the genes lat- estwas version interrupted were then by 4 used to 16 for introns further (Table analys S2).is. TheThe proteincoding region length of of these GmHMAs soybean ranged P1B from 505 to 1096 amino acids (Table S2). All of the GmHMA proteins were predicted to contain a E1-E2 ATPase domain (Figure S1). Twelve GmHMA proteins were predicted to contain one to three heavy-metal-associated domains (Figure S1). All GmHMA proteins were predicted to contain two to eight transmembrane helices by using the TMHMM2.0 program (http://www.cbs.dtu.dk/services/TMHMM/), with the exception that there was no transmembrane helix for GmHMA4 (Table S2), which is consistent with a previous report [65]. An unrooted phylogenetic analysis was conducted to explore the evolutionary relationships of HMAs in soybean, Arabidopsis and rice plants. The topology of the phylogenetic tree revealed that the P1B subfamily can be divided into six clades (I–VI) (Figure3 ). In each clade, genes from the three plant species were present, suggesting a common ancestor of the same clade in these plants. Agronomy 2021, 11, x FOR PEER REVIEW 7 of 25

ATPase genes was interrupted by 4 to 16 introns (Table S2). The protein length of GmHMAs ranged from 505 to 1096 amino acids (Table S2). All of the GmHMA proteins were predicted to contain a E1-E2 ATPase domain (Figure S1). Twelve GmHMA proteins were predicted to contain one to three heavy-metal-associated domains (Figure S1). All GmHMA proteins were predicted to contain two to eight transmembrane helices by using the TMHMM2.0 program (http://www.cbs.dtu.dk/services/TMHMM/), with the exception that there was no transmembrane helix for GmHMA4 (Table S2), which is consistent with a previous report [65]. An unrooted phylogenetic analysis was conducted to explore the evolutionary relationships of HMAs in soybean, Arabidopsis and rice plants. The to- Agronomy 2021, 11, 71 pology of the phylogenetic tree revealed that the P1B subfamily can be divided into7 of six 25 clades (I–VI) (Figure 3). In each clade, genes from the three plant species were present, suggesting a common ancestor of the same clade in these plants.

100 GmHMA9 (Glyma.08g013600) 96 GmHMA20 (Glyma.19g140000) 81 GmHMA1 (Glyma.01g219000) 98 AtHMA5 100 GmHMA2 (Glyma.01g219100) 100 OsHMA5 Ⅲ OsHMA4 GmHMA3 (Glyma.03g086000) 100 100 100 GmHMA17 (Glyma.16g088300) 100 OsHMA6 OsHMA9 AtHMA7 100 100 GmHMA7 (Glyma.05g132900) Ⅳ 99 100 GmHMA11 (Glyma.08g087300) 100 GmHMA12 (Glyma.09g052000) 100 GmHMA15 (Glyma.15g158300) 100 GmHMA4 (Glyma.04g056100) 97 GmHMA8 (Glyma.06g056300) Ⅵ 100 AtHMA8 OsHMA8 100 OsHMA7 AtHMA6 100 GmHMA6 (Glyma.05g117400) 98 Ⅴ 100 GmHMA10 (Glyma.08g072400) 97 OsHMA2 OsHMA3 GmHMA13 (Glyma.09g055600) 100 100 GmHMA14 (Glyma.13g099600) Ⅱ 97 GmHMA18 (Glyma.17g060400) AtHMA2 98 AtHMA3 100 63 AtHMA4 OsHMA1 AtHMA1 Ⅰ 100 GmHMA5 (Glyma.05g098800) 99 GmHMA19 (Glyma.17g166800) 0.1 100

FigureFigure 3.3.Phylogenetic Phylogenetic tree tree of of P P1B1Bfamily family proteins proteins from from soybean soybean (Glycine (Glycine max max),), rice rice (Oryza (Oryza sativa sativa), and), Arabidopsisand Arabidopsis thaliana thaliana: the full-length: the full-length amino amino acid acid sequences sequences were were aligned aligned by ClustalW. by ClustalW. The neighbor-The joiningneighbor-joining phylogenetic phylogenetic tree was constructed tree was constructed by MEGA6 by with MEGA6 1000 with bootstrap 1000 bootstrap replications. replications. GmHMAs GmHMAs are denoted by red circles, AtHMAs are denoted by purple triangles, and OsHMAs are are denoted by red circles, AtHMAs are denoted by purple triangles, and OsHMAs are denoted by denoted by green diamonds. The locus name of a soybean gene is shown in brackets. Roman nu- green diamonds. The locus name of a soybean gene is shown in brackets. Roman numerals designate merals designate the subfamilies. the subfamilies.

3.3.3.3. P2P2 SubfamilySubfamily GenesGenes inin SoybeanSoybean TheThe P2P2 subfamilysubfamily ATPasesATPases cancan bebe classifiedclassified inin twotwo groups:groups: P2AP2A (ECA)(ECA) andand P2BP2B (ACA).(ACA). Previously,Previously, we we identified identified 8 8 P2A P2A subfamily subfamily genes genes and and 25 25 P2B P2B subfamily subfamily genes genes in in thethe soybeansoybean genome [66]. [66]. The The soybean soybean P2 P2 ATPase ATPase proteins proteins were were predicted predicted to contain to contain five five to ten transmembrane helices (Table S3). Domain prediction analysis showed that five domains characteristic for P2 subfamily ATPase, such as N-terminal autoinhibitory domain, N-terminus cation transporter/ATPase, E1-E2 ATPase, haloacid dehalogenase-like hydrolase, and C-terminus cation transporter/ATPase, were predicted to exist in most of the soybean P2 subfamily proteins [66]. Most of the soybean P2B proteins were predicted to contain an N-terminal autoinhibitory domain [66]. A phylogenetic tree was constructed using deduced amino acid sequences to analyze the evolutionary relatedness among the members of P2 subfamily from soybean, rice, and Arabidopsis (Figure4). The phylogenetic analysis suggested that the P2 subfamily formed five distinct clades; P2B can be divided into four clades, i.e., P2B-I, P2B-II, P2B-III, and P2B-IV (Figure4). Gene members in the same clade have a high degree of evolutionary relatedness in these three plant species, suggesting the common ancestry of P2 type ATPases in these plants. Agronomy 2021, 11, x FOR PEER REVIEW 8 of 25

to ten transmembrane helices (Table S3). Domain prediction analysis showed that five domains characteristic for P2 subfamily ATPase, such as N-terminal autoinhibitory domain, N-terminus cation transporter/ATPase, E1-E2 ATPase, haloacid dehalogenase-like hydrolase, and C-terminus cation transporter/ATPase, were predicted to exist in most of the soybean P2 subfamily proteins. [66]. Most of the soybean P2B proteins were predicted to contain an N-terminal autoinhibitory domain [66]. A phylogenetic tree was constructed using deduced amino acid sequences to analyze the evolutionary relatedness among the members of P2 subfamily from soybean, rice, and Arabidopsis (Figure 4). The phylogenetic analysis suggested that the P2 subfamily formed five distinct clades; P2B can be divided into four clades, i.e., P2B-I, P2B-II, P2B-III, and P2B-IV (Figure 4). Gene members in Agronomy 2021, 11, 71 the same clade have a high degree of evolutionary relatedness in these three plant species,8 of 25 suggesting the common ancestry of P2 type ATPases in these plants.

FigureFigure 4.4.Phylogenetic Phylogenetic tree tree of of P2 P2 subfamily subfamily proteins proteins from from soybean soybean (Glycine (Glycine max max), rice), rice (Oryza (Oryza sativa sativa), and), andArabidopsis Arabidopsis thaliana thali-: theana full-length: the full-length amino amino acid sequences acid sequences were alignedwere aligned by ClustalW. by ClustalW. The neighbor-joining The neighbor-joining phylogenetic phylogenetic tree was tree constructed was con- bystructed MEGA6 by with MEGA6 1000 with bootstrap 1000 replications.bootstrap replications. Soybean genes Soybean are denotedgenes are by denoted red circles, by Arabidopsisred circles, Arabidopsis genes are denoted genes byare denoted by purple triangles, and rice genes are denoted by green diamonds. The locus name of a soybean gene is shown purple triangles, and rice genes are denoted by green diamonds. The locus name of a soybean gene is shown following the following the gene name. The P2 subfamily is divided into six clades, i.e., P2A, and P2B-I to P2B-IV. gene name. The P2 subfamily is divided into six clades, i.e., P2A, and P2B-I to P2B-IV. 3.4. P3 Subfamily Genes in Soybean 3.4. P3A Subfamilytotal of 24 Genes putative in Soybean genes encoding PM H+-ATPases which belong to the P3 sub- familyA totalwere ofidentified 24 putative in soybean genes encoding (Table S4). PM These H+-ATPases genes were which designated belong to as the GmHA1 P3 sub- to familyGmHA24 were. The identified intron number in soybean of GmHA (Table genes S4). These varied genes from were 8 to 21, designated and the protein as GmHA1 lengthto GmHA24of GmHAs. The ranged intron from number 888 ofto GmHA1017 aminogenes acid varieds (Table from S4). 8 to All 21, of and the the GmHA protein proteins length ofwere GmHAs predicted ranged to contain from 888 6 toto 101710 transmembrane amino acids (Table helices S4). (Table All of S4). the All GmHA of the proteins GmHA wereproteins predicted were predicted to contain to 6 contain to 10 transmembrane an N-terminus helicescation transporter/ATPase (Table S4). All of the domain, GmHA a proteins were predicted to contain an N-terminus cation transporter/ATPase domain, a E1-E2 ATPase domain, and a haloacid dehalogenase-like hydrolase domain, with the exception that there is no N-terminus cation transporter/ATPase domain for GmHA18 (Figure S2). By sequencing alignment, the protein sequences of PM H+-ATPases were found to be highly conserved among the P3 subfamily proteins in Arabidopsis, rice and soybean (Figure S3). All of the 24 GmHA proteins possess a penultimate Thr, a conserved region I, and a conserved region II in the C-terminal regions with the exception of GmHA13 (Figure 5). Region I and region II have been identified to be important for autoinhibitory effects on the PM H+-ATPase [67]. The phosphorylation of penultimate Thr or Ser is required for 14-3-3 protein binding, which results in the activation of PM H+-ATPases [32,68]. A phylogenetic tree was constructed using full-length amino acid sequences of all the PM H+-ATPase in soybean, Arabidopsis, and rice. These proteins could be grouped into five distinct clades (Figure6). In each clade, PM H +-ATPase genes from soybean, Arabidopsis, and rice are all included (Figure6). Agronomy 2021, 11, x FOR PEER REVIEW 9 of 25

E1-E2 ATPase domain, and a haloacid dehalogenase-like hydrolase domain, with the exception that there is no N-terminus cation transporter/ATPase domain for GmHA18 (Fig- ure S2). By sequencing alignment, the protein sequences of PM H+-ATPases were found to be highly conserved among the P3 subfamily proteins in Arabidopsis, rice and soybean (Figure S3). All of the 24 GmHA proteins possess a penultimate Thr, a conserved region I, and a conserved region II in the C-terminal regions with the exception of GmHA13 (Figure 5). Region I and region II have been identified to be important for autoinhibitory effects on the PM H+-ATPase [67]. The phosphorylation of penultimate Thr or Ser is required for 14-3-3 protein binding, which results in the activation of PM H+-ATPases [32,68]. A phylogenetic tree was constructed using full-length amino acid sequences of all the PM H+- ATPase in soybean, Arabidopsis, and rice. These proteins could be grouped into five dis- Agronomy 2021, 11, 71 9 of 25 tinct clades (Figure 6). In each clade, PM H+-ATPase genes from soybean, Arabidopsis, and rice are all included (Figure 6).

Region I Region II 14-3-3

970 980 990 1000 1010 1020 1030 1040 1050 ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| .... GmHA1 NNLLENKTAFTTKKDYGKEEREAQWALAQRTLHGLQPPETSNIFNEKSSYRELTEIAEQAKRRAEVARLRELHTLKGHVESVVKLKGLDIDTIQQHYTV GmHA2 NNLLENKTAFTTKKDYGKEEREAQWALAQRTLHGLQPPETSNIFNEKSSYRELTEIAEQAKRRAEVARLRELHTLKGHVESVVKLKGLDIDTIQQHYTV GmHA3 VNMLENKTAFTTKKDYGKEEREAQWAHAQRTLHGLQPPETSGIFNEKNSYRELSEIAEQAKRRAEVARLRELHTLKGHVESVVKLKGLDIDTIQQHYTV GmHA4 VNMLENKTAFTTKKDYGKEEREAQWAHAQRTLHGLQPPETSGIFNEKNSYRELSEIAEQAKRRAEVARLRELHTLKGHVESVVKLKGLDIDTIQQHYTV GmHA5 DNLLENKTAFTTKKDYGKEEREAQWATAQRTLHGLQPPETTNLFNDKNSYRELSEIAEQAKRRAEVARLRELHTLKGHVESVVKLKGLDIDTIQQHYTV GmHA6 DNLLENKTAFTTKKDYGKEEREAQWATAQRTLHGLQPPETTNLFNDKNSYRELSEIAEQAKRRAEVARLRELHTLKGHVESVVKLKGLDIDTIQQHYTV GmHA7 DNLLENKTAFTTKKDYGKEEREAQWAAAQRTLHGLQPPETSNLFNDKNSYRELSEIAEQAKRRAEVARLRELHTLKGHVESVVKLKGLDIDTIQQHYTV GmHA8 DNLLENKTAFTTKKDYGKEEREAQWAAAQRTLHGLQPPETSNLFNDKNSYRELSEIAEQAKRRAEVARLRELHTLKGHVESVVKLKGLDIDTIQQHYTV AtAHA2 LNLFENKTAFTMKKDYGKEEREAQWALAQRTLHGLQPKEAVNIFPEKGSYRELSEIAEQAKRRAEIARLRELHTLKGHVESVVKLKGLDIETP-SHYTV GmHA9 NNITENKTAFTTKKDYGKEEREAQWATAQRTLHGLNPPETEQIFSEKNNYRELSEIADQARKRAEVARLRELHTLKGHVESVVKLKGLDIDTIQQHYTV GmHA10 NNITENKTAFTTKKDYGKEEREAQWVTAQRTLHGLNPPETEQIFSEKNKYRELSDIADQARKRAEVARLRELYTLKGHVESVVKLKGLDIGTIQQHYTV GmHA11 NNITENRVAFTTKKDYGKGEREAQWAAAQRTLHGLNPPETEEMLNEKNNYRELSELAEQAKKRAEVARLRELHTLKGHVESVVKLKGLDIETMQQHYTV GmHA12 NTITENRVAFTTKKDYGKGEREAQWAAAQRTLHGLNPPETEEMLNEKNNYRELSELAEQAKKRAEVARLRELHTLKGHVESVVKLKGLDIETMQQHYTV GmHA14 DNMLENKTAFTTKKDYGRGEREAEWAVAQRTLHGLQVGESNK-ANQHD----QSEIAEQAKRRAEAARLRELHTLKGHVESVVKLKGIDIDTIQQHYTL GmHA15 DNMLDNKTAFTTKKDYGRGEREAEWAVAQRTLHGLQVGESNK-AKQHE----QSEIAEQAKRRAEAARLRELHTLKGHVESVVKLKGIDIDTIQQHYTL GmHA13 DNMHQNKV------AtAHA8 DNMINQKTAFTTKKDYGKGEREAQWALAQRTLHGLPPPEAMFNDNKNE----LSEIAEQAKRRAEVARLRELHTLKGHVESVVKLKGLDIDTIQQHYTV GmHA24 KLIFERKTAFTYKKDYGKEERAAKEENGRGS------SLIAEKARRRAEIARLGEIHSLRGHVQSVLRLKNFDQNLIQSAHTV GmHA18 DLVIEQRIAFTRKKDFGKEERELKWAHAQRTLHGLHPPETKM-FNERTSYTELNQMAEEARRRAEIARLRELHTLKGRVESVVRLKGLNIDTIQQAYTV GmHA19 DLVIEQRIAFTRKKDFGKEERELKWAHAHRTLHGLHPPETKM-FNERTSYTELNQMAEEARRRANIARLRELHTLTGHVESVFRLKGLDIDTIQQAYTI GmHA20 ELVIEQRIAFTRQKDFGKEQRELQWAHAQRTLHGLQPPDTKM-FTERTHFNELNQMAEEAKRRAEIARLRELHTLKGHVESVLKLKGIDVDTIQQAYTV GmHA21 ELVIEQRIAFTRQKDFGKEQRELQWAHAQRTLHGLQPPDTKM-FTERPHFNELNQMAEEAKRRAEIARLRELHTLKGHVESVLKLKGIDVDTIQQAYTV GmHA22 DLVIEQRIAFTRQKDFGKEQRELQWAHAQRTLHGLQPADTK--FNERTHVSELNQMAEEAKRRAEIARLRELHTLKGHVESVVRLKGLDIDTIQQAYTL GmHA23 DLVIEQRIAFTRQKDFGKEQRELQWAHAQRTLHGLQPADTK--FNERTHVNELNQMAEEAKRRAEIARLRELHTLKGHVESVVRLKGLDIDTIQQAYTV OsA1 DLVIEQRIAFTRKKDFGKEERELKWAHAHRTLHGLQPPDAKP-FPEKTGYSELNQMAEEAKRRAEIARLRELHTLKGHVESVVKLKGLDIDTIHQSYTV GmHA16 NTVINQRTAFTNKNDFGKEAREAAWATEQRTLHGLQSAESKG-FTDKHTFREINTLAEEARRRAEIARLRELHTLKGRVESFAKLRGLDIDAMNGHYTV GmHA17 NTVINQRTAFINKNDFGKEAREAAWATEQRTLHGLQSAESKG-FTDKHTFREINTLAEEARRRAEIARLRELHTLKGRVESFAKLRGLDIDAMNGHYTV Clustal Consensus : :.:

Figure 5. Alignment of the C-terminal regions of 24 soybean PM H+-ATPases and three representative PM H+-ATPases Figure 5. Alignment of the C-terminal regions of 24 soybean PM H+-ATPases and three representative PM H+-ATPases fromfrom Arabidopsis Arabidopsis andand ricerice (AHA2,(AHA2, AHA8, and OsA1): OsA1): region region I, I, region region II, II, and and the the 14-3-3 14-3-3 prot proteinein binding binding site site within within the the C- C-terminalterminal regions regions are are indicated indicated by by lines. lines. Region Region I Iand and region region II II in the C-terminal region have beenbeen identiﬁedidentified toto bebe criticalcritical forfor the the autoinhibitory autoinhibitory effects effects on on the the H+ -ATPase.H+-ATPase. The The phosphorylation phosphorylation of penultimate of penultimate Thr or Thr Ser isor requiredSer is required for 14-3-3 for protein 14-3-3 binding,protein binding, which results which in results the activation in the activation of H+-ATPases. of H+-ATPases.

3.5. P4 Subfamily Genes in Soybean In this study, 27 P4 subfamily genes were identiﬁed in the soybean genome (Table S5). These genes were designated as GmALA1 to GmALA27 according to their positions in the phylogenetic tree (Figure7). The intron number of GmALA genes varied from 6 to 24, and the protein length of GmALAs ranged from 943 to 1297 amino acids (Table S5). All the P4 ATPase proteins in soybean were predicted to contain seven to ten transmembrane helices (Table S5). All of these proteins were predicted to contain several conserved domains of P4-ATPases, such as an N-terminal phospholipid-translocating P-type ATPase domain, a E1-E2 ATPase domain, a cation-transport ATPase domain, a haloacid dehalogenase-like hydrolase, and a C-terminal phospholipid-translocating P-type ATPase domain (Figure8). A phylogenetic tree was constructed using the neighbor-joining method to determine the homology between the P4 subfamily genes in soybean, Arabidopsis and rice. The topology of the phylogenetic tree showed that the P4 subfamily can be divided into ﬁve clades (I–V) (Figure7). Genes coming from the monocot rice, the dicotyledon Arabidopsis, and the legume soybean were all included in each of these clades (Figure7), suggesting common ancestors of P4 ATPases in these angiosperms.

3.6. P5 Subfamily Genes in Soybean By homolog searching, two putative genes were identiﬁed to encode P5 subfamily ATPases in soybean; they were named GmP5a and GmP5b (Table S6). The coding regions of these two genes are both interrupted by 20 introns. The encoded proteins of these two genes were predicted to contain 10 transmembrane helices (Table S6). GmP5a and GmP5b were both predicted to contain a E1-E2 ATPase domain and a haloacid dehalogenase-like hydrolase domain (Figure9A). The protein sequences of GmP5a and GmP5b are similar to the P5 proteins in Arabidopsis and rice (Figure S4). A phylogenetic tree suggested that GmP5a and GmP5b are closely related to their orthologous proteins in Arabidopsis and rice (Figure9B). AgronomyAgronomy 20212021, 11, ,11 x, FOR 71 PEER REVIEW 10 of 25 10 of 25

100 GmHA1 (Glyma.09g056300) 100 GmHA2 (Glyma.15g162600) GmHA3 (Glyma.13g097900) 27 100 GmHA4 (Glyma.17g061800) AtAHA3 75 AtAHA1 100 100 AtAHA2 OsA7 AtAHA5 Ⅱ 59 27 100 GmHA5 (Glyma.04g075100) 98 GmHA6 (Glyma.06g076100) 99 GmHA7 (Glyma.14g134100) 100 GmHA8 (Glyma.17g199200) OsA5 99 100 GmHA9 (Glyma.07g025900) 99 GmHA10 (Glyma.08g216500) 85 GmHA11 (Glyma.13g369300) 100 GmHA12 (Glyma.15g004300) AtAHA9 100 OsA6 56 62 OsA10 OsA4 Ⅳ 99 AtAHA6 55 AtAHA8 GmHA13 (Glyma.15g207900) 88 GmHA14 (Glyma.13g156000) 100 100 GmHA15 (Glyma.17g103700) 100 GmHA16 (Glyma.03g113400) 61 GmHA17 (Glyma.07g113500) 100 OsA8 AtAHA7 Ⅴ 100 OsA1 99 OsA3 98 OsA2 100 GmHA18 (Glyma.13g065400) 100 GmHA19 (Glyma.19g019200) 99 AtAHA4 85 AtAHA11 Ⅰ 100 GmHA20 (Glyma.04g174800) 98 GmHA21 (Glyma.06g189900) 99 GmHA22 (Glyma.05g030700) 100 GmHA23 (Glyma.17g096100) OsA9 GmHA24 (Glyma.03g262600) 100 90 AtAHA10 Ⅲ 0.05

FigureFigure 6. 6.Phylogenetic Phylogenetic tree soybeantree soybean (Glycine ( maxGlycine), rice max (Oryza), rice sativa (Oryza), and Arabidopsis sativa), and thaliana Arabidopsisof the thaliana of the P3AP3A subfamily: subfamily: the the full-length full-length amino amino acid sequences acid sequence were aligneds were by ClustalW.aligned by The ClustalW. phylogenetic The phylogenetic neighbor-joiningneighbor-joining tree wastree made was bymade MEGA6 by MEGA6 with 1000 bootstrapwith 1000 replications. bootstrap GmAHAs replications. are denoted GmAHAs are de- bynoted red circles, by red AtAHAs circles, are AtAHAs denoted by are purple denoted triangles, by whilepurple OsAs triangles, are denoted while by green OsAs diamonds. are denoted by green Thediamonds. locus name The of alocus soybean name gene of is showna soybean in brackets. gene Romanis shown numerals in brackets. designate Roman the subfamilies. numerals designate the subfamilies.

3.5. P4 Subfamily Genes in Soybean In this study, 27 P4 subfamily genes were identified in the soybean genome (Table S5). These genes were designated as GmALA1 to GmALA27 according to their positions in the phylogenetic tree (Figure 7). The intron number of GmALA genes varied from 6 to 24, and the protein length of GmALAs ranged from 943 to 1297 amino acids (Table S5). All the P4 ATPase proteins in soybean were predicted to contain seven to ten transmembrane helices (Table S5). All of these proteins were predicted to contain several conserved domains of P4-ATPases, such as an N-terminal phospholipid-translocating P-type ATPase domain, a E1-E2 ATPase domain, a cation-transport ATPase domain, a haloacid dehalogenase-like hydrolase, and a C-terminal phospholipid-translocating P-type ATPase domain (Figure 8). A phylogenetic tree was constructed using the neighbor-joining method to determine the homology between the P4 subfamily genes in soybean, Arabidopsis and rice. The topology of the phylogenetic tree showed that the P4 subfamily can be divided into five clades (I–V) (Figure 7). Genes coming from the monocot rice, the dicotyledon Arabidopsis, and the legume soybean were all included in each of these clades (Figure 7), suggesting common ancestors of P4 ATPases in these angiosperms. AgronomyAgronomy 20212021, 11, 11, x, 71FOR PEER REVIEW 11 of 25 11 of 25

100 GmALA1 (Glyma.01g092900) 85 GmALA2 (Glyma.02g129500) 100 GmALA3 (Glyma.04g166100) 100 GmALA4 (Glyma.06g196400) GmALA5 (Glyma.08g268900) 99 100 GmALA6 (Glyma.16g099700) 100 AtALA9 AtALA12 100 97 AtALA10 100 AtALA11 100 GmALA7 (Glyma.04g144900) 53 GmALA8 (Glyma.06g208900) Ⅱ 96 100 GmALA9 (Glyma.05g015400) 100 GmALA10 (Glyma.17g123800) 75 OsALA5 OsALA4 100 AtALA8 100 GmALA11 (Glyma.05g006800) 100 GmALA12 (Glyma.19g007400) OsALA6 100 100 AtALA4 100 AtALA5 AtALA6 100 AtALA7 100 100 OsALA7 Ⅲ 100 GmALA13 (Glyma.07g007700) 52 GmALA14 (Glyma.08g190400) 100 GmALA15 (Glyma.13g348200) 100 GmALA16 (Glyma.15g025800) AtALA3 OsALA8 100 Ⅳ 50 GmALA17 (Glyma.08g293900) 100 GmALA18 (Glyma.18g129200) 100 GmALA19 (Glyma.09g273900) 100 100 GmALA20 (Glyma.18g213100) GmALA21 (Glyma.16g220100) OsALA1 96 OsALA2 100 OsALA3 Ⅰ AtALA1 100 100 GmALA22 (Glyma.08g228800) 100 GmALA23 (Glyma.15g215400) 100 GmALA24 (Glyma.04g129200) 100 GmALA25 (Glyma. 06g315100) OsALA10 OsALA9 100 AtALA2 100 Ⅴ 0.1 100 GmALA26 (Glyma.12g205800) 100 GmALA27 (Glyma.13g295100)

FigureFigure 7. Phylogenetic7. Phylogenetic tree of tree soybean of soybean (Glycine max (Glycine), rice (Oryza max), sativa rice), ( andOryzaArabidopsis sativa), thaliana and Arabidopsisof the thaliana of P4the subfamily: P4 subfamily: the neighbor-joining the neighbor-joi phylogeneticning phylogenetic tree was made tree using was MEGA6 made with using 1000 bootstrapMEGA6 with 1000 boot- replicationsstrap replications by aligning by full-length aligning amino full-length acid sequences amino withacid ClustalW. sequences GmALAs with ClustalW. are denoted GmALAs by are de- rednoted circles, by AtALAs red circles, are denoted AtALAs by purple are denoted triangles, by while purple OsALAs triangles, are denoted while by greenOsALAs diamonds. are denoted by The locus name of the soybean gene is shown in brackets. Roman numerals designate the subfamilies. green diamonds. The locus name of the soybean gene is shown in brackets. Roman numerals designate the subfamilies. AgronomyAgronomy 20212021, 11, 11, x, 71FOR PEER REVIEW 12 of 25 12 of 25

Figure 8. Schematic representation of functional domains of soybean P4 subfamily proteins. Figure 8. Schematic representation of functional domains of soybean P4 subfamily proteins.

3.6. P5 Subfamily Genes in Soybean By homolog searching, two putative genes were identified to encode P5 subfamily ATPases in soybean; they were named GmP5a and GmP5b (Table S6). The coding regions of these two genes are both interrupted by 20 introns. The encoded proteins of these two genes were predicted to contain 10 transmembrane helices (Table S6). GmP5a and GmP5b were both predicted to contain a E1-E2 ATPase domain and a haloacid dehalogenase-like hydrolase domain (Figure 9A). The protein sequences of GmP5a and GmP5b are similar to the P5 proteins in Arabidopsis and rice (Figure S4). A phylogenetic tree suggested that GmP5a and GmP5b are closely related to their orthologous proteins in Arabidopsis and rice (Figure 9B). AgronomyAgronomy 20212021, ,1111, ,x 71 FOR PEER REVIEW 1313 of of 25 25

FigureFigure 9. 9. SchematicSchematic representation representation of functional of functional domains domains of soybean of soybean P5 subfamily P5 subfamily proteins proteins (A) (A) andand the the phylogenetic phylogenetic tree tree of ofP5 P5subfamily subfamily genes genes from from soybean soybean (Glycine (Glycine max), max rice), ( riceOryza (Oryza sativa sativa), ), andand ArabidopsisArabidopsis thaliana thaliana (B):(B ):the the neighbor-joining neighbor-joining phylogenet phylogeneticic tree tree was was made made using using MEGA6 MEGA6 with with 1000 bootstrap replications by aligning full-length amino acid sequences with ClustalW. GmP5s 1000 bootstrap replications by aligning full-length amino acid sequences with ClustalW. GmP5s are denoted by red circles, AtP5 is denoted by a purple triangle, while OsP5 is denoted by a green are denoted by red circles, AtP5 is denoted by a purple triangle, while OsP5 is denoted by a green diamonds. The locus name of the soybean gene is shown in brackets. diamonds. The locus name of the soybean gene is shown in brackets.

3.7.3.7. Chromosomal Chromosomal Distribution Distribution and and Gene Gene Du Duplicationplication of of Soybean Soybean P-Type P-Type ATPase ATPase Genes Genes TheThe identified identified 105 105 putative putative genes encoding P-type ATPasesATPases werewere distributed distributed on on 19 19 of ofthe the 20 20 chromosomes chromosomes in in soybean soybean (Figure (Figure 10 10).). There There is is no no P-type P-type ATPase ATPase gene gene on on chromo- chro- mosomesome 20. 20. On On each each of theof the other other 19 chromosomes, 19 chromosomes, the number the number of P-type of P-type ATPase ATPase genes rangedgenes rangedfrom one from to one eleven; to eleven; only one only gene one (GmACA10 gene (GmACA10) was found) was on found chromosome on chromosome 10, while 10, 11 whilegenes 11 were genes found were on found chromosome on chromosome 8 (Figure 108 ).(Figure The duplication 10). The duplication of genes resulted of genes in genere- sultedfamily in expansion. gene familyGmHMA1 expansion.and GmHMA1GmHMA2 andwere GmHMA2 found to were be tandem foundduplicated to be tandem because du- plicatedtheir distance because is lesstheir than distance 100 kb is and less no than intervening 100 kb geneand wasno intervening found (Figure gene 10). was In addition, found (Figure47 segmentally 10). In addition, duplicated 47 segmentally gene pairs were dupl identifiedicated gene with pairs higher were bootstrap identified values with higher (>90%) bootstrap(Table S7). values The synonymous (>90%) (Table substitution S7). The syno ratesnymous (Ks), the substitution nonsynonymous rates (Ks), substitution the nonsyn- rates onymous(Ka) and substitution the Ka/Ks ratiorates for(Ka) the and 47 the duplicated Ka/Ks ratio gene for pairs the indicated47 duplicated high gene similarities pairs inin dicatedtheir coding high similarities sequence alignments. in their coding The Kssequence values ofalignments. these 47 gene The pairs Ks values ranged of from these 0.056 47 genefor genepairs pair rangedGmALA26 from 0.056/GmALA27 for geneto 0.372pair GmALA26 for gene pair/GmALA27GmACA21 to /0.372GmACA22 for genewith pair an GmACA21average Ks/GmACA22 of 0.115 (Table with S7).an average The Ka/Ks Ks of of 0.115 all the (Table 47 duplicated S7). Thegene Ka/Ks pairs of all were the found 47 du- to plicatedbe between gene 0.023 pairs and were 0.338 found (Table to S7),be between suggesting 0.023 the and influence 0.338 of(Table purifying S7), suggesting selection on the the influenceevolution of of purifying these gene selection pairs, because on the a evolution pair of genes of these having gene Ka/Ks pairs, < 1because could indicate a pair pu-of genesrifying having selection Ka/Ks enforcing < 1 could on indicate the different purifying protein selection coding enforcing genes during on the the different evolution pro- [53]. teinThe coding duplication genes time during for eachthe evolution gene pairs [53]. was The calculated duplication based time on thefor divergenceeach gene pairs rate ofwasλ = − calculated6.1 × 10 9basedproposed on the for divergence soybean [53 rate]. All of λ the = 6.1 segmentally × 10−9 proposed duplicated for soybean gene pairs [53]. showed All the a segmentallytime frame betweenduplicated 4.59 gene and pairs 30.52 showed MYA, having a time an frame average between time of 4.59 9.4 MYAand 30.52 (Table MYA, S7). having an average time of 9.4 MYA (Table S7). 3.8. Tissue Expression Patterns of Soybean P-Type ATPase Genes The tissue expression patterns of putative genes encoding P-type ATPase proteins in soybean were analyzed by using two published RNA-seq datasets. The first dataset contains nine tissues, i.e., leaves, shoot apical meristem (SAM), flower, green pods, root, root tip, mature nodules, and root hair cells isolated 84 and 120 h after sowing (HAS) [56]. The second dataset contains 14 tissues, i.e., three vegetative tissues (leaves, root, and nodules) and whole seed at 11 stages of reproductive tissue development (flower, pod, and seeds) [55]. In the first dataset, with the exception of GmACA18 and GmACA24, which do not have corresponding locus names of version 1.0 (Wm82.a1.v1, used in the published RNA-seq data), the expression of the other 103 putative P-type ATPase genes could be detected in at least one of the nine tissues (Figure 11 and Table S8), suggesting authentic expression of these identified P-type ATPase genes. In the second dataset, the expression of 93 putative P-type ATPase genes could be detected in at least one of the 14 tissues, while the expression of ten genes (GmHMA20, GmACA3/4/7/8, and GmHA9/10/11/12/16) Agronomy 2021, 11, 71 14 of 25

could not be found (Figure S5). Consistently, the expression of the ten genes showed very low expression levels in tissues of soybean in the first dataset (Figure 11 and Table S8), suggesting that these genes could be expressed under specific conditions and that their functions may be very weak under normal conditions. It is notable that the previously identified GmHMA16 could not be detected in any of these two datasets, suggesting that it is a pseudogene. As shown in both datasets, many genes were expressed ubiquitously in various tissues, such as GmHMA5-8/10-12/14/15/19, GmACA1/2/5/6/9-14/19/20/23/25/26, GmP5a/b, and GmHA3-8/20-23, whereas some genes were tissue-specifically expressed. For example, GmACA3/4, and GmHA11-15 were predominantly expressed in flowers; Agronomy 2021, 11, x FOR PEER REVIEW 14 of 25 GmHMA1/2 and GmHA1/2 were strongly expressed in roots; and GmHMA18, GmACA21/22, and GmALA15/16 were predominantly expressed in root nodules (Figure 11 and Figure S5).

0 GmACA5 GmACA6 GmECA3 GmHMA8 GmHMA12 GmHMA4 GmHMA9 GmHMA13 GmHA5 GmHA6 GmACA13 GmALA13 GmACA21 GmHA1 GmALA11 GmHA9 GmHMA10 GmACA17 10 GmHMA11 GmALA9 GmECA4 GmHA22 GmP5b GmECA2 GmALA2 GmHA21 GmALA24 GmALA4 GmHA17 GmALA14 GmALA8 GmHA10 20 GmALA1 GmACA14 GmALA22 GmACA10 GmALA7

30 GmHMA3 GmHMA5 GmACA9 GmACA3 GmHA16 GmALA5 GmALA3 GmHMA6 40 GmACA11 GmHMA7 GmACA24 GmHA20 GmALA17 GmACA1 GmECA6 GmECA1 GmP5a GmACA7 GmHMA1 GmHMA2 GmALA25 GmALA19 50 GmHA24

Chr1 Chr2 Chr3 Chr4 Chr5 Chr6 Chr7 Chr8 Chr9 Chr10 60 (Mb)

GmACA16 GmHA12 GmALA12 GmALA16 GmHA19 GmHMA GmACA26 GmACA23 GmALA GmHMA14 GmACA GmHMA15 GmALA18 GmHA GmACA8 GmACA20 GmHA2 GmECA GmHA4 GmACA18 GmECA5 GmHMA18 GmP5 GmHA23 GmHA15 GmACA2 GmALA10 GmHA18 Tandem GmACA22 GmHMA17 GmACA4 GmHA7 GmHMA19 duplication GmHA3 GmACA19 GmHA13

GmHA14 GmALA6 GmALA23

GmACA12 GmHA8 GmHMA20 GmACA25 GmALA20 GmECA7 GmALA27 GmALA26 GmALA15 GmHA11 GmALA21 GmACA15 Chr11 Chr12 Chr13 Chr14 Chr15 Chr16 Chr17 Chr18 Chr19 Chr20

FigureFigure 10. Graphical 10. Graphical representation representation of of locations locations for for all thethe P-typeP-type ATPase ATPase genes genes on eachon each chromosome chromosome of soybean: of soybean: genes genes belongingbelonging to the to same the same family family are areshown shown in inthe the same same color. color. Genes Genes within within a a light light golden golden box areare putativelyputatively tandemly tandemly duplicated.duplicated.

3.8. Tissue Expression Patterns of Soybean P-Type ATPase Genes The tissue expression patterns of putative genes encoding P-type ATPase proteins in soybean were analyzed by using two published RNA-seq datasets. The first dataset contains nine tissues, i.e., leaves, shoot apical meristem (SAM), flower, green pods, root, root tip, mature nodules, and root hair cells isolated 84 and 120 h after sowing (HAS) [56]. The second dataset contains 14 tissues, i.e., three vegetative tissues (leaves, root, and nodules) and whole seed at 11 stages of reproductive tissue development (flower, pod, and seeds) [55]. In the first dataset, with the exception of GmACA18 and GmACA24, which do not have corresponding locus names of version 1.0 (Wm82.a1.v1, used in the published RNA- seq data), the expression of the other 103 putative P-type ATPase genes could be detected in at least one of the nine tissues (Figure 11 and Table S8), suggesting authentic expression of these identified P-type ATPase genes. In the second dataset, the expression of 93 putative P-type ATPase genes could be detected in at least one of the 14 tissues, while the expression of ten genes (GmHMA20, GmACA3/4/7/8, and GmHA9/10/11/12/16) could not be found (Figure S5). Consistently, the expression of the ten genes showed very low expression levels in tissues of soybean in the first dataset (Figure 11 and Table S8), suggesting that these genes could be expressed under specific conditions and that their functions may be very weak under normal conditions. It is notable that the previously identified GmHMA16 could not be detected in any of these two datasets, suggesting that it is a pseudogene. As shown in both datasets, many genes were expressed ubiquitously in various tissues, such as GmHMA5-8/10-12/14/15/19, GmACA1/2/5/6/9-14/19/20/23/25/26, GmP5a/b, and GmHA3-8/20-23, whereas some genes were tissue-specifically expressed. Agronomy 2021, 11, x FOR PEER REVIEW 15 of 25

For example, GmACA3/4, and GmHA11-15 were predominantly expressed in flowers; GmHMA1/2 and GmHA1/2 were strongly expressed in roots; and GmHMA18, Agronomy 2021, 11, 71 15 of 25 GmACA21/22, and GmALA15/16 were predominantly expressed in root nodules (Figure 11 and Figure S5).

FigureFigure 11.11. Heat mapmap representationrepresentation for for tissue tissue expression expressi patternson patterns of the of putativethe putative P-type P-type ATPase ATPase superfamilysuperfamily genesgenes in in soybean soybean according according to to Illumina Illumina transcriptome transcriptome data: data: the Reads/Kb/Millionthe Reads/Kb/Million (RPKM)(RPKM) normalized normalized log2 log2 transformed transformed counts counts were visualizedwere visualized in the heat in the map. heat The map. red colors The indicatered colors indi- cateexpression expression intensity, intensity, and grey and color grey indicates color noindica detectedtes no expression. detected HAS:expression. hours after HAS: sowing. hours SAM: after sowing.shoot SAM: apical shoot meristem. apical meristem. Agronomy 2021, 11, 71 16 of 25

3.9. Analysis of Cis-Acting Element in the Promoter Regions of Soybean P-Type ATPase Genes The presence of 10 classes of cis-elements related to hormonal signal and/or stress responses was surveyed in the 1.5 kb promoter regions upstream from the predicted transcription start sites of these putative P-type ATPase genes. These cis-elements include dehydration and cold response element DRE/CRTE, abscisic acid response element ABRE, ethylene responsive element GCC-box, auxin signaling component ARF1 binding site (AuxRE), salicylic acid-responsive element SARE, phosphate starvation signaling transcription factor PHR1 binding site (P1BS), sulfur-responsive element (SURE), stress-related CAMTA transcription factor binding site CG-box, and stress-related WRKY transcription factor binding site W-box (Table S9) [69–78]. The positions of cis-elements located in the promoter regions are shown in Table S10. With the exception of GmALA4 and GmALA21, all of the other P-type ATPase genes contained at least one of the ten cis-elements in their promoter regions (Figure S6). More than 60 of these genes contained more than four kinds of cis-elements in their promoters, especially for GmACA20 and GmHA6; each of them contained eight kinds of cis-elements (Figure S6). Among the P-type ATPase genes, more than half of them contained SARE, W-box and/or SURE, while only around 15% of them contained DRE/CRT or P1BS (Figure S6). Some cis-elements have more than one copy in the promoter region. For example, four ABRE elements were found in the promoter of GmHA11; seven SARE elements were found in the promoter of GmACA14; and nine SURE elements were found in the promoter of GmHMA17 (Figure S6 and Table S10).

3.10. Expression Analysis of Soybean P-Type ATPase Genes in Response to Stresses and Rhizobia Inoculation The expression profiles of soybean P-type ATPase genes in response to various environmental stresses, like salt, drought/dehydration, cold, flooding, and phosphate starvation, were retrieved from published microarray or RNA-seq datasets [57–60]. Among all the P-type ATPase genes in soybean, the expressions of 51 genes (48.6%) were found to be significantly changed under at least one of these environmental stresses (Figure 12). Five GmHMA genes (GmHMA6/7/10/11/15) were induced by cold, while six GmHMA genes (GmHMA2/5/8/13/14/18) were repressed by salt or dehydration stress. GmACA1/2/23/25 were strongly induced by multiple stresses, like cold, salt and drought. The expression of GmHA1/3/19 was increased, while the expression of GmHA6/16/17/20/21/22/23/24 was repressed under cold, drought, and/or salt stress. The expression profile of P-type ATPase genes in response to rhizobia (Bradyrhizobium japonicum) inoculation (12–48 h after inoculation (HAI)) was also analyzed in soybean root hairs by acquiring published transcriptome data [61]. With the exception of GmHA12, GmHA14, GmACA4, GmACA22, and GmECA2, almost all of the P-type ATPase genes (98 genes) could be detected in the root hairs with or without rhizobia inoculation (Figure 13 and Table S11). It is interesting that some of these genes were significantly affected during the inoculation of rhizobia. For example, GmHMA2 was significantly induced at 12 and 24 HAI; GmACA19 and GmALA1 were remarkably repressed at 12 HAI and 48 HAI, respectively; and GmALA7 was dramatically increased at 12 HAI (Figure 13). Two P-type ATPase genes, GmACA1 and GmACA2, were suggested to be responsive to phosphorus deficiency by RNA-seq (Figure 12). In order to confirm their transcriptional responses to phosphorus deficiency, we analyzed their expression patterns under phosphorus deficiency stress in soybean leaves and roots by qRT-PCR. Consistent with the RNA-seq data, GmACA1 and GmACA2 were found to be significantly induced by phosphorus deficiency one day and seven days after phosphorus deficiency treatment in both leaves and roots (Figure 14). Agronomy 2021, 11, 71 17 of 25 Agronomy 2021, 11, x FOR PEER REVIEW 17 of 25

FigureFigure 12. 12. HeatmapHeatmap representation representation for for the the expression expression pr proﬁlesofiles of ofputative putative genes genes encoding encoding P-type P-type ATPaseATPase proteins under environmental stresses,stresses, likelike short-termshort-term saltsalt stressstress (1 (1 h, h, 6 6 h, h, and and 12 12 h) h) in in roots, roots, 1 d cold stress in shoots, 4 d dehydration stress in shoots, 7 d flooding stress in leaves, 7 d 1 d cold stress in shoots, 4 d dehydration stress in shoots, 7 d ﬂooding stress in leaves, 7 d drought drought stress in leaves, and 7 d phosphate starvation stress in roots: the intensities of the color stress in leaves, and 7 d phosphate starvation stress in roots: the intensities of the color represent represent the relative magnitude of fold changes in log2 values according to microarray or high- throughputthe relative sequencing magnitude ofdata fold (fold changes change in > log2 2, and values p-value according < 0.05). to Red microarray color indicates or high-throughput induction, bluesequencing color indicates data (fold repression, change >and 2, andgrayp color-value me

FigureFigure 13. 13. HeatmapHeatmap representation representation for forthe theexpression expression profiles proﬁles of putative of putative genes genesencoding encoding P-type P-type ATPase proteins in root hairs (RH) and stripped roots inoculated (IN) and mock-inoculated (UN) ATPase proteins in root hairs (RH) and stripped roots inoculated (IN) and mock-inoculated (UN) with Bradyrhizobium japonicum at 12, 24, and 48 h after inoculation (HAI) according to Illumina transcriptomewith Bradyrhizobium data: the japonicum Reads/Kb/Millionat 12, 24, (RPKM) and 48 no hrmalized after inoculation log2-transformed (HAI) accordingcounts were to visu- Illumina alizedtranscriptome in the heatmap. data: The the Reads/Kb/Millionasterisk indicates a significant (RPKM) difference normalized between log2-transformed rhizobia-inoculated counts were RHvisualized and mock-inoculated in the heatmap. RH The at the asterisk same indicatestime point a (an signiﬁcant absolute difference fold change between of IN/UN rhizobia-inoculated > 2, p- valueRH and < 0.05 mock-inoculated by binomial test). RH at the same time point (an absolute fold change of IN/UN > 2, p-value < 0.05 by binomial test). Agronomy 2021, 11, x FOR PEER REVIEW 19 of 25

Two P-type ATPase genes, GmACA1 and GmACA2, were suggested to be responsive to phosphorus deficiency by RNA-seq (Figure 12). In order to confirm their transcriptional responses to phosphorus deficiency, we analyzed their expression patterns under phosphorus deficiency stress in soybean leaves and roots by qRT-PCR. Consistent with the RNA-seq data, GmACA1 and GmACA2 were found to be significantly induced by phos- Agronomy 2021, 11, 71 phorus deficiency one day and seven days after phosphorus deficiency treatment in19 both of 25 leaves and roots (Figure 14).

70.0 * Leaf Root 60.0 50.0 40.0 * 30.0 20.0 10.0 5.0 * * 4.0 * 3.0 * * Relative expression level expression Relative 2.0

1.0

0.0 +P 1d -P 1d +P 7d -P 7d +P 1d -P 1d +P 7d -P 7d GmACA1 GmACA2

FigureFigure 14. 14. RelativeRelative expression expression levels levels of of GmACA1GmACA1 andand GmACA2GmACA2 inin the the roots roots and and leaves leaves of of soybean soybean seedlingsseedlings after after phosphorus-deficiency phosphorus-deficiency ( (−P)P) treatment treatment for for one one day day (1 (1 d) d) and and 7 7 days days (7 (7 d): d): the the rela- relative tiveexpression expression levels levels of GmACA1 of GmACA1and andGmACA2 GmACA2in roots in roots and and leaves leaves under under phosphorus-sufficient phosphorus-sufficient condi- conditionstions (+P) were(+P) were normalized normalized to one. to one. Relative Relative expression expression levels levels of these of these genes genes in soybean in soybean leaves and leavesroots wereand roots detected were by detected qRT-PCR. by Data qRT-PCR. presented Data are presented mean ± SDare frommean three ± SD biologicalfrom three replicates. biological The replicates. The asterisks indicate an absolute fold change > 2 (−P/+P) and p-value < 0.05 by Stu- asterisks indicate an absolute fold change > 2 (−P/+P) and p-value < 0.05 by Student’s t-test. dent’s t-test. 4. Discussion 4. Discussion The P-type ATPase superfamily comprises a large number of ATP-hydrolyzing trans- portersThe ofP-type cations ATPase and phospholipids superfamily comprises [1]. Based a large on their number evolutionary of ATP-hydrolyzing relationships trans- and porterstransporting of cations targets, and the phospholipids superfamily [1]. genes Based are dividedon their intoevolutionary five subfamilies, relationships e.g., P1and to transportingP5 [2,3]. Many targets, P-type the ATPase superfamily genes havegenes been are di indicatedvided into to befive involved subfamilies, in various e.g., P1 cellular to P5 [2,3].and physiologicalMany P-type processesATPase genes during have the been whole indicated life of plants to be [involved6–9,37]. Thein various P-type cellular ATPase andsuperfamily physiological genes processes have been during characterized the whole genome-wide life of plants in the[6–9,37]. model The plants P-type Arabidopsis ATPase superfamilyand rice, as genes well as have in somebeen characterized other plants [genome-wide3,12,41,42,79– 81in]. the The model total plants number Arabidopsis of P-type andATPase rice, genes as well in Arabidopsisas in some other and rice plants is 47 [3,12,41,42,79–81]. and 45, respectively The [11 –total13]. Innumber this study, of P-type a total ATPaseof 105 genes genes were in Arabidopsis identified toand encode rice is putative 47 and 45, P-type respectively ATPases [11–13]. in the soybean In this study, genome a total(Figure of 1105 and genes Tables were S1–S6). identified Phylogenetic to encode analysis putative suggested P-type thatATPases soybean in the P-type soybean ATPase ge- nomegenes (Figure can be 1 grouped and Table into S1–S6). five subfamilies, Phylogenetic i.e., analysis P1B, P2A/B, suggested P3A, that P4, soybean and P5, P-type which ATPaseis consistent genes with can thatbe grouped in Arabidopsis into five and subfamilies, other flowering i.e., P1B, plants P2A/B, [11, 41P3A,]. The P4, P2C- and andP5, whichP2D-type is consistent Na+ pumps with exist that in in Chlorophyceae Arabidopsis and (e.g., otherOstreococcus flowering tauriplantsand [11,41].Chlamydomonas The P2C- andreinhardtii P2D-type) and Na moss+ pumps (e.g., existPhyscomitrella in Chlorophyceae patens) but (e.g., appear Ostreococcus to have beentauri lostand inChlamydo- vascular monasplants reinhardtii [3]. ) and moss (e.g., Physcomitrella patens) but appear to have been lost in vascular Theplants number [3]. of the total P-type ATPase genes in soybean is about 2.2 and 2.3 times higherThe than number that inof Arabidopsisthe total P-type and ATPase rice, respectively genes in soybean (Figure2 is). Soybeanabout 2.2 is and a paleopoly- 2.3 times higherploid withthan 75%that ofin theArabidopsis genes present and rice, in multiple respectively copies (Figure [43]. The 2). Soybean larger size is ofa paleopoly- the P-type ploidATPase with gene 75% family of the in genes soybean present could in be multiple attributed copies to the [43]. two The whole-genome larger size of duplicationthe P-type ATPaseevents occurredgene family approximately in soybean could 59 and be 13 attributed MYA [43 to]. Amongthe two thewhole-genome P-type ATPase duplication genes in eventssoybean, occurred one pair approximately of genes (GmHMA1 59 and 13and MYAGmHMA2 [43]. Among) was foundthe P-type to be ATPase tandemly genes dupli- in cated, and 47 pairs of genes were possibly duplicated segmentally (Figure 10 and Table S7). The time frame of segmental duplication of these genes was estimated to be between 4.59 and 30.52 MYA (Table S7). Thus, the whole genome segmental duplication could play a major role in the expansion of P-type ATPase genes during the evolution of soybean. Other gene families like SWEET transporters, calcium transporters, and WRKY transcription factors were also found to be expanded mainly by segmental duplication in the soybean genome [52,66,82]. Duplicated genes could enhance the adaptation to various environmental conditions during the evolution of soybean. Agronomy 2021, 11, 71 20 of 25

In this study, nearly all of the P-type ATPase genes could be detected in at least one of the soybean tissues and most of them were expressed in multiple tissues (Figure 11 and Figure S5), suggesting that these genes are authentic and that they may have functions during the growth and development of soybean. Some genes in the same subfamily showed different expression patterns in various tissues, suggesting that they may have distinct functions during the growth and development of soybean. It is notable that many of the duplicated gene pairs showed similar tissue expression patterns, suggesting that they may have redundant functions. However, they may have potential different roles at specific developmental stages or under specific environmental stresses. The ion transport of each P-type ATPase is dependent on the ion binding capabilities and affinity constants [15,83]. Further biochemical experiments are needed to address the specific roles of homologous proteins. In addition, a number of genes were found to be constitutively expressed in various tissues (Figure 11 and Figure S5), such as GmHA3-8/20-23, GmHMA5- 8/10-12/14/15/19, and GmACA1/2/5/6/9-14/19/20/23/25/26, suggesting that they may have general functions during the whole life of soybean. However, some soybean P-type ATPase genes were expressed in a tissue-specific manner (Figure 11 and Figure S5). The genes predominantly expressed in the flowers, like GmACA3/4 and GmHA11-15, could play potential roles in reproductive growth of soybean. Genes strongly expressed in the roots, like GmHMA1/2 and GmHA1/2, could have putative functions in nutrient uptake and translocation. Some P-type ATPase genes have been suggested to be expressed strongly in specific tissues and to play critical roles in diverse physiological processes, such as stomatal regulation, root development, pollen tube growth, and flower coloration [25,40,84–87]. For example, Arabidopsis PM H+-ATPase genes AHA1 and AHA2 are the dominating isoforms in the majority of tissues, whereas AHA8, AHA7, AHA6, and AHA9 exhibited high expression levels in pollen [88]. Recently, it has been found that the simultaneous loss of function mutation of AHA6, AHA8, and AHA9 delays pollen germination, causes pollen tube growth defects, and drastically reduces fertility in Arabidopsis [84]. Some P-type ATPase genes play important roles in plant symbiosis with arbuscular mycorrhizal fungi and rhizobial bacteria [89–92]. For example, Medicago truncatula P2A- ATPase gene MCA8 has been suggested to be critical for the generation of Ca2+ spiking in the nucleus, which is essential for legume symbiosis [91]; tomato PM H+-ATPase SlHA8 is critical for arbuscule development and energizing the periarbuscular membrane for symbiotic phosphate and nitrogen uptake [92]. It is interesting that almost all of the putative P-type ATPase genes could be detected in soybean root hairs with or without rhizobia inoculation (Bradyrhizobium japonicum) (Figure 13). Some genes, like GmHMA18, GmACA21/22, and GmALA15/16 were predominantly expressed in root nodules (Figure 11 and Figure S5). In addition, the expression of some genes, like GmACA19, GmHMA2, and GmALA1/7 were significantly changed at 12 or 24 HAI (Figure 13). These results suggest that P-type ATPase genes could participate in the establishment of symbiosis and nodule development in soybean. Further research is needed to investigate their potential roles in symbiosis. The production of soybean all over the world is frequently influenced by various environmental stresses. By retrieving published microarray and RNA-seq transcriptome data, the expression of 51 putative P-type ATPase genes was found to be remarkably affected by environmental stresses like salt, drought, cold, flooding, and/or phosphate starvation. These genes included five GmHMA genes (GmHMA6/7/10/11/15) that were induced by cold; six GmHMA genes (GmHMA2/5/8/13/14/18) that were repressed by salt or dehydration; four GmACA genes (GmACA1/2/23/25) and three GmHA genes (GmHA1/3/19) that were induced cold, salt, and/or drought; and eight GmHA genes (GmHA6/16/17/20/21/22/23/24) that were repressed by cold, drought, and/or salt (Figure 12). Modification of the expression of HMA genes could affect the transport and distribution of Cu and Zn in plant cells and could further affect the activities of Cn/Zn superoxide dismutase, which are closely related to stress tolerance. Many P-type ATPase genes have been indicated to be involved in stress response and tolerance in plants [26,39,93,94]. For example, AtHMA1, Agronomy 2021, 11, 71 21 of 25

an Arabidopsis P1B-type ATPase localized in the chloroplast envelope, is involved in Cu and Zn transport, and the loss of function mutant hma1 is sensitive to high light and excess Zn stress [95,96]; rice OsACA6 is involved in salt and drought tolerance by increasing reactive oxygen species scavenging [24]; and PM H+-ATPase plays a key role in salt and alkali resistance [97,98]. Ca2+ signals play a very important role in plant response and tolerance to various stresses. The Ca2+-ATPase has been indicated to be associated with the generation of Ca2+ signaling and cellular Ca2+ homeostasis [8]. It is interesting that many Ca2+-ATPase genes were found to be induced by stresses like salt, cold, drought, and phosphate starvation (Figure 12). By using qRT-PCR, GmACA1 and GmACA2 were conﬁrmed to be induced by phosphate starvation after treatment for short and moderate periods (Figure 13). Similarly, the expression of a tomato Ca2+-ATPase gene LCA1 was also increased by phosphate starvation [99]. Whether the induction of Ca2+-ATPase genes is involved in the generation of Ca2+ signaling under phosphate starvation, and whether the increased expression of speciﬁc Ca2+-ATPases can enhance phosphate starvation adaptation deserve further investigations.

5. Conclusions In this study, 105 putative P-type ATPase genes were identified in the soybean genome. According to the phylogenetic relationships and the conserved protein domains, they were classified into five subfamilies. All of the P-type ATPase genes identified included nineteen P1B, seven P2A, twenty-six P2B, twenty-four P3A, twenty-seven P4, and two P5 genes. Comprehensive bioinformatic and expression analyses of the P-type ATPase superfamily provide a foundation for future exploration of the potential cellular and physiological functions of P-type ATPase genes in growth and development, symbiosis establishment, and environmental stress tolerance in soybean. Considering the important roles of P-type ATPases in stress resistance and nutrient utilization in model plants like Arabidopsis and rice, further research is needed to develop stress-resistant and nutrient-efficient plants by modulating the expression of P-type ATPase genes in soybean.

Supplementary Materials: The following data is available online at https://www.mdpi.com/20 73-4395/11/1/71/s1. Figure S1: Schematic representation of functional domains of soybean P1B subfamily proteins; Figure S2: Schematic representation of functional domains of soybean P3A subfamily proteins; Figure S3: Alignment of the amino acid sequences of 24 soybean PM H+-ATPases and three representative PM H+-ATPases from Arabidopsis and rice (AHA2, AHA8, and OsA1); Figure S4: Alignment of the amino acid sequences of P5 ATPases from soybean, Arabidopsis, and rice; Figure S5: Heat map representation for tissue-speciﬁc expression patterns of the predicted P-type ATPase family genes in soybean according to Illumina RNA sequencing data (https://www.soybase. org/soyseq/); Figure S6: The distribution of cis-elements in the 1.5 kb promoter regions of soybean P-type ATPase family genes, Table S1: A list of all the putative genes encoding P-type ATPase proteins in the soybean genome; Table S2: A list of putative P1B subfamily ATPase genes and their sequence details in soybean; Table S3: A list of putative P2 subfamily ATPase genes and their sequence details in soybean; Table S4: A list of putative P3 subfamily ATPase genes and their sequence details in soybean; Table S5: A list of putative P4 subfamily ATPase genes and their sequence details in soybean; Table S6: A list of putative P5 subfamily ATPase genes and their sequence details in soybean; Table S7: Identiﬁcation of substitution rates for soybean P-type ATPase superfamily gene pairs; Table S8: Expression patterns of soybean P-type ATPase genes in 9 different tissues; Table S9: Summary of cis-acting elements that are related to plant hormone and stress response; Table S10: Distribution of hormone- and stress-related cis-elements in promoters of putative P-type ATPase genes in soybean; Table S11: Expression pattern of soybean P-type ATPase genes in root hair (RH) and stripped roots in response to B. japonicum. Author Contributions: H.Z. and H.C. conceived the study and wrote the manuscript. B.Z., H.W., W.X., W.Z. and H.Z. performed the bioinformatic analyses and the experiments. H.Z., X.C. and Y.Z. analyzed the data. All authors have read and agreed to the published version of the manuscript. Agronomy 2021, 11, 71 22 of 25

Funding: This work was supported by the National Key Research and Development Program of China (No. 2018YFE0112200), the Zhejiang Provincial Natural Science Foundation of China (No. LY20C150002), the Key Research and Development Project of Jiangsu Province (No. BE2019376), and the Agriculture Project of Hangzhou Science and Technology Bureau (No. 20180432B11). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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