Heavy Metal Transporters-Associated Proteins in Solanum Tuberosum: Genome-Wide Identiﬁcation, Comprehensive Gene Feature, Evolution and Expression Analysis

G C A T T A C G G C A T genes

Article Heavy Metal Transporters-Associated Proteins in Solanum tuberosum: Genome-Wide Identiﬁcation, Comprehensive Gene Feature, Evolution and Expression Analysis

Guandi He 1, Lijun Qin 1, Weijun Tian 2, Lulu Meng 2, Tengbing He 2,3,* and Degang Zhao 1,4,*

1 The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Institute of Agro-Bioengineering and College of Life Sciences, Guizhou University, Guiyang 550025, China; [email protected] (G.H.); [email protected] (L.Q.) 2 Agricultural College, Guizhou University, Guiyang 550025, China; [email protected] (W.T.); [email protected] (L.M.) 3 Institute of New Rural Development of Guizhou University, Guiyang 550025, China 4 Guizhou Academy of Agricultural Science, Guiyang 550025, China * Correspondence: [email protected] (T.H.); [email protected] (D.Z.)

Received: 25 September 2020; Accepted: 25 October 2020; Published: 28 October 2020

Abstract: Plants have evolved a number of defense and adaptation responses to protect themselves against challenging environmental stresses. Genes containing a heavy metal associated (HMA) domain are required for the spatiotemporal transportation of metal ions that bind with various enzymes and co-factors within the cell. To uncover the underlying mechanisms mediated by StHMA genes, we identified 36 gene members in the StHMA family and divided them into six subfamilies by phylogenetic analysis. The StHMAs had high collinearity and were segmentally duplicated. Structurally, most StHMAs had one HMA domain, StHIPPc and StRNA1 subfamilies had two, and 13 StHMAs may be genetically variable. The StHMA gene structures and motifs varied considerably among the various classifications, this suggests the StHMA family is diverse in genetic functions. The promoter analysis showed that the StHMAs had six main cis-acting elements with abiotic stress. An expression pattern analysis revealed that the StHMAs were expressed tissue specifically, and a variety of abiotic stresses may induce the expression of StHMA family genes. The HMA transporter family may be regulated and expressed by a series of complex signal networks under abiotic stress. The results of this study may help to establish a theoretical foundation for further research investigating the functions of HMA genes in Solanum tuberosum to elucidate their regulatory role in the mechanism governing the response of plants to abiotic stress.

Keywords: gene duplication; phylogenetic analysis; abiotic stress; expression analysis

1. Introduction Potato (Solanum tuberosum) is an important food crop worldwide and a good source of vitamins, minerals and dietary ﬁber as well as phytochemicals, which beneﬁts human body as nutrients supplementary and antioxidants [1]. Due to its special geological conditions, Guizhou has most of its soil developed from carbonatite. The parent material that determines the Cd content in local soil remains quite high [2], which severely undermines the growth, yield, quality, and production of S. tuberosum. During their growth and development, plants absorb such essential microelements as cuprum (Cu), zinc (Zn), and manganese (Mn), as well as some non-essential metal elements like Cadmium

Genes 2020, 11, 1269; doi:10.3390/genes11111269 www.mdpi.com/journal/genes Genes 2020, 11, 1269 2 of 21

(Cd), plumbum (Pb), and mercury (Hg) [3]. When excessively accumulated, non-essential elements can not only affect the growth and development of plants, but also gather in the human body and undermine human health through the effect of food chain [4,5]. Recent studies have revealed that, during plants’ growth and development, a mechanism that can finely regulate extracellular metallic ion concentration is formed to guarantee the uptake of essential trace metal elements and reduce plants’ tolerance of non-essential heavy metal ions at the same time [6]. According to related studies, Cd absorption within plants involves a string of procedures, including Cd absorption by root, Cd loading/unloading by xylem, transport of Cd from xylem to phloem, Cd redistribution among aerial parts, such as stem and leaves, and Cd accumulation in seeds [7,8]. The transport of Cd in scattered vascular bundles at stem nodes from the xylem to phloem is considered as a key factor determining the Cd content in aerial parts [9]. In recent years, several genes or quantitative trait locus (QTLs) have been identified as being related to Cd absorption and transport, including heavy metal ATPase (HMA) [10], ATP-binding cassette (ABC) [11], iron transporter (IRT) [12,13], metal tolerance protein (MTP) [14], natural resistance associated macrophage protein (NRAMP) [15], cation-efflux transporter (CET) [16], cation/H+ antiporters (CAX) [17,18], Zn-regulated transporter-like protein (ZNT/ZIP) [19,20], and low-affinity cation transporter (LCT) [21]. Among those gene families, P-type ATPase is kind of ion pump that utilizes the energy resulting from ATP hydrolysis to achieve transmembrane transport of ions and is widely seen in bacteria, plants and human body [22]. It can be further divided into five subfamilies by the transported substrate, namely heavy metal ATPase (HMA/P1B), Ca2+-ATPase (P2A/P2B), H+-ATPase (P3A), ALA (P4), and P5-ATPase (P5) with unknown substrate specificity [23]. Among them, HMAs/P1B are some channel proteins that associate with plants’ absorption of trace metal nutrients, which function in xylem load in case of long-distance transport of metal ions by binding with metal ions in large quantities depending on P-type ATPase activity and completing the transport to aerial parts [24]. This family is able to achieve transport through cytomembrane by hydrolyzing ATP [22]. As revealed by studies concerning genetic functions of the HMAs family, for rice, OsHMA2 located in plasma membrane is involved in the loading of Cd and Zn of xylem [25–27], OsHMA5 takes a part in loading Cu onto the xylem of root and other organs [28], OsHMA3 on tonoplast is charge of fixing Cd in vacuoles of the root [29], and OsHMA4 acts to separate Cu into root vacuoles and limit its accumulation in seeds [30]. Different stresses such as cold, drought, and high salinity share some common features with regards to their impacts on plants and the ways by which plants perceive them. For instance, The stress sensing mechanisms, including receptor-like kinases, cytoskeleton-related mechanosensors, stretch-dependent ion (calcium) channels and redox-mediated system, act at multidimensional levels in order to integrate the perception of a specific environmental cue [31]. These signal transduction pathways lead to the activation of well characterized proteins involved in the biosynthesis of proteases, transporters, and chaperones as well as ROS detoxification enzymes (alternative oxidase, glutathione peroxidase, glutathione reductase, copper-zinc superoxide dismutase and glutathione S transferase) [32]. The molecular mechanisms and signal transduction pathways of the HMA gene family under abiotic and biotic stress remain poorly understand. The HIPP gene family containing HMA domain has made some progress against abiotic and biotic stress. Roles of HIPP proteins in heavy metal homeostasis and/or detoxification [33], cold- and salt-stress responses [34], virus long-distance movement [35], and plant–pathogen interactions [36] have been demonstrated. The plant perception of overall stress induces a coordinated response mediated by complex signaling networks [31]. Plants have evolved a finely tuned response system enabling coordinated cross-talk between specific defense pathways involving shared elements and common outputs [32]. However, there is a dearth of research in the HMA gene family. This study sets out to analyze the genetic structure, chromosome distribution and phylogenetic relationship of HMA gene family in the hope to support the understanding of the evolution and diversification of HMAs in S. tuberosum species at a genome-wide scale. The potential functions of the potato HMA genes were predicted through their expression profiles of 6 abiotic stresses analysis, Genes 2020, 11, 1269 3 of 21 will lay a theoretical foundation for further study on the functions and abiotic stress (like heavy metal, high salt, high temperature, low temperature, flooding, and drought stress) resistant response mechanism of HMA gene family in S. tuberosum.

2. Results

2.1. Genome-Wide Identification, Chromosome Assignment, and Collinearity Analysis In this study, 36 candidate S. tuberosum HMA genes, respectively, were identified by a hidden Markov model for the HMA domain. In keeping with their locations on the chromosome, these genes were named StHMA1-StHMA36 in turn. A bioinformatic analysis of the amino acid sequences of these 36 family members showed that their molecular weights ranged from 7.69 to 138.88 kDa, and 19 genes were located on the minus strand with 70–1002 amino acids (StHMA6 remains the smallest with 70 amino acids, whereas StHMA9 has over 1000 encoded amino acids). The isoelectric points of HMA proteins are in the range of 4.72–9.98. The StHMA family members consist of 22 basic proteins, 13 acidic proteins and 1 neutral protein. The difference in isoelectric points indicates the diversity of the three-dimensional spatial structure of the protein sequence. Supplementary Table S2 lists the physicochemical properties of the StHMA proteins. The forecast of subcellular localization suggests that in the HMA gene family of S. tuberosum, 26 StHMAs exist in the cytoplasm, 22 in the cell nucleus, 26 in the chloroplast, seven in the plasma membrane, 10 in the mitochondria, and nine in the cytoplasmic matrix; furthermore, StHMA30 and StHMA23 are in the vacuoles, StHMA9 is in the endoplasmic reticulum, StHMA1 and StHMA4 are in the peroxisome, and StHMA4 and StHMA9 are in the plasma membrane (Supplementary Table S3). For HMA genes obtained from S. tuberosum, their locations on chromosomes were determined by searching the S. tuberosum database (http://plants.ensembl.org/Solanum_tuberosum/Info/Index). After all the sequences were located on S. tuberosum, a location map of those genes was constructed using Mapinspect software (Figure1). An analysis revealed that 36 genes in this family are distributed on 11 chromosomes of S. tuberosum, with 1–8 being on each chromosome at varying locations. All the genes are primarily located at the two ends of the chromosomes. There are eight genes on chromosome 1, four genes on chromosomes 3, 4, 5 and 11, three genes on chromosomes 2 and 10, two genes on chromosome 6, and one gene on chromosomes 8, 9, and 12, respectively. However, no member of this gene family has been detected on chromosome 7. In summary, StHMAs are primarily located on Chr1-5 and Chr11. Genes 2020, 11, 1269 4 of 21 Genes 2020, 11, x FOR PEER REVIEW 4 of 21

Figure 1. ChromosomalChromosomal locations locations of of StHMA genes in Solanum tuberosum. Black Black bars bars represent represent the the chromosomes.chromosomes. Chromosome numbersnumbers areare shown shown at at the the tops tops of of the the bar. bar.StHMA StHMAgenes genes are are labeled labeled at the at theleft left and and right right of the of the chromosomes. chromosomes. Scale Scale bar bar on theon the left left indicates indicates the the chromosome chromosome lengths lengths (Mb). (Mb).

Six pairs of genes exhibited massive chromosome multiplication events (segmental duplication) Six pairs of genes exhibited massive chromosome multiplication events (segmental duplication) in keeping with S. tuberosum intraspecific collinearity analysis, and the pairs are StHMA11 & StHMA12, in keeping with S. tuberosum intraspecific collinearity analysis, and the pairs are StHMA11 & StHMA11 & StHMA16, StHMA31 & StHMA28, StHMA31 & StHMA26, StHMA18 & StHMA19, StHMA12, StHMA11 & StHMA16, StHMA31 & StHMA28, StHMA31 & StHMA26, StHMA18 & and StHMA18 & StHMA22. Tandem duplication is observed with StHMA19 and StHMA20. The results StHMA19, and StHMA18 & StHMA22. Tandem duplication is observed with StHMA19 and indicate that some genes may cause HMA family members to multiply on different chromosomes StHMA20. The results indicate that some genes may cause HMA family members to multiply on through gene duplication, and repeats are localized to chromosomes 2–6, 9, and 10 (Figure2). To further different chromosomes through gene duplication, and repeats are localized to chromosomes 2–6, 9, analyse the homologous relationships between S. tuberosum and other species in terms of the HMA gene and 10 (Figure 2). To further analyse the homologous relationships between S. tuberosum and other family, six species were chosen for collinearity analysis, including Solanum tuberosum, Arabidopsis thaliana, species in terms of the HMA gene family, six species were chosen for collinearity analysis, including Ipomoea triloba, Nicotiana attenuata, Zea mays, and Oryza sativa (Figure3, Supplementary Table S4). S. tuberosum, A. thaliana, I. triloba, N. attenuate, Z. mays, and O. sativa (Figure 3, Supplementary Table Among 49 pairs of orthologous genes discovered in the analysis, S. tuberosum shares only one HMA S4). Among 49 pairs of orthologous genes discovered in the analysis, S. tuberosum shares only one orthologous gene with O. sativa, four with Z. mays, six with N. attenuata, 20 with I. triloba, and 18 HMA orthologous gene with O. sativa, four with Z. mays, six with N. attenuata, 20 with I. trilobaand, with A. thaliana. Thus, it can be assumed that the collinearity between S. tuberosum and A. thaliana and 18 with A. thaliana. Thus, it can be assumed that the collinearity between S. tuberosum and A. and I. triloba is more significant than that observed between S. tuberosum and O. sativa, or Z. mays thaliana and I. triloba is more significant than that observed between S. tuberosum and O. sativa, or Z. and N. attenuata. In addition, HMA genes in both I. triloba and A. thaliana have corresponding mays and N. attenuata. In addition, HMA genes in both I. triloba and A. thaliana have corresponding paralogous genes in S. tuberosum, and they largely have more than two such paralogous genes in paralogous genes in S. tuberosum, and they largely have more than two such paralogous genes in S. S. tuberosum. This association may have played a central role in the gene duplication of the HMA tuberosum. This association may have played a central role in the gene duplication of the HMA gene gene family. The foregoing results show that numerous HMAs could be produced by gene replication. family. The foregoing results show that numerous HMAs could be produced by gene replication. We We calculated the Ka/Ks ratios of the HMA homologous gene pairs to determine the selective pressure calculated the Ka/Ks ratios of the HMA homologous gene pairs to determine the selective pressure on the HMA gene family (Supplementary Table S4). Most of the gene pairs had Ka/Ks < 1. However, on the HMA gene family (Supplementary Table S4). Most of the gene pairs had Ka/Ks < 1. However, some homologous gene pairs showed Ka/Ks > 1, indicating that these genes might have been subjected some homologous gene pairs showed Ka/Ks > 1, indicating that these genes might have been to positive selective pressure. subjected to positive selective pressure.

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FigureFigure 2. 2. SchematicSchematic representations representations of ofsegmental segmental duplications duplications of S. of tuberosumSolanum tuberosumStHMA genes.StHMA Different genes. grayDiﬀerent lines grayindicate lines all indicate synteny all blocks synteny in blocks potato in genome potato genome between between each chromosome. each chromosome. Thick red Thick lines red indicatelines indicate segmental segmental duplication duplication heavy-metal-associ heavy-metal-associatedated gene gene pair, pair, and and thick thick blue blue lines lines indicate indicate tandemtandem duplicationduplication gene gene pair. pair. The The chromosome chromosome number number is indicated is indicated at the bottom at the of eachbottom chromosome. of each chromosome.Scale bar marked Scale on bar the marked chromosome on the indicatingchromosome chromosome indicating lengthschromosome (Mb). lengths (Mb).

Genes 2020, 11, 1269 6 of 21 Genes 2020, 11, x FOR PEER REVIEW 6 of 21

Figure 3. Collinearity analysis of heavy-metal-associated genes between Solanum tuberosum, Figure 3. Collinearity analysis of heavy-metal-associated genes between S. tuberosum, I. triloba, A. Ipomoea triloba, Arabidopsis thaliana, Nicotiana attenuata, Zea mays, and Oryza sativa. Gray lines in thaliana, N. attenuate, Z. mays, and O. sativa. Gray lines in the background indicate the collinear blocks the background indicate the collinear blocks within potato and other plant genomes, while the red lines within potato and other plant genomes, while the red lines highlight the syntenic StHMA pairs of highlight the syntenic StHMA pairs of orthologous genes. The species names with the prefixes “I. triloba”, “orthologousA. thaliana”, genes. “N. attenuate The specie”, “Z.s names mays”, with and the “O. prefixes sativa” indicate“I. trilobaIpomoea,” “A. thaliana triloba,,”Arabidopsis “N. attenuate thaliana,” “Z., Nicotianamays,” and attenuata “O. sativa, Zea” maysindicate, and IpomoeaOryza sativa triloba, respectively., Arabidopsis Bluethaliana color, Nicotiana bars represent attenuata the, chromosomesZea mays, and ofOryza different sativa species., respectively. The chromosome Blue color number bars represent is labeled the at the chromosomes top or bottom of of different each chromosome. species. The chromosome number is labeled at the top or bottom of each chromosome. 2.2. Multiple Sequence Alignment, Phylogenetic Analysis, and Classification 2.2. Multiple Sequence Alignment, Phylogenetic Analysis, and Classification To extend the study on the phylogenetic relationship among HMAs, 8, 12 and 22 HMAs were selectedTo extend from A. the thaliana study, O.on sativathe phylogenetic, and Solanum relationship lycopersicum among, respectively, HMAs, to8, 12 be and incorporated 22 HMAs into were a maximumselected from likelihood A. thaliana phylogenetic, O. sativa tree, and together S. lycopersicum with the 36, respectively, HMA amino to acid be sequencesincorporated identified into a frommaximumS. tuberosum likelihood. According phylogenetic to the tree structures together and with functions the 36 HMA of these amino genes, acid they sequences were divided identified into 6from subfamilies S. tuberosum named. According StHIPPa, StHIPPb,to the structures StHIPPc, and StCCH, functions StPAA1 of these and StRAN1. genes, they Some were members divided that into had 6 beensubfamilies previously named confirmed StHIPPa, as RAN1, StHIPPb, PAA1, StHIPPc, CCH, and StCCH, HIPP StPAA1 were assigned and StRAN1. to corresponding Some members subfamilies, that had been previously confirmed as RAN1, PAA1, CCH, and HIPP were assigned to corresponding

Genes 2020, 11, 1269 7 of 21 Genes 2020, 11, x FOR PEER REVIEW 7 of 21 subfamilies,including AtHMA7, including AtHMA4, AtHMA7, and AtHMA4, AtHMA8 and of A.AtHMA8 thaliana ,of OsHMA2, A. thaliana OsHMA8,, OsHMA2, and OsHMA8 OsHMA12, and of OsHMA12O. sativa, and of SlHMA3,O. sativa, and SlHMA2, SlHMA3, and SlHMA2, SlHMA8 ofandS. SlHMA8 lycopersicum of S.(Figure lycopersicum4). (Figure 4).

Figure 4. Phylogenetic relationships among 78 heavy-metal-associated proteins in Solanum tuberosum, Figure 4. Phylogenetic relationships among 78 heavy-metal-associated proteins in S. tuberosum, A. Arabidopsis thaliana, Oryza sativa and Solanum lycopersicum. The maximum likelihood tree was created thaliana, O. sativa and S. lycopersicum. The maximum likelihood tree was created using MEGA X using MEGA X (bootstrap value = 1000) and the bootstrap value of each branch is displayed. The blue (bootstrap value = 1000) and the bootstrap value of each branch is displayed. The blue solid circles, solid circles, brown solid circles, red solid circles and pink solid circles represent heavy-metal-associated brown solid circles, red solid circles and pink solid circles represent heavy-metal-associated proteins proteins from potato, Arabidopsis, rice and tomato, respectively. from potato, Arabidopsis, rice and tomato, respectively. When the amino acid sequences of different StHMA subfamilies were aligned (Figure5), When the amino acid sequences of different StHMA subfamilies were aligned (Figure 5), StHIPPc and StRNA1 subfamilies were determined to contain two highly conserved core sequences, StHIPPc and StRNA1 subfamilies were determined to contain two highly conserved core sequences, “CXXC” (in which X indicates different amino acids) and metal binding domains (MBD), while StCCH, “CXXC” (in which X indicates different amino acids) and metal binding domains (MBD), while StHIPPa, and StHIPPb were observed to have only one. In contrast to other subfamilies, StRNA1 exhibits StCCH, StHIPPa, and StHIPPb were observed to have only one. In contrast to other subfamilies, an A-domain “TGES” aa motif and a P-domain “DKTGT” aa motif at the C-end [37]. The A-domain StRNA1 exhibits an A-domain “TGES” aa motif and a P-domain “DKTGT” aa motif at the C-end [37]. regulates the absorption and release of heavy metal ions, similar to a gateway. In RAN1-1 of A. thaliana, The A-domain regulates the absorption and release of heavy metal ions, similar to a gateway. In when the A-domain exhibits variation, the transport power is notably impaired [38]. The amino acid RAN1-1 of A. thaliana, when the A-domain exhibits variation, the transport power is notably impaired sequence of the P-domain is homologous to the Haloacid dehalogenase (HAD) family, as they can [38]. The amino acid sequence of the P-domain is homologous to the Haloacid dehalogenase (HAD) both be phosphorylated [39,40]. Mutations of the sequence motif aspartic acid significantly inhibits family, as they can both be phosphorylated [39,40]. Mutations of the sequence motif aspartic acid the transport capacity of AtHMA3 [41]. Therefore, StHMA9 may exhibit a complete set of transport significantly inhibits the transport capacity of AtHMA3 [41]. Therefore, StHMA9 may exhibit a

Genes 2020, 11, 1269 8 of 21 Genes 2020, 11, x FOR PEER REVIEW 8 of 21 andcomplete detoxication set of transport functions and against detoxication heavy metals. functions StPAA1 against and heavy StCCH metals. display StPAA1 a more and conserved StCCH structuraldisplay a more domain. conserved In the StHIPPa,structural StHIPPb, domain. andIn the StHIPPc StHIPPa, families, StHIPPb, MBD and is StHIPPc usually characterizedfamilies, MBD by is mdCdusually/eC, characterized iHCdgC and by mdCd/eC, m/l/fHCegC iHCdgC aa, but and variation m/l/fHCegC may occur aa, but in variation 13 family may members, occur in including 13 family StHMA31members, (withincluding one MBDStHMA31 missing), (with StHMA27 one MBD (with missin oneg), moreStHMA27 cysteine (with residue; one more presented cysteine as CCSGC)residue; andpresented StHMA29 as CCSGC) (with HCEGCaaand StHMA29 changed (with to HCEGCaa be HCPKCaa) changed (Figure to be5 ).HCPKCaa) For more (Figure details, 5). please For more see Figuredetails,5 .please In the see StHIPPb Figure family, 5. In the except StHIPPb for StHMA35 family, except and StHMA30, for StHMA35 the remainingand StHMA30, six subfamilies the remaining are allsix locatedsubfamilies at the are N-terminus. all located Taken at the together, N-terminus. these Ta ﬁndingsken together, indicated these that findings each subfamily indicated has that its each own uniquesubfamily conserved has its own MBD unique and may conserved exert di ﬀMBDerent and functions. may exert different functions.

80 * 100 * 120 * 540 * 560 * StHMA9 : -LRRIQVRVTGMTCAACSTSVEGALMGVNGVVKASVALLQNKADVVFDP StHMA9 : NEGMVTGESAPVVKEIDSVVIGGTINLHGSLHIQGTKVGSNTVLSQIIS AtHMA7 : GLRKIQVGVTGMTCAACSNSVEAALMNVNGVFKASVALLQNRADVVFDP AtHMA7 : NESMVTGESVPVSKEVDSPVIGGTINMHGALHMKATKVGSDAVLSQIIS OsHMA2 : --RRVQVRVTGMTCSACTAAVEAAVSARRGVGGVAVSLLQSRARVVFDP OsHMA2 : DESMVTGESAPISKEVSSIVIGGTMNLHGILHIQATKVGSGTVLSQIIS OsHMA8 : --EEAHVRVTGMTCSACTSAVEGAVSARRGVRRVAVSLLQNRAHVVFDP OsHMA8 : NESMITGESAPIPKEVSSAVIGGTMNLHGVLHIQANKVGSETVLSQIIS AtHMA3 : -ISRAVFQVLGMTCSACAGSVEKAIKRLPGIHDAVIDALNNRAQILFYP AtHMA3 : NESMITGEARPVAKRKGDTVIGGTLNENGVLHVKVTRVGSESALAQIVR OsHMA3 : -TRKVMFNVRGISCASCAVSIETVVAGLKGVESVSVSPLQGQAVVQYRP OsHMA3 : NESMITGEARPIAKKPGDKVIGGTVNDNGCIIVKVTHVGSETALSQIVQ OsHMA7 : -EKVAVFEVSGMTCAACAGSVEKAVKRLQGIHDAAVDVLGGRAQVVFYP OsHMA7 : NESMITGESRPVAKRKGDTVIGGTVNENGVLHVRATFVGSESALAQIVR V GmtC aC vE a G v L A v f P nEsM TGES/A P K VIGGT N G lh t VGS L QI

160 * 180 * 200 680 * 700 * 720 StHMA9 : ASRTNPHGTVVGQFTIGGMTCAACVNSVEGILKNLPGVRKAVVALATSL StHMA9 : GGDALERAQKISHVIFDKTGTLTQGNAKVTTVKIFNEMDRGEFLTLVAS AtHMA7 : T-----QATLVGQFTIGGMTCAACVNSVEGILRDLPGVKRAVVALSTSL AtHMA7 : GGDALEKAHKVKYVIFDKTGTLTQGKATVTTTKVFSEMDRGEFLTLVAS OsHMA2 : VSQPKLQNTLSGQFRIGGMTCAACVNSVEGILKKLPGVKRAVVALATSL OsHMA2 : GGDALERAQNVKYVIFDKTGTLTQGKATVTSTKVFSGIDLGDFLTLVAS

StRNA1 OsHMA8 : ISQPKAQKTLSAQFRIGGMTCANCVNSVEGILKRLSGVKGAVVALATSL OsHMA8 : GGDALERAQNVNYVIFDKTGTLTQGKAVVTTAKVFSGMDLGDFLTLVAS AtHMA3 : NE----RSRQVCRIRINGMTCTSCSSTIERVLQSVNGVQRAHVALAIEE AtHMA3 : GGQALERAHKVNCIVFDKTGTLTMGKPVVVKTKLLKNMVLREFYELVAA OsHMA3 : QE----QEIAVCRLQIKGMACTSCSESVERALQMVPGVKKAAVGLALEE OsHMA3 : GGNALEKAHKVKAIIFDKTGTLTVGKPSVVQTKVFSKIPLLELCDLAAG OsHMA7 : KE----KNILVCRLHIKGMTCTSCASTVESILQVVPGVQRASVALATEE OsHMA7 : GGQALESAQKVDCIVFDKTGTLTIGKPVVVNTRLLKNMVLREFYAYVAA I GMtC C vE L GV A VaLa GG ALE A v FDKTGTLT Gk V k f lvA * 20 * 40 160 * 180 * 200 StHMA33 : -MSQTVVLKVGMSCQGCVGAVNRVLGKMEGVESFDIDIKEQ OsHMA6 : ADVILLRVGGMSCGGCAASVKRILESQPEVTSATVDFEKKTAAVWTTPE SlHMA18 : -MSQTVVLKVGMSCQGCVGAVNRVLGKMEGVESFDIDIKEQ AtHMA4 : SDIIILDVGGMTCGGCSASVKKILESQPQVASASVNLTTETAIVWPVPE OsHMA4 : MAAETVVLKVGMSCQGCAGAVRRVLTKMEGVETFDIDMEQQ StHMA7 : SDVIILDVTGMTCGGCASSVKRILESQPQVSSATVNLTTETAIVWPVSD StHMA24 : -MSQTVVLKVGMSCEGCVGAVKRVLGKMEGVETFDIDLKEQ SlHMA2 : SDVIILDVTGMTCGGCASSVKRILESQPQVSSATVNLTTETAIVWPVSD SlHMA8 : -MSQTVVLKVGMSCEGCVGAVKRVLGKMEGVETFDIDLKEQ sDvIiLdV GMtCGGCa SVKrILESQPqV SAtVnltteTAiVWpv OsHMA11 : -MAETVVLRVGMSCEGCVGAVKRVLGKMQGVESFDVDIKEQ m TVVLkVGMSC GCvGAV RVLgKMeGVE FDiD keQ

StPAA1 * 60 * 80 * StHMA19 : IATVSIKIR-MDCEGCARKVKNALSKVKGAKSVDVDLKQQKATVTG * 60 * 80 StHMA27 : LQTVELKVR-MCCSGCERVIKDAVYKLRGVESVSVELEMEKVTVVG StHMA25 : KKEAKPEPPPPPPEIVLRVFMHCEGCARKVRKSLKGFQGVE StHMA13 : SIMVEVRVPNLDCEGCASKLRKAIFKLKGVETIDIDMETQKVTARG StHMA14 : KTEEAPAPPPPPQEIVLRVYMHCEGCARKVRKSLKGFQGVE StHMA1 : SRVTEIQVR-MDCNGCVQKIKKALHSVHGIHDLYIDFPQQKITIVG StHMA17 : ------IQEIVLKVDMHCEACARKVARSLRGFQGVE StHMA22 : FHTVELKVR-MDCDGCELKVKKTLSSLSGVKSVEINRKQQKVTVTG StHMA32 : ------AQEMVLKVDMHCEGCARKVARALKGFQGVE StHMA11 : LQTVEIKVK-MDCDGCERRVKNAVTRMKGVKTVEVIRKQHRVIVSG StHMA15 : EEEEKKEEPKPPSPFVLYVDLHCVGCAKKIERSISKIRGVE StHMA16 : MQTVEIKVK-MDCDGCERKVTNAVSSIKGVKSVDVSRKQSKVTVSG StHMA29 : ------AVYKVGLHCPKCAHDIRKPLLTTQGVH StHMA18 : FTTVELKVR-MDCEGCEMKVKNTLSSMSGVKSVEINRKQQKVTVTG StHMA28 : G-DAAGKKDAGPTAIVLKLDLHCEGCAKKVKRFIRHFEGVE StHMA12 : TSTVEIKVK-MDCDGCERRIKNSVKHMKGVKSVEVIRKQSKVIING StHMA8 : LQTVEIKVK-MDCEGCERKVRRSVEGMKGVSSVTIEPKQHKLTVVG StHMA31 : GKNDAG---GGIATILLKLDLHCEGCAQKVRRSVRHFEGVE StHMA10 : ------MDCEGCERRVRKSVEGMRGVTKVEVEPKKHKLTVIG StHMA26 : -KNEQN-----NRNVVLKVEFHCEGCVRKILKVVRSFEGVE ve v mdC GC Gv v k t G StHMA34 : EKKNDG-----NLTVVLKSDFHCEGCVTKVVKAIKSVGGVE * 200 * 220 * vl M/L/F HCegCa k GVe StHMA35 : P-----GEQVVELRVSIHCKGCEGKVRKHISRMEGVKSFNIDLASK StHMA30 : PPPSASANQVVVLRVSLHCRGCERKMRKHISRMQGVKSFNIDFAAK * 180 * 200 P QVV LRVS HC GCE K RKHISRM GVKSFNID A K StHMA25 : KEEP-QVITVVLKVHMHCEACAQEIKRR------VENAEPD * 100 * 120 StHMA14 : KEEPPQVITVVLKVHMHCEACSQEIKRRIQKMKGVENAEPD StHMA36 : RVSIHCNGCARKVEKHISKMEGVDMYQVDLETKKVVVIGDI StHMA17 : KDEPPPVITVKMTVQMHCEACAQVMQKRIRKIQGVESVTTD StHMA3 : KVNIHCDGCKHKVKKILQKIEGVYKTNIDSEQGKVTVSGNV StHIPPcStHMA32 : KD StCCH EPLPEVTLVLNVQMHCDACALVLQKKIRKIKGIV----- StHMA21 : RVNIHCDGCKQKVKKLLQRIEGVYQVNIDFEHQKVTVYGSV StHMA15 : ASQVSGLTTVELNVNMHCEACAEQLKKKILRMKGVRSAETE StHMA5 : KVSIHCEGCKRKVKKILQQVAGVHTIDIDLKQQKVTVTGNV StHMA29 : QLKKETIKTTTLNVYMHCNKCEVDLERRLLKHKGIYSVKTN StHMA4 : KVNIHCDGCKQKVKKKLQKIEGVYTVKIDSDQSKVTVTGNV StHMA28 : KPKEPQVSTVVLKIRLHCDGCAHKIKRIIKKIDGVEEVKVD StHMA2 : KVSIHCPGCKRKVKKVLQSIEGVYTTDIDPQQQKVIVTGNV StHMA31 : ------StHMA23 : KLQVHCKYCARELEKELLNIKGVRSVKINQELGKVIISGKV StHMA26 : KEKEPPVTTVILKLHLHCEGCIQKIQKTITKNKGYKEMKID StHIPPbSt StHIPPa HMA6 : RVSLHCQGCAGKVKKHLSKMEGVTSFSIDLDSQRVTVMGHV StHMA34 : KEKEPPITTAVLKVHLHCQGCVQKISKIVTKCKGYKEMKID v iHC gC kvkK l eGV id kV v G v t l M/L hc c g Figure 5. Multiple Alignment of StHMAs Metal binding domains amino acid sequences. “StRNA1”, Figure 5. Multiple Alignment of StHMAs Metal binding domains amino acid sequences. “StRNA1,” “StPAA1”, “StCCH”, “StHIPPa”, “StHIPPb”, and “StHIPPc” represent different StHMA proteins “StPAA1,” “StCCH,”“StHIPPa,” “StHIPPb,” and “StHIPPc” represent different StHMA proteins classification. The putative xxCxxC, TGEx, and DKTGT motifs locations were highlighted in red classification. The putative xxCxxC, TGEx, and DKTGT motifs locations were highlighted in red boxes. Comparison of MBD motifs in Solanum tuberosum, Arabidopsis thaliana, Oryza sativa and boxes. Comparison of MBD motifs in S. tuberosum, A. thaliana, O. sativa and S. lycopersicum HMA Solanum lycopersicum HMA proteins. proteins. 2.3. Analysis of Conserved Motifs and Gene Structure 2.3. Analysis of Conserved Motifs and Gene Structure By referring to the HMA genetic structures and sequence motifs of A. thaliana, O. sativa and S. lycopersicumBy referring, the to di thefferences HMA ingenetic the structures structures and and sequence sequence motifs motifs of of di ffA.erent thaliana HMA, O. subfamilies sativa and ofS. S.lycopersicum tuberosum, werethe differences determined. in the The structures number ofand exons sequen ofce the motifs HMA of gene different family HMA in S. subfamilies tuberosum were of S. determinedtuberosum were to range determined. from 1 to The 9, which number is in of keeping exons withof the the HMA classification gene family of HMAs in S. in tuberosum each subgroup. were Specifically,determined StRAN1to range was from determined 1 to 9, which to have is thein mostkeeping exons with followed the classification by StHIPPc, of StPAA1, HMAs StHIPPa,in each StHIPPb,subgroup. and Specifically, StCCH. StHMA9 StRAN1 was was determined determined to have to have the highest the most number exons of exons.followed The by members StHIPPc, in theStPAA1, same StHIPPa, branch exhibited StHIPPb, a commonand StCCH. property: StHMA9 exon was length determined was determined to have tothe correspond highest number with the of numberexons. The (Figure members6). in the same branch exhibited a common property: exon length was determined to correspond with the number (Figure 6).

Genes 2020, 11, 1269 9 of 21 Genes 2020, 11, x FOR PEER REVIEW 9 of 21

Figure 6. Exon-intron structure,phylogenet structure, phylogeneticic relationships, relationships, and and protein protein domain domain analysis of StHMAs genes in potato. Phylogenetic relationships and ex exon-intronon-intron structure of StHMA genes. Green lines indicate introns.introns. BlueBlue boxesboxes represent represent upstream upstream/d/downstream-untranslatedownstream-untranslated regions. regions. The The scale scale bar bar at the at thebottom bottom estimates estimates the lengths the lengths of the exons, of the introns, exons, and introns, untranslated and untranslated regions. Phylogenetic regions. Phylogenetic relationships relationshipsand motif structures and motif of structures HMA proteins of HMA between proteinsSolanum between tuberosum S. tuberosum, Arabidopsis, A. thaliana thaliana, O., sativaOryza and sativa S. lycopersicumand Solanum. lycopersicumThe maximum. The likelihood maximum tree likelihood was generate tree wasd with generated MEGA with X using MEGA the X usingNJ method. the NJ StRAN1,method. StRAN1,StPAA1, StPAA1,StCCH, StHIPPa, StCCH, StHIPPa, StHIPPb, StHIPPb, and StHI andPPc StHIPPc are marked are marked with different with di ﬀcolors.erent colors.Motif analysisMotif analysis was performed was performed using using the MEME the MEME program. program. Boxes Boxes of ofdifferent diﬀerent colors colors represent represent the the various various motifs. Their location in each sequence is marked. Motif Motif sequence logo is shown in Table S5. The scale bar at the bottom indicates the lengthslengths of the HMA protein sequences.

The motif distribution of HM HMAA gene family members members of of S.S. tuberosum tuberosum,, A.A. thaliana thaliana,, S.S. lycopersicum lycopersicum,, and O. sativa was studied using MEME online software, and a total of 15 motifs were identifiedidentified (Figure6 6).). AsAs revealedrevealed by by the the results, results, the the subclasses subclasses vary vary significantly. significantly. Motifs Motifs 1 and 1 and 3 were 3 were identified identified as asHMA HMA structural structural domains domains that that exist exist in all in StHMA all StHMA genes. genes. The aminoThe amino acid sequence acid sequence of StRAN1 of StRAN1 is clearly is clearlyhigher thanhigher that than of otherthat of subclasses, other subclasses, and it was and also it was determined also determined to have the to highesthave the number highest of number motifs, ofincluding motifs, including 10 specific 10 motifs specific (Motifs motifs 2, 4,(Motifs 5, 7, 9, 2, 10, 4, 11,5, 7, 12, 9, 14,10, and11, 12, 15). 14, These and motifs15). These may motifs function may to functionregulate theto regulate absorption the and absorption release of and heavy release metal of ions, heavy undergo metal phosphorylation,ions, undergo phosphorylation, and provide energy and providefor transmembrane energy for transmembrane transport [37,42 transport,43]. Thus, [37,42 the StHMA,43]. Thus, proteins the StHMA in StRAN1 proteins may in be StRAN1 completely may befunctional completely in undertaking functional in heavy undertaking metal transport heavy metal and detoxication.transport and detoxication. 2.4. Expression Profiles in Various Tissues and Organs 2.4. Expression Profiles in Various Tissues and Organs To investigate the expression profiles of different genes at various transcriptomic levels, To investigate the expression profiles of different genes at various transcriptomic levels, the the expression profiles of HMA gene family RNA-seq data downloaded from the Potato Genome expression profiles of HMA gene family RNA-seq data downloaded from the Potato Genome Sequencing Consortium 2001 were analysed. These data cover 36 genes from the HMA family Sequencing Consortium 2001 were analysed. These data cover 36 genes from the HMA family expressed in 15 tissues, such as root, stem, leaf, flower, stamen and stolon, accompanied by a heat

Genes 2020, 11, 1269 10 of 21

Genes 2020, 11, x FOR PEER REVIEW 10 of 21 expressed in 15 tissues, such as root, stem, leaf, flower, stamen and stolon, accompanied by a heat map (Figuremap (Figure7). All 7). HMA All HMA genes genes were determinedwere determined to be expressed to be expressed in at least in at one least tissue one ortissue organ, or organ, while somewhile weresome observedwere observed to be to more be more strongly strongly expressed expressed in somein some specific specific tissues tissues and and organs. organs. ForFor instance, StHMAStHMA 11/2/5/11/17/18/24/28/35/2/5/11/17/18/24/28/35 were were observed observed to to be beexpressed expressed inin roots, StHMA 3/12/15/33/36 3/12/15/33/36 inin stems, StHMAStHMA 3030/32/32 in in leaves, leaves,StHMA StHMA 2/4/ 82/4/8/9/9 in petioles, in petioles,StHMA StHMA 15/29 in 15/29 ripe tubers, in ripeStHMA tubers, 3/ 34StHMAspecifically 3/34 inspecifically tuber sprouts, in tuberStHMA25 sprouts,in StHMA25 piths, pellicles in piths, and pellicles cortex and of tubers,cortex ofStHMA tubers, 4 StHMA/11/10 notably 4/11/10 in notably tuber buds,in tuberStHMA buds, 6 /StHMA13/17/21 /266/13/17/21/26notably in stolons,notably StHMAin stolons, 7/22 StHMAspecifically 7/22in specifically stem tips, andin stemStHMA tips, 28 and/31 specificallyStHMA 28/31 in specifically stamens. Even in stamens. the same Even genes the weresame observedgenes were to observed be expressed to be diexpressedfferently indifferently various tissuesin various and organs.tissues and For example,organs. ForStHMA8 example,and StHMA18StHMA8 /and27 were StHMA18/27 observed towere be more observed highly to expressed be more inhighly the petiole expressed and in root, the respectively,petiole and root, but lessrespective stronglyly, expressedbut less strongly in other expressed tissues and in other organs. tissues Overall, and theorgans. genes Overall, in the HMAthe genes family in werethe HMA determined family towere be moredetermined highly expressedto be more in highly underground expressed parts in thanunderground in aerial parts. parts Thesethan in genes aerial were parts. observed These genes to be were strongly observed expressed to be in strongly roots, stolons, expressed and petioles;in roots, thesestolons, genes and werepetioles; observed these togenes be leastwere strongly observed expressed to be least in strongly flowers; expressed and they werein flowers; observed and to they be expressedwere observed similarly to be in expressed the pith, surface,similarly and in the cortex pith, of surface, tubers. and cortex of tubers.

Figure 7. Tissue-specific expression profiles of StHMA genes in different Solanum tuberosum organs. TheFigure transcription 7. Tissue-specific levels ofexpression StHMA genesprofiles in diof ffStHMAerent tissues. genes Heatmapsin different were S. tuberosum generated organs. using The the TBtoolstranscription (Toolbox levels for of biologists StHMA genes V.1.046) in softwaredifferent fromtissues. the Heatmaps normalized were value generated by row forusing the the signatures TBtools in(Toolbox transcripts for biologists per million V.1.046) (TPM), software and gradient from color the normalized from green tovalue red by was row transcript for the levelssignatures of each in StHMAtranscripts gene. per StHMA million family (TPM), were and acquired gradient from color the fr geneom green expression to red profiles was transcript database (ArrayExpress,levels of each https:StHMA//www.ebi.ac.uk gene. StHMA/arrayexpress family were) (accession acquired number: from E-MTAB-552), the gene expression are listed inprofiles Table S7. database (ArrayExpress, https://www.ebi.ac.uk/arrayexpress) (accession number: E-MTAB-552), are listed in Table S7.

Genes 2020, 11, 1269 11 of 21

2.5. Analysis of Abiotic Stress-Related Cis-Acting Elements and Expression Pattern of Partial StHMA Members A region 2000 bp upstream of the StHMAs’ genomic sequences was selected for an analysis of cis-elements in the abiotic stress-related promoter sequences of StHMAs (Figure8). StHMAs have several cis-elements that are related to biotic stress. Among these elements, six cis-acting elements are predominant. Drought (MYB, MYC and MBS), low temperature (LTR), abscisic acid (ABRE), and disease resistance (W-box) were induced to cause stress reactions in plants. It was assumed that the HMA gene family of S. tuberosum would react to multiple abiotic stresses. As a result, 15 StHMA genes were chosen to be processed with 6 abiotic stresses. An analysis of qRT-PCR results indicates (Figure9) that when processed with di fferent abiotic stresses, StHMAs were distinctly expressed in various tissues. Compared with the control group, the expression levels of StHMA16 and StHMA31 stressed by CdCl2, that of StHMA19 and StHMA36 stressed by PEG6000, that of StHMA17, StHMA31 and StHMA36 stressed by flood, that of StHMA 1/16/19/31 stressed by NaCl, that of StHMA 2/4/19/31 stressed by 40 ◦C, and that of StHMA 2/4/17/19/22/31/36 stressed by 4 ◦C displayed significant variations at one time point. When stressed by specific abiotic factors, some genes exhibited higher transcription levels in specific tissues. For instance, in response to CdCl2 stress, StHMA1 and StHMA16 maintained high expression levels in aerial parts; in response to PEG6000 stress, StHMA36 was highly expressed in roots; in response to 40 ◦C stress, StHMA2 and StHMA4 were highly expressed in stems and roots, respectively; in response to 4 ◦C stress, StHMA19 was highly expressed in aerial parts, whereas StHMA 8/17/25/36 were highly expressed in stems. Aside from those results, in all tissues, StHMA31 expression 2+ + was significantly elevated at all time points when stressed by Cu , Na , 40 ◦C and 4 ◦C in contrast to the tissues in the control group. This gene may have been activated by several promoter elements to play roles in more than one abiotic stress. When processed with Cd2+, StHMA 1/4/16/19/22/31/33/34/36 were observed to actively react to this stress at least one time point in different tissues (Figure9). When processed with PEG6000, except for StHMA19 and StHMA36, which present an active response to stress, all the remaining StHMAs were observed to remain actionless. When subjected to flooding, StHMA 17/19/25/31/36 presented an active response to stress by being mostly expressed in the stem and root. In contrast, StHMA 1/2/4/8/16/22/35 were not observed to be significantly elevated, regardless of time and location. When processed with Na+, StHMA 1/2/4/8/16/19/22/31/34 in the root and stem were observed to actively respond to stress, StHMA 16/19/22/31 in the leaf were determined to be highly expressed, and the expression levels of StHMA 16/19/31 were observed multiplied by over 30-fold compared with those at 0 h. When stressed at 40 ◦C, StHMA2, StHMA19, and StHMA31 displayed significantly high expression, and StHMA2 was determined to be primarily expressed at 6 h. When stressed at 4 ◦C, except for StHMA 2/4/16/22, all other genes were observed to be remarkably expressed in the stem, and StHMA19 and StHMA31 were also determined to be highly expressed in the leaf. In summary, since some StHMA members were observed to respond to different abiotic stresses in varying degrees, it can be speculated that the StHMAs not only exerts certain effects on heavy metal stress, but also plays an important role in reacting to external abiotic stresses. Furthermore, the members of this family vary from one another in terms of their expression profiles and functional patterns. Genes 2020, 11, 1269 12 of 21 Genes 2020, 11, x FOR PEER REVIEW 12 of 21

Figure 8. Predicted cis-elements in the promoter regions of the Solanum tuberosum StHMA genes. Figure 8. Predicted cis-elements in the promoter regions of the S. tuberosum StHMA genes. Abiotic Abiotic stress-related promoter sequences ( 2000 bp upstream genomic sequence) were analyzed. stress-related promoter sequences (−2000 bp− upstream genomic sequence) were analyzed. The scale The scale bar at the bottom indicates the length of promoter sequence. Diﬀerent cis-elements were bar at the bottom indicates the length of promoter sequence. Different cis-elements were labeled by labeled by rectangle of diﬀerent color. Abiotic stress-related cis-acting elements are shown in Table S6. rectangle of different color. Abiotic stress-related cis-acting elements are shown in Table S6.

Genes 2020, 11, 1269 13 of 21 Genes 2020, 11, x FOR PEER REVIEW 13 of 21

2+ 0h Cd 20 32 6h ** 14 ** ** 28 12h 12 16 24h 10 24 ** ** ** 20 8 8 ** ** ** ** 12 **

6 ** 6 10 ** ** ** * * * **

8 **

4 * * 4 * ** * ** *

6 * ** * ** * * * * ** * ** * * * ** 4 * 2

2 * 2

Relative Gene Expression 0 0 0

4 8 16 17 19 31 35 36 4 8 16 17 31 35 36 4 8 16 17 31 35 36 MA2 MA2 MA2 H HMA MA25 MA34 H HMA MA25 MA34 H HMA MA22MA25 MA34 tHMA HMAtHMA H tHMA H HMAtHMA tHMA HMAtHMA H H tHMAtHMA tHMA tHMAtHMA H H H tHMAtHMA StHMA1St St S StHMA11St S StHMAStHMA22St S StHMA33St St S StHMA1St St S StHMA11St S StHMA19StHMA22St StHMAStHMA33St S S StHMA1St St S StHMA11S S StHMA19St St StHMAStHMA33St S S Root Stem Leaf 0h PEG6000 6h 18 18

36 ** ** 12h ** 16 24h 16

32 8 ** ** 6 ** 4 4

4 * * * * * * * * * * * * 2 2 2

Relative Gene Expression Gene Relative 0 0 0 1 6 2 5 3 4 5 1 6 9 2 5 3 4 5 1 6 9 2 5 3 4 5 1 2 1 2 2 3 3 1 2 1 1 2 2 3 3 1 2 1 1 2 2 3 3 MA MA MA H HMA4 MA MA H HMA4 MA MA H HMA4 MA MA MA tHMAt t HMA1tHMA17 H tHMA31 H HMA3tHMA36 tHMAt t HMA1tHMA17 H H tHMA3tHMA36 tHMAt t tHMA1tHMA17 H H H tHMA3tHMA36 S S S StHMA8StHMASt S StHMA19StHMASt S StHMASt St S S S S StHMA8StHMASt S StHMAStHMASt StHMA31StHMASt S S S S S StHMA8StHMAS S StHMASt St StHMA31StHMASt S S Root Stem Leaf 0h Flooding stress 7 24 ** 6h 28 6 12h ** ** ** ** 24h 20 ** 5 ** 24 4 ** 3 * * ** * 6 ** ** **

** 4 2 * * * 4 * * * * * 2 2 1

Relative Gene Expression Gene Relative 0 0 0

4 8 16 17 19 31 35 36 4 8 16 17 31 35 36 4 8 16 17 31 35 36 MA2 MA2 MA2 H HMA MA11MA MA MA MA22 MA MA33 MA MA H HMA MA11MA MA MA19MA22 MA MA33 MA MA H HMA MA11MA MA MA19 MA MA33 MA MA tHMAH tH H H tH H tH tHMAH tH H H H H tH tH tHMAH tH tH H H H tH tH StHMA1St St S St StH S St St StHMA25S St StHMA34StH S StHMA1St St S St StH S St St StHMA25St St StHMA34S S StHMA1St St S St S S St StHMA22StHMA25St St StHMA34S S Root Stem Leaf

+ 0h 40 Na 6h **

40 **

48 ** 12h

** 36 * 32 * 24h **

40 ** 28

24 ** **

20 ** **

** 24 **

16 24 ** ** ** 8 12 ** ** ** ** ** 12 ** 6 ** ** 8 ** ** ** ** 8

** 4 ** ** ** * * * ** ** ** ** * * * * * * * * *

4 * * * 4 * 2

Relative Gene Expression Gene Relative 0 0 0

** 40℃ 6h ** 48 **

105 ** 12h

48 ** 90

** 24h ** ** 75 28 40 ** * * 24 60 ** 16 ** ** **

20 45 ** 12 ** ** **

** 8 8 20 ** ** ** ** ** ** ** ** * * ** ** * * * * * ** ** *

4 * * * ** * *

4 ** **

10 ** * * * * * * * * * * * Relative Gene Expression Gene Relative 0 0 0 1 6 2 5 3 4 5 1 6 9 2 5 3 4 5 1 6 9 2 5 3 4 5 1 2 1 2 2 3 3 1 2 1 1 2 2 3 3 1 2 1 1 2 2 3 3 MA MA MA H HMA4 MA MA H HMA4 MA MA H HMA4 MA MA MA tHMAt t tHMAtHMA1 tHMA19tHMAtH tHMAtH tHMA3 tHMAt t tHMAtHMA1 tHMAtHMAtH tHMA31tHMAtH tHMAt t tHMA tHMAtH tH tHMA31tHMAtH S S S StHMA8S S StHMA17S S S StHMA31S S S StHMA36 S S S StHMA8S S StHMA17S S S S S S StHMA3StHMA36 S S S StHMA8S StHMA1StHMA17S S S S S S StHMA3StHMA36 Root Stem Leaf 0h 28 4℃ 6h

** 40 ** ** 32 12h 24 35 24h 30 ** ** ** ** 20

12 ** ** 25

10 ** **

20 16 **

8 ** ** ** 15 ** ** ** ** **

6 ** ** **

** 6 ** **

* 10 ** **

4 * * ** * ** * *

4 * * * * * * * 2 5 *

* 2

Relative Gene Expression Gene Relative 0 0 0

1 2 16 17 19 31 35 36 1 2 16 17 31 35 36 1 2 16 17 31 35 36 MA MA MA H HMA4 MA25 MA34 H HMA4 MA25 MA34 H HMA4 MA22MA25 MA34 tHMA8 HMAtHMA H tHMA H HMAtHMA tHMA8 HMAtHMA H H tHMAtHMA tHMA8 tHMAtHMA H H H tHMAtHMA StHMASt St S StHMA11St S StHMAStHMA22St S StHMA33St St S StHMASt St S StHMA11St S StHMA19StHMA22St StHMAStHMA33St S S StHMASt St S StHMA11S S StHMA19St St StHMAStHMA33St S S Root Stem Leaf

FigureFigure 9. 9.Expression Expression profiles proﬁles of of 15 15 selected selected StHMAStHMAs ins response in response to 40 to°C, 40 4 °C,◦C, 30% 4 ◦ C,PEG6000 30% PEG6000 (mass (massratio), ratio), 25% 25%NaCl NaCl (mass (mass ratio), ratio),and 1g/L and CdCl 1g/2L stresses CdCl2 instresses 2-months in 2-monthsold potato oldseedlings potato after seedlings treatment after treatmentfor 6, 12,and for 6, 24 12, h. and Data 24 represent h. Data represent means (±SD) means of th ( reeSD) biological of three biologicalreplicates. replicates.Vertical bars Vertical indicate bars ± indicatestandard standard deviations. deviations. Asterisks Asterisks indicate indicate corresponding corresponding genes genes significantly signiﬁcantly upregulated upregulated or or downregulateddownregulated between between the the treatment treatment and and control control (n(n= = 15, * p < 0.05;0.05; ** ** pp < <0.01;0.01; Student’s Student’s t-test).t-test).

Genes 2020, 11, 1269 14 of 21

3. Discussion The HMA protein contains a heavy metal binding domain [37]. We first identified 36 StHMA genes from the HMA gene family of S. tuberosum and divided them into six classes according to their structural features (Figure4). A collinearity analysis of the StHMA family revealed 6 pairs of segmental duplication regions, including StHMA11 & StHMA12 and StHMA11 & StHMA16, and tandem duplications, such as StHMA10 & StHMA20. These gene duplications suggest that segmental and tandem sequences matter greatly in extending the species specificity of gene families [44]. This finding provides a new perspective for further functional genomic studies and molecular design-based breeding of S. tuberosum. Intraspecies collinearity analysis revealed 49 pairs of homologous genes; among them, 18 were in A. thaliana, six were in N. attenuata, four were in Zea mays, and one was in O. sativa, indicating different evolutionary branches between dicotyledons and monocotyledons during their phyletic evolution [45]. According to an alignment of HMA domain sequences, there is one core sequence in the HMA structural domain of each group, namely, the highly conserved CXXC (Figure5). The StHMA subfamilies have their own unique conserved sequence motifs. For instance, StHMA9 has an A-domain “TEGS” aa motif and a P-domain “DKTGT” aa motif at the C-terminus that can regulate the absorption and release of heavy metal ions and promote phosphorylation [37]. These motifs indicate that StHMA9 may be functionally complete in terms of heavy metal transport and detoxication. During evolution, 13 members of the StHMA family, including StHMA31, StHMA27, and StHMA29, may undergo natural variation due to environmental stresses or genetic recombination and show some unique functions in StHMAs, which may be employed in future population genetics or functional genomics studies [46–48]. The genetic structure and sequence motif analysis demonstrated that genes in the same subfamily are relatively conserved (Figure6), but StHMA family proteins vary significantly under di fferent classifications. This finding may be attributed to the high number of exons in StHMAs. Exon shuffling and alternative splicing enrich the diversity of proteins in the StHMA family [49]. The findings of StHMAs expression profile analysis suggest that the expression levels of StHMAs are tissue-specific. This finding is in keeping with the results of previous studies [50,51]. Thus, these HMAs may be specifically involved in stress reaction-associated biological functions. StHMA31 (OsHIPP3-1; AtHIPP5) has a higher transcription level in the stamen than other genes, while StHMA11 (AtPAA1) is more heavily transcribed in tuber sprouts than other StHMAs. StHMAs are largely expressed in roots (StHMA 1/2/5/18/24/27/35) and stolons (StHMA 6/13/14/17/21/26), which may be connected with their possible role in heavy metal absorption and transport. Nevertheless, the transcription levels of these genes remain low in some organs. According to previous studies, the downregulation of some genes may be helpful in preserving their ancestral functions [52]. In other words, those HMAs may be conserved from their ancestors, which cannot be induced unless in certain special conditions appear. Most HMA genes contain abiotic stress-related cis action elements [50,53]. When analysing the promoters of the StHMA family, we discovered 6 major promoter elements that function in response to abiotic stress (Figure8). Transcription factors (TFs) and co-transcriptional regulators can precisely adjust the molecular response in various signaling cascades. Among them are various members belonging to the MYB, WRKY, NAC, DOF, AREB/ABF (ABA response-element binding factor), GBFs (G-box binding factors), and AP2/ERF families [54]. Therefore, this study investigated the expression profiles of 15 StHMA family members in different organs in response to various abiotic stresses. Only a few StHMAs react to all abiotic stresses, as StHMA16, StHMA19, and StHMA36 actively respond to all six abiotic stresses (Figure9). When stressed by Cd 2+, 15 StHMA genes respond actively. When processed with PEG6000, an active response is detected only in StHMA19 and StHMA36. Against flood stress, six StHMAs respond actively and are primarily expressed in the stem and root. When processed with Na+, 12 StHMAs present an active response, while StHMA 16/19/31 are significantly expressed in different parts and are expressed almost 30-fold more strongly than that in the control group. For high-temperature stress, significant expression was observed in StHMA2, StHMA19, and StHMA31, and for low-temperature stress, 15 StHMAs were significantly expressed. Previous studies on the HMA Genes 2020, 11, 1269 15 of 21 gene family have focused on the mechanism of heavy metal transport. Our results show that a variety of abiotic stresses may induce the expression of StHMA family genes. Plants exposed to different abiotic stresses cause significant changes the cellular redox status (leading to an increase in reactive nitrogen), the HMA family may play an important role, as they are necessary for plants to survive in extremely oxidizing environments [55], and play a signaling role in mediating defense responses, including genes in the heavy metal associated (HMA) domain [55]. With respect to plant perception of different stresses inducing a coordinated response mediated by complex signaling networks, plants specifically sense environmental cues and consequently activate signaling cascades for assembling an overall response with the final aim of surviving [31]. We speculate that the HMA gene family is also closely related to abiotic stress. However, little information is available about their roles in the protection of plants against pathogens and environmental stresses. AcHMA1 in the yeast expression system can improve yeast cell resistance to such stresses as salt, alkali, drought, and oxygen [56]. Previous studies have shown that some similar proteins play a role in the transportation of other heavy metals and together with antioxidative enzymes are responsible for cross tolerance mechanism. They participate in plant adaptation to other stresses [35,36,57]. The HIPP gene family containing HMA domain has some preliminary studies on abiotic stress. HvFP1 from barley shows a complex expression pattern with induction under different abiotic stress conditions (e.g., cold, drought, and heavy-metal exposure) during leaf senescence and in response to abscisic acid [34]. Like HvFP1, HIPP26 from Arabidopsis can also be induced by cold, salt, and drought stresses [58]. This study performed a comprehensive analysis of StHMA family genes against abiotic stress for the first time and provided a basis for further research investigating the functions of HMA genes faced with abiotic stress.

4. Materials and Methods

4.1. Plant Growth Conditions and Treatments S. tuberosum cultivar “YunShu505” plants were micropropagated in vitro on Murashige and Skoog 1 (MS) medium plus 30 gL− sucrose and 0.8% agar (Sigma-Aldrich, St. Louis, MO, USA), with the pH being adjusted to 5.8. Potato tissue culture seedlings were transplanted in the seedling tray after 3 weeks. Two months later, the plants with uniform growth and health were selected for processing (which was repeated three times). The plants were processed with 40 ◦C, 4 ◦C, 30% PEG6000 (mass ratio), 25% NaCl (mass ratio), and 1 g/L CdCl2. 500 mL processing liquids(drought stress, 30% PEG6000; salt stress, 25% NaCl; heavy metal stress, 1 g/L CdCl2) were uniformly sprayed on the plants with a watering pot (For simulating rain irrigation). High temperature stress (at 40 ◦C) and cold stress (at 4 ◦C) experiment were ﬁnished in artiﬁcial climate incubator (TAISITE RGX-400E, Tianjin Taisite Instrument Co., Ltd., Tianjin, China) The plants grown under a 16 h: 8 h, high light intensity: darkness regime. Samples were collected at 0 h, 6 h, 12 h, and 24 h after processing. In the control group, plants were sprayed with nd rd 500 mL clean water at 25 ◦C and sampled at corresponding time points. The 2 to 3 leaf, full stem, and root were cut from the S. tuberosum plants for quick freezing with liquid nitrogen and preservation at 80 C for later use. − ◦ 4.2. RNA Extraction and Quality Control Total RNA was extracted from the S. tuberosum processed as mentioned above by following the manufacturer’s instructions for the RNAplant (RTR2303, Real-Times (Beijing) Biotechnology Co., Ltd., Beijing, China) reagent kit. RNA purity (OD260/OD280), concentration and integrity were tested with an ultralow volume nucleic acid tester NanoDrop 2000 (Shanghai Chuangmeng Biological Technology Co., Ltd., Shanghai, China) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The RNA passing the test was preserved at 80 C for later use. − ◦ Genes 2020, 11, 1269 16 of 21

4.3. Identification, Physicochemical Properties Analysis, Chromosome Distribution Characteristics and Collinearity Analysis of the HMA Gene Family in S. tuberosum S. tuberosum genome data files were downloaded from the plant genome EnsemblPlants database (http://plants.ensembl.org/Solanum_tuberosum/Info/Index)[59]. From the Pfam database (http://pfam. xfam.org/)[60], the hidden Markov Model (PF00403) of HMA structural domains was acquired and then combined with HMMBUILD and HMMSEARCH in localized HMM (http://www.hmmer.org/) to search candidate members for the HMA family from S. tuberosum with default thresholds. For HMA structural domains of candidate family members, SMART (http://smart.embl-heidelberg.de/) and NCBI CDD (https://www.ncbi.nlm.nih.gov/cdd/) were applied for further integrated analysis and identification to determine the final members of the HMA gene family. The screened HMA gene family members of S. tuberosum were analysed in terms of physicochemical properties, such as amino acid number in sequence, gene position, theoretical isoelectric point, and molecular weight, using ExPASy (http://expasy.org/)[61]. Online software MG2C (http://mg2c.iask.in/mg2c_v2.0/) was employed to plot the chromosome distribution of the HMA gene family. Genome-wide sequences of A. thaliana, I. triloba, N. attenuata, Z. mays and O. sativa were downloaded from EnsemblPlants database (http: //plants.ensembl.org). The detection and study of the gene duplication events in HMA genes were per formed with the use of multiple collinear scanning toolkits (MCScanX) [62]. Corresponding results were visualized using Circos software [63]. A computation of their Ka (Synonymous), Ks (Nonsynchronous substitution), and their ratio with KaKs_Calculator2.0 software [64].

4.4. Multi-Sequence Alignment of HMA Gene Family and Phylogenetic Tree Construction in S. tuberosum To determine the evolutionary relations among members of the HMA family in S. tuberosum, amino acid sequences of A. thaliana, O. sativa and S. lycopersicum were acquired from The Ensembl Genome Browser [59] and aligned with that of S. tuberosum in HMA amino acid sequences. The alignment results were used to form a phylogenetic tree using Neighbor-Joining [65] 1000 bootstrap in MEGA X software [66].

4.5. Genetic Structure and Motif Analysis of the HMA Gene Family in S. tuberosum After CDS region sequences and HMA genome-wide sequences of StHMA family members were retrieved from the phytozome database, online software GSDS2.0 (http://gsds.cbi.pku.edu.cn/index. php)[67] was used to predict the distribution of their introns, CDS region, and 5’ and 3’ untranslated regions. Conversely, the motif structures of HMA family members in S. tuberosum were forecast with MEME (http://meme-suite.org/) with the number of target motifs set to 15 [68,69]. Their intron and exon sequences were analysed and visualized with TBtools.

4.6. Cis-Element and Expression Analysis of StHMA Gene Family The expression profiles of 36 members in the StHMA family were acquired from the gene expression profile database (ArrayExpress, https://www.ebi.ac.uk/arrayexpress) (accession number: E-MTAB-552) [70]. In this study, an analysis was performed on the expression levels of 36 genes in different tissues or organs against diverse stresses. Heatmaps were generated with HemI from the normalized value by row for the signatures in transcripts per million (TPM). The analytic results were visualized using TBtools software in the form of a heat map [71]. From S. tuberosum genome data files (http://plants.ensembl.org/Solanum_tuberosum/Info/ Index)[59], the upstream 2000 bp sequences of transcription start sites of all the members were downloaded for a predictive analysis of abiotic stress-related cis acting elements with TBtools (V.1.046) [71]. Many studies have indicated massive functional redundancy among StHMA members. Therefore, based on the phylogenetic tree, 2–3 members were chosen from the StHMA family to undergo an expression analysis with real-time quantitative PCR against abiotic stress. With CDS sequences of the HMA gene family acquired from the S. tuberosum genome database, primers (Supplementary Genes 2020, 11, 1269 17 of 21

Table S1) were designed with Primer6.0 software and then delivered to SANGON Biotech (Shanghai, China) for synthesis. The template cDNA synthesis was ﬁnished with Primer ScriptTM RT regent Kit and gDNA Eeaser kit (TaKaRa, Beijing, China) during the quantiﬁcation. With root, stem and leaf sampled at 0 h, 6 h, 12 h and 24 h, respectively, after processing, as the raw materials, total RNA was extracted and reverse transcribed into cDNA. With the S. tuberosum EF1α (elongation factor 1-alpha) gene as the internal reference, the relative expression of each gene in all parts of S. tuberosum was ∆∆Ct determined using the 2− method.

5. Conclusions HMA genes are involved in many biological processes in plants. In this paper, we identified 36 StHMAs from S. tuberosum. Gene replication analysis indicated that HMA genes tend to be conserved in different plants, and some of them may come from the same ancestor. Based on phylogenetic analysis, the HMAs were grouped into six subfamilies. HMA genetic structures and conserved domains vary greatly with species. As revealed by the expression profile analysis, StHMA expression is tissue-specific and plays a role in Cd stress, salt stress, and some abiotic stresses. This study may help to elucidate the biological functions of StHMAs.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/2073-4425/11/11/1269/s1. Table S1: qRT-PCR primers for expression analysis of StHMA gene family in Solanum tuberosum; Table S2: The IDs of 36 HMA genes as well as their properties in S. tuberosum; Table S3: Subcellular localization prediction of StHMA gene family in potato; Table S4: Ka/Ks ratios of homologous gene pairs between potato and different species; Table S5: Motif sequences of heavy-metal associated proteins in S. tuberosum, A. thaliana, O. sativa and S. lycopersicum; Table S6: Abiotic stress-related cis-acting elements in the promoter region of StHMA genes; Table S7: RNA-seq data of potato StHMA genes in different tissues. Author Contributions: Conceptualization, G.H. and T.H.; methodology, G.H.; software, G.H.; validation, L.Q., D.Z. and T.H.; formal analysis, W.T.; investigation, G.H.; resources, T.H.; data curation, D.Z.; writing—original draft preparation, G.H., W.T. and L.M.; writing—review and editing, G.H. and L.Q.; visualization, G.H. and W.T.; supervision, D.Z. and T.H.; project administration, D.Z.; funding acquisition, T.H. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Genetically Breeding Major Project of the Ministry of Agriculture of China, grant number (2016ZX08010-003); the Program Foundation of Institute for Scientific Research of Karst Area, grant number NSFC-GZGOV(U1612442); the Guizhou Science and Technology Agency Fund for Basic-condition Platform, grant number ([2019]5701). Acknowledgments: All the authors thank the raw data download from the public database. We thank all the colleagues in our laboratory for providing useful discussions and technical assistance. We would like to thank both the editor and reviewers for evaluating and providing constructive comments on this manuscript. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ABC ATP-binding cassette CAX cation/H+ antiporters CET cation-eﬄux transporter HAD Haloacid dehalogenase HMA Heavy metal ATPase IRT iron transporter LCT low-aﬃnity cation transporter MBD metal binding domains MTP metal tolerance protein NRAMP natural resistance associated macrophage protein ZIP Zn-regulated transporter-like protein NJ Neighbor-joining Genes 2020, 11, 1269 18 of 21

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