Acta agriculturae Slovenica, 120/2, 1–15, Ljubljana 2024 doi:10.14720/aas.2024.120.2.12559 Original research article / izvirni znanstveni članek Identification of metal tolerance proteins (MTP) and their gene expres- sion under drought stress in potato (Solanum tuberosum L.) Zahra HAJIBARAT 1, Abbas SAIDI 1, 2 Received March 02, 2023; accepted June 10, 2024. Delo je prispelo 2. marca 2023, sprejeto 10. junija 2024. 1 Department of Cell and Molecular biology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran 2 Corresponding author, e-mail: abbas.saidi@gmail.com Identification of metal tolerance proteins (MTP) and their gene expression under drought stress in potato (Solanum tu- berosum L.) Abstract: Metal tolerance proteins (MTPs) are as metal efflux transporters, existing extensively at all plant sections and play significant roles in regulation of the metal levels in biologi- cal processes. In the current study, phylogenetic relationships, gene structures, conserved motifs, and StMTP domains were analyzed. Here, 12 MTP genes in S. tuberosum were detected and categorized in three major clusters namely Fe/Zn-MTP, Zn-MTP, and Mn-MTP and seven groups (1, 5, 6, 7, 8, 9, and 11) according to phylogenetic relationships. Based on in silico and qPCR analysis, all of StMTPs included a cation diffusion facilitator (CDF) domains and the putative Mn-MTP harbored the ZT-dimer. An evolutionary analysis indicated that StMTP genes had undergone gene duplication leading to gene loss and gene expansion events. Analysis of transcription factor binding sites (TFBS) and microRNA in promoter region and coding se- quence of StMTP genes revealed the presence of 5312 putative TFBS and 13 StmiRNAs. The analysis of promoter regions of StMTP genes possess various frequencies of TFBS, illustrating various responses in different growth and developmental stages as well as under abiotic stress. Expression profile analysis re- vealed that the StMTP9 were up-regulated in leaves and stem, while, StMTP8 up-regulated in leaves. Both genes down-regu- lated in tubers, roots as well as under drought stress. These re- sults will provide a better insight for functional characterization of StMTP genes and can be helpful to elucidate the biological structure of their genes in potato. Key words: biological processes, transporter, metal toler- ance proteins, S. tuberosum, gene expression Določanje na kovine tolerantnih beljakovin (MTP) in izražanje njihovih genov v razmerah sušnega stresa pri krom- pirju (Solanum tuberosum L.) Izvleček: Na kovine tolerantni proteini (MTPs) so tran- sporterji kovin iz celice, ki so prisotni v velikem številu pri vseh rastlinah in igrajo pomembno vlogo pri uravnavanju količine kovin v bioloških procesih. V raziskavi so bila analizirana fi- logenetska razmerja, zgradbe genov, ohranjena zaporedja in StMTP domene. V krompirju je bilo ugotovljeno 12 MTP ge- nov, ki so bili razporejeni v tri glavne skupine in sicer Fe/Zn- -MTP, Zn-MTP ter sedem skupin Mn-MTP genov (1, 5, 6, 7, 8, 9, in 11) glede na filogenetska razmerja. Na osnovi in silico in qPCR analize so se vsi StMTPs geni vključevali domene za olaj- šanje difuzije kationov (CDF) in gene z domnevno isto funkci- jo (Mn-MTP), ki so vsebovali ZT-dimer. Evolucijska analiza je pokazala, da so StMTP geni prešli podvojevanje, kar je vodilo do izgube genov in njihovega povečevanja. Analiza mest vezave transkripcijskega faktorja (TFBS) in mikroRNK v promotorski regiji in kodirajočih sekvencah StMTP genov je odkrila priso- tnost 5312 možnih TFBS in 13 StmiRNAs. Analiza promotor- ske regije StMTP genov je pokazala, da ti vsebujejo različne fre- kvence TFBS, kar kaže na različne odzive v različnih rastnih in razvojnih fazah kot tudi učinke abiotičnega stresa. Analiza iz- ražanja profila je odkrila, da so geni StMTP9 bolj aktivni v listih in steblu med tem, ko so geni StMTP8 bolj aktivni v listih. Obe skupini genov sta manj aktivni v gomoljih in koreninah kot tudi v razmerah sušnega stresa. Ti rezultati prispevajo boljši vpogled v funkcionalno opredeljevanje StMTP genov in bi lahko bili ko- ristni za razjasnitev biološke zgradbe genov v krompirju. Ključne besede: biološki procesi, transporter, proteini to- lerance na kovine, S. tuberosum, izražanje genov Acta agriculturae Slovenica, 120/2 – 20242 Z. HAJIBARAT and A. SAIDI 1 INTRODUCTION Transition metals participate in many biological and physiological processes. Since they act as essential cofactors for many enzymes, they are components of transcription factors and other proteins and are impor- tant for both mitochondrial and chloroplast functions. However, high concentration together with non-essential metals can lead to extremely toxicity and can cause oxi- dative damages or compete with other essential ions. The physiological range of transition metals from deficiency to toxicity is extremely narrow and therefore a network to control the micronutrient fluctuations is required for all organisms. Since transmission metals are also essential components in reaction centers of enzymes, deficiency will also cause stress symptoms (Ducic and Polle, 2005). To regulate toxic effects of high and low concentration metals, it is necessary that plants maintain metal homeo- stasis at cellular levels (Hall and Williams, 2003). Special transporters were encoded by multigenic families which are responsible for the uptake and secretion of metal cati- ons in different organelles (Montanini et al., 2007). Transporters of the cation diffusion facilitator (CDF) family are namely Zn2+, Co2+, Fe2+, Ni2+, Cd2+, and Mn2+, first detected by Nies and Silver (1995). CDFs are divided into three substrate-specific clades Zn-CDF, Fe/ Zn-CDF, and Mn-CDF. These transporters are ubiqui- tous and spanning in all three kingdoms of organisms: Archaea, Eubacteria, and Eukaryotes. Expressed funda- mentally in both root sand shoots, AtMTP1 is over-ex- pressed conferring Zn tolerance in rice (Zhang and Liu, 2017). OsMTP1, a cation transporter localized in tono- plast, possesses low affinity to Co, Fe, and Cd, controlling ion hemostasis in rice (Menguer et al., 2013). AtMTP5 and AtMTP12, other functional complex members of Zn-CDF proteins, were found to transport Zn into the Golgi apparatus (Fujiwara et al., 2015). In Mn-CDF groups, there were four AtMTP proteins (AtMTP8-11), which both AtMTP9 and AtMTP11 functioned as a Mn transporter. AtMTP11 was involved in maintaining Mn hemostasis and localized in pre-vacuolar compartment and/or trans-Golgi. Mutation of atmtp11 display Mn sen- sitivity and higher levels of Mn in shoots and roots than the wild-type plants were accumulated. Five Mn-CDF members (OsMTP8.1/8.2/9/11/11.1) with known func- tions are classified into groups 8 and 9. ShMTP8, another member of Mn-CDF, is isolated from the Mn-tolerant legume Stylosanthes hamata (L.) Taub.. ShMTP8 is lo- calized in tonoplast, exhibiting Mn-tolerance when ex- pressed in A.thaliana (L.) Heynh. (Delhaize et al., 2003). In Arabidopsis and cucumber, AtMTP7, CsMTP7 act as Fe transporter member and are localized in plant mito- chondrial (Migocka et al., 2018). CsMTP8 was found in vacuolar membrane and participated in the maintenance of Mn hemostasis (Migocka et al., 2014). CsMTP9 is in- volved in the efflux of Mn+2 and Cd+2 from cucumber root cells using H+-coupled with manganese and cadmium antiporter (Migocka et al., 2015). Potato (S. tuberosum L.) is one of the largest non- cereal food crop worldwide and sequencing of its entire genome is completed (Zhang et al., 2017). Potato can be utilized for molecular plant biological research and to fa- cilitate gene discovery and comparative genetics (Jaillon et al. 2007). There has been few relevant research on the StMTP genes in potato. The present genome-wide sur- vey was conducted to identify the MTP gene family in S. tuberosum and systematically analyzed their sequence and structural characteristics as well as evolutionary re- lationships. Besides, the transcription factor binding sites distributions, and the potential microRNA target sites in StMTP genes were predicted. In addition, the expression profiles of StMTP genes in different potato tissues and in response to abiotic and biotic stresses were analyzed using a microarray data approach. Results in this study could provide a better insight into the biological func- tions of StMTP proteins and the molecular mechanisms underlying these metal transporters and the homeostasis maintained by them in potato. 2 MATERIAL AND METHODS 2.1 IDENTIFICATION OF MTP GENES IN S. tubero- sum The MTP genes of A.thaliana and O.sativa L. were taken from TAIR and RAP-DB databases, respectively. To detect the potential StMTP genes in potato, the HMM file of the MTP domain (PF01545) was taken from the Pfam database and utilized to perform the HMMER search. Then, the resulting MTP sequences were adopted for tBLASTn. Finally, following the removal of redundant predicted sequences, the sequences all putative MTPs were further confirmed using InterProScan. 2.2 SEQUENCE ALIGNMENT AND PHYLOGE- NETIC Sequence similarity analysis of MTPs proteins be- tween S. tuberosum and A. thaliana were performed in blastp at NCBI. Each protein sequence of MTPs in Arabidopsis and O.sativa was used at the query, and all 12 StMTP protein sequences were used as the subject se- quence. For phylogenetic analysis, multiple sequence align- Acta agriculturae Slovenica, 120/2 – 2024 3 Identification of metal tolerance proteins (MTP) and their gene expression under drought stress in potato (Solanum tuberosum L.) ments at protein levels were performed by ClustalX, and MEGA 6.0. Phylogenetic tree construction was es- tablished by the Maximum likelihood method (Tamura et al., 2013). The MTPs sequence from S. tuberosum, A. thaliana, and O. sativa were downloaded from the above databases, as described by Liu et al (2019). 2.3 AMINO ACID PROPERTIES AND STRUCTURE CHARACTERISTICS OF MTP PROTEINS The molecular weight, Pi, and peptide length were evaluated using the ProParam software and prediction of protein transmembrane helices was examined using protter. Sub-cellular localization was predicated using Plant-Mploc server (Hall 2002). MEME program was utilized to detect the conserved motifs (Bailey et al., 2009; Finn et al., 2016). Motifs functions were determined us- ing the hmmscan tool. Then, detected MTP sequences were aligned using Muscle, and identity residue was cal- culated. The exon-intron structures of StMTP genes were characterized using GSDS program. 2.4 TFBS ANALYSIS AND MIRNA TARGET SITES PREDICTION The promoter regions (up-stream 1000 bp) of St- MTP genes were extracted to predict the TFBS using PlantPAN. The miRNA target sites of StMTPs were ex- amined using small RNA target analysis server. 2.5 PLANT GROWTH AND QRT-PCR OF StMTP GENES To analyze specific expression in root, stem and leaf tissue, samples were taken from two-week-old seedlings. Three tubers were planted in the pot. To analyze the ex- pression under drought stress, two treatments of drought and irrigation were used. Each treatment was in a com- pletely randomized block design with three blocks. In the first six weeks, all the plants in each two treatments were watered equally. After 2 weeks of stress, leaf and tuber samples were taken under the mentioned conditions. Then the leaves and tubers were immersed in liquid ni- trogen and kept at -80 temperature until RNA extraction. RNA extraction was performed using the Synaclone kit. Then cDNA synthesis was performed as follows. Potato EF-1α gene was used as internal control. All primers used in gene expression analysis are listed in Table S1. Real time was done using SYBR Green Supermix. Relative ex- pression was determined via 2-∆∆Ct. 3 RESULT 3.1 IDENTIFICATION AND CLASSIFICATION OF MTP GENES IN POTATO Using 12 and 10 AtMTP and OsMTP protein se- quences as the queries, a total of 12 MTP genes were detected in S. tuberosum. Subsequently, HMM verifica- tion was performed in 12 MTP sequences, including the cation efflux domain in the potato genome. According to sequence identity, cover value, and orthologous rela- tionship, the 12 StMTP proteins were designated as St- MTP1 to StMTP11. For each AtMTP protein, there was at least one MTP homolog in S.tuberosum except for At- MTP2, AtMTP10, and AtMTP12, where no correspond- ing StMTP was found. To understand the evolutionary relationships of MTP gene family members among po- tato, Arabidopsis, and rice 35 MTP protein sequences from three species were comprehensively analyzed and a phylogenetic tress was constructed. According to the classification of previous surveys (Montanini et al., 2007; Shirazi et al., 2019), 35 MTP proteins were divided into three substrate-specific groups (Zn-CDFs, Zn/Fe-CDFs, and Mn-CDFs) and seven primary groups (1, 5, 6, 7, 8, 9, and 11) that were similar to the AtMTPs and OsMTPs. Of the seven groups, group 1 had the maximum StMTP with 12 members, whereas groups 5, 6, and 7 contained the minimum StMTP with three members each. There are four, five, and five StMTP members in groups 11, 8, and 9, respectively. StMTP1, StMTP3, StMTP4, and StMTP5 belonged to Zn-CDF family; StMTP6 and St- MTP7 to Fe/Zn-CDF family and StMTP8, StMTP8.1, St- MTP9, StMTP9.1, StMTP9.2 and StMTP11 to Mn-CDF family (Fig 1). 3.2 STRUCTURE AND CHARACTERISTIC ANALY- SIS OF STMTP GENES The characteristics of the StMTP genes were ana- lyzed in detail. The length of protein sequences of StMTP genes ranged from 86 to 503 amino acids. The molecu- lar weights and pIs of these potato proteins ranged from 9781.63 to 55006.92 KDa and 4.98-10.45, respectively (Table 1). Most of the StMTP proteins included five to six Acta agriculturae Slovenica, 120/2 – 20244 Z. HAJIBARAT and A. SAIDI putative transmembrane domains (TMDs), StMTP8 and StMTP11 had only four TMDs, StMTP7 contained three TMDs, and StMTP6 carried twelve TMDs. Particularly, StMTP5 and StMTP8.1 proteins lacked any of the TMDs. To examine differences in StMTP genes, the exon and intron structures of 12 potato MTP genes were com- pared. As illustrated in Figure 2, the number of introns in the StMTP genes ranged from 1 to 12. Further, the results showed that most of the StMTP in same groups exhib- ited similar exon-intron compositions. Most members of Mn-CDF had six exons, all of StMTP in Fe/Zn included two exons, and more members in ZN-CDF possessed variable number of exons (Fig 2a). To obtain more insight into the structure character- istics of the StMTP proteins and conserved motif analy- sis, their amino acid sequences were submitted to MEME program. As shown in Figure 3 and Table 2, ten motifs were in total identified in StMTP family members, while only four of them were explored to encode functional domains when subjected to Pfam. Motif 1, 4, and 8 were annotated as cation_efflux (PF01545), motif 3 as ZT- dimer (PF16916) while motifs 2, 5, 6, 7, 9, and 10 were not assigned by the Pfam. Highly similarity motifs are expected to have similar functions. StMTPs belonged to Mn-CDF group included three motifs sequences namely, motif 1 and 4. StMTPs relevant to Zn-CDF contained three motif (4, 8, and 9). StMTPs (1/3/4/5) included both motifs 4 and 8 cation-efflux. Whereas, StMTP 6 and St- MTP7 had only one of 4 motif which belonged to Zn/ Fe-CDF groups (Fig 2b). As explained earlier, the cation efflux domain is a typical feature of the MTP transporters. Hence, the domain architec- tures in StMTP proteins were analyzed. Results showed that all the StMTP proteins included the cation efflux domain. However, the mem- bers of groups 8, 9, and 11 (except to 9.2) pos- sessed a ZT.dimer which is a significant zinc transporter dimerization domain. 3.3 MULTIPLE SEQUENCE ALIGNMENT, CON- SERVED MOTIFS, AND DOMAIN ARCHITEC- TURES IN STMTPS PROTEINS To evaluate the sequence of the StMTP proteins, the amino acid sequences of the AtMTPs, OsMTPs, and St- MTPs from the three substrate-specific groups were mul- tiple aligned by ClustalX, respectively. Results revealed that total of the AtMTPs, OsMTPs, and StMTPs proteins has one and two conserved HxxxD residues in Zn/Fe- Figure 1: Phylogenetic relationship of MTP proteins in three main plants of Arabidopsis, rice, and potato. The tree was constructed using the MEGA 6.0 software by the Maximum likelihood method. The identical proteins were categorized into three sub-families (Mn-MTPs, Zn-MTPs, and Zn/Fe-MTPs). The Zn-MTP sub-family (red line) contains 1: (MTP1:4), 5: MTP5 (red); Zn/Fe-MTPs (yellow line) includes (MTP6-7); and Mn-MTP (green line) contains (MTP8-11) Acta agriculturae Slovenica, 120/2 – 2024 5 Identification of metal tolerance proteins (MTP) and their gene expression under drought stress in potato (Solanum tuberosum L.) Table 1: MTP proteins information for potato Gene Accession number Peptide lenght MW (kDa) pI No. of TMDs N to C Subcellular localization StMTP9 PGSC0003DMG400004287 317 36293.33 6.41 5/into in Cell memebrane. Vaculoe. StMTP9.1 PGSC0003DMG400009656 86 9781.63 10.45 5/into in Cell membrane. Vacuole. StMTP9.2 PGSC0003DMG400011247 413 47080.09 6.22 5/into in Vacuole. StMTP8.1 PGSC0003DMG400001111 373 42328.02 4.98 0 Cell membrane. Vacuole. StMTP8 PGSC0003DMG400032189 405 45429.20 5.07 4/into out Vacuole. StMTP7 PGSC0003DMG400026506 463 50752.05 6.36 3/into out Vacuole. StMTP6 PGSC0003DMG402011364 503 55006.92 6.19 12/out to out Vacuole. StMTP5 PGSC0003DMG400014975 387 43337.68 6.67 0 Vacuole. StMTP4 PGSC0003DMG400030333 380 42495.88 5.85 6/into in Vacuole. StMTP3 PGSC0003DMG400030740 385 42861.52 5.86 6/into in Vacuole. StMTP1 PGSC0003DMG400030701 415 45972.68 6.05 6/into in Vacuole. StMTP11 PGSC0003DMG400023516 401 45240.14 5.03 4/ into out Vacuole. Figure 2: A) Distributions of the conserved domains in StMTP proteins. B) Conserved motifs detected by MEME and displayed in different colored boxes Acta agriculturae Slovenica, 120/2 – 20246 Z. HAJIBARAT and A. SAIDI CDFs and Zn-CDFs, respectively, and two DxxxD resi- dues were explored in the Mn-CDF subgroups (Fig 3). 3.4 POTENTIAL MicroRNA TARGET SITES IN StMTP GENES MicroRNA (miRNAs) are small non-coding RNA molecules that can play key roles in gene expression (Zhang and Chen, 2013). With the expectation score lower than 3.0, a total of 13 StmiRNAs comprising target sites in three StMTP genes were detected (Table 3). Two members of group 1 can be targeted by stu-miR7992-3p. Moreover, StMTP5 was targeted by stu-miR5303g, stu- miR5303i, stu-miR5303h, stu-miR5303j, stu-miR156e, stu-miR156f-5p/g-5p/h-5p/i-5p/j-5p/k-5p, and stu- miR5303f. All identified miRNAs-targeted StMTP genes were predicted to be silenced by cleavage inhibition. Giv- en that miRNA regulate a large section of mRNA tran- scripts, resulting nearly all biological events are affected by miRNAs (Bartel, 2009). The findings showed that the UPE ranged from 18.379 (stu-miR7992-3p/ StMTP3) to 23.914 (stu-miR5303f/ StMTP5) (Table 3). 3.5 ANALYSIS OF THE TFBS IN THE PROMOTER REGIONS OF STMTP GENES TF binding sites (TFBS), regions of DNA binding sites in promoter, are important in transcription initia- tion of its target genes (Yu et al., 2016). As shown in Ta- ble 4, 7 TFBS groups, containing elements associated to biotic and abiotic stresses, light response, developmental response, cell cycle, basic transcription, phytohormo- nal response, and other binding sites were annotated. Among the more common TFBS, MYB and bZIP ap- peared to be the most frequent elements (with 1046 and 606 numbers, respectively), and were commonly estab- lished by all StMTP genes. Notably, elements involved in light control was distributed in the promoter regions of all StMTP genes. While, elements involved in hormone responsiveness were less abundant than the others (Table 4), it appears that the presence of these elements are an indication that StMTP genes could be transcriptionally regulated by different hormones (Table 4). Table 2: The sequences and the Pfam annotations of conserved motifs in StMTP proteins Motif ID Motif sequence Length Pfam Motif 1 YCRSFGNEIVRAYAQDHFFDVVTNVVGLVAAVLADRFYWWIDPVGAIIJALYTISTWSGT 60 Cation-efflux, Pfam, PF01545 Motif 2 AIIASTLDSLLDLLSGFILWFTSLAMKSPNQYKYPIGKKRMQP 43 No motif was found in Pfam Motif 3 KHIDTVRAYTFGVLYFVEVDIVLPEDMPLKEAHNIGETLQEKLEQLPEVERAFVHJDFEC 60 ZT_dimer, PF16916 Motif 4 EKKKKQRNINVQGAYLHVLGDCIQSIGVMIGGAIIWYKPEWKIIDLICTLIFSVIVLATT 60 Cation-efflux, Pfam, PF01545 Motif 5 VLENVVSLIGRSAPPEFLQKLTYLVWNHH 29 No motif was found in Pfam Motif 6 SERIAIHISNIANVVLFIAKVYASVKSGSL 30 No motif was found in Pfam Motif 7 BESHPKMTKEQEKWLIGIMVSVTVVKFVLW 30 No motif was found in Pfam Motif 8 SYGYFRJEILGALVSIQMIWLLAGILVYEAIARLIHDTGEVKGFLM 46 Cation-efflux, Pfam, PF01545 Motif 9 LCEMEEVVAIHELHIWAITVGKVLLACHVKIKPDADADMVLDKVVDYIRREYNISHVTIQ 60 No motif was found in Pfam Motif 10 VGIIVFASVMATLGLQILFES 21 No motif was found in Pfam Acta agriculturae Slovenica, 120/2 – 2024 7 Identification of metal tolerance proteins (MTP) and their gene expression under drought stress in potato (Solanum tuberosum L.) Figure 3: Multiple sequence alignment of StMTP, AtMTP, and OsMTP proteins. The signature sequences and the consensus sequence HXXXD or DXXXD (X = any amino acid) are indicated with black line and open boxes, respectively Acta agriculturae Slovenica, 120/2 – 20248 Z. HAJIBARAT and A. SAIDI Ta bl e 3: Th e po te nt ia l m iR N A ta rg et si te s i n St M TP g en es m iR N A _A cc . Ta rg et _A cc .E xp ec ta tio n U PE m iR N A- le ng th T ar ge t St ar t- En d m iR N A a lig ne d fr ag m en t Ta rg et a lig ne d fr ag m en t In hi bi tio n St M TP 3 st u- m iR 79 92 -3 p St M TP 3 3 18 .3 79 22 13 42 -1 36 4 U G U C U A G AU G U G C AU U U C A A A G U U C C AU G A AU U G C A C AU U U G G G C G C le av ag e St M TP 1 st u- m iR 79 92 -3 p St M TP 1 3 20 .7 77 22 11 01 -1 12 3 U G U C U A G AU G U G C AU U U C A A A G U U C C AU G A AU U G C A C AU U U G G G C G C le av ag e St M TP 5 st u- m iR 53 03 g St M TP 5 1 20 .8 55 23 13 27 8- 13 30 1 AU AU U U U U G A A G A G U C U G A G - C A A C G U U G C U C G G A C U C U U C A A A A AU - G U C le av ag e St M TP 5 st u- m iR 53 03 i St M TP 5 1 20 .8 55 23 13 27 8- 13 30 1 AU AU U U U U G A A G A G U C U G A G - C A A C G U U G C U C G G A C U C U U C A A A A AU - G U C le av ag e St M TP 5 st u- m iR 53 03 h St M TP 5 1. 5 19 .9 62 23 13 27 9- 13 30 2 A A C AU U U U U G A A G A G U C U G A G - C A A U U G C U C G G A C U C U U C A A A A AU - G U C C le av ag e St M TP 5 st u- m iR 53 03 j St M TP 5 2 19 .9 62 23 13 27 9- 13 30 2 A AU AU U U U U G A A G A G U C U G A G - C A A U U G C U C G G A C U C U U C A A A A AU - G U C C le av ag e St M TP 5 st u- m iR 15 6e St M TP 5 3 19 .5 33 19 16 07 1- 16 09 0 U G A C A G A A G A G A G U G A G C A C A A G C C U A C U C U U U U C U G U C A C le av ag e St M TP 5 st u- m iR 15 6f -5 p St M TP 5 3 18 .5 72 19 16 07 2- 16 09 1 C U G A C A G A A G A G A G U G A G C A A G C C U A C U C U U U U C U G U C A C C le av ag e St M TP 5 st u- m iR 15 6g -5 p St M TP 5 3 19 .5 33 19 16 07 1- 16 09 0 U G A C A G A A G A G A G U G A G C A C A A G C C U A C U C U U U U C U G U C A C le av ag e St M TP 5 st u- m iR 15 6h -5 p St M TP 5 3 19 .5 33 19 16 07 1- 16 09 0 U G A C A G A A G A G A G U G A G C A C A A G C C U A C U C U U U U C U G U C A C le av ag e St M TP 5 st u- m iR 15 6i -5 p St M TP 5 3 19 .5 33 19 16 07 1- 16 09 0 U G A C A G A A G A G A G U G A G C A C A A G C C U A C U C U U U U C U G U C A C le av ag e St M TP 5 st u- m iR 15 6j -5 p St M TP 5 3 19 .5 33 19 16 07 1- 16 09 0 U G A C A G A A G A G A G U G A G C A C A A G C C U A C U C U U U U C U G U C A C le av ag e St M TP 5 st u- m iR 15 6k -5 p St M TP 5 3 19 .5 33 19 16 07 1- 16 09 0 U G A C A G A A G A G A G U G A G C A C A A G C C U A C U C U U U U C U G U C A C le av ag e St M TP 5 st u- m iR 53 03 f St M TP 5 3 23 .9 14 23 13 30 8- 13 33 1 AU U U U U G G A G A AU C U - G A C A C G G G U G U G C AU G G C G A AU U C U C C A A A A- G U C le av ag e Acta agriculturae Slovenica, 120/2 – 2024 9 Identification of metal tolerance proteins (MTP) and their gene expression under drought stress in potato (Solanum tuberosum L.) Ta bl e 4: S um m ar y of th e tr an sc rip tio n fa ct or b in di ng si te s ( TF BS ) d et ec te d in th e pr om ot er re gi on s o f S tM TP g en es TF BS re la te d to h or m on e/ tis su e- sp ec ifi c/ st re ss re sp on se /b in di ng si te N am e of TF BS St M TP 5 St M TP 3 St M TP 1 St M TP 6 St M TP 8 St M TP 8. 1S tM TP 9. 1 St M TP 9. 2S tM TP 9 St M TP 11 St M TP 4 St M TP 7E xp ec te d fu nc tio n Tf s r el at ed to h or m on e re sp on se A P2 87 92 78 51 53 14 5 19 15 39 28 37 Et hy le ne -r es po ns iv e el em en t Tf s r el at ed to h or m on e re sp on se BB R- BP C 6 2 0 0 0 0 0 0 0 0 0 0 C yt ok in in -r es po ns iv e el em en t Tf s r el at ed to h or m on e re sp on se BE S1 4 3 0 2 0 0 0 1 2 0 0 2 S tr ig ol ac to ne a nd Br as sin os te ro id s -r e- sp on siv e el em en t Tf s r el at ed to h or m on e re sp on se A RF 2 1 0 0 0 0 0 0 0 0 0 0 A ux in -r es po ns iv e el e- m en t Tf s r el at ed to h or m on e re sp on se EI N 3 ; E IL 8 3 7 5 4 2 0 0 0 0 3 0 In vo lv ed in e th yl en e an d JA si gn al in g Tf s r el at ed to h or m on e re sp on se V O Z 9 3 0 0 0 0 0 0 0 0 1 0 G ib be re lli n -r es po ns iv e el em en t TF s r el at ed to li gh t re sp on se bH LH 28 17 8 36 36 14 5 37 38 7 34 34 Li gh t- re sp on siv e e le m en t TF s r el at ed to li gh t re sp on se D of 36 54 51 16 11 12 9 13 15 13 11 15 Li gh t- re sp on siv e e le m en t TF s r el at ed to li gh t re sp on se G AT A 30 26 26 17 18 13 4 7 15 13 13 11 Li gh t- re sp on siv e e le m en t TF s r el at ed to ti ss ue - sp ec ifi c l oc al isa tio n AT -H oo k 30 18 18 29 19 21 3 13 27 23 11 26 Va sc ul at ur e- sp ec ifi c ex pr es sio n TF s r el at ed to ti ss ue - sp ec ifi c l oc al isa tio n SB P 26 5 5 21 19 2 2 18 13 16 3 17 In vo lv ed in fl ow er a nd fr ui t d ev el op m en t TF s r el at ed to ti ss ue - sp ec ifi c l oc al isa tio n LO B 1 0 0 0 0 0 0 0 0 0 0 0 In vo lv ed in la te ra l o rg an de ve lo pm en t TF s r el at ed to ti ss ue - sp ec ifi c l oc al isa tio n M A D S bo x 8 3 0 0 0 0 0 0 3 0 0 8 In vo lv ed in fl ow er in g de ve lo pm en t TF s r el at ed to ti ss ue - sp ec ifi c l oc al isa tio n M A D F 16 0 0 0 0 0 0 3 1 0 1 1 In vo lv ed in fl ow er a nd fr ui t d ev el op m en t Co nt in ue d on th e n ex t p ag e Acta agriculturae Slovenica, 120/2 – 202410 Z. HAJIBARAT and A. SAIDI TF s r el at ed to ti ss ue - sp ec ifi c l oc al isa tio n TC R 14 0 6 4 3 4 0 4 0 0 4 0 In vo lv ed in d ev el op - m en t m al e an d fe m al e re pr od uc tiv e tis su es / tis su e- sp ec ifi c e xp re s- sio ns TF s r el at ed to ti ss ue - sp ec ifi c l oc al isa tio n W O X 9 0 2 0 0 1 0 1 0 0 1 1 Ti ss ue -s pe ci fic e xp re s- sio ns TF s r el at ed to ce ll cy cl e E2 F/ D P 3 1 0 1 0 0 0 0 0 2 0 0 In vo lv ed in ce ll po lifi ca - tio n TF s r el at ed to st re ss re sp on se M YB 25 9 21 4 16 5 70 58 37 9 47 46 49 32 60 re sp on siv e to e nv iro n- m en ta l s tr es s TF s r el at ed to st re ss re sp on se W RK Y 10 6 55 3 33 27 34 2 13 20 23 33 27 in vo lv ed in d ev el op m en - ta l a nd p hy sio lo gi ca l pr oc es se s TF s r el at ed to st re ss re sp on se H SF 20 2 2 2 0 3 0 0 0 0 0 20 In vo lv ed in ce ll di ffe re nt ia tio n, a nd pr ol ife ra tio n TF s r el at ed to st re ss re sp on se C 2H 2 50 37 37 14 11 9 4 9 7 9 11 9 re sp on siv e to st re ss a nd th e ho rm on e sig na l tr an sd uc tio n Tf s r el at ed to b as ic tr an sc rip tio n N F- Y 7 2 3 2 1 1 4 1 2 2 3 3 In vo lv ed in tr an sc rip - tio n by r ec og ni zi ng a nd bi nd in g to a C C A AT m ot if in p ro m ot er s ot he r t fs b in di ng si te s W RC ;G RF 4 0 0 0 0 1 1 1 1 0 0 0 In vo lv ed in st em a nd le af de ve lo pm en t ot he r t fs b in di ng si te s So x 7 2 4 4 0 2 0 2 3 3 0 3 In vo lv ed in ce ll cy cl e re gu la tio n ot he r t fs b in di ng si te s FA R1 1 0 0 0 1 0 0 1 0 0 0 0 Li gh t- re sp on siv e e le m en t ot he r t fs b in di ng si te s SR S 4 0 2 4 0 0 0 0 0 2 2 2 In vo lv ed in st yl e an d st ig m a de ve lo pm en t ot he r t fs b in di ng si te s N A C 17 5 75 16 7 18 6 0 6 6 12 2 11 In vo lv ed in d ev el op m en - ta l p ro ce ss a nd st re ss re sp on se s ot he r t fs b in di ng si te s bZ IP 12 1 87 66 51 53 26 5 34 48 56 22 37 D ev el op m en ta l a nd ph ys io lo gi ca l p ro ce ss es Co nt in ue d on th e n ex t p ag e Acta agriculturae Slovenica, 120/2 – 2024 11 Identification of metal tolerance proteins (MTP) and their gene expression under drought stress in potato (Solanum tuberosum L.) 3.6 EXPRESSION PATTERNS OF STMTP GENES UNDER DROUGHT STRESS AND TISSUE- SPECIFIC ANALYSIS 3.6.1 The expression patterns of StMTP under drought stress To better understand the expression of StMTP genes under the influence of drought stress, two StMTP genes were selected and their expression levels were checked by qPCR in leaves and tubers under stress. The expres- sion levels of drought and normal treatments are given in Figure 1. The analysis results showed that StMTP8 and StMTP9 showed the highest level of expression in leaf and tuber (natural) under normal treatment, while both genes decreased under drought conditions. In leaves and tubers, the expression level of StMTP9 was higher than StMTP8 in both leaves and tubers (normal) (Fig 4a). 3.6.2 The expression patterns of StMTP in tissue-spe- cific The tissue expression patterns of StMTPs were in- vestigated based on the qPCR data. As shown in Fig 1B and C, both genes (StMTP8 and StMTP9) were expressed in the four determined tissues. The results of qPCR analysis revealed that the StMTP9 gene significantly had higher expression levels as compared with StMTP8 in all tissues such as root, stem, leaf, and tuber. StMTP9 and StMTP8 genes exhibited maximum levels of gene expres- sion in leaf whereas, the minimum levels had in tuber. Moreover, the high expression of StMTP9 gene was ob- served in the stem (Fig 4b,c). 4 DISCUSSION In the present study, a total of 12 StMTPs were de- tected in potato. The MTPs were named based on the sequence similarities and orthologous relationships be- tween them and the AtMTPs. First, the phylogenetic re- lationships of the MTP proteins between S.tuberosum, A. thaliana, and O.sativa were assessed. Based on previous studies, A.thaliana included 12 MTPs (AtMTP1-12). Contrasted with Arabidopsis, S.tuberosum genome car- ried multiple MTP homologs for each AtMTP, but the homologs for AtMTP2 and AtMTP3 were absent. There were two, four, and six StMTP genes belonging to Fe/Zn- CDFs, Zn-CDFs, and Mn-CDFs, respectively. It is estab- lished that phylogenetic relationships can be utilized to infer structure and functional roles among species (Va- tansever et al. 2017). This finding could provide clues to ot he r t fs b in di ng si te s H om eo do - m ai n 69 42 56 30 30 31 1 27 26 36 29 27 In vo lv ed in ce ll fa te a nd di ffe re nt ia tio n ot he r t fs b in di ng si te s St or e- ke ep er 4 0 0 1 0 0 0 0 0 2 0 1 In vo lv ed in su cr os e in du ci bl e ex pr es sio n of pa ta tin g en e ot he r t fs b in di ng si te s B3 37 28 13 21 18 12 9 13 13 21 7 20 In vo lv ed in d ev el op m en - ta l p ro ce ss ot he r t fs b in di ng si te s Tr ih el ix 24 6 0 0 0 0 0 0 0 0 3 1 Ti ss ue -s pe ci fic e xp re s- sio ns ot he r t fs b in di ng si te s TC P 22 5 0 14 8 2 6 9 4 23 9 5 In vo lv ed in p la nt m or - ph ol og y ot he r t fs b in di ng si te s ZF -H D 13 13 12 0 0 0 1 0 0 0 0 0 In vo lv ed in sp ik e de ve l- op m en t Acta agriculturae Slovenica, 120/2 – 202412 Z. HAJIBARAT and A. SAIDI Figure 4: A) The qPCR expression of the potato StMTP8 and StMTP9 genes from tuber and leaf samples under drought stress. B, C) The expression of the potato StMTP8 and StMTP9 genes among different tissues in organs such as root, leaf, stem, tuber under control condition. Tuber normal, (TN), Tuber stress (TS), Leaf normal, (LN), Leaf stress (LS) discover the functional characteristics, particularly the substrate-specificities of StMTP proteins. Montanini et al. (2007) identified a modified signature available in the trans-membrane regions of the metal tolerance proteins, and proposed a functional role the conserved group-resi- dues in metal selectivity (Montanini et al., 2007). Further, the signature sequences HxxxD (x = any amino acid) and DxxxD were detected to illustrate the sequence charac- teristics of the both Zn-CDFs and Fe/Zn-CDFs, and Mn- CDFs, respectivey. Features of the StMTP genes including peptide length, MW, Pi, sub-cellular, and TMD localization were analyzed. Our results agree with previous stud- ies in wheat and tobacco (Vatansever et al., 2017; Liu et al., 2019), StMTP proteins were mainly predicted to be localized to vacuole, whereas some others are localized in cellular membrane and nucleus. It is suggested that StMTPs could function as the vacuole-localized cation transporters. Other studies in Arabidopsis revealed that AtMTP1 and AtMTP3 are involved in the transport of excess Zn into vacuoles, regulating cellular Zn hemosta- sis (Kobae et al. 2004; Arrivault et al. 2006). Although all of the StMTPs were identified the cation efflux domain and the modified features, however, some other motifs were not present in some StMTP members. StMTP6 and StMTP8.1 do not possess any TMD, a common signature of membrane proteins, which may have distinct biologi- cal functions and novel roles except other transporters. Besides the transmembrane region, the modified signature sequence between TMDs I and II, and the characteristics C-terminal cation-efflux domain are two structural features of MTP proteins. Our findings re- vealed that all the StMTP proteins included two typical structural characteristics. Further, the signature sequenc- es HxxxD and DxxxD were also detected in associated members of three main substrate-specific groups, which were in accordance with consensus residues. Also, these results provided a precious support for our phylogeny tree. Moreover, ZT-dimer was as molecule of zinc trans- porter that formed a homodimer during activity (Lu and Fu, 2007). The existence of ZT-dimer in specific StMTPs suggested that these proteins could require to organize heterodimers and homodimers when ministering as met- al ion transporters. In this study, the ZT-dimer was iden- Acta agriculturae Slovenica, 120/2 – 2024 13 Identification of metal tolerance proteins (MTP) and their gene expression under drought stress in potato (Solanum tuberosum L.) tified in members of groups 8, 9 (except for StMTP9.2), and 11. Overall, these structure features of StMTP pro- teins were consistant with the structure characteristics of MTP transporters. These results revealed that there are structural similarity of StMTPs within the same groups. The regulatory mechanisms controlling StMTP gene expression were evaluated at two levels, transcriptional and post-transcriptional using TFBS and the microRNA target sites in the promoter regions and the coding se- quences of StMTP genes, respectively. A total of 5312 putative TFBS involved in multiple biological processes and thirteen StmiRNAs were detected. Former studies have revealed that some of these detected miRNAs were implicated in abiotic and biotic stress response. For ex- ample, the expression of stu-miR156e, stu-miR5303f, stu-miR5303g, stu-miR5303h, and stu-miR5303j would be up-regulated under late blight infection in potato (Ku- mar et al., 2018). In addition, mir156 possesses various functional roles in response to heat, cold, drought, and hypoxia (Stief et al., 2014). Stu-miR7992-3p was up-reg- ulated in defense-related miRNAs to virus (Kondhare et al., 2018). Stu-miR5303g might also respond to Li+ stress through regulating their target genes (Kwenda et al., 2016). Thus, it would be of interest to discover the func- tions of StMTP genes in this biological and physiological processes in latter studies. The importance of StMTP function in potato growth and developmental stages could be identified through tissue expression profile analysis. For example, StMTP8 is highly expressed in leaf, an indication that it might be vital for potato leaf development. On the other hand, the expression levels of StMTP8 was most abundant in all three types of leaf structures during leaf formation, indicating it might be involved in regulating leaf devel- opment. StMTP9 was exclusively expressed in leaf and stem, indicating they have important roles in leaf and stem growth and development stages in potato. StMTP9, was slightly expressed in root and tuber, demonstrated a non-significant role in root growth and development. Also, StMTP8 was not or rarely induced in the evaluated root and tubers. In Populus trichocarpa, PtrMTP9 is ex- pressed in roots and is sharply up-regulated by excess Fe (Gao et al., 2020). Earlier study has shown that OsMTP9 knockout significantly decreased Mn uptake and root-to- shoot translocation (Sasaki et al., 2016). Drought treatment expression analysis showed that the StMTP8 and StMTP9 genes were down-regu- lated. Previous studies suggested that both AtMTP9 and VvMTP9 share two identical orthologues in potato, which StMTP9 is expected to be down-regulated in response to drought stress (Shirazi et al., 2019). However, both genes (AtMTP9 and VvMTP9) were upregulated in response to drought, salinity, osmotic shock, and hormonal stresses. AtMTP8 is expressed in root while it is orthologues with StMTP8 in potato. StMTP8 gene is expressed in leaves whereas, it is slightly expressed in roots, stem, and tubers. AtMTP 1 and AtMTP3 were up-regulated in response to ABA, but were down-regulated in response to biotic and abiotic stresses. Additionally, AtMTP2 was down-reg- ulated in response to all stresses. Other AtMTPs varied with respect to expression, up and down regulation. This result showed that MTP genes have diverse roles in adap- tation of plants under various stresses. Using analysis of each StMTP genes promoter re- gions, different elements were identified that may regu- late gene expression in developmental stages and drought stress in potato. In cucumber, MTP8 is a Mn transporter which maintain Mn homeostasis in root. CsMTP7 is con- stitutively induced in all cucumber tissues during plant development, a putative Fe/Zn transporter (Migocka et al., 2014). OsMTP1 was widely expressed in mature leaves and stems. Analysis of expression profiles revealed that StMTP might be involved in several aspects of pota- to development, and also be significant in leaf and shoot development. Further, StMTP genes may play significant roles drought and abiotic stress. The expression profiles of the MTP genes under drought stress could reflect differences in the type and number of TFBS in the promoter region of the genes. As a result, different genes can respond to various stresses (Vatansever et al. 2017; Saidi et al., 2020a, b). The MYB and bZIP were two common TFBS found in the upstream regions of StMTP genes at a high frequency. MYB plays a key role in plants under metal stress. In Arabidopsis, MYB4 is induced following exposure to Cd and Zn while MYB43, MYB48, and MYB124, member of MYB family were found to be particularly expressed in roots in response to Cd stress. MYB28 is as another member of MYB family which is induced after Cd-stress. Moreover, MYB, bZIP, AP2 play vital role in regulating the specific response of plants under Cd stress through modulating the particular responsive genes (Wu et al., 2012; Hajibarat and Saidi, 2022 a, Hajibarat et al., 2022b). BZIP has been identified as one of the most TFBS in Arabdiopsis, bean, sesame, and wheat, involved in adapt- ing to zinc deficiency through inducing the expression of members of membrane transporters (ZIPs) (Wang et al., 2018; Saidi et al., 2020a, b). In the current study, diverse MTPs revealed drought responses to stress conditions, the lowest level expression was observed for StMTP9 in response to drought stress. These results did not agree with our findings in Arabi- opsis. Previous studies showed that some genes were up/ down-regulated with similar stress with corresponding genes to same group (Li et al., 2018; Shirazi et al., 2019; Saidi and Hajibarat, 2019). AtMTP9, 10, 11 were up-reg- Acta agriculturae Slovenica, 120/2 – 202414 Z. HAJIBARAT and A. SAIDI ulated in plants exposed to drought, cold and salt stresses, regulating gene expression and functional proteins to en- hance stress tolerance. In addition, drought stress lead to changes in plant metals concentration in Brachypodium (Chen et al., 2018). In general, our findings could provide significant evidence for highlighting the metal transport mechanism mediated by StMTP proteins in growth and developmental stages and drought stress. 5 CONCLUSION Twelve MTPs in S. tuberosum were identified in the current study. Using bioinformatics tools, comprehen- sive analysis of StMTP genes were performed includ- ing protein properties, analysis of TFBS and structure, MicroRNA analysis, and analysis of gene expression in developmental and growth and drought stress. Based on phylogenic study, StMTPs were clustered into three sub- families and seven groups (1, 5, 6, 7, 8, 9, and 11), similar to the MTP genes in Arabidopsis, rice, and tobacco. The MTP genes may have apparently been underwent gene loss and expansion via tandem duplication after poly- ploidization. All StMTPs contained cation-efflux and signature sequence, while, few of them also possess the ZT-dimer. 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