Identification, structural characterization, and in silico expression analysis of the sucrose transporter ‘SWEET’ gene family in peanut (Arachis hypogaea)

Life ScienceS | Agriculture Vietnam Journal of Science, Technology and Engineering62 September 2020 • Volume 62 Number 3 Introduction Peanut (Arachis hypogaea) is considered to be one of the most important legume crops and is mainly cultivated in tropical and subtropical areas. These legumes provide a good source of protein, monounsaturated fats, and antioxidants [1]. Several peanut by-products such as peanut meal, peanut skin, peanut hull, and peanut vine can be used by the food processing industry and consequently play an essential role in food security [2]. However, peanut production and quality are severely affected by abiotic stress [3]. It has been confirmed that the concentration of soluble sugars (predominantly sucrose) can be boosted when plants are exposed to abiotic stress [4, 5]. of our interest, a group of sucrose transporters, so-called “Sugars Will Eventually be Exported Transporters” or SWEETs, are reported as the functional proteins involved in the translocation of sucrose [6, 7]. Thus, SWEETs regulate numerous biological processes in the growth and development of plants such as nectar secretion, phloem loading and development, and seed filling [7, 8]. Previously, some studies have identified and characterized SWEET genes in many main crops such as rice (Oryza sativa) [9], soybean (Glycine max) [10], sorghum (Sorghum bicolor) [11], rapeseed (Brassica napus) [12], cotton (Gossypium spp.) [13], wheat (Triticum aestivum) [14, 15], and litchi (Litchi chinensis) [16]. Meanwhile, information from the SWEET gene family in peanut is lacking. Therefore, in this study, the SWEET gene family in peanut is identified and characterized based on a bioinformatics approach. Specifically, a comprehensive survey of all putative SWEET genes was conducted in the Identification, structural characterization, and in silico expression analysis of the sucrose transporter ‘SWEET’ gene family in peanut (Arachis hypogaea) Ha Duc Chu1*, Quynh Thi Ngoc Le2, Yen Hai Thi Hoang3, Thu Phuong Pham3, Hong Viet La3, Nguyet Minh Thi Nguyen1, Anh Xuan Duong4, Thao Duc Le1, Linh Hung Le1, Hoi Xuan Pham1 1Agricultural Genetics Institute, Vietnam Academy of Agricultural Sciences 2Faculty of Chemistry and Environment, Thuyloi University 3Faculty of Biology, Agricultural Technology, Hanoi Pedagogical University 2 4Padworth College, United Kingdom Received 2 March 2020; accepted 29 May 2020 *Corresponding author: Email: hachu_amser@yahoo.com Abstract: SWEET (Sugars Will Eventually be Exported Transporter) proteins are well known to play pivotal roles in the growth and development of plants. Here, we report the presence of 43 members of the AhSWEET family in peanut (Arachis hypogaea) and determine their general characteristics including chromosomal localization, gene structure, and numerous physical and chemical features of the proteins. We found that the AhSWEET genes were unevenly distributed among the peanut’s 20 chromosomes. The AhSWEET proteins were hydrophobic with a grand average hydropathicity >0 while a majority of the proteins were basic with isoelectric points >7.0. Additionally, most of the AhSWEET genes contained 6 exons and 5 introns. The expression profiles of the AhSWEET genes were explored based on the previous transcriptome atlas. Interestingly, we found that the AhSWEET genes exhibited differential expression patterns across various organs and tissues during the growth and development of peanut plants. Our study provides a solid foundation of the AhSWEET gene family for further functional characterization of AhSWEET genes in the regulation of peanut growth and development. Keywords: bioinformatics, expression profiles, genome- wide, peanut, sucrose transporter, SWEET. Classification number: 3.1 Doi: 10.31276/VJSTE.62(3).62-67 Life ScienceS | Agriculture Vietnam Journal of Science, Technology and Engineering 63September 2020 • Volume 62 Number 3 peanut genome. Subsequently, the expression profiles of the SWEET genes in various organs were generated based on a previous transcriptome atlas. Materials and methods Materials The latest reference genome, proteome, and transcriptome of the peanut (‘Tifrunner’ cultivar) [17, 18] from the Legume information System [19] and PeanutBase [18] were used as the platforms for our in silico analyses. Methods Identification and annotation of the SWEET genes: to identify SWEET proteins in the peanut, we conducted a study in which the domain of the plant’s SWEET, namely ‘PF03083’ [6, 7], obtained from the Pfam server [20], was acquired to search against the recent peanut assembly (BioProject: PRJNA419393) [17] published in NCBi and the Legume Information System [19]. The identified protein sequences were then subjected to a BlastP search against the proteome of the peanut [17] to obtain their necessary annotated information, which includes coding DNA sequence (CDS), genomic DNA sequence (gDNA), and chromosomal localization. Analysis of characteristics of SWEET proteins: the full- length protein sequences of SWEET proteins were searched against the ExPASy Protparam to obtain general features such as molecular mass, length, instability index, isoelectric point, and grand average of hydropathicity (GRAVY) [21]. An instability index score of 40 indicates potential stability and instability, respectively. GRAVY values of <0 and >0 suggest hydrophilic and hydrophobic characteristics, respectively [21]. Phylogenetic analysis and gene organization of SWEET genes: a neighbour-joining phylogenetic tree comprised of the full-length amino acid sequences of all identified SWEET proteins was constructed with the aid of MEGA (Molecular Evolutionary Genetics Analysis) 7.0 [22] using the following essential criteria: a gap extension penalty of 0.2 and a gap open penalty of 10 [23]. Bootstrapping was performed with 1,000 replications. The exon/intron structure of each SWEET gene was analysed by subjecting the CDS and corresponding gDNA to the GSDS (Gene Structure Display Server) 2.0 tool [24]. Expression profiles of SWEET genes: the PeanutBase database was explored to provide a previous transcriptome atlas of different tissues/organs in the peanut [18]. Particularly, data from seven vegetative plant parts, including vegetative shoot tips, reproductive shoot tips, primary stem leaves, seedling leaves, lateral stem leaves, roots, and nodules were collected. The cluster heatmap for the relative expression of the SWEET gene was visualized in R software with the gplots package [25]. Results and discussion Identification and annotation of the SWEET gene family in peanut To identify all potential members of the SWEET family in the peanut, a comprehensive search of a well-established conserved domain of SWEETs [6, 7] against the newest peanut database [17] was performed. As a result, a total of 43 members of the SWEET family were found in the peanut. The annotation of these identified proteins, including protein identifiers and locus name, are subsequently explored and listed in Table 1. Previously, the SWEET gene family has been reported in several plant species. More specifically, 21 members of the OsSWEET family have been investigated in rice [9], while 52 and 23 SWEET genes have been found in soybean and sorghum, respectively [10, 11]. Recently, it has been reported that the SWEET gene family in rapeseed contained 68 members [12]. in the cotton species, the members of the SWEET gene family varied from 22 to 60 [13]. our results indicated that the number of SWEET genes in plant species is highly variable. Next, to annotate the chromosomal localization of the SWEET genes, we matched their corresponding gDNA sequence to the peanut genome [17]. We found that all members of the SWEET genes were randomly distributed among the 20 chromosomes of the peanut genome and no SWEET gene was localized in unplaced scaffolds (Fig. 1). Among them, chromosome Arahy.13 and Arahy.03 share the highest members of the SWEET family by 6 and 5 genes, respectively (Fig. 1). Additionally, there are 4 SWEET genes found in chromosome Arahy.08, while the chromosomes Arahy.14, 15, 16, 17, and 18 have 3 SWEET genes (Fig. 1). We also found that 2 SWEET genes were mapped on each of chromosomes Arahy.05, 06, and 20, while only 1 SWEET gene was reported in chromosomes Arahy.01, 04, 07, 09, 10, 11, and 19 (Fig. 1). it is also noted that no SWEET gene was localized in chromosomes Arahy.02 and 12 (Fig. 1). The entire 43 SWEET genes set was based on the order of the occurrences on the chromosomes (Table 1, Fig. 1). Life ScienceS | Agriculture Vietnam Journal of Science, Technology and Engineering64 September 2020 • Volume 62 Number 3 Table 1. General information on SWEET gene family in the peanut. # Gene name Protein code Locus code Size MM pI II GRAVY 1 AhSWEET01 XP_025675869.1 LoC112776072 227 25.81 8.82 43.35 0.96 2 AhSWEET02 XP_025689387.1 LoC112790966 279 31.48 8.99 38.23 0.46 3 AhSWEET03 XP_025689047.1 LoC112790726 253 27.92 9.37 46.63 0.56 4 AhSWEET04 XP_025676850.1 LoC112776807 159 18.23 9.74 33.58 0.87 5 AhSWEET05 XP_025691262.1 LoC112792298 293 32.86 8.49 31.46 0.48 6 AhSWEET06 XP_025691265.1 LoC112792300 220 25.04 9.68 28.98 1.05 7 AhSWEET07 XP_029153458.1 LoC112795203 175 20.21 9.83 38.61 0.82 8 AhSWEET08 XP_025697629.1 LoC112799834 250 27.59 9.19 32.87 0.72 9 AhSWEET09 XP_025697627.1 LoC112799832 242 26.85 8.68 37.49 0.80 10 AhSWEET10 XP_025606390.1 LoC112697429 244 26.70 8.91 32.98 0.78 11 AhSWEET11 XP_025603714.1 LoC112695553 312 34.23 9.15 21.70 0.41 12 AhSWEET12 XP_025610780.1 LoC112703520 235 26.20 8.38 44.42 0.84 13 AhSWEET13 XP_025612828.1 LoC112705985 246 27.12 9.30 32.96 0.64 14 AhSWEET14 XP_025612772.1 LoC112705945 262 29.03 9.02 27.45 0.51 15 AhSWEET15 XP_025616058.1 LoC112708127 301 34.32 8.84 51.86 0.10 16 AhSWEET16 XP_025616059.1 LoC112708128 285 32.77 9.05 39.49 0.40 17 AhSWEET17 XP_025616659.1 LoC112708960 320 35.26 8.34 34.39 0.70 18 AhSWEET18 XP_025622395.1 LoC112714914 292 32.46 8.82 33.82 0.66 19 AhSWEET19 XP_025629145.1 LoC112722363 226 25.74 8.82 43.49 0.95 20 AhSWEET20 XP_025641347.1 LoC112736207 261 29.73 6.99 41.81 0.53 21 AhSWEET21 XP_025637471.1 LoC112732876 278 31.37 9.09 38.33 0.48 22 AhSWEET22 XP_025637469.1 LoC112732875 249 28.29 9.71 33.13 0.84 23 AhSWEET23 XP_025636904.1 LoC112732406 253 27.90 9.37 46.29 0.56 24 AhSWEET24 XP_025639618.1 LoC112734493 293 32.90 8.97 32.73 0.48 25 AhSWEET25 XP_025639626.1 LoC112734496 225 25.30 9.66 31.43 1.00 26 AhSWEET26 XP_025650623.1 LoC112745021 274 30.31 8.95 35.74 0.60 27 AhSWEET27 XP_025646602.1 LoC112741728 200 21.71 8.46 30.60 0.58 28 AhSWEET28 XP_025650514.1 LoC112744946 261 29.73 6.99 41.08 0.54 29 AhSWEET29 XP_025655604.1 LoC112750900 248 27.61 8.72 36.50 0.68 30 AhSWEET30 XP_025655423.1 LoC112750786 250 27.76 9.18 33.60 0.71 31 AhSWEET31 XP_025650947.1 LoC112747164 242 26.89 8.84 38.20 0.80 32 AhSWEET32 XP_025659195.1 LoC112755367 268 29.33 9.16 24.33 0.44 33 AhSWEET33 XP_025662459.1 LoC112758096 310 34.17 9.22 25.22 0.36 34 AhSWEET34 XP_025659173.1 LoC112755353 244 26.62 9.22 32.26 0.77 35 AhSWEET35 XP_025667967.1 LoC112766275 262 28.88 9.01 28.76 0.54 36 AhSWEET36 XP_025664711.1 LoC112763193 104 11.33 6.51 38.82 0.65 37 AhSWEET37 XP_025666957.1 LoC112765256 246 26.95 9.30 34.20 0.64 38 AhSWEET38 XP_025672359.1 LoC112771761 235 26.10 7.62 43.28 0.86 39 AhSWEET39 XP_025673457.1 LoC112772695 301 34.38 8.71 57.28 0.10 40 AhSWEET40 XP_025672677.1 LoC112772012 287 33.01 9.05 39.29 0.38 41 AhSWEET41 XP_025679981.1 LoC112779842 281 31.26 8.47 40.94 0.78 42 AhSWEET42 XP_025685723.1 LoC112786568 248 27.49 8.84 36.64 0.81 43 AhSWEET43 XP_025683874.1 LoC112784769 292 32.52 8.81 35.55 0.65 Note: mm: molecular mass (kDa), pI: isoelectric point, II: instability index, GrAVY: grand average of hydropathicity. Life ScienceS | Agriculture Vietnam Journal of Science, Technology and Engineering 65September 2020 • Volume 62 Number 3 Structural analysis of the SWEET gene family in peanut The exon/intron organization of each AhSWEET gene was first analysed in order to gain insight into the AhSWEET gene family. As shown in Fig. 2, the most common motif of the gene structure of the AhSWEET family was 6 exons/5 introns. only AhSWEET41 and AhSWEET36 contained 2 exons/1 intron and 3 exons/2 introns, respectively, while 3 genes, including AhSWEET04, 07, 17 had 4 exons/3 introns (Fig. 2). Our findings were also confirmed by previous studies [10, 12-15, 26]. More specifically, a total of 34 (out of 52) GmSWEETs was recorded to contain 6 exons/5 introns [10], while the majority of BnSWEETs (51 out of 68) also had 6 exons/5 introns [12]. This phenomenon was also reported in other plant species such as cotton [13], wheat [14, 15], and litchi [16]. Taken together, it would be a reliable assumption that the general structure of SWEET genes in higher plant species is 6 exons/5 introns. Next, the full-length protein sequence of each SWEET was used for retrieval from the ExPASY Protparam [21] in order to analyse the general features of the SWEET family in the peanut. The length of the SWEET proteins varied Fig. 1. Chromosomal distribution of AhSWEET genes in peanut. Chromosomal localization of 43 AhSWEET genes was based on the latest physical map described in NCbI and the legume Information System. Fig. 2. Gene structure of AhSWEET gene family. An unrooted neighbour-joining tree was derived from the full-length AhSWeet sequences (left) and exon/intron organization analysis (right). Life ScienceS | Agriculture Vietnam Journal of Science, Technology and Engineering66 September 2020 • Volume 62 Number 3 from 104 (AhSWEET36) to 320 residues (AhSWEET17) with their molecular masses ranging from 11.33 to 35.26 kDa, respectively (Table 1). The pi values of a majority of the SWEET proteins were >7, which revealed that these proteins were basic whereas only AhSWEET36 was acidic (pi=6.51) (Table 1). The two remaining SWEET proteins, AhSWEET20 and 28, were neutral (pI≈7) (Table 1). We also found that 32 SWEET proteins were stable (instability score <40) (Table 1). Furthermore, all 43 SWEET proteins were hydrophobic with a GRAVY value >0 (Table 1). Previously, the characteristics of SWEET proteins have also been investigated in other plant species. For example, the SWEET proteins in rapeseed varied from 56 to 303 residues, while their molecular weight ranged from 6.5 to 33.45 kDa [12]. A total of 63 members (out of 68) of SWEET proteins were basic [12]. Additionally, most of the identified cotton’s SWEET proteins ranged between 180 and 311 residues, while the molecular masses and isoelectric values of these proteins varied from 9.93 to 38.04 kDa and from 5.47 to 10.08, respectively [13]. in wheat, the molecular weights of SWEET proteins ranged from 10.93 to 33.86 kDa, while a majority of members in the SWEET family exhibited pi values >7 (basic) [14, 15]. Recently, the sizes and molecular weights of the LcSWEET proteins have been found to vary from 229 to 300 residues and from 25.6 to 33.6 kDa, respectively, while the pi values ranged from 7.66 to 9.81 [16]. Our findings suggest a diversity of molecular features of SWEETs in the peanut and perhaps in the plant species. Expression profiles of AhSWEET genes in various tissues To understand the expression patterns of the AhSWEET gene family, we visualized the transcriptome data obtained from 7 tissues respectively taken from vegetative shoot tip, reproductive shoot tip, main stem leaf, seedling leaf, lateral stem leaf, root, and nodule [18] by R programming with the gplots package [25]. We found that 17 genes, including AhSWEET03, 04, 07, 10, 14, 17, 18, 20, 21, 23, 31, 34, 35, 36, 39, 41, and 42, had no information on the expression profiles. The expressions of the remaining AhSWEET genes are displayed in Fig. 3. Among them, 11 AhSWEET genes had no changes in the transcriptional levels of the 7 collected tissues (Fig. 3). interestingly, AhSWEET02 was noted to exclusively express in 3 samples of leaves and the reproductive shoot tip, while AhSWEET15 was also strongly induced in lateral stem leaves, seeding leaves, and main stem leaves (Fig. 3). AhSWEET27 was found to be strongly up-regulated in both reproductive and vegetative shoot tips (Fig. 3). in some cases, the AhSWEET genes were down-regulated in organs/ tissues during the growth and development of the peanut plants. For example, AhSWEET13 and 37 were recorded to be strongly reduced in lateral stem leaves, seeding leaves, and main stem leaves (Fig. 3). Taken together, the AhSWEET genes displayed differential transcription patterns in the investigated organs. our results suggest that AhSWEET proteins might have diverse functions in controlling the development of various organs in peanut plants. Conclusions in this study, 43 AhSWEET genes have been identified in the peanut genome. Structural analyses revealed that the AhSWEET proteins were highly variable. our expression re-analysis showed that the AhSWEET genes displayed differential expression levels in various organs. Two genes, AhSWEET02 and 15, were noted to strongly express in leaves and AhSWEET27 was strongly induced in shoot tips, which indicate that these genes might play crucial roles Fig. 3. Expression profiles of the AhSWEET genes in various tissues. the heat map was generated using r software with the gplots package. the detailed microarray data were obtained from the peanut gene atlas database. Life ScienceS | Agriculture Vietnam Journal of Science, Technology and Engineering 67September 2020 • Volume 62 Number 3 in these organs during the growth and development of the peanut. The authors declare that there is no conflict of interest regarding the publication of this article. REFERENCES [1] o.T. Toomer (2018), “Nutritional chemistry of the peanut (Arachis hypogaea)”, Crit. Rev. Food Sci. Nutr., 58(17), pp.3042- 3053. [2] X. Zhao, J. Chen, F. Du (2012), “Potential use of peanut by- products in food processing: a review”, J. Food Sci. Technol., 49(5), pp.521-529. [3] D.M. Kambiranda, et al. (2011), “impact of drought stress on peanut (Arachis hypogaea L.) productivity and food safety”, Plants Environ., Tech Publisher, pp.249-272. [4] P.V. Minorsky (2003), “Raffinose oligosaccharides”, Plant Physiol., 131(3), pp.1159-1160. [5] M.R. Morsy, L. Jouve, J.F. Hausman, L. Hoffmann, J.M. Stewart (2007), “Alteration of oxidative and carbohydrate metabolism under abiotic stress in two rice (Oryza sativa L.) genotypes contrasting in chilling tolerance”, J. Plant Physiol., 164(2), pp.157-167. [6] B.T. Julius, K.A. Leach, T.M. Tran, R.A. Mertz, D.M. Braun (2017), “Sugar transporters in plants: new insights and discoveries”, Plant Cell Physiol., 58(9), pp.1442-1460. [7] R.F. Baker, K.A. Leach, D.M. Braun (2012), “SWEET as sugar: new sucrose effluxers in plants”, Mol. Plant, 5(4), pp.766-768. [8] L.Q. Chen (2014), “SWEET sugar transporters for phloem transport and pathogen nutrition”, New Phytol., 201(4), pp.1150-1155. [9] M. Yuan, S. Wang (2013), “Rice MtN3/saliva/SWEET family genes and their homologs in cellular organisms”, Mol. Plant, 6(3), pp.665-674. [10] G. Patil, et al. (2015), “Soybean (Glycine max) SWEET gene family: insights through comparative genomics, transcriptome profiling and whole genome re-sequence analysis”, BMC Genomics, 16, Doi: 10.1186/s12864-015-1730-y. [11] H. Mizuno, S. Kasuga, H. Kawahigashi (2016), “The sorghum SWEET gene family: stem sucrose accumulation as revealed through transcriptome profiling”, Biotechnol. Biofuels, 9, Doi: 10.1186/ s13068-016-0546-6. [12] H. Jian, et al. (2016), “Genome-wide analysis and expression profiling of the SUC and SWEET gene families of sucrose transporters in oilseed rape (Brassica napus L.)”, Front. Plant Sci., 7, Doi: 10.3389/fpls.2016.01464. [13] L. Zhao, et al. (2018), “A genome-wide analysis of SWEET gene family in cotton and their expressions under different stresses”, J. Cotton Res., 1(1), Doi: 10.1186/s42397-018-0007-9. [14] Y. Gao, et al. (2018), “Genome-wide identification of the SWEET gene family in wheat”, Gene, 642, pp.284-292. [15] T. Gautam, et al. (2019), “Further studies on sugar transporter (SWEET) genes in wheat (Triticum aestivum L.)”, Mol. Biol. Reps., 46(2), pp.2327-2353. [16] H. Xie, et al. (2019), “Genome-wide identification and expression analysis of SWEET gene family in Litchi chinensis reveal the involvement of LcSWEET2a/3b in early seed development”, BMC Plant Biol., 19(1), Doi: 10.1186/s12870-019-2120-4. [17] D.J. Bertioli, et al. (2019), “The genome sequence of segmental allotetraploid peanut Arachis hypogaea”, Nat. Genet., 51(5), pp.877-884. [18] J. Clevenger, Y. Chu, B. Scheffler, P. Ozias-Akins (2016), “A developmental transcriptome map for allotetraploid Arachis hypogaea”, Front. Plant Sci., 7, Doi: 10.3389/fpls.2016.01446. [19] S. Dash, et al. (2016), “Legume information system (Legumeinfo.org): a key component of a set of federated data resources for the legume family”, Nucleic Acids Res., 44(D1), pp.1181-1188. [20] S. El-Gebali, et al. (2019), “The pfam protein families database in 2019”, Nucleic Acids Res., 47(D1), pp.427-432. [21] E. Gasteiger, A. Gattiker, C. Hoogland, i. ivanyi, R.D. Appel, A. Bairoch (2003), “ExPASy: the proteomics server for in- depth protein knowledge and analysis”, Nucleic Acids Res., 31(13), pp.3784-3788. [22] S. Kumar, G. Stecher, K. Tamura (2016), “MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets”, Mol. Biol. Evol., 33(7), pp.1870-1874. [23] H.D. Chu, K.H. Nguyen, Y. Watanabe, D.T. Le, T.L.T. Pham, K. Mochida, L.P. Tran (2018), “Identification, structural characterization and gene expression analysis of members of the nuclear factor-Y family in chickpea (Cicer arietinum L.) under dehydration and abscisic acid treatments”, Int. J. Mol. Sci., 19(11), Doi: 10.3390/ijms19113290. [24] B. Hu, J. Jin, A.Y. Guo, H. Zhang, J. Luo, G. Gao (2015), “GSDS 2.0: an upgraded gene feature visualization server”, Bioinformatics, 31(8), pp.1296-1297. [25] Y. Liao, G.K. Smyth, W. Shi (2019), “The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads”, Nucleic Acids Res., 47(8), Doi: 10.1093/nar/gkz114. [26] H. Miao, et al. (2017), “Genome-wide analyses of SWEET family proteins reveal involvement in fruit development and abiotic/ biotic stress responses in banana”, Sci. Rep., 7(1), Doi: 10.1038/ s41598-017-03872-w.