Evolutionary analysis and expression profiling of the sweet sugar transporter gene family in cassava (manihot esculenta crantz)

91 HNUE JOURNAL OF SCIENCE DOI: 10.18173/2354-1059.2017-59 Chemical and Biological Science 2017, Vol. 62, Issue 10, pp. 91-99 This paper is available online at EVOLUTIONARY ANALYSIS AND EXPRESSION PROFILING OF THE SWEET SUGAR TRANSPORTER GENE FAMILY IN CASSAVA (Manihot esculenta Crantz) Chu Duc Ha 1 , Le Xuan Dac 2 , Tran Thi Thanh Huyen 3 , Pham Thi Ly Thu 1 1 Agricultural Genetics Institute, Vietnam Academy of Agricultural Sciences 2 Institute of Tropical Ecology, Vietnam-Russia Tropical Center 3 Faculty of Biology, Hanoi National University of Education Abstract. SWEETs play important roles in various biological processes, including plant growth, development, and response to environmental stimuli. Previously, we comprehensively found a total of 28 members of MeSWEET gene family in cassava (Manihot esculenta Crantz). Most of MeSWEET genes had 6 exons and 5 introns. In this study, 6 MeSWEET duplicated pairs, including 5 segmental and 1 tandem duplication events were manually predicted in cassava genome. Promoter analysis showed that most members of SWEET genes are responsive to stress conditions. The distributions of various hormone- and/or stress- responsive cis- regulatory elements in the promoter regions of MeSWEET genes clearly indicated their involvement in stress response and plant signaling. Finally, expression analysis of SWEET genes in 4 types of tissues were performed based on the available RNA-seq data. MeSWEET7 was likely to expressed in FEC (friable embryogenic callus) and OES (organized embryogenic structure), while MeSWEET18 was strongly expressed in RAM (root apical meristem). Both of 2 genes, MeSWEET26 and MeSWEET27 were recorded to be expressed in RAM and SAM (shoot apical meristem). Our study was valuable evidences for further functional studies of SWEET sucrose transporters in cassava. Keywords: Cassava, sucrose transporter, in silico, evolution, expression profile. 1. Introduction Sucrose, one of the most important carbohydrates, was found in only oxygenic photosynthetic organisms [1]. In plants, the transportation of sucrose is considered to 2 functional protein systems, sucrose transporters/sucrose carriers (SUTs/SUCs) and SWEETs (Sugars Will Eventually be Exported Transporters). Of our interest, SWEET family has significant roles in transporting glucose across a membrane along a concentration gradient, therefore be considered to multiple biological processes, such as the signaling pathway [2], pollen nutrition [3] and pathogen susceptibility [2]. For example, Received May 26, 2017. Revised September 7, 2017. Accepted September 14, 2017. Contact Chu Duc Ha, e-mail address: hachu_amser@yahoo.com Chu Duc Ha, Le Xuan Dac, Tran Thi Thanh Huyen, Pham Thi Ly Thu 92 Arabidopsis thaliana AtSWEET8 was shown to be essential for pollen viability [4], while AtSWEET17 was recorded to control fructose content in leaves [5]. In rice (Oryza sativa), OsSWEET11 and OsSWEET14 are targets of disease-causing microbes, which switch sugars in plant for pathogen use [6]. Thus, understanding of SWEET family could get insight into the sucrose metabolite and signaling pathway in plants. To date, SWEET genes have been well established in A. thaliana [7, 8] and in several important crops, such as rice [9], sweet orange (Citrus sinensis) [10], tomato (Solanum lycopersicum) [11], soybean (Glycine max) [12]. Recently, SWEET gene family was also identified and characterized in oilseed rape (Brassica napus) [13] and sorghum (Sorghum bicolor) [14]. In our first phase, 28 members of MeSWEET gene family were found in the cassava (Manihot esculenta Crantz) genome. These genes were distributed in 13 chromosomes out of 18 ones in cassava. Our structural analysis revealed that most of MeSWEET genes had 6 exons and 5 introns. We also suggested that a majority of MeSWEET proteins may be located on the secretory pathway. Thus, it would be more interesting if we could initially provide the roles of SWEET genes in the regulation of the biological processes and stress responses of cassava plants. Here, we reported our results on the prediction of duplication events in MeSWEET gene family in cassava. The calculation of approximate time of duplication event of SWEET genes was provided. These evolutionary analyses will bring us insight into the expansion of SWEET gene family in cassava. We also analyzed the structures, conserved domains, and phylogenetic relationships of MeSWEET proteins. Next, the presences of cis- regulatory elements in the promoter regions of MeSWEET genes were carried out to imply their roles in the regulation under stress conditions. Finally, we analyzed the expression profiles of MeSWEET genes in various tissues based on previous RNA-seq data. Our study would significantly provide the understanding of SWEET genes in the cassava plants. 2. Content 2.1. Materials and methods * Materials: The cassava genome database of AM560-2 [15] available in the Phytozome v12.0 [16]. * Methods: - Gene duplication analysis and Ka/Ks calculation Genomic sequences of MeSWEET genes were obtained from the cassava genome database [15] based on their identifiers. Gene duplication was manually predicted using pairwise sequence comparison algorithm as previously described [17]. In this study, a pair of duplicated genes was defined as sharing > 50% homology at nucleotide level. The number of nonsynonymous substitutions per nonsynonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks) of duplicated genes were determined by using DnaSP v5.10.1 software [18] as previous report [19]. Ka/Ks < 1.0 indicates the possibility of natural selection associated with the limitation of deleterious mutations, whereas Ks/Ks > 1.0 reveals that natural selection has positively accelerated evolution by changing the protein [20, 21]. Evolutionary analysis and expression profiling of the sweet sugar transporter gene family 93 - Prediction of cis- regulatory elements The promoter regions (1 kb upstream from start codon site) of SWEET genes were collected from the cassava genome database [15]. The presence of cis-regulatory elements were predicted through PlantCARE web- based tool [22]. - Expression analysis of the MeSWEET genes Expression data available for MeSWEET genes were retrieved from the publicly microarray database (GEO accession: GSE82279) [23]. Following the criteria of identification of uniquely expressed genes, FPKM values of 1 and 10 were represented 'below the limit of detection' and 'expressed', respectively [23]. 2.2. Results and discussions 2.2.1. Gene duplication event in MeSWEET gene family in cassava genome To understand how 28 MeSWEET genes were amplified in the genome during the evolution, the gene duplication events were firstly analyzed by using pairwise algorithm. In this study, two genes shared > 50% homology at nucleotide level were considered as a duplicated pair. As shown in Table 1 and Figure 1, 6 duplicated pairs of SWEET genes were found in cassava genome. Table 1. The gene duplication found in SWEET gene family in cassava Duplicated pair Duplication event Homology Level Ks value Ka value Ka/Ks ratio MeSWEET5 (Manes.02G174100) MeSWEET28 (Manes.18G086400) Segmental 58.0 0.42 0.37 0.87 MeSWEET10 (Manes.06G123500) MeSWEET21 (Manes.14G047600) Segmental 55.1 1.47 1.38 0.94 MeSWEET11 (Manes.06G123400) MeSWEET22 (Manes.14G047700) Segmental 70.1 0.92 0.93 1.02 MeSWEET12 (Manes.06G123300) MeSWEET13 (Manes.06G123200) Tandemly 68.1 0.14 0.14 0.99 MeSWEET16 (Manes.08G116200) MeSWEET18 (Manes.09G172000) Segmental 56.5 0.37 0.37 1.00 MeSWEET19 (Manes.12G006700) MeSWEET20 (Manes.13G006800) Segmental 59.3 0.31 0.26 0.87 The Ks value represents the number of non-synonymous substitution per non- synonymous site. The Ka value represents the number of synonymous substitution per synonymous site. The homologous identities of duplicated MeSWEET genes were ranged from 55.1 % (MeSWEET10/MeSWEET21) to 70.1 % (MeSWEET11/MeSWEET22) (Table 1). Five segmental pairs were found for the following MeSWEET genes: MeSWEET5/MeSWEET28, MeSWEET10/ MeSWEET21, MeSWEET11/MeSWEET22, MeSWEET16/MeSWEET18 and MeSWEET19/MeSWEET20; only one tandemly Chu Duc Ha, Le Xuan Dac, Tran Thi Thanh Huyen, Pham Thi Ly Thu 94 duplicated pair (MeSWEET12/MeSWEET13) was identified in cassava genome. Recently, 21 SWEET sister pairs were phylogenetically identified in soybean, while 23 BnSWEET gene pairs (including 2 tandem duplication events) were shown in oilseed rape [13]. Although the criterion of determining two duplicated genes were slightly different, but these comparisons suggested that the segmental duplication events are the major reason for the expansion of the SWEET gene family in cassava. Figure 1. The chromosomal locations of MeSWEET genes Next, the nonsynonymous substitution rate (Ka), the synonymous substitution rate (Ks) and their ratio (Ka/Ks) were generally used to aid in understanding the direction of evolution and the selective pressures on genes [24]. As provided in Table 1, the Ka/Ks ratio of 3 duplicated pairs (MeSWEET19/MeSWEET20, MeSWEET10/MeSWEET21 and MeSWEET5/MeSWEET28) were less than 1.0, suggesting that the purifying selection was appeared to maintain the preservation of SWEET protein as is. During the evolution of SWEET genes in cassava, the differences caused by deleterious mutation between these duplicated SWEET proteins were eliminated in most the time selection of the evolution. Additionally, the Ka/Ks ratios of 3 pairs (MeSWEET16/MeSWEET18, MeSWEET11/MeSWEET22 and MeSWEET12/MeSWEET13) likely equal to 1.0, showing the neutral (or no) selection on these genes. 2.2.2. The prediction of cis-regulatory elements in the promoter regions of the MeSWEET genes in cassava To investigate the putative function and regulation of SWEET family in cassava, the cis-regulatory elements were determined in the promoter (1000-bp-upstream regions from the start codon site) of each gene. The CAAT-box and TATA-box were recognized in these promoter regions as the milestones to confirm the transcription start site. As provided in Table 2, a number of well-known stress responsive regulatory elements were Evolutionary analysis and expression profiling of the sweet sugar transporter gene family 95 identified in these promoter regions, indicating that SWEET genes may be involved in stress responses. Table 2. The presence of well-known cis-regulatory elements in the promoter region of MeSWEET genes Gene name The presences of well-known stress responsive cis-regulatory elements ABRE CACGTG HSE RAAAATTYS TC-rich repeats ATTTTCTTCA MBS TGACCG LTRE GCCGAC DRE CCGAC MeSWEET1 (+) (+) MeSWEET2 (+) MeSWEET3 (+) (+) MeSWEET4 (+) MeSWEET5 MeSWEET6 (+) (+) MeSWEET7 (+) MeSWEET8 (+) (+) MeSWEET9 MeSWEET10 (+) MeSWEET11 (+) MeSWEET12 (+) (+) (+) MeSWEET13 (+) MeSWEET14 (+) (+) (+) (+) MeSWEET15 MeSWEET16 (+) (+) MeSWEET17 (+) (+) MeSWEET18 (+) MeSWEET19 (+) (+) MeSWEET20 (+) (+) MeSWEET21 (+) (+) MeSWEET22 (+) MeSWEET23 (+) (+) (+) MeSWEET24 (+) MeSWEET25 (+) (+) MeSWEET26 (+) (+) MeSWEET27 (+) (+) MeSWEET28 (+) Chu Duc Ha, Le Xuan Dac, Tran Thi Thanh Huyen, Pham Thi Ly Thu 96 ABRE: Abscisic acid responsive element; HSE: Heat stress element; MBS: MYB binding site; LTRE: Low temperature responsive element; DRE: Dehydration responsive element; (+): Presence. The promoters of 25 MeSWEET genes contained at least one type of regulatory elements related to the stress responsiveness. For example, ABRE (abscisic acid responsive element) and MBS (MYB binding site) were found in the promoter region of MeSWEET19 (Manes.12G006700), while the distributions of HSE (heat stress element) and ABRE were also recorded on the promoter of 3 genes, MeSWEET6 (Manes.03G197300), MeSWEET8 (Manes.05G067400) and MeSWEET12 (Manes.06G123300). These findings suggested that these genes might be involved in the defense mechanism to heat and drought stress via the ABA-dependent pathway [8]. In contrast, no stress- responsive elements were found in the promoter regions of MeSWEET5 (Manes.02G174100), MeSWEET9 (Manes.06G123600) and MeSWEET15 (Manes.06G157700). Previously, the existences of various stress responsive elements, including TC-rich repeats, MBS, LTRE and HSE were also recorded to be located on the promoter regions of SWEET genes of B. napus [13]. The presences of various stress- responsive elements in the promoter regions of SWEET gene family in cassava indicated that these MeSWEET genes may be associated with the hormonal signaling pathway, and perhaps in the response to the abiotic stresses. Furthermore, their promoters could be used in genetic engineering of cassava plants against the adverse environmental conditions. 2.2.3. Expression profiling of MeSWEET genes in various tissues To understand how MeSWEET genes play roles in cassava, we analyzed their expression profiles using available transcriptome data (GSE82279) [23]. In that study, 4 different samples, including FEC (friable embryogenic callus), OES (organized embryogenic structure), RAM (root apical meristem) and SAM (shoot apical meristem) were isolated from tissues of 3-month-old 'TME 204' cassava plants [23]. The expression profiles of MeSWEET genes were analyzed and provided in Figure 2. Expression patterns of various SWEET genes were found in this study. Among them, the transcript expression profiles of 6 genes, MeSWEET1, MeSWEET5, MeSWEET7, MeSWEET26, MeSWEET27 and MeSWEET28 were calculated and identified in all provided tissues. Interestingly, MeSWEET7 (Manes.03G078800) was likely to expressed in FEC and OES, while MeSWEET18 (Manes.09G172000) was strongly expressed in RAM. Both of 2 genes, MeSWEET26 (Manes.15G011300) and MeSWEET27 (Manes.17G062900) were recorded to be expressed in RAM and SAM. Recently, the expression patterns of GmSWEET genes were also determined by the RNA-seq analysis [12]. Among them, GmSWEET5, GmSWEET10, GmSWEET23 and GmSWEET48 were found to be highly expressed in the seeds, suggesting that these genes might be specific in seed. Additionally, GmSWEET12, GmSWEET 21 and GmSWEET40 also had higher expression in pods [12]. Taken together, our finding strongly suggested that the SWEET genes play a diverse functional role during plant development of cassava. The high expression of MeSWEET genes in specific tissues also shown that these genes might function in the growth and development of tissues. Evolutionary analysis and expression profiling of the sweet sugar transporter gene family 97 Figure 2. Expression patterns of MeSWEET genes in some tissues in cassava plants 3. Conclusion Six MeSWEET duplicated pairs, including 5 segmental and 1 tandemly duplication events were predicted in cassava genome. The analysis of Ka/Ks ratio of duplicated pairs suggested that the purifying selection was appeared to maintain the preservation of SWEET19/20, SWEET10/21 and SWEET5/28 proteins as are, whereas the neutral (or no) selection were likely appeared on SWEET16/18, SWEET11/22 and SWEET12/13 genes. The distributions of many stress responsive elements in the promoter regions of most members of SWEET family suggested that these genes are responsive to stress conditions. Their promoters could be used in genetic engineering of cassava plants against the adverse environmental conditions. Expression analysis of SWEET genes showed that MeSWEET7 was likely to expressed in FEC and OES, while MeSWEET18 was strongly expressed in RAM. Both of 2 genes, MeSWEET26 and MeSWEET27 were recorded to be expressed in RAM and SAM. These genes might be involved in the growth and development of tissues. To conclude, our study significantly contributed to expand our understanding on SWEET sucrose transporters in cassava. These results were of value for both conventional and biotechnological improvement programs. In further studies, the functions of several interest MeSWEET genes will be characterized in order to see how these genes play roles in the sucrose transportation in cassava plants. Chu Duc Ha, Le Xuan Dac, Tran Thi Thanh Huyen, Pham Thi Ly Thu 98 REFERENCES [1] Wind J., Smeekens S., Hanson J., 2010. Sucrose: metabolite and signaling molecule. 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