Genome-wide identification and expression profile analysis of SWEET genes in Chinese jujube

Genome-wide identification of SWEET family genes in Chinese jujube

The protein sequences of Arabidopsis SWEETs (Chen et al., 2010) were used as the query sequences to identify the members of the SWEET gene family in Chinese jujube (Ziziphus jujuba Mill.). Based on the obtained Arabidopsis SWEET gene, BLASTP analysis was performed on the jujube genome database (https://www.ncbi.nlm.nih.gov/assembly/GCF_000826755.1) to determine candidate genes. The conservative domain analysis of the candidate members by the Conserved Domain Database (CDD) (Marchler-Bauer et al., 2009) and Pfam (http://pfam.xfam.org/) (Finn et al., 2006) confirmed the existence of MtN3/saliva domain in each member. The chromosomal positions, open reading frames (ORFs), and amino acid sequences were obtained from NCBI database. TMHMM Server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM) and ProtParam in ExPASy were used to analyze the transmembrane region, isoelectric point (PI), and molecular weight of each member.

Gene structure, multi-sequence alignment, and phylogenetic analyses

The exon/intron structure of the gene was analyzed using GSDS (http://gsds.cbi.pku.edu.cn/) (Guo et al., 2007). The conserved motifs of SWEETs protein were analyzed using online MEME tools (https://meme-suite.org/meme/tools/meme). The MEME parameter settings were as follows: the number of motifs was 10, and the length of motifs was 5 to 50.

The amino acid sequences of 17 Arabidopsis thaliana SWEET genes, 20 Malus domestica SWEET genes (Velasco et al., 2010; Wei et al., 2014) and 19 jujube SWEET genes were used to construct an unrooted phylogenetic tree. SWEET proteins from Arabidopsis thaliana and Malus domestica were downloaded from the NCBI (https://www.ncbi.nlm.nih.gov/) (File S4). The amino acid sequences of the members of SWEET gene family were analyzed by ClustalW of MEGA5.2. The parameters of alignment were as follows: gap opening penalty, 10.00; gap extension penalty, 0.20 (both in pairwise alignment and in multiple alignments); protein weight matrix, gonnet; residue-specific penalties, on; hydrophilic penalties, on; gap separation distance, 4; end-gap separation, on; use negative matrix, off; and delay divergent cutoff (%), 30. Phylogenetic trees were constructed by the maximum-likelihood based algorithms of MEGA5.2. The parameters chosen for the constructed trees were as follows: statistical method, maximum-likelihood; test of phylogeny, bootstrap method; number of bootstrap replications, 1,000; substitution types, amino acid; model/method, Jones-Taylor-Thornton (JTT) model; rates among sites, gamma distributed; ML heuristic method, nearest-neighbor-interchange; initial tree for ML, make initial tree automatically (Tamura et al., 2011). The analysis of 19 ZjSWEET genes in Chinese jujube and the construction of the evolutionary tree are the same as above (Figs. 1 and 2).

Cis -element analysis of putative promoters

The 2 Kb base sequences were taken from the NCBI database at the upstream position of the transcription start sites to form the promoter sequences of each ZjSWEET gene. The promoter sequences were input into the PlantCare (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) online tool to analyze their cis-acting elements (Lescot et al., 2002).

Plant materials and treatments

The tissue materials for specific gene expression analysis were obtained from the root, stem, leaf, flower, and fruit of the plants. All samples were collected from three different sampling trees and were repeated three times.

The fruits used to analyze gene expression in different developmental stages were taken from the young fruit stage (Y), early white mature fruit stage (EWM), white mature stage (WM), half-red fruit stage (HR), and full-red fruit stage (FR) of Ziziphus jujuba Mill. ‘Linyilizao’ and ‘Beijingjidanzao’, which were planted in the national jujube germplasm repository in the Shanxi province. Among the different varieties of jujube, ‘Beijingjidanzao’ has a higher sugar content, and ‘Linyilizao’ has a lower sugar content. These trees were planted in the same garden under the same cultivation conditions.

The material used to investigate the response of ZjSWEETs to the abiotic and biotic stresses was the callus of Ziziphus jujuba Mill. Guanyangchangzao. The calluses were placed at 4 °C for cold treatment and at 25 °C for the control. The callus tissues were subjected to 150 mM NaCl and NaHCO₃-NaOH solution (pH 9.5) for salinity and alkaline treatments. All controls were kept in an aqueous solution (Guo et al., 2017; Wang et al., 2020). The treated and control materials were collected at 0 h, 1 h, 6 h, and 24 h after treatment, respectively, and all of the collected samples were immediately stored in liquid nitrogen for next use.

RNA isolation and qRT- PCR analysis

The above materials include jujube fruits at different development stages, callus tissues treated with low temperature, alkali, salt, and their corresponding controls were used for RNA extraction. The total RNA was extracted using the RNA prep Pure Plant Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol, then the DNA in total RNA was removed with DNase I (Tiangen Biotech, Beijing, China). The concentration and purity were checked using the NanoDrop2000 spectrophotometer. First-strand cDNA synthesis with reverse transcriptase (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol using 1 µg of RNA template.

The expression analysis was carried out by quantitative reverse-transcription PCR (qPCR) on the Bio-Rad iQ™5 using TransStart Top Green qPCR SuperMix AQ131 (TransGen Biotech, Beijing, China). The entire reaction system measured 20 µL and was comprised of 10 µL of 2 ×SYBR Premix ExTaq™, 0.4 µL each of 10µM primers, 1 µL diluted cDNA, and 8.2 µL ddH₂O. The conditions were: 3 min at 95 °C, followed by 40 cycles of 5 s at 95 °C, 15 s at 55–62 °C and 15 s at 72 °C. Three biological replicates were performed for analysis. Relative expression levels of ZjSWEETs were calculated according to the 2^−ΔΔCt method (Livak & Schmittgen, 2003). The ZjACT was used as a reference gene (Bu, Zhao & Liu, 2016). The primers were listed in File S6 designed by using Primer 5.

Gel semi-quantification

Gel bands were quantified by using ImageJ software. The ACT from each tissue (root, stem, leaf, flower, fruit) was used as a control to calculate the bands of each gene.

Heatmap construction

The expression profiles of all ZjSWEETs in different developmental stages were taken from the young fruit stage (Y), early white mature fruit stage (EWM), white mature stage (WM), half-red fruit stage (HR), and full-red fruit stage (FR) of Ziziphus jujuba Mill. ‘Linyilizao’ and ‘Beijingjidanzao’ are illustrated by a colour gradient heatmap. The heatmap was constructed by TBtools (Chen et al., 2020), and cluster rows were selected.

The expression profiles of all ZjSWEETs in response to low temperature, alkaline, and salt stresses are illustrated by a colour gradient heatmap. The heatmap was constructed by TBtools. The values shown in the heat map were: experimental group/ 0 h calluses.

Genome-wide identification of ZjSWEETs in Chinese jujube

ZjSWEET genes were excavated based on the sequenced genomes of ‘Dongzao’. The homology search and conservative domain analysis were combined to identify SWEET genes in the jujube genome. A total of 19 SWEET genes were found. The members of the jujube SWEET gene family were named ZjSWEET1-19 referring to Arabidopsis thaliana. Their amino acid length, protein molecular weight, isoelectric point, and chromosome localization were forecasted and analyzed (Table 1). The amino acid length (aa) encoded by the members of the SWEET gene family in jujube ranged from 163 to 292, of which 15 genes encode more than 200 amino acids (Table 1). The molecular weight of SWEET protein varied from 18.2 to 33 kDa, and the range of protein isoelectric point varied from 4.88 to 9.84. The chromosome locations of the SWEET genes in jujube were analyzed and were found to be distributed on chromosomes 1, 2, 3, 4, 5, 7, 11 and 12 of the jujube genome, while 6 ZjSWEETs (2, 9, 10, 11, 16, 17) were not localized in the assembled regions.

Gene structure and motif analysis

In order to further understand the biological function of the members of the SWEET gene family, the number and location of introns and exons of the 19 genes were analyzed using the online GSDS (Fig. 1). The comparative analysis of the coding sequence and genome sequence of jujube SWEET family members showed that all the gene sequences of the members contained introns. Most ZjSWEETs contained four to five introns; ZjSWEET4, 5, 6, 16, 17, and 18 contained four introns; ZjSWEET1, 9, 10, 12, 15 contained five introns; ZjSWEET3, 8, 14 contained three introns; ZjSWEET7, 13 contained six introns; and ZjSWEET11 contained only two introns.

In eukaryotes, SWEET proteins have seven transmembrane α-helical domains, 7-TMs, which consist of tandem repeats of two 3-TMs units separated by a TM unit, containing a functional transporter consisting of at least four TMs (Chen et al., 2010; Livak & Schmittgen, 2003). To confirm the presence of the transmembrane domain, the protein sequences of ZjSWEETs were submitted to the HMMER online website, as shown in Table 1. The results showed that 10 ZjSWEETs contained seven TMs, five ZjSWEETs contained six TMs, while the rest had 5 TMs (ZjSWEET8, 18), or four TMs (ZjSWEET14, 16) (Table 1). All the ZjSWEETs had two MtN3/saliva domains except ZjSWEET8 and 18 which had only one (Table 1). Furthermore, 10 motifs in ZjSWEETs were predicted using the MEME database (Fig. 2). Motif analysis found that motif 3(LVITINSIGCVIETIYIAJFLIYAPKKKR) was present in all jujube SWEET proteins, motif 1 was absent only in ZjSWEET14 protein, and motif 5 was absent only in ZjSWEET6 protein. Motif 4 was present in other proteins except the ZjSWEET8 protein and ZjSWEET16 protein, and motif 6 was only present in the ZjSWEET6 and ZjSWEET11 proteins.

Sequence alignments and phylogenetic analysis

To better understand the evolutionary origin and function of jujube SWEET gene, a phylogenetic tree was computed by using the full-length sequence of jujube SWEET protein with Arabidopsis thaliana and Malus domestica to comprehend their phylogenetic relationship. The results showed that the 19 jujube SWEET genes could be divided into four groups according to previously reported classes for Arabidopsis thaliana. In detail, the four subfamilies were named class I-IV, which respectively contained four, five, seven and three SWEET genes (Fig. 3). Four ZjSWEETs (ZjSWEET2, 3, 12, 13), 3 AtSWEETs (AtSWEET1-3) and 9 MdSWEETs (MdSWEET1.1, 1.2, 1.4, 1.5, 1.6, 1.9, 1.10, 1.12, 1.13) were clustered in class I, while 5 ZjSWEETs (ZjSWEET4, 5, 7, 9, 16), 5 AtSWEETs (AtSWEET4-8), and 3 MdSWEETs (MdSWEET2.2, 2.3, 2.4) belonged to class II. Class III contained 7 ZjSWEETs (ZjSWEET6, 10, 11, 14, 17, 18, 19), seven AtSWEETs (9–15), six MdSWEETs (MdSWEET3.1, 3.3, 3.4, 3.5, 3.10, 3.11). A total of three ZjSWEETs (ZjSWEET1, 8, 15), two AtSWEETs (AtSWEET16, 17), and MdSWEET4.1 were included in class IV.

Prediction of cis-acting regulatory elements

It has been reported that the SWEET gene family plays important roles in the biotic and abiotic stress responses of plants (Seo et al., 2011; Li et al., 2018; Zhou et al., 2018; Klemens et al., 2013; Qin et al., 2020). We analyzed the cis-regulatory elements of the upstream sequence located 2 Kb from the ATG codon to reveal the function of ZjSWEETs. Promoter sequence analysis by PlantCARE revealed different cis-elements (Table 2). The results showed that the promoter sequences of ZjSWEETs contained 9 types of phytohormone-responsive cis-regulatory elements and 6 environmental factors, including ABRE, the CGTCA and TGACG motifs, ERE, the GARE motif, P-box, TATC-box, TCA element, TGA element, ARE, LTR, MBS, TC-rich repeats, WUN-motif, and Circadian-motif, suggesting that ZjSWEET genes may be involved in diverse stress responses. Among them, the promoter region of ZjSWEET8 contained 10 types of phytohormone responsive cis-elements, while ZjSWEET3 and ZjSWEET17 contained the least types. The promoters of the 19 ZjSWEETs contained at least five phytohormone-responsive cis-elements (ZjSWEET17), while the promoter of ZjSWEET8 contained the most (24 cis-elements). The aerobic-responsive cis-element (ARE) was present in all the ZjSWEET genes, suggesting important roles for these genes in anaerobic stress responses. The ABRE motif is involved in the ABA response and was included in all the promoters of the 19 ZjSWEETs except ZjSWEET4 and ZjSWEET5. These data suggest that ZjSWEETs may be involved in the response to environmental stress through a complex mechanism, and that each ZjSWEET gene can be induced by different environmental stresses.

Expression profiles of ZjSWEETs in various organs and different developmental stages of fruit

qPCR testing was used to determine their expression patterns in five plant organs (root, shoot, leave, flower, fruit) of ‘Dongzao’ to investigate the expression profiles of the jujube SWEET genes. The qPCR signal for ZjSWEET5, 7, 9, 10, 14, 16, 17, and 19 was not detected in the five tissues of jujube suggesting that they may not be expressed, or that the expression level is low. Other genes were expressed multiple tissues, but their expression levels varied considerably (Fig. 4). For example, ZjSWEET1 and ZjSWEET11 were highly expressed in all organs, while ZjSWEET3, ZjSWEET6 and ZjSWEET12 were hardly expressed in fruit. ZjSWEET18 had very low expression in flowers, but high in stems, leaves, and fruit tissues. These results showed that different ZjSWEET genes had different tissue-specific expression patterns, which indicated that ZjSWEETs play different roles in multiple organs.

Existing studies have shown that ZjSWEET5, 7, 9, 14, 16, 17, and 19 had little expression in jujube fruits (Liu et al., 2014). In order to understand the expression of sugar transporter genes in jujube, the famous high sugar variety ‘Beijingjidanzao’ and low sugar variety ‘Linyilizao’ were used. As shown in Fig. 5, the expression patterns of some ZjSWEETs during fruit development were consistent in ‘Linyilizao’ and ‘Beijingjidanzao’, in which the expression levels of ZjSWEET11 and ZjSWEET18 increased with fruit ripening and reached higher levels in the half-red fruit stage (HR) and full-red fruit stage (FR). The expression level of ZjSWEET13 gradually reached its highest level in the white mature stage (WM), and decreased with the fruit ripening. However, the expression levels of the remaining few genes were different between the two jujube varieties. The expression level of ZjSWEET1 in ‘Linyilizao’ was the highest at the ripening stage of fruit, but it was the highest in white mature stage (WM) in ‘Beijingjidanzao’. The expression of ZjSWEET4, 11, 18 increased along with the ripening of the fruit; the expression of ZjSWEET11 and ZjSWEET18 increased to a very high level at the whole red stage. However, the expression of some genes (ZjSWEET8, 12, 13, 15) decreased with the development of fruit. The expression of ZjSWEET2 was higher at the young fruit stage and full red fruit stage than that in the white mature stage.

Expression profiles of ZjSWEETs under abiotic stresses

In order to verify the sugar transport gene under stress, the callus of high sugar variety ‘Guanyangchangzao’ was selected for verification. We detected the expressions of ZjSWEETs in low temperature conditions to determine its role under abiotic stress. The results showed that all of the ZjSWEET genes were downregulated except ZjSWEET2, 4, 6, and 12 (Fig. 6). Compared with the control, the expression level of ZjSWEET18 decreased and reached the lowest value at 1 h (about 0.2 times), while the expression levels of ZjSWEET1, 11, and 15 reached the lowest value at 6 h. The expression of ZjSWEET2, 12, and 4 increased at 1 h, but ZjSWEET6 was induced at 6 h. Therefore, there may be different mechanisms for these ZjSWEETs involved in the response of jujube to low temperature stress.

In addition, these genes all responded to alkaline stress (Fig. 6). Most of the ZjSWEET genes were up-regulated except ZjSWEET2, 4, 15 and 18 after alkaline treatment. For example, ZjSWEET1, 8, and 13 were highly increased at 1 h, while ZjSWEET6 and 12 were enhanced at 24 h. This indicates that ZjSWEETs have a different mechanism in the tolerance of jujube alkali.

We also found that there were differences in the expression of different ZjSWEETs under salt stress, among which the expression of ZjSWEET1, 13, and 15 were down-regulated, while ZjSWEET8 and 12 were highly induced (Fig. 6). We also made a significant analysis of the data, as shown in File S8.

Under low temperature, alkali, and salt stress, the expressions of ZjSWEET12 all increased, indicating that ZjSWEET12 may play an important role in resistance to various stresses. In summary, ZjSWEET genes were induced or repressed by the above treatments indicating their indispensable regulation role in adapting abiotic stresses of jujube.