Plants of oilseed rape (Brassica napus Zhongshuang11) were grown in field conditions in Chongqing, China. To analyse transcripts of BnSUC/BnSWEET members in different tissues, roots (R), stems (ST), senescent leaves (SL), extended leaves (EL), buds (B), flowers (F), stalk (ST), silique walls (21 DAF, SW), seeds (21 DAF, SE), and main inflorescences (MI) were collected from “ZS11”. All tissues were quickly frozen in liquid nitrogen and stored at −80°C until use.

For analysis of BnSUC/BnSWEET members under various abiotic or exogenous hormone stresses, oilseed rape seedlings (at the four-leaf stage) were grown in a greenhouse under long-day conditions (16-h light, 8-h dark) and transferred to 1/2 Hoagland solution 24 h before the induction of different stresses. A final concentration of 200 mM NaCl or 20% polyetheleneglycol-6000 (PEG-6000) was used for salt or drought stress, respectively. For heat stress, seedlings were transferred to a growth chamber at 40°C. For Sclerotinia sclerotiorum stress, methods were in accord with Li et al. (2015). For hormone treatment, 50 μM ABA (GenTel, Beijing, China), 100 μM GA3 (GenTel, Beijing, China), 10 μM BR (GenTel, Beijing, China), 75 μM NAA (GenTel, Beijing, China), 75 μM 6-BA (GenTel, Beijing, China), and 2 mM SA (GenTel, Beijing, China) were employed (Yang et al., 2009; Gao et al., 2010). Leaves were collected at 0, 3, 6, 12, 24, 48, and 72 h after salt, drought and heat treatments and 0, 0.5, 1, 3, 6, 12, and 24 h after various exogenous hormone treatments and immediately frozen in liquid nitrogen for storage at −80°C until use.

Brassica rapa and Brassica oleracea sucrose transporters SUC and SWEET were identified by performing a BLASTP analysis (Altschul et al., 1997) with the BRAD database (http://brassicadb.org/brad/index.php) and the B. napus genome (http://www.genoscope.cns.fr/brassicanapus/) at a cut-off value of < E⁻²⁰ using the A. thaliana sucrose transporter SUC and SWEET amino acid sequences as the query sequences, respectively.

To gain insights into the evolutionary relationships of oilseed rape SUC and SWEET proteins, we performed multiple alignments of the SUC and SWEET proteins of certain species (A. thaliana, B. rapa, B. oleracea, and B. napus). Multiple sequence alignments of the deduced amino acid sequences of the SUC and SWEET proteins were performed using the default parameters of ClustalW (Eom et al., 2015). Dendrograms were generated by the MEGA 6 program (Tamura et al., 2013) using the neighbor-joining (NJ) method and bootstrap analysis (1000 replications).

The molecular weight (MW) and isoelectric points (pI) of the presumed sucrose transporter proteins were predicted by the online ExPASy proteomics server database (http://expasy.org/). The Gene Structure Display Server (GSDS 2.0, http://gsds.cbi.pku.edu.cn/index.php) was used to generate the exon/intron organization. Motifs were identified using the MEME program (http://meme-suite.org/). The maximum number of motifs was 25 and 29 in the BnSUC and BnSWEET proteins, respectively, and the optimum width of the motifs was set from 6 to 50. Furthermore, all identified motifs were annotated according to InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/).

The promoter sequences (1.5 kb upstream of the translation start site) of the BnSUC and BnSWEET genes were obtained from the B. napus genome (http://www.genoscope.cns.fr/brassicanapus/). PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to analyse the BnSUC and BnSWEET gene promoters and identify their cis-elements (Rombauts et al., 1999; Lescot et al., 2002).

Total RNA was extracted from all samples with the RNeasy extraction Kit (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from 1 μg of total RNA using M-MLV transcriptase (TaKaRa Biotechnology, Dalian, China) according to the manufacturer's instructions after the contaminated genomic DNA was removed by DNase I treatment. Real-time PCR was used to determine the expression levels of BnSUCs and BnSWEETs in different tissues and in response to various stresses. Quantitative real-time PCR was performed according to methods described in Wei et al. (2016). To obtain precise and reproducible results, each sample was replicated three times. As many studies have suggested only SWEET9 through SWEET15 function as sucrose transporters, we only performed expression analysis of BnSWEET9–BnSWEET15 in various tissues and stresses. Because of their high similarities, 18 BnSUC and 16 BnSWEET gene primers were designed for quantitative RT-PCR analysis (Table S1).

To identify all putative SUC and SWEET protein sequences in B. napus, B. rapa, and B. oleracea, BLASTP analysis was conducted. Through this approach, 22, 9, and 8 SUC genes were identified. The 22 predicted BnSUCs proteins ranged from 478 (BnaSUC1- 8) to 540 (BnaSUC3-1) amino acid (aa) residues in length, with an average length of 512 aa. The relative molecular mass varied from 50.91 kDa (BnSUC1-8) to 57.19 kDa (BnSUC3-2). The pI values ranged from 6.06 (BnSUC3-2) to 9.40 (BnSUC4-1), with 17 members exhibiting pI values >7 (Table S1). Sixty-eight, 26 and 16 SWEET proteins were identified in the B. napus, B. rapa, and B. oleracea genomes, respectively. The 68 predicted BnSWEET proteins ranged from 56 (BnSWEET3-2) to 303 (BnSWEET4-4) aa in length, with an average length of 245.5 aa. The relative molecular mass varied from 6.5 kDa (BnSWEET3-2) to 33.45 kDa (BnSWEET4-4). The pIs ranged from 4.68 (BnSWEET3-2) to 9.75 (BnSWEET10-3), with 92.6% (63) members exhibiting pI values >7, and the other three having pI values < 7 (Table 1).

To study the evolutionary relationships among oilseed rape SUC and SWEET proteins and known SUCs and SWEETs from A. thaliana, B. rapa and B. oleracea, an unrooted neighbor-joining phylogenetic tree was created using the amino acid sequences of the SUC and SWEET family proteins from oilseed rape, A. thaliana, B. rapa, and B. oleracea. Based on our multiple sequence alignment and phylogenetic analysis, SUCs cluster into three types, consistent with previous studies using angiosperms SUCs (Figure 1). Briefly, 26 SUC proteins, including 13 BnSUCs, seven AtSUCs, four BrSUCs, and two BoSUCs, were most likely type I SUCs. AtSUC3, five BnSUC3s, two BrSUC3s, and two BoSUC3s belonged to type II. Type III consisted of four BnSUC4s, two BrSUC4s, two BoSUC4s, and AtSUC4. Although the SWEET gene family is large, 126 members from A. thaliana, B. napus, B. rapa, and B. oleracea were clustered into four clades (I, II, III, and IV), containing 21, 36, 56, and 13 members, respectively (Figure 2).

The locations of the BnSUC and BnSWEET genes are shown in Figure 3. Approximately 50% of the two gene families are located on the A or C genomes, and the precise chromosomal positions of the BnSUC and BnSWEET genes are listed in Table S1. The 22 BnSUCs are located on 14 chromosomes in B. napus. Chromosome A07 contains 3 SUC genes, whereas chromosomes A02, A03, A06, C02, C07, and C08 each contain two SUC genes. Chromosomes A09, C03, C05, C06, A09_random, C05_random and Cnn_random each contain one SUC gene (Figure 3). As shown in Figure 3, 68 BnSWEET genes are located on A09_random, C09_random, Cnn_random, and on 18 of the 19 chromosomes (the exception is chromosome C06). Chromosome A03 contains the largest number (8) of BnSWEET genes, followed by chromosome C03, which contains six members. Chromosome A02 and C02 each contain five members. Five chromosomes each contain four genes, and another five chromosomes (A09, A10, C05, C08, and Cnn_random) each contain three members. Chromosome A05 and A08 each contain two members, and the other five chromosomes (A04, A07, C04, C03_random, and C09_random) each contain only one member.

Previous studies have determined that gene divergence and duplication events are the major reasons for evolutionary momentum (Vision et al., 2000; Bowers et al., 2003). In our studies, eight pairs of segmental duplications were identified for the following BnSUCs: BnSUC1-1/BnSUC1-2, BnSUC1-3/BnSUC1-4, BnSUC1-5/BnSUC1-6, BnSUC1-8/BnSUC1-9, BnSUC2-1/BnSUC2-4, BnSUC2-2/BnSUC2-3, BnSUC4-1/BnSUC4-2, and BnSUC4-3/BnSUC4-4 (Figure 4). Twenty-three sister gene pairs (including two tandem duplicates: BnSWEET5-1/BnSWEET5/2 and BnSWEET5-4/BnSWEET5/5) were identified for the BnSWEET gene family and are shown in Figure 5.

Gene structural diversity and conserved motif divergence play important roles in the evolution of the SWEET/SUC gene family (Hu et al., 2010; Xu et al., 2012). To further examine the structural features of oilseed rape SUC and SWEET genes, we compared their exon/intron organizations. As shown in Figure 4, the most closely related BnSUC genes within the same type shared similar gene structures in terms of either intron numbers or exon lengths. For example, type I genes had one to four exons. Seven of the 13 type I genes had three exons, and the rest had five exons, except BnSUC2-1, which had only one exon. By contrast, all type III genes had five exons, and type II members had 15 exons, except BnSUC3-2, which had 11 exons. We analyzed the gene structures of BnSWEET members using the same approach (Figure 5). Most members (75%) had six exons, and 1, 2, 6, 5, 2, and 1 had 8, 7, 5, 4, 3, and 1 exons, respectively. Overall, 83.3, 57.9, 83.3, and 71.4% of the members in clades I, II, III, and IV contained six exons, respectively.

Twenty-five and 29 putative protein motifs were predicted using the MEME program for oilseed rape BnSUC (Figure 6) and BnSWEET (Figure 7) proteins, respectively. For BnSUC proteins, motifs 1-13 were observed in all 22 BnSUC proteins. Motifs 15, 17, 22, and 23 were only detected in the type I subgroup. Motifs 14, 19, and 25 were detected only in type II. Motif 16 was only detected in the type III subgroup. According to InterProScan annotation, motifs 1-8 were SUC1-RELATED sucrose transport proteins. Motifs 1-5 were observed in almost all of the BnSWEET proteins and accounted for 92.6, 89.7, 91.2, 92.6, and 85.3% of the motifs, respectively. Motifs 1-3 were annotated as SWEET sugar transporters. Notably, BnSWEET3-2, BnSWEET8-2, BnSWEET8-4, BnSWEET9-2, BnSWEET10-2, BnSWEET16-3, and BnSWEET17-2 were the most diverse, consistent with their gene structures. Generally, proteins with similar motif compositions were clustered in the same class, indicating similar functions among members of the same class.

Cis-regulatory elements, which act as binding sites for TFs, are critical for determining the expression patterns of genes (Liu et al., 2013). To further understand the transcriptional regulation and potential functions of BnSUCs and BnSWEETs, the 1500 bp regions upstream of the transcriptional start codons were analyzed using the PLACE database to identify cis-regulatory elements. Ninety-eight types of cis-regulatory elements were detected in the promoters of BnSUC genes, including the TATA-box, CAAT-box, and light responsive elements. In addition, some types of elements are potentially responsive to stresses, including the heat shock element (HSE), the low temperature-responsive element (LTR), the defense and stress-responsive element (TC-rich repeats), the MYB binding site involved in drought-inducibility (MBS), and elements that act in response to hormones, such as the gibberellin-responsive element (GARE-motif), the SA response (TCA-element), the MeJA response (CGTCA motif), and the ABA response (ABRE). All 22 BnSUC genes contained 5-19 cis-elements related to stress or hormone responses. Nineteen and 17 BnSUC genes contained the GARE motif (gibberellin-responsive element) and the CGTCA motif (MeJA response) in their promoter regions, respectively, suggesting that BnSUC genes may play key roles in responses to gibberellin and MeJA. In addition, 18, 16, and 15 BnSUC genes contained TC-rich repeats (defense and stress-responsiveness), MBSs (drought stress), and HSEs (heat stress), respectively. Among the 22 BnSUC genes, BnSUC1-2 may be involved in responses to low temperature stress, as implied by the presence of 10 LTR elements (Table S2).

As listed in Table S3, the top seven elements involved in stress and hormone responses were detected in the promoters of BnSWEETs. There were 90 instances of the heat shock element (HSE), 103 instances of the defense and stress-responsive element (TC-rich repeats), 80 instances of the MYB binding site involved in drought-inducibility (MBS), 42 instances of the gibberellin-responsive element (GARE-motif), 61 instances of the SA response (TCA-element), 73 instances of the MeJA response (CGTCA motif), and 58 instances of the ABA response element (ABRE).

To investigate the functions of BnSUC and BnSWEET genes, their gene expression profiles in different tissues were determined by qRT-PCR. Tissues were sampled from roots, stems, senescent leaves, extended leaves, buds, flowers, stalk, silique walls, seeds, and main inflorescences of “ZS11” to explore the expression patterns of selected BnSUCs and BnSWEETs. As illustrated in Figure 8, the selected BnSUC genes including 11 type I, 5 type II, and 2 type III members were mainly expressed in extended leaves and flowers, except 2 type II genes (BnSUC3-1, which was mainly expressed in roots and stalks, and BnSUC3-4, which was mainly expressed in main inflorescences and stems). Therefore, BnSUC3 might be involved in different functions with other members.

The expression patterns of selected BnSWEETs in different tissues varied. Three BnSWEET9 genes and all BnSWEET14 genes were mostly expressed in flowers, indicating that BnSWEET9 and BnSWEET14 function in flower development. All BnSWEET15 members were highly expressed in developing seeds. The rest of the selected BnSWEET genes were expressed in a variety of tissues, particularly in extended leaves, developing seeds, stalks and main inflorescences. The three BnSWEET10 genes had different expression patterns, that is, BnSWEET10-1, and BnSWEET10-3 were mainly expressed in developing seeds, buds, flowers, and extended leaves, whereas BnSWEET10-2 was highly expressed in extended leaves and flowers but was not expressed in developing seeds and other tissues. Neither BnSWEET11 nor BnSWEET12 expression was detected in stems and silique walls, whereas BnSWEET13-1, and BnSWEET13-2 transcripts were not detected in developing seeds and silique walls. The high expression of BnSWEETs in specific tissues may indicate specific roles in the corresponding tissues.

To obtain insight on the roles of BnSUC and BnSWEET genes responding to various stresses, oilseed rape seedlings of ZS11 were subjected to salt, drought, heat, and S. sclerotiorum stresses. As shown in Figure 9, only BnSUC1-2, BnSWEET10-3 and BnSWEET12 were upregulated under salt stress, and the remaining genes were downregulated. BnSUC1-2, BnSWEET10-3, BnSWEET12, and BnSWEET14 were upregulated after 3 h under drought stress and then immediately decreased, except BnSWEET12. For heat stress, 11 of 16 genes were upregulated after 3 h and decreased immediately, except BnSUC1-2 and BnSUC2-4. For S. sclerotiorum infections, BnSUC1-1, BnSUC1-5, BnSUC2-2, BnSUC2-4, BnSWEET9-2, and BnSWEET11 were upregulated after 48 h.

Plant hormones such as SA, auxin, NAA, BR, GA, and ABA play important roles in the regulation of developmental processes (Yang et al., 2012; Curaba et al., 2014). In our study, hormone treatments resulted in a wide variety of changes in the transcript levels of sucrose transporter genes in oilseed rape as determined by qRT-PCR. Briefly, all members were upregulated under GA stress, particularly BnSUC1-2, BnSUC3-5, BnSUC4-3, BnSWEET10-3, BnSWEET12, and BnSWEET13-2. These genes were also induced by ABA, BR, and 6-BA stresses, whereas only BnSUC1-2 and BnSWEET10-3 were induced under SA stress, and only BnSUC1-2 and BnSWEET12 were upregulated by NAA stress at some time points (Figure 9). These results indicate that hormones are involved in the regulation of sucrose transporters in oilseed rape.

Polyploidization is considered an outstanding power of eukaryotic evolution and the main contributor to evolutionary events (Dun et al., 2014). After the Brassica genus of plants formed from A. thaliana, it triplicated its genome, with an expansion in gene numbers (Lysak et al., 2005, 2007). Generally, three syntenic copies of each gene in A. thaliana should be detected in a diploid Brassica species, such as B. rapa (Wang et al., 2011; Cheng et al., 2013; Dun et al., 2014), and B. oleracea (Cheng et al., 2014; Liu et al., 2014). Thus, the allotetraploid B. napus, which was generated from the spontaneous hybridization of B. rapa and B. oleracea approximately 7500–12,500 years ago (Chalhoub et al., 2014), should contain six copies of each Arabidopsis gene. However, the B. rapa (Mun et al., 2009; Wang et al., 2011), and B. napus (Chalhoub et al., 2014; Cheng et al., 2014) genomes only contain 1.5–2 and 2–6 copies of each gene in Arabidopsis, respectively, because of genome shrinkage and gene loss. In our study, a total of 22 SUC and 68 SWEET genes were identified in the genome of the oilseed rape.

Our molecular characterizations revealed great variations. The molecular masses and pIs varied markedly among subfamilies, whereas the SUC and SWEET proteins clustered in the same subfamily and shared closer molecular masses and pIs. Consistent with previous studies (Reinders et al., 2012; Chong et al., 2014), the members of the SUC and SWEET gene families in the genomes of A. thaliana, B. napus, B. rapa, and B. oleracea were classified into three and four groups, respectively. Variation in exon–intron structure plays a significant role in the evolution of gene families (Rogozin et al., 2005; Xu et al., 2012). Our studies of the SUC and SWEET gene families provide an explanation for this diversification in gene structure. Type II BnSUC3s contain more exons than type I and type III BnSUCs, indicating their diverse functions. These data, along with detected motif data, suggest similar origins, and evolutionary patterns for the SUC and SWEET genes of different species.

Comprehensive gene expression analyses of sucrose transporter family genes have revealed that SUCs have distinct expression patterns in various tissues of Arabidopsis, maize, wheat and grape (Meyer et al., 2004; Slewinski et al., 2009; Afoufa-Bastien et al., 2010; Mukherjee et al., 2015). Consistent with these previous results, BnSUCs also exhibited differential expression patterns in various tissues. Notably, BnSUC2 in the type I subclass was highly expressed in different tissues, particularly flowers and extended leaves, suggesting specific roles required in these tissue types. According to previous studies, AtSUC3, which was clustered into type II with the BnSUC3s, functions as a sugar signal-transducer. However, SUC1/2 were only observed in eudicot species, and these genes are necessary for phloem loading (Srivastava et al., 2008; Gould et al., 2012) and normal pollen function (Sivitz et al., 2008). Type III SUCs act as sucrose carriers and are localized to the vacuolar membrane (Endler et al., 2006; Schneider et al., 2012).

All clade III members of BnSWEETs were highly expressed in the extended leaves, flowers and developing seeds. Similar patterns have been observed in other plant species (Xuan et al., 2013; Lin et al., 2014). Some BnSWEETs exhibited high expression levels in specific tissues, implying specific roles required in these tissue types. For example, BnSWEET9-1/9-2/9-3, and BnSWEET14 were highly specifically expressed in flowers, and BnSWEET15 was abundantly expressed in developing seeds.

Several studies have examined regulators of sucrose transporters. Meyer et al. (2004) observed that AtSUC3 expression is strongly induced upon wounding of Arabidopsis tissues. Chincinska et al. (2008) subsequently determined that StSUT4 expression in wild-type plants was induced by GA and ET. Mukherjee et al. (2015) demonstrated that ABA negatively regulates sucrose import into the endosperm by repressing TaSUT1 in wheat using physiological, molecular and biochemical approaches. In our study, expression changes in 16 selected genes were detected under heat, drought, salt, S. sclerotiorum, and six hormone stresses. Notably, sucrose transporter genes in oilseed rape were positively regulated by cytokinin (6-BA) and GA, consistent with previous reports (Chincinska et al., 2008). By contrast, these selected genes were mostly downregulated under ABA, NAA, SA, NaCl, and PEG stresses. These results indicate that sucrose transporter genes (BnSUCs and BnSWEETs) are regulated by plant hormones and abiotic and biotic stresses. These results may facilitate improvements in crop yield.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.