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Article

Genome-Wide Identification of Sucrose Transporter Genes and Functional Analysis of RsSUC1b in Radish (Raphanus sativus L.)

by
Xiaofeng Zhu
1,†,
Xiaoli Zhang
1,†,
Yang Cao
1,†,
Ruixian Xin
1,
Yinbo Ma
2,
Lun Wang
2,
Liang Xu
1,
Yan Wang
1,
Rui Liu
1 and
Liwang Liu
1,2,*
1
National Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Horticultural Crop Biology and Genetic Improvement (East China), Ministry of Agriculture and Rural Affairs of P.R.C., College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
2
College of Horticulture and Landscape Architecture, Yangzhou University, Yangzhou 225009, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2022, 8(11), 1058; https://doi.org/10.3390/horticulturae8111058
Submission received: 12 September 2022 / Revised: 18 October 2022 / Accepted: 7 November 2022 / Published: 10 November 2022
(This article belongs to the Special Issue Germplasm Resource Identification and Genetic Improvement of Horticultural Crop)
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Abstract

:
In most higher plants, sucrose is the significant form of carbohydrate for long-distance transportation. Sucrose transporters/sucrose carriers (SUTs/SUCs) are involved in the loading and unloading of sucrose in phloem and play an important role in the growth and development of plants. In this study, 12 RsSUC genes were first identified from the radish genome, and their phylogenetic relationships, gene structure, and conserved motifs were further analyzed. RT-qPCR results indicated that RsSUC genes exhibited various expression patterns in different tissues and development stages of the radish. Overexpression of RsSUC1b in Arabidopsis significantly improved the uptake efficiency of exogenous sucrose, and promoted leaves and lateral root growth. In addition, the transgenic plants flowered significantly earlier than wild-type (WT) plants, and the soluble sugar contents (SSCs) including sucrose, glucose, and fructose in the mature leaves and pods were increased. It could be inferred that RsSUC1b is a plasma membrane sucrose transporter and plays a vital role in sucrose transportation and sugar accumulation during plant growth and development. These findings provided novel insights into the biological function of RsSUC genes and facilitate dissecting the molecular mechanism underlying sugar transport during radish development.

1. Introduction

The radish (Raphanus sativus L.) is widely cultivated worldwide, and its thickened taproot is not only a product organ, but also an important sink organ [1]. Radish taproot is rich in nutrients, including protein, carbohydrates, minerals, vitamins, lipids, and amino acids. Carbohydrates mainly include glucose, sucrose, and fructose [2]. Sucrose is the main product of photosynthesis in higher plants and the main form of assimilated transport in phloem [3]. Sucrose export directly affects the transportation and distribution of photosynthate, which plays a critical role in the formation and development of radish taproot [4]. Therefore, understanding the molecular mechanism of sucrose transportation in radishes is of great significance to improve the yield and quality of radishes.
Sucrose is a kind of soluble sugar, which is the main photosynthate in the leaves of higher plants. It can maintain the metabolism of source organs; most products are loaded into phloem and distributed to plant sink organs, such as the stem, seed, and taproot [5,6]. The transport of sucrose involves symplast and apoplast pathways in phloem [7]. In the apoplast pathway, sucrose loading or unloading requires the participation of sucrose transporters/sucrose carriers (SUTs/SUCs) [8,9].
SUCs are a class of carrier proteins that can mediate sucrose transport, and widely exist in tissues and cells of higher plants [10]. SUCs were members of a subfamily of the glycoside-pentoside-hexuronide (GPH) cation family in the major facilitator superfamily (MFS) [11] and played a critical role in the transportation of soluble sucrose. SUC gene was first isolated from spinach, and it was found that the gene had sucrose transport ability through the transformation of INV-deficient yeast experiment [12]. Until now, lots of SUC genes were successfully isolated from various plants including sorghum [13], oilseed rape [14], pomegranate [15], and blackberry [16]. SUCs in dicotyledons could be classified into three subgroups, SUT1, SUT2, and SUT4 [17]. The influence of SUT1 members on plant growth and development has been verified in a variety of plants by gain or loss of function analysis. The Arabidopsis sucrose transporter AtSUC1 was found to function in sucrose transport during development of pollen and roots [18,19]. Overexpressing of the spinach SoSUT1 gene in potatoes could promote the export of sucrose in leaf and significantly increase the content of soluble sugar in tubers [20]. Additionally, inhibiting the expression of the maize ZmSUT1 gene resulted in a large amount of soluble sugar accumulation in leaves, and the leaves withered and became senescent rapidly, and plant flowering was delayed [21]. These results demonstrated the important role of SUC genes in regulating sucrose transport during plant growth and development.
In this study, to further investigate the function of RsSUC genes involved in regulating sucrose transport in radish, SUC family members were first identified from the radish genome, and the sequence characteristics, gene structure, conserved motifs, transmembrane regions, and phylogenetic relationships were analyzed. In addition, the expression profiles of RsSUC genes were explored in the leaves and taproots at different developmental stages. Additionally, the function of RsSUC1b was further investigated by overexpression in Arabidopsis. These results provided novel insight into the function of SUC genes in sucrose transport during radish taproot formation.

2. Materials and Methods

2.1. Plant Materials

An advanced inbred line, NAU-ZYQ, was sown in the NAU (Baima) experimental station. Tissue samples of leaf and root were harvested every 20 days after sowing (20 DAS) until harvest (100 DAS). The phenotypes during the development stages of the radish were shown in Figure S1. The growth cycle of radishes is about three months. According to the phenotypic changes in each stage, the development stages of radishes could be divided into cortex split stage (20–40 DAS), early stage of taproot thickening (40–60 DAS), middle stage of taproot thickening (60–80 DAS), and late stages of taproot thickening (80–100 DAS). An approximately 0.3 g sample was rapidly frozen in liquid nitrogen and stored at −80 °C for gene expression analysis, and the remaining sample was dried at 80 °C for soluble sugar content determination.
Tobacco (Nicotiana benthamiana) was utilized for the subcellular localization analysis. A. thaliana (ecotype: Col-0) was utilized for genetic transformation.

2.2. Identification of SUC Gene Family in Radish

The SUC protein sequences containing conserved domain (PF13347) in the radish genome (WK10039, http://radish-genome.org/) (accessed on 5 July 2021) were searched by Hmmer3.0 software [22]. Subsequently, NCBI (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) (accessed on 12 July 2021), SMART (http://smart.embl-heidelberg.de/) (accessed on 12 July 2021), and Pfam (http://Pfam.sanger.ac.uk/) (accessed on 12 July 2021) were employed to further verify the conserved domain of candidate SUC proteins.

2.3. Sequence Characteristic and Phylogenetic Analysis

The ExPASy ProtParam (http://web.expasy.org/protparam/) (accessed on 20 August 2021) was performed to predict relative molecular weight, theoretical pI, instability index, aliphatic index, and grand average of hydropathicity of RsSUC proteins [23]. Moreover, the transmembrane regions were analyzed by Hidden Markov Models Server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/) (accessed on 21 August 2021) [24]. The Arabidopsis SUC protein sequences are from the TAIR database (http://www. arabidopsis.org/) (accessed on 3 September 2021) (Table S2). The SUC protein sequences of Brassica oleracea, Oryza sativa, and Zea mays were obtained from NCBI (https://www.ncbi.nlm.nih.gov/) (accessed on 9 September 2021) according to the reported SUC genes accession numbers (Table S2). The phylogenetic tree including five species was constructed by MEGA6.0 with the neighbor-joining (NJ) and bootstrap value set to 1000 replicates [25]. Additionally, the gene structure was analyzed by TBtools software and conservative motifs were analyzed by MEME (http://meme.sdsc.edu/meme/) (accessed on 15 September 2021) [26].

2.4. RNA Extraction and RT-qPCR Analysis

Total RNA was extracted by using the TRIzol reagent RNA simple total RNA kit (Tiangen, Beijing, China). and reverse transcribed into cDNA by using the PrimeScript™ RT reagent kit (Takara, Dalian, China) according to the instructions. The cDNA was used for RT-qPCR to analyze the expression of RsSUC genes. RT-qPCR analysis was performed on LightCycler® 480 System (Roche, Mannheim, Germany). RsActin was an internal reference gene, and the 2−∆∆CT formula was used to calculate the relative expression level [27,28]. The RT-qPCR primers were shown in Table S3.

2.5. Subcellular Localization of RsSUC1b

The CDS of RsSUC1b was isolated and deposited into GenBank (Acc. No.: OP653780). The gene construct 35::RsSUC1b-GFP was transformed into Agrobacterium tumefaciens GV3101 strain and was injected into N. benthamiana leaves with 35S::GFP and 35S::AUX1-RFP, which were used as the negative control and nuclear marker, respectively [29,30,31]. The subcellular localization of 35S::RsSUC1b-GFP in the N. benthamiana leaf cell was observed under a laser confocal microscope (LSM 800, Zeiss, Jena, Germany). The primer sequences for 35::RsSUC1b-GFP construction were listed in Table S4.

2.6. Construction of Expression Vector and Genetic Transformation

The fusion plasmids 35S::RsSUC1b was transferred into Arabidopsis by using A. tumefaciens-mediated transformation with the floral dip method [32]. The transformed seeds were sown on MS solid medium supplemented with 36 mg·L−1 hygromycin. Hygromycin-resistant seedlings (T1) were grown to maturity for seed collection. T2 seeds were obtained from selfing T1 plants, and a portion of the T2 seeds was germinated on MS solid medium supplemented with 36 mg·L−1 hygromycin. T3 seeds were harvested from T2 plants and used in this study.

2.7. Determination of Soluble Sugar Contents

After adding 0.05 g dried samples and 5 mL extracting solution, ultrasonic extraction was performed for 20 min. After an 80 °C water bath for 30 min and 5000 g spinning for 15 min, the supernatant was filtered through a 0.45-μm water system filter membrane. The content of soluble sugar was determined by UPLC [3]. Total sugar contents were calculated as the sum of sucrose, glucose, and fructose content.

2.8. Statistical Analysis

Data processing was performed by using Microsoft Excel. A Student’s t-test was used to determine statistical significance between two groups. One-way analysis of variance with least significant difference (LSD) tests were used to determine statistical significance among multiple range tests. Bar graphs were created by using Graphpad Prism software.

3. Results

3.1. Identification of the Radish SUC Gene Family

A total of 12 RsSUC genes on chromosome R1, R2, R4, R5, R6, R7, R9, and RUS were identified from the radish genome (WK10039) according to the combination of Blastp and Hmmer3.0. These RsSUCs were named as RsSUC1a to RsSUC4b (Table S1). The physical and chemical properties of the RsSUC proteins were analyzed. The sizes of RsSUC proteins ranged from 392 to 540 amino acid (aa) with a molecular weight range from 41.94 to 62.41 kDa, and the theoretical pI ranged from 5.49 to 9.40. The instability index ranged from 29.04 to 40.89, and the aliphatic index varied from 96.64 to 111.00. In addition, according to the grand average of hydropathicity range (0.342~0.545), which suggests that all RsSUC proteins were hydrophobic proteins. TMHMM was used to predict the transmembrane regions of RsSUC proteins. The number of transmembrane regions of RsSUC proteins ranged from 7 to 12, RsSUC2a, RsSUC2b, RsSUC4a, and RsSUC4b contained typical 12 transmembrane regions, whereas RsSUC2c and RsSUC3b had fewer transmembrane regions (Table S1).

3.2. Phylogenetic Analysis of RsSUC Proteins

An unrooted phylogenetic tree from radish, Arabidopsis, B. oleracea, O. sativa, and Z. mays was created to understand the SUC gene evolutionary relationships (Figure 1). It was indicated that the SUC proteins from monocotyledon and dicotyledon species can be classified into five subgroups including SUT1, SUT2, SUT3, SUT4, and SUT5. The largest was the SUT1 subgroup and was unique to dicotyledons, which consisted of seven members of radish, seven members of Arabidopsis, and eight members of B. oleracea. SUT3 and SUT5 were specific subgroups of monocotyledons, which consisted of three members of O. sativa and four members of Z. mays. SUT2 and SUT4 contained both dicotyledon and monocotyledon, which contained five members of radish, two members of Arabidopsis, four members of B. oleracea, two members of O. sativa, and two members of Z. mays. These RsSUC proteins were clustered closely with the dicotyledon and were significantly distant from the monocotyledon.

3.3. Gene Structure and Conserved Motifs Analysis of RsSUCs

RsSUC proteins were classified into three subgroups, SUT1, SUT2, and SUT4 (Figure 2A). The 15 conserved motifs in RsSUC proteins were predicted (Figure 2B). Most RsSUC members of the same subgroup have similar motif compositions. For instance, motif 8 and motif 14 were distributed in SUT1, motif 15 was distributed in SUT2 and SUT4, and motif 12 was only detected in SUT2. These specific motifs might imply diverse functions of the SUC family in radish. The exon-intron structures were further analyzed to understand the structural characteristics of RsSUC genes (Figure 2C). The structures of genes in the same subgroup were similar, whereas the number of the exon and intron was different. The number of RsSUC exons ranged from 1 to 14. The RsSUC genes in SUT1 contained 1 to 5 exons; The RsSUC genes in SUT4 all contained 5 exons; the majority of RsSUC genes in SUT2 contained 14 exons, of which RsSUC3b contained 8 exons. The exon numbers of RsSUC genes in SUT2 were more than those in SUT1 and SUT4.

3.4. Expression Analysis of RsSUC Genes during Radish Taproot Development

SUT1 is the largest subgroup among SUC gene family in radish. To elucidate the functions of SUT1 members in radish, the dynamic expression patterns of SUT1 members in developing leaves and taproots were analyzed by RT-qPCR (Figure 3). The results showed that RsSUC1a, RsSUC1b, RsSUC2a, RsSUC2b, and RsSUC2c were predominately expressed in leaves, whereas RsSUC1c and RsSUC1d were expressed in the leaves and taproots. The expression level of RsSUC1a and RsSUC1b increased significantly at 60 days after sowing (DAS) and decreased significantly at 100 DAS. RsSUC2a, RsSUC2b, and RsSUC2c were expressed at a higher level in leaves at 20 DAS, whereas the expression level of these genes appeared to be down-regulated afterward. The expression level of RsSUC1c and RsSUC1d increased significantly in leaves at 80 days after sowing (DAS) and decreased significantly at 100 DAS. In the taproots, RsSUC1c was expressed at a lower level at 20 DAS, whereas the expression appeared to be up-regulated afterward. However, the expression level of RsSUC1d increased significantly at 40 DAS and 80 DAS, respectively, decrease significantly at 60 DAS and 100 DAS.
In this study, RsSUC1b showed a high level of expression during the development of leaves, and it might play a vital role in sucrose transport in leaves. In addition, the nucleotide sequence identity between RsSUC1b and AtSUC1 show as high as 83.40%. Therefore, RsSUC1b was selected for further investigation of the function in sugar transport during radish taproot formation.

3.5. Subcellular Localization of RsSUC1b

To confirm the intracellular localization of RsSUC1b protein, 35S::RsSUC1b-GFP expression vector containing green fluorescent protein (GFP) reporter gene was constructed. The fusion vector 35S::RsSUC1b-GFP and a plasma membrane marker protein, auxin transporter 35S::AUX1-RFP fusion protein, co-infiltrated in tobacco leaf epidermal cells by Agrobacterium mediated transformation. The results showed that the GFP signals transiently expressed by 35S::RsSUC1b-GFP overlapped completely with the 35S::AUX1-RFP, whereas the control displayed GFP signal in the plasma membrane and nucleus of cells, indicating that RsSUC1b might function in the plasma membrane (Figure 4).

3.6. Effects of RsSUC1b Overexpression on Seedling Growth under Different Sucrose Treatments

The transgenic Arabidopsis plants overexpressing RsSUC1b were generated to further investigate the biological function of RsSUC1b. Homozygous plants were obtained from three generations (T3) of transgenic lines and confirmed higher transcript levels of RsSUC1b (Figure S2). To further verify the role of RsSUC1b in sucrose transport, the seedling growth of transgenic and wild-type (WT) plants was examined under different sucrose concentration treatments (0%, 1%, 3% and 6%). All transgenic and WT plants could not grow normally in media without sucrose, indicating the importance of sucrose for the growth of plants. Furthermore, the phenotype has no significant difference between transgenic and WT plants were planted in the media without sucrose. Under the conditions of 1%, 3%, and 6% sucrose concentration, transgenic plants grew more vigorously than WT plants, and showed that the larger rosette leaves and the number of lateral roots was increased. The transgenic seedlings may be subject to osmotic stress on the medium with sucrose concentration of 6%, showing smaller rosette leaves and shorter lateral roots compared with 1% and 3% sucrose concentration (Figure 5A,B). Notably, the number of transgenic lateral roots increased in the medium with a relatively high sugar content compared with WT. As compared with WT plants, the relative expression levels of several root development-related genes, AtWOX4, AtKNAT1, and AtLBD3, were increased in the transgenic plants under sucrose treatments of 6% (Figure 5C). These results show that the overexpression of RsSUC1b could increase the uptake efficiency of exogenous sucrose.

3.7. Overexpression of RsSUC1b in Arabidopsis Resulted in Early Flowering, Increased Height, and Higher SSCs of Plants

The transgenic lines grew faster and flowered earlier than WT plants (Figure 6A). After 45 d of growth, the height of transgenic plants was significantly higher than that of WT plants (Figure 6B and Figure S3). When transgenic lines and WT plants were planted and grown under the same conditions for 45 d, the soluble sugar including sucrose, fructose, glucose, and total sugar contents were analyzed (Figure 6C). Overexpression of RsSUC1b significantly increased the soluble sugar contents (SSCs) in the leaves and pods of the transgenic lines. The total sugar contents increased by 23.08% in the leaves and by 88.36% in the pods compared with the WT. In the transgenic lines, the sucrose content was also considerably increased by 30.13% in the leaves and by 92.89% in the pods. The hexose (fructose and glucose) content was considerably increased in transgenic lines. The fructose and glucose content was increased by 6.69% and 12.32% in the leaves, respectively, and by 28.70% and 133.50% in the pods, respectively, indicating that overexpression of RsSUC1b in Arabidopsis resulted in phenotypic and SSC variations during both vegetative and reproductive growth stages.

4. Discussion

4.1. Characterization of SUC Gene Family Members in Radish

The growth and development of plants depend on the transportation and utilization of sugars produced during photosynthesis. Sucrose transporters (SUTs/SUCs) are a group of membrane proteins that involve in the loading and unloading of sucrose in phloem and play essential roles in plant growth, development and stress response [33,34]. With the completion of genome sequencing, SUC family members have been reported in various plant species. Herein, totally 12 SUC genes were identified in radishes based on the genome database in this study (Table S1). The radish genome contains more RsSUC genes than the maize [11], sorghum [13], rice [35], and wheat [36] genome, indicating specific expansion events of SUC family may have occurred in radish evolution.
The duplicated genes could obtain new functions to improve plant adaptability to the environment in the process of plant evolution. Phylogenetic analysis showed that RsSUC genes were classified into five subgroups. SUT1 subgroup was dicotyledon-specific, and the largest number is in radishes, indicating that it could play a critical role in sugar loading and transportation [17]. The phylogenetic relationship of RsSUCs was also supported by both their conserved motif and gene structure. The RsSUC members in each subgroup shared several unique motifs, indicating that the RsSUC proteins within the same subgroups may have certain functional similarities, and the motif distribution suggested that these genes were largely conserved during evolution [37]. The characteristics of gene structure also had an important influence on the evolution of gene families. The gene structure analysis showed that each subfamily displayed similar exon–intron organizations. Interestingly, SUT1 members contained lower exons, whereas has the longest length, and SUT2 members displayed the opposite result. This might be the result of chromosomal rearrangements and fusions and led to functional diversification of polygenic families [38]. The characterization of the radish SUC gene family through genome-wide analysis provided valuable information for further investigating the functions of RsSUC genes during radish taproot formation.

4.2. Expression Patterns and Functional Diversity of RsSUC Genes

Previous studies revealed that SUC genes have distinct expression patterns in various tissues and developmental stages of plants [5]. In this study, SUT1 members showed different expression patterns in leaves and taproots at various points (Figure 3). RsSUC1a and RsSUC1b showed the highest relative expression levels in leaves at 60 DAS, then declined sharply, indicating that they may play positive roles in regulating the loading of sucrose during leaf development. The weak expression of RsSUC1c was observed during leaf and taproot development. RsSUC1d was significantly higher in leaves and taproots at 80 DAS than in any other stages, suggesting that it may be involved in the transportation of sucrose during the late developmental stages of leaves and roots. RsSUC2a, RsSUC2b, and RsSUC2c showed higher relative expression levels in leaves at 20 DAS, indicating that they might function on the transportation of sucrose during the early developmental stages of leaves. These results showed that there were differences in expression patterns and functions of RsSUC genes, whereas they all participated in the distribution of photosynthetic products and jointly regulated plant growth and development.

4.3. Sucrose Transporters Play Fundamental Roles in Plant Growth and Development

Soluble sugar is crucial for plant growth and development, and it usually needs to be transported to sinks [39]. SUCs are involved in transmembrane transport during phloem loading and unloading, for which the function of SUCs in the loading of source organs has been verified in a variety of plants. For instance, overexpressing spinach SoSUT1 in potatoes promoted the export of sucrose in leaves and significantly increased the contents of soluble sugar in tubers [20]. Inhibiting the expression of potato StSUT4 resulted in plant growth retardation, a large amount of soluble sugar and starch accumulated in leaves, and finally reduced tuber yield [40]. In this study, RsSUC1b showed the highest relative expression level in leaves than taproots of radishes, and RsSUC1b protein was localized on the plasma membrane. In Arabidopsis and other species, SUT1 gene functions in the cell membrane [19,41]. Therefore, it could be speculated that RsSUC1b gene might function in the cell membrane and participate in the transmembrane transport of sucrose.
The transgenic plants grew more robustly than WT plants on the medium with sucrose concentrations of 1%, 3%, and 6%. With the increase of sucrose concentration, transgenic plants had more lateral roots than WT. In root crops, the yields of storage roots are mainly determined by secondary growth driven by the vascular cambium. It was reported that AtWOX4, AtKNAT1, and AtLBD3 were positive regulators of cambial activities in Arabidopsis [42]. In this study, as compared with WT, the lateral root number of transgenic plant was increased in the medium with high sucrose concentration (6%). As compared with WT plants, the relative expression levels of these genes were up-regulated in the transgenic plants. These results demonstrated overexpression of RsSUC1b increased the absorption efficiency of exogenous sucrose, and the source organ (leaves) obtained more carbohydrates which could be transported to the sink organ (root). Therefore, it is reasonable to infer that the RsSUC1b gene could promote sugar accumulation in the roots of plants, which induces the higher expression level of several root development-related genes such as AtWOX4, AtKNAT1, and AtLBD3 in transgenic plants.
In addition, the overexpression of RsSUC1b resulted in the increased SSCs in leaves and pods, indicating that overexpression of RsSUC1b could influence sucrose metabolism and sugar accumulation. SUCs not only are involved in sucrose transport but also play essential roles in pollen germination, fruit ripening, and reproductive growth in various plant species [35,43]. In this study, overexpression of RsSUC1b produced a visible phenotype in the shoot, transgenic Arabidopsis plants had the feature of early bolting and flowering. The role of SUC gene in flowering has been verified in several species. The overexpression of apple MdSUT2 gene in Arabidopsis led to early flowering and increased plant height [44]. The antisense inhibition of sucrose transporter activity in phloem of tobacco resulted in delayed flowering.

5. Conclusions

In conclusion, the systematic genome characterization of RsSUC gene family was investigated in this study, and the expression patterns of each member were different during radish growth and development. RsSUC1b might be a plasma membrane transporter. In addition, overexpression of RsSUC1b affected sucrose transport and sugar accumulation in Arabidopsis and promoted vegetative and reproductive growth. These results might lead to a better understanding of the function of SUC genes in sugar transportation during radish taproot formation.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/horticulturae8111058/s1, Table S1: List of the 12 RsSUC genes identified in this study; Table S2: List of SUC proteins used for the construction of the phylogenetic tree; Table S3: The primer sequences of RT-qPCR; Table S4: Primer sequences for 35::RsSUC1b-GFP construction; Figure S1: The phenotype during the development of radish. Figure S2: RT-PCR analysis of WT and over-expression T3 transgenic Arabidopsis plants; Figure S3: The plant height of WT and over-expression of RsSUC1b Arabidopsis plants.

Author Contributions

Conceptualization, X.Z. (Xiaofeng Zhu), X.Z. (Xiaoli Zhang) and L.L.; writing—original draft preparation, X.Z. (Xiaofeng Zhu) and Y.C.; writing—review and editing, Y.M., L.W., L.X., Y.W. and L.L.; visualization, X.Z. (Xiaofeng Zhu), X.Z. (Xiaoli Zhang) and R.X.; validation, X.Z. (Xiaofeng Zhu) and X.Z. (Xiaoli Zhang); project administration, L.L. and R.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Jiangsu Agricultural S&T Innovation Fund [CX(21) 2020], the “JBGS” Project of Seed Industry Revitalization in Jiangsu Province (JBGS(2021)071), and the earmarked fund for Jiangsu Agricultural Industry Technology System [JATS(2022)463], the Guidance Foundation, the Hainan Institute of Nanjing Agricultural University(NAUSY-MS02), the Project Founded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

  1. Fan, L.X.; Xu, L.; Wang, Y.; Tang, M.J.; Liu, L.W. Genome-and transcriptome-wide characterization of bZIP gene family identifies potential members involved in abiotic stress response and anthocyanin biosynthesis in radish (Raphanus sativus L.). Int. J. Mol. Sci. 2019, 16, 6334. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Masakazu, H.; Daiki, T.; Tatsuo, A.; Ikuo, T. Variations in the soluble sugar and organic acid contents in radish (Raphanus sativus L.) cultivars. Int. J. Food Sci. Technol. 2011, 46, 2387–2392. [Google Scholar]
  3. Kang, J.N.; Kim, J.S.; Lee, S.M.; Won, S.Y.; Seo, M.S.; Kwon, S.J. Analysis of phenotypic characteristics and sucrose metabolism in the roots of Raphanus sativus L. Front. Plant Sci. 2021, 12, 716782. [Google Scholar] [CrossRef] [PubMed]
  4. Mitsui, Y.; Shimomura, M.; Komatsu, K.; Namiki, N.; Shibata-Hatta, M.; Imai, M.; Katayose, Y.; Mukai, Y.; Kanamori, H.; Kurita, K.; et al. The radish genome and comprehensive gene expression profile of tuberous root formation and development. Sci. Rep. 2015, 5, 10835. [Google Scholar] [CrossRef] [Green Version]
  5. Liu, Y.; Yin, X.; Yang, Y.; Wang, C.; Yang, Y. Molecular cloning and expression analysis of turnip (Brassica rapa var. rapa) sucrose transporter gene family. Plant Divers. 2017, 20, 123–129. [Google Scholar] [CrossRef]
  6. Chen, Q.; Hu, T.; Li, X.; Song, C.P.; Zhu, J.K.; Chen, L.; Zhao, Y. Phosphorylation of SWEET sucrose transporters regulates plant root:shoot ratio under drought. Nat. Plants 2022, 8, 68–77. [Google Scholar] [CrossRef]
  7. Ma, S.; Li, Y.X.; Li, X.; Sui, X.L.; Zhang, Z.X. Phloem unloading strategies and mechanisms in crop fruits. J. Plant Growth Regul. 2019, 38, 494–500. [Google Scholar] [CrossRef]
  8. Reuscher, S.; Akiyama, M.; Yasuda, T.; Makino, H.; Aoki, K.; Shibata, D.; Shiratake, K. The sugar transporter inventory of tomato: Genome-wide identification and expression analysis. Plant Cell Physiol. 2014, 55, 1123–1141. [Google Scholar] [CrossRef] [Green Version]
  9. Dinant, S.; Le, H.R. Delving deeper into the link between sugar transport, sugar signaling, and vascular system development. Physiol. Plant. 2022, 174, e13684. [Google Scholar] [CrossRef]
  10. Sauer, N. Molecular physiology of higher plant sucrose transporters. FEBS Lett. 2007, 581, 2309–2317. [Google Scholar] [CrossRef] [Green Version]
  11. Usha, B. Diverse expression of sucrose transporter gene family in Zea mays. J. Genet. 2015, 94, 151–154. [Google Scholar] [CrossRef]
  12. Riesmeier, J.W.; Willmitzer, L.; Frommer, W.B. Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast. EMBO J. 1992, 11, 4705–4713. [Google Scholar] [CrossRef]
  13. Milne, R.J.; Byrt, C.S.; Patrick, J.W.; Grof, C.P. Are sucrose transporter expression profiles linked with patterns of biomass partitioning in Sorghum phenotypes? Front. Plant Sci. 2013, 4, 223. [Google Scholar] [CrossRef] [Green Version]
  14. Jian, H.; Lu, K.; Yang, B.; Wang, T.; Zhang, L.; Zhang, A.; Wang, J.; Liu, L.; Qu, C.; Li, J. 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. 2016, 28, 1464. [Google Scholar] [CrossRef] [Green Version]
  15. Poudel, K.; Luo, X.; Chen, L.; Jing, D.; Xia, X.; Tang, L.; Li, H.; Cao, S. Identification of the SUT gene family in pomegranate (Punicagranatum L.) and functional analysis of PgL0145810.1. Int. J. Mol. Sci. 2020, 21, 6608. [Google Scholar] [CrossRef]
  16. Yan, Z.X.; Yang, H.Y.; Zhang, C.H.; Wu, W.L.; Li, W.L. Functional analysis of the blackberry sucrose transporter gene RuSUT2. Russ. J. Plant Physiol. 2021, 68, 246–253. [Google Scholar] [CrossRef]
  17. Kühn, C.; Grof, C.P. Sucrose transporters of higher plants. Curr. Opin. Plant Biol. 2010, 13, 288–298. [Google Scholar] [CrossRef]
  18. Stadler, R.; Truernit, E.; Gahrtz, M.; Sauer, N. The AtSUC1 sucrose carrier may represent the osmotic driving force for anther dehiscence and pollen tube growth in Arabidopsis. Plant J. 1999, 19, 269–278. [Google Scholar] [CrossRef]
  19. Sivitz, A.B.; Reinders, A.; Ward, J.M. Arabidopsis sucrose transporter AtSUC1 is important for pollen germination and sucrose-induced anthocyanin accumulation. Plant Physiol. 2008, 147, 92–100. [Google Scholar] [CrossRef]
  20. Leggewie, G.; Kolbe, A.; Lemoine, R.; Roessner, U.; Lytovchenko, A.; Zuther, E.; Kehr, J.; Frommer, W.B.; Riesmeier, J.W.; Willmitzer, L.; et al. Overexpression of the sucrose transporter SoSUT1 in potato results in alterations in leaf carbon partitioning and in tuber metabolism but has little impact on tuber morphology. Planta 2003, 217, 158–167. [Google Scholar] [CrossRef]
  21. Baker, R.F.; Leach, K.A.; Boyer, N.R.; Swyers, M.J.; Benitez-Alfonso, Y.; Skopelitis, T.; Luo, A.; Sylvester, A.; Jackson, D.; Braun, D.M. Sucrose transporter ZmSut1 expression and localization uncover new insights into sucrose phloem loading. Plant Physiol. 2016, 172, 1876–1898. [Google Scholar] [CrossRef] [PubMed]
  22. Jeong, Y.M.; Kim, N.; Ahn, B.O.; Oh, M.; Chung, W.H.; Chung, H.; Jeong, S.; Lim, K.B.; Hwang, Y.J.; Kim, G.B.; et al. Elucidating the triplicated ancestral genome structure of radish based on chromosome-level comparison with the Brassica genomes. Theor. Appl. Genet. 2016, 129, 1357–1372. [Google Scholar] [CrossRef] [PubMed]
  23. Elisabeth, G.; Alexandre, G.; Christine, H.; Ivan, I.; Ron, D.A.; Amos, B. ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2003, 31, 3784–3788. [Google Scholar]
  24. Chen, Y.J.; Yu, P.; Luo, J.C.; Jiang, Y. Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mamm. Genome 2003, 14, 859–865. [Google Scholar] [CrossRef] [PubMed]
  25. Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 2013, 30, 2725–2729. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Machanick, P.; Bailey, T.L. MEME-ChIP: Motif analysis of large DNA datasets. Bioinformatics 2011, 27, 1696–1697. [Google Scholar] [CrossRef] [Green Version]
  27. Xu, Y.; Zhu, X.; Gong, Y.; Xu, L.; Wang, Y.; Liu, L. Evaluation of reference genes for gene expression studies in radish (Raphanus sativus L.) using quantitative real-time PCR. Biochem. Biophys. Res. Commun. 2012, 424, 398–403. [Google Scholar] [CrossRef]
  28. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
  29. Wu, Z.; Li, T.; Liu, X.Y.; Yuan, G.Z.; Hou, H.Z.; Teng, N.J. A novel R2R3-MYB transcription factor LlMYB305 from Lilium longiflorum plays a positive role in thermos tolerance via activating heat-protective genes. Environ. Exp. Bot. 2021, 184, 104399. [Google Scholar] [CrossRef]
  30. Zhang, W.T.; Li, J.X.; Dong, J.H.; Wang, Y.; Xu, L.; Li, K.X.; Yi, X.F.; Zhu, Y.L.; Liu, L.W. RsSOS1 responding to salt stress might be involved in regulating salt tolerance by maintaining Na+ homeostasis in radish (Raphanus sativus L.). Horticulturae 2021, 7, 458. [Google Scholar] [CrossRef]
  31. Yu, C.L.; Sun, C.D.; Shen, C.J.; Wang, S.K.; Liu, F.; Liu, Y.; Chen, Y.L.; Li, C.Y.; Qian, Q.; Aryal, B.; et al. The auxin transporter, OsAUX1, is involved in primary root and root hair elongation and in Cd stress responses in rice (Oryza sativa L.). Plant J. 2015, 83, 818–830. [Google Scholar] [CrossRef]
  32. Clough, S.J.; Bent, A.F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 2010, 16, 735–743. [Google Scholar] [CrossRef]
  33. Li, W.; Sun, K.; Ren, Z.Y.; Song, C.X.; Pei, X.Y.; Liu, Y.A.; Wang, Z.Y.; He, K.L.; Zhang, F.; Zhou, X.J.; et al. Molecular evolution and stress and phytohormone responsiveness of SUT genes in Gossypium hirsutum. Front. Genet. 2018, 9, 494. [Google Scholar] [CrossRef]
  34. Vanbel, A.; Gamalei, Y.V. Ecophysiology of phloem loading in source leaves. Plant Cell Environ. 2010, 15, 265–270. [Google Scholar]
  35. Sun, L.; Deng, R.; Liu, J.; Lai, M.; Wu, J.; Liu, X.; Shahid, M.Q. An overview of sucrose transporter (SUT) genes family in rice. Mol. Biol. Rep. 2022, 49, 5685–5695. [Google Scholar] [CrossRef]
  36. Deol, K.K.; Mukherjee, S.; Gao, F.; Brûlé-Babel, A.; Stasolla, C.; Ayele, B.T. Identification and characterization of the three homeologues of a new sucrose transporter in hexaploid wheat (Triticum aestivum L.). BMC Plant Biol. 2013, 13, 181. [Google Scholar] [CrossRef] [Green Version]
  37. Wang, Y.; Chen, Y.; Wei, Q.; Wan, H.; Sun, C. Phylogenetic relationships of sucrose transporters (SUTs) in plants and genome-wide characterization of SUT genes in Orchidaceae reveal roles in floral organ development. Peer. J. 2021, 9, e11961. [Google Scholar] [CrossRef]
  38. Xu, G.; Guo, C.; Shan, H.; Kong, H. Divergence of duplicate genes in exon-intron structure. Proc. Natl. Acad. Sci. USA 2012, 109, 1187–1192. [Google Scholar] [CrossRef] [Green Version]
  39. Julius, B.T.; Leach, K.A.; Tran, T.M.; Mertz, R.A.; Braun, D.M. Sugar transporters in plants: New insights and discoveries. Plant Cell Physiol. 2017, 58, 1442–1460. [Google Scholar] [CrossRef] [Green Version]
  40. Chincinska, I.A.; Liesche, J.; Krügel, U.; Michalska, J.; Geigenberger, P.; Grimm, B.; Kühn, C. Sucrose transporter StSUT4 from potato affects flowering, tuberization, and shade avoidance response. Plant Physiol. 2008, 146, 515–528. [Google Scholar] [CrossRef] [Green Version]
  41. Kühn, C.; Hajirezaei, M.R.; Fernie, A.R.; Roessner-Tunali, U.; Czechowski, T.; Hirner, B.; Frommer, W.B. The sucrose transporter StSUT1 localizes to sieve elements in potato tuber phloem and influences tuber physiology and development. Plant Physiol. 2003, 131, 102–113. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Hoang, N.V.; Park, C.; Kamran, M.; Lee, J.Y. Gene regulatory network guided investigations and engineering of storage root development in root crops. Front. Plant Sci. 2020, 17, 762. [Google Scholar] [CrossRef] [PubMed]
  43. Wang, L.F.; Qi, X.X.; Huang, X.S.; Xu, L.L.; Jin, C.; Wu, J.; Zhang, S.L. Overexpression of sucrose transporter gene PbSUT2 from Pyrus bretschneideri, enhances sucrose content in Solanum lycopersicum fruit. Plant Physiol. Biochem. 2016, 105, 150–161. [Google Scholar] [CrossRef] [PubMed]
  44. Ma, Q.J.; Sun, M.H.; Liu, Y.J.; Hu, D.G.; Hao, Y.J. Molecular cloning and functional characterization of the apple sucrose transporter gene MdSUT2. Plant Physiol. Bioch. 2016, 109, 442–451. [Google Scholar] [CrossRef]
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Figure 1. Phylogenetic analysis of SUC genes in radish, Arabidopsis, B. oleracea, O. sativa, and Z. mays. SUC proteins were divided into five subgroups (SUT1, SUT2, SUT3, SUT4, and SUT5), and each color group represented a subgroup. Stars, circles, checkmarks, and triangles represent SUC proteins in radish, B. oleracea, Arabidopsis, O. sativa, and Z. mays, respectively.
Figure 1. Phylogenetic analysis of SUC genes in radish, Arabidopsis, B. oleracea, O. sativa, and Z. mays. SUC proteins were divided into five subgroups (SUT1, SUT2, SUT3, SUT4, and SUT5), and each color group represented a subgroup. Stars, circles, checkmarks, and triangles represent SUC proteins in radish, B. oleracea, Arabidopsis, O. sativa, and Z. mays, respectively.
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Figure 2. Conserved motifs and gene structure analysis of RsSUC genes. (A) Phylogenetic tree of RsSUC proteins. RsSUC proteins were divided into three subgroups (SUT1, SUT2 and SUT4), and each color group represented a subgroup. (B) Conserved motifs of RsSUC proteins. The 15 motifs were identified by using the MEME program, with each number of colored boxes representing a different motif; (C) Exon–intron structure of RsSUC genes. Boxes and lines represent exons and introns, respectively.
Figure 2. Conserved motifs and gene structure analysis of RsSUC genes. (A) Phylogenetic tree of RsSUC proteins. RsSUC proteins were divided into three subgroups (SUT1, SUT2 and SUT4), and each color group represented a subgroup. (B) Conserved motifs of RsSUC proteins. The 15 motifs were identified by using the MEME program, with each number of colored boxes representing a different motif; (C) Exon–intron structure of RsSUC genes. Boxes and lines represent exons and introns, respectively.
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Figure 3. Expression patterns of RsSUC genes during radish development. (A–G) display the relative expression levels of RsSUC1a, RsSUC1b, RsSUC1c, RsSUC1d, RsSUC2a, RsSUC2b, and RsSUC2c in leaves and taproots at 20, 40, 60, 80, and 100 days after sowing (DAS), respectively. Each bar shows the mean ± SD (n = 3). Values with different letters indicate a significant difference at p < 0.05 according to Duncan’s multiple range tests.
Figure 3. Expression patterns of RsSUC genes during radish development. (A–G) display the relative expression levels of RsSUC1a, RsSUC1b, RsSUC1c, RsSUC1d, RsSUC2a, RsSUC2b, and RsSUC2c in leaves and taproots at 20, 40, 60, 80, and 100 days after sowing (DAS), respectively. Each bar shows the mean ± SD (n = 3). Values with different letters indicate a significant difference at p < 0.05 according to Duncan’s multiple range tests.
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Figure 4. The subcellular localization of RsSUC1b in tobacco epidermal cells. The plasmids 35S::GFP and 35S::AUX1-RFP were used as the negative control and cell membrane marker, respectively. Scale bars = 50 μm.
Figure 4. The subcellular localization of RsSUC1b in tobacco epidermal cells. The plasmids 35S::GFP and 35S::AUX1-RFP were used as the negative control and cell membrane marker, respectively. Scale bars = 50 μm.
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Figure 5. RsSUC1b-overexpressing (OE) Arabidopsis plants response to the different sucrose concentrations. The morphology (A) and morphological index (B) were 20-day-old wide type (WT) and transgenic line seedlings after exogenous sucrose treatment. (C) The expression profiles of plant development-related genes of AtWOX4, AtKNAT1, and AtLBD3 in WT and transgenic plants under sucrose treatments of 6%. Each bar shows the mean ± SD (n = 3). Values with different letters indicate a significant difference at p < 0.05 according to Duncan’s multiple range tests. * indicates significant difference at p < 0.05, and ** indicates significant difference at p < 0.01 according to Student’s t-test.
Figure 5. RsSUC1b-overexpressing (OE) Arabidopsis plants response to the different sucrose concentrations. The morphology (A) and morphological index (B) were 20-day-old wide type (WT) and transgenic line seedlings after exogenous sucrose treatment. (C) The expression profiles of plant development-related genes of AtWOX4, AtKNAT1, and AtLBD3 in WT and transgenic plants under sucrose treatments of 6%. Each bar shows the mean ± SD (n = 3). Values with different letters indicate a significant difference at p < 0.05 according to Duncan’s multiple range tests. * indicates significant difference at p < 0.05, and ** indicates significant difference at p < 0.01 according to Student’s t-test.
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Figure 6. The phenotypes of RsSUC1b-overexpressing (OE) transgenic Arabidopsis plants. (A) Morphological phenotypes of WT and three independent transgenic lines in vegetative growth stage (A) and in bolting stage (B), bar = 1 cm. (C) The soluble sugar (total sugar, sucrose, fructose and glucose) contents of the transgenic lines and WT plants in leaves and pods. DW, dry weight. Each bar shows the mean ± SD (n = 3). Value with different letters indicates a significant difference at p < 0.05 according to Duncan’s multiple range tests.
Figure 6. The phenotypes of RsSUC1b-overexpressing (OE) transgenic Arabidopsis plants. (A) Morphological phenotypes of WT and three independent transgenic lines in vegetative growth stage (A) and in bolting stage (B), bar = 1 cm. (C) The soluble sugar (total sugar, sucrose, fructose and glucose) contents of the transgenic lines and WT plants in leaves and pods. DW, dry weight. Each bar shows the mean ± SD (n = 3). Value with different letters indicates a significant difference at p < 0.05 according to Duncan’s multiple range tests.
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Zhu, X.; Zhang, X.; Cao, Y.; Xin, R.; Ma, Y.; Wang, L.; Xu, L.; Wang, Y.; Liu, R.; Liu, L. Genome-Wide Identification of Sucrose Transporter Genes and Functional Analysis of RsSUC1b in Radish (Raphanus sativus L.). Horticulturae 2022, 8, 1058. https://doi.org/10.3390/horticulturae8111058

AMA Style

Zhu X, Zhang X, Cao Y, Xin R, Ma Y, Wang L, Xu L, Wang Y, Liu R, Liu L. Genome-Wide Identification of Sucrose Transporter Genes and Functional Analysis of RsSUC1b in Radish (Raphanus sativus L.). Horticulturae. 2022; 8(11):1058. https://doi.org/10.3390/horticulturae8111058

Chicago/Turabian Style

Zhu, Xiaofeng, Xiaoli Zhang, Yang Cao, Ruixian Xin, Yinbo Ma, Lun Wang, Liang Xu, Yan Wang, Rui Liu, and Liwang Liu. 2022. "Genome-Wide Identification of Sucrose Transporter Genes and Functional Analysis of RsSUC1b in Radish (Raphanus sativus L.)" Horticulturae 8, no. 11: 1058. https://doi.org/10.3390/horticulturae8111058

APA Style

Zhu, X., Zhang, X., Cao, Y., Xin, R., Ma, Y., Wang, L., Xu, L., Wang, Y., Liu, R., & Liu, L. (2022). Genome-Wide Identification of Sucrose Transporter Genes and Functional Analysis of RsSUC1b in Radish (Raphanus sativus L.). Horticulturae, 8(11), 1058. https://doi.org/10.3390/horticulturae8111058

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