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Article

Genome-Wide Identification and Expression Profiling Reveal the Potential Functions of the SWEET Gene Family during the Sink Organ Development Period in Apple (Malus × domestica Borkh.)

by
Peixian Nie
1,2,3,
Gongxun Xu
1,2,
Bo Yu
1,2,
Deguo Lyu
1,2,
Xiaomin Xue
3 and
Sijun Qin
1,2,*
1
College of Horticulture, Shenyang Agricultural University, Shenyang 110866, China
2
Key Lab of Fruit Quality Development and Regulation of Liaoning Province, Shenyang Agricultural University, Shenyang 110866, China
3
Shandong Institute of Pomology, Taian 271000, China
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(8), 1747; https://doi.org/10.3390/agronomy12081747
Submission received: 6 July 2022 / Revised: 16 July 2022 / Accepted: 22 July 2022 / Published: 25 July 2022
(This article belongs to the Section Horticultural and Floricultural Crops)
Browse Figures
Versions Notes

Abstract

:
Sugars Will Eventually be Exported Transporters (SWEETs) play important roles during plant growth and development. Bioinformatics revealed 27 SWEET genes in the apple (Malus × domestica Borkh.) genome and classified them into four clades. These genes are unevenly located on 12 chromosomes, and 8 tandem-repeat genes and 18 fragment-repeat genes are present in the MdSWEET family. PlantCARE-based prediction of cis-acting elements of the MdSWEET promoter suggested that most MdSWEETs may be expressed specifically in the phloem and respond to hormones and stresses. qRT-PCR results showed that expression patterns of MdSWEETs displayed pronounced differences in different tissues/organs and different stages of apple fruit development. MdSWEET5, MdSWEET7, and MdSWEET21 were strongly expressed in mature leaves, MdSWEET2, MdSWEET12, MdSWEET13, MdSWEET14, MdSWEET17, and MdSWEET22 were highly expressed in peduncles, MdSWEET4 was highly expressed in young leaves, MdSWEET3, MdSWEET11, MdSWEET15, MdSWEET16, MdSWEET19, MdSWEET24, and MdSWEET25 were highly expressed in different parts of flowers, and MdSWEET1, MdSWEET6, MdSWEET8, MdSWEET9, MdSWEET10, MdSWEET18, MdSWEET20, MdSWEET23, and MdSWEET26 were strongly expressed in fruits. MdSWEET8 showed higher expression in the early stage of fruit development, MdSWEET1, MdSWEET10, and MdSWEET27 were highly expressed in the middle stage of fruit development, and MdSWEET6, MdSWEET9, MdSWEET18, MdSWEET20, MdSWEET23, and MdSWEET26 were sharply upregulated in the late developmental period. Our study could facilitate SWEET functional analysis in different tissue/organs and in sugar accumulation throughout the development and ripening of apple fruits. These findings provide potential opportunities to increase sugar accumulation in fruit, thereby improving fruit quality and yield.

1. Introduction

In higher plants, photosynthates are produced in the mesophyll cells of photosynthesizing leaves and are then transported via the phloem to different sink organs, such as the roots, stems, reproductive structures, and vegetative storage organs to provide the carbon skeletons and energy needed for growth and synthesis of storage reserves. Phloem unloading is a crucial step during photoassimilate transport and partitioning into sink organs. Generally, there are three potential pathways for phloem unloading, i.e., the symplasmic pathway, the apoplasmic pathway, and a third that alternates between apoplasmic and symplasmic unloading. Sugar transporters play an important role in the apoplasmic phloem-unloading strategy. To date, three types of sugar transporters have been characterized, i.e., monosaccharide transporters (MSTs) [1], sucrose transporters (SUTs) [2], and Sugars Will Eventually be Exported Transporter (SWEET) proteins [3]. SWEETs, first identified in Arabidopsis by Chen et al. [4], are a new type of sugar transporters. Unlike the major facilitator superfamily (MST and SUT), SWEETs are described as uniporters/facilitators that mediate both uptake and efflux of different sugar substrates across cell membranes along a concentration gradient [5]. In addition, these transport processes are pH- and energy-independent [5]. In angiosperms, the SWEET protein family consists of 20 paralogs on average, whose members perform different physiological functions during plant growth and development [6]. In Arabidopsis, 17 members of this family are divided into four clades. Clades I and II include AtSWEET1–3 and AtSWEET4–8, respectively, which primarily mediate glucose uptake; moreover, clade III includes AtSWEET9–15, which primarily transport sucrose; lastly, clade IV includes AtSWEET16 and AtSWEET17, which are localized in the tonoplast membrane and predominantly transport fructose [7]. To date, genome-wide identification and characterization of the SWEET gene family have been performed for a variety of plants [3,8,9,10,11].
SWEETs are involved in the transport and distribution of photoassimilates as they play important roles in phloem loading and unloading. Specifically, during apoplasmic loading, SWEETs reportedly mediate sugar efflux from the phloem parenchyma into the phloem apoplasm [3,6]. Moreover, SWEETs play an indispensable role in sugar unloading from phloem complexes to sink tissues through the apoplastic pathway. In tomatoes, SlSWEET1a is highly expressed in the veins of young leaves and regulates glucose accumulation in the parenchyma cells [12]. SlSWEET1a virus-induced silencing causes a significant reduction in the levels of fructose and glucose in young leaves, concomitant with a significant increase in the concentrations of these sugars in mature leaves [12]. High expression of SlSWEET1a has also been observed in flowers [12]. Similarly, AtSWEET11, AtSWEET12, and AtSWEET15 are highly expressed during the filling stage of seed development and are involved in transporting sucrose from the seed coat to the embryo [7]. Lastly, AtSWEET9 was found to be an important factor in nectar secretion [13].
Fleshy fruits are the end product of most horticultural crops and they also serve as a strong sink organ. The relationship between SWEETs and sugar accumulation in fruits has been studied in various horticultural crops, such as apples [14,15], tomatoes [9], cucumbers [11], pineapples [16], grapes [8], and pears [17]. VvSWEET4, VvSWEET7, VvSWEET10, VvSWEET11, VvSWEET15, and VvSWEET17 are highly expressed in grapes, where they are presumed to play a role in sugar transport and accumulation [8]. The pear (Pyrus bretschneideri) genome contains 18 SWEET genes [17], among which PbSWEET5 has a negative correlation with sucrose levels during pear fruit development [17]. In the Ussurian pear (Pyrus ussuriensis), the WRKY transcription factor PuWRKY31 is bound to the PuSWEET15 promoter and induces its transcription [18]. The tomato genome contains 29 SWEET genes [9], among which SlSWEET7a and SlSWEET14 encode proteins that are localized to the plasma membrane, where they actively transport fructose, glucose, and sucrose, and are closely associated with sugar transport and accumulation in the fruit [19]. Hu et al. [11] identified 17 SWEET genes from the cucumber genome and reported that CsSWEET7a, CsSWEET7b, and CsSWEET15 may be involved in sugar accumulation in fruit. Similarly, 39 SWEET genes have been identified in the pineapple genome, wherein AnmSWEET5 and AnmSWEET11 display the highest transcript abundance during fruit development [16]. ClSWEET3, encoding a hexose transporter located in the plasma membrane of parenchyma cells in watermelon, is one of the key genes that is responsible for the derivation of the modern sweet watermelon from a nonsweet accession during the domestication of this species [20].
Apples (Malus domestica) are one of the most popular and commonly cultivated fruit trees that are grown in temperate regions worldwide, and their genome sequences have been released [21]. As for the apple, Wei et al. [14] identified 29 SWEET genes and studied their expression in the early-ripening “Gala” variety of apple. Furthermore, Zhen et al. [15] identified 25 of the apple SWEET members and compared their expression levels between the high-sugar variety “K9” and the low-sugar variety “Lion Mountain 2” at three developmental stages (30, 60, and 90 days after anthesis). “Fuji” is one of the most popular and widely produced apple varieties worldwide. “Hanfu”, a cultivar bred from “Dongguang” and “Fuji”, has the advantages of strong cold resistance, high yield and self-fruitfulness [22]. As a late-ripening variety, “Hanfu” takes approximately 160 days from flowering to harvest, and its characteristic developmental rhythm and sugar accumulation pattern are different from those of the early-ripening varieties. In this study, the expression and distribution of MdSWEET genes in the different tissues/organs of Fuji apple cultivar “Hanfu” were analyzed in detail using qRT-PCR. Furthermore, the correlation between the level of MdSWEET expression and sugar accumulation during fruit development and ripening was investigated at 15-day intervals after bloom. Our study provides the basis for future analysis of the function of SWEET genes during phloem unloading in sink organs and in sugar accumulation in fruits during the development of Fuji apples.

2. Materials and Methods

2.1. Identification and Characterization of SWEET Family Genes from the Apple Plant

The genome of M. domestica (GDDH13 Version 1.1), gene_models_20170612.gff3, GDDH13_1-1_prot.fasta, GDDH13_1-1_mrna.fasta, and GDDH13_1-1_formatted.fasta files, were downloaded from the Genome Database for Rosaceae. The GDDH13_1-1_cds.fasta file was obtained using the TBtools v1.075 software based on information from the gene_models_20170612.gff3 files [23]. Two methods were used to identify SWEET family members in apple: Hidden Markov Model search (HMMER) with the Hidden Markov Model of MtN3/saliva domain (PF03083) from PFAM database (http://pfam.xfam.org/ (accessed on 25 November 2020)), and BLASTp searches using the protein sequences of SWEET from Arabidopsis downloaded from the TAIR database (https://www.arabidopsis.org/ (accessed on 27 November 2020)) as queries. Candidate protein sequences obtained using these two methods were submitted to the Conserved Domains Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi/ (accessed on 7 December 2020)) and PFAM (http://pfam.xfam.org/ (accessed on 7 December 2020)) to verify the structure of MtN3/saliva; thereafter, the redundant hits were removed to obtain candidate proteins. The parameters (molecular weight, theoretical isoelectric point (pI), instability index, aliphatic index, and grand average of hydropathicity) of SWEET proteins were determined by the online ProtParam tool (http://web.expasy.org/protaram/ (accessed on 9 December 2020)) using amino acid sequences.

2.2. Multi-Sequence Alignment and Evolutionary Tree Construction of SWEETs

Clustal X2 was used to compare MdSWEET protein sequences with those of the OsSWEET2b protein, the only eukaryotic SWEET protein whose 3D structure has been resolved [24]. Thereafter, the results were submitted to the Essprint online website (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi/ (accessed on 10 December 2020)) for visualization. MEGA X [25] was used to construct a phylogenetic tree of the SWEET family from Arabidopsis, tomato, and apple. The parameter settings were as follows: alignment by Clustal W; phylogeny analysis based on the neighbor-joining (NJ) method; test of phylogeny based on the bootstrap method using 1000 bootstrap replications; and use of the Poisson model. The resulting file was submitted to the EVOLVIEW V2 website (http://www.evolgenius.info/evolview/ (accessed on 10 December 2020)) for mapping.

2.3. Prediction of the Secondary and 3D Model Structure of MdSWEET Proteins

The secondary structures of MdSWEET proteins were predicted using the SOPMA secondary structure prediction method (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_%20sopma.html/ (accessed on 15 December 2020)). The 3D model structure of the MdSWEET proteins was predicted using Protein Homology/Analogy Recognition Engine V 2.0 to obtain predicted structures [26].

2.4. Gene Structure, Conserved Motif, Chromosomal Location, and Duplication Analysis

TBtools v1.075 was used to map the exon–intron structure of the SWEET genes in the chromosome [23]. The conserved motifs of the SWEET proteins were analyzed using MEME v5.4.1 (https://meme-suite.org/meme/tools/meme/ (accessed on 15 December 2020)) and mapped by TBtools [25]. MCscanX was used to determine the duplication events for MdSWEET genes of apple [27].

2.5. Promoter Cis-Element Analysis of MdSWEETs

A 2000 bp sequence upstream of the transcription start site of the MdSWEET genes was extracted from the GDDH13_1-1_formatted.fasta file and submitted to PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 10 January 2021)) for analysis of cis-acting regulatory elements. The results were visualized using TBtools v1.075 [23].

2.6. Plant Materials and Experimental Treatments

Ten-year-old “Hanfu” apple trees (M. domestica) grafted on interstock GM256 and rootstock M. baccata were grown at a spacing of 2 (row) × 4 (interval) m in North–South rows at the experimental station of Shenyang Agricultural University. Tissues of young leaves, flowers, peduncles, mature leaves, and fruits were sampled randomly. Petals, stamens, styles (including stigma), and ovaries (including receptacle and nectaries) were sampled separately. Mature leaves were collected 30 days after bloom (DAB). Fruits were sampled at 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, and 160 DAB. Tissue disks (4 mm diameter × 4 mm thick) of the sepal bundle (SB) vascular region, petal bundle (PB) vascular region, and flesh parenchyma (FP) cell region were excised from the equatorial portion of the apple fruit (140 DAB) as shown in Figure S1. All samples were immediately frozen in liquid nitrogen, and each was sampled three independent times.

2.7. Measurement of Sugar Content

The fructose, glucose, sucrose, and sorbitol contents of apple fruits were determined using high-performance liquid chromatography (HPLC; 1260 Series, Agilent Technologies, Beijing, China), as described by Li et al. [18]. The total soluble sugar of fruits was determined using the anthrone-sulfuric acid colorimetry method [28].

2.8. RNA Extraction and Quantitative Real-Time PCR

Total RNA was extracted from the samples using a polysaccharide, polyphenol plant total RNA extraction kit (TIANGEN, Beijing, China). Complementary DNA (cDNA) was synthesized using a one-step gDNA removal and cDNA synthesis SuperMix kit (AT311, TRANSGEN, Beijing, China). qRT-PCR was performed in a total volume of 20 μL, containing 10 μL of 2 × PerfectStart™ Green qPCR SuperMix (AQ601, TRANSGEN, Beijing, China), 1 μL cDNA, and 1 μM primer for each gene, using CFX96 Real-Time System (CFX96, Bio-Rad, Hercules, CA, USA). Primers used for qRT-PCR are shown in Table S1. A housekeeping gene, the apple β-actin gene, was used as a reference gene. Primers were synthesized by Shanghai Sangon Biotechnology Co., Shanghai, China. The following conditions were used for the PCR procedure: 94 °C pre-denaturation for 30 s; 94 °C for 5 s, 58 °C for 30 s, 72 °C for 1 min, 40 cycles; and 72 °C for 10 min. Relative mRNA expression was calculated using the 2−∆∆CT method [29,30].

2.9. Statistical Analysis

Data were collated using MS Excel 2010, and statistical analyses were performed for data correlation using Duncan’s multiple range test in IBM SPSS Statistics 23, and data were graphed with Sigma Plot 10.0. Each treatment included three independent biological replicates. Significant and highly significant differences were indicated by * p < 0.05 and ** p < 0.01, respectively.

3. Results

3.1. Identification and Characterization of SWEET Gene Family Members in Apple

A total of 27 putative SWEET proteins were identified in apple tissues using HMMER and BLASTp, which were confirmed to possess the structure of MtN3/saliva (according to CDD and PFAM databases; Table 1). These 27 MdSWEET genes were named MdSWEET1-27 according to their chromosomal location. The length of these SWEET proteins ranged from 215 (MdSWEET11) to 340 (MdSWEET18) amino acid (aa) residues. The molecular weight varied from 24,637.52 (MdSWEET11) to 38,117 Da (MdSWEET18). The pI values of putative SWEET family members ranged from 5.13 (MdSWEET15) to 9.90 (MdSWEET8); of these, 21 members (77.8%) had a pI higher than 7.0, suggesting that these proteins were slightly alkaline. The instability index of SWEETs was estimated between 20.71 (MdSWEET13) and 47.41 (MdSWEET17); among them, the instability index values of 11 members were greater than 40, suggesting that these proteins were unstable. The grand average of hydrophilicity ranged from 0.381 (MdSWEET20) to 0.959 (MdSWEET7), indicating that SWEETs were all hydrophobic proteins. Furthermore, most of the SWEETs had seven transmembrane (TM) domains, with the exception of MdSWEET 1, 11, and 18, which had six, six, and nine TM domains, respectively.

3.2. Multi-Sequence Alignment and Phylogenetic Analysis of the SWEET Proteins

The conserved amino acid residues of MdSWEETs were analyzed using the rice OsSWEET2b (PDB ID: 5xpd.1A) as a template [24]. The results showed that all 16 amino acid residues were conserved; among them, T30, P47, Y48, Y61, and N77 were on triple-helix bundles 1 (THB1), and R131, G136, P150, V163, M166, F168, S171, N197, G200, Q207, and Y211 were on THB2, revealing that THB2 was more conserved than THB1 (Figure 1). In addition, the proline residues of TM1, TM2, TM5, and TM7 were conserved, except for TM1 of MdSWEET1 and TM7 of MdSWEET18, in which proline was substituted by cysteine and serine.
To evaluate the evolutionary relationship and the classification of the MdSWEET gene family members, a phylogenetic tree of the SWEETs was constructed based on the amino acid sequences of Arabidopsis (17), tomatoes (22), and apples (27; Figure 2). Based on phylogenetic analysis, the 27 putative MdSWEET proteins were divided into four clades: clade Ι (12 genes), clade II (5 genes), clade III (9 genes), and clade IV (one gene). The sizes of clade Ι and III were significantly larger than those of the other two clades.

3.3. Secondary and 3D Model Structure of MdSWEET Proteins

Secondary structure prediction showed that apple MdSWEET proteins were primarily α-helices (35.74–53.97%) and random coils (25.10–42.62%), followed by extension chains and β-turns (Table 2). Further, 23 SWEET proteins had similar tertiary structures, except for MdSWEET2, 7, 17, and 27 (Figure 3).

3.4. Gene Structural and Conservative Motif Analyses of the MdSWEET Gene Family in Apple

The MEME program was used to predict the sequence structure information of MdSWEET genes (Figure 4). A total of 10 conserved motifs were obtained; the apple SWEET members included 6 to 8 motifs, whereas motifs 1, 3, 4, and 5 were observed in all MdSWEET members. In addition, the SWEET members in clade III lack motif 7 and instead had their specific motifs (i.e., motifs 9 and 8). The exons and introns of MdSWEETs were analyzed to distinguish the differences in the structure of the MdSWEETs. The number of introns in the 27 MdSWEETs ranged from three to nine, and the number of exons was between four and six. As shown in Figure 4C, 21 MdSWEET genes had five introns, except for MdSWEET1 with three, MdSWEET8, 18, and 22 with four, and MdSWEET2 and 17 with six introns.

3.5. Chromosome Localization and Gene Duplication Analysis of MdSWEET Genes

Chromosomal localization analysis was performed to understand the distribution of the MdSWEETs on chromosomes (Figure 5). The MdSWEETs are unevenly distributed on the 17 chromosomes of the apple genome (2 n  =  34). Chromosome 10 contains most MdSWEET genes (5), whereas chromosomes 1, 3, 4, 12, 16, and 17 contain one gene each. Notably, many MdSWEET genes were concentrated at both ends of the chromosomes. Gene duplication plays a key role in the occurrence of novel functions and gene family expansion; therefore, we analyzed the duplication events that occurred in the MdSWEET gene family using the MCScanX software (Table 3). Nineteen SWEET gene pairs were detected as duplicated gene pairs, among which four pairs were tandem duplications and fifteen were associated with a segmental duplication event. Segmental duplication events may be one of the main reasons for the origin of the MdSWEET gene family during evolution. In addition, the duplication events of MdSWEET genes occurred primarily in clades I and III.

3.6. Cis-Acting Elements in the Promoter Regions of MdSWEET Genes

To investigate the possible regulatory functions of MdSWEET family genes in relation to hormone and abiotic stress, the promoter regions of 27 MdSWEET genes were analyzed using the PlantCARE online tool to identify the putative stress-associated and plant hormone-related cis-acting elements. The results showed that the promoter of MdSWEET genes contained not only a large number of core elements (CAAT-box and TATA-box) and light-responsive elements, but also a variety of elements related to hormones and stress. The elements (not including core elements and light-responsive elements) can be generally classified into three types: plant growth and development, hormone responses, and stress responses (Figure 6). A total of 10 types of elements related to plant growth and development, including AC-II, as-1, meristem expression (CAT-box), endosperm expression (GCN4-motif), zein metabolism regulation (O2-site), palisade mesophyll cell-associated (HD-zip 1), flavonoid biosynthetic gene regulation (MBSI), cell cycle regulation (MSA-like), seed-specific regulation (RY-element), and circadian control (circadian) were predicted in the promoters of MdSWEET genes. These contained six types of stress-responsive elements, including anaerobic induction (ARE), anoxic specific inducibility (GC-motif), low-temperature responsive (LTR), MYB binding site involved in drought inducibility (MBS), wound-responsive (WUN-motif), and defense and stress responsiveness (TC-rich), and 11 types of hormone-responsive elements, including abscisic acid-responsive (ABRE), CGTCA-MeJA, ERE, GARE-motif, P-box, TATC-box, TCA-element, TGACG-MOTIF, TGA-element, W box, and AuxRR-core. A total of 486 cis-acting elements (not including core elements and light-responsive elements) were predicted, and hormone-responsive elements accounted for 53.1%. It was apparent that the expression of apple MdSWEET genes was regulated by hormones.
In addition, we performed a tissue-specific analysis of 27 MdSWEET genes and found that 19 MdSWEET genes contained a total of 42 phloem tissue-specific cis-acting elements (as-Ι), 17 genes contained a total of 24 root-specific expression cis-acting elements (W box), 4 genes contained four seed-specific expression cis-acting elements (RY-element and AACA_motif), 2 genes contained two endosperm-specific expression cis-acting elements (GCN4_motif), and 1 gene contained one vascular bundle-specific expression cis-acting element (AC-II; Figure 6). Among these, 14 genes contained multiple tissue-specific expression cis-acting elements simultaneously.

3.7. Expression Patterns of MdSWEET Genes in Different Apple Tissues/Organs and Developmental Stages

To comprehensively decipher the potential functions of MdSWEETs, the expression of 27 MdSWEET genes in different tissues/organs and developmental stages of apple was detected by qRT-PCR (Figure 7). The results showed that expression of MdSWEET genes was tissue-specific. For example, MdSWEET5, 7, and 21 were strongly expressed in mature leaves (source organ); MdSWEET5 and 7 belong to clade Ι, whereas MdSWEET21 belongs to clade IV. MdSWEET14, 13, 22, 12, 2, and 17 were strongly expressed in peduncles (transport system); all of them belong to clade Ι, except for MdSWEET14 (clade III). MdSWEET4 belongs to clade Ι and was highly expressed in young leaves (sink organs). In turn, MdSWEET24, 11, 16, 19, 3, 25, and 15 were highly expressed in flowers (sink organs), and all of them belong to clade IΙ or clade III, except for MdSWEET15, which belongs to clade Ι. These seven genes that were predominantly expressed in flowers were selected and their expression levels were analyzed in different floral tissues (Figure 8A). The results showed that MdSWEET15 was predominantly expressed in petals; MdSWEET3 and 19 were highly expressed in ovaries, and MdSWEET11, 16, 24, and 25 had the highest expression level in stamens. MdSWEET1, 6, 8, 9, 10, 18, 20, 23, and 26 were strongly expressed in fruits (sink organs). To gain further insight into the possible roles of these nine genes in phloem unloading in apple fruit, we analyzed the expression of these genes in different regions of the fruit (Figure 8B). MdSWEET8, 10, and 18 were highly expressed in PB, whereas MdSWEET1, 9, 20, and 23 showed the highest expression level in SB, and MdSWEET6 and 26 were primarily expressed in FP.
To better understand the potential functions of MdSWEET genes in different developmental stages of apple fruit, the expression patterns of all 27 MdSWEET genes were analyzed every 15 DAB (Figure 7). The expression patterns of MdSWEET genes displayed evident differences during apple fruit development. For example, MdSWEET8 showed a high expression in the early stage of fruit development (15 DAB), whereas MdSWEET10, 27, and 1 were highly expressed in the middle stage of fruit development (45–90 DAB); in turn, the expressions of MdSWEET18, 23, 9, 6, 26, and 20 were sharply upregulated in the late developmental period (150–160 DAB).

3.8. Relationship between the Expression of MdSWEET Genes and the Concentration of Major Sugars during Fruit Development of the “Hanfu” Apple

During the whole fruit development period, the total soluble sugar content increased continuously, and the content of fructose and glucose showed a similar tendency, whereas the content of sucrose increased gradually from 105 DAB (Figure 9A). Among the four sugars, the percentage of fructose and glucose was always at a high level, and the percentage of sucrose reached 30.10% in mature fruits (Figure 9B). Unlike these three sugars, the content and percentage of sorbitol were highest at 15 DAB after which they remained at a lower level. The relationship between the expression of MdSWEET genes and concentration of major sugars during fruit development of the “Hanfu” apple was studied. Correlation analysis showed that the expression of MdSWEET4, 6, 9, 12, 13, 19, 20, 23, and 26 was significantly and positively correlated with total sugar accumulation during fruit ripening (Table 4).

4. Discussion

The SWEET gene family plays important roles in various physiological and biochemical processes, such as phloem loading [3,31,32], seed filling [33,34], and nectar secretion [13]. Genome-wide studies of the SWEET family genes have been conducted in many plants such as Arabidopsis [4], rice [35], apples [14,15], tomatoes [9], soybean [36], cucumbers [11], cotton [37], and pineapples [16]. In the present study, we identified 27 MdSWEET genes in the apple genome, a number that differs from that reported by Wei et al. [14] and Zhen et al. [15], likely because of the different criteria used in each case to identify the candidate members. The criteria used herein were that the candidate members contain at least 200 amino acids and each of them have two MtN3/saliva structural domains. For this part, Wei et al. [14] used a number of α-transmembrane helices greater than four as the criterion to determine the members of the SWEET gene family. Thus, two genes (Md09G1175800 and Md14G1079800), which contain more than four α transmembrane helices but have only one MtN3_slv structural domain, were not identified as SWEET genes in this study. We identified two other genes, namely, MdSWEET1 (MD01G1215700) and MdSWEET12 (MD10G1012200), as members of the SWEET gene family, in addition to the 25 SWEET genes identified by Zhen et al. [15]. The proteins of these two genes contain two MtN3/saliva domains, as analyzed using CDD and PFAM (Table S2), and their 3D structures are similar to those of other family members (Figure 3). Therefore, these two genes were identified as candidate members of the SWEET gene family in this study.
The membership in a clade can slightly define the substrate specificity of SWEETs. All 27 apple SWEETs were phylogenetically divided into four clades based on the classifications of SWEETs in Arabidopsis. Based on the research on Arabidopsis, members in clades I (MdSWEET 1, 2, 4–7, 13–17) and II (MdSWEET 8, 11, 22, 25, and 27) may transport monosaccharides, those in clade III (MdSWEET 3, 9, 10, 18–20, 23, 24, and 26) are predominantly involved in sucrose uptake, and the one gene in clade IV (MdSWEET16) may mediate fructose transport [4,7,38,39]. Gene duplication, which can lead to the expansion of gene families and the acquisition of new gene functions, is important in the evolution of plant gene families as it improves the adaptability of plants [40,41]. There were eight tandem-repeat genes and 18 fragment-repeat genes in the MdSWEET family. We hypothesized that together, tandem and segmental duplication contributed to the expansion of the MdSWEET gene family, and that segmental duplication may play a dominant role. This result was consistent with that of a study in cotton [37].
Plant SWEETs are differentially expressed in several tissues. In photosynthetic organs, SWEETs participate in the export of sucrose to the apoplast. Sucrose transporters AtSWEET11 and 12 are highly expressed in a subset of leaf phloem parenchyma cells and play key roles during phloem loading [3]. Consistently, StSWEET11 in potato [42] and ZjSWEET2.2 in jujube [32] were also demonstrated to play roles in sugar loading processes. In turn, MdSWEET5, 7, and 21 were highly expressed in mature leaves (Figure 7), and the promoter regions of these genes all contained phloem tissue-specific expression elements (Figure 6). In addition, MdSWEET5 and 7 belong to clade Ι, whereas MdSWEET21 belongs to clade IV. These three genes might be involved in sugar transport within source tissues, but further studies are needed to elucidate their specific functions. SWEETs are also involved in sugar unloading from phloem to sink tissues through the apoplastic pathway. In tomatoes, SlSWEET1a is strongly expressed in the veins of young leaves (sink organs) as a glucose transporter; it plays a key role in the uptake of glucose from the apoplast to the parenchyma cells [12]. MdSWEET4 was abundantly expressed in young leaves (Figure 7), indicating that it may play a similar role in regulating sugar accumulation in young leaves as SlSWEET1a. SWEETs also play important roles in the growth and development of reproductive organs, such as flowers, fruits, and seeds, by mediating phloem unloading of sugar. In this study, there were seven SWEET genes (MdSWEET24, 11, 16, 19, 3, 25, and 15) highly expressed in flowers (Figure 7). Specifically, MdSWEET15 was predominantly expressed in petals, MdSWEET3 and 19 were highly expressed in ovaries, and MdSWEET11, 16, 24, and 25 were abundantly expressed in stamens. In comparison, in Arabidopsis, eight SWEET genes (AtSWEET15, 14, 13, 8, 7, 5, 4, and 1) were highly expressed in flowers [4,43]; grape VvSWEET5a and 5b were also highly expressed in floral organs [8]; and seven SWEETs are sequentially expressed during flower development in Jasminum sambac [44]. AtSWEET15 is continuously expressed during pollen maturation and germination [45]; AtSWEET8 is highly expressed in the tapetum and is involved in glucose transport [46]; AtSWEET5 is specifically expressed in pollen grains and functions as a transporter of glucose and galactose [47]; these SWEETs are all involved in the transport of sugar for pollen nutrition during pollen development. The paralogous homologous gene pairs MdSWEET11/25, homologous to AtSWEET5, were highly expressed in stamens (Figure 8A), suggesting roles in generative cell development. AtSWEET9 mediates sugar efflux in the nectary parenchyma [13]; NEC1, homologous to AtSWEET9, is also nectary specific, and its silencing leads to male sterility [48,49]. MdSWEET3/19, a AtSWEET9 homolog in apple, were highly expressed in the ovary (including nectaries; Figure 8A), suggesting that they might have similar functions.
SWEET genes also play important functions during the development of fleshy fruits. Although research on the identification and function of these genes has only recently begun, substantial progress has been made. At present, most researchers study the function of SWEET genes in flesh fruit primarily by comparing gene expression between plant materials with different fruit sugar contents. For example, by comparing the transcriptomes of the “Nanguo” pear and its bud sport with higher sucrose content than the “Nanguo” pear, researchers found that PuSWEET15 was expressed at higher levels in the fruit of the bud sport as a sucrose transporter [18]. Overexpression of PuSWEET15 in the “Nanguo” pear fruit increased the fruit’s sucrose content, whereas its silencing in the bud sport fruit reduced the sucrose content [18]. In watermelons, a cultivated line with high-sugar content and a nonsweet wild accession were studied [20]. The results of this study revealed that ClSWEET3, which mediates hexose uptake into fruit cells from the intercellular space, played a key role in the derivation of the modern sweet watermelon from nonsweet accession during domestication [20]. Similarly, EjSWEET15 expression in fruits of mutant white-flesh loquats was significantly higher than that in the wild-type red-flesh loquats [50]. In apples, comparative research was conducted in the high-sugar variety “K9” and the low-sugar variety “Lion Mountain 2”. The results showed that MdSWEET9b and MdSWEET15a, which were significantly associated with fruit sugar content, were likely candidates for the regulation of sugar accumulation in apple fruit [15]. However, more studies are needed to determine the idiographic function of MdSWEET9b and MdSWEET15a during flesh fruit development in apple. In contrast, we paid more attention to the function of SWEETs over the entire developmental period of apple fruits to detect the expression of MdSWEET genes and investigate its relationship with sugar accumulation throughout the development and ripening of “Hanfu” apple fruits.
Apple fruit development consists of an early fruit growth phase involving exponential cell division, followed by mid- and late-fruit linear growth phases during which the fruit enlarges to its mature size through extensive cell expansion. Early fruit growth extends from ~8 until ~30 DAB, whereas mid- and late-fruit growth may extend up to 120–180 DAB. In apples, fruit growth is particularly sensitive to carbohydrate limitation during the early stages of fruit development. Significantly, MdSWEET8 was highly expressed in apple fruit 15 DAB (Figure 7), suggesting that it might play a role in fruit-set and early fruit development. Plasma membrane–localized SlSWEET7a and SlSWEET14, which were also primarily expressed in the early stage of fruit growth, could be directly or indirectly involved in sucrose unloading in tomato fruits [19]. From 30 and until 60 DAB, fruits continued to grow because of cell expansion, and starch content increased rapidly but low levels of sugars accumulated in apple fruits. MdSWEET10 was highly expressed in fruits 45 DAB, suggesting that this gene might be involved in the rapid accumulation of starch in apple fruit during this period. Beginning at approximately 60 DAB and until fruits are fully ripe, the level of starch declines concomitant with an increase in soluble sugar, especially fructose. MdSWEET27 and 1 were highly expressed in fruit at 75 and 90 DAB, respectively. The former belongs to clade II and the latter to clade I; furthermore, their proteins may both transport monosaccharides. Hence, they might play roles in the rapid accumulation of sugar in the fruit by mediating the unloading of monosaccharides in apple fruits. At the late stage of fruit development, starch breaks down, and soluble sugars accumulate massively as fruits ripen faster; the expressions of MdSWEET6, 9, 20, 23, and 26 were sharply upregulated in fruits at 150 and 160 DAB; thus, these genes might be involved in the progress of apple fruit maturation.
In fleshy fruits that accumulate high sugar concentrations, the apoplasmic unloading pathway effectively prevents the return of sugars from the fruit sieve elements. Phloem unloading of photoassimilate follows the apoplasmic unloading pathway in “Golden Delicious” apple fruits [51], and the unloading pathway does not change with varieties [28]. Compared with many other species, apples and other fruits in Rosaceae are unique in that both sorbitol and sucrose are the primary carbohydrates transported over long distances; specifically, sorbitol accounts for nearly 80% of long-distance sugar transport [52,53]. The concentrations of sorbitol (1.3–11.6 mM) and sucrose (0.17–0.41 mM) that reach the vascular bundles of the fruit [52] are both much lower than the concentrations of sorbitol (36 mM) and sucrose (16 mM) in the free space [53]. The plasmodesmata between the sieve element/companion cell (SE/CC) complex and the surrounding parenchyma cells are absent. Therefore, sugars (sucrose and sorbitol) in the SE/CC complex are pumped to the free space by transporters such as sucrose transporter/sorbitol transporter (SUT/SOT) localized to the plasma membrane of the SE/CC complex [3,54] against a concentration gradient. Sugars in the free space are broken down to hexoses by cell wall acid invertase localized on the sieve element and companion cell walls. Hexoses then enter the phloem parenchyma cells assisted by the hexose transporters localized in the plasma membrane of the phloem parenchyma cells [51]. However, the emergence of SWEETs has provided a new perspective on phloem unloading. SWEETs have been investigated in detail during phloem loading of Arabidopsis leaves and phloem unloading in seeds, but the role of the unloading process in fleshy fruits has not been thoroughly analyzed. Unloading and loading are generally considered as two opposite processes. Arabidopsis genes AtSWEET11 and AtSWEET12 of clade III, localized in the plasma membrane of phloem parenchyma cells in the leaves, mediated export from parenchyma cells into the cell wall space [3]. Similar findings have been observed in pear leaves [55]. During apple fruit development, many SWEETs are highly expressed, and some of them are significantly and positively correlated with total sugar accumulation. MdSWEETs may mediate the transport of sucrose or sorbitol in the free space of the phloem to the phloem parenchyma cells along the sugar concentration gradient. However, according to our investigation, there is no direct experimental evidence to test this hypothetical pathway in apple or other flesh fruits yet. In this study, MdSWEET9 and 23, with high homology to AtSWEET11–14, were highly expressed in the sepal bundle vascular regions, and they all belong to clade III. The expression of these two genes was significantly and positively correlated with total sugar accumulation, suggesting that they may be involved in sucrose unloading in apple fruit. However, the functions of MdSWEETs in sugar unloading in apple fruit still need to be extensively studied.

5. Conclusions

Overall, a hypothetical model for sugar transport from leaves to sink tissues/organs was proposed based on the expression pattern of MdSWEET genes in different apple tissues. As shown in Figure 10, when sucrose and sorbitol are produced in leaves through photosynthesis, MdSWEET5, 7, and 21 contribute to photoassimilate efflux from the leaves, and then MdSWEET2, 12, 13, 14, 17, and 22 contribute to the unloading of sugar in peduncles (stems). Finally, sugars are transported to fruits and other sink tissues/organs. MdSWEET4 may be involved in regulating sugar accumulation in young leaves (sink organs). In turn, MdSWEET11 and 25 may play a role in generative cell development, whereas MdSWEET3 and 19 may mediate sugar efflux in the nectary parenchyma. Meanwhile, MdSWEET1, 6, 8, 9, 10, 18, 20, 23, 26, and 27 may play roles at different fruit developmental stages. Thus, MdSWEET8 may play a role in fruit-set and early fruit development, MdSWEET10, MdSWEET27, and MdSWEET1 might be involved in starch and sugar accumulation in the mid- and late-fruit growth stages, and MdSWEET6, 9, 20, 23, and 26 may be involved in the progress of apple fruit maturation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12081747/s1, Figure S1: Tissue disks indicate the sampled regions; Table S1: Primers used for gene expression; Table S2: Results of candidate proteins analyzed using the Conserved Domains Database and the PFAM database.

Author Contributions

P.N. performed most of the experiments, analyzed the data, and compiled the original manuscript. G.X. participated in the bioinformatics analysis of gene families. B.Y. assisted with the qRT-PCR experiments. D.L. designed the experiments and helmed the project. X.X. contributed suggestions and discussion. S.Q. designed the experiments and reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the National Key Research and Development Program of China (2020YFD1000201), the China Agriculture Research System of MOF and MARA (CARS-27), the Agricultural Research and Industrialization Project of Liaoning Province (2020JH2/10200028), and the Key Research and Development Program of Shandong Province (2021CXGC010802).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Sequence alignment of MdSWEETs and OsSWEET2b. Sequences of SWEETs were aligned using Clustal X2, and the results were submitted to the Essprint online website (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi/ (accessed on 10 December 2020)) for visualization. Based on the OsSWEET2b structure, the positions of the seven transmembrane (TM) structural domains (TM1 to TM7) are indicated by wavy lines above the sequence. Conserved amino acid sequences are indicated in red.
Figure 1. Sequence alignment of MdSWEETs and OsSWEET2b. Sequences of SWEETs were aligned using Clustal X2, and the results were submitted to the Essprint online website (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi/ (accessed on 10 December 2020)) for visualization. Based on the OsSWEET2b structure, the positions of the seven transmembrane (TM) structural domains (TM1 to TM7) are indicated by wavy lines above the sequence. Conserved amino acid sequences are indicated in red.
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Figure 2. Phylogenetic relationship between SWEET proteins in Arabidopsis, tomatoes, and apples. The phylogenetic tree was constructed based on the amino acid sequences by MEGA X using the NJ method with 1 000 bootstrap replicates. The resulting file was submitted to the EVOLVIEW V2 website (http://www.evolgenius.info/evolview/ (accessed on 10 December 2020)) for mapping. Based on the grouping of SWEET members in Arabidopsis, 27 MdSWEETs were divided into four clades that were labeled as I, II, III, and IV, which are represented by yellow, green, red, and purple, respectively. At, Arabidopsis; Sl, tomatoes; Md, apples.
Figure 2. Phylogenetic relationship between SWEET proteins in Arabidopsis, tomatoes, and apples. The phylogenetic tree was constructed based on the amino acid sequences by MEGA X using the NJ method with 1 000 bootstrap replicates. The resulting file was submitted to the EVOLVIEW V2 website (http://www.evolgenius.info/evolview/ (accessed on 10 December 2020)) for mapping. Based on the grouping of SWEET members in Arabidopsis, 27 MdSWEETs were divided into four clades that were labeled as I, II, III, and IV, which are represented by yellow, green, red, and purple, respectively. At, Arabidopsis; Sl, tomatoes; Md, apples.
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Figure 3. Three-dimensional models of MdSWEET proteins. The 3D model structures of the MdSWEET proteins were predicted using Protein Homology/Analogy Recognition Engine V 2.0. The square, circle, triangle, and star at the left of each protein name represent clade I, II, III, and IV, respectively.
Figure 3. Three-dimensional models of MdSWEET proteins. The 3D model structures of the MdSWEET proteins were predicted using Protein Homology/Analogy Recognition Engine V 2.0. The square, circle, triangle, and star at the left of each protein name represent clade I, II, III, and IV, respectively.
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Figure 4. Analysis of conserved motifs and gene structure in the phylogenetic tree of the MdSWEET gene family. (A) Phylogenetic tree of MdSWEETs. Different clades are marked with different colors. Frames in red, green, purple, and blue indicate clades I, II, III, and IV. (B) Conserved motifs predicted in MdSWEET proteins. The conserved motifs were analyzed using MEME v5.4.1 (https://meme-suite.org/meme/tools/meme/ (accessed on 15 December 2020)), and the 10 motifs are represented by squares of different colors and numbered 1–10. (C) Exon–intron structures of MdSWEET genes. Yellow bars: exons; lines: introns; green bars: untranslated region.
Figure 4. Analysis of conserved motifs and gene structure in the phylogenetic tree of the MdSWEET gene family. (A) Phylogenetic tree of MdSWEETs. Different clades are marked with different colors. Frames in red, green, purple, and blue indicate clades I, II, III, and IV. (B) Conserved motifs predicted in MdSWEET proteins. The conserved motifs were analyzed using MEME v5.4.1 (https://meme-suite.org/meme/tools/meme/ (accessed on 15 December 2020)), and the 10 motifs are represented by squares of different colors and numbered 1–10. (C) Exon–intron structures of MdSWEET genes. Yellow bars: exons; lines: introns; green bars: untranslated region.
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Figure 5. Chromosome localization of MdSWEETs. The bars indicate the apple chromosomes, and the chromosome numbers are shown at the left of each chromosome. The scale ruler on the left side shows the physical distance of the chromosomes. The relative positions of MdSWEETs are marked on the chromosomes.
Figure 5. Chromosome localization of MdSWEETs. The bars indicate the apple chromosomes, and the chromosome numbers are shown at the left of each chromosome. The scale ruler on the left side shows the physical distance of the chromosomes. The relative positions of MdSWEETs are marked on the chromosomes.
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Figure 6. Analysis of cis-acting elements in the promoter regions of MdSWEET genes. The three common cis-acting elements identified were related to plant growth and development, phytohormone response, and stress response. AC-II, vascular bundle-specific expression element; as-1, phloem tissue-specific element; GCN4_motif, endosperm-specific expression element; O2-site, zein metabolism regulation element; HD-zip 1, palisade mesophyll cell-associated element; MBSI, flavonoid biosynthetic gene regulation element; MSA-like, cell cycle regulation element; RY-element, seed-specific regulation element, ABRE, abscisic acid-responsive element; ERE, ethylene-responsive element; GARE-motif, gibberellin-responsive element; W box, root-specific expression element; ARE, anaerobic induction element; GC-motif, anoxic specific inducibility element; LTR, low-temperature responsive element; MBS, MYB binding site; WUN-motif, wound-responsive element; TC-rich, defense and stress responsiveness element. AC-II, as-1, GCN4_motif, O2-site, HD-zip 1, MBSI, MSA-like, RY-element, and circadian are involved in plant growth and development; ABRE, CGTCA-MeJA, ERE, GARE-motif, P-box, TATC-box, TCA-element, TGACG, TGA-element, and AuxRR-core are involved in hormone responses; ARE, GC-motif, LTR, MBS, WUN-motif, and TC-rich are involved in stress responses. The heatmap and color columns indicate the numbers of cis-acting elements.
Figure 6. Analysis of cis-acting elements in the promoter regions of MdSWEET genes. The three common cis-acting elements identified were related to plant growth and development, phytohormone response, and stress response. AC-II, vascular bundle-specific expression element; as-1, phloem tissue-specific element; GCN4_motif, endosperm-specific expression element; O2-site, zein metabolism regulation element; HD-zip 1, palisade mesophyll cell-associated element; MBSI, flavonoid biosynthetic gene regulation element; MSA-like, cell cycle regulation element; RY-element, seed-specific regulation element, ABRE, abscisic acid-responsive element; ERE, ethylene-responsive element; GARE-motif, gibberellin-responsive element; W box, root-specific expression element; ARE, anaerobic induction element; GC-motif, anoxic specific inducibility element; LTR, low-temperature responsive element; MBS, MYB binding site; WUN-motif, wound-responsive element; TC-rich, defense and stress responsiveness element. AC-II, as-1, GCN4_motif, O2-site, HD-zip 1, MBSI, MSA-like, RY-element, and circadian are involved in plant growth and development; ABRE, CGTCA-MeJA, ERE, GARE-motif, P-box, TATC-box, TCA-element, TGACG, TGA-element, and AuxRR-core are involved in hormone responses; ARE, GC-motif, LTR, MBS, WUN-motif, and TC-rich are involved in stress responses. The heatmap and color columns indicate the numbers of cis-acting elements.
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Figure 7. Expression heatmaps of SWEET genes in four tissues/organs and eleven different fruit development stages. Relative mRNA expression was measured using qRT-PCR. MdSWEET expression data were normalized and the heatmap was generated using the Tbtools V1.075 software. YL indicates young leaf; F indicates flower; P indicates peduncle; ML indicates mature leaf; DAB indicates days after bloom.
Figure 7. Expression heatmaps of SWEET genes in four tissues/organs and eleven different fruit development stages. Relative mRNA expression was measured using qRT-PCR. MdSWEET expression data were normalized and the heatmap was generated using the Tbtools V1.075 software. YL indicates young leaf; F indicates flower; P indicates peduncle; ML indicates mature leaf; DAB indicates days after bloom.
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Figure 8. Expression pattern analysis of MdSWEET genes in different tissues of flowers and fruits. (A) Expression of MdSWEET genes in four different flower tissues. (B) Expression of MdSWEET genes in different fruit tissues. PB indicates petal bundle vascular region; SB indicates sepal bundle vascular region; and FP indicates fleshy parenchyma cell. The significances of the gene expression differences between tissues are indicated, and different lowercase letters indicate the significant difference at 0.05 level by Duncan’s multiple range test.
Figure 8. Expression pattern analysis of MdSWEET genes in different tissues of flowers and fruits. (A) Expression of MdSWEET genes in four different flower tissues. (B) Expression of MdSWEET genes in different fruit tissues. PB indicates petal bundle vascular region; SB indicates sepal bundle vascular region; and FP indicates fleshy parenchyma cell. The significances of the gene expression differences between tissues are indicated, and different lowercase letters indicate the significant difference at 0.05 level by Duncan’s multiple range test.
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Figure 9. Analysis of the content and proportion of sugar components at different fruit development stages of the “Hanfu” apple. (A) Sugar contents in different fruit stages. Fructose (Fru), glucose (Glu), sucrose (Suc), and sorbitol (Sor) contents were determined by HPLC. Data represent the mean of three biological replicates. (B) Proportions of sugar components in different fruit stages. Error bars: standard deviation (n = 3).
Figure 9. Analysis of the content and proportion of sugar components at different fruit development stages of the “Hanfu” apple. (A) Sugar contents in different fruit stages. Fructose (Fru), glucose (Glu), sucrose (Suc), and sorbitol (Sor) contents were determined by HPLC. Data represent the mean of three biological replicates. (B) Proportions of sugar components in different fruit stages. Error bars: standard deviation (n = 3).
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Figure 10. Schematic models of gene expression and roles of MdSWEET proteins in different apple tissues/organs and development stages. The figure shows representative genes that are highly expressed in each tissue. These genes may be involved in sugar transport in young leaves, mature leaves, stamens, ovaries, petals, pedicels, and fruits. Gene names under tissue names indicate that they are highly expressed in these tissues.
Figure 10. Schematic models of gene expression and roles of MdSWEET proteins in different apple tissues/organs and development stages. The figure shows representative genes that are highly expressed in each tissue. These genes may be involved in sugar transport in young leaves, mature leaves, stamens, ovaries, petals, pedicels, and fruits. Gene names under tissue names indicate that they are highly expressed in these tissues.
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Table
Table 1. Characterization of MdSWEET proteins in apple. The molecular weight, theoretical pI, instability index, aliphatic index, and grand average of hydropathicity were determined by the online ProtParam tool (http://web.expasy.org/protaram/ (accessed on 9 December 2020)). The number of transmembrane domains was predicted by PFAM (http://pfam.xfam.org/ (accessed on 25 November 2020)).
Table 1. Characterization of MdSWEET proteins in apple. The molecular weight, theoretical pI, instability index, aliphatic index, and grand average of hydropathicity were determined by the online ProtParam tool (http://web.expasy.org/protaram/ (accessed on 9 December 2020)). The number of transmembrane domains was predicted by PFAM (http://pfam.xfam.org/ (accessed on 25 November 2020)).
GeneAccession
Number		Number of Amino Acids (aa)Molecular
Weight (Da)		Theoretical
pI		Instability
Index		Aliphatic
Index		Grand Average of
Hydropathicity		Transmembrane
Domain
MdSWEET1MD01G121570026729,800.198.9741.01112.280.4906
MdSWEET2MD03G125060023526,042.159.3445.93121.960.8637
MdSWEET3MD04G123600026729,846.59.3428.59117.490.6427
MdSWEET4MD05G101220026328,799.019.6834.91100.110.5117
MdSWEET5MD05G129310023525,952.919.0235.46123.620.9117
MdSWEET6MD05G129320023926,752.735.4244.92116.570.7767
MdSWEET7MD05G129330023225,370.248.7134.52129.780.9597
MdSWEET8MD06G111200026129,363.149.9040.37126.210.6137
MdSWEET9MD06G113650029833,191.838.2143.04103.050.4627
MdSWEET10MD06G113660029533,115.425.8044.98124.20.6817
MdSWEET11MD06G117680021524,637.529.2343.46130.330.8236
MdSWEET12MD10G101220024326,504.459.5421.66105.140.6927
MdSWEET13MD10G101310024827,134.189.5420.71104.190.6647
MdSWEET14MD10G126910022925,204.898.8128.44122.970.9137
MdSWEET15MD10G126930023826,746.475.1343.03111.760.7287
MdSWEET16MD10G126940023225,374.178.5536.2127.280.9347
MdSWEET17MD11G127080023525,969.737.5947.41121.530.8457
MdSWEET18MD11G129920034038,1177.5640.54109.530.5339
MdSWEET19MD12G125500026830,385.119.4730.74117.840.6277
MdSWEET20MD13G112430030534,116.928.5234.31108.660.3817
MdSWEET21MD13G116680028131,083.46.7347.41112.380.4847
MdSWEET22MD14G113340026129,467.119.6334.66118.350.5887
MdSWEET23MD14G115130029833,491.478.8232.7108.520.4977
MdSWEET24MD14G115140029533,290.716.8338.63120.20.7067
MdSWEET25MD14G118300023626,576.968.9338.41136.060.9817
MdSWEET26MD16G112530030534,303.16.530.25114.030.4267
MdSWEET27MD17G103520025027,611.869.0331.52117.720.6747
Table
Table 2. Secondary structural statistics of MdSWEET proteins. The secondary structures were predicted based on the amino acid sequences using the SOPMA secondary structure prediction method (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_%20sopma.html/ (accessed on 15 December 2020)).
Table 2. Secondary structural statistics of MdSWEET proteins. The secondary structures were predicted based on the amino acid sequences using the SOPMA secondary structure prediction method (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_%20sopma.html/ (accessed on 15 December 2020)).
ProteinAlpha Helix (%)Extended Strand (%)Beta Turn (%)Random Coil (%)
MdSWEET140.0718.733.7537.45
MdSWEET242.1320.855.1131.91
MdSWEET344.5717.601.5036.33
MdSWEET435.7420.154.1839.92
MdSWEET541.7022.983.4031.91
MdSWEET653.9717.573.3525.10
MdSWEET743.122.843.4530.6
MdSWEET837.1620.694.2137.93
MdSWEET936.9118.462.0142.62
MdSWEET1045.7617.971.3634.92
MdSWEET1141.4022.794.1931.63
MdSWEET1237.8623.464.1234.57
MdSWEET1339.5221.376.4532.66
MdSWEET1445.8520.525.6827.95
MdSWEET1547.0620.172.1030.67
MdSWEET1649.1417.243.0230.60
MdSWEET1741.7022.553.8331.91
MdSWEET1839.4120.291.7638.53
MdSWEET1943.6616.042.9937.31
MdSWEET2040.6616.391.3141.64
MdSWEET2145.2018.861.4234.52
MdSWEET2242.5321.073.8332.57
MdSWEET2344.6315.102.6837.58
MdSWEET2450.5115.251.6932.54
MdSWEET2540.2523.315.0831.36
MdSWEET2638.6920.333.6137.38
MdSWEET2736.8021.203.6038.40
Table
Table 3. Basic information of MdSWEET homologous genes. The duplication events were detected using the MCScanX software. Nineteen SWEET gene pairs were detected as duplicated gene pairs, of which four were tandem duplications and fifteen were segmental duplications.
Table 3. Basic information of MdSWEET homologous genes. The duplication events were detected using the MCScanX software. Nineteen SWEET gene pairs were detected as duplicated gene pairs, of which four were tandem duplications and fifteen were segmental duplications.
Duplication TypeDuplicated Gene PairsChrClade
Tandem duplicationMdSWEET5/MdSWEET6Chr05/05I/I
MdSWEET9/MdSWEET10Chr06/06III/III
MdSWEET15/MdSWEET16Chr10/10I/I
MdSWEET23/MdSWEET24Chr14/14III/III
Segmental duplicationMdSWEET15/MdSWEET17Chr10/11I/I
MdSWEET3/MdSWEET19Chr04/12III/III
MdSWEET2/MdSWEET17Chr03/11I/I
MdSWEET5/MdSWEET14Chr05/10I/I
MdSWEET4/MdSWEET12Chr05/10I/I
MdSWEET11/MdSWEET25Chr06/14II/II
MdSWEET11/MdSWEET27Chr06/17II/II
MdSWEET8/MdSWEET22Chr06/14II/II
MdSWEET9/MdSWEET20Chr06/13III/III
MdSWEET9/MdSWEET23Chr06/14III/III
MdSWEET9/MdSWEET26Chr06/16III/III
MdSWEET25/MdSWEET27Chr14/17II/II
MdSWEET20/MdSWEET23Chr13/14III/III
MdSWEET20/MdSWEET26Chr13/16III/III
MdSWEET23/MdSWEET26Chr14/16III/III
Table
Table 4. Correlation analysis of expression levels and main sugar or soluble sugar content during the fruit developmental period. Correlation analysis of the expression levels and main sugar or soluble sugar content during the fruit developmental period was performed using Duncan′s multiple range test. Significant and highly significant differences are indicated by * p < 0.05 and ** p < 0.01, respectively.
Table 4. Correlation analysis of expression levels and main sugar or soluble sugar content during the fruit developmental period. Correlation analysis of the expression levels and main sugar or soluble sugar content during the fruit developmental period was performed using Duncan′s multiple range test. Significant and highly significant differences are indicated by * p < 0.05 and ** p < 0.01, respectively.
FruGluSucSorSoluble Sugar FruGluSucSorSoluble Sugar
MdSWEET1−0.005−0.084−0.115−0.141−0.082MdSWEET15−0.599−0.387−0.4890.036−0.720 *
MdSWEET2−0.109−0.135−0.2350.274−0.129MdSWEET160.3430.2370.259−0.2360.157
MdSWEET3−0.383−0.295−0.267−0.077−0.184MdSWEET17−0.264−0.262−0.2950.440−0.205
MdSWEET40.614 *0.4700.635 *0.1130.612 *MdSWEET180.4460.5420.634 *0.1300.472
MdSWEET5−0.403−0.347−0.1210.299−0.178MdSWEET190.658 *0.5550.707 *−0.1310.627 *
MdSWEET60.663 *0.5550.686 *−0.0320.653 *MdSWEET200.683 *0.5900.786 **0.0620.707 *
MdSWEET70.2250.3550.176−0.2140.335MdSWEET21−0.491−645 *−0.4090.811 **−0.527
MdSWEET8−0.474−0.576−0.3260.932 **−0.396MdSWEET22−0.519−633 *−0.3860.914 **−0.470
MdSWEET90.790 **0.748 **0.934 **0.0830.835 **MdSWEET230.765 **0.726 *0.948 **0.0750.897 **
MdSWEET10−0.532−0.300−0.3610.303−0.513MdSWEET24−0.471−0.592−0.3330.938 **−0.396
MdSWEET110.4080.3080.2240.1270.480MdSWEET250.3000.223−0.034−0.044−0.042
MdSWEET120.773 **0.775 **0.921 **0.0070.821 **MdSWEET260.668 *0.5750.751 **0.0170.684 *
MdSWEET130.660 *0.5540.720 *0.0210.674 *MdSWEET270.047−0.099−0.084−0.075−0.166
MdSWEET140.204−0.087−0.017−0.249−0.104
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Nie, P.; Xu, G.; Yu, B.; Lyu, D.; Xue, X.; Qin, S. Genome-Wide Identification and Expression Profiling Reveal the Potential Functions of the SWEET Gene Family during the Sink Organ Development Period in Apple (Malus × domestica Borkh.). Agronomy 2022, 12, 1747. https://doi.org/10.3390/agronomy12081747

AMA Style

Nie P, Xu G, Yu B, Lyu D, Xue X, Qin S. Genome-Wide Identification and Expression Profiling Reveal the Potential Functions of the SWEET Gene Family during the Sink Organ Development Period in Apple (Malus × domestica Borkh.). Agronomy. 2022; 12(8):1747. https://doi.org/10.3390/agronomy12081747

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Nie, Peixian, Gongxun Xu, Bo Yu, Deguo Lyu, Xiaomin Xue, and Sijun Qin. 2022. "Genome-Wide Identification and Expression Profiling Reveal the Potential Functions of the SWEET Gene Family during the Sink Organ Development Period in Apple (Malus × domestica Borkh.)" Agronomy 12, no. 8: 1747. https://doi.org/10.3390/agronomy12081747

APA Style

Nie, P., Xu, G., Yu, B., Lyu, D., Xue, X., & Qin, S. (2022). Genome-Wide Identification and Expression Profiling Reveal the Potential Functions of the SWEET Gene Family during the Sink Organ Development Period in Apple (Malus × domestica Borkh.). Agronomy, 12(8), 1747. https://doi.org/10.3390/agronomy12081747

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