XlAO Qian-lin ,Ll Zhen ,WANG Ya-yun ,HOU Xian-bin ,WEl Xi-mei ,ZHAO Xiao ,HUANG Lei,GUO Yan-jun,LlU Zhi-zhai

1 College of Agronomy and Biotechnology,Southwest University,Chongqing 400715,P.R.China

2 State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Sichuan Agricultural University,Chengdu 611130,P.R.China

3 College of Agriculture and Food Engineering,Baise University,Baise 533000,P.R.China

4 College of Animal Science and Technology,Southwest University,Chongqing 400715,P.R.China

Abstract Sugar transporters are essential for osmotic process regulation,various signaling pathways and plant growth and development.Currently,few studies are available on the function of sugar transporters in sorghum (Sorghum bicolor L.).In this study,we performed a genome-wide survey of sugar transporters in sorghum.In total,98 sorghum sugar transporters(SSTs) were identified via BLASTP.These SSTs were classified into three families based on the phylogenetic and conserved domain analysis,including six sucrose transporters (SUTs),23 sugars will eventually be exported transporters(SWEETs),and 69 monosaccharide transporters (MSTs).The sorghum MSTs were further divided into seven subfamilies,including 24 STPs,23 PLTs,two VGTs,four INTs,three pGlcT/SBG1s,five TMTs,and eight ERDs.Chromosomal localization of the SST genes showed that they were randomly distributed on 10 chromosomes,and substantial clustering was evident on the specific chromosomes.Twenty-seven SST genes from the families of SWEET,ERD,STP,and PLT were found to cluster in eight tandem repeat event regions.In total,22 SSTs comprising 11 paralogous pairs and accounting for 22.4% of all the genes were located on the duplicated blocks.The different subfamilies of SST proteins possessed the same conserved domain,but there were some differences in features of the motif and transmembrane helices (TMH).The publicly-accessible RNA-sequencing data and real-time PCR revealed that the SST genes exhibited distinctive tissue specific patterns.Functional studies showed that seven SSTs were mainly located on the cell membrane and membrane organelles,and 14 of the SSTs could transport different types of monosaccharides in yeast.These findings will help us to further elucidate their roles in the sorghum sugar transport and sugar signaling pathways.

Keyword: sorghum (Sorghum bicolor L.),sugar transporter,SUT,SWEET,MST,phylogenetic analysis

1.lntroduction

Sugars,the end product of photosynthesis,are the basic carbon skeletons for the biosynthesis of cellular compounds,and they play important roles in osmotic processes and participate in various signaling pathways(Lastdrageret al.2014;Ruan 2014;Chen L Qet al.2015).Sugar transport from autotrophic tissues/cells to heterotrophic tissues/cells is a complex process including phloem loading,unloading,metabolism,and signaling pathways (Braunet al.2014;Ruan 2014).This process is also controlled by multiple transporters,i.e.,sucrose transporter (SUT) (Riesmeieret al.1992;Hiroseet al.1997),sugars will eventually be exported transporter(SWEET) (Chenet al.2010),and monosaccharide transporter (MST) (Büttner and Sauer 2000).

SUTs,the H+/sucrose symporters,have been extensively documented in plants and can load sucrose into the phloemviathe proton motivation on the plasma membrane (Divyaet al.2003;Carpanetoet al.2005;Kühn and Grof 2010;Juliuset al.2017).Spinach SUT was the first cloned SUT family member in plants,and it was found to be able to transport sucrose in yeast(Riesmeieret al.1992).The SUT proteins have been widely documented inArabidopsis(AtSUCs),and the reportedAtSUCs exhibit special affinity to sucrose for transportation or sucrose-related responses (Chandranet al.2003;Sivitzet al.2006,2008;Liet al.2012;De Molineret al.2018).In addition,theAtSUCs have been expressed or located in different cells/tissues,such as plasma membrane,cells adjacent to the vascular tissue,and endosperm,and in response to wounding,seed development and germination,and other conditions(Meyeret al.2001;Baudet al.2005;Sivitzet al.2006;Pommerreniget al.2013).

Many SUTs have been widely documented in crops.In rice,the OsSUT1 was presumed to be involved in the transportation of sucrose from sink tissue to developmental tissue,and was further proven to load sucrose into the phloem in germinating seedings (Hiroseet al.1997;Matsukuraet al.2000;Ishimaruet al.2001).ZmSUT1,located on the plasma membrane of companion cells,participates in the loading of sucrose into the sieve element-companion cell complex in maize leaves(Slewinski and Braun 2009;Slewinskiet al.2010;Bakeret al.2016).Several SUT genes have been cloned from hexaploid wheat and potato (Riesmeieret al.1993;Weiseet al.2000;Aokiet al.2002).In sorghum,SUT was the earliest reported sugar transporter (Braun and Slewinski 2009),and six coding genes have been identified in previous studies with different patterns of expression and function (Milneet al.2013,2017;Bihmidineet al.2015).

SWEETs are membrane proteins closely related to sugar transport which containα-helical and MtN3 domains(Chenet al.2010,2012;Xuanet al.2013),and they have been widely reported inArabidopsis(Chenet al.2010),rice (Yuan and Wang 2013),and tomato (Fenget al.2015).These proteins exhibit diverse biological functions,including the regulation of plant growth,seed and plant development (Sossoet al.2015;Patilet al.2015),pollen development (Guanet al.2008;Sunet al.2013),leaf senescence (Quirinoet al.1999),and response to the biotic stress of pathogen infection (Yanget al.2006;Yuet al.2010) and some abiotic stresses (Guoet al.2014;Fenget al.2015).For example,AtSWEET11 and AtSWEET12 play important roles in phloem loading(Chenet al.2012),OsSWEET11 participates in sugar transport in the early grain-filling stage (Maet al.2017),and OsSWEET4 and ZmSWEET4 can affect the grain filling by mediating hexose transport (Sossoet al.2015).In sorghum,the SWEET family includes 23 coding genes (SbSWEETs) which exhibit different expression patterns (Bihmidineet al.2016;Mizunoet al.2016).For example,SbSWEET8-1is strongly expressed in leaf and stem,SbSWEET4-4is mainly expressed in panicle,SbSWEET9-1is highly expressed in leaf,andSbSWEET9-3andSbSWEET3-6are only expressed in primary panicle (Mizunoet al.2016).Aside from the detailed documentation of gene identification and expression,further functional dissection of the sorghum SWEETs still remains unexplored.

MSTs include more coding genes than SUTs and SWEETs,and theMSTgenes can be divided into seven subfamilies,i.e.,STP(sugartransporterprotein),PLT(polyol/monosaccharidetransporter),VGT(vacoular glucosetransporter),INT(inositoltransporter),pGlcT/SBG1(plastidicglucosetransporter/suppressorof Gproteinbeta1),TMT(tonoplasticmonosaccharide transporter),andERD6-like(earlyresponseto dehydration-like) (Johnsonet al.2006;Johnson and Thomas 2007).The different types of MST proteins carry specific substrates in the process of sugar transport(Slewinski 2011).AtSTP1 was the first identified MST and it participates in the transport of fructose and other several hexoses (Saueret al.1990;Booreret al.1994).AtPLT5,a member of PLT,mediates H+-symport with myo-inositol,glycerol,and ribose (Klepeket al.2005).AtTMT1 is located on the vacuole membrane,and it mediates the transport of glucose and fructose (Wormitet al.2006).In sorghum,only TMT/TST (tonoplastic monosaccharide transporter) have been identified and namedSbTST1andSbTST2(Bihmidineet al.2016).SbTST1andSbTST2showed higher expression levels in the stem of sweet sorghum than in grain sorghum,and they could transport sucrose,glucose and fructose on the vacuole membrane (Bihmidineet al.2016).In summary,the answers to the questions of the numbers and functions of MSTs involved in sugar signaling pathways,physiological regulation and plant growth and development by participating in sugar transporting remain to be further studied in sorghum.

In the present study,in order to reveal the genome-wide distribution of sorghum sugar transporters (SSTs),and provide informative reference for further functional profiling and the potential utilization of SSTs in sorghum variety improvement,we conducted genome-wide identification and analysis of SSTs.In addition,the expression patterns of candidateSSTs were analyzed based on the publiclyavailable RNA-sequence data,and further confirmed by RT-PCR in different tissues of sorghum cultivar BTx623.The functions of several candidate genes were further studied by yeast complementary experiments and subcellular location analysis in onion epidermal cells and leaf protoplasts.The results of this study will lay a foundation for the further dissection of both the biological functions and sugar transport mechanisms of sugar transporters in sorghum.

2.Materials and methods

2.1.Plant materials

BTx623,a sorghum cultivar provided by Rice and Sorghum Institute,Sichuan Academy of Agricultural Sciences (Luzhou,Sichuan,China),was used for the experimental analysis.The seeds were planted in the college farm (College of Agronomy and Biotechnology,Southwest University,Chongqing,China) with standard irrigation and fertilization treatments.Fresh tissue samples were collected at different developmental stages,including jointing,flowering and maturity,and were immediately immersed in liquid nitrogen and then stored at -80°C prior to gene expression analysis.Three biological replicates were collected for each sample.

2.2.Sequence profiling and phylogenetic analysis

A total of 79 sugar transporter proteins inArabidopsis(Appendices A and B) were compiled from previous studies (Büttner 2007;Chenet al.2010;Kühn and Grof 2010) and used as the index to identify the candidate sugar transporter proteins in the sorghum genome.BLASTP queries were performed to search the sorghum genome in the Gramene database (http://www.gramene.org/) and NCBI (https://www.ncbi.nlm.nih.gov/) by using theArabidopsissugar transporter protein sequences.Further analysis only focused on the retained opening reading frame (ORF) of the candidate sequences after removing redundant and incomplete protein sequences.ClustalW1.83 was used for the multiple sequence alignment and SMART (http://smart.embl-heidelberg.de/) was applied to confirm each protein containing the conserved functional domains.MEGA (v5.10) was used to construct the phylogenetic treesviathe neighborjoining (NJ) method combined with ap-distance model,and the bootstrap replicates were set to 1 000 (Tamuraet al.2011).

Both coding sequences (CDS) and genomic sequences of the candidate genes were downloaded from the reference genome (Patersonet al.2009).Gene Structure Display Server (GSDS,v2.0,http://gsds.cbi.pku.edu.cn/)was used to define the gene structure (Guoet al.2007).The intron distribution pattern,intron phases and intron/exon boundaries of the candidate genes were obtained and the results were aligned with the 5′ terminus.The chromosome locations,duplication and gene clusters were analyzed by local BLASTP and McscanX (Wanget al.2012),and Circos was used to display the results(Krzywindskiet al.2009).TMHMM Server (v2.0) was used for predicting the transmembrane helixes (TMH) of candidate sugar transporter proteins.Conserved motifs were investigatedviaMEME (http://meme.nbcr.net/meme/cgi-bin/meme.cgi),and the minimum and maximum motif widths,and number of different motifs were specified as 6,50,and 20,respectively.

2.3.Expression and co-expression analysis of SST genes

The gene expression information of theSSTcoding gene was obtained from the Expression Alta database (https://www.ebi.ac.uk/gxa/home).The transcripts per kilobase of exon model per million mapped reads (TPM) values for all sorghum genes were downloaded from three independent baseline experiments (Davidsonet al.2012;Makitaet al.2015).The expression data of allSSTgenes reflect their expression in different tissues under normal planting conditions.Cluster v3.0 was used to perform hierarchical clustering based on the TPM value,and TreeView was used for graphical display.

The co-expression analysis ofSSTswas performed according to the TPM of three independent baseline experiments.The expression data of all predictedSSTgenes were compared in pairs,and Pearson correlation coefficient (PCC) values between them and graphical display were completed using TBtools (Chenet al.2020).

2.4.RNA extraction and real-time PCR

During the developmental stages of BTx623,total RNA was extracted from the different tissues using an RNA extraction kit (Tiangen,Beijing,China),and used for reverse transcription to obtain the cDNA.Real-time PCR(RT-PCR) was performed through a Bio-Rad CFX96 Realtime System in a total reaction volume of 10 μL Hieff qPCR SYBR Green Master Mix (Yeasen,China).The internal control was sorghumActin(SORBI_3008G047000).All experiments were conducted four times,with three samples taken at each developmental stage.The PCR products were sequenced to verify the specificity of the reaction.The relative transcription levels were calculatedviathe 2-ΔΔCTmethod.All primers used for real-time PCR are named as SbSTsQF (forward primer) and SbSTsQR(reverse primer) (Appendix C).

2.5.Sub-cellular localization of SSTs

The sub-cellular localizations of SSTs were confirmed by transient expression of a fusion construct containing enhanced green fluorescent protein (eGFP) in onion epidermal cells and protoplasts of sorghum leaf.The construction of the SbSTs-eGFP fusion protein was mainly carried out by fragment recombination through ClonExpress II One Step Cloning Kit (Vazyme,Nanjing,China).The sevenSbSTs were amplified withKpnI andXbaI restriction sites without the termination codon,and then sub-cloned into pCAMBIA2300-35S-eGFP to construct the final carrier.A Biolistic PDS-1000/He Particle Delivery System (Bio-Rad,CA,USA) was used to deliver gold particles coated with the constructed vector into the onion epidermal cells using 900 psi He pressure(Huet al.2012).The preparation of sorghum protoplasts was improved according to the method ofArabidopsis(Wuet al.2009).The PEG-Ca2+conversion method was performed to transform the plasmids as described by Chenet al.(2016).The subcellular localization of the GFP fusion proteins was visualized with an ECLIPSE 80i florescence microscope (Nikon,Tokyo,Japan) under excitation with blue light at 488 nm.

2.6.Complementary functional analysis of SSTs in yeast strains

The cDNA served as the template for cloning theSSTgenes (Appendix C).The yeast expression vector pDR196 was constructedviaClonExpress II One Step Cloning Kit (Vazyme,Nanjing,China),and all the recombinant primers are listed in Appendix C.The PEGLiAc method was used for yeast transformation,and salmon essence was used to carry the plasmids.The constructed yeast HTX5 sugar transporter was used as the positive control (Diderichet al.2001),and empty pDR196 served as the negative control.

All transformants were first grown on syntheticdeficient (SD) media without Uridine (URA) but containing 2% maltose.The clones of uniform size were dissolved directly in 200 μL of sterilized ddH2O,and further identified through a yeast colony PCR reaction.The positive clones were diluted into different concentrations in a gradient,and dotted onto URA3 deficient medium with 2% maltose,glucose,fructose,mannitol or galactose.The colonies were grown under dark conditions for 3 days at 28°C.

3.Results

3.1.Characterization of SST genes

A total of 98SSTgenes were identified after removing the truncated versions without the start codon and retaining the single longest protein sequenceviascreening(Appendices A and B).Based on their chromosomal locations,we preliminarily named the genes of different families from chromosomes 1 to 10 (Appendix D).The phylogenetic results showed that the SUT,SWEET and MST ofArabidopsisand sorghum were clustered into independent evolutionary branches (Appendix E).The SUT,SWEET,and MST also formed relatively independent branches when the phylogenetic analysis of the SSTs was carried out independently (Fig.1),which was highly consistent with the phylogenetic analysis of theArabidopsissugar transporters alone (Appendix B).In addition,the SWEETs,STPs,and PLTs were further divided into three corresponding groups (Appendix F).The lengths of the coded SUT proteins ranged from 501 (SbSUT4) to 596 (SbSUT6) amino acids (aa),with molecular weights of 53.21 kDa (SbSUT3) to 63.34 kDa(SbSUT2),andpI values from 6.00 (SbSUT2) and 8.95(SbSUT6).All of these sorghum MSTs could be divided into seven subfamilies (Fig.1;Appendix E).

Fig.1 Phylogenetic and conserved domain analysis of sorghum sugar transporters.aa,amino acid.

Twenty-three sorghum SWEET proteins were identified,consistent with a previous study (Mizunoet al.2016).The lengths of the sorghum SWEET proteins ranged from 213 aa (SbSWEET5) to 336 aa (SbSWEET15),with molecular weights of 23.69 kDa (SbSWEET5) to 35.69 kDa(SbSWEET15),andpI values from 4.53 (SbSWEET5) to 9.69 (SbSWEET19).The MSTs were also further divided into seven subfamilies of STP,PLT,VGT,INT,pGlcT/SBG1,TMT,and ERD (Fig.1;Appendix E).STP and PLT included 24 and 23 coding genes,respectively,indicating they are the two largest subfamilies.Among the other five subfamilies,ERD included eight coding genes,which was more than the other four (ranging from two to five)(Appendix G).Three reported MST genes belonging to the TMT subfamily (SbTST1,SbTST2,andSbTST3) were also identified in the present research,corresponding toSbTMT1,SbTMT2andSbTMT5,respectively (Bihmidineet al.2016;Appendix D).Among the MST proteins,SbTMT5 exhibited the greatest length (767 aa) and the highest molecular weight (81.65 kDa),while SbPLT11 was the shortest (166 aa) with the lowest molecular weight(17.66 kDa;Appendix G).ThepI values of the MST proteins ranged from 4.76 (SbTMT5) to 10.40 (SbPLT10).Further information on the SSTs,including their cDNA lengths and chromosomal distributions,is listed in detail in Appendices D and H.

3.2.Chromosomal distribution and gene structure of SSTs

Based on the physical positions of the SST coding genes,their chromosomal locations were determined among the 10 chromosomes of sorghum (Fig.2;Appendix H).Eighty-seven of the 98 genes (88.78%) are located on seven chromosomes,chromosome 1 (Chr1) to Chr6 and Chr9,while the remaining 11 are located on Chr7 (3),Chr8 (6),and Chr10 (2),indicating an unequal genomewide distribution (Fig.3).In addition,more genes are located on the distal long arm ends of Chr1,Chr2,Chr5,Chr6,Chr8,and Chr9,away from the centromeres.However,Chr3 and Chr4 possessed similar genes on both the long and short arms,exhibiting relatively even distribution trends.

Gene duplications,including tandem and segmental duplication,are considered to be one of the main forces in the evolution and expansion of gene families (Lynch and Conery 2000).In our study,substantial clustering of theSSTgenes was evident on the specific chromosomes(Fig.2).Twenty-sevenSSTgenes from the families of SWEET,ERD,STP,and PLT,were clustered in eight tandem repeat event regions on Chr1,Chr2,Chr5,Chr6,Chr8,and Chr9 (Fig.2).In addition,22 out of the 98SSTs (22.45%) formed 11 paralogous pairs,which were located on the duplicated blocks.Among these paralogous pairs,one pair belonged to each of SUT,ERD,and TMT,while two pairs belonged to STP,and both SWEET and PLT included three pairs.

Fig.2 Genome localization,duplication and collinearity analysis of sorghum sugar transporters.Solid line at the edge of a box indicates the location and connects the name of the gene;tandem duplication is marked with a solid red-line outside the circle;colinear genes are connected by the different solid lines inside the circle.

In addition to the differential chromosomal distribution,allSSTs presented distinct gene structures (Fig.3).ForSUTs,the number of exons ranged from 5 (SbSut4)to 14 (SbSut2),exhibiting an irregular distribution pattern (Fig.3-A).The exons ofSWEETs were mainly concentrated from four to six,exceptSbSWEET6andSbSWEET23,which contained only three exons.Besides,four members ofSWEET,i.e.,SbSWEET2,SbSWEET7,SbSWEET12,andSbSWEET13,all contained introns greater than 2 kb at their 3′ ends (Fig.3-B).Unlike the SUTs and SWEETs,the coding genes of ERD,pGlcT/SGB,and VGT consisted of more than 12 exons,and someERDs even contained 18 exons (Fig.3-C and D).The remaining four families,includingPLT,TMT,INT,andSTP,only contained several exons,i.e.,SbPLTpossessed 2 to 4 exons,SbSTP1 to 5,SbINT2 to 6,andSbTMT1 to 6 (Fig.3-C,E,and F).Interestingly,two special genes,SbTMT3andSbSTP22,were found to possess only one exon,unlike all otherSSTs (Fig.3-C and F).

3.3.Characterization of SST proteins

The Sugar_tr domain is a vital functional region of sugar transporters.In SST proteins,both of the monosaccharide transporters INT and TMT possessed Sugar_tr domains on their N-and C-terminals,while SUT and all other monosaccharide transporters contained only one Sugar_tr domain.The SWEET proteins contained two MtN3_slv domains at the N-terminal,playing important roles for sugar efflux transport,especially in the mediation of glucose transport.

Different types of SST proteins contained different motif distribution patterns,but the motifs of a given type of SSTs were indeed consistent (Appendices I and J).SWEETs contained two combinations of motif 1,motif 2 and motif 3,which were located at the N-and C-terminal,respectively (Appendix I-a).SUTs contained 10 conserved motifs,which were highly consistent among the six proteins (Appendix I-b).Except for SbSUT3,the other five SbSUTs contained different motifs (Appendix I-b).However,the distribution of conserved motifs was more distinct in MST.Sorghum STPs usually exhibited 16 conservative motifs and formed two aggregated parts of Nand C-terminals (Appendix I-c).However,SbSTP7 lacked five conserved motifs at the C-terminal,representing the absence of three TMHs at the C-terminal (Appendix K).

Eleven relatively conservative motifs were identified among PLTs,while SbPLT9,SbPLT10 and SbPLT11 presented only a few of them (Appendix I-d).The results showed that motif 2,motif 6,motif 10 and motif 11 were present in SbPLT11,while SbPLT9 and SbPLT10 exhibited their unique motif 18 and motif 20 (Appendix I-d).Sorghum ERDs usually possessed seven conserved motifs at the C-terminal,except for ERD2 which lacked two motifs (Appendix I-e).The N-terminal of ERDs exhibited greater variability,while SbERD5-8 shared the same conservative motif at the N-terminal,unlike the other four sorghum ERDs (Fig.1).Four INTs showed almost identical conservative motifs,and the different motifs were mainly found at the end of their protein sequences(Appendix I-f).

Among the five TMTs,SbTMT3 possessed the unique motif 17 while it lacked five motifs at the C-terminal.SbTMT4 showed greater variability in the middle sequence and failed to form a conserved motif that was consistent with the other proteins.The SbTMT1,2,and 5 showed good consistency in their conserved motifs(Appendix I-g).Unlike the others,the two VGTs and three pGlcT/SGBs exhibited highly conserved motifs to form a conserved domain,and they also possessed significantly different motifs to form their unique features (Appendix I-h and i)

3.4.ldentification of TMHs and subcellular localization

Except for SbSUT2 and SbSUT3,four SUTs contained 12 TMHs,while SbSUT2 and SbSUT3 contained 11 and 10 TMHs,respectively (Appendices K and L).Most of the SWEET proteins contained seven TMHs,while SWEET6,7 and 11 only contained six TMHs.In the MST family,the distribution of TMHs exhibited more diverse trends (Appendices K and L).For example,VGT and INT exhibited 10 and 11 TMHs,respectively.Three pGlcT/SGB proteins possessed the corresponding THM counts of 10,11,and 12.The number of THMs in TMT was 11,while SbTMT5 exhibited only five.The ERDs mainly contained 12 THMs,except for SbERD1 (10) and SbERD2 (7).For SbSTP and SbPLT,the numbers of TMHs were mainly 10,11 or 12,while a few members contained 7,8 or 9 TMHs.The SbPLT11 only contained three TMHs,less than all other SSTs.Additionally,the distribution patterns of TMHs are also different among identified SSTs (Appendix K).These diverse TMH structures might suggest the binding features and potential functions of the SST proteins.

The results from the subcellular localization showed that the fluorescent signals of seven SSTs were mainly observed on the cell membrane and organelles,and the fluorescent signal was separated with plasmolysis of the onion epidermis cells (Fig.4).No fluorescence signal was detected in the nucleus of the onion epidermal cells.In summary,these results suggested that SbSWEET11 and SbSTP3 are mainly distributed on the cell membrane,while SbWEET18,SbINT3,SbPLT15,SbpGlcT1 and SbEDR3;1 are mainly distributed on the membrane-bound organelles (Fig.4).

Fig.4 Sub-cellular location analysis of some sorghum sugar transporters in onion epidermal cells and protoplasts of sorghum leaves.The pCAMBIA2300-35S-eGFP was bombarded into onion epidermal cells as a control.The subcellular localization of GFP fusion proteins was visualized with a florescence microscope ECLIPSE 80i (Nikon,Tokyo,Japan) under blue excitation light at 488 nm.The label of SbSST-eGFP corresponding the line indicates the distribution of SbST-eGFP under fluorescence field,bright field and merged field illumination.Normal represents normal onion epidermal cells,0.3% NaCl means that concentration of NaCl was used for the treatment,and Protoplast is the protoplasts of sorghum leaf.

3.5.Expression profiling of SSTs

According to the expression Alta (https://www.ebi.ac.uk/gxa/home) of sorghum,the expression patterns of SSTs were divided into five classes,i.e.,Classes I to V(Fig.5).Almost all genes in Class I exhibited median to low expression levels in the tissues of leaf,shoot,and vegetative meristem,while median to high levels were found in seed and some seed-related traits,i.e.,seed 5 days after pollination,seed 10 days after pollination,and endosperm (Fig.5).While someSSTs in Class II presented median to high expression levels in the leaf tissue,they had lower levels in seed and some seedrelated tissues (Fig.5).Similarly,genes from both Classes I and II exhibited median to low levels in vegetative meristem (Fig.5).Consistently high expression level trends were observed in the tissues of anther,flower,and spikelet among theSSTs of Class IV,while median to low levels were observed in the seed-related tissues,which was the opposite to that in Class I (Fig.5).

The co-expression analysis ofSSTs was based on the expression data in different tissues (Appendix M).Obviously,some SST genes shared the same expression pattern (Fig.5),their PCC values were ＞0.6,and only a few genes presented PCC values ＜-0.6.Positive correlations of expression patterns were mainly found in two parts.The first part mainly consisted of genes from Class II,while the second part mainly included genes from Class IV.There are also gene pairs with PCC＞0.6 among other genes that cannot be centralized in these two parts,which also has reference value for additional functional research.

Fig.5 The expression analysis of sorghum sugar transporter coding genes.Groups I,II,and III indicate that the expression data are derived from three independent baseline experiments.Group I data did not have duplicates,group II had two to four duplicates,and group III has three duplicates for each data point.Classes I-V represent classification results based on expression patterns.

Sixteen SSTs were further detected in different tissues of BTx623 by real-time PCR (Fig.6).For example,SbPLT3was mainly expressed in stems;whileSbPLT5,SbSTP3andSbSTP9possessed more transcripts in leaf than in the other tissues.The genes ofSbPLT6/8,SbINT3,SbTMT2,SbSWEET11/18/21,SbSGB2andSbERD3were highly expressed in tissues of both leaf and stem.SbSWEET14/15andSbpGlcT1were highly expressed in three tissues,i.e.,relatively higher expression levels ofSbSWEET14were observed among tissues of root,leaf and stem,and the corresponding tissues forSbSWEET15were stem,inflorescence,and seed,while those forSbpGlcT1were leaf,stem,and seed.

Fig.6 The expression pattern analysis of sorghum sugar transporter coding genes in different tissues of BTx623.JS,jointing stage;HS,heading stage.stem.AP,stem apex.DAP,the days after pollination.SbActin is the internal reference gene,and three independent biological replicates were checked separately for the real-time PCR analysis.

3.6.Transport activities of SSTs in yeast

To investigate the potential roles of the different subfamilies of SSTs,14 members were selected for expression in a yeast(Saccharomycescerevisiae)mutant,EBY.VW4000 (Fig.7).Compared with the control,the yeast cells containing different sugar transporters exhibited significant differences in growth status on various sugar-containing media.The expression ofSbSWEET11and18could effectively complement the mutant phenotype in galactose medium,butSbSWEET21could not.Meanwhile,the expression ofSbSWEET11,18and21in EBY.VW4000 could not complement the mutant phenotype in the defective media containing glucose,fructose or mannitol.Yeast with the expression ofSbPLT5and6could efficiently grow on the medium supplemented with fructose.The transformed strains ofSbSTP8and9could only grow well on the defective medium containing glucose,while those ofSbSTP3could not grow on the defective media with glucose,fructose,galactose or mannitol.The strains transformed withSbTMT2could grow on the defective medium containing fructose.The transformation ofSbINT3affected the growth on defective media with glucose or mannitol.The transformed strains ofSbSGB2grew well on the defective medium containing fructose,whileSbpGlcT1could grow well on defective medium with glucose.SbERD3;1andSbERD3;2were different transcripts of the same gene.SbERD3;1grew well on defective media with glucose or galactose,whileSbERD3;2could not grow on defective media with glucose,fructose,galactose or mannitol.These results suggested that SbSWEET11 and 18 could transport galactose;SbPLT5/6,SbTMT2 and SbSGB2 could transport fructose;SbpGlcT1 and SbSTP8/9 could transport glucose;and SbINT3 could transport glucose and mannitol in yeast,indicating diverse and various potential functions of the sugar transporters in sorghum plants.

Fig.7 Complementary analysis of biological functions of the sorghum sugar transporters in Saccharomyces cerevisiae hexose deficient strain,EBY.VW4000.

4.Discussion

4.1.Types and potential functions of sugar transporters in sorghum

Sugar transporters are widely involved in transporting different sugars,and their functions mainly include phloem loading,unloading,and metabolism (Braunet al.2014;Ruan 2014),while the proteins involved in the sugar transport family are generally classified into SUT(Riesmeieret al.1992;Hiroseet al.1997),SWEET (Chenet al.2010),and MST (Büttner and Sauer 2000).Some other types of sugar transporters have been reported in different plants,such asArabidopsis,rice,and maize(Divyaet al.2003;Carpanetoet al.2005;Klepeket al.2005;Schneideret al.2006;Chenet al.2012).In sorghum,only a small number of sugar transporters have been reported thus far,i.e.,SbSWEETs,SbSUTs,and SbTMT (Braun and Slewinski 2009;Bihmidineet al.2016;Mizunoet al.2016).Therefore,the overview of sugar transporters in sorghum,including the total number,types,genome-wide distribution,sequence characterizations,potential functions,and so on,still remains incomplete.

The results of the present study revealed the whole genome distribution of sugar transporters in sorghum,including the previously reported members and all the subfamilies that have been reported inArabidopsisand other plants (Fig.1;Appendix B) (Braun and Slewinski 2009;Milneet al.2013;Bihmidineet al.2015;Mizunoet al.2016).In some other plants,such asArabidopsis,rice,and maize,the reported members of SUT and SWEET all possessed conserved domains composed of multiple TMH (Scofieldet al.2007;Chen 2014;Yuanet al.2014;Bakeret al.2016).In our present work,we also found such features among the SUT and SWEET members in sorghum,which might suggest similar functions of these SSTs in sorghum.

Additionally,in rice andArabidopsis,the members belonging toMSTwere divided into different subfamilies(Johnson and Thomas 2007).The three members in sorghum,i.e.,SbTMT1,SbTMT2,and SbTMT5,belong to the TMT subfamily of MST and were reported to mediate the transport of sucrose,glucose and fructose on the vacuole membrane in sorghum (Bihmidineet al.2016),yet the full overview of MSTs in sorghum remains unknown.In the present study,we revealed seven subfamilies of MST in sorghum,and also identified some members that were never reported before,including SbTMT3 and SbTMT4 (Fig.1;Appendix D).These results provide informative references for the further functional dissection ofMSTs in sorghum.The sequence characteristics of diverse subfamilies were different,while they exhibited high conservation within the same subfamily (Appendices N-V).Combined with the SUT and SWEET families,the SSTs are also divided into nine subfamilies,which is consistent with the results of sugar transporters found in different species (Büttner 2010;Slewinski 2011;Chen 2014;Juliuset al.2017;Milneet al.2017).The phylogenic classificationsviasequence features could serve as the foundation for in-depth studies of the functions of sugar transporters.

4.2.Organ/tissue specific expression patterns of SST genes

The differential expression patterns of sugar transporters are closely related to their biological functions (Yuanet al.2014;Bakeret al.2016).Based on the results of independent experiments in sorghum,the expression patterns of the identified sugar transporters were divided into five classes,each with a different tissue/organspecific expression pattern (Fig.5;Appendix M).The expression patterns of someSSTs in the present study were consistent with those reported for theSWEETandSUTgenes in sorghum (Mizunoet al.2016).Differential expression patterns have suggested diverse biological functions.For example,the high expression ofAtSWEET2in root restricted both carbon sequestration andPythiuminfection (Chen H Yet al.2011);whileOsSWEET11was highly expressed during caryopsis development,and played a key role in early grain filling(Maet al.2017).SbTST1,SbTST2andSbTST3are represented by different transcripts between grain sorghum and sweet sorghum,and functional studies found that they are involved in transporting sucrose,glucose and fructose (Bihmidineet al.2016).In the present study,the results of real-time PCR analysis further showed thatSbPLT5,SbSTP3andSbSTP9are mainly expressed in leaves,but the remaining genes are expressed in different tissues and development stages(Fig.6).High expression ofSbPLT6,SbINT3,SbSTP8,SbTMT2,SbSWEET11,SbSWEET18,SbSWEET21,SbSGB2andSbERD3(SbERD3;1) in root and leaf might indicate that they are mainly involved in sugar transport or other functions in those tissues.Besides sugar transportation,further studies are also needed to clarify the other biological functions related to their specific expression in tissues.

4.3.Diverse sugar transporting functions of SSTs

Transmembrane domains (TMDs) and loops are the typical structures of SUT proteins (Shiratake 2007).In this study,we found that all sugar transporters in sorghum contain different numbers of TMHs (Appendices K and L),which correspond to their locations on the membranes.Meanwhile,we studied the locations of seven sugar transporters in onion epidermal cells,and all of them were found to be located on the membrane or membrane bound organelles (Fig.4).These results were consistent with previous studies in maize,sorghum,Arabidopsis,and wheat.In maize,ZmSUT1 was reported to be localized to the plasma membrane (Bakeret al.2016),and in wheat,the TaSUT1 protein was also located on the plasma membrane of phloem sieve elements in all classes of veins (Aokiet al.2004).In sorghum,SbSUT1 and SbSUT5 were also found to be located on the plasma membrane,while SbSUT4 was on the tonoplast (Milneet al.2017).Meanwhile,the subcellular localization results of SWEET and PMT/PLT proteins inArabidopsisalso support the membrane localization results for the onion epidermal cells in the present study (Klepeket al.2010;Xuanet al.2013;Tianet al.2017).The differences in protein locations also confirmed the diversity of their functions.For example,ZmSUT1 plays an important role in the apoplasmic phloem (Bakeret al.2016),and TaSUT1 is responsible for loading photo assimilates and for axial transport in veins (Aokiet al.2004).

A preliminary study on the functions of 14 sorghum sugar transporters was carried out by utilizing the complementarity of the EBY.VW4000 mutant (Yuanet al.2014).The results of similar methods in the present study indicated that galactose could be transported by SbSWEET11 and SbSWEET18 in yeast,but not by SbSWEET21,which is similar to the finding that not all SWEET proteins in rice were involved in galactose transport in yeast (Yuanet al.2014).Although few studies have reported the MST family proteins in sorghum,results similar to those for the MSTs in present study were also documented in other plants.For example,ArabidopsisPLT subfamily members AtPMT1 and AtPMT2 were found to participate in the transport of fructose and xylitol in pollen (Klepeket al.2010).TheSbPLT5andSbPLT6identified in the present study were highly expressed in leaves (Figs.5 and 6),and results from complementarity of EBY.VW4000 suggested that both of them might participate in fructose transport (Fig.7),similar to those inArabidopsis.Furthermore,it was reported that sugar transport protein AtSTP13 affected the glucose transport activity in leaves ofArabidopsisplants (Lemonnieret al.2014).For sorghum,we also observed that SbSTP8 and SbSTP9 presented functions of transporting glucose in yeast.

Interestingly,we also found some specific functions of the sugar transporters.For example,SbSGB2 and SbpGlcT1 exhibited the functions of transporting both fructose and glucose in EBY.VW4000,but VvpGLT only served as a glucose transporter inSaccharomyces cerevisiae(Zenget al.2014).Bhimidine and colleagues reported that in sorghum,SbTMT1,SbTMT2,and SbTMT5 mediated the transport of sucrose,glucose,and fructose on the vacuole membrane (Bihmidineet al.2016),while in the present study,we found that SbTMT2 was only involved in fructose transport in yeast (Fig.7).Similar functional differences were also observed among SbINT3,SbERD3;1,and SbERD3;2 (Fig.7).In summary,the diverse functional profiles of sugar transporters in sorghum might indicate the potential utilization of these transporters in yield and quality improvements of sorghum varieties.

5.Conclusion

In this study,a total of 98 sorghum sugar transporters(SSTs) are identified and classified into three families(SUTs,SWEETs and MSTs) according to phylogenetic analysis.The MSTs are further divided into seven subfamilies,STPs,PLTs,VGTs,INTs,pGlcT/SBG1s,TMTs,and ERDs.Twenty-seven SST genes are clustered in eight tandem repeats,and 22 SSTs comprise 11 paralogous pairs.Although the SST proteins exhibit the same conserved domain in different subfamilies,there are some differences in the features of gene structure,motifs and transmembrane helices (TMH).The SST genes exhibit various expression patterns,and their coding proteins are mainly located on the cell membrane and membrane organelles.Meanwhile,the SST proteins can transport various types of monosaccharides in yeast.These results can increase our understanding of theSSTgenes and provide valuable information for further elucidating their functions in sugar transport and sorghum development.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (32001607) and the Fundamental Research Funds for the Central Universities of Southwest University,China (SWU118087).We are grateful to the researcher who provided the sorghum expression data in the public database (https://www.ebi.ac.uk/gxa/home).We thank Prof.Huang Yubi (State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China and Agronomy College,Sichuan Agricultural University) for providing yeast strains EBY.VW4000 and the plasmid.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm