Genome-wide identi cation and expression analysis of SWEET gene family in daylily (Hemerocallis fulva) and functional analysis of HfSWEET17 in response to cold stress

Background The Sugars Will Eventually be Exported Transporters (SWEETs) are a newly discovered family of sugar transporters whose members exist in a variety of organisms and are highly conserved. SWEETs have been reported to be involved in the growth and development of many plants, but little is known about SWEETs in daylily (Hemerocallis fulva), an important perennial ornamental flower. Results In this study, 19 daylily SWEETs were identified and named based on their homologous genes in Arabidopsis and rice. Phylogenetic analysis classified these HfSWEETs into four clades (Clades I to IV). The conserved motifs and gene structures showed that the HfSWEETs were very conservative during evolution. Chromosomal localization and synteny analysis found that HfSWEETs were unevenly distributed on 11 chromosomes, and there were five pairs of segmentally duplicated events and one pair of tandem duplication events. The expression patterns of the 19 HfSWEETs showed that the expression patterns of most HfSWEETs in different tissues were related to corresponding clades, and most HfSWEETs were up-regulated under low temperatures. Furthermore, HfSWEET17 was overexpressed in tobacco, and the cold resistance of transgenic plants was much higher than that of wild-type tobacco. Conclusion This study identified the SWEET gene family in daylily at the genome-wide level. Most of the 19 HfSWEETs were expressed differently in different tissues and under low temperatures. Overexpression further suggests that HfSWEET17 participates in daylily low-temperature response. The results of this study provide a basis for further functional analysis of the SWEET family in daylily. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-022-03609-6.


Introduction
As the substrate of carbon and energy metabolism, sugar provides energy sources for plant growth and development [1] . Sugar also participates in various signal transduction pathways in plants [2] . But sugar can not independently cross the plant biomembrane system, assistance of the transport function of corresponding sugar transporters is needed [3] . SWEET protein is a kind of sugar transporter that is largely pHindependent and bidirectional transmembrane transport of sugar along concentration gradient [4][5] . It can selectively transport monosaccharides or disaccharides in cells or plasma membrane [6] . It is widely found in prokaryotes, plants, humans and other animals [7][8] . The typical structures of eukaryotic SWEET proteins consist of seven transmembrane helices, harboring two MtN3/saliva domains, also known as PQ-looprepeat [9] . However, the SWEET protein in prokaryotes contains only three transmembrane helical proteins and one MtN3/saliva domain [10] . This difference may indicate that the eukaryotic SWEET protein evolved by replicating and fusing the basic 3-TM unit present in the prokaryotic semi-SWEET protein [11] .
The rst identi ed plant SWEET transporter is AtSWEET1, which acts as a single glucose transporter and involves in ower development by supplying nutrients to the gametophyte or nectaries [6] . Arabidopsis (Arabidopsis thaliana) AtSWEET8 has also been shown to be critical for plant pollen viability [12] . In addition to, numerous studies have shown that SWEET genes are involved in multiple biological processes, such as reproductive development, modulating gibberellins, disease resistance, abiotic stress, seed and fruit development [13][14][15][16] . Genome-wide identi cation and analysis of SWEET gene family have been reported in a variety of plant species, such as Gossypium hirsutum, Arabidopsis, Sorghum bicolor, rice (Oryza sativa), Litchi chinensis, Glycine max and so on [9,11,17−19] .
Daylily (Hemerocallis fulva) is a herbaceous perennial plant, with edible, medicinal and ornamental value, it is widely cultivated worldwide. In recent years, daylily has attracted biological investigation, there is a growing number of reports on daylily molecular mechanisms and gene function analysis [20][21][22][23] .
Our previous study analyzed the characteristics of daylily HfSWEET2a gene and its expression level changes at low temperature [24] . Besides, there is no report on SWEET gene in daylily. In the current study, given the molecular mechanism of daylily gene family remains poorly understood, we performed whole genome-wide analysis to identify SWEET genes in daylily and analyze the phylogenetic relationships, gene structures, chromosomal localization, conserved motifs and domains of the SWEET genes in detail. Then the expression characteristics of daylily SWEET gene family members at different low temperatures stages were studied. Beyond that HfSWEET17 gene was introduced into tobacco by Agrobacterium-mediated method to investigate its function. The results of this study provided some clues for understanding the function and cold response of SWEET gene.

Identi cation of the daylily SWEET Gene Family
Through the screening, a total of 19 SWEET genes were obtained in daylily (GenBank accession No. OM264165-OM264183, Additional le 1: Table S1), named as HfSWEET1-HfSWEET17 according to their identity percentage with Arabidopsis AtSWEETs and rice OsSWEETs. Gene characteristics, including the complete ORFs, number of amino acids (AA), molecular weight (MW), isoelectric point (pI) and so on were analyzed ( Table 1). The results showed that the ORFs of the 19 HfSWEET genes ranged from 699 bp to 900 bp in length, encoding proteins 232 aa to 299 aa. HfSWEET7 protein had the smallest MW with 25.764 kDa, and the largest one was HfSWEET16 with 32.976 kDa. The pI ranged from 4.74 (HfSWEET17) to 9.64 (HfSWEET16), which indicated that most of the HfSWEET protein were basic proteins. The instability index ranged from 27.15 (HfSWEET1b) to 48.05 (HfSWEET12), both stable and unstable proteins were present in HfSWEET protein. All of the HfSWEET proteins were hydrophobic protein (Grand average of hydropathicity, GRAVY > 0). The results of the number of transmembrane helix analysis showed that majority of the HfSWEET proteins contained 7 transmembrane domains, only HfSWEET4a contained 6 transmembrane domains. These results indicated that the basic properties of the proteins encoded by members of the daylily HfSWEET gene family were different.

Phylogenetic Analysis of the HfSWEET Gene Family
In order to investigate the evolutionary relationships among HfSWEET proteins and SWEET proteins from Arabidopsis and rice (Additional le 2: Table S2), a neighbor-joining phylogenetic tree was constructed using MEGA 7 software. Results showed that the HfSWEET proteins were clearly divided into four clades (Clades I, II, III, and IV) (Fig. 1). The largest clade was Clade II, which consisted of seven HfSWEET proteins (HfSWEET4a/4b/4c/5/6a/6b/7); The second clade is Clade III, which contained six HfSWEET proteins (HfSWEET12/13a/13b/14a/14b/15); Clade I contained four HfSWEET proteins (HfSWEET1a/1b/2a/3b); and Clade III were the fewest, containing only HfSWEET proteins (HfSWEET16/17). Compared with Arabidopsis, the similarity of SWEET gene between daylily and rice is higher, indicating that SWEET genes in daylily was more closely related to rice than to Arabidopsis. The conserved motifs and conserved domains were analyzed to further understand the characteristics of HfSWEETs. The results of conserved motif analysis showed that a total of 10 motifs were identi ed and named motif 1 to 10 (  Table 1. In addition, there are no other conserved domains.

Gene structure analysis of HfSWEETs
In order to elucidate the structural characteristics of daylily SWEET genes, the exon-intron organization was analyzed. The result showed that ve or six exons were exist in most HfSWEETs (Fig. 3). The HfSWEET genes in the Clade I, III and IV all contained 6 exons; Majority HFSWEET genes in the Clade II contained 5 exons, while HFSWEET5 and HFSWEET6a containing 6 exons, and HFSWEET7 containing 7 exons. In general, the introns length of HfSWEET members in Clade II and IV were longer than that in Clade I and III. These results revealed that HfSWEET genes in the same clade share similar gene structure.

Chromosomal localization and synteny analysis of HfSWEETs
According to the gene loci information, the 18 HfSWEET genes were unevenly distributed in eleven chromosomes of daylily and the detailed chromosomal locations were shown in the Figure 4. By contrast, HfSWEET16 was distributed on a scaffold whose exact locations on chromosome was not determined. Chromosome 2 and 9 had the largest number of HfSWEETs (three genes), followed by chromosomes 1, 3, 4, 5 and 10 (two genes on each chromosome), and the minimum number was found on chromosome 8 and 11 (one gene). Except for the HfSWEET7 gene, other HfSWEET genes were located in the middle and lower part of the chromosomes.
According to the results of collinearity analysis, there was ve pairs of segmental duplicated events of HfSWEET genes in daylily genomes. The most frequently duplicated gene was HFSWEET13a, which duplicated three times, which corresponding to HFSWEET13b, HFSWEET14b, and HFSWEET15, respectively. HFSWEET4a/7 and HFSWEET4a/4b/4c may also be generated by fragment duplication. In addition to, HFSWEET14a/14b was clustered into tandem duplication events. Based on the above results, some HfSWEET genes were probability generated by gene segmental or tandem duplication. The results of collinearity analysis between daylily and Arabidopsis and rice show that seven HfSWEET homologous protein genes appear in the last three chromosomes of Arabidopsis ( Fig. 5), but there were nine HfSWEET genes that can nd corresponding paralogous genes on six chromosomes on rice. It can be seen that the relationship between daylily and rice is closer than that of Arabidopsis.

Expression pro les of HfSWEETs under low temperature
To obtain insights into the physiological functions of the HfSWEETs in response to low temperature stress, the expression patterns of 19 HfSWEETs under different temperature (25 ℃ as CK, low temperature treatments: 10 ℃, 5 ℃ and 0 ℃) were measured by qRT-PCR analysis. The results showed that the expression patterns were different among the 19 HfSWEETs (Fig. 6). Compared with the CK (25 ℃), with the decrease of temperature, the relative expression levels of nine HfSWEETs increased rst and then decreased, but three HfSWEETs showed contrary expression trend. The relative expression levels of ve HfSWEETs (HfSWEET3b, HfSWEET5, HfSWEET14b, HfSWEET16, HfSWEET17) were higher than CK at all lower temperatures. Among them, the expression level of HfSWEET5 and HfSWEET17 rose steadily as the temperature drops. However, three HfSWEETs (HfSWEET1a, HfSWEET12, HfSWEET13b) were lower than CK at all lower temperatures and the expression level of HfSWEET1a gradually decreased with the decrease of temperature. In general, the relative expression of majority HfSWEETs were up-regulated by low-temperature treatment, and most of them was highest at 10 ℃ or 0 ℃, which were 1.43-57.95 times than CK.

Subcellular localization analysis of HfSWEET17 protein
The HFSWEET17 had the highest relative expression level in the daylily SWEET family, and the expression level of it gradually increased with the decrease of temperature. In order to explore the function of HFSWEET17 in daylily, the subcellular localization of HfSWEET17 protein was studied. HfSWEET17 protein was transiently expressed as translational GFP (green uorescent protein) fusion proteins in tobacco leaf epidermalcells. Confocal images of transient expression of GFP fusion protein in protoplasts was showed that 35S:HfSWEET17-GFP fusion protein was mainly presented in the cytoderm (Fig. 7). This result suggested that HfSWEET17 protein was cytoderm-localized.

Ectopic expression of HfSWEET17
To further explore the function of HFSWEET17 in the response to low temperature stress, it was chosen to be ectopical expressed in tobacco through Agrobacterium-mediated transformation. Under normal conditions (25 ℃), the leaf size of transgenic plants was signi cantly larger than those of the WT plants (Fig. 8). When exposed to cold stress condition, all lines received mild cold injury, chlorosis and leaf margins slightly curled before the temperature drops to 5 ℃, but no signi cant difference between transgenic and WT plants was observed. When the temperature reached 0 ℃, all lines were wilted, but transgenic plants showed signi cantly better status than the WT plants under low temperature treatment (Fig. 8).
The level of REL and the activity of POD were measured. In normal condition, the REL and POD were not

Discussion
Plant SWEETs play signi cant roles in physiological metabolism, growth, and development by regulating sugar transport and distribution [5] . For example, they are involved in pollen wall formation, anther dehiscence, seed development and responses of various abiotic stresses [25][26][27] . Recently, the SWEET gene families from some plants species have been reported. A growing number of evidence suggests SWEETs play important roles in low-temperature response [16,28] . In the present study, we identi ed and characterized SWEET gene family in daylily by genome-wide analysis and investigated their expression patterns under low temperature treatment.

SWEET gene family in daylily
In this study, we successfully identi ed 19 HfSWEETs based on daylily genome and named them as HfSWEET1-HfSWEET17 based on their homologies in Arabidopsis and rice ( Table 1). The length of HfSWEET proteins ranged from 232 aa to 299 aa, which was similar to that has been reported in other plants, such as 229-300 aa in litchi, 233-308 aa in tomato, and 234-301 aa in Gossypium hirsutum [8,11,19] . Phylogenetic analysis divided 19 HfSWEETs into four clades (Clade I to IV) which is consistent with the results in Arabidopsis, Litchi chinensis, and Gossypium hirsutum [6,8,19] . Each clade contained 4, 7, 6, and 2 HfSWEET members in daylily, respectively (Fig. 1), which was similar to other plants [29][30] . The results of the intron-exon location analysis showed that the number and distribution of the introns and exons of HfSWEETs were highly conserved, and most HfSWEETs possessed ve or six exons (Fig. 3). It has been indicated that the results of conserved motif analysis were similar to those of phylogenetic analysis [30][31] .
Our results were consistent with those reports. The HfSWEETs in each clade harbored some special conserved motifs (Fig. 2), which suggested that they might have different functions in daylily.
Further chromosomal localization and synteny analysis showed that 18 HfSWEETs were unevenly distributed on eleven chromosomes of daylily, and only one (HfSWEET16) was distributed on scaffold. Collinearity analysis showed there were segmental duplicated events and tandem duplication events in the daylily HfSWEET gene family (Fig. 4). This suggested that HfSWEETs in daylily might have evolved from gene duplication. Gene duplication, including whole-genome duplication, tandem gene duplication and segmental duplication events, can be a crucial factor for plant gene family evolution [32] , and the latter two have been suggested to represent the main causes of gene family expansion in plants [33] . Following gene duplication, duplicated gene pairs can undergo different functions [34] . Combined with the above analysis of the characteristics of HfSWEETs, we speculated that the expansion of HfSWEET genes might play an important role in various gene functions of HfSWEET [34] .
Expression patterns and function diversity of HfSWEETs in response to low temperature stress The differential expression analysis of SWEETs can help to explore the special functions of SWEET proteins. The expression of SWEET genes has been shown to change in response to chilling stress in several plant species [28,31] . Analyzing the expression pattern of 19 HfSWEETs under low temperature treatment, we found that compared with the control group (25 ℃), the expression levels of all HfSWEETs in the low temperature (10 ℃, 5 ℃, 0 ℃) treatment group were changed and the relative expression levels of most HfSWEETs were increased (Fig. 6), suggesting that more than one HfSWEET gene were responsive to low-temperature stress. The expression patterns of 19 HfSWEETs were different, the relative expression of most of them was highest at 10 ℃ or 0 ℃, suggesting that these genes may have functional redundancy.
Retained duplication genes were generally believed to be those involved in neofunctionalization, subfunctionalization, and nonfunctionalization, among which, neofunctionalizationn and subfunctionalization can lead to the differential spatial and temporal expression of duplication genes [35] . In the present study, the expression patterns of the pairs of duplicated genes in daylily under low temperature stress were various. For example, some duplicated genes, such as HfSWEET4a/4b and HfSWEET4a/7 were the same, whereas that of some duplicated genes like HfSWEET13a/13b and HfSWEET14a/14b were signi cantly different. These results indicated that some duplicated HfSWEET genes were functionally similar may due to nonfunctionalization during gene replication, while some duplicated HfSWEET genes may have developed neofunctions or subfunctions and were functionally different [32,35] . These results were consistent with results reported for litchi and apple [19,36] .

Ectopic expression of HfSWEET17 improved cold stress tolerance in transgenic tobacco
In Arabidopsis, AtSWEET17 is a vacuolar fructose transporter that participates in the regulation of fructose levels and controls leaves fructose content [37][38] , and is critical for root development and drought tolerance [39] . DsSWEET17 from Dianthus spiculifolius affected the sugar metabolism and conferred multiple tolerance in transgenic Arabidopsis [40] . In the present study, the HfSWEET17 gene was highly expressed under low temperature treatment (Fig. 6). To further evaluate the roles of HfSWEET17 gene in response to cold stress condition, the HfSWEET17 from daylily was transformed into tobacco. Morphological observations revealed that, the leaf size of the HfSWEET17-overexpressed lines was obviously larger than those of the WT plants under the non-stress growth condition (Fig. 7), indicating that HfSWEET17 may promote nutrition and reproductive growths by transporting and utilizing sugars, which was consistent with the experimental results of Yao et al [41] . Under 0 ℃ treatment condition, HfSWEET17-overexpressed plants showed signi cantly better status than the WT plants (Fig. 8), indicating that transgenic plants were less damaged by chilling.
Analysis of physiological indices showed that the HfSWEET17-overexpressed tobacco exhibited lower REL and higher POD under cold stresses compared to the WT plants, which conferred cold tolerance in transgenic tobacco. These results indicated that HfSWEET17 from daylily positively regulates cold stress in tobacco. Similar studies have previously reported that the overexpression of CsSWEET17 gene from Camellia sinensi increased sugar transport in Arabidopsis, thus affecting plant germination and growth, and improving freezing resistance [41] . However, the roles of HfSWEET17 was limited such as the transgenic plants did not produce enough in uence to reverse tobacco performance under cold stress in this study. The effect may be made even more pronounced through the formation of homo-or heterodimers by oligomerization [10] .
However, the biological function of this potential interaction remain to be further investigated.

Conclusions
In summary, this study identi ed the SWEET gene family in daylily at the genome-wide level. Nineteen HfSWEET genes were identi ed and comprehensively characterized, including phylogenetic analysis, conserved motifs prediction, exon-intron structure, chromosomal localization, and synteny analysis.
Phylogenetic analysis classi ed 19 HfSWEET genes into four clades (Clade I to IV). We also focused on the expression patterns of all the HfSWEETs under low temperature treatments, which indicated that they may involve in low temperature stress signaling pathway regulation. Furthermore, the overexpression of HfSWEET17 gene improved cold stress tolerance in transgenic tobacco. This study laid the foundation for elucidate the functions of the HfSWEET genes in daylily under low-temperature response.  [42] , was used to retrieve daylily genome database (unpublished) by HMMER3.0 and SPDE software [43][44] . The results were sequentially sorted to remove redundancy, and candidate genes of daylily SWEET gene family members were preliminarily obtained. Then, the candidate sequences were identi ed by SMART (http://smart.embl-heidelberg.de) and NCBI-CCD (https://www.ncbi.nlm.nih.gov/cdd) [45][46] .

Materials And Methods
Leaf total RNA was extracted using the Quick RNA isolation Kit and the quality of the RNA was analyzed by 1.5% (w/v) agarose gel electrophoresis and NanoDrop One. The rst-strand cDNA was synthesized using M-MuLV First Strand cDNA Synthesis Kit. The coding sequences of daylily SWEET genes were ampli ed from cDNA using gene-speci c primers (Additional le 3: Table S3). PCR ampli cation was carried out using Taq DNA Polymerase Kit in a PCR Thermal Cycler (Bio-Rad, S1000, USA). All PCR products were puri ed with the Prep Column PCR Product Puri cation Kit, then the puri ed PCR products were sequenced and the consensus sequences were deposited in GenBank (Additional le 1: Table S1). All the above kits and primers were provided by Sangon, Shanghai, China.

Sequence analyses
ProtParam (https://web.expasy.org/protparam/) was used to analyz the amino acids, molecular weights, and theoretical isoelectric point of daylily SWEET gene family members. Transmembrane domains was predicted by TMHMM Server v2.0, and the MtN3/saliva (PQ-loop repeat) domain position was searched by NCBI-CCD.
Gene structure analysis and prediction of conserved motifs and domains The exon-intron structures were analyzed by GSDS (http://gsds.cbi.pku.edu.ch). The MEME (http://memesuite.org/) was used for conserved protein motif prediction, and the NCBI conserved domain database was used to predict the conserved domains of SWEET gene family members of daylily.

Chromosomal distribution and gene synteny analysis
The positions of daylily SWEET genes on chromosomes were obtained from the daylily genome annotation les (unpublished). Arabidopsis and rice genome were both obtained from Ensembl plants (https://plants.ensembl.org/index.html). Furthermore, the synteny analysis among members of the daylily SWEET family members and the synteny analysis between daylily and Arabidopsis and rice were constructed using the MCScanX and TBtools [49][50] .

Expression pro les of SWEET genes in daylily
The 'Golden Doll' daylily was moved to an indoor incubator at a constant temperature and cultured at 25 ℃ (control group, CK), 10 ℃, 5 ℃ and 0 ℃ with a 12 h photoperiod for 24 h respectively. Samples were collected from fully expanded functional leaves. All samples were frozen in liquid nitrogen immediately after collection and stored at -80 ℃. Primer based on cDNA sequences of daylily SWEET family members were design by Primer5 (https://sg.idtdna.com/pages/tools/primerquest) (Additional le 3: Table S3). UBQ was used as the internal reference for real-time quantitative PCR (qRT-PCR) [51] .
Total RNA was extracted from leaves by the Quick RNA isolation Kit (Sangon, Shanghai), and the rst-strand cDNA was synthesized using M-MuLV First Strand cDNA Synthesis Kit (Sangon, Shanghai). Real-time quantitative PCR ampli cation was performed by AceQ qPCR SYBR Green Master Mix (Vazyme Biotech).
Ampli cation was initiated with a denaturation step of 5 min at 95 ℃, followed by 40 cycles of 95 ℃ for 10 s and 60 ℃ for 30 s. Fluorescence signals were detected at the end of every cycle. All reactions were performed using the Real-Time PCR Detection System (QuantStudio 5, USA), and data were analyzed using QuantStudio TM Design and Analysis Software. All reactions were performed in triplicate. Changes in gene expression were calculated using the 2 −ΔΔCt method [52] . Statistical analysis was performed by SPSS 20 software, and one-way ANOVA was performed for the relative expression of HfSWEETs under different temperatures.

Construction of HfSWEET17 transient expression vectors and subcellular localization
The open reading frame (ORF) of HfSWEET17 was ampli ed using primers containing the EcoR I/Spe I restriction sites, and the expression vector 35S:HfSWEET17-YFP was constructed using pc131-YFP vector framework. The recombined plasmids were then transformed into Agrobacterium Tumefaciens strain GV3101 through shock transformation [53] . Agrobacterium tumefaciens was cultured and injected into tobacco (Nicotiana benthamiana), and the uorescence distribution in leaf cells was observed under confocal laser microscope (Leica STELLARIS 5, Germany) after 48 h dark culture. The primers used were listed in Additional le 3: Table S3.

Generation of transgenic plants with HfSWEET17
The ORF of HfSWEET17 was inserted into the EcoR I/Sal I restriction sites of pCAMBIA1301. The obtained plasmid was transformed into Agrobacterium tumefaciens strain GV3101. Generation of transgenic tobacco was performed following the of leaf dish transformation method. Transgenic plants were selected using hygromycin B (50 mg/L) and con rmed by PCR analysis.

Declarations
Ethics approval and consent to participate All methods used in the manuscript were performed in accordance with relevant guidelines and regulations.

Consent for publication
Not applicable.

Availability of data and materials
The datasets generated and analysed during the current study are available in the GenBank repository, GenBank accession is No. OM264165-OM264183 and all sequences were provide in Additional le 1.

Competing interests
The authors declare that there are no con ict of interest.  Figure 1 Phylogenetic tree of SWEETs from daylily, Arabidopsis, and rice

Figures
The protein sequences of the 54 SWEETs from daylily, Arabidopsis, and rice were aligned by Clustal Omega, and the phylogenetic tree was constructed by the MEGA7.0 using the neighbor-joining method with 500 bootstrap replicates. SWEETs of daylily, Arabidopsis, and rice were pre xed with Hf, At, and Os, respectively, and represented by Black circles, hollow circles, and ve-pointed start, respectively.   Locations and duplications of HfSWEETs on daylily chromosomes.
The chromosome location of HfSWEET genes were shown by short grey lines. The red lines indicate segmentally duplicated genes, and the tandemly duplicated genes are boxed.

Figure 5
The synteny analysis between daylily and Arabidopsis and rice genome.
Syntenic relationship between daylily and Arabidopsis and rice genome shown on the chromosome maps.
The gray line represents the collinearity among all members, and the green line represents the collinearity among the members of the SWEET family.  Subcellular localization of HfSWEET17 protein.
HfSWEET17-GFP fusion protein and GFP alone (as control) were constructed using pc131-YFP vector framework and transiently expressed in Nicotiana benthamiana leaves using shock transformation. Protein localization was examined 48 h after dark culture and representative images are shown. Bar = 10 μm.

Figure 8
Phenotypic changes of WT and transgenic plants under cold treatment.