Identification 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 file 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 file 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.
Table 1
Information about daylily SWEET genes
Gene name
|
ORF length (bp)
|
AA (aa)
|
MW(kDa)
|
pI
|
II
|
AI
|
GRAVY
|
THM
|
MtN3/saliva (PQ-loop repeat) domain position
|
HfSWEET1a
|
735
|
244
|
27.162
|
9.26
|
40.21
|
111.80
|
0.656
|
7
|
7-95, 132-214
|
HfSWEET1b
|
762
|
253
|
28.351
|
9.01
|
27.15
|
105.49
|
0.519
|
7
|
7-95, 132-214
|
HfSWEET2a
|
699
|
232
|
25.908
|
8.81
|
42.55
|
124.78
|
1.018
|
7
|
18-100, 138-218
|
HfSWEET3b
|
714
|
237
|
26.322
|
9.44
|
38.27
|
117.22
|
0.700
|
7
|
7-98, 132-217
|
HfSWEET4a
|
774
|
257
|
28.370
|
9.30
|
28.83
|
127.35
|
0.800
|
6
|
10-95, 133-217
|
HfSWEET4b
|
735
|
244
|
26.993
|
8.89
|
37.65
|
130.82
|
0.942
|
7
|
10-97, 133-217
|
HfSWEET4c
|
735
|
244
|
26.959
|
8.95
|
34.37
|
132.87
|
0.941
|
7
|
10-98, 133-217
|
HfSWEET5
|
714
|
237
|
25.932
|
8.63
|
31.03
|
131.52
|
0.891
|
7
|
11-96, 134-213
|
HfSWEET6a
|
714
|
237
|
26.235
|
9.21
|
44.96
|
133.59
|
0.957
|
7
|
9-98, 133-217
|
HfSWEET6b
|
840
|
279
|
30.460
|
9.03
|
35.24
|
118.14
|
0.669
|
7
|
11-96, 133-217
|
HfSWEET7
|
711
|
236
|
25.764
|
9.22
|
36.30
|
134.49
|
1.106
|
7
|
10-95, 133-213
|
HfSWEET12
|
786
|
261
|
29.196
|
8.94
|
48.05
|
122.53
|
0.721
|
7
|
14-98, 132-218
|
HfSWEET13a
|
825
|
274
|
30.706
|
9.20
|
29.62
|
118.43
|
0.667
|
7
|
12-99, 133-214
|
HfSWEET13b
|
870
|
289
|
32.278
|
5.74
|
34.67
|
116.99
|
0.516
|
7
|
12-82, 134-215
|
HfSWEET14a
|
861
|
286
|
32.412
|
8.80
|
33.51
|
123.36
|
0.593
|
7
|
12-99, 133-215
|
HfSWEET14b
|
861
|
286
|
32.276
|
8.80
|
31.49
|
122.38
|
0.596
|
7
|
12-99, 133-215
|
HfSWEET15
|
870
|
289
|
32.116
|
5.49
|
38.42
|
121.45
|
0.724
|
7
|
12-98, 134-215
|
HfSWEET16
|
900
|
299
|
32.976
|
9.64
|
34.03
|
112.04
|
0.442
|
7
|
7-91,128-211
|
HfSWEET17
|
723
|
240
|
26.934
|
4.74
|
43.23
|
119.67
|
0.730
|
7
|
7-90,128-212
|
AA: Number of amino acids; MW: Molecular weight(kDa); pI, isoelectric point |
II: Instability index; AI: Aliphatic index; GRAVY: Grand average of hydropathicity |
THM: Prediction of the number of transmembrane helix |
Conserved motifs and conserved domains analyses of HfSWEETs
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 identified and named motif 1 to 10 (Fig. 2). Motif 1 to 5 were detected in all HfSWEET proteins except HfSWEET12, which lacking Motif 5. Motif 6 was detected in two members in each of Clade I, Ⅱ, and III, but not in any member of Clade Ⅳ. Motif 7 was detected in the members of the Clade Ⅱ and Clade Ⅳ. Motif 8 and motif 9 were only detected in some members of the Clade Ⅱ. Motif 10 was only detected in HfSWEET3b, HfSWEET12 and HfSWEET16 protein.
The results of conserved domain analysis showed that all the HfSWEET proteins harbored two MtN3/saliva domains or PQ-loop superfamily at the similar positions (Fig. 2). These MtN3/saliva domains ranged from 70 aa to 91 aa, and most of them were approximately 85 aa in length. The position of the MtN3/saliva domains in the protein were shown in 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 five 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 five 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 find 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 profiles 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 first and then decreased, but three HfSWEETs showed contrary expression trend. The relative expression levels of five 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 fluorescent 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 significantly 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 significant difference between transgenic and WT plants was observed. When the temperature reached 0 ℃, all lines were wilted, but transgenic plants showed significantly 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 significantly different between the WT and transgenic plants. With the decrease of temperature, REL and POD of leaves from all transgenic and WT plant leaves showed a trend of first increasing and then decreasing (Fig. 9). The transgenic plants showed significantly lower REL values of 1.13-, 1.26-, 1.23-, and 1.08-fold under 15 ℃, 10 ℃, 5 ℃, 0 ℃, respectively, than that of WT plants. The activity of POD was significantly increased in transgenic plants compared with that of WT plants and was 1.32-, 1.51-, 1.15-, 1.42-, and 1.2-fold higher under 20 ℃, 15 ℃, 10 ℃, 5 ℃, 0 ℃, respectively.