Genome-wide identification of SUT genes in Orchidaceae species
The SUTs are prevalent in plants and play fundamental roles in plant growth, development and stress tolerance [19,41-42]. To understand the potential roles of SUTs in orchids, the three sequenced Orchidaceae species, D. officinale, P. equestris, and A. shenzhenica, were used for genome-wide identification and characterization of SUT genes. The HHM profile of the SUT protein was used as a query to perform an HMMER search against the genome assemblies of the three species. Bioinformatics analysis identified a total of 22 SUTs from the three species, which were designated ‘DenSUT’ for D. officinale, ‘PeqSUT’ for P. equestris, and ‘ApoSUT’ for A. shenzhenica, with a serial number (Table 1, Table S1). Among them, D. officinale had 8 genes (DenSUT01-08), P. equestris had 8 genes (PeqSUT01-08) and A. shenzhenica had 6 genes (PeqSUT01-06). The results agree with previous reports that plant sucrose transporters are encoded by relatively small gene families.
According to the phylogenetic tree, the 22 SUT genes from three orchids were classified into four subgroups, subgroups A, B2.1, B2.2 and C (Fig. 1, Table 1). Subgroup A included three genes, DenSUT01, PeqSUT01 and ApoSUT01. There were also four genes in subgroup C (DenSUT03, DenSUT04, PeqSUT08 and ApoSUT02) and three genes in subgroup B2.2 (DenSUT02, PeqSUT03 and ApoSUT03), respectively. However, the SUT genes were significantly expanded in the monocot-specific subgroup B2.1 which comprised 12 genes. Phlogenetically, sucrose transporters of D. officinale were more close to that of P. equestris than A. shenzhenica.
The molecular weights of the SUT genes ranged from 51.22 to 106.90 kD with pI values ranged between 4.95 and 10.12. Most of these genes were ~500 aa or ~600 aa in length with 11-13 introns and 12-14 extrons, whereas there were several genes with only 4-5 introns/extrons. Previous studies indicate that plant sucrose transporters are usually consisted of 500-600 aa, with molecular weight of 55-60 kD [15,43], which is consistent with the findings in the present study. Detail information of the SUT genes, including name, coding protein, CDS length, molecular weight and PI value, is shown in Table 1.
Table 1
Physical and molecular characteristics of SUT genes in A. shenzhenica, D. officinale, and P. equestris
Gene Name
|
Scaffold location
(bp)
|
Subgroup
|
Length
(bp)
|
Size
(aa)
|
MW
(kDa)
|
pI
|
Extron
|
Intron
|
ApoSUT01
|
1215667
|
1232090
|
A
|
16423
|
458
|
49879.49
|
7.5
|
12
|
11
|
ApoSUT02
|
644564
|
658003
|
C
|
13439
|
532
|
56556.26
|
9.6
|
5
|
4
|
ApoSUT03
|
1021836
|
1041725
|
B2.2
|
19889
|
589
|
64293.55
|
5.39
|
13
|
12
|
ApoSUT04
|
328657
|
335790
|
B2.1
|
7133
|
488
|
51878.16
|
8.85
|
13
|
12
|
ApoSUT05
|
534128
|
538346
|
B2.1
|
4218
|
477
|
51224.4
|
9.05
|
14
|
13
|
ApoSUT06
|
314008
|
320943
|
B2.1
|
6935
|
499
|
52812.97
|
8.36
|
14
|
13
|
DenSUT01
|
1939695
|
1953824
|
A
|
14147
|
716
|
78785.55
|
8.55
|
13
|
12
|
DenSUT02
|
11559848
|
11579293
|
B2.2
|
19445
|
571
|
62989.08
|
4.95
|
14
|
13
|
DenSUT03
|
83114
|
90826
|
C
|
7712
|
216
|
22730.49
|
10.12
|
5
|
4
|
DenSUT04
|
74903
|
90826
|
C
|
7712
|
216
|
22730.49
|
10.12
|
5
|
4
|
DenSUT05
|
10800848
|
10810268
|
B2.1
|
9420
|
984
|
106898.82
|
8.93
|
14
|
13
|
DenSUT06
|
3505256
|
3512511
|
B2.1
|
7255
|
177
|
18812.02
|
8.79
|
14
|
13
|
DenSUT07
|
29089
|
32887
|
B2.1
|
3798
|
492
|
52983.52
|
9.06
|
14
|
13
|
DenSUT08
|
397815
|
406603
|
B2.1
|
8788
|
470
|
50102.76
|
8.38
|
14
|
13
|
PeqSUT01
|
247532
|
268499
|
A
|
20967
|
461
|
50349.07
|
7.51
|
13
|
12
|
PeqSUT02
|
14129
|
22513
|
B2.1
|
8384
|
240
|
26208.15
|
9.3
|
8
|
7
|
PeqSUT03
|
3166955
|
3194083
|
B2.2
|
27128
|
611
|
65924.14
|
6.19
|
14
|
13
|
PeqSUT04
|
2062452
|
2066080
|
B2.1
|
3628
|
499
|
53546.81
|
8.32
|
14
|
13
|
PeqSUT05
|
58510
|
76466
|
B2.1
|
17956
|
500
|
53040.17
|
8.24
|
14
|
13
|
PeqSUT06
|
523303
|
531370
|
B2.1
|
8067
|
492
|
52933.39
|
9.11
|
14
|
13
|
PeqSUT07
|
659976
|
662642
|
B2.1
|
2666
|
489
|
52440.8
|
9.17
|
12
|
11
|
PeqSUT08
|
10082423
|
10107132
|
C
|
24709
|
413
|
43952.87
|
9.02
|
7
|
6
|
Phylogenetic relationship of the SUT proteins in major plant species
To provide insight into the evolution of SUT gene families, we performed phylogenetic analysis using 24 representative plant species, including green alga, mosses, lycophytes, gymnosperms, monocots and dicots. Detailed information of the SUTs see Methods. The SUT domain sequence and neighbour-joining method was used with 1000 bootstraps to construct the phylogenetic tree. In this study, the SUT genes of several eukaryotic chlorophyta were clustered in a special branch, which was defined as outgroups. The SUTs from the 24 species were classified into three groups and five subgroups: subgroups A, B1, B2.1, B2.2, and C (Fig. 1). Group A contained at least one member from mosses, lypophytes and angiosperms including both monocots and dicots. Group B was the largest group which is divided into three subgroups; subgroup B1 was made up of SUT genes from exclusively dicot species, corresponding to the SUT1 clade by Lalonde & Frommer [20]. Subgroup B2.2 contained both monocot and dicot species that are also present in the SUT2 group reported by Lalonde & Frommer [20]. Whereas subgroup B2.1 was monocot-specific expansion clade containing members from SUT3 and SUT5 reported by Kühn & Grof [2]. Group C contained mosses, lypophytes and angiosperms including both monocots and dicots, corresponding to SUT4 clade [20]. According to previous studies, the SUT1 and SUT2 proteins mainly play roles in phloem loading and unloading, sucrose transport to sink cells, and sucrose exchanges with microbes [2, 30,31,44-45]. While SUT4 proteins are involved in various physiological processes such as circadian rhythms and responses to dehydration and photosynthesis [46-47]. In recent studies, the SUTs were classified into two subfamilies (Ancient Group 1 and Ancient Group 2) and three types (Type I, Type II and Type III) [18,27]. Type I clade is dicot specific which corresponds to the SUT1 group [2] and Type III clade contains both monocots and dicots which corresponds to the SUT4 group [20]. Type II (A) is composed of monocot and dicot species that are also reported in the SUT2 group by Lalonde & Frommer [20], whereas the monocot specific Type IIB contains members from SUT3 and SUT5 reported by Kühn & Grof [2].
Sucrose transporters have been identified in primary terrestrial plants including both lypophyte and moss, with 6 SUTs in Selaginella lepidophylla and 7 SUTs in Physcomitrella patens; however, none was identified in green alga Chlamydomonas reinhardtii [18]. There were 6-10 SUT genes in monocot see crops such as rice (6 genes), maize (10 genes) and sorghum (8 genes). In contrast, in another monocot species, Ananas comosus, only 3 SUTs were identified. For most dicot species, 4-9 SUTs were identified. These results revealed that the number of sucrose transporters remains largely stable during the evolution from lower plants to terrestrial plants. This conclusion is consistent with previous studies on SUT gene identification and evolution [18,20,27]. However, the SUTs were expanded in several species such as Triticum aestivum (18 genes) and Glycine max (14 genes), which may be the result of whole genome polyploidization. The SUTs of some monocot species were expanded in subgroups B2.1; for example, there were 5 ZmaSUTs in subgroup B2.1, whereas 3 ZmaSUTs were identified in subgroup A, and only one was identified in subgroup B2.2 and C. Likewise, the SUTs from dicot species were expanded in subgroup B1 such as GmaSUTs, AtSUTs and DcaSUTs. The characean algae Chlorokybus atmosphyticus contains one SUT homolog which is basal to all the streptophyte SUTs [18]. We also identified one SUT (VcaSUT01) in chlorophyta Volvox carteri. Thereby, the origin of the sucrose transporters predates the divergence between green alga and the ancestors of terrestrial plants.
Conserved motifs analyses of the SUT genes
The diversity of motif compositions in sucrose transporters of Orchidaceae species was assessed using the MEME programme; a total of 10 conserved motifs were identified. The distribution of these 10 motifs in the SUT proteins is shown in Figure 2. The motif, motif2 was the most conserved SUT domain, which was identified in all of the SUT proteins except PeqSUT08 and DenSUT06. Besides, motif10 was observed in 17 SUT proteins, whereas absent from PeqSUT08, ApoSUT05, DenSUT06, DenSUT08, and PeqSUT02. All the three members in group A contained the same four motifs, motif10, motif2, motif5 and motif9. Moreover, Group B members shared the same motif, motif5, except for DenSUT07; likewise, motif4 was also commonly owned by all group B SUTs except for ApoSUT04 (Fig. 2). Among the 12 SUTs in subgroup B2.1, three motifs were commonly owned, i.e. motif2, motif3, and motif5. There were eight sucrose transporters that had all 10 motifs, among which five for P. equestris (PeqSUT03, PeqSUT04, PeqSUT05, PeqSUT06 and PeqSUT07), two for A. shenzhenica (ApoSUT02 and ApoSUT06), whereas D. officinale had only one (DenSUT05). The sucrose transporters in each subgroup shared several unique motifs, indicating that the SUT proteins within the same subgroups may have certain functional similarities. In addition, motif distribution of the SUTs suggested that those genes were largely conserved during evolution.
Water-soluble sugar content in D. officinale
Photosynthetically produced sugar is not just a resource of carbon skeletons but also an energy vector and signaling molecule, which has major impacts on plant growth, development and physiology [48-49]. After synthesis in mesophyll cells of leaves, sucrose needs to be loaded to the phloem parenchyma cells or apoplasm of mesophyll cells, then transported in specialized networks [i.e. sieve element/companion cell complexes (SE/CCC)], and finally unloaded to distal sink organs [2,30,49]. Unlike other monocot crops such as maize, rice, and wheat that uses seeds as main storage sinks, the endosperms of most orchid seeds are significantly degenerated. As a result, Orchidaceae plants are highly dependent on symbiotic fungi to complete their life cycle, especially at the stage of seed germination and seedling growth due to nutrient deficiency [50-52]. To shed light into sucrose partitioning and functions of sucrose transporters in Orchidacea species, we analysed water-soluble sugar content in different tissues of D. officinale, including leaves, stems, flowers and roots, using the GC-MS/MS method. The results showed that the content of water-soluble polysaccharides varies significantly among different tissues (Fig. 3). The amount of total water-soluble polysaccharides was highest in the stems of D. officinale, with approximately 116.17 mg/g, followed by leaves with approximately 113.23 mg/g; flowers had approximately 88.08 mg/g, whereas roots have a significantly lower level of water-soluble polysaccharides, only ~26.66 mg/g (Fig. 3a). These indicated that stems were the major sink organs for sugar storage in D. officinale. Because the D. officinale is an epiphytic plant in its natural habitation which usually experiences drought stress [53-54], thus the high amount of sugar in stems may help to maintain osmic pressure to improve drought tolerance. The content of sucrose also varies greatly among different tissues. Nonetheless, sucrose content was highest in flowers, approximately 28.1 mg/g, followed by leaves (with ~18.13 mg/g) which are the major source tissues for the photosynthetically assimilated sucrose. The amount of sucrose in stems is ~13.77 mg/g, and that of roots is also the lowest containing only ~7.82mg/g (Fig. 3b). Previous studies show that the developing pollen grains are strong sink tissues, which require sucrose to provide energy for maturing, germination and growth [55-56]. These results showed that although total sugar content was highest in the stems, the photoassimilated sucrose was mainly transported to support the growth and physiology of floral organs in D. officinale.
Expression patterns of the SUT genes in different tissues of D. officinale
Sucrose transport systems play vital roles in carbon partitioning, plant development, inter-/intra cellular communications and environmental adaptations. The SUT genes are not only involved in sucrose transport, but also play essential roles in pollen germination, fruit ripening, and ethylene biosynthesis in many species [10,28-29,47]. To further understand the roles of the SUT genes in orchids, we investigated expression profiles of DenSUT genes in D. officinale. RNA sequencing (RNA-seq) was performed using different tissues of D. officinale including leaves, stems, flowers and roots. The FPKM expression of DenSUT genes in four different tissues is provided in Table S2. The expression levels of different DenSUT genes in the four D. officinale tissues are represented in different colours and are shown in Fig. 4.
The Arabidopsis AtSUC1 is expressed in germinating pollens where it is translationally regulated and facilitates anthocyanin accumulation; while the mutant atsuc9 promotes floral transition by manipulating sucrose uptake [28,32]. AtSUC1 is also expressed in the parenchymatic cells of the style and anthers, which guides modulates water availability around the region and finally results in pollen-tube towards the ovule and anther opening [55]. Recent studies have also described the roles of NtSUT3 and LeSUT2 in sucrose uptake during pollen development and pollen tube growth [9,56]. In the present study, RNA-seq showed that most of the DenSUTs were expressed in flowers, among which three genes, DenSUT01, DenSUT08, and DenSUT06 had significantly high expression levels. In agreement with the expression profiles, sucrose accumulation also predominantly occurs in the flowers, with approximately 28.1 mg/g. These result indicated that these genes mainly took function in the cellular machinery and development of floral organs. Phylogenetically, DenSUT01, DenSUT08, and DenSUT06 were classified in subgroup A and the monocot-specific expanded subgroup B2.1, respectively.
In leaves, sucrose is mainly synthesized in the mesophyll cytoplasm, maybe also in organelles such as vacuoles and plastids [57]. Once released to the leaf apoplasm, sucrose is actively loaded into the SE-CCCs via a sucrose/H+ mechanism in apoplasmic loading species [58]. The analysis of transgenic and mutant plants indicates that dicot members of the SUT1 clade and monocot members of the SUT3 clade are essential for apoplasmic loading of SE-CCC [59-61]. In maize, ZmSUT1 plant an important role in efficient phloem loading [61]. The inhibition of sucrose transporters results in starch accumulation in the epidermal cell [62]. The sucrose transporter SUC2 is crucial for sucrose allocation, the null mutant of which in Arabidopsis led to compromise health of the plant [63]. After loaded into the SE-CCC, energy-driving reloading is required along the whole process of long terminal sucrose transport from source to sink. In D. officinale, sucrose content was ~18.13 mg/g in the leaves, ranked second among four tissues. However, only one gene, DenSUT02, was significantly expressed in the leaves, which was also expressed in flowers and roots. We deduce that DenSUT02 may play a potential role in phloem loading in D. officinale. Nonetheless, other sugar transporters such as SWEETs and MSTs are also likely involved in sucrose transport.
In well studied grass stems, immature internodes are considered as utilization sinks, whereas the fully elongated mature internodes are storage sinks where the sucrose accumulates [64-66]. The plasmamembrane-localized sucrose transporters are promising candidates for sucrose uptake in stems. For example, all of the SbSUTs in sorghum are active in sucrose uptake, although the expressed sites of different SUTs in internodes may vary [19,66-67]. The SbSUTs are localized to sieve elements in both developing and mature sorghum stems [68], which is consistent with the localization of wheat TaSUT1 and rice OsSUT1 proteins in SE-CCCs in mature stems [69-70]. In the present study, three genes, DenSUT03, DenSUT05 and DenSUT07, were expressed in Dendrobium stems. DenSUT05 and DenSUT07 were both moderately expressed in stems and flowers, whereas DenSUT03 was slightly expressed in the stems and significantly expressed in the roots. In addition, DenSUT01 and DenSUT08 were also slightly expressed in the roots. The expression of DenSUTs was also analysed in flower, stem and leaf D. officinale using qRT-PCR (Fig. 5). The results were largely consistent with that from RNA-seq analysis. However, the functions of SUT genes in D. officinale and the other two Orchidaceae species remain to be verified.