To identify the Dof gene family members in Spinach, all proteins from the spinach genome were scanned by using HMMER-3.2 and 22 genes were predicted as Dof gene family members in spinach. These Dof candidate genes were named as SoDof1-SoDof22 (Table 1). Then the predicted proteins were further confirmed to contain the conserved Dof domain. Similarly, 36 Dof genes had been identified in the Arabidopsis database. The full-length of the coding sequence (CDS) ranged from 492 bp to 1485 bp with an average length of 1060 bp. The quantity of aa (amino acids) for SoDof varied from 163 (SoDof12) to 494 (SoDof13) aa, with average protein length of ~352 aa. The molecular weights (MW) fluctuated between 18.5 Kilodalton (kDa) (SoDof12) and 54.5 kDa (SoDof13), and the theoretical isoelectric points (pI) ranged from 4.6 (SoDof20) to 8.92 (SoDof9). Over half SoDofs were alkaline with all members in Group B, and Group D1 contained most members with a wide wave in MWs (Table 1).
Table 1
Spinach Dof genes and their related information
Gene name
|
Gene ID
|
Chromosome
|
Location
|
Gene DNA(bp)
|
CDS(bp)
|
Protein length(aa)
|
Molecular weight
|
Theoretical pI
|
Dof domain
|
Intron
|
Class
|
SoDof1
|
Spo01218
|
chr2
|
58115820..58118612 forward
|
2793
|
1104
|
367
|
40642.53
|
8.52
|
57-114
|
1
|
C2.1
|
SoDof2
|
Spo26525
|
chr4
|
115910084..115910743 reverse
|
660
|
660
|
219
|
23339.72
|
8.47
|
23-79
|
0
|
A
|
SoDof3
|
Spo14528
|
chr3
|
51468026..51469123 forward
|
1098
|
1098
|
365
|
39514.46
|
7.32
|
41-96
|
0
|
B2
|
SoDof4
|
Spo15329
|
chr5
|
13015823..13016842 forward
|
1020
|
1020
|
339
|
37310.74
|
5.59
|
52-108
|
0
|
A
|
SoDof5
|
Spo26037
|
chr6
|
40210301..40212930 forward
|
2630
|
1197
|
398
|
44408.07
|
6.25
|
58-115
|
1
|
C2.1
|
SoDof6
|
Spo25524
|
SpoScf_02134
|
33891..35945 reverse
|
2055
|
1287
|
428
|
46606.00
|
8.80
|
90-146
|
1
|
B2
|
SoDof7
|
Spo19252
|
chr5
|
6739988..6741368 reverse
|
1381
|
1110
|
369
|
39234.09
|
6.93
|
47-104
|
1
|
C1
|
SoDof8
|
Spo19232
|
SpoScf_01574
|
110099..110860 reverse
|
762
|
762
|
253
|
25482.25
|
8.12
|
28-83
|
0
|
D2
|
SoDof9
|
Spo13986
|
SpoScf_01503
|
63276..64439 reverse
|
1164
|
1165
|
387
|
41004.88
|
8.92
|
79-135
|
0
|
A
|
SoDof10
|
Spo20892
|
Super_scaffold_114
|
1245494..1248131 reverse
|
2638
|
1326
|
441
|
46968.23
|
8.21
|
95-150
|
1
|
B1
|
SoDof11
|
Spo08108
|
chr5
|
10912882..10916291 forward
|
3410
|
1344
|
447
|
49445.56
|
5.39
|
108-164
|
1
|
D1
|
SoDof12
|
Spo04353
|
SpoScf_01506
|
92311..92802 forward
|
492
|
492
|
163
|
18468.93
|
8.87
|
44-99
|
0
|
D1
|
SoDof13
|
Spo05430
|
SpoScf_01199
|
340472..345369 forward
|
4898
|
1485
|
494
|
54499.48
|
5.63
|
154-210
|
1
|
D1
|
SoDof14
|
Spo16539
|
SpoScf_00408
|
13249..16754 forward
|
3506
|
1059
|
352
|
38506.78
|
6.46
|
99-155
|
1
|
D1
|
SoDof15
|
Spo26832
|
chr6
|
26503975..26505054 reverse
|
1080
|
1080
|
359
|
40449.97
|
6.23
|
28-82
|
0
|
C2.2
|
SoDof16
|
Spo22565
|
chr1
|
19149992..19151942 reverse
|
1951
|
1098
|
365
|
39747.75
|
8.50
|
84-138
|
1
|
B1
|
SoDof17
|
Spo22229
|
SpoScf_01420
|
149590..151164 forward
|
1575
|
1101
|
366
|
40015.00
|
8.51
|
87-141
|
1
|
B1
|
SoDof18
|
Spo07164
|
SpoScf_08285
|
1203..2777 forward
|
1575
|
1101
|
366
|
40027.05
|
8.51
|
87-141
|
1
|
B1
|
SoDof19
|
Spo25703
|
Super_scaffold_205
|
553984..554928 reverse
|
945
|
945
|
314
|
35306.63
|
8.53
|
58-111
|
0
|
B2
|
SoDof20
|
Spo00332
|
Chr4
|
83899644..83900468 reverse
|
825
|
825
|
274
|
30538.30
|
4.60
|
34-88
|
0
|
C2.2
|
SoDof21
|
Spo10686
|
chr1
|
41630415..41632583 forward
|
2169
|
1305
|
434
|
47592.39
|
5.74
|
149-205
|
1
|
D1
|
SoDof22
|
Spo16511
|
SpoScf_00982
|
142499..143254 forward
|
756
|
756
|
251
|
27368.16
|
7.60
|
44-98
|
0
|
C3
|
To better understand the distribution of SoDof genes on the Spinach chromosome, we performed MG2C to draft the chromosomal map. The 22 putative Dof genes were found to be distributed in 6 chromosomes, and unplaced contigs (Fig.1). Only 50% SoDofs genes were anchored in chromosomes. The largest number of SoDof members was located in chromosome 5, which contains SoDof 7, 11 and 4. Compared with the gap of SoDof on other chromosomes, these three genes were closer with each other, especially SoDof11 and SoDof4. There were 2 SoDof genes in chromosomes 1, 4, and 6, respectively. SoDof1 and SoDof3 were located in chromosomes 2 and 3, respectively.
Calculating the value of Ka and Ks aims to identify duplication event for each SoDof gene. The duplication of SoDof genes originated from about 5.66 Mya (Ks=0.793) to 41.27 Mya (Ks=5.778) with an average of 16.12 Mya (Table S1). All values of Ka/Ks were below 1 and even almost all of them were below 0.5. Especially, the Ka/Ks values for five segmental duplication were extremely low (below 0.1) (Fig.2).
Multiple sequence alignment, phylogenetic analysis and classification
Multiple sequence alignment showed a Dof conserved motif of 52 amino acid located in 22 SoDof genes, with a single Cys2/Cys2 zinc finger structure at the N-terminal (Fig.3). Phylogenetic tree was constructed between 22 SoDof genes and 36 Dofs in Arabidopsis (Fig.4). A total of 22 SoDof TFs from spinach were classified into four main groups (Groups A to D), which could be divided into multiple subgroups, A, B1, B2, C1, C2.1, C2.2, C3, D1 and D2. The quantity of SoDofs in Group B, Group C and Group D was similar with a total number of 18. Specifically, Group B (contained the most number among all groups) could be divided into subgroup B1 and subgroup B2 with SoDof10, SoDof16, SoDof17, SoDof18 in subgroup B1 and SoDof3, SoDof6, SoDof19 in subgroup B2 (Fig.4). Subgroup D1 had the second largest number of SofDofs (SoDof11, SoDof12, SoDof13, SoDof14, SoDof21). SoDof2, SoDof4 and SoDof9 belonged to Group A (Fig. 4).
Gene structure and motif analysis of SoDof genes
Candidate SoDof genes were analyzed using Gene Structure Display Sever to investigate the characterization of exon-intron structure. Remarkably, there were one or no intron occurred in SoDofs (Fig.5). SoDofs showed closed position in the phylogenetic tree, displayed similar intron-exon distribution, indicating the similar function within subgroup. For example, there were only one exon appeared in Group A.
To further reveal the diversification of SoDof genes, we performed the MEME program to detect motif patterns, and 25 distinct motifs were identified (Fig.6). The schematic distribution of the 25 motifs showed that only the Dof region (motif1) was highly conserved in all SoDof proteins. Notably, SoDofs shared similar conserved motif composition. Motif 10 and 7 were highly conserved in Group B. And in subgroup B1, motif 4 jointly related to the N-terminal Dof domain. Interestingly, motif 9 and 11 were prominently conserved in the subgroup D1 (contained the most SoDof members among all subgroups). Specifically, motif 9 presented at the N-terminal and motif 11 jointed the N-terminal Dof region.
Cis-regulatory element analysis
PlantCARE was used to analyse the cis-regulatory element for each SoDof gene by retrieving the 2kb upstream sequence of each candidate, except for SoDof18 because of lack of 2kb upstream sequence on its scaffold location (Supplementary Data). Dof gene family in spinach had TATA-box, CAAT-box and typical eukaryotic switch elements. SoDof genes may also be controlled by many phytohormones, such as methyl jasmonate (MeJA), gibberellins (GA), ethylene, auxin, and salicylic acid (SA). We also detected many other important cis-elements on Dof gene family that involve in plant growth and development. For example, there were a large amount of elements associated with physiological process, such as light responsiveness, circadian control, endosperm expression, meristem and flower meristem expression, root specific and seed-specific regulation. Some elements, participated in some small molecule pathway, also had been found, such as zein metabolism regulation, maximal elicitor-mediated activation and flavonoid biosynthetic genes regulation. Additionally, eight cis-elements (WUN-motif, STRE, TC-rich repeats e.g.) were also predicted, which were related to defense and stress responsiveness.
Tissue-specific expression analysis of SoDof genes
We isolated RNA samples from organs, roots, stems, leaves, male flowers, and female flowers, and detected expression of all SoDof genes in spinach using qRT-PCR. A heatmap to visualize a expression profile of the SoDof genes was generated, revealing nine SoDofs that exhibited their highest transcript level in reproductive organs and eight SoDofs in vegetative leaves (Fig.7A). Only two SoDofs (SoDof1 and SoDof5) expressed in roots and stems, respectively. Notably, SoDof10 and SoDof15 had extremely high expression in leaves; SoDof22 showed high expression in male flowers (Fig.7B). Comparing with the expression in leaves or inflorescences, the transcript level of these three genes in other tissues was neglectable, indicating that their expression were tissue-specific such as in leaves and flowers. There were three homologous genes (SoDof16, SoDof17 and SoDof18) with same mRNA sequence, and their expression patterns were not analyzed.
Expression patterns of SoDof genes under abiotic stresses
To investigate the different stress responsiveness and expression pattern for each SoDof gene within different gender of spinach, we treated female plants, male plants and plants at vegetative stage by three types of abiotic stress (low temperature 4℃, high temperature 40℃ and drought 20%PEG4000). Then the spinach functional leaves were collected at 0h, 2h, 4h, 7h, 12h, 24h after treatment and detected by qRT-PCR.
Over half SoDof genes in female plants were up regulated under low temperature (Fig.8A). The greatest increase in expression occurred in SoDof22 at female plants (Fig.8B). The SoDof22 was also up-regulated and expressed most compared to other SoDofs in plants at vegetative stage, and its extreme expression reached to the top at 7h and then went down. However, in male plants, the expression of SoDof3 reached the highest level (Fig.8B). In vegetative plants, 84% SoDof genes (more than those in male or female plants) were up-regulated and its highest expression appeared at 7h (Fig.8A). There were the most number of SoDofs (SoDof5, SoDof6 and SoDof9) down-regulated in male plants, indicating that SoDofs in males showed more negative response under 4℃.
Under high temperature, most SoDofs was up-regulated and only five SoDofs (SoDof4, SoDof5, SoDof6, SoDof11 and SoDof21) were down-regulated in female plants. In plants at vegetative stage, the down-regulated SoDofs were SoDof1, SoDof4, SoDof6, SoDof11 and SoDof21. Additionally, the expression of SoDof6, SoDof11 and SoDof21 were also suppressed in male plants (Fig.8C). The expression of SoDof21 and SoDof6 in vegetative plants was constrained until 24h when the expression was almost equal to that before treatment (Fig.8C). In addition, over half SoDofs showed the highest transcript level at 24h in plant at vegetative stage, and over half SoDofs showed the highest transcript level at 7h in male plants.
To investigate the expression profile for each SoDofs when they suffered from the drought condition, we simulated the drought condition by using 20% (mass fraction) PEG 4000. SoDof21 was down-regulated (Fig.8C) and SoDof15 (Fig.8B) expressed at the highest level in three types of spinach. All SoDofs were up-regulated in females and vegetative plants except SoDof21. In male plants, five SoDofs, SoDof5, SoDof6, SoDof11, SoDof20 and SoDof21, exhibited suppressed expression, and the expression of all SoDofs are lower than in female and vegetative plants.