Identification of LBD genes in Sweet Potato
The 53 sweet potato LBD genes were assigned names from IbLBD1 to IbLBD53 based on their chromosomal positions. The proteins encoded by the 53 LBD genes contained 149 (IbLBD31) to 520 (IbLBD50) amino acids with molecular weights (MWs) ranging from 16.16 kD (IbLBD31) to 57.51 kD (IbLBD50). Predicted protein isoelectric points (pI) ranged from 4.58 (IbLBD24) to 10.08 (IbLBD6). Among them, 28 proteins had isoelectric points greater than 7, resulting in a positive charge in acidic solutions. The hydrophilicity of the proteins ranged from − 1.033 (IbLBD45) to 0.361 (IbLBD49), indicating different degrees of hydrophilicity. In addition, Cell-PLoc subcellular localization predictions showed that all LBD proteins were located in the nucleus (Table 1).
Table 1 Identification of LBD genes and analysis of physicochemical properties of proteins in sweet potato
Gene name
|
Accession Number
|
Chromosome
Location
|
Size (aa)
|
Mw
(kD)
|
pI
|
GRAVY
|
Predicted Location
|
IbLBD1
|
OR133645
|
1687306
|
1688873
|
188
|
20.72
|
6.5
|
-0.311
|
Nucleus
|
IbLBD2
|
OR133646
|
8494464
|
8496120
|
249
|
27.22
|
6.6
|
-0.447
|
Nucleus
|
IbLBD3
|
OR133647
|
27014760
|
27015755
|
209
|
23.87
|
5.21
|
-0.431
|
Nucleus
|
IbLBD4
|
OR133648
|
271534
|
275058
|
174
|
19.07
|
8.97
|
-0.411
|
Nucleus
|
IbLBD5
|
OR133649
|
1039868
|
1043206
|
167
|
18.27
|
7.72
|
-0.411
|
Nucleus
|
IbLBD6
|
OR133650
|
1069879
|
1074143
|
191
|
21.44
|
10.08
|
-0.717
|
Nucleus
|
IbLBD7
|
OR133651
|
3938571
|
3939920
|
215
|
23.25
|
6.14
|
-0.039
|
Nucleus
|
IbLBD8
|
OR133652
|
5639554
|
5640802
|
191
|
20.68
|
6.99
|
-0.117
|
Nucleus
|
IbLBD9
|
OR133653
|
5829105
|
5830345
|
242
|
25.65
|
7.66
|
0.173
|
Nucleus
|
IbLBD10
|
OR133654
|
7327214
|
7328603
|
236
|
26.03
|
6.2
|
-0.534
|
Nucleus
|
IbLBD11
|
OR133655
|
36568489
|
36573862
|
334
|
35.22
|
9.28
|
-0.256
|
Nucleus
|
IbLBD12
|
OR133656
|
1456672
|
1458605
|
214
|
23.11
|
8.36
|
-0.817
|
Nucleus
|
IbLBD13
|
OR133657
|
1466969
|
1469187
|
225
|
24.32
|
6.48
|
-0.534
|
Nucleus
|
IbLBD14
|
OR133658
|
3005283
|
3007545
|
160
|
17.77
|
6.71
|
0.322
|
Nucleus
|
IbLBD15
|
OR133659
|
3278659
|
3280029
|
172
|
18.94
|
7.63
|
-0.211
|
Nucleus
|
IbLBD16
|
OR133660
|
22684760
|
22686086
|
224
|
24.46
|
8.6
|
-0.628
|
Nucleus
|
IbLBD17
|
OR133661
|
4128151
|
4129743
|
277
|
31.01
|
6.16
|
0.000
|
Nucleus
|
IbLBD18
|
OR133662
|
5451750
|
5452974
|
226
|
25.48
|
4.68
|
-0.700
|
Nucleus
|
IbLBD19
|
OR133663
|
5662166
|
5664439
|
197
|
22.23
|
8.15
|
-0.278
|
Nucleus
|
IbLBD20
|
OR133664
|
27801512
|
27802278
|
180
|
19.99
|
8.28
|
-0.445
|
Nucleus
|
IbLBD21
|
OR133665
|
27967462
|
27968594
|
214
|
23.51
|
8.6
|
-0.445
|
Nucleus
|
IbLBD22
|
OR133666
|
1121586
|
1126383
|
246
|
26.19
|
8.67
|
0.122
|
Nucleus
|
IbLBD23
|
OR133667
|
1261129
|
1262521
|
212
|
23.44
|
5.79
|
-0.584
|
Nucleus
|
IbLBD24
|
OR133668
|
2016696
|
2019488
|
307
|
34.00
|
4.58
|
-0.080
|
Nucleus
|
IbLBD25
|
OR133669
|
27558292
|
27560598
|
187
|
20.18
|
9.27
|
-0.284
|
Nucleus
|
IbLBD26
|
OR133670
|
28842217
|
28843398
|
204
|
21.82
|
8.17
|
-0.417
|
Nucleus
|
IbLBD27
|
OR133671
|
29266026
|
29267217
|
236
|
25.74
|
8.52
|
-0.528
|
Nucleus
|
IbLBD28
|
OR133672
|
33791108
|
33797053
|
326
|
36.47
|
6.66
|
-0.462
|
Nucleus
|
IbLBD29
|
OR133673
|
34877261
|
34878276
|
210
|
23.58
|
6.65
|
-0.745
|
Nucleus
|
IbLBD30
|
OR133674
|
850688
|
852583
|
218
|
22.77
|
8.47
|
-0.250
|
Nucleus
|
IbLBD31
|
OR133675
|
4610655
|
4611562
|
149
|
16.16
|
8.75
|
-0.017
|
Nucleus
|
IbLBD32
|
OR133676
|
3472142
|
3475757
|
322
|
36.23
|
9.58
|
-0.739
|
Nucleus
|
IbLBD33
|
OR133677
|
6217307
|
6221397
|
366
|
39.29
|
4.59
|
-0.306
|
Nucleus
|
IbLBD34
|
OR133678
|
14820911
|
14822874
|
214
|
23.46
|
9.11
|
-0.261
|
Nucleus
|
IbLBD35
|
OR133679
|
4501947
|
4503162
|
226
|
24.89
|
5.39
|
-0.273
|
Nucleus
|
IbLBD36
|
OR133680
|
4532985
|
4534460
|
245
|
27.27
|
4.97
|
-0.233
|
Nucleus
|
IbLBD37
|
OR133681
|
3579351
|
3580312
|
209
|
22.47
|
8.67
|
-0.101
|
Nucleus
|
IbLBD38
|
OR133682
|
13130843
|
13131707
|
201
|
22.17
|
7.64
|
-0.336
|
Nucleus
|
IbLBD39
|
OR133683
|
22497885
|
22500062
|
240
|
26.20
|
5.47
|
-0.062
|
Nucleus
|
IbLBD40
|
OR133684
|
39950925
|
39952093
|
223
|
25.26
|
8.77
|
-0.966
|
Nucleus
|
IbLBD41
|
OR133685
|
8697591
|
8699166
|
202
|
21.89
|
6.11
|
-0.084
|
Nucleus
|
IbLBD42
|
OR133686
|
9909480
|
9911649
|
214
|
23.43
|
9.25
|
-0.284
|
Nucleus
|
IbLBD43
|
OR133687
|
28316625
|
28318692
|
175
|
18.91
|
6.71
|
-0.007
|
Nucleus
|
IbLBD44
|
OR133688
|
29434436
|
29438687
|
183
|
19.95
|
8.58
|
-0.578
|
Nucleus
|
IbLBD45
|
OR133689
|
11950627
|
11951867
|
280
|
30.17
|
5.64
|
-1.033
|
Nucleus
|
IbLBD46
|
OR133690
|
18216174
|
18217557
|
314
|
34.94
|
5.46
|
-0.580
|
Nucleus
|
IbLBD47
|
OR133691
|
19821743
|
19824082
|
185
|
20.18
|
6.88
|
-0.728
|
Nucleus
|
IbLBD48
|
OR133692
|
291788
|
295234
|
475
|
52.48
|
6.25
|
0.084
|
Nucleus
|
IbLBD49
|
OR133693
|
8713728
|
8714718
|
157
|
17.29
|
9.97
|
0.361
|
Nucleus
|
IbLBD50
|
OR133694
|
26782324
|
26785743
|
520
|
57.51
|
8.81
|
-0.730
|
Nucleus
|
IbLBD51
|
OR133695
|
5492804
|
5494838
|
227
|
24.66
|
8.56
|
-0.495
|
Nucleus
|
IbLBD52
|
OR133696
|
5850533
|
5851542
|
213
|
22.89
|
5.47
|
-0.056
|
Nucleus
|
IbLBD53
|
OR133697
|
18062560
|
18063739
|
277
|
31.14
|
9.69
|
-0.403
|
Nucleus
|
MW, molecular weight; pI, isoelectric point; GRAVY, grand average of hydropathicity score.
Motif compositions and gene structure of the LBD gene family in Sweet Potato
Through a conservative motif analysis on the LBD protein sequence of sweet potato based on the MEME online tool, ten conservative motifs were predicted and several differences in the number and distribution of motifs were found for different sweet potato LBD proteins (Fig. 1A-B). Each gene contained three to six motifs and all of the sweet potato LBD proteins contained conservative LOB domain Motif1 and Motif2. The N-terminal of sweet potato LBD protein was highly conservative and for most of the proteins, the N-terminal contained Motif2 and Motif3, while the C-terminal was less conservative. In all Class Ⅰ members, Motif10 exclusively appeared in Class Ia, Motif8 exclusively appeared in Class Ic, and Motif7 was present in both Class Ia and Class Ic. In addition, the Motif3 and Motif4 domains appeared in most Class Ⅰ members but both did not appear in Class II. Motif6 and Motif9 domains were only found in Class Ⅱ, indicating that LBD gene family members were not only highly conservative but also had several differences. Different subclasses contained motifs varying in type and order, which contributed to the functional diversity of the LBD gene family.
We analyzed the composition of the exon–intron structures of the coding sequences of all 53 LBD genes with a yellow box indicating the CDS sequence of LBD gene family members (Fig. 1C). As shown, the LBD gene family members of sweet potato contained 1 ~ 5 CDS sequences. The number of introns ranged from 0 to 4, but most of the genes (70%) contained one intron. After further analysis, all of the genes in Class Ⅱ were found to have only one intron except for ⅠbLBD26, which contained two introns. Similarly, this pattern was observed in the subclasses of Class I. The results indicate that most of the LBD genes in sweet potato were similar in structure while a few were differentiated.
Conserved sequence alignment of LBD gene family in Sweet Potato
Multiple sequence alignment was performed for 53 IbLBD proteins using ClustalW. The results showed that all the LBD proteins have a highly conserved LOB region at the N-terminal end consisting of approximately 100 amino acids (Fig. 2A), of which 45 IbLBDs (84.90%) belonged to class I and eight IbLBDs (15.10%) belonged to class II. Among them, all proteins contained the CX2CX6CX3C motif and the leucine zipper-like domain (LX6LX3LX6L) is only present in Class I IbLBD proteins, similar to the results of other plants studied (Fig. 2B), further indicating that the two classes of IbLBD proteins were different in biological function.
Phylogenetic analysis
To explore the evolutionary relationship between the LBD gene family of sweet potato and Arabidopsis, We reconstructed a phylogenetic tree of LBD protein sequences (Fig. 3). The results showed that Class I is divided into four subclasses, Ia, Ib, Ic and Id with twenty, six, twelve and seven LBD gene family members, respectively. Class II is divided into two subclasses, IIa and IIb, with three and five LBD gene family members, respectively. The bootstrap values of Class Ⅰa (ⅠbLBD20/AT3G11090), Class Ⅰb (ⅠbLBD51/AT1G65620) and Class Ⅰd (ⅠbLBD40/AT1G06280) orthologous gene pairs were greater than 90, and the ⅠbLBD proteins containing the same motif were assigned to the same branch. These results indicated that the LBD gene in adjacent branches had similar functions.
Chromosome locations and gene duplication analysis
Through an analysis of the chromosomal localization, 53 LBD genes were distributed on 13 of the 15 chromosomes (Fig. 4). Among them, chromosomes 2 (LG2) and 7 (LG7) had the most indispensable genes, containing eight LBD genes followed by chromosomes 3 (LG3) and 5 (LG5), containing five LBD genes. Chromosomes 11 and 12 contained four LBD genes. In contrast, other chromosomes had the fewest members, with only 1 ~ 3 genes localized at the chromosomes. At the same time, the LBD gene contained three tandem duplication gene pairs, namely chromosome 2 (ⅠbLBD5/ⅠbLBD6), chromosome 3 (ⅠbLBD12/ⅠbLBD13) and chromosome 10 (ⅠbLBD35/ⅠbLBD36), which were substantially close to each other on their chromosomes. They were also clustered together on the phylogenetic tree, which might be caused by the replication of gene fragments, suggesting their similar functions. Tandem duplication and segment duplication have been identified as the main mechanism of gene family expansion [38, 39]. Through MCScanX collinearity analysis, there were 14 duplicate gene pairs of the LBD gene segment, distributed on different chromosomes (Fig. 5), indicating the occurrence of tandem duplication and segment duplication during the expansion of LBD genes.
Evolutionary analysis of the IbLBD genes
To further investigate the evolutionary relationships between the LBD family and other species, an evolutionary relationship analysis of LBD genes between sweet potato and other species was performed (Fig. 6) including three dicotyledonous plants (Arabidopsis, tomato, and pepper) and two monocotyledonous plants (maize and rice). As shown in Fig. 6, there were 38 collinear genes in sweet potato and Arabidopsis, while there were 52, 50, 19, and 12 collinear genes in tomato, capsicum, rice and maize respectively. These results indicated that the LBD gene of sweet potato had a close evolutionary relationship with that of dicotyledonous plants and had a closer evolutionary relationship with tomato and capsicum of Solanaceae.
Analysis of cis‑regulatory element distribution in IbLBD promoters
To investigate the potential biological functions of the IbLBDs, the genomic sequence of 2000 bp upstream of the IbLBDs was extracted as a hypothetical promoter sequence for cis-acting element analysis (Fig. 7). As shown in the figure, various types of cis-acting elements were found in the IbLBD gene family, including elements related to plant growth and development, hormones responses and abiotic stress responses. Among the analysed genes, it was found that 43 genes contained either one or seven abscisic acid response elements, 38 genes contained either two or 14 methyl jasmonate response elements, nine genes contained one gibberellin response element, and 13 genes contained either one or two zeatin metabolic regulatory elements. Most of the genes contained a large number of light response elements and anaerobic induction elements. The light response elements included G-Box and G-box types and the anaerobic induction elements were all of the ARE type, while the remaining two types of environmental change response elements were less distributed. Overall, various types of cis-elements were found in the promoter regions, suggesting that these IbLBDs may be involved in various biological processes and regulatory pathways.
Expression patterns of IbLBD gene family in Sweet Potato
To explore the influence of the LBD gene on plant tissue growth and development, transcriptome data were used to analyze the expression patterns of seven different tissues (flower, fruit, leaf, stem, primary root, pencil root, and tuber root). The gene expression levels were quantified as Fragments Per Kilobase of exon model per Million mapped fragments (FPKM). The results show that 52 IbLBD genes (the expression levels of IbLBD40 in tissues were extremely low) had significantly different expression patterns in tissues (Fig. 8A). The cluster analysis results reveal that the most highly expressed genes were in the stem, including seven genes with relative expression levels above two. The relative expression levels of these genes were low in other parts. However, in flowers, three genes showed high expression levels while in fruits, six genes were highly expressed. Such results suggest that these genes may have important roles in the development of flowers and fruits. Notably, IbLBD33 exhibited high expression specifically in leaves, while the other genes displayed low expression levels in leaves. Meanwhile, the expression levels of IbLBD44 and IbLBD37 were all up-regulated in different roots, suggesting that they might play an important role in root growth. In addition to these specific genes, IbLBD in the same subclass showed similar expression patterns in the development of some tissues. For instance, some of the genes in Ia, Ib and Ic all showed high expression levels in the stem, as well as an increasing trend, while their expression levels in other parts were relatively low. Most of the members of Class Ⅱ were highly expressed in the roots and leaves, showing an up-regulated expression result in the roots and a down-regulated expression result in the leaves. The results indicate that IbLBD genes were differentially expressed in various tissues. Different subclasses of IbLBD genes may play roles in the development of the same tissue, suggesting functional redundancy while some genes may have specific functions in corresponding tissues.
The expression levels of IbLBD genes in sweet potato tissues were analysed under conditions of salt stress and drought stress (Fig. 8B). A total of 45 IbLBD genes were detected in tissues under salt stress and drought stress (the expression levels of the other eight genes under different stress conditions were extremely low). Under both stress conditions, IbLBD genes were mostly expressed on primary roots, followed by stems with leaves having the least number of genes expressed. There were 16 genes with relatively high relative expression levels in sweet potato roots under salt stress. Six genes had relative expression levels greater than two including IbLBD26 and IbLBD27 which were highly expressed in stems and IbLBD33 that was highly expressed in leaves. There were 20 genes with relatively high expression levels in sweet potato roots under drought stress and four genes with relative expression levels higher than two. Among them IbLBD41 was highly expressed in leaves and IbLBD24 was highly expressed in stems. There were seven genes in Class Ⅰ and four genes in Class Ⅱ respectively that were up-regulated in primary roots. The results indicate that these genes had a protective mechanism for roots under stress conditions. Overall, most of the genes responded under different stress conditions.
Quantitative qPCR analysis of IbLBD genes in different tissues
To verify the reliability of the transcriptome data, 12 genes with significant expression differences under salt and drought stresses were selected for qRT-PCR analysis (Fig. 9). Among the genes, the expression analysis of IbLBD genes in different parts of sweet potato were consistent with the transcriptome data. Overall, the expression of these IbLBD genes was mostly detected in the primary root and leaves of sweet potato. Meanwhile, there were significant differences in the expression of these IbLBD genes in different parts. IbLBD genes in Class I were highly expressed in the primary root, pencil root, and leaves of sweet potato. Among them, IbLBD7 and IbLBD21 had also a high expression level in the stem and flower of sweet potato. IbLBD genes in Class II were highly expressed in the primary root.
Quantitative qPCR analysis of IbLBD genes under abiotic stresses
To validate the LBD gene expression levels in different tissues of sweet potato under different stress conditions, qRT-PCR was used to analyze the expression level of sweet potato treated at different times. (Fig. 10A-C). The results showed that salt and drought stress induced the expression of LBD genes in different parts of sweet potato. Under these two stresses, the expression levels of most IbLBD genes in the primary root and stem of sweet potato were up-regulated first and then down-regulated, presenting a consistent expression trend. However, the expression of these IbLBD genes in leaves presented a downward trend. The sensitivity of DEGs in roots was higher than that in the stems with the lowest sensitivity detected in the leaves. Further, the expression of DEGs was the highest in stems, followed by roots, and the lowest expression of these genes was found in leaves. Notably, the maximum expression level of IbLBD7 in roots was 82 times higher than the control group at 6h under salt stress and 13 times higher than the control group at 12h under drought stress. The maximum expression level of IbLBD12 in the stem was 628 times higher than the control group at 6h under salt stress and 46 times higher than the control group at 12h under drought stress.
Regulatory network in Sweet Potato
We used the STRING database to predict potential interactions among the IbLBD proteins (Fig. 11). There were 27 nodes in the IbLBD protein interaction network, each of which interacted with other nodes. Some proteins exhibited direct interactions, such as IbLBD1 and IbLBD40, whereas others exhibited more complex multigene interactions, such as IbLBD16, IbLBD18, and IbLBD38. Notably, IbLBD40 was predicted to be central nodes, radiating eight and nine connections to other genes, respectively.