Identification of LBD family in wheat
Using the method as described in Method section, a total of 90 genes were remained as putative LBD proteins, which represented the most abundant LBD genes in plant species reported up to now, indicating that LBD gene family expanded significantly in wheat (Tables 1 and 2). Chromosome location analysis found that these 89 TaLBD genes were unevenly distributed on all of the 21 chromosomes in wheat and only one (Ta-U-LBD90) was not mapped on chromosome, of which 4B and 4D had the most abundant PYL genes with each containing 11 TaLBD genes, followed by 4A with 8, 3A with 7, 3B, 3D and 5A with 6 respectively while 6B, 7B and 7D only possessed one TaLBD gene (Fig. 1). Totally, 31, 29 and 29 LBD genes are non-randomly distributed in the wheat A, B and D sub-genome respectively, indicating that no significant variation was occurred on the LBD abundance at subgenome level. Since there is no standard nomenclature, the predicted wheat LBD genes were then named based on their chromosome location and multiple alignment.
Table 1
Comparison of the abundance of LBD genes in different plant species
Species
|
Class Ⅰ
|
Class Ⅱ
|
Total
|
Wheat
|
60
|
15
|
90
|
Arabidopsis
|
36
|
6
|
42
|
Rice
|
29
|
6
|
35
|
Barley
|
19
|
5
|
24
|
Brachypodium
|
24
|
4
|
28
|
Maize
|
37
|
7
|
44
|
Pepper
|
36
|
9
|
45
|
Tomoto
|
40
|
6
|
46
|
Table 2
Basic information of the LBD genes identified in wheat
Gene
name
|
EnsemblPlants
ID
|
Gene length
(bp)
|
ORF length
(bp)
|
Deduced Protein
|
Splice
Variants
|
Subcellular location
|
Size (aa)
|
MW
(KDa)
|
pI
|
GRAVY
|
Ta-1A-LBD1
|
TraesCS1A02G062200
|
647
|
621
|
206
|
21.2059
|
6.7
|
0.037378
|
1
|
Nuclear
|
Ta-1A-LBD2
|
TraesCS1A02G111700
|
1888
|
714
|
237
|
24.7437
|
8.74
|
-0.11772
|
1
|
Nuclear
|
Ta-1A-LBD3
|
TraesCS1A02G222300
|
808
|
570
|
189
|
20.4461
|
6.76
|
-0.18307
|
1
|
Nuclear
|
Ta-1A-LBD4
|
TraesCS1A02G260000
|
3986
|
795
|
264
|
27.1022
|
7.78
|
-0.02046
|
1
|
Nuclear
|
Ta-1B-LBD5
|
TraesCS1B02G080600
|
936
|
648
|
215
|
22.0948
|
6.61
|
0.012093
|
1
|
Nuclear
|
Ta-1B-LBD6
|
TraesCS1B02G131900
|
1847
|
711
|
236
|
24.6616
|
8.73
|
-0.13136
|
2
|
Nuclear
|
Ta-1B-LBD7
|
TraesCS1B02G235700
|
1028
|
570
|
189
|
20.4461
|
7.07
|
-0.18519
|
1
|
Nuclear
|
Ta-1B-LBD8
|
TraesCS1B02G270400
|
4076
|
792
|
263
|
27.0902
|
7.78
|
-0.03384
|
1
|
Nuclear
|
Ta-1D-LBD9
|
TraesCS1D02G110300
|
744
|
636
|
211
|
22.6064
|
8.38
|
-0.50237
|
1
|
Nuclear
|
Ta-1D-LBD10
|
TraesCS1D02G113100
|
1930
|
708
|
235
|
24.6746
|
8.49
|
-0.18596
|
1
|
Nuclear
|
Ta-1D-LBD11
|
TraesCS1D02G224000
|
996
|
570
|
189
|
20.4742
|
7.07
|
-0.18836
|
1
|
Nuclear
|
Ta-1D-LBD12
|
TraesCS1D02G259900
|
4097
|
792
|
263
|
27.0611
|
7.78
|
-0.03726
|
1
|
Nuclear
|
Ta-2A-LBD13
|
TraesCS2A02G194500
|
1442
|
705
|
234
|
24.2473
|
8.41
|
0.004274
|
1
|
Nuclear
|
Ta-2A-LBD14
|
TraesCS2A02G271300
|
1415
|
924
|
307
|
33.541
|
5.97
|
-0.64691
|
1
|
Nuclear
|
Ta-2B-LBD15
|
TraesCS2B02G212400
|
1205
|
714
|
237
|
24.4175
|
8.41
|
0.002954
|
1
|
Nuclear
|
Ta-2B-LBD16
|
TraesCS2B02G289800
|
1303
|
909
|
302
|
32.9763
|
6.29
|
-0.63576
|
1
|
Nuclear
|
Ta-2D-LBD17
|
TraesCS2D02G008400
|
1174
|
648
|
215
|
22.0328
|
6.61
|
0.058139
|
1
|
Nuclear
|
Ta-2D-LBD18
|
TraesCS2D02G193400
|
1336
|
732
|
243
|
25.7559
|
12.11
|
-0.61276
|
2
|
Nuclear
|
Ta-2D-LBD19
|
TraesCS2D02G270100
|
1619
|
912
|
303
|
33.0934
|
5.78
|
-0.62277
|
1
|
Nuclear
|
Ta-3A-LBD20
|
TraesCS3A02G093200
|
1898
|
774
|
257
|
26.492
|
7.72
|
0.02179
|
1
|
Nuclear
|
Ta-3A-LBD21
|
TraesCS3A02G170500
|
1200
|
723
|
240
|
25.0127
|
9.88
|
-0.54125
|
1
|
Nuclear
|
Ta-3A-LBD22
|
TraesCS3A02G205500
|
764
|
765
|
254
|
27.5746
|
7.09
|
-0.57126
|
1
|
Nuclear
|
Ta-3A-LBD23
|
TraesCS3A02G295100
|
1007
|
696
|
231
|
24.6465
|
7.07
|
-0.3316
|
1
|
Nuclear
|
Ta-3A-LBD24
|
TraesCS3A02G346300
|
1516
|
753
|
250
|
26.3556
|
6.51
|
-0.0492
|
1
|
Chloroplast
|
Ta-3A-LBD25
|
TraesCS3A02G402300
|
2718
|
780
|
259
|
26.5147
|
8.12
|
-0.02162
|
1
|
Nuclear
|
Ta-3A-LBD26
|
TraesCS3A02G492000
|
1346
|
1146
|
381
|
41.3431
|
4.57
|
-0.61076
|
1
|
Nuclear
|
Ta-3B-LBD27
|
TraesCS3B02G106900
|
1375
|
888
|
295
|
30.8182
|
6.42
|
-0.24644
|
1
|
Chloroplast
|
Ta-3B-LBD28
|
TraesCS3B02G108500
|
2073
|
774
|
257
|
26.49
|
7.72
|
0.046303
|
1
|
Nuclear
|
Ta-3B-LBD29
|
TraesCS3B02G196100
|
1152
|
747
|
248
|
25.7215
|
9.88
|
-0.56532
|
1
|
Nuclear
|
Ta-3B-LBD30
|
TraesCS3B02G378100
|
1559
|
879
|
292
|
30.6535
|
6.34
|
0.024657
|
1
|
Chloroplast
|
Ta-3B-LBD31
|
TraesCS3B02G435700
|
2926
|
780
|
259
|
26.5588
|
8.12
|
-0.00888
|
1
|
Nuclear
|
Ta-3B-LBD32
|
TraesCS3B02G553000
|
1455
|
1155
|
384
|
41.5733
|
4.93
|
-0.62708
|
1
|
Nuclear
|
Ta-3D-LBD33
|
TraesCS3D02G091700
|
1349
|
894
|
297
|
30.9474
|
7.07
|
-0.22054
|
1
|
Nuclear
|
Ta-3D-LBD34
|
TraesCS3D02G093500
|
2246
|
774
|
257
|
26.46
|
7.72
|
0.04786
|
1
|
Chloroplast
|
Ta-3D-LBD35
|
TraesCS3D02G308000
|
1127
|
741
|
246
|
26.4426
|
8
|
-0.32195
|
1
|
Nuclear
|
Ta-3D-LBD36
|
TraesCS3D02G340000
|
1516
|
747
|
248
|
26.0512
|
6.23
|
-0.02339
|
1
|
Chloroplast
|
Ta-3D-LBD37
|
TraesCS3D02G397200
|
3190
|
780
|
259
|
26.5377
|
8.12
|
-0.02317
|
1
|
Nuclear
|
Ta-3D-LBD38
|
TraesCS3D02G498100
|
1359
|
1155
|
384
|
41.8898
|
4.74
|
-0.61953
|
1
|
Nuclear
|
Ta-4A-LBD39
|
TraesCS4A02G067300
|
2149
|
801
|
266
|
27.3337
|
7.85
|
-0.22481
|
1
|
Nuclear
|
Ta-4A-LBD40
|
TraesCS4A02G107300
|
963
|
672
|
223
|
23.9169
|
6.88
|
-0.21794
|
1
|
Nuclear
|
Ta-4A-LBD41
|
TraesCS4A02G235100
|
1920
|
867
|
288
|
31.3771
|
7.36
|
-0.58854
|
1
|
Nuclear
|
Ta-4A-LBD42
|
TraesCS4A02G236200
|
2184
|
702
|
233
|
24.0108
|
7.56
|
-0.0588
|
1
|
Extracellular
|
Ta-4A-LBD43
|
TraesCS4A02G297500
|
3326
|
522
|
173
|
18.4588
|
8.14
|
-0.1659
|
1
|
Nuclear
|
Ta-4A-LBD44
|
TraesCS4A02G312500
|
993
|
660
|
219
|
24.1458
|
5.96
|
-0.43425
|
1
|
Nuclear
|
Ta-4A-LBD45
|
TraesCS4A02G415300
|
923
|
558
|
185
|
19.6217
|
6.98
|
-0.27838
|
1
|
Nuclear
|
Ta-4A-LBD46
|
TraesCS4A02G415400
|
1296
|
870
|
289
|
30.2694
|
6.74
|
-0.25813
|
1
|
Nuclear
|
Ta-4B-LBD47
|
TraesCS4B02G001200
|
1110
|
720
|
239
|
26.3001
|
5.97
|
-0.42134
|
1
|
Nuclear
|
Ta-4B-LBD48
|
TraesCS4B02G016200
|
2197
|
528
|
175
|
18.658
|
8.38
|
-0.196
|
1
|
Nuclear
|
Ta-4B-LBD49
|
TraesCS4B02G078800
|
1904
|
693
|
230
|
23.6684
|
7.57
|
-0.04957
|
1
|
Extracellular
|
Ta-4B-LBD50
|
TraesCS4B02G079900
|
4020
|
855
|
284
|
30.8945
|
7.34
|
-0.58099
|
1
|
Nuclear
|
Ta-4B-LBD51
|
TraesCS4B02G197100
|
1167
|
672
|
223
|
24.021
|
5.64
|
-0.19776
|
1
|
Nuclear
|
Ta-4B-LBD52
|
TraesCS4B02G224600
|
2045
|
789
|
262
|
27.1386
|
7.86
|
-0.1813
|
1
|
Nuclear
|
Ta-4B-LBD53
|
TraesCS4B02G316100
|
741
|
564
|
187
|
19.7849
|
6.7
|
-0.21177
|
1
|
Nuclear
|
Ta-4B-LBD54
|
TraesCS4B02G316200
|
1319
|
867
|
288
|
30.3994
|
7.29
|
-0.38854
|
1
|
Nuclear
|
Ta-4B-LBD55
|
TraesCS4B02G346500
|
578
|
507
|
168
|
18.3305
|
5.52
|
-0.42738
|
1
|
Nuclear
|
Ta-4B-LBD56
|
TraesCS4B02G361100
|
1179
|
882
|
293
|
30.5339
|
7.77
|
-0.19898
|
1
|
Chloroplast
|
Ta-4B-LBD57
|
TraesCS4B02G361200
|
1155
|
834
|
277
|
28.6916
|
7.79
|
-0.23357
|
1
|
Chloroplast
|
Ta-4D-LBD58
|
TraesCS4D02G001700
|
1042
|
654
|
217
|
23.8554
|
6.27
|
-0.44608
|
1
|
Nuclear
|
Ta-4D-LBD59
|
TraesCS4D02G014600
|
2210
|
885
|
294
|
31.8152
|
8.94
|
-0.23027
|
1
|
Nuclear
|
Ta-4D-LBD60
|
TraesCS4D02G077600
|
1454
|
693
|
230
|
23.7696
|
7.56
|
-0.03783
|
1
|
Extracellular
|
Ta-4D-LBD61
|
TraesCS4D02G078800
|
3868
|
855
|
284
|
30.8425
|
7.36
|
-0.55669
|
1
|
Nuclear
|
Ta-4D-LBD62
|
TraesCS4D02G197400
|
1112
|
672
|
223
|
23.9208
|
5.64
|
-0.21525
|
1
|
Nuclear
|
Ta-4D-LBD63
|
TraesCS4D02G225200
|
2152
|
783
|
260
|
26.9293
|
7.85
|
-0.20692
|
1
|
Nuclear
|
Ta-4D-LBD64
|
TraesCS4D02G312700
|
879
|
564
|
187
|
19.7428
|
6.7
|
-0.2246
|
1
|
Nuclear
|
Ta-4D-LBD65
|
TraesCS4D02G312800
|
1393
|
861
|
286
|
30.3505
|
7.21
|
-0.35699
|
1
|
Nuclear
|
Ta-4D-LBD66
|
TraesCS4D02G341500
|
621
|
522
|
173
|
19.1583
|
5.51
|
-0.49422
|
1
|
Nuclear
|
Ta-4D-LBD67
|
TraesCS4D02G354100
|
1203
|
897
|
298
|
31.1127
|
6.92
|
-0.17282
|
1
|
Chloroplast
|
Ta-4D-LBD68
|
TraesCS4D02G354200
|
1107
|
828
|
275
|
28.8449
|
7.77
|
-0.29891
|
1
|
Chloroplast
|
Ta-5A-LBD69
|
TraesCS5A02G152200
|
1695
|
534
|
177
|
19.7023
|
7.37
|
-0.29831
|
1
|
Nuclear
|
Ta-5A-LBD70
|
TraesCS5A02G191900
|
1574
|
1092
|
363
|
38.5621
|
5.72
|
-0.38843
|
1
|
Nuclear
|
Ta-5A-LBD71
|
TraesCS5A02G284000
|
652
|
567
|
188
|
20.1495
|
5.3
|
-0.42447
|
1
|
Nuclear
|
Ta-5A-LBD72
|
TraesCS5A02G515300
|
846
|
540
|
179
|
19.719
|
4.89
|
-0.42123
|
1
|
Nuclear
|
Ta-5A-LBD73
|
TraesCS5A02G529300
|
1168
|
870
|
289
|
30.2026
|
7.77
|
-0.23114
|
1
|
Chloroplast
|
Ta-5A-LBD74
|
TraesCS5A02G529400
|
806
|
807
|
268
|
27.9669
|
7.09
|
-0.23769
|
1
|
Chloroplast
|
Ta-5B-LBD75
|
TraesCS5B02G150800
|
1044
|
534
|
177
|
19.7163
|
7.37
|
-0.29774
|
1
|
Nuclear
|
Ta-5B-LBD76
|
TraesCS5B02G191200
|
1415
|
1140
|
379
|
40.1759
|
5.3
|
-0.39077
|
1
|
Nuclear
|
Ta-5B-LBD77
|
TraesCS5B02G282700
|
658
|
573
|
190
|
20.7041
|
4.94
|
-0.50684
|
1
|
Nuclear
|
Ta-5D-LBD78
|
TraesCS5D02G157400
|
1151
|
534
|
177
|
19.7183
|
7.37
|
-0.32542
|
1
|
Nuclear
|
Ta-5D-LBD79
|
TraesCS5D02G199000
|
1593
|
1155
|
384
|
40.6975
|
5.44
|
-0.36406
|
1
|
Nuclear
|
Ta-5D-LBD80
|
TraesCS5D02G291300
|
655
|
570
|
189
|
20.2766
|
5.28
|
-0.40106
|
1
|
Nuclear
|
Ta-6A-LBD81
|
TraesCS6A02G053700
|
1078
|
714
|
237
|
26.0791
|
6.49
|
-0.31814
|
1
|
Nuclear
|
Ta-6A-LBD82
|
TraesCS6A02G398400
|
1043
|
621
|
206
|
21.2541
|
7.99
|
0.151942
|
1
|
Nuclear
|
Ta-6B-LBD83
|
TraesCS6B02G072200
|
978
|
714
|
237
|
26.105
|
6.42
|
-0.36245
|
1
|
Nuclear
|
Ta-6B-LBD84
|
TraesCS6B02G438700
|
1033
|
636
|
211
|
21.7136
|
8
|
0.120379
|
1
|
Nuclear
|
Ta-6D-LBD85
|
TraesCS6D02G382600
|
1109
|
630
|
209
|
21.4853
|
7.99
|
0.190909
|
1
|
Nuclear
|
Ta-7A-LBD86
|
TraesCS7A02G066100
|
350
|
351
|
116
|
12.542
|
4.73
|
-0.17759
|
1
|
Nuclear
|
Ta-7A-LBD87
|
TraesCS7A02G228900
|
1559
|
690
|
229
|
24.6203
|
6.65
|
-0.38079
|
1
|
Nuclear
|
Ta-7B-LBD88
|
TraesCS7B02G195100
|
1394
|
690
|
229
|
24.5704
|
6.49
|
-0.27773
|
1
|
Nuclear
|
Ta-7D-LBD89
|
TraesCS7D02G229900
|
1392
|
690
|
229
|
24.5012
|
6.66
|
-0.35808
|
1
|
Nuclear
|
Ta-U-LBD90
|
TraesCSU02G132900
|
*
|
708
|
235
|
26.0411
|
6.73
|
0.102128
|
1
|
Nuclear
|
As reported in rice and Arabidopsis, the LBD gene family could be divided into two major groups, class I and class II according to the specific conserved domain [3, 8]. We further investigated the conserved signature motif in these TaLBDs. Results showed that all the putative wheat LBDs possessed conserved cysteine rich C-motif (CX2CX6CX3C) signature motifs (Figure S1). Among them, 73 TaLBD genes shared cysteine rich C-motif, GAS-block and leucine zipper-like structure basically, which could categorized into class I, while the remaining 17 TaLBD genes had a reminiscent of these motifs, belonging to the class II. Furthermore, the length of putative TaLBD proteins ranged from 116 to 384 amino acids, with the putative molecular weight (Mw) ranging from 12.5 to 41.9 KDa and theoretical isoelectric point (PI) ranging from 4.57 to 12.11, respectively. Meanwhile, the subcellular localization prediction found that most of the TaLBDs (76) localized in nuclear, despite of 11 in chloroplast and 3 in extracellulary (Table 2)
Phylogenetic relationship, conserved motif and gene structure analysis
To evaluate the evolutionary relationships of TaLBDs, phylogenetic analysis was further conducted based on multiple protein sequence alignment of all of the TaLBD together with rice and Arabidopsis LBD proteins. Phylogenetic tree clustered these LBD genes into two major clades (class I, class II), which was consistent with the categorization depending on their domain composition as described above (Figure 2). The class I clades could further divide into 8 groups (IA-IH), and class II divided into two group according to the phylogenetic relationship. It is obvious that the phylogenetic tree was monophyletic and the TaLBDs clustered together with their orthologous counterpart in rice and Arabidopsis in each subgroup, respectively (Figure 3A). Furthermore, a clear paralogous expansion by gene tandem duplication was occurred in wheat LBD gene family and each orthologous loci had two or three homoecologous copies, indicating that the allohexaploidization together with tandem duplication contributed to the expansion of wheat LBD gene family [21, 22]. However, compared to rice and Arabidopsis, no significant paralogous expansion was happened on wheat LBD family. Interestingly, the IE subgroup only contained AtLBDs without rice and wheat orthologous genes, suggesting this subgroup might be lost in rice and wheat.
Furthermore, all of the deduced TaLBD proteins were submitted to MEME web server and 15 conserved motifs were identified. Result showed that motif 1 and motif 3 were most conserved among 15 motifs which inserted in LBD domain and shared by all of LBD proteins (Figure 3B). Further analysis found that the class I and class II LBD proteins possessed the specific motif composition. All of class I proteins harbored motif 1, motif 2 and motif 3, however, class II proteins contained motif 1, motif 3 and motif 5 (Figure 3B and Figure S2). Interestingly, we found that motif 5 was uniquely in class II and could be used as the reference to divide the two class. It is noteworthy that proteins from the same subgroup are likely to share one or more motifs that outside the LBD domain region. For example, motif 9 and 10 were shared by 3 members in the 1E subfamily while motif 13 was specifically shared by 9 members in the class II subfamily. These results confirmed our phylogeny-based groupings.
Gene structure can provide important clues for analyzing evolution feature and phylogenetic relationships of gene family [23]. Therefore, we analyzed the intron and exon structure of TaLBD genes. Result showed that, most of the genes were intron-less and the number of exons ranged from 1 to 4 (Figure 3C). Totally, 25 TaLBD genes had no exon, 42 had one exon and 23 genes had multiple exons. The subfamilyⅠA has more sophisticated structure than other subfamily because of the various number of intron. Furthermore, the members within the same subfamily shared the similar intron-exon structure and gene size, which supported their close evolutionary relationship and the stated classification of subfamilies. Through comparing the LBD gene structure between wheat with barley and Arabidopsis [3, 6], we found that they shared similar intron/exon structures, proving that LBD gene family was rather conserved. Based on phylogenetic tree and gene structure, the homoeologous groups of these 90 TaLBDs were identified and a total of 22 group with each containing A, B and D homoeologous copies were identified in wheat LBD gene family, and 8 gene pairs with a copy on only 2 of the 3 homoeologous chromosomes were also found, while the remaining 8 genes were not found homologous copy in wheat genome. This result demonstrated homoeologous copy loss event might occurred in LBD gene family during wheat polyploidization. The specific retention and dispersion patterns of TaLBDs in homoeologous chromosomes provided the important insight on the mechanism of wheat chromosome evolution and interaction [24]. Interestingly, although the chromosome location of Ta-U-LBD90 was unknown, its location could be deduced to on 6D based on the homoeologous grouping.
Cis-elements and miRNA targets analysis
The Cis-elements are the important regulatory factor that involved in the transcriptional regulation of genes during plant growth and development as well as stress response [25]. The 1.5kb genomic sequences upstream from 5’-UTR of the 90 TaLBD genes were extracted from wheat genome sequences and used to identify cis-elements. A total of 36 cis-elements were identified belonging to the different functional categories (Table S1 and S2). . Obviously, a lot of phytohormone-responsive elements were widely found in the promoters of the wheat LBD genes. In detail, 79 TaLBDs contained ABA-responsive element (ABRE) in their promoters, suggesting these TaLBD genes were probably induced by ABA. And 62 TaLBDs contained MeJA-responsive element (TGACG-motif and CGTCA-motif) (Figure S2). In addition, three gibberellin-responsive elements, including GARE-motif, P-box motif and TATC-box were also found, , which was consistent with previous study that LBD genes may involve in signaling transduction such as gibberellin pathway [10]. Furthermore, it is also shown that TaLBD promoters were highly enriched in light related responsive elements, for example, sp1 and G-box. We found that 68 out 90 TaLBDs had the G-box motif. According to previous study, light-responsive element combined with other cis-element such as ABA-related, may provide a link to the light and stress mediated signaling in plant defense responses [26].
The microRNAs (miRNAs) are a class of small non-coding regulatory RNAs that involved in gene expression through guiding target mRNA cleavage or translational inhibition [27]. Recently, some miRNA was found to involve in response to abiotic stress in different plants through targeting on transcription factors [28-30] In order to investigate the potential regulatory association between LBD transcription factor and miRNA, the putative miRNA-TaLBDs relationship were predicted. Results showed that and a total of 30 TaLBDs were predicted to be targeted by21 miRNAs (Figure 4), of which tae-miR5384-3p could targeted on 11 TaLBDs, tae-miR444, tae-miR9677b and tae-miR9659-3P could target on 3 TaLBDs Most of TaLBDs were targeted by one miRNA, but Ta-4A-LBD39, Ta-3A-LBD25 and Ta-3B-LBD31as well as Ta-3D-LBD37 were targeted by 3 miRNAs. What’s more, Ta-3A-LBD25, Ta-3B-LBD31 and Ta-3D-LBD37 belonged to the same homoeologous group but targeted by 3 different miRNAs. The specific miRNA-LBD pairs provide the crucial insight for precisely manipulating these LBD genes’ function through miRNA-based method. Although miRNA inhibition mostly involved the transcript cleavage, some genes such as Ta-4B-LBD55 was predicted to be inhibited by translation. In addition, we found that the homoeologous group genes Ta-6A-LBD82, Ta-6B-LBD84 and Ta-6D-LBD85 were regulated by tae-miR444a and tae-miR444b. , According to previous study, miR444 family played important role in root development, tiller formation and stress response[31], indicating that they might involve in wheat development and stress tolerance through miRNA regulation. The miRNAs-LBD complex identified in this study would be useful in interpreting the post-transcriptional control of gene expression during various stress-induced physiological and cellular processes in wheat as well as other cereal crops.
Regulatory network between LBDs and other genes in wheat
LBDs transcription factor superfamily plays the crucial role in regulating the complex processes of plant growth, development and stresses response through interacting with other functional genes [10, 13]. To get the preliminary information about the interaction relationship between LBD and other genes in wheat, we constructed the interaction network they involved in based on the orthology-based prediction followed the network in Arabidopsis (Figure 5). Results showed that these TaLBDs widely interacted with the functional genes associated with organ development and morphogenesis (KNAT, EXPA17 and PRR1 ) and also interacted with other transcription factors to form signal transduction cascades (ARF19, NAC075 and WOX). Among them, Ta-4A-LBD40 was found to interact with KAN and KAN2 gene, which has been reported to involve in the molecular mechanism of lateral axis-dependent development of lateral organs in seed plants [32], suggesting that Ta-4A-LBD40 might act on regulator in wheat lateral axis-dependent development. Previous study found that LBD18/ASL20 could up-regulated EXPA17 to promote lateral root formation through auxin signaling in Arabidopsis [33, 34]. Here, we found that Ta-3A-LBD21, the orthology of AtLBD18, could interacted with EXPA17, indicating that it might regulate root development in wheat. In conclusion, the co-expression network analysis of LBD genes provided the vital information for the better understanding LBD transduction pathways in wheat.
Expression profiles of TaLBD genes
The spatio-temporal expression specificity of genes will provide the helpful information to understand their function in growth and development [35]. In this study, the tissue-specific expression profiles of the 90TaLBD genes in different developmental organs (grain, leaf, root, shoot and spike ) were investigated using RNA-Seq data based on the FPKM analysis (Figure 6 ). Based on the log10-transformed (PFKM + 1) values, we found that the expression levels of the TaLBD genes varied significantly in different tissues and showed obvious tissue specificity. In detail, a total of 72 TaLBD genes were detected to express in at least one of the tested tissues, while 18 showed no expression in all these tissues. . We deduced that during long time evolution, these genes may undergone functional differentiation and redundancy. Ta-1A-LBD2, Ta-6B-LBD84 and Ta-4B-LBD47 displayed specially high expression in root. Ta-5A-LBD73, Ta-4B-LBD56 and Ta-4D-LBD67 highly expressed in spikes, but little expression in other tissues, suggesting they might involve in seed development and mature. Ta-3A-LBD25, Ta-3D-LBD35 and Ta-4D-LBD61 showed specific expression in spike. Ta-3B-LBD28, Ta-4A-LBD46, Ta-4B-LBD54 specially expressed in stem with relative low level, and Ta-2B-LBD18 displayed high expression in leave but lower expression in other tissues. By comparison, there is much less genes showing specific expression in stem and leave and most of TaLBD genes were much more highly expressed in the root and grain. Furthermore, most of the homologous genes shared similar expression pattern among different tissues, nevertheless, we also found that some homologous genes showed different express pattern, such as Ta-4D-LBD61 showed special expression in spike, while Ta-4A-LBD41, Ta-4B-LBD50 did not detected any expression in spike.
Previous studies have showed that the LBD genes especially Class Ⅱ genes were generally considered to be also involved in stress tolerance [36, 37]. To gain insight into the expression and putative functions of LBD genes in response to stress, the expression patterns of TaLBDs in four abiotic (cold, heat, drought and salt) stresses were investigated using RNA-seq (Figure 7). Results revealed that most of the TaLBDs showed differential expression patterns under these abiotic stress, particularly under salt stress. Under salt stress, 54 TaLBDs were found to show differential expression at different time points. Ta-6B-LBD81, Ta-4B-LBD51 and Ta-U-LBD90 displayed high expression level at all 4 time points, while Ta−2B−LBD16, Ta-2D-LBD19 and Ta-1B-LBD5 showed specifically up-regulated expression at 6h. Ta−5A−LBD69, Ta−4B−LBD54, Ta−4D−LBD65 expressed specifically at 24h after salt stress. Furthermore, the expression levels of Ta-1A-LBD1 and Ta-4D-LBD62 increased continually from 6h to 24h, while Ta-lA-LBD2, Ta-1D-LBD10 and Ta-4B-LBD47 possessed high expression at 6h, 12h and 24h and then declined to little expression at 48h. These results suggested that LBD widely involved in salt stress induction in wheat and the different members played the differential roles in regulating salt stress response, Under cold stress, Ta-4A-LBD40 and Ta-4D-LBD62 were induced to highly express at 4 while Ta-4B-LBD49 specially expressed at 23. In addition, Ta-2A-13, Ta-2B-LBD15 and , Ta-2D-LBD18 showed up-regulated expression under drought stress, while Ta-5A-LBD72, Ta-5D-LBD79, Ta-5B-LBD76 and Ta-3D-LBD37 showed up-regulated firstly by heat stress induction at 1h and then were detected to be down-regulated after expose to heat stress 6h..The spatio-temporal expression profiles of these TaLBDs provided the useful information to better understand the roles of TaLBD playing in the growth and development as well as stress tolerance.
Validation of the expression of wheat LBD genes using qRT-PCR
Based on the expression patterns, 12 TaLBDs belonging to 4 homoeologous groups with significantly differential expression based on RNA-seq analysis were selected to investigate their expression levels under salt stress conditions by qPCR analysis (Figure 8). The expression divergence among homoeologous genes was also analyzed. The group A (comprising of Ta-1A-LBD1, Ta-1B-LBD5 and Ta-2D-LBD17) and the group B (comprising Ta-2A-LBD14, Ta-2B-LBD16 and Ta-2D-LBD19) showed up-regulated expression under salt stress, while the group A showed highest expression at 6h and then decreased expression continually but group B showed higest expresion at 24h after salt stress. Comparing to their A and D homoeologous genes, Ta-1B-LBD5 and Ta-2B-LBD16 showed the higher expression levels at all of 4 tested time point under salt stress (Figure 8A and 8B). At the same time, the 6 genes in group C (Ta-4A-LBD44, Ta-4B-LBD47 and Ta-4D-LBD58) and group D (Ta-6A-LBD81,Ta-6B-LBD83,Ta-U-LBD90) were induced to down-regulated under salt stress except for Ta-4A-LBD44 at 6h (Figure 8C and 8D). The homoeologous genes also showed differential expressions. For group C, Ta-4A-LBD44 had higest expression level at 6h and 12h while its B homoeologous copy Ta-4B-LBD47 showed highest expression level at 24h and 48h (Figure 8C). In group D, Ta-6B-LBD83 had a higher expression level compared to its homoeologous gene Ta-6A-LBD81 and Ta-U-LBD90 at all of the 4 time points (Figure 8D). These results demonstrated the overall expression trend of these genes obtained by qRT-PCR analysis was basically consistent with that of RNA-seq analysis. Furthermore, the expression levels of homoeologous genes showed significantly divergent, suggested that subfunctionalization has occurred in these homoeologous genes when responded to salt stress. Further studies on the underlying effect of subfunctionalization will contribute to better understand the function of LBD genes and the mechanism of salt tolerance in wheat.
Genetic diversity of wheat LBD genes
To obtain the information on genetic variations of wheat LBD genes, we further investigated their genetic diversities using the available wheat resequencing data [38]. A total of 404 (A:183, B:172 and D:49) and 550 (A:231, B: 218 and D: 101) SNPs were found in LBD genes of wheat landrace and variety populations, respectively (Table S3). The average genetic diversity (Pi value) of landrace population was 0.889E-04, compared to that of variety population with 0.886E-04 (Figure 9). No significant genetic variation was observed between them, suggesting no severe bottleneck was occurred on LBD gene family during wheat improvement. Furthermore, at subgenome level, the average genetic diversity of A, B and D subgenome in landrace population was 1.31E-04, 1.15E-04 and 2.08E-05 respectively, while in variety population was 1.06 E-04, 1.31E-04 and 2.87E-05 (Table S4). The D subgenomes of varieties showed higher diversity than that of the landraces, which was consistent with the previous study using the whole genome resequencing data [38]. It demonstrated that during modern wheat breeding processes, introgressive hybridization with its wild D relatives had the genetic effect on LBD gene family to enrich its diversity [39]. At the same time, the B subgenomes of varieties also had higher diversity than that of the landraces, which was not consistent with the general finding landrace with higher diveristy on B subgenome [38-40]. It suggested that some specific alien introgression events might occur in the wheat LBD genes on B subgenome. Further association the variations with the agronomic traits will not only contribute to the function of TaLBDs and also to better understand the evolutionary mechanism of this family.