3.1 Identification of Poplar BTB Domain Gene Family
Through conducting a BLAST search of Arabidopsis BTB proteins against poplar genomes, refined by the BTB conserved domain, we identified 89 family members in poplar. These BTB proteins were named based on subsequent subfamily branches of the evolutionary tree. We analyzed the characteristics of these 89 BTB genes in poplar. Detailed information for each PaBTB, including gene name, Protein ID, Transcript ID, Gene Description, length of coding sequence and protein, theoretical isoelectric point, and molecular weight, is presented in Table 1. The deduced proteins exhibited diverse lengths, resulting in varied isoelectric points and molecular weights. The coding sequences (CDS) ranged from 819 bp (PaBTB35) to 3060 bp (PaBTB41), corresponding to protein lengths spanning from 273 (PaBTB35) to 1020 (PaBTB41) amino acids. The average molecular weight ranged from 30.54 kDa (PaBTB35) to 115.419 kDa (PaBTB41), and the theoretical isoelectric point ranged from 4.77 (PaBTB89) to 9.21 (PaBTB61). Among the 89 proteins, 62 had isoelectric points below 7.0.
Gene Name
|
Protein ID
|
Transcript ID
|
CDS(bp)
|
Peptide(aa)
|
Molecular weight (average)
|
Theoretical pI
|
PaBTB1
|
XP_034887108.1
|
rna-XM_035031217.1
|
1278
|
426
|
47267.18
|
5.2
|
PaBTB2
|
XP_034893008.1
|
rna-XM_035037117.1
|
1302
|
434
|
48106.78
|
5.93
|
PaBTB3
|
XP_034901062.1
|
rna-XM_035045171.1
|
1191
|
397
|
43467.36
|
6.23
|
PaBTB4
|
XP_034895770.1
|
rna-XM_035039879.1
|
1230
|
410
|
45648.97
|
6.64
|
PaBTB5
|
XP_034930383.1
|
rna-XM_035074492.1
|
1227
|
409
|
45261.51
|
6.36
|
PaBTB6
|
XP_034919073.1
|
rna-XM_035063182.1
|
1221
|
407
|
44953.25
|
5.9
|
PaBTB7
|
XP_034928498.1
|
rna-XM_035072607.1
|
1224
|
408
|
45035.47
|
5.97
|
PaBTB8
|
XP_034915024.1
|
rna-XM_035059133.1
|
2112
|
704
|
77757.31
|
6.48
|
PaBTB9
|
XP_034931121.1
|
rna-XM_035075230.1
|
2088
|
696
|
76877.46
|
6.92
|
PaBTB10
|
XP_034911440.1
|
rna-XM_035055549.1
|
2151
|
717
|
79317.69
|
6.11
|
PaBTB11
|
XP_034923574.1
|
rna-XM_035067683.1
|
2151
|
717
|
79656.28
|
6.53
|
PaBTB12
|
XP_034898733.1
|
rna-XM_035042842.1
|
2628
|
876
|
95739.53
|
5.76
|
PaBTB13
|
XP_034901911.1
|
rna-XM_035046020.1
|
2889
|
963
|
108647.87
|
5.51
|
PaBTB14
|
XP_034913644.1
|
rna-XM_035057753.1
|
2871
|
957
|
108442.64
|
5.78
|
PaBTB15
|
XP_034923420.1
|
rna-XM_035067529.1
|
2505
|
835
|
95036.29
|
5.74
|
PaBTB16
|
XP_034886832.1
|
rna-XM_035030941.1
|
2736
|
912
|
103762.09
|
6.1
|
PaBTB17
|
XP_034931056.1
|
rna-XM_035075165.1
|
2682
|
894
|
100902.12
|
5.59
|
PaBTB18
|
XP_034915193.1
|
rna-XM_035059302.1
|
2667
|
889
|
100642.84
|
5.6
|
PaBTB19
|
XP_034887120.1
|
rna-XM_035031229.1
|
903
|
301
|
33397.81
|
6.25
|
PaBTB20
|
XP_034897945.1
|
rna-XM_035042054.1
|
909
|
303
|
33609.27
|
6.66
|
PaBTB21
|
XP_034894693.1
|
rna-XM_035038802.1
|
1434
|
478
|
52590.6
|
5.89
|
PaBTB22
|
XP_034887922.1
|
rna-XM_035032031.1
|
1431
|
477
|
52407.18
|
5.84
|
PaBTB23
|
XP_034907679.1
|
rna-XM_035051788.1
|
1338
|
446
|
50177.51
|
5.5
|
PaBTB24
|
XP_034895516.1
|
rna-XM_035039625.1
|
1338
|
446
|
49850.98
|
5.44
|
PaBTB25
|
XP_034899513.1
|
rna-XM_035043622.1
|
1386
|
462
|
50248.95
|
5.28
|
PaBTB26
|
XP_034926170.1
|
rna-XM_035070279.1
|
1080
|
360
|
41583.3
|
8.39
|
PaBTB27
|
XP_034890544.1
|
rna-XM_035034653.1
|
1140
|
380
|
43338.22
|
9
|
PaBTB28
|
XP_034928393.1
|
rna-XM_035072502.1
|
1221
|
407
|
46054.67
|
8.9
|
PaBTB29
|
XP_034929542.1
|
rna-XM_035073651.1
|
1221
|
407
|
46339.18
|
8.84
|
PaBTB30
|
XP_034889040.1
|
rna-XM_035033149.1
|
825
|
275
|
31124.15
|
5.28
|
PaBTB31
|
XP_034931549.1
|
rna-XM_035075658.1
|
822
|
274
|
31100.16
|
5.07
|
PaBTB32
|
XP_034899593.1
|
rna-XM_035043702.1
|
990
|
330
|
37379.67
|
5.76
|
PaBTB33
|
XP_034888967.1
|
rna-XM_035033076.1
|
981
|
327
|
37715.3
|
5.82
|
PaBTB34
|
XP_034893131.1
|
rna-XM_035037240.1
|
1056
|
352
|
40351.2
|
6.11
|
PaBTB35
|
XP_034903424.1
|
rna-XM_035047533.1
|
819
|
273
|
30540.18
|
5.48
|
PaBTB36
|
XP_034904619.1
|
rna-XM_035048728.1
|
1755
|
585
|
66277.44
|
5.07
|
PaBTB37
|
XP_034904867.1
|
rna-XM_035048976.1
|
1659
|
553
|
62368.43
|
5.16
|
PaBTB38
|
XP_034926213.1
|
rna-XM_035070322.1
|
1659
|
553
|
62493.72
|
5.24
|
PaBTB39
|
XP_034894827.1
|
rna-XM_035038936.1
|
1683
|
561
|
63466.22
|
5.68
|
PaBTB40
|
XP_034896281.1
|
rna-XM_035040390.1
|
1683
|
561
|
63471.41
|
5.8
|
PaBTB41
|
XP_034914749.1
|
rna-XM_035058858.1
|
3060
|
1020
|
115419.27
|
7.31
|
PaBTB42
|
XP_034931454.1
|
rna-XM_035075563.1
|
2418
|
806
|
92051.63
|
5.48
|
PaBTB43
|
XP_034905526.1
|
rna-XM_035049635.1
|
2403
|
801
|
91586.45
|
5.77
|
PaBTB44
|
XP_034913316.1
|
rna-XM_035057425.1
|
1767
|
589
|
65538.79
|
5.87
|
PaBTB45
|
XP_034887663.1
|
rna-XM_035031772.1
|
1758
|
586
|
65253.68
|
6.12
|
PaBTB46
|
XP_034927243.1
|
rna-XM_035071352.1
|
1758
|
586
|
65271.72
|
6.12
|
PaBTB47
|
XP_034891412.1
|
rna-XM_035035521.1
|
1761
|
587
|
65361.9
|
6.31
|
PaBTB48
|
XP_034915068.1
|
rna-XM_035059177.1
|
1773
|
591
|
66152.16
|
6.15
|
PaBTB49
|
XP_034931163.1
|
rna-XM_035075272.1
|
1734
|
578
|
64846.84
|
6.03
|
PaBTB50
|
XP_034892332.1
|
rna-XM_035036441.1
|
1329
|
443
|
48822.9
|
6.3
|
PaBTB51
|
XP_034920071.1
|
rna-XM_035064180.1
|
1443
|
481
|
52977.46
|
6.14
|
PaBTB52
|
XP_034890011.1
|
rna-XM_035034120.1
|
1644
|
548
|
62109.55
|
8.43
|
PaBTB53
|
XP_034904755.1
|
rna-XM_035048864.1
|
1926
|
642
|
71464.38
|
8.8
|
PaBTB54
|
XP_034932491.1
|
rna-XM_035076600.1
|
1893
|
631
|
70510.3
|
8.87
|
PaBTB55
|
XP_034913246.1
|
rna-XM_035057355.1
|
1803
|
601
|
66913.33
|
7.1
|
PaBTB56
|
XP_034912285.1
|
rna-XM_035056394.1
|
1821
|
607
|
67711.67
|
5.77
|
PaBTB57
|
XP_034915940.1
|
rna-XM_035060049.1
|
1830
|
610
|
68931.65
|
6.64
|
PaBTB58
|
XP_034908343.1
|
rna-XM_035052452.1
|
1725
|
575
|
64135.89
|
8.52
|
PaBTB59
|
XP_034921371.1
|
rna-XM_035065480.1
|
1737
|
579
|
64676.41
|
8.44
|
PaBTB60
|
XP_034898247.1
|
rna-XM_035042356.1
|
1785
|
595
|
67316.3
|
8.46
|
PaBTB61
|
XP_034892699.1
|
rna-XM_035036808.1
|
1998
|
666
|
75714.49
|
9.21
|
PaBTB62
|
XP_034909886.1
|
rna-XM_035053995.1
|
1884
|
628
|
69945.2
|
8.7
|
PaBTB63
|
XP_034900308.1
|
rna-XM_035044417.1
|
1884
|
628
|
70083.36
|
8.97
|
PaBTB64
|
XP_034927831.1
|
rna-XM_035071940.1
|
1839
|
613
|
67812.73
|
6.5
|
PaBTB65
|
XP_034923296.1
|
rna-XM_035067405.1
|
1842
|
614
|
67957.88
|
7.88
|
PaBTB66
|
XP_034910354.1
|
rna-XM_035054463.1
|
1878
|
626
|
69319.11
|
6.43
|
PaBTB67
|
XP_034917574.1
|
rna-XM_035061683.1
|
1836
|
612
|
69035.28
|
8.86
|
PaBTB68
|
XP_034916341.1
|
rna-XM_035060450.1
|
1833
|
611
|
68849.23
|
8.29
|
PaBTB69
|
XP_034907483.1
|
rna-XM_035051592.1
|
1911
|
637
|
71929.29
|
5.96
|
PaBTB70
|
XP_034891911.1
|
rna-XM_035036020.1
|
1923
|
641
|
72575.23
|
6.96
|
PaBTB71
|
XP_034903215.1
|
rna-XM_035047324.1
|
2028
|
676
|
76176.56
|
8.18
|
PaBTB72
|
XP_034892198.1
|
rna-XM_035036307.1
|
1755
|
585
|
65577.59
|
6.75
|
PaBTB73
|
XP_034928962.1
|
rna-XM_035073071.1
|
1884
|
628
|
69875.39
|
5.66
|
PaBTB74
|
XP_034919263.1
|
rna-XM_035063372.1
|
1881
|
627
|
70245.89
|
5.23
|
PaBTB75
|
XP_034910556.1
|
rna-XM_035054665.1
|
1872
|
624
|
69697.82
|
5.08
|
PaBTB76
|
XP_034925093.1
|
rna-XM_035069202.1
|
1887
|
629
|
69947.32
|
6.92
|
PaBTB77
|
XP_034896790.1
|
rna-XM_035040899.1
|
1896
|
632
|
70250.8
|
6.46
|
PaBTB78
|
XP_034933095.1
|
rna-XM_035077204.1
|
1968
|
656
|
73263.97
|
5.4
|
PaBTB79
|
XP_034913910.1
|
rna-XM_035058019.1
|
1971
|
657
|
73551.92
|
5.43
|
PaBTB80
|
XP_034929025.1
|
rna-XM_035073134.1
|
1881
|
627
|
70598.49
|
8.08
|
PaBTB81
|
XP_034910082.1
|
rna-XM_035054191.1
|
1887
|
629
|
70518.73
|
7.82
|
PaBTB82
|
XP_034892672.1
|
rna-XM_035036781.1
|
1671
|
557
|
63805.27
|
5.26
|
PaBTB83
|
XP_034923717.1
|
rna-XM_035067826.1
|
1641
|
547
|
60824.41
|
8.79
|
PaBTB84
|
XP_034894446.1
|
rna-XM_035038555.1
|
1698
|
566
|
63059.18
|
8.98
|
PaBTB85
|
XP_034906775.1
|
rna-XM_035050884.1
|
1761
|
587
|
65462.36
|
8.75
|
PaBTB86
|
XP_034922619.1
|
rna-XM_035066728.1
|
1599
|
533
|
59777.09
|
8.66
|
PaBTB87
|
XP_034903082.1
|
rna-XM_035047191.1
|
1626
|
542
|
60898.53
|
8.98
|
PaBTB88
|
XP_034904576.1
|
rna-XM_035048685.1
|
2019
|
673
|
76167.56
|
8.88
|
PaBTB89
|
XP_034887357.1
|
rna-XM_035031466.1
|
1308
|
436
|
49228.38
|
4.77
|
Table 1 Identification of the BTB gene family in poplar.
3.2 Conserved domains, Phylogenetic analysis and classification of poplar BTB gene family
To explore the phylogenetic relationship and divergence of the BTB family in poplar, a phylogenetic tree was constructed using MEGA11 according to the aligned 89 BTB protein sequences (Figure 1). Based on the evolutionary relationship and distribution of protein domains, we divided poplar BTB family into seven major families (A, B, C, D, E, F, H) according to the classification of Arabidopsis and rice BTB families (Gingerich et al. 2005; Gingerich et al. 2007). It was then further divided into 16 subfamilies (A2, B1, B2, B3, B4, C1, C2, C3, C4, D, E1, E2, E3, E4, F, H). The poplar has the same number of families and subfamilies as Arabidopsis, but the number of members in each family is different. Rice (Oryza sativa) possesses 8 major families (A, B, C, D, E, F, G, H) and 18 subfamilies (A1, A2, A3, B1, B2, B3, B4, C1, C2, C3, C4, D, E1, E3, E4, F, G, H). In comparison, poplar lacks the family G and certain subfamilies within the family A (A1 and A3), while additionally has the E2 subfamily. The poplar gene families exhibit varying subfamily compositions: Family A includes only the A2 subfamily with seven members. Family B comprises four subfamilies—B1 (5), B2 (6), B3 (2), and B4 (5). Family C is composed of four subfamilies—C1 (4), C2 (2), C3 (3), and C4 (1). Family D consists solely of the D subfamily with one member. Family E encompasses four subfamilies—E1 (4), E2 (1), E3 (2), and E4 (8). Family F includes only the F subfamily, which comprises 27 members.
All BTB subfamilies of poplars have at least one BTB domain, except PaBTB36 of the D subfamily and PaBTB42, PaBTB43 of the E3 subfamily with two BTB domains, other PABTBs have one BTB domain. Most of these BTB domains are located in the N-terminal, and only a few are distributed in the C-terminal or intermediate region. Moreover, the poplar BTB proteins contained many other domains. In addition, poplar BTB protein also contains many other domains, and different subfamilies have obvious differences or characteristics in the composition of the domain. Members of the A2 subfamily are characterized by significant conservation of the N-terminal MATH domain and C-terminal BACK domain. The B1 subfamily shares a conserved N-terminal BACK domain and additionally possesses the SRP1, PLN03200, and HEAT domains. Most members of the B2 subfamily feature the LapB domain along with additional domains such as TPR, Spy, and NlpI. The B3 subfamily is primarily characterized by the conserved YjpI domain. In the B4 subfamily, members other than PaBTB22 contain solely a conserved BTB domain, while PaBTB22 also includes a WD40 domain. The C1 subfamily is distinguished by the presence of conserved TAZ and BACK domains. One member of the C2 subfamily carries a C-terminal BACK domain, whereas another member possesses only a BTB domain without additional domains. The C3 subfamily exclusively contains the BTB domain. In the C4 subfamily, one member features a C-terminal BACK domain. One member of the D subfamily bears an ANK domain. Members of the E1 subfamily all exhibit a conserved C-terminal BACK domain. Within the E2 subfamily, one member has only a BTB domain. The E3 subfamily displays a conserved Methyltransf FA domain at the N-terminal and additionally features BACK and F5_F8_type_C domains at the C-terminal. In the E4 subfamily, most members carry DUF3420 and ANK domains, aside from PaBTB50 and PaBTB51, other members also possess an NPR1 domain. Members of the F subfamily, excluding PaBTB52, possess a conserved C-terminal NPH3 domain, while PaBTB52 additionally includes a Cnn 1N domain. The sole member of the H subfamily exclusively contains a BTB domain.
3.3 Gene Structure
Using TBtools software, we analyzed the gene structure of the poplar BTB gene family (Figure 2). The exon-intron architecture of the poplar BTB gene exhibits notable diversity across its various subfamilies. Apart from B1, B3, and E3 subfamilies, which display more than 10 exons each, the majority of BTB genes are characterized by 1-8 exons. Notably, all members of the A family consist of precisely four exons, with the exception of PaBTB2, which possesses three exons. The B1 subfamily stands out with 19 exons, contrasting with the B2, B3, and B4 subfamilies, which typically feature 4-5, 11, and 1-2 exons, respectively. Members of the C1 subfamily are structured with five exons, while the C2, C3, and C4 subfamilies exhibit two, four, and three exons, respectively. The D subfamily uniformly presents with 5 exons, whereas the E1 subfamily is characterized by 6 exons, the E2 subfamily by 8 exons, the E3 subfamily by 12 exons, and the E4 subfamily by 4 exons. The F subfamily displays a diverse range of 2-7 exons per gene, predominantly featuring 4 exons. Finally, members of the H subfamily uniformly possess a single exon.
3.4 Identification of cis-acting element in the Promoter Region
In order to predict the potential biological function of BTB gene, the cis-acting elements of the promoter were analyzed. Cis-acting elements were found abundantly predicted in the promoters of all BTB genes, totaling 103(Table S1). Each gene's promoter includes elements associated with plant growth, hormone responses, and stress responses (Figure 3). Cis-acting elements relevant to plant growth and development are widely present within the poplar BTB gene promoters. These motifs include CAT-box associated with meristematic tissue expression, O2-site linked to maize protein metabolism regulation, AACA motif and GCN4motif associated with endosperm expression, HD-Zip 1 relevant to epidermal differentiation, MBSI involved in flavonoid biosynthesis gene regulation, RY-element specific to seed regulation, and motifs related to circadian rhythm regulation. Furthermore, Cis-acting elements responsive to hormones and stress stimuli are notably abundant in these promoters. Specifically, elements associated with abscisic acid response (ABRE), ethylene response (ERE), and anaerobic induction (ARE) are prevalent in the majority of poplar gene promoters. Additionally, most BTB gene promoters contain cis-acting elements involved in drought and stress responses such as MYB binding sites (MBS), multiple stress-responsive elements (STRE), defense and stress response elements (TC-rich repeats), non-biological stress and defense response WRKY binding sites (W-box), as well as wound response elements (WRE3, WUN-motif, and box S).The presence of these cis-acting element suggests that the poplar BTB gene family may regulate diverse gene expressions under various conditions.
3.5 Expression Patterns of BTB Domain Genes in Different Tissues
To characterize the expression dynamics of poplar BTB (Bric-a-Brac/Tramtrack/Broad complex) genes across various tissues and developmental stages, we utilized RNA-seq data from Populus trichocarpa on Phytozome to generate a heatmap (Figure 4). The expression patterns of poplar BTB genes exhibits notable diversity. Within apical buds, the majority of BTB genes display a gradual decline in expression levels during spring, followed by a gradual increase post-winter. This phenomenon may be associated with the protection of internal tissues within apical buds, with these genes potentially implicated in the regulation of bud dormancy and differentiation. However, a select few individual BTB genes exhibit higher expression levels towards the end of spring, juxtaposed with lower expression levels during other periods, hinting at their potential involvement in reproductive organ development. During mid-stage male catkin development, certain BTB genes demonstrate heightened expression levels, notably PtBTB58 and PtBTB59 within the F subfamily, suggesting their potential role in pivotal pathways underlying catkin development. Moreover, a minority of BTB genes display elevated expression levels in stems during both spring and winter, with the majority exhibiting a trend of decreased expression in spring and increased expression in winter, potentially indicating their involvement in the stem's response to low-temperature stress.
3.6 BTB Response to Exogenous Hormones
Hormones play a crucial role in regulating plant growth and development, with BTB proteins significantly involved in hormone response and signaling pathways in plants. To investigate the response of BTB genes to exogenous hormones in poplar, quantitative real-time PCR (qRT-PCR) analysis was employed to assess the transcriptional levels of representative BTB genes (PaBTB7, PaBTB17, PaBTB27, PaBTB36, PaBTB44, PaBTB65, PaBTB89, from different subfamily) subject to the treatments with SA, MeJA, ABA, IAA, and GA3 (Figure 5).
After SA treatment, the expression levels of BTB genes showed varied responses. Specifically, PaBTB17 and PaBTB65 exhibited decreased expression across all sampling points, with PaBTB17 notably reduced to 0.048-fold of the 0-hour control level. In contrast, PaBTB7, PaBTB36, PaBTB44, and PaBTB89 displayed increased expression after 6 hours of treatment, while other sampling points showed decreased expression, indicating fluctuation in transcriptional activity following SA stimulation.
After MeJA treatment, significant increases in expression were observed for PaBTB27 and PaBTB36 after 1 hour, with fold changes of 5.238 and 7.959, respectively, demonstrating a strong MeJA response. Conversely, PaBTB17 and PaBTB65 exhibited decreased expression at all sampling points. PaBTB7, PaBTB44, and PaBTB89 showed increased expression levels at 6 hours, 1 hour, and 12 hours after MeJA treatment, respectively, whereas other sampling points showed slight decreases.
After ABA treatment, most BTB genes showed an overall increase in expression. PaBTB7, PaBTB17, PaBTB36, PaBTB44, and PaBTB65 displayed elevated expression levels across all sampling points, with PaBTB44 showing the most significant increase, reaching 77.714-fold at 6 hours post-treatment. However, PaBTB27 exhibited decreased expression at 1 hour, and PaBTB89 showed a decline after 12 hours.
Following IAA treatment, BTB gene expression levels exhibited varied responses. PaBTB17, PaBTB65, and PaBTB89 showed significant decreases at all sampling points, particularly reducing to 0.241, 0.224, and 0.203-fold after 1 hour, respectively. In contrast, PaBTB36 and PaBTB44 displayed substantial increases after 24 hours, with fold changes of 24.545 and 15.921, respectively. PaBTB7 showed a slight increase at 1 hour, while other sampling points showed decreased expression. PaBTB27 exhibited a decrease from 1 to 12 hours, followed by an increase at 24 hours.
After GA3 treatment, most BTB genes showed increased expression levels. Particularly, PaBTB36 and PaBTB65 displayed significant increases at 12 hours, with fold changes of 5.559 and 3.497, respectively. PaBTB17 showed increased expression at all sampling points. PaBTB7 and PaBTB89 initially decreased in expression at 1 hour post-treatment but gradually increased thereafter. PaBTB27 exhibited sustained decreased expression until 24 hours post-treatment, followed by a subsequent increase. PaBTB44 showed decreased expression at 1 hour, increased at 6 hours, and decreased again by 24 hours.
3.7 BTB Response to Drought Stress
To further explore the function of poplar BTB genes respond to stress, we subjected two-month-old poplar to 20% PEG6000 treatments at different time points, followed by sample collection and qRT-PCR analysis. Under drought stress conditions, various subfamily members exhibited diverse expression level changes (Figure 5). The BTB genes in poplar displayed heterogeneous expression patterns under drought stress. Following drought treatment, the expression levels of PaBTB7, PaBTB17, PaBTB36, PaBTB44, and PaBTB65 decreased after 1 hour, showed a slight recovery at 6 hours, and then declined again. In contrast, PaBTB27 and PaBTB89 exhibited significantly higher expression levels at 1 hour and 12 hours post-drought treatment compared to the baseline at 0 hours, while expression levels at other time points were comparatively lower.