3.1 M. acuminata genome has 50 GST genes that belong to seven canonical GST classes
With the pBLAST search of M. acuminata DH- Pahang v.4 genome at Banana Genome Hub, a total of 61 GST genes were obtained. The identified GST protein sequences were validated for the presence of conserved N and C-terminal domain through NCBI CD search, Pfam search and SMART database search. Out of 61 GSTs, 11 GST proteins that did not possess the conserved N or C-terminal domain were omitted from the study. 50 full length banana GST genes containing both the domains were named as MaGSTs belonging to seven established classes namely tau, phi, zeta, lambda, DHAR, EF1G and GHR. Elongation factor 1-gammma possessed an additional EF1G domain with the canonical N and C-terminal domain (Fig. 1). Plant specific tau and phi GSTs were highest in the number 24 and 11 respectively and theta GSTs were missing in the banana genome (Table 1). The nomenclature of banana GST protein was done as MaGSTs by taking the prefix Ma from M. acuminata, as proposed by Dixon et al. 2010 for A. thaliana. All the classes of MaGSTs were named MaGSTU, MaGSTF, MaGSTZ, MaGSTL, MaDHAR, MaGHR and MaEF1G. The numbering for each members of the class was done based on their chromosomal localization in the ascending order.
Table.1
List of identified GST members in Musa acuminata along with their detailed genomic information, physicochemical features and subcellular localization
S. No.
|
Locus ID
|
Gene Name
|
Chr. No.
|
Start
|
End
|
Strand
|
Gene (bp)
|
Protein (aa)
|
pI
|
Mol Wt (kDa)
|
GRAVY
|
AI
|
Subcellular Localization
|
1
|
Macma4_01_g17560.1
|
MaGSTU1
|
chr01
|
12645908
|
12646720
|
-
|
812
|
226
|
5.67
|
25.37
|
-0.104
|
95
|
Cya,b, Cpc
|
2
|
Macma4_01_g17570.1
|
MaGSTU2
|
chr01
|
12662270
|
12663054
|
-
|
784
|
217
|
5.1
|
24.43
|
-0.061
|
94.52
|
Cya,b,c
|
3
|
Macma4_01_g17580.1
|
MaGSTU3
|
chr01
|
12673915
|
12674696
|
-
|
781
|
226
|
5.52
|
25.57
|
-0.087
|
90.27
|
Cya,b, Cpc
|
4
|
Macma4_01_g17600.1
|
MaGSTU4
|
chr01
|
12679873
|
12686709
|
-
|
6836
|
233
|
8.85
|
25.77
|
-0.152
|
89.7
|
Cya,b, Cpc
|
5
|
Macma4_01_g17620.1
|
MaGSTU5
|
chr01
|
12699355
|
12700135
|
-
|
780
|
226
|
5.66
|
25.55
|
-0.037
|
92.43
|
Cya,b, Cpc
|
6
|
Macma4_01_g17610.1
|
MaGSTU6
|
chr01
|
12711730
|
12712528
|
-
|
798
|
226
|
5.53
|
25.71
|
-0.124
|
88.14
|
Cya,b,c
|
7
|
Macma4_01_g17590.1
|
MaGSTU7
|
chr01
|
12718722
|
12723573
|
-
|
4851
|
203
|
5.27
|
22.53
|
-0.08
|
91.82
|
Cya,b,c
|
8
|
Macma4_03_g27660.1
|
MaGSTU8
|
chr03
|
38741904
|
38742965
|
+
|
1061
|
254
|
4.96
|
28.03
|
-0.106
|
93.31
|
Cya,b,c
|
9
|
Macma4_04_g25520.1
|
MaGSTU9
|
chr04
|
33447586
|
33448535
|
+
|
949
|
227
|
5.26
|
25.45
|
0.02
|
104.45
|
Cya,b,c
|
10
|
Macma4_05_g07780.1
|
MaGSTU10
|
chr05
|
5643248
|
5644184
|
-
|
936
|
224
|
5.38
|
25.75
|
-0.189
|
92.77
|
Cya,b,c
|
11
|
Macma4_05_g10530.1
|
MaGSTU11
|
chr05
|
7490268
|
7491372
|
+
|
1104
|
232
|
5.4
|
25.64
|
0.003
|
99.22
|
Cya,b,c
|
12
|
Macma4_05_g10520.1
|
MaGSTU12
|
chr05
|
7492167
|
7493157
|
+
|
990
|
239
|
5.92
|
26.86
|
0.115
|
108.54
|
Cya,b,c
|
13
|
Macma4_05_g10540.1
|
MaGSTU13
|
chr05
|
7496933
|
7497902
|
+
|
969
|
230
|
5.59
|
25.54
|
0.088
|
100.96
|
Cya,b,c
|
14
|
Macma4_05_g31120.1
|
MaGSTU14
|
chr05
|
45082780
|
45084014
|
+
|
1234
|
244
|
5.11
|
27.38
|
-0.139
|
93.11
|
Cya,b,c
|
15
|
Macma4_08_g05550.1
|
MaGSTU15
|
chr08
|
33881616
|
33882956
|
-
|
1340
|
259
|
4.84
|
28.52
|
-0.082
|
91.47
|
Cya,b,c
|
16
|
Macma4_09_g28170.1
|
MaGSTU16
|
chr09
|
43993848
|
43997243
|
+
|
3395
|
241
|
5.38
|
27.23
|
-0.177
|
90.21
|
Cya,b,c
|
17
|
Macma4_09_g28180.1
|
MaGSTU17
|
chr09
|
44018622
|
44019703
|
+
|
1081
|
256
|
5.11
|
29.25
|
-0.2
|
85.35
|
Cya,b,c
|
18
|
Macma4_09_g28210.1
|
MaGSTU18
|
chr09
|
44010622
|
44011745
|
+
|
1123
|
224
|
5.4
|
25.08
|
-0.11
|
91.43
|
Cya,b,c
|
19
|
Macma4_09_g28220.1
|
MaGSTU19
|
chr09
|
44029677
|
44032258
|
+
|
2581
|
235
|
5.02
|
26.69
|
-0.134
|
96.64
|
Cya,b
|
20
|
Macma4_09_g28230.1
|
MaGSTU20
|
chr09
|
44043122
|
44047786
|
+
|
4664
|
302
|
5.54
|
34.82
|
-0.236
|
94.3
|
Cya,b,c
|
21
|
Macma4_09_g28240.1
|
MaGSTU21
|
chr09
|
44048790
|
44049518
|
+
|
728
|
214
|
5.28
|
24.24
|
-0.071
|
93.41
|
Cya,b, Cpc
|
22
|
Macma4_09_g28250.1
|
MaGSTU22
|
chr09
|
44051483
|
44052263
|
-
|
780
|
221
|
5.14
|
25.24
|
-0.203
|
82.94
|
Cya,b,c
|
23
|
Macma4_10_g01170.1
|
MaGSTU23
|
chr10
|
1342950
|
1344013
|
+
|
1063
|
234
|
5.21
|
26.41
|
-0.026
|
100.85
|
Cya,b, Cpc
|
24
|
Macma4_10_g13200.1
|
MaGSTU24
|
chr10
|
26157275
|
26158656
|
+
|
1381
|
223
|
5.91
|
25.71
|
-0.371
|
87.89
|
Cya,b,c
|
25
|
Macma4_01_g08790.1
|
MaGSTF1
|
chr01
|
6268539
|
6277061
|
-
|
8522
|
223
|
9.08
|
24.80
|
-0.27
|
81.03
|
Cya,b, Cpc
|
26
|
Macma4_02_g05750.1
|
MaGSTF2
|
chr02
|
20914202
|
20915401
|
-
|
1199
|
216
|
5.71
|
23.84
|
-0.087
|
87.59
|
Cya,b, Cpc
|
27
|
Macma4_02_g14640.1
|
MaGSTF3
|
chr02
|
26910761
|
26911782
|
-
|
1021
|
258
|
9.32
|
28.88
|
-0.267
|
80.89
|
Mta,b, Cpc
|
28
|
Macma4_03_g29570.1
|
MaGSTF4
|
chr03
|
39940354
|
39943419
|
+
|
3065
|
314
|
6.16
|
36.02
|
-0.404
|
85.06
|
Nua,c, Cyb
|
29
|
Macma4_04_g05170.1
|
MaGSTF5
|
chr04
|
4043451
|
4047857
|
+
|
4406
|
457
|
6.53
|
51.16
|
-0.23
|
87.26
|
Cya,b, Cpc
|
30
|
Macma4_04_g12310.1
|
MaGSTF6
|
chr04
|
8782518
|
8783723
|
-
|
1205
|
221
|
6.32
|
25.19
|
-0.243
|
92.58
|
Cya,b,c
|
31
|
Macma4_04_g23890.1
|
MaGSTF7
|
chr04
|
32148837
|
32149900
|
-
|
1063
|
221
|
7.64
|
25.24
|
-0.38
|
90
|
Cya,b, Cpc
|
32
|
Macma4_04_g38330.1
|
MaGSTF8
|
chr04
|
42452829
|
42453742
|
+
|
913
|
221
|
8.38
|
25.57
|
-0.414
|
90.45
|
Cya,b, Cpc
|
33
|
Macma4_05_g16910.1
|
MaGSTF9
|
chr05
|
14241272
|
14242352
|
-
|
1080
|
221
|
6.43
|
24.52
|
-0.278
|
78.24
|
Cya,b, Cpc
|
34
|
Macma4_07_g04070.1
|
MaGSTF10
|
chr07
|
3080875
|
3083111
|
+
|
2236
|
549
|
6.54
|
58.66
|
-0.685
|
58.71
|
Nua,c, Cyb
|
35
|
Macma4_11_g19810.1
|
MaGSTF11
|
chr11
|
30559356
|
30560881
|
+
|
1525
|
215
|
5.44
|
23.57
|
0.095
|
102.56
|
Cya,b, Cpc
|
36
|
Macma4_06_g31870.1
|
MaGSTZ1
|
chr06
|
37035146
|
37040197
|
-
|
5051
|
225
|
5.83
|
25.16
|
-0.132
|
95.91
|
Cpa, Cyb, Nuc
|
37
|
Macma4_08_g30570.1
|
MaGSTZ2
|
chr08
|
47426590
|
47431263
|
-
|
4673
|
218
|
5.82
|
24.49
|
-0.267
|
95.87
|
Cya,b,c
|
38
|
Macma4_08_g30590.1
|
MaGSTZ3
|
chr08
|
47436224
|
47440775
|
-
|
4551
|
218
|
5.1
|
24.36
|
-0.204
|
98.12
|
Cya,b,c
|
39
|
Macma4_10_g20220.1
|
MaGSTZ4
|
chr10
|
30806066
|
30810276
|
+
|
4210
|
261
|
5.85
|
29.08
|
-0.102
|
90.46
|
Cpa,b,c
|
40
|
Macma4_06_g36420.1
|
MaGSTL1
|
chr06
|
40281165
|
40283472
|
+
|
2307
|
287
|
6.21
|
32.12
|
-0.22
|
83.38
|
Cpa,b,c
|
41
|
Macma4_08_g22450.1
|
MaGSTL2
|
chr08
|
41703039
|
41705386
|
-
|
2347
|
248
|
5.24
|
28.27
|
-0.371
|
88.1
|
Cya,c, Cpb
|
42
|
Macma4_09_g04240.1
|
MaGSTL3
|
chr09
|
2799886
|
2802861
|
-
|
2975
|
266
|
5.15
|
30.15
|
-0.382
|
80.04
|
Cpa,b,c
|
43
|
Macma4_10_g01880.1
|
MaGSTL4
|
chr10
|
2621522
|
2624676
|
+
|
3154
|
242
|
5.41
|
27.88
|
-0.524
|
77.02
|
Cya,b
|
44
|
Macma4_06_g38000.1
|
MaDHAR1
|
chr06
|
41146917
|
41149619
|
-
|
2702
|
220
|
4.93
|
24.26
|
-0.117
|
90.36
|
Cya,b,c
|
45
|
Macma4_11_g23820.1
|
MaDHAR2
|
chr11
|
33111424
|
33114740
|
-
|
3316
|
218
|
5.52
|
24.29
|
-0.211
|
98.3
|
Cya,b,c
|
46
|
Macma4_02_g17210.1
|
MaEF1G1
|
chr02
|
28568385
|
28569603
|
-
|
1218
|
263
|
5
|
29.64
|
-0.307
|
81.29
|
Cya,b,c
|
47
|
Macma4_09_g11850.1
|
MaEF1G2
|
chr09
|
8034508
|
8039435
|
-
|
4927
|
419
|
5.7
|
47.86
|
-0.333
|
83.99
|
Cya,b,c
|
48
|
Macma4_09_g27020.1
|
MaEF1G3
|
chr09
|
42919531
|
42930570
|
-
|
11039
|
619
|
8.48
|
69.69
|
-0.345
|
81.42
|
Cpa,b,c
|
49
|
Macma4_09_g18510.1
|
MaGHR1
|
chr09
|
13698126
|
13701543
|
+
|
3417
|
425
|
8.37
|
46.44
|
-0.204
|
80.59
|
PMa, Cpb,c
|
50
|
Macma4_10_g19250.1
|
MaGHR2
|
chr10
|
30181763
|
30186696
|
-
|
4933
|
424
|
6.04
|
47.64
|
-0.381
|
70.64
|
Exa, Cpb, Cyc
|
3.2 50 MaGST genes are clustered on 11 banana chromosomes and evolutionarily conserved. Tandem duplication was the driving force for MaGST gene family expansion
On the basis of the M. acuminata DH-Pahang v.4 annotation, 50 MaGST genes were assigned to the eleven chromosomes ranging from 1 (Chr7) to 11 (Chr9). Chromosome 11 contained the highest nine MaGST genes followed by Chr1 holding eight MaGST genes. The pattern of chromosomal allocation of GSTs was also noticeable i.e. mainly on proximal or distal end of the banana chromosome as depicted in Fig. 2. A phylogenetic tree was constructed using M. acuminata, Arabidopsis, rice, soybean (angiosperm), P. patens (a bryophyte) and L. kaempferi (a gymnosperm) GST protein sequences. The different GST classes branched out into their individual clades, with the members of each class clustering together. Two major clades are of plant specific tau and phi GSTs that made two superclades under which small clades were noticed (Fig. 3). The individual class of GST from different plants that belong to separate sub-groups of the plant kingdom were clustered together and an indicative of their divergent evolution from a common ancestor. Phylogenetic analysis of MaGSTs with angiosperm (Arabidopsis and rice), gymnosperm (L. kaempferi) and bryophyte (P. patens) was carried out. The outcome revealed that the evolution of plant GSTs might be earlier than their division into individual groups such as bryophyte, pteridophyte, gymnosperm and angiosperm and also the each GST classes have diverged prior to the division of monocot and dicot. Additionally, the numbers of each class of GSTs expanded in a species specific manner independently and irrespective of their genome size. Additionally, the gene pairs under tandem and segmental duplication were close together in a phylogenetic tree showing close relatedness with each other.
To elucidate the gene family expansion in MaGSTs the duplication mechanism were analyzed. A total of 21 duplication events were noticed in banana GST gene family expansion and evolution. Tandem duplication was found to play a major role as 16 gene pairs were involved in tandem duplication event creating 16 gene clusters on Chr 1, 5, 8 and 9. Among them 15 gene pairs of tau class genes were majorly duplicated. MaGSTZ2/ MaGSTZ3 gene pair was also tandem duplicated gene. Five gene pairs composed of MaGSTU8/ MaGSTU14, MaGSTU8/ MaGSTU15, MaGSTU14/ MaGSTU15, MaGSTF1/ MaGSTF9 and MaGSTL2/ MaGSTL4 genes were segmental duplicated. The duplication event majorly occurred on banana chr1, 5, 8, 9 and 10. Moreover, the dN/dS values of duplicated genes were calculated and found to be less than 1, which is an indicative of purifying selection. Lastly, the divergence time was also calculated for these duplicated genes. The estimated divergence time of these gene pairs were approximately 2.51~145.41 million year ago (MYA) (Table. 2).
Table. 2
Estimated dN/dS ratios and divergence times of the duplicated MaGST genes
S. No.
|
Gene Name 1
|
Chr No.
|
Gene Name 2
|
Chr No.
|
Percent Identity
|
dN
|
dS
|
dN/dS
|
Duplication time (Mya)
|
Duplication Type
|
Selection type
|
1
|
MaGSTU1
|
chr01
|
MaGSTU2
|
chr01
|
93.09%
|
0.0302
|
0.1293
|
0.2334
|
9.95
|
Tandem
|
Purifying
|
2
|
MaGSTU1
|
chr01
|
MaGSTU3
|
chr01
|
91.59%
|
0.0341
|
0.1636
|
0.2082
|
12.58
|
Tandem
|
Purifying
|
3
|
MaGSTU1
|
chr01
|
MaGSTU5
|
chr01
|
90.71%
|
0.0396
|
0.1267
|
0.3126
|
9.75
|
Tandem
|
Purifying
|
4
|
MaGSTU1
|
chr01
|
MaGSTU6
|
chr01
|
91.15%
|
0.0376
|
0.1035
|
0.3635
|
7.96
|
Tandem
|
Purifying
|
5
|
MaGSTU2
|
chr01
|
MaGSTU3
|
chr01
|
95.39%
|
0.0188
|
0.0738
|
0.2545
|
5.68
|
Tandem
|
Purifying
|
6
|
MaGSTU2
|
chr01
|
MaGSTU4
|
chr01
|
81.02%
|
0.1055
|
0.6032
|
0.175
|
46.40
|
Tandem
|
Purifying
|
7
|
MaGSTU2
|
chr01
|
MaGSTU5
|
chr01
|
93.09%
|
0.0303
|
0.065
|
0.4658
|
5.00
|
Tandem
|
Purifying
|
8
|
MaGSTU2
|
chr01
|
MaGSTU6
|
chr01
|
94.93%
|
0.0207
|
0.0418
|
0.494
|
3.22
|
Tandem
|
Purifying
|
9
|
MaGSTU3
|
chr01
|
MaGSTU5
|
chr01
|
94.69%
|
0.0231
|
0.0326
|
0.7076
|
2.51
|
Tandem
|
Purifying
|
10
|
MaGSTU3
|
chr01
|
MaGSTU6
|
chr01
|
95.13%
|
0.0197
|
0.0717
|
0.2751
|
5.52
|
Tandem
|
Purifying
|
11
|
MaGSTU4
|
chr01
|
MaGSTU7
|
chr01
|
89.32%
|
0.0591
|
0.1798
|
0.3287
|
13.83
|
Tandem
|
Purifying
|
12
|
MaGSTU5
|
chr01
|
MaGSTU6
|
chr01
|
92.92%
|
0.0306
|
0.042
|
0.7292
|
3.23
|
Tandem
|
Purifying
|
13
|
MaGSTU11
|
chr05
|
MaGSTU13
|
chr05
|
84.72%
|
0.0889
|
0.1082
|
0.8214
|
8.32
|
Tandem
|
Purifying
|
14
|
MaGSTU12
|
chr05
|
MaGSTU13
|
chr05
|
86.90%
|
0.069
|
0.0909
|
0.7584
|
6.99
|
Tandem
|
Purifying
|
15
|
MaGSTU17
|
chr09
|
MaGSTU20
|
chr09
|
82.73%
|
0.1048
|
1.2164
|
0.0861
|
93.57
|
Tandem
|
Purifying
|
16
|
MaGSTZ2
|
chr08
|
MaGSTZ3
|
chr08
|
95.41%
|
0.021
|
0.0377
|
0.5572
|
2.90
|
Tandem
|
Purifying
|
17
|
MaGSTU8
|
chr03
|
MaGSTU14
|
chr05
|
88.00%
|
0.0844
|
0.9435
|
0.0895
|
72.58
|
Segmental
|
Purifying
|
18
|
MaGSTU8
|
chr03
|
MaGSTU15
|
chr08
|
83.08%
|
0.0796
|
0.6984
|
0.1139
|
53.72
|
Segmental
|
Purifying
|
19
|
MaGSTU14
|
chr05
|
MaGSTU15
|
chr08
|
85.65%
|
0.0994
|
0.9109
|
0.1091
|
70.07
|
Segmental
|
Purifying
|
20
|
MaGSTF1
|
chr01
|
MaGSTF9
|
chr05
|
81.00%
|
0.1521
|
1.8903
|
0.0805
|
145.41
|
Segmental
|
Purifying
|
21
|
MaGSTL2
|
chr08
|
MaGSTL4
|
chr10
|
81.40%
|
0.0972
|
0.4392
|
0.2214
|
33.78
|
Segmental
|
Purifying
|
3.3 MaGST proteins are highly stable, hydrophilic and found majorly in the cytoplasm
The protein length of MaGSTs ranged from 203 (MaGSTU7) to 619 (MaEF1G3) with their corresponding molecular weight of 22.53 kDa to 69.69 kDa respectively. The isoelectric point (pI) ranged from 4.84 (MaGSTU15) to 9.32 (MaGSTF3). Among 50 MaGSTs, seven MaGSTs were basic and 43 MaGSTs were acidic in nature. The grand average of hydropathy values of most of the MaGST proteins of all the classes were negative indicating that all the MaGST proteins were hydrophilic having good interaction with water. The aliphatic index of MaGSTs ranged from 58.71 (MaGSTF10) to 108.54 (MaGSTU12). Most of the MaGSTs were having the AI value below 100 and hence these are hydrophilic in nature. The subcelluar localization prediction results showed that MaGSTs were centrally localized in the cytoplasm followed by chloroplast, nucleus, mitochondria, plasma membrane and extracellular (Table. 1, Fig. 4).
3.4 MaGST proteins are characterized by the presence of many class specific motifs and gene architecture among tau, phi, zeta, lambda and DHAR class is highly conserved
The conserved motif analyses identified many class specific motifs and few motifs were found to be distributed among all the GST classes. The tau class MaGSTs possessed the highest number of eight motifs i.e. motif 1, 2, 3, 4, 5, 7, 8 and 13 whereas MaDHAR possessed least number of motifs i.e. 4 and 6. Motif 4 was found to be distributed across all the MaGSTs except MaEF1G members likewise motif 1 was also present in all the MaGSTs omitting MaDHAR and MaGHR members. Motif 11 was found only in MaGSTF members. Motif 14 and 15 was found in MaGSTLs. Motif 12 was found only in MaGSTZ and MaEF1G class. Motif 10 was found in MaGSTF, MaGSTZ and MaEF1G. Motif 6 was found in all the MaGST classes except tau and lambda. Motif 3 was found in MaGSTU and MaGSTZ members (Fig. 5).
MaGST genes possessed two to ten exons. All the MaGSTU possessed two-exons/ one-intron except MaGSTU5/12/16/19/20. Except MaGSTF1/4/5, all the phi members contained three-exons/ two-introns. All the genes in the zeta and DHAR members possessed nine and six exons respectively. In the lambda class MaGSTL1/2/3 possessed nine exons whereas MaGSTL4 possessed eight exons. Two MaGHR genes possessed two and seven exons respectively whereas three MaEF1G genes contained five, six and nine exons correspondingly. The numbers of exons were highly variable in MaEF1G and MaGHR class (Fig. 6). The conservation in the number of exons can be correlated with the expansion of MaGST gene family.
3.5 Ser and Cys catalytic residues are highly conserved in MaGST classes
Multiple sequence alignment was performed by taking the GST protein sequences of M. acuminata, A. thaliana, G. max and O. sativa to identify the conserved residues and catalytic residue among different GST classes. The position of catalytic residues and their signature sequences have been depicted in Fig.. The position of active site residue varied among the classes. Tau and phi GSTs possessed Ser active site residue at the position 17 and 12 respectively whereas zeta hold active site Ser residue at position 41. Lambda and DHAR contained active site Cys residue at position 20. The GHR also possessed Cys active site residue at position 46.
3.6 MaGSTs are predominantly composed of α-helices
The secondary structure of plant GSTs is characterized by the dominance of alpha helix followed by coil, beta strand and beta turns. The percent of alpha helix was highest in all the MaGSTs especially tau proteins, except MaGSTF4/ MaGSTF10/ MaGSTZ4/ MaGSTL1 and MaDHAR2, in which the percent of coil was highest. The MaGSTU11 contained the 61.92 alpha helix which was highest among all the MaGSTs. Both the MaGHRs also possessed highest percent of coil than alpha helix (Fig. Table.S1).
3.7 Phosphorylation, as a major post translational modification in MaGSTs
Post translational modification plays an important role in protein structural modification and its functioning. In the post translational alteration prediction in banana GSTs, only 5 out of 50 MaGSTs possessed the glycosylation sites (Table S2). The phosphorylation prediction study revealed that Ser and Thr residues are highly phosphorylated accounting for 46.76% and 46% followed by Tyr that is 29.28% (Fig. 9; Table S3).
3.8 Five types of functional Cis-regulatory elements are present in the promoter region of MaGST genes
Cis-acting regulatory elements (CAREs) are found in the promoter region of target genes. They are basically short motif of 5-20 bp length, non-coding DNA that binds the transcription factors and regulate the gene transcription. In addition to four core elements (AT~TATA-box, CAAT-box, TATA-box, TATA), the current study identified 36 cis-elements categorized on the basis of its importance in plants physiology i.e. light responsive element, hormone responsive element, stress responsive element, cellular development related elements and other elements. The light responsive elements were highest in the number followed by stress and hormone responsive element (Fig. 10). The MaGSTU24 possessed the highest number of 57 cis-elements whereas MaGSTU6 possessed only 6 cis-elements in its promoter region. MYB and MYC cis-elements were highest in number 140 and 178 respectively. Abscisic acid responsiveness elements were than highest in number accounting for 127. The high number of stress responsive elements (MYB and MYC) in the promoter region can be associated with their role in combating against diverse stress in banana plant by upregulating the MaGST transcripts and total enzyme activity.
3.10 RNA expression profiling of MaGSTs showed most of the MaGST genes get up-regulated during fruit development stage
To predict the role of MaGSTs in fruit development, the expression level of 45 MaGST genes expressed in endocarp tissue were analyzed during 0 days, 20 days and 80 days after flowering (DAF) based on its RNA-seq data. Among the banana GSTs tau members the MaGSTU1 was highly up-regulated initially than down-regulated at 20 DAF and then up regulated at 80 DAF and could be involved in fruit ripening and at the phase of ripening the activity of the ethylene signal transduction pathway is also significantly increased (Dhar et al. 2019). Likewise the expression of MaGSTU9, MaGSTU11, and MaGSTU21 was also up-regulated at 80 DAF and they might be involved in fruit ripening. MaGSTU12, MaGSTU19 and MaGSTU20 were found to be down-regulated at 80 DAF and might not be involved in fruit development. Additionally, MaGSTU3, MaGSTU4, MaGSTU5, MaGSTU8, MaGSTU9, MaGSTU10, MaGSTU11, MaGSTU12, MaGSTU13, MaGSTU15, MaGSTU16, MaGSTU18 and MaGSTU22 genes were found to be up-regulated at 20 DAF. MaGSTF2 and MaGSTF11 were found to be down-regulated from 0 DAF to 80 DAF whereas MaGSTF3, MaGSTF4, MaGSTF6 and MaGSTF7 up-regulated at 20 DAF. MaGSTF5 was up-regulated at 80 DAF during fruit development. Gene expression level of MaGSTZ were found to be variable i.e. MaGSTZ2 was up-regulated from 0 to 20 DAF and than down-regulated at 80 DAF whereas MaGSTZ3 showed low level of expression at 0 to 80 DAF. The MaGSTZ4 showed increased expression at 0 DAF and then down-regulated from 20 DAF to 80 DAF. The MaGSTL1 showed a low expression level whereas MaGSTL3 and MaGSTL4 were highly expressed. All the MaGSTL, MaGHR genes and MaEF1G3 showed high level of expression at 20 DAF. The expression level of MaDHAR2 was high at 0 DAF and then low at 80 DAF. MaGSTU24, MaGSTF1, MaGSTF9, MaGSTF10 and MaGSTZ1 were down-regulated during the process of fruit development (Fig. 11, 12).