1. IDENTIFICATION AND CHARACTERIZATION OF OsDHSRP GENES.
The characterized OsDHSRP sequence from Arabidopsis thaliana utilized as a query sequence for BlastP search against Oryza Sativa genome for identification of OsDHSRP genes. Among all the hits, 15 genes were determined as potential candidates OsDHSRP gene family members. All these 15 sequences containing C3HC4 zinc finger domain which are renamed as OsDHSRP1, OsDHSRP2, OsDHSRP3, ……...OsDHSRP15 with respect to their location on pseudo chromosomes. (Table 1). Characterization of genes was done and the protein with longest peptide sequence was OsDHSRP4 (709 a.as) and protein with shortest one is OsDHSRP6 (197 a.as). The molecular weight of candidate proteins was analysed, all predicted OsDHSRP proteins ranges in between 20 kDa to 76 KDa. We analysed protein parameters include pI ranged from 3.85 to 9.33, GRAVY ranged from − 0.068 to + 0.325. (Table 2). Gene structure prediction shown that among 15 OsDHSRP genes OsDHSRP4, OsDHSRP8,9,1,11,13,14 has 4/5 intron to exon ratio; OsDHSRP2,3,10 has 3/4 intron to exon ratio; OsDHSRP6,12,15 has no introns and the intron to exon ratio was almost similar among members of same group. (Fig. 1b). The subcellular localization of OSDHSRP homologs were investigated; OsDHSRP1, OsDHSRP2, OsDHSRP3, OsDHSRP4, OsDHSRP7, OsDHSRP10, OsDHSRP12, OsDHSRP13, OsDHSRP14 are localized in nucleus; OsDHSRP6, OsDHSRP8, OsDHSRP9, OsDHSRP15 found in chloroplast; OsDHSRP5 and OsDHSRP11are localized in vacuole.
TABLE – 1
Characterisation of OsDHSRP homologues genes in rice.
Gene Name
|
Transcript ID
|
START END
|
Chr
|
CDS bp
|
Introns/Exons
|
Domain
|
Localization
|
>OsDHSRP1
|
>LOC_Os02g05692
|
2791241..2795471
|
2
|
580
|
4,5
|
ZINCFINGERC3HC4
|
NUCLEUS
|
>OsDHSRP2
|
>LOC_Os08g14320
|
8582060..8584105
|
8
|
1050
|
3,4
|
ZINCFINGERC3HC4
|
NUCLEUS
|
>OsDHSRP3
|
>LOC_Os04g51400
|
30439466..30442859
|
4
|
1104
|
3,4
|
ZINCFINGERC3HC4
|
NUCLEUS
|
>OsDHSRP4
|
>LOC_Os04g10680
|
5802054..5811151
|
4
|
2130
|
4,5
|
ZINCFINGERC3HC4
|
NUCLEUS
|
>OsDHSRP5
|
>LOC_Os04g55510
|
33022715..33028387
|
4
|
2004
|
5,6
|
ZINCFINGERC3HC4
|
VACUOLE
|
>OsDHSRP6
|
>LOC_Os01g53500
|
30730644..30731238
|
1
|
594
|
0,1
|
ZINCFINGERH2
|
CHLOROPLAST
|
>OsDHSRP7
|
>LOC_Os01g06590
|
3099427..3105235
|
1
|
1506
|
5,6
|
ZINCFINGERC3HC4
|
NUCLEUS
|
>OsDHSRP8
|
>LOC_Os01g49770
|
28582842..28586527
|
1
|
606
|
4,5
|
ZINCFINGERC3HC4
|
CYTOPLASM
|
>OsDHSRP9
|
>LOC_Os01g47740
|
27303110..27312308
|
1
|
1575
|
4,5
|
ZINCFINGERC3HC4
|
CHLOROPLAST
|
>OsDHSRP10
|
>LOC_Os03g07790
|
3960942..3966307
|
3
|
753
|
3,4
|
ZINCFINGERC3HC4
|
NUCLEUS
|
>OsDHSRP11
|
>LOC_Os06g48040
|
29059686..29063780
|
6
|
1740
|
4,5
|
ZINCFINGERC3HC4
|
VACUOLE
|
>OsDHSRP12
|
>LOC_Os12g05270
|
2346442..2347276
|
12
|
834
|
0,1
|
ZINCFINGERC3HC4
|
NUCLEUS
|
>OsDHSRP13
|
>LOC_Os05g48970
|
28085119..28092428
|
5
|
1503
|
4,5
|
ZINCFINGERC3HC4
|
NUCLEUS
|
>OsDHSRP14
|
>LOC_Os05g47670
|
27317412..27320555
|
5
|
1095
|
4,5
|
ZINCFINGERC3HC4
|
NUCLEUS
|
>OsDHSRP15
|
>LOC_Os03g57410
|
32736205..32737399
|
3
|
657
|
0,1
|
ZINCFINGERH2
|
CHLOROPLAST
|
TABLE-2
Protein parameters of OsDHSRP Homologs
pI- isoelectric point
GRAVY-Grand average of hydropathicity index
Gene Name
|
Protein length (A.A)
|
Protein Molecular Weight(kDa)
|
GRAVY
|
PI
|
OsDHSRP1
|
580
|
64.278
|
-0.757
|
7.94
|
OsDHSRP2
|
349
|
38.586
|
-0.85
|
9.33
|
OsDHSRP3
|
367
|
41.0323
|
-0.886
|
8.82
|
OsDHSRP4
|
709
|
77.5573
|
-0.65
|
6.09
|
OsDHSRP5
|
667
|
72.641
|
-0.619
|
5.92
|
OsDHSRP6
|
197
|
20.5115
|
0.325
|
7.57
|
OsDHSRP7
|
501
|
54.6499
|
-0.579
|
8.59
|
OsDHSRP8
|
201
|
23.2885
|
-0.649
|
6.6
|
OsDHSRP9
|
524
|
57.7528
|
-0.591
|
6.1
|
OsDHSRP10
|
250
|
28.678
|
-0.665
|
5.91
|
OsDHSRP11
|
579
|
64.7518
|
-0.858
|
6.94
|
OsDHSRP12
|
277
|
29.126
|
-0.218
|
3.85
|
OsDHSRP13
|
500
|
54.536
|
-0.413
|
5.86
|
OsDHSRP14
|
364
|
39.6005
|
-0.606
|
9.2
|
OsDHSRP15
|
208
|
20.88264
|
-0.068
|
6.28
|
2. PREDICTION OF MOTIFS AND CONSERVED DOMAIN.
Using MEME tool, motifs which are found in OsDHSRP genes were predicted, a total of 10 motifs were identified. Out of 10 motifs found, motifs-1,2&3 are conserved across all OsDHSRP genes. The majority of motifs are limited to specific members of the OsDHSRP gene family. Motif 4 found only in OsDHSRP 2&3, motif 10 in OsDHSRP8&14, motif 5 and 7 in OsDHSRP 1&11 and motif 9 is exclusively located in OsDHSRP 9&13. Almost the motif pattern is comparable within the same clade. (Fig. 1c). The results of motif pattern and the conserved domain analysis of candidate genes showed that Ring H2 domain and Zinc finger C3HC4 domain are conserved among all genes and located towards N- terminal end of peptide. (Fig. 1e). The pattern of zinc finger C3HC4 domain was similar in all proteins. The zinc finger C3HC4 domain (Fig. 1d) is present in all OsDHSRP gene homologs. This suggests that all predicted genes were belongs to OsDHSRP gene family. The identical motif patterns among genes of same groups correlates with evolutionary relationships.
3. THE EVOLUTIONARY ASPECTS AND SYNTENY ANALYSIS.
The evolutionary relationship among OsDHSRP genes was analysed and phylogenetic tree was constructed (Fig. 1a) and all candidate genes were grouped into 4 clades. A total of 6 pairs of paralogs were observed among 15 genes. OsDHSRP6 and OsDHSRP11 is a paralog pair from group 1. OsDHSRP4 and OsDHSRP5 from group 2. OsDHSRP2 and OsDHSRP3; OsDHSRP1 and OsDHSRP12 from group 3. OsDHSRP15 and OsDHSRP19 is a paralog pair from group 4.
The phylogenetic analysis of OsDHSRP proteins among closely related species such as Sorghum bicolor, Zea mays, Arabidopsis thaliana and Triticum aestivum and Oryza sativa showed 4 ortholog pairs with Sorghum they are OsDHSRP2 and SbDHSRP10; OsDHSRP4 and SbDHSRP13; OsDHSRP14 and SbDHSRP4; OsDHSRP13 and SbDHSRP7; There is one ortholog pair each with Zm and At includes OsDHSRP15 and ZmDHSRP7; OsDHSRP10 and AtDHSRP14 respectively. Based on above results it is clear that Oryza sativa OsDHSRP proteins are closely related with Sorghum.
Collinearity analysis of genes among Os, Zm, At. Genomes was done using TB tools and there is no syntenic regions found between Os and At for OSDHSRP homologs. The collinearity between Zm and Os shown that Os chromosome 1 shows 2, 3, 4 homologs each with Zm chromosome 3, chromosome 6, chromosome 8 respectively; Os chromosome 2 shows 2 homologs with Zm chromosome 5; Os chromosome3 shows 2,1,1,1 homolog each with Zm chromosome1, chromosome7 and chromosome9 respectively; Os chromosome shows 2,2 homologue each with Zm chromosome 10 and chromosome 2; Os chromosome 5 shows 1,2,3 Homologs with Zm chromosome 3, chromosome 6 and chromosome 8 respectively; Os chromosome 6 shows 2 homologues with Zm chromosome 5; Os chromosome 8 shows. 2. 1. Homologs with Zm chromosome 10 and 2; Os chromosome 12 shows one homologue with Zm chromosome 4. (Fig. 2a).
Synteny analysis results of Miscanthus sinesis versus Oryza sativa displayed that Os chromosome1 shows homology of 2, 2 with Ms chromosome 5 and chromosome 6; Os chromosome 2 shows one homology each with Ms Chromosome 7 and Chromosome 8; Os chromosome3 shows 2,2 homologies with Ms chromosome 1 and Ms Chromosome 2; Os chromosome 4 shows 1,2 homology with Ms chromosome 11 and chromosome 12 respectively. Chromosome 5 shows one homology each with Ms Chromosome 16 and chromosome 17; Os chromosome 6 shows 1 homology each with Ms chromosome 18 and chromosome 19 respectively; Os chromosome8 showed one homology with Ms chromosome 13.
Synteny analysis results of Oryza sativa versus Panicum virgatum revealed that Os chromosome1 Shows 4 homologs with Pv chromosome 5; Os chromosome 2 shows 2 homologies with Pv chromosome1; Os chromosome 3 shows 4 homologs with Pv chromosome 9; Os chromosome 4 shows 4 homologs with PV chromosome 7; Os chromosome 5 shows 2 homologues with Pv chromosome 3; Os chromosome 6 shows 2 homologs with Pv chromosome 4; Os chromosome a shows 2 homologues with Pv chromosome 6.
The outcome of collinearity mapping of genes among Oryza sativa and Setaria italica are most of the homology was found in scaffold region of Setaria Italica. (Fig. 3).
4. MULTIPLE SEQUENCE ALIGNMENT AMONG SPECIES AND Ka/Ks ANNOTATION.
Multiple sequence alignment with protein sequences of OsDHSRP1, OsDHSRP5, Zm00001d014609, sobic.004G042100.1. P, Traes_7AL_ADB7BCC89.1, AT5G10650.1 was analysed using Mega 11.0 by ClustalW and visualised through Snap gene viewer. The alignment showed conserved blocks among all these genes, suggests that these OsDHSRP genes are conserved across the species. (Fig. 2b).
According to Ka/Ks annotated evolutionary tree, 6 paralog pairs were observed. The paralog pair OsDHSRP14, OsDHSRP18 has the lowest Ka/Ks value. All Paralogs displayed Ka/Ks ratio less than one, which explains specific purifying selection. MYA was calculated to predict the duplication distance. The estimation of divergence time for 6 pairs showed that gene duplication occurred between 11.2–71.8 MYA. The Ka/KS values, Mya values, paralog pair are tabulated. (Table 3).
TABLE − 3
Ks-synonymous substitution; Ka-non-synonymous substitution; T(MYA)-Evolution time in Million years ago. Time calculated based on T = Ks/2x where x is 6.56x10− 9 formula.
GENE PARALOGS
|
KA
|
KS
|
KA/KS
|
MYA
|
OsDHSRP7,OsDHSRP5
|
0.39945
|
0.51475
|
0.7760078
|
30445884.15
|
OsDHSRP1,OsDHSRP11
|
0.093260335
|
0.1746
|
0.5341371
|
7108257.241
|
OsDHSRP10,OsDHSRP12
|
0.1471
|
0.4224
|
0.3482481
|
11211890.24
|
OsDHSRP3,OsDHSRP2
|
0.363
|
0.4433
|
0.8188586
|
27667682.93
|
OsDHSRP9,OsDHSRP4
|
0.3731
|
0.4914
|
0.7592593
|
28437500
|
OsDHSRP14,OsDHSRP8
|
0.082964555
|
0.2423
|
0.3424043
|
6323517.912
|
5. PROMOTER ANALYSIS AND CHROMOSOMAL LOCATIONS.
The predicted Cis regulatory elements found in promoter regions of OsDHSRP homologs are as follows- Light responsive element, MYB drought responsive, meristem expression, circadian cycle, cell cycle regulation, MYB binding site, Low temperature responses, Endosperm specific negative regulation, seed specific regulation. Plant hormone related such as auxin, Methyl Jasmonate, gibberellin, salicylic acid and abscisic acid responsive and Defence and stress responsive elements against biotic stress. Some abiotic stress elements include drought and salt, low temperature responsive and metal responsive. Among stress related elements most of them include abscisic acid responsive element which determines their function in abiotic stress response. (Fig. 4a). The presence of light responsive elements specifies they are regulated by light. Metal responsive element suggests the genes are involved in heavy metal stress. Based on this data OsDHSRP genes may play key roles under drought, salinity and heat or cold stress. Some unique elements show diversity among gene homologs in their roles. Physical mapping of OsDHSRP genes on Oryza genome revealed that these genes are scattered across all chromosomes and most of the genes are localized on chromosome 1 of Oryza (Fig. 4b).
6. PROTEIN 2D & 3D STRUCTURE MODELING AND PPI ANALYSIS
The 2-D structure prediction showed different forms of proteins including beta turns, extended strands, alpha helices, and random coils. The proportion distribution of these were tabulated. (Table 4), (Fig. 5b).
TABLE − 4
Secondary structure of OsDHSRP protein homologs.
Gene/ protein Name
|
Alpha helix %
|
Extended Stand %
|
Beta turn %
|
Random coil%
|
>OsDHSRP1
|
25.69
|
7.59
|
2.76
|
63.97
|
>OsDHSRP2
|
24.64
|
12.89
|
6.3
|
56.16
|
>OsDHSRP3
|
33.51
|
11.17
|
5.45
|
49.86
|
>OsDHSRP4
|
19.46
|
8.32
|
2.96
|
69.25
|
>OsDHSRP5
|
16.49
|
9
|
3.3
|
71.21
|
>OsDHSRP6
|
36.04
|
12.69
|
6.09
|
45.18
|
>OsDHSRP7
|
16.17
|
8.18
|
2.4
|
73.25
|
>OsDHSRP8
|
42.79
|
2.99
|
1.49
|
52.74
|
>OsDHSRP9
|
12.79
|
11.07
|
2.1
|
74.05
|
>OsDHSRP10
|
23.56
|
21.15
|
8.17
|
47.12
|
>OsDHSRP11
|
22.45
|
5.7
|
2.94
|
68.91
|
>OsDHSRP12
|
12.88
|
15.52
|
11.19
|
44.4
|
>OsDHSRP13
|
15.2
|
9.8
|
3.4
|
71.6
|
>OsDHSRP14
|
37.64
|
6.87
|
6.32
|
49.18
|
>OsDHSRP15
|
14.4
|
17.6
|
4.4
|
63.6
|
The 3-dimensional structure of OsDHSRP proteins were predicted using Swiss model server. The models were selected based on maximum percentage of identity with standard PDB structures. The selected structure PDB ID, protein ID, percentage identity was tabulated. (Table 2). 3D structures are given in (Fig. 5c).
The template 6W9a.1.B- E3 ubiquitin ligase, Ubiquitin conjugating enzyme E2 showed highest structural similarity with most of the homologous including OsDHSRP2, OsDHSRP5, OsDHSRP9, OsDHSRP1, OsDHSRP12. Template ID 6W9a.2.B- E3 ubiquitin ligase RLIM shown similarity with OsDHSRP3, OsDHSRP9, OsDHSRP15. Furthermore, template 6W7z.1.B - E3 ubiquitin ligase, E2 ubiquitin conjugating enzyme D2, Showed similarity with OsDHSRP4, OsDHSRP6, OsDHSRP7, OsDHSRP10, OsDHSRP14. OsDHSRP13.
Using string database, the proteins which interact with OsDHSRP1 protein was analysed. The interpretation of result suggests that OsDHSRP1 protein interacting with ARF-16, MP-ARF11 auxin responsive element that precisely binds to DNA sequence 5'-TGTCTC-3' found in the auxin-responsive promoter elements (Aux REs). Additionally, other interacting proteins include BHLH 156- transcription factor is involved in negative regulation of iron acquisition genes for mugineic acid (MA) family Phyto siderophores biosynthesis, S- adenosylmethionine cycle and iron transport (Fig. 5a), Os03g0383800 SAP domain containing protein, Os06g0666400, Os02g0173200, Os04G0663100, Os01g0687600, - which are uncharacterized proteins. Another protein Os07g0626200 is AcinusL protein-like; The result of string interaction analysis among the 15 candidate genes shown they are non- interactive with each other.
7. miRNA AND TRANCRIPTION FACTORS PREDICTION-
The results of transcriptional factor identification revealed that candidate genes are regulated by 35 distinct types of transcriptional factors. These includes MYB, WOX, FAR1, EIL, TALE, Trihelix, CAMTA, C2H2, LBD, ERF, MYB-related, CPP, HD-zip, HSF, BZIP, GATA, SBP, MIKC-MADS, AP2, NAC, WRKY, C3H, YABBY, RAV, bHLH, G2-like are mostly abiotic stress related transcriptional factors and are involved in plant growth and development. (Fig. 6a). (Kaplan et al,2012; Xiao et al, 2021; Han et al,2020). Dof, C2H2, YABBY, GATA, C3H are zinc domain containing proteins whereas other transcriptional factors are related in plant growth and development and some are hormone related responsive Tfs.
The miRNA was identified, which can be used to target these homologous genes and regulate their gene expression patterns. OsDHSRP1 shows 6 miRNA targets which includes Osa-miRN2282, Osa-miR444c, Osa-miRN2222, Osa-miR528, Osa-miR440, Osa-miRN2376. (Fig. 7). Additionally, we found that Osa-miR5508 is a potential target for both OsDHSRP13 and OsDHSRP9 genes.
8. GENE ONTOLOGY AND CO EXPRESSION ANALYSIS-
The GO terms are analysed for all the candidate genes. The results showed that in molecular function category, most of the genes exhibited similar functions that is ubiquitin protein ligase activity-GO:0061630, metal ion binding-GO:0046872, and ligase activity-GO:0016874 was displayed by OsDHSRP1, OsDHSRP2, OsDHSRP3, OsDHSRP4, OsDHSRP5, OsDHSRP9, OsDHSRP8, OsDHSRP10, OsDHSRP11, OsDHSRP13, OsDHSRP14. The gene OsDHSRP10 showed acyl transferase activity-GO:0016746 alongside with above functions and OsDHSRP15 showed only acyl transferase activity. OsDHSRP7 has only the ubiquitin protein ligase activity and metal ion binding without ligase activity. OsDHSRP6 has transferase activity-GO:0016740 along with metal ion binding. OsDHSRP14 have an additional helicase activity of GO:0004386 and OsDHSRP12 showed zinc ion binding ubiquitin-GO:0008270 and protein ligase activity.
Co-expression analysis for OsDHSRP1 gene was done and the results were tabulated and a network was created using Cytoscape tool. (Fig. 6b). Several genes were co expressed along with OsDHSRP1 gene includes Os01g0936200 is probable feruloyl esterase A an enzyme showing highest rank with OsDHSRP1 expression. Several other E3 Ubiquitin protein ligases such RHB1A, AIRP2, atg7, AIRP2-LIKE, CHIP, At3g022g0 were also expressed. Furthermore, Ubiquitin receptor RAD23d is also expressed along with OsDHSRP1 gene.
9. INSILICO EXPRESSION PATTERN ANALYSIS
According to In silico expression data (Xia et al,2017) the expression of OsDHSRP1 gene is high in root and embryo compared to other tissues/organs. (Fig. 8e) and low in leaf blade, endosperm, and reproductive leaf sheath. Among organs comparatively, expression of OsDHSRP1 is more in leaf than in stem. (Fig. 8f).
Under sodium dihydro phosphate treatment at different time periods (Fig. 8b) (Secco et al,2013), OsDHSRP1, OsDHSRP2, OsDHSRP4 exhibited strong expression patterns.
When rice plants exposed to heavy metal stress (cadmium sulphate) (Oono et al,2014) (Fig. 8a)- OsDHSRP1 showed moderate expression. Moreover, OsDHSRP7 and OsDHSRP9 displayed highest expression under cold stress or low temperature stress. (Zhang et al, 2012) (Fig. 8c). Additionally, OsDHSRP1showed an moderate expression, whereas OsDHSRP10 and OsDHSRP8 had highest expression.
10. q-RTPCR ANALYSIS
According to qRTPCR analysis, OsDHSRP1 exhibited strong expression pattern in leaf tissue under cold stress, in contrast to drought and salt stress. Compared to leaf tissue, expression of OsDHSRP1 gene was high in stem tissue under salt stress. The comparative expression of OsDHSRP1 gene shows low in untreated plant tissues both stem and leaf in relative abiotic stress conditions. When exposed to stress, the OsDHSRP1 gene's relative expression up-regulated by 18.74, 12.81, and 11.27 -fold in cold leaves, drought leaves, and salt stem tissues, respectively. (Fig. 8d).
OsDHSRP2 Gene shows high expression levels (17.03- fold change) under salt in stem tissue. The relative expression of this gene also suppressed in untreated plants than plants subjected to stress. OsDHSRP8 gene showed strong expression (13.68 folds) under salt conditions in stem tissue and moderate expression in drought stressed leaf tissue and cold conditions. Overall, the expression of OsDHSRP homologs displayed high expression patterns under various abiotic stresses compared to natural environmental conditions.