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Sequence and functional analysis of cis-elements associated with MIR159 loci from Brassica juncea reveal functional diversification and complex transcriptional regulation

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Abstract

Identification and functional analysis of promoters is critical towards gaining insights into transcriptional regulation, and helps in identifying interacting trans-factors. Analysis of MIRNA promoter can differentiate the function of paralogs and homeologs where identifying the role of members of MIRNA family is difficult based on small RNA blots due to near-identical mature sequences. The role of miRNA as regulators of development and adaptation in polyploid species-Brassica, and the impact of polyploidy on divergence among paralogs and homeologs especially with reference to transcriptional regulation remains unexplored. We identified homologs of MIR159 from 137 species across plants based on their homology to pre-MIR159A, MIR159B, and MIR159C from Arabidopsis and retrieved their promoter sequences. Promoter sequence analysis revealed high level of divergence; albeit clades of closely related taxonomic units were obtained. We detected biased, but no lineage-specific distribution of transcription factor binding motifs (TFBS) in families. Phylogenetic reconstruction in Brassicaceae revealed genome- and sub-genome specific clades. We isolated three homeologs of MIR159A, and one of MIR159C from the A-genome of B. juncea. Functional characterization involved transgenic lines in A. thaliana Col-0 with promoter::reporter transcriptional fusions (p-MIR159::GUS::ter) and monitoring of reporter activity during development, abiotic stresses, and after administering hormones. Comparison of reporter activity of full-length promoters across homeologs and paralogs reveal extensive functional diversification. Comparative analysis of reporter activity of nested deletions, and with distribution of TFBS did not yield a clear correlation. In summary, analysis of GUS during development, hormone treatment and abiotic stresses reveal complex transcriptional regulation of MIR159 expression.

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Acknowledgements

The research was supported by a Department of Biotechnology, Govt. Of India (DBT) grant (Grant No. BT/PR14532/AGR/36/673/2010) to SD, and a DBT project Junior Research Fellowship (JRF) to CC. GJ is supported by JRF/SRF from University Grants Commission (UGC). SD would also like to acknowledge financial assistance received from Delhi University under R&D grant support.

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  1. Department of Botany, University of Delhi, New Delhi, Delhi, 110 007, India

    Chetan Chauhan, Gauri Joshi & Sandip Das

  2. Centre for Biotechnology, Maharshi Dayanand University, Rohtak, India

    Chetan Chauhan & Darshna Chaudhary

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  1. Chetan Chauhan
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  2. Gauri Joshi
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Chetan Chauhan, Darshana Chaudhary and Sandip Das were involved in conceptualizing and planning of the study. Chetan Chauhan and Gauri Joshi performed the experiments and, collected data. CC and SD analysed and wrote the manuscript. All authors have read and approve of the manuscripts.

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Accession Numbers: MN165009, MN165018.

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10725_2020_578_MOESM1_ESM.ai

Supplementary Figure 1 Average sequence identity (as percentage) between promoters of MIR159 homologs identified across plants kingdom presents. Supplementary file 1 (AI 2606 kb)

10725_2020_578_MOESM2_ESM.pdf

Supplementary Figure 2 Phylogenetic reconstruction based on ca. 550 bp of putative promoter region associated with MIR159 homologs across plants. Supplementary file 2 (PDF 2324 kb)

10725_2020_578_MOESM3_ESM.tif

Supplementary Figure 3 Phylogenetic reconstruction based on ca. 550 bp of putative promoter region associated with MIR159 homologs across plants. The homologs and homeologs of MIR159 in members of Brassicaceae are boxed. Supplementary file 3 (TIF 210 kb)

10725_2020_578_MOESM4_ESM.tif

Supplementary Figure 4 Cis-regulatory motif analysis using MEME server (http://meme-suite.org/) and STAMP (http://www.benoslab.pitt.edu/stamp/). STAMP is an alignment base tool to identify statistically significant motif. Alignment file of retrieved sequences were processed on MEME server to identify conserve motifs. A list of motifs was further analyzed on STAMP server to identify statistically significant motif based on experimental evidence and functional significance. A: X-axis represents TFBS motifs and Y-axis represent the number of sequences containing this motif. B: X-axis represents functional category of motif and Y-axis represent the frequency of occurrence of the TFBS of a functional category across MIR159 promoters. Supplementary file 4 (TIF 8104 kb)

10725_2020_578_MOESM5_ESM.tif

Supplementary Figure 5 Analysis of cis-regulatory motif module distributed along the phylogenetic tree reconstructed using the promoter sequences of MIR159 gene from different species. Cis-motifs involved in different processes like cell differentiation (CD)- ACTCTCTATC, cell proliferation (CP)- ACAATGTCAG, Drought responsive (DR)- ACGCKTAGGG, Flower development (FD)- CACTTSTYCC; TTGACKTGTC, Flower timing (FT)- YTTGTYYTTC; AAACCACTAT, Meristematic responsive (MR)- CCCATGTCTT; CTTCATCTHT, Seed development (SD)- GAYGCATGTG; GCATGCATGC, Gibberellin responsive (GR)- CCGGCCSTTR were identified (black boxes represents presence of individual motifs) acoss the various members of the clade and marked. Supplementary file 5 (TIF 13414 kb)

10725_2020_578_MOESM6_ESM.pdf

Supplementary Figure 6 Presence or absence of cis-regulatory motif in the promoter sequences of MIR159 among various plant species. Cis-motifs involved in different processes like cell differentiation (CD)- ACTCTCTATC, cell proliferation (CP)- ACAATGTCAG, Drought responsive (DR)- ACGCKTAGGG, Flower development (FD)- CACTTSTYCC; TTGACKTGTC, Flower timing (FT)- YTTGTYYTTC; AAACCACTAT, Meristematic responsive (MR)- CCCATGTCTT; CTTCATCTHT, Seed development (SD)- GAYGCATGTG; GCATGCATGC, Gibberellin responsive (GR)- CCGGCCSTTR were identified (black boxes indicate presence of individual motifs). Supplementary file 6 (PDF 192 kb)

10725_2020_578_MOESM7_ESM.tif

Supplementary Figure 7 Comparative study of intergenic space and upstream region of MIR159A-02 (upper left), MIR159A07-1 (upper right), MIR159A07-2 (lower left) and MIR159C-A05 (lower right) between Arabidopsis thaliana and Brassica rapa var chiifu. Sliding window analysis using DnaSP reveals conserved (valleys) and divergent (peaks) region among promoters of Arabidopsis thaliana and Brassica rapa var chiifu. The line below the sliding window analysis shows position of cis-regulatory motifs on the promoter region (see supplementary file 7 for details about individual motifs; and supplementary figure 7) on the promoter of Arabidopsis thaliana and Brassica rapa var chiifu). Arrow marked as F1, F2, F3 and R denote position of primers for cloning of full-length and nested-deletion series. Supplementary file 7 (TIF 7205 kb)

10725_2020_578_MOESM8_ESM.pptx

Supplementary Figure 8 Diagrammatic representation of the promoter::reporter constructs and Multiple sequence alignment between promoter homologs of Arabidopsis thaliana col-0 and Brassica juncea var. Varuna. A: MIR159A; B: MIR159B; C: MIR159C. Supplementary file 8 (PPTX 3139 kb)

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Chauhan, C., Joshi, G., Chaudhary, D. et al. Sequence and functional analysis of cis-elements associated with MIR159 loci from Brassica juncea reveal functional diversification and complex transcriptional regulation. Plant Growth Regul 90, 279–306 (2020). https://doi.org/10.1007/s10725-020-00578-4

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