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<p>Delineating relative contribution of homologs of regulatory genes is a major challenge in polyploid Brassicas. Homeostasis of multiple copies of <i>MIR160</i> and target <i>ARF10/16/17</i> regulatory module is plausibly achieved by intricate feedback loops underlying biological processes. Despite centrality of promoters in gene regulation, amplification and homolog diversification, knowledge base in polyploids remains limited. Herein, we characterise 127 promoters of Brassicaceae <i>MIR160</i> paralogs and homeologs using phylogenetic approaches, comparative expression analyses and TFBS profile to decipher evolutionary trends. A total of 17 <i>MIR160</i> homologs were identified from <i>Brassica juncea</i> representing 5 <i>MIR160a</i> paralogs (2 from AA and 3 BB progenitor genome), 8 <i>MIR160b</i> paralogs (4 each from AA and BB genomes) and 4 <i>MIR160c</i> paralogs (3 from AA and 1 from BB genome). Retention of all copies of LF, MF1 and MF2 sub-genome–specific homeologs, corresponding to <i>MIR160a</i>, <i>b</i> and<i> c</i>, suggested cruciality of miR160 functions. Comprehensive expression analyses of <i>MIR160</i> paralog, and homeolog-specific transcript levels in <i>B. juncea</i> cv. Varuna, involving 48 datasets from 16 spatio-temporal windows, revealed promoter diversification. <i>GUS</i> assays validated diversification of promoter homeologs of <i>MIR160a</i>. Interestingly, LF sub-genome–specific expression studies, comparative analyses of TFBS profiles and phylogenetics validated ancestral nature and distinctness of <i>MIR160a.</i> Presence of binding sites of ARF10, 16 and 17 on <i>MIR160</i> promoters, confirmed using Y1H, suggested a regulatory feedback mechanism. This led us to propose two hypothetical models depicting (i) complex interaction patterns among <i>MIR160</i> and 19 members of ARF family and (ii) feedback loop between <i>MIR160</i> and <i>ARF10/16/17</i> for homeostasis of miR160 and ARF protein levels. This is a first ever study on assessment of steady-state levels of primary and mature miRNA transcripts for demonstrating regulatory diversification in <i>B. juncea</i>. Experimental validation of regulatory feedback mechanism and impact on functional diversification needs to be undertaken gain molecular insights for crop improvement.</p>

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Differential Expression Pattern of Brassica juncea MIR160 Paralogs and Homeologs and In Silico Analysis of Brassicaceae Promoters Uncover Evolutionary Distinctness of MIR160a

  • 期刊论文

  • 发表日期：

    2025年1月27日

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  • Swati Singh,
  • Swetannita Chattopadhyay,
  • Simran Kaur,
  • Sandip Das,
  • Anandita Singh

摘要

Delineating relative contribution of homologs of regulatory genes is a major challenge in polyploid Brassicas. Homeostasis of multiple copies of MIR160 and target ARF10/16/17 regulatory module is plausibly achieved by intricate feedback loops underlying biological processes. Despite centrality of promoters in gene regulation, amplification and homolog diversification, knowledge base in polyploids remains limited. Herein, we characterise 127 promoters of Brassicaceae MIR160 paralogs and homeologs using phylogenetic approaches, comparative expression analyses and TFBS profile to decipher evolutionary trends. A total of 17 MIR160 homologs were identified from Brassica juncea representing 5 MIR160a paralogs (2 from AA and 3 BB progenitor genome), 8 MIR160b paralogs (4 each from AA and BB genomes) and 4 MIR160c paralogs (3 from AA and 1 from BB genome). Retention of all copies of LF, MF1 and MF2 sub-genome–specific homeologs, corresponding to MIR160a, b and c, suggested cruciality of miR160 functions. Comprehensive expression analyses of MIR160 paralog, and homeolog-specific transcript levels in B. juncea cv. Varuna, involving 48 datasets from 16 spatio-temporal windows, revealed promoter diversification. GUS assays validated diversification of promoter homeologs of MIR160a. Interestingly, LF sub-genome–specific expression studies, comparative analyses of TFBS profiles and phylogenetics validated ancestral nature and distinctness of MIR160a. Presence of binding sites of ARF10, 16 and 17 on MIR160 promoters, confirmed using Y1H, suggested a regulatory feedback mechanism. This led us to propose two hypothetical models depicting (i) complex interaction patterns among MIR160 and 19 members of ARF family and (ii) feedback loop between MIR160 and ARF10/16/17 for homeostasis of miR160 and ARF protein levels. This is a first ever study on assessment of steady-state levels of primary and mature miRNA transcripts for demonstrating regulatory diversification in B. juncea. Experimental validation of regulatory feedback mechanism and impact on functional diversification needs to be undertaken gain molecular insights for crop improvement.