MYB transcription factors, their regulation and interactions with non-coding RNAs during drought stress in Brassica juncea

Abstract

Background: Brassica juncea (L.) Czern is an important oilseed crop affected by various abiotic stresses like drought, heat, and salt. These stresses have detrimental effects on the crop's overall growth, development and yield. Various Transcription factors (TFs) are involved in regulation of plant stress response by modulating expression of stress-responsive genes. The myeloblastosis (MYB) TFs is one of the largest families of TFs associated with various developmental and biological processes such as plant growth, secondary metabolism, stress response etc. However, MYB TFs and their regulation by non-coding RNAs (ncRNAs) in response to stress have not been studied in B. juncea. Thus, we performed a detailed study on the MYB TF family and their interactions with miRNAs and Long non coding RNAs.

Results: Computational investigation of genome and proteome data presented a comprehensive picture of the MYB genes and their protein architecture, including intron-exon organisation, conserved motif analysis, R2R3 MYB DNA-binding domains analysis, sub-cellular localization, protein-protein interaction and chromosomal locations. Phylogenetically, BjuMYBs were further classified into different subclades on the basis of topology and classification in Arabidopsis. A total of 751 MYBs were identified in B. juncea corresponding to 297 1R-BjuMYBs, 440 R2R3-BjuMYBs, 12 3R-BjuMYBs, and 2 4R-BjuMYBs types. We validated the transcriptional profiles of nine selected BjuMYBs under drought stress through RT-qPCR. Promoter analysis indicated the presence of drought-responsive cis-regulatory components. Additionally, the miRNA-MYB TF interactions was also studied, and most of the microRNAs (miRNAs) that target BjuMYBs were involved in abiotic stress response and developmental processes. Regulatory network analysis and expression patterns of lncRNA-miRNA-MYB indicated that selected long non-coding RNAs (lncRNAs) acted as strong endogenous target mimics (eTMs) of the miRNAs regulated expression of BjuMYBs under drought stress.

Conclusions: The present study has established preliminary groundwork of MYB TFs and their interaction with ncRNAs in B. juncea and it will help in developing drought- tolerant Brassica crops.

Keywords: Brassica juncea; Cis-regulatory elements; MYB domain; Abiotic stress; Expression profile; lncRNAs; miRNA targets.

Fig. 1

The chromosomal positions of BjuMYBs in B. juncea sub-genomes A and B were mapped by TBtools software. The chromosome numbers are shown on the left side of each chromosome (a vertical bar in black) in blue, and the gene name is shown on the right side of each chromosome in red. The scale bar is in megabytes (Mb) on the left side

Fig. 2

Phylogenetic analysis of MYB proteins from B. juncea (Blue square) and A. thaliana (Red triangle). A neighbour-joining phylogenetic tree was constructed via MEGA7 utilising 1000 bootstrap replication. (a) R2R3-MYB proteins divided into 36 (B1-B36) subgroups, which are represented by different colours of clades; (b) 1R-MYB proteins divided into 36 (J1-J36) subgroups, which are represented by different colours of clades; (c) 3R-MYB and 4R-MYB proteins divided into 5 (A1-A5) subgroups, which are represented by different colours of clades

Fig. 3

Sequence logos in MYB repeat R2 (a) and R3 (b) were generated from multiple alignments of all R2R3-BjuMYB domains. The overall height of each stack represents the sequence conservation at that position, and the bit score represents the relative frequency of the relevant amino acid residues. The conserved tryptophan residues (denoted as Trp, W) in the MYB domain are marked by red arrows, whereas the substituted amino acid residues in the R3 repeat are marked by blue arrows

Fig. 4

Protein-protein network analysis of BjuMYB proteins in B. juncea with reference species Arabidopsis

Fig. 5

Differential expression analysis of BjuMYBs in response to drought stress. (a) In-silico analysis revealed 19 differentially expressed genes in drought stress and represented as heat map using TBtools software. The scale bar on the right displays the transcript abundance from low level (Green) to high level (Red), (b) The relative expression level of BjuMYBs in B. juncea leaf tissues at different time points during drought stress was evaluated using RT-qPCR. The relative expression was determined by referencing transcription levels in control leaf tissues. The error bars represent the mean and standard deviation of triplicate samples, and their statistical significance relative to the control plant are noted at (*** means p ≤ 0.001, ** p ≤ 0.01 and * means p ≤ 0.05)

Fig. 6

Evaluation of cis-regulatory element in the promoters of 19 differentially expressed BjuMYB in response to abiotic, biotic and hormone responses. The number of cis-acting elements in the promoter region is indicated by a number inside a square box. The scale bar on the right displays the abundance of cis-regulatory element from low level (blue) to high level (Red)

Fig. 7

miRNA regulatory network of BjuMYBs represented using Cytoscape software. Conserved Bju- miRNAs (purple box), novel Bju- miRNAs (orange box) and BjuMYB targets (green box) are represented in the network

Fig. 8

Regulatory network analysis of (a) lncRNA (Blue), miRNAs (Red), and MYB TFs (Green) in B. juncea in response to drought stress was constructed by Cytoscape Software. lncRNAs in pink act as eTMs of Bju-miRNAs and co-expressing partners with MYB TFs, while rest of the lncRNAs (in blue) represent the targets of miRNAs, (b) The relative expression level of lncRNAs-miRNAs-BjuMYBs in B. juncea leaf tissues at different time points during drought stress was evaluated using RT-qPCR. The relative expression was determined by referencing transcription levels in control leaf tissues. The error bars represent the mean and standard deviation of triplicate samples, and their statistical significance relative to the control plant are noted at (*** means p ≤ 0.001, and * means p ≤ 0.05)