Integrative RNA- and miRNA-Profile Analysis Reveals a Likely Role of BR and Auxin Signaling in Branch Angle Regulation of B. napus

Abstract

Oilseed rape (Brassica napus L.) is the second largest oilseed crop worldwide and one of the most important oil crops in China. As a component of plant architecture, branch angle plays an important role in yield performance, especially under high-density planting conditions. However, the mechanisms underlying the regulation of branch angle are still largely not understood. Two oilseed rape lines with significantly different branch angles were used to conduct RNA- and miRNA-profiling at two developmental stages, identifying differential expression of a large number of genes involved in auxin- and brassinosteroid (BR)-related pathways. Many auxin response genes, including AUX1, IAA, GH3, and ARF, were enriched in the compact line. However, a number of genes involved in BR signaling transduction and biosynthesis were down-regulated. Differentially expressed miRNAs included those involved in auxin signaling transduction. Expression patterns of most target genes were fine-tuned by related miRNAs, such as miR156, miR172, and miR319. Some miRNAs were found to be differentially expressed at both developmental stages, including three miR827 members. Our results provide insight that auxin- and BR-signaling may play a pivotal role in branch angle regulation.

Keywords: Brassica napus; auxin; branch angle; brassinosteroid; deep sequencing; miRNA.

Figure 1

Phenotypes of 6098B and Purler showing variation in branch angle. (A) 6098B and Purler at early flowering. (B) Upper panel: 6098B showing lax branch angle (52°), lower panel: erect branch angle of Purler (22°).

Figure 2

Number and functional classification of differentially expressed genes (DEGs). (A) The number of DEGs detected between 6098B and Purler at bolting (blue), early flowering (light pink), and both (dark pink) developmental stages. (B) Functional classification of DEGs identified at both development stages by Gene Ontology (GO) categorization. Bold colours indicate the representation in the whole genome, the light colours indicate representation in the DEGs.

Figure 3

Pathway analysis of DEGs based on the KEGG database. (A) Analysis of DEGs at the bolting and (B) early flowering stages. The X-axis indicates the pathways, the Y-axis indicates the numbers of annotated genes.

Figure 4

Schematic diagrams of (A) the BR biosynthesis pathway, (B) BR signal transduction pathway, and (C) auxin signaling transduction pathway. Genes marked in red indicate those differentially expressed between the two lines.

Figure 5

Heat maps of DEGs related to the auxin and BR signaling pathways showing (A–D) the cluster of DEGs involved in auxin signaling transduction and (E) the cluster of DEGs involved in BR biosynthesis and transduction. Tissue samples from 6098B (C1, C3) and Purler (C2, C4) at the bolting and early flowering stages, respectively. Color key represents log2 transformed FPKM (fragments per kilobase of exon per million fragments mapped) values, from low (blue) to high (red) expression.

Figure 6

Nucleotide preference of small RNAs in B. napus showing (A) nucleotide preference at each position; (B) number of 19- to 24-nucleotide (nt) small RNAs in all the identified small RNAs and (C) the first nucleotide bias of 19- to 24-nucleotide (nt) small RNA.

Figure 7

Number of differentially expressed miRNAs and the expression profile of miRNA-targets. (A) The number of differentially expressed miRNAs detected between 6098B and Purler at bolting (blue), early flowering (light pink), and both (dark pink) developmental stages. (B) The heat map of differentially expressed miRNAs and targets showing greater than 2-fold change between 6098B (S1, S3) and Purler (S2, S4) at bolting or early flowering. SPL (SQUAMOSA promoter binding protein-like), ARF (auxin response factor), HD-ZIP III (Homeodomain leucine zipper III), AP2 (APETALA2), SULTR (sulfate transporter), TCP (TEOSINTE-BRANCHED/CYCLOIDEA/PCF). Color key represents log2 transformed FPKM values, from low (blue) to high (red) expression.

Figure 8

Validation of DEGs involved in (A) auxin signaling pathway and (B) BR signaling transduction and biosynthesis, by RT-PCR in 6098B (B) and Purler (P). Actin was amplified with 27 cycles, other genes were amplified with 34 cycles.

Figure 9

Validation of selected differentially expressed miRNAs by qRT-PCR in the leaf (L), flower bud (F), branching site at bolting (Br1), and branching site at early flowering (Br2) in 6098B (solid line) and Purler (dotted line), performed on the same tissue samples used for verifying gene expression. Data represent means (three biological replicates) ± standard deviation.