The initial deficiency of protein processing and flavonoids biosynthesis were the main mechanisms for the male sterility induced by SX-1 in Brassica napus

Abstract

Background: Rapeseed (Brassica napus) is an important oil seed crop in the Brassicaceae family. Chemical induced male sterility (CIMS) is one of the widely used method to produce the hybrids in B. napus. Identification of the key genes and pathways that involved in CIMS were important to understand the underlying molecular mechanism. In the present report, a multi-omics integrative analysis, including of the proteomic, transcriptomic and miRNAs, combined with morphological and physiological analysis were conducted.

Results: Earlier degeneration of the tapetosomes and elaioplasts, aberrantly stacking in tapetal cells and incompletely deposition in tryphine of pollen wall were observed in chemical hybridization agent (CHA) of SX-1 treated B. napus through SEM and TEM analysis. It was revealed that the deficiencies in protein processing in endoplasmic reticulum (ER) and flavonoids biosynthesis were occurred at early stage in the SX-1 treated materials. Subsequently, plant hormone signal transduction, biosynthesis of amino acids, fatty acids and steroid in anther at later stages were identified down-regulated after SX-1 treatment. 144 transcript factors (TFs) were also indentified to down-regulated at early stage, which suggested the early regulation in anther and pollen wall development were disordered in CHA treated B. napus. In addition, 7 important miRNAs were identified and 2 of the predicted target genes of miRNAs were Rf-like genes.

Conclusions: Taken together, an interaction network of candidate genes and the putative metabolism pathways were constructed based on the multi-omics integrative analysis, it provided a new insight into the male sterility induced by CHA of SX-1 in B. napus.

Keywords: Brassica napus; Chemical hybridization agent; Flavonoids biosynthesis; Male sterility; Protein processing; Proteome; Transcript factor; Transcriptome; Tryphine; miRNA.

Fig. 1

Phenotypic characterization of fertile and sterile buds and the dynamic SEM analysis of pollen grains. a, CK, control materials. CHA, the materials treated with SX-1. b, Length of anther, filament and pistil, and width of petal in opened flowers (Student’s t test, *P < 0.05, **P < 0.01). c-f, k-n, s and u, The pollen grains in control. g-j, o-r, t and v, The pollen grains in CHA treated materials. Scale bar in h-j, p-r,10 μm. Scale bar in c-g, k-o, s and t, 5 μm. Scale bar in u and v, 2 μm

Fig. 2

The dynamic TEM analysis of the developmental pollen grains and tapetum cell of B. napus. a-d and i-l, The developing pollen grains of control groups. e-h and m-p, The developing pollen of CHA treated materials. q, Pollen coats in control groups. r, Enlarged pollen coats in CHA treated materials. s-v, Tapetum in control groups. w-z, Tapetum in CHA treated materials. Red arrow, the degraded tapetal cells. Scale bar, 2 μm

Fig. 3

Representative 2-DE images of control and CHA groups and the enlarged area of 2-DE gel of some representative DEPs. a, c, e and g, Representative 2-DE images of control at SA, MA, LA and LA2 stage respectively. b, d, f and h, Representative images of CHA treated materials at SA, MA, LA and LA2 stage respectively. i, The enlarged area of 2-DE of some representative DEPs

Fig. 4

Protein expression profiles of the DEPs between CHA treated materials and control. a, The hierarchical clustering of the quantitative change proteins. b, Protein functional classification of four clusters

Fig. 5

The heatmap for the DEGs between CHA treated materials and control and biological process analysis of down-regulated DEGs. a, Up-regulated genes. b, Down-regulated genes. c, The heatmap analysis of all DEGs at different stage. d, The yellow and red circles indicate the common and different biological processes among the four stages

Fig. 6

The heatmap analysis of miRNA expression between CHA treated materials and control and miRNA-mRNA regulatory network analysis in B. napus. a, The heatmap of miRNAs expression. b, miRNA-mRNA regulatory network analysis. Pink triangles, miRNA. Blue circles, functional genes. Green diamonds, TFs

Fig. 7

The interaction analysis of candidate genes. Node size are showed according to the interaction counts. Edge size are showed according to combined score. Gene associated with special type of pathway is shown using special color. Green node represents key gene in the interaction network

Fig. 8

A proposed model for male sterility induced by SX-1 in B. napus. ER: endoplasmic reticulum, PE: primexine, Pla: plasma membrane, Te: tectum, Ba: bacula, In: intine, Ne: nexine, Pc: pollen coat (tryphine), Cy: cytoplasm, Vac: vacuole, LBs: lipid bodies, AA: amino acid, Fla.: flavonoids, UN: unknown nutrients, UTPs: unknown transport proteins, PMC: pollen mother cell. Up arrow, up-regulation. Down arrow, down-regulation