Comparative transcriptome analysis of isonuclear-alloplasmic lines unmask key transcription factor genes and metabolic pathways involved in sterility of maize CMS-C

Abstract

Although C-type cytoplasmic male sterility (CMS-C) is one of the most attractive tools for maize hybrid seed production, the detailed regulation network of the male sterility remains unclear. In order to identify the CMS-C sterility associated genes and/or pathways, the comparison of the transcriptomes between the CMS-C line C48-2 and its isonuclear-alloplasmic maintainer line N48-2 at pollen mother cell stage (PS), an early development stage of microspore, and mononuclear stage (MS), an abortive stage of microspore, were analyzed. 2,069 differentially expressed genes (DEGs) between the two stages were detected and thought to be essential for the spikelet development of N48-2. 453 of the 2,069 DEGs were differentially expressed at MS stage between the two lines and thought to be participated in the process or the causes of microspore abortion. Among the 453 DEGs, 385 (84.99%) genes were down-regulated and only 68 (15.01%) genes were up-regulated in C48-2 at MS stage. The dramatic decreased expression of the four DEGs encoding MYB transcription factors and the DEGs involved in "polyamine metabolic process", "Cutin, suberine and wax biosynthesis", "Fatty acid elongation", "Biosynthesis of unsaturated fatty acids" and "Proline metabolism" might play an important role in the sterility of C48-2. This study will point out some directions for detailed molecular analysis and better understanding of sterility of CMS-C in maize.

Keywords: Cytoplasmic male sterility; Differentially expressed gene; Maize; Metabolic pathway; RNA-seq; Transcription factor.

Figure 1. Statistical analysis of gene expression detected by RNA-seq.

(A) Venn diagram of gene counts expressed at PS and MS stages for C48-2 and N48-2; (B) Number of total DEGs and down- or up- regulated DEGs, respectively; (C) Venn diagram displaying the relationship of DEGs between the two comparisons “MS-N vs PS-N” and “MS-C vs MS-N”. MS-C, PS-C, MS-N and PS-N represent the mononuclear stage of C48-2, pollen mother cell stage of C48-2, mononuclear stage of N48-2 and pollen mother cell stage of N48-2, respectively.

Figure 2. qRT-PCR verification on RNA-seq results and DEGs identification.

x-axis represents genes ID, y-axis represents the logarithm of fold change; the blue column and red column respectively represents the qRT-PCR results and RNA-seq results of relative expression levels fold change between the two lines; |Log2Fold Change| ≥ 1 represents the gene is differentially expressed between the two lines.

Figure 3. Comparative analyses of the five transcriptional factors expression levels between C48-2 and N48-2 along with the microspore development.

(A) The expression level of LOC100191533; (B) The expression level of LOC100274206; (C) The expression level of LOC100272446; (D) The expression level of LOC100216895; (E) The expression level of LOC732739. PS, DS, TS and MS represents pollen mother cell stage, dyad stage, tetrad stage and mononuclear stage, respectively.

Figure 4. Phylogenetic analysis of the four MYB transcription factors with the 13 MYBs of Arabidopsis.

Multiple sequence alignments of proteins were generated using ClustalW (version 7.2.5), and the phylogenetic tree was constructed with the neighbor-joining algorithm in MEGA (version 5.1). Bootstrap analysis was carried out with 1,000 replicates.