Transcriptome analysis to identify genes related to programmed cell death resulted from manipulating of BnaFAH ortholog by CRISPR/Cas9 in Brassica napus

Abstract

Abstract Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of the tyrosine degradation pathway. In this study, we isolated and characterized two homologous BnaFAH genes in Brassica napus L. variant Westar, and then used CRISPR/Cas9-mediated targeted mutagenesis to generate a series of transgene-free mutant lines either with single or double-null bnafah alleles. Among these mutant lines, the aacc (bnafah) double-null mutant line, rather than the aaCC (bnaa06fah) mutant line, exhibited programmed cell death (PCD) under short days (SD). Histochemical staining and content measurement confirmed that the accumulation of reactive oxygen species (ROS) in bnafah was significantly higher than that in bnaa06fah. To further elucidate the mechanism of PCD, we performed transcriptomic analyses of bnaa06fah and bnafah at different SD stages. A heatmap cluster of differentially expressed genes (DEGs) revealed that PCD may be related to various redox regulatory genes involved in antioxidant activity, ROS-responsive regulation and calcium signaling. Combined with the results of previous studies, our work revealed that the expression levels of BnaC04CAT2, BnaA09/C09SAL1, BnaA08/C08ACO2, BnaA07/C06ERO1, BnaA08ACA1, BnaC04BIK1, BnaA09CRK36 and BnaA03CPK4 were significantly different and that these genes might be candidate hub genes for PCD. Together, our results underscore the ability of different PCD phenotypes to alter BnaFAH orthologs through gene editing and further elucidated the molecular mechanisms of oxidative stress-induced PCD in plants.

Keywords

• Fumarylacetoacetate hydrolase
• Programmed cell death
• Hydrogen peroxide
• Calcium signaling