Identification of post-transcriptional regulation reveals complexity in peanut pod development by Direct RNA

Peanut (Arachis hypogaea) is widely cultivated worldwide as an important source of edible vegetable oil and protein. Peanut seed pods develop below ground from a gynophore that forms above ground and then penetrates the soil surface to bury the developing pod. Numerous studies have explored transcriptional regulation during peanut pod development. Here, we explored post-transcriptional regulation, including polyadenylation, alternative splicing, and RNA adenosine methylation (m6A), in peanut pods across four developmental stages by performing direct RNA sequencing. This produced 70.43 million long reads with average lengths of 890–1,136 nucleotides (nt) from 12 samples across four developmental stages, yielding a total of 14,627 newly identified transcripts. We detected a negative relationship between poly(A) tail lengths and transcript abundance, with the shortest poly(A) tails at the subterranean peg and expanded pod 1 stages, and longest poly(A) tails at the aerial gynophore and expanded pod 2 stages. Moreover, throughout pod development, from the penetration of the gynophore into the soil to pod enlargement, the splicing machinery utilized more proximal than distal alternative polyadenylation sites in the transcripts. The date showed no correlation between m6A modification and gene expression in peanut, but found more transcripts with alternative first and last exon types of alternative splicing events. Transcripts that were differentially abundant across developmental stages were primarily enriched in the Gene Ontology terms photosynthesis, response to oxidative stress, response to auxin, plant-type cell wall organization, and lignin catabolism. This study lays a foundation for revealing the roles of epigenetics and post-transcriptional regulation in pod development in peanut.