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Callus induction in plants is similar to pluripotent stem cell induction in animals and can incite global changes in gene expression. Alternative splicing is considered a key factor underlying functional complexity and function in the induction of pluripotent stem cells in animals. However, the role of alternative splicing in plant callus induction remains unclear. We performed high-throughput RNA sequencing of maize callus induced on a callus induction medium (CIM) at different stages and recorded global alternative splicing changes. At every stage, over 20,000 alternative splicing events were detected, and the numbers slightly increased with callus induction. In total, 648 to 2580 genes were found to exhibit significant alternative splicing changes during callus induction—mainly those that belong to the spliceosome, metabolic pathways, and mRNA surveillance pathways. It was also found that alternative splicing can function alongside transcriptional regulation to contribute to callus induction. Especially, ZmWOX11, whose orthologous gene in Arabidopsis plays a vital role in the first-step cell fate transition of callus induction, was found to exhibit alternative splicing and expression level changes during callus induction. Our results establish a foundation from which researchers can further explore the molecular mechanism of callus induction in maize, especially at alternative splicing level.

Callus induction is a dedifferentiation process that accompanies a cell fate transition, and epigenetic regulation plays a crucial role in the process. N6‐methyladenosine (m6A) methylation is an important mechanism in post‐transcriptional epigenetic regulation and functions in cell reprogramming. However, the function of m6A methylation during callus induction is still unknown. Here, we performed transcriptome‐wide m6A‐seq on immature maize embryos after culturing for 2, 4, or 8 days with or without the auxin analogue 2,4‐D. A total of 26,794 unique m6A peaks were detected from 17,456 maize genes; and 2,338 specific, 2,4‐D‐induced m6A peaks (D‐specific m6A) were detected only in embryos cultured with 2,4‐D. Furthermore, a positive correlation between m6A methylation and mRNA abundance was discovered in the genes with D‐specific m6A deposition, especially at the beginning of callus induction. Key genes involved in callus induction, i.e. BABY BOOM and LBD transcription factors, underwent m6A methylation, increasing their transcript levels, thus improving callus induction. These results revealed the importance of m6A methylation during the early stage of callus induction and provided new insights into the molecular mechanism of callus induction at an epitranscriptomic level.

Callus induction is a dedifferentiation process that accompanies a cell fate transition, and epigenetic regulation plays a crucial role in the process. N

Maize transformation is highly based on the formation of embryonic callus, which is mainly derived from scutellum cells of the immature maize embryo. However, only a few genes involved in callus induction have been identified in maize. To reveal the potential genes involved in the callus induction of maize, we carried out a high-throughput RNA sequencing on embryos that were cultured for 0, 1, 2, 4, 6, and 8 days, respectively, on a medium containing or lacking 2,4-dichlorophenoxyacetic acid. In total, 7,525 genes were found to be induced by 2,4-dichlorophenoxyacetic acid and categorized into eight clusters, with clusters 2 and 3 showing an increasing trend related to signal transmission, signal transduction, iron ion binding, and heme binding. Among the induced genes, 659 transcription factors belong to 51 families. An AP2 transcription factors, ZmBBM2, was dramatically and rapidly induced by auxin and further characterization showed that overexpression of ZmBBM2 can promote callus induction and proliferation in three inbred maize lines. Therefore, our comprehensive analyses provide some insight into the early molecular regulations during callus induction and are useful for further identification of the regulators governing callus formation.