Dynamic Transcriptome Analysis Reveals Uncharacterized Complex Regulatory Pathway Underlying Dose IBA-Induced Embryogenic Redifferentiation in Cotton

The somatic embryogenesis (SE) process of plants is regulated by exogenous hormones. During the SE, different genes sensitively respond to hormone signals through complex regulatory networks to exhibit plant totipotency. When cultured in indole-3-butyric acid (IBA) concentration gradient medium supplemented with 0 mg dm^-3, 0.025 mg dm^-3, and 0.05 mg dm^-3 IBA, the callus differentiation rate first increased then decreased in cotton. To characterize the molecular basis of IBA-induced regulating SE, transcriptome analysis was conducted on embryogenic redifferentiation. Upon the examination of the IBA's embryogenic inductive effect, it was revealed that pathways related to plant hormone signal transduction and alcohol degradation were significantly enriched in the embryogenic responsive stage (5 days). The photosynthesis, alcohol metabolism and cell cycle pathways were specifically regulated in the pre-embryonic initial period (20 days). Upon the effect of the IBA dose, in the embryogenic responsive stage (5 days), the metabolism of xenobiotics by the cytochrome P450 pathway and secondary metabolism pathways of steroid, flavonoid, and anthocyanin biosynthesis were significantly enriched. The phenylpropanoid, brassinosteroid, and anthocyanin biosynthesis pathways were specifically associated in the pre-embryonic initial period (20 days). At different developmental stages of embryogenic induction, photosynthesis, flavonoid biosynthesis, phenylpropanoid biosynthesis, mitogen-activated protein kinase (MAPK) signaling, xenobiotics metabolism by cytochrome P450, and brassinosteroid biosynthesis pathways were enriched at low a IBA concentration. Meanwhile, at high IBA concentration, the carbon metabolism, alcohol degradation, circadian rhythm and biosynthesis of amino acids pathways were significantly enriched. The results reveal that complex regulating pathways participate in the process of IBA-induced redifferentiation in cotton somatic embryogenesis. In addition, collections of potential essential signaling and regulatory genes responsible for dose IBA-induced efficient embryogenic redifferentiation were identified. Quantitative real-time PCR (qRT-PCR) was performed on the candidate genes with different expression patterns, and the results are basically consistent with the RNA-seq data. The results suggest that the complicated and concerted IBA-induced mechanisms involving multiple cellular pathways are responsible for dose-dependent plant growth regulator-induced SE. This report represents a systematic study and provides new insight into molecular signaling and regulatory basis underlying the process of dose IBA-induced embryogenic redifferentiation during SE.