Date Published: July 3, 2017
Publisher: Public Library of Science
Author(s): Fei Ge, Xing Huang, Hongmei Hu, Yanling Zhang, Zhaoling Li, Chaoying Zou, Huanwei Peng, Lujiang Li, Shibin Gao, Guangtang Pan, Yaou Shen, Maoteng Li.
The induction efficiency of maize embryonic callus is highly dependent on the genotype, and only a few lines possess a high capacity for callus formation. Although certain genes and pathways have been reported to contribute to the regulation of callus induction, to the best of our knowledge, the functions of the small interfering RNAs (siRNAs) involved in this process remain unknown. In this study, we identified 861 differentially expressed siRNAs and 576 target genes in the callus induction process. These target genes were classified into 3 clusters, and their functions involve controlling metalloexopeptidase activity, catalase activity, transcription regulation, and O-methyltransferase activity. In addition, certain genes related to auxin transport and stem cell or meristem development (e.g., PLT5-like, ARF15, SAUR-like, FAS1-like, Fea3, SCL5, and Zmwox2A) were regulated by the differentially expressed siRNAs. Moreover, zma-siR004119-2 directly cleaves the 5’ UTR of Homeobox-transcription factor 25, which further leads to the down-regulation of its expression. Twelve 24-nt-siRNAs led to the hyper-methylation of GRMZM2G013465, which further decreases its expression. These results suggest that differentially expressed siRNAs regulate callus formation by controlling the expression of their target genes.
Transgenic maize (Zea mays L.) is currently widely used to study gene functions and breeding. Maize embryonic calli that are induced from immature embryos are the most important explants for transformation. However, only a few maize lines have a high frequency of embryonic callus induction [1,2]. According to a previous study, the dedifferentiation efficiency is determined by the genotype . Previous studies using RNA-seq , small RNA-seq  and proteomic analyses of maize embryonic calli [5–7] have revealed that most differentially expressed genes are involved in many processes, such as regulating pyruvate biosynthesis , hormone transduction , stress response , and cell proliferation . Recently, several genes have been shown to control embryonic callus induction, including BABY BOOM , the WUSCHEL-CLAVATA3 feedback loop [8,9], and LEAFY COTYLEDON .
We identified 861 DE-siRNAs and 576 corresponding differentially expressed target genes. Based on the cluster analysis and GO function enrichment analysis, these target genes were classified into 3 clusters, and most of these genes function in controlling metalloexopeptidase activity, catalase activity, transcription regulation, and O-methyltransferase activity. Among these differentially expressed siRNAs, zma-siR001849-2, zma-siR001071-1, zma-siR003715-1-1, zma-siR004720-2, zma-siR002558-2, zma-siR000403-2, and zma-siR002521-1 target PLT5-like, ARF15, SAUR-like, FAS1-like, Fea3, SCL5, and Zmwox2A, respectively. These genes also showed a significantly changed expression (FDR < 0.001) during callus induction and were previously reported to function in controlling auxin transportation, stem cell development, and meristem development. Homeobox-transcription factor 25 was identified as the target gene of zma-siR004119-2, which was confirmed by our degradome sequencing data. Twelve 24-nt siRNAs were identified to target the 3’ UTR of GRMZM2G013465, leading to DNA methylation near the target site and further resulting in decreased expression. Source: http://doi.org/10.1371/journal.pone.0180567