Transcriptome and small RNA analysis unveils novel insights into the C₄ gene regulation in sugarcane

Main Conclusion: This study reveals miRNA indirect regulation of C₄ genes in sugarcane through transcription factors, highlighting potential key regulators like SsHAM3a. Abstract: C₄ photosynthesis is crucial for the high productivity and biomass of sugarcane, however, the miRNA regulation of C₄ genes in sugarcane remains elusive. We have identified 384 miRNAs along the leaf gradients, including 293 known miRNAs and 91 novel miRNAs. Among these, 86 unique miRNAs exhibited differential expression patterns, and we identified 3511 potential expressed targets of these differentially expressed miRNAs (DEmiRNAs). Analyses using Pearson correlation coefficient (PCC) and Gene Ontology (GO) enrichment revealed that targets of miRNAs with positive correlations are integral to chlorophyll-related photosynthetic processes. In contrast, negatively correlated pairs are primarily associated with metabolic functions. It is worth noting that no C₄ genes were predicted as targets of DEmiRNAs. Our application of weighted gene co-expression network analysis (WGCNA) led to a gene regulatory network (GRN) suggesting miRNAs might indirectly regulate C₄ genes via transcription factors (TFs). The GRAS TF SsHAM3a emerged as a potential regulator of C₄ genes, targeted by miR171y and miR171am, and exhibiting a negative correlation with miRNA expression along the leaf gradient. This study sheds light on the complex involvement of miRNAs in regulating C₄ genes, offering a foundation for future research into enhancing sugarcane's photosynthetic efficiency.