Improved laser capture microdissection combined with transcriptome sequencing reveals gene expression reprogramming during rice panicle branch meristems differentiation

BACKGROUND: The number of branches per panicle exhibits a strong positive correlation with the grain number per panicle, which is a key agronomic trait determining rice yield. Efficient and precise identification of genes regulating the development of panicle branch meristems will contribute to high-yield rice breeding. METHOD: Here, we present an optimized laser capture microdissection (LCM) method capable of extracting high-quality mRNA, along with 3': 5' assay for assessing mRNA integrity in rice LCM samples. Base on this method, we successfully isolated high integrity mRNA from inflorescence meristems, primary branch meristems and secondary branch meristems, which were suitable for RNA-seq. RESULTS: We observed extensive transcriptome reprogramming during the differentiation of inflorescence meristems into branch meristems. Numerous phytohormone biosynthesis, transport, and response genes exhibited significant differential expression across the three types of meristems. The results of LCM-qPCR validated the accuracy of the transcriptome sequencing data. We selected three genes from the aforementioned differentially expressed genes for RNA in situ hybridization analysis, the results demonstrated their elevated expression in both primary and secondary panicle branch meristems, suggesting their likely role in regulating the differentiation trajectory from inflorescence meristems to branch meristems. CONCLUSION: We have established an optimized LCM protocol that enables the isolation of high-quality RNA from young rice panicles, roots, and stems. Using this method, we successfully isolated inflorescence meristems, primary branch meristems, and secondary branch meristems from young rice panicles for transcriptome sequencing. Among the differentially expressed genes identified by transcriptome analysis, a number of key genes known to regulate panicle branch meristems development were detected, demonstrating the feasibility of our established LCM method and its applicability in identifying key genes regulating rice panicle development. The combined use of improved laser capture microdissection and transcriptome sequencing enables rapid identification of key regulatory genes, such as OsMADS32, represents a promising candidate for yield improvement its expression is elevated.