Transcriptome and hormonal analyses reveal an important role of auxin and cytokinin in regulating rice ratooning ability

• Ratooning ability (RA) in rice is influenced by both genetic and environmental factors. • With common genotypes of the known ratooning ability genes RRA3 and qRA2 , 23 rice varieties showed diverse RA phenotypes. • RNA-seq reveals key DEGs enriched in auxin and cytokinin pathways, with greater expression of cytokinin biosynthesis genes and lower expression of auxin biosynthesis genes in high RA varieties than in low RA variety. • High RA varieties have lower auxin and higher cytokinin levels, together with different expression of their biosynthesis genes, suggesting auxin and cytokinin play key roles in rice ratooning. Ratooning refers to the rapid regeneration of rice tillers from dormant buds located on the stubble following the harvest of the main crop. Rice ratoon cropping could significantly reduce labor and inputs demands, making it an attractive cultivation strategy in areas with suitable climatic conditions. A crucial determinant of rice yield is ratooning ability (RA), defined as the ratio of panicle number of the second to first crop. To date, only two genes associated with ratooning ability (RA), RRA3 and qRA2 , have already been identified; however, the genetic network and underlying biological mechanisms regulating RA remain largely unexplored. RRA3 has been shown to inhibit RA of rice by suppressing cytokinin signal transduction, while qRA2 promotes RA. In the present study, the RA trait was evaluated in 23 rice varieties cultivated across three different locations in Hainan Province, showing substantial phenotypic variation. Despite this variation, genotyping analysis showed that all 23 varieties had a common RRA3 allele, and there was no correlation between the qRA2 genotype and RA values. Based on these findings, two high RA and one low RA varieties were selected for further analysis. Transcriptome sequencing was performed on these selected varieties, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The results showed that genes involved in plant hormone biosynthesis and response pathways were significantly enriched among the differentially expressed genes. Quantitative real-time (qRT-PCR) analysis further confirmed that expression levels of genes involved in the auxin signaling pathway was significantly lower in the two high RA varieties compared to the low RA variety. The opposite trend was observed for the expression of genes involved in cytokinin signaling. Transcription levels of RRA3 and qRA2 observed in the present study were consistent with their previously reported roles in the regulation of cytokinin biosynthesis. Further hormonal analysis showed that the two high RA varieties had significantly lower auxin and higher cytokinin levels than the low RA variety. Collectively, these findings suggest that in addition to the well-established role of cytokinin, auxin is also involved in the regulation of rice ratooning, with its level showing a negative correlation with RA.