Relationship between reduction in rice (Nipponbare) leaf blade size under elevated CO₂ and miR396–GRF module

Elevated CO₂ (eCO₂; 1000 ppm) influences developing rice leaf formation, reducing leaf blade length and width as compared to rice grown under ambient CO₂ (aCO₂; 400 ppm). Since micro RNAs (miRNAs) are known to play multiple roles in plant development, we hypothesized that miRNAs might be involved in modulating leaf size under eCO₂ conditions. To identify miRNAs responding to eCO₂, we profiled miRNA levels in developing rice leaves (P4; plastochron number of the fourth-youngest leaf) under eCO₂ using small RNA-seq. We detected 18 mature miRNA sequences for which expression levels varied more than two-fold between the eCO₂ and aCO₂ conditions. Among them, only miR396e and miR396f significantly differed between the two conditions. Additionally, the expression of growth-regulating factors (GRFs), potential target mRNA of miR396s, were repressed under the eCO₂ condition. We used an antisense oligonucleotide approach to confirm that single-strand DNA corresponding to the miR396e sequence effectively downregulated GRF expression in developing leaves, reducing the leaf blade length, such as for rice grown under eCO₂. These results suggest that the miR396–GRF module is crucially relevant to controlling rice leaf blade length in eCO₂ environments.