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.