Relationship between reduction in rice (Nipponbare) leaf blade size under elevated CO 2 and miR396- GRF module.

Elevated CO ₂ (eCO ₂ ; 1000 ppm) influences developing rice leaf formation, reducing leaf blade length and width as compared to rice grown under ambient CO ₂ (aCO ₂ ; 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 eCO ₂ conditions. To identify miRNAs responding to eCO ₂ , we profiled miRNA levels in developing rice leaves (P4; plastochron number of the fourth-youngest leaf) under eCO ₂ using small RNA-seq. We detected 18 mature miRNA sequences for which expression levels varied more than two-fold between the eCO ₂ and aCO ₂ 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 eCO ₂ 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 eCO ₂ . These results suggest that the miR396- GRF module is crucially relevant to controlling rice leaf blade length in eCO ₂ environments.