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.