The Secondary Structure Of MiRNA/miRNA* Is Important For The Regulation Pattern Of NFYA5 By MiRNA169

Micro RNAs, which are processed from primary mi RNAs with hairpin structures, regulate gene expression at the transcriptional and post-transcriptional levels. As the largest mi RNA family in Arabidopsis, MIRNA169 has 14 members and can be divided into four subgroups based on their mature mi RNA sequences. Although previous research showed that expression of both mi RNA169 and its target, NFYA5, are downregulated by drought stress, the current study showed that expression of mi RNA169 and NFYA5 are induced by dehydration shock. Unlike overexpression of MIRNA169a/b, overexpression of MIRNA169 l did not decrease NFYA5 transcripts but increased NFYA5 protein levels. mi RNA secondary structure was demonstrated to affect accurate micro RNA processing and mature mi RNA amounts. We deduced that mi RNA secondary structure might be important for the regulatory pattern of NFYA5 by different members in mi RNA169 family. The main results are as follows:1. Overexpression of MIRNA169 l or MIRNA169 i improves drought resistance in Arabidopsis. we grew WT, 35S::MIRNA169l and 35S::MIRNA169i transgenic plants for 3 weeks in soil before withholding water for 12 days. Most WT plants wilted, and their leaves became purple, while 35S::MIRNA169l and 35S::MIRNA169i transgenic plants remained turgid, and their leaves remained green. When data were analyzed from three independent experiments, 28% of the WT plants survived the drought stress treatment, while ~80% of 35S::MIRNA169l transgenic plants and ~53% of 35S::MIRNA169i transgenic plants survived the drought stress treatment.2. The bulges at the 3′ end of miRNA169 l are essential for its up-regulation of NFYA5 protein abundance. We introduced two “As” into the mi RNA169l* sequence to smooth the two bulges at the 3′ end of mi RNA169 l, causing the secondary structure of mi RNA169l/mi RNA169l* to be similar to that of mi RNA169a/mi RNA169a*. The level of NFYA5 transcripts in the 35S::MIRNA169l*-mut transgenic plants was ~25% lower than that in the 35S::MIRNA169l transgenic plants, and was ~50% lower than that in the WT plants. To further characterize the potential function of mi RNA169/mi RNA169* complementarity, another construct resembling the secondary structure of the mi RNA169l/mi RNA169l* duplex was designed by introducing two bulges in mi RNA169a/mi RNA169a* duplex at the 5′ end of mi RNA169a*. Contrary to the efficient repression of NFYA5 transcripts in 35S::MIRNA169a transgenic plants and despite the significant up-regulation of mi RNA169 in 35S::MIRNA169a*-mut transgenic plants, the relative m RNA and protein levels of NFYA5 did not differ substantially between WT and 35S::MIRNA169a*-mut transgenic plants.3. Overexpression of MIRNA169l*-mut, like overexpression of MIRNA169 a, increases sensitivity to drought stress in Arabidopsis. We grew WT and 35S::MIRNA169l*-mut transgenic plants for 3 weeks in soil before withholding water for 12 days. Nearly all of the WT plants but fewer than 10% of the 35S::MIRNA169l*-mut plants survived the drought stress and recovered from the stress after rewatering. We also evaluated the drought resistance of 35S::MIRNA169a*-mut transgenicplants, 35S::MIRNA169a*-mut transgenic plants did not show hypersensitivity to drought stress as plants overexpressing mi RNA169 a, and the difference between WT and 35S::MIRNA169a*-mut plants was not statistically significant.4. Both #11 and #16 bulges are essential for the miRNA169l-guided up-regulation of NFYA5 protein abundance. In order to test which bulge in mi RNA169l/mi RNA169l* duplex mainly contributed to the mi RNA169l-guided up-regulation of NFYA5 protein abundance, 35S::MIRNA169l*-11-mut, 35S::MIRNA169l*-16-mut, 35S::MIRNA169a*-11-mut and 35S::MIRNA169a*-16-mut four transgenic plants were designed. In 35S::MIRNA169l*-11-mut and 35S::MIRNA169l*-16-mut transgenic plants, the up-regulation of NFYA5 protein abundance in 35S::MIRNA169l plants was not observed. Contrary to NFYA5 expression levels in 35S::MIRNA169a transgenic plants, NFYA5 transcripts in 35S::MIRNA169a*-11-mut and 35S::MIRNA169a*-16-mut transgenic plants did not show obvious difference from that in WT plants. These results strongly suggest that both #11 and #16 bulges are essential for the mi RNA169l-guided up-regulation of NFYA5 protein abundance.