miR164g-<i>MsNAC022</i> acts as a novel module mediating drought response by transcriptional regulation of reactive oxygen species scavenging systems in apple

Abstract

Under drought stress, reactive oxygen species (ROS) overaccumulate as a secondary stress that impairs plant performance and thus severely reduces crop yields. The mitigation of ROS levels under drought stress is therefore crucial for drought tolerance. MicroRNAs (miRNAs) are critical regulators of plant development and stress responses. However, the complex molecular regulatory mechanism by which they function during drought stress, especially in drought-triggered ROS scavenging, is not fully understood. Here, we report a newly identified drought-responsive miRNA, miR164g, in the wild apple species <i>Malus sieversii</i> and elucidate its role in apple drought tolerance. Our results showed that expression of miR164g is significantly inhibited under drought stress and it can specifically cleave transcripts of the transcription factor <i>MsNAC022 in M. sieversii</i>. The heterologous accumulation of miR164g in <i>Arabidopsis thaliana</i> results in enhanced sensitivity to drought stress, while overexpression of <i>MsNAC022</i> in Arabidopsis and the cultivated apple line 'GL-3' (<i>Malus domestica</i> Borkh<i>.</i>) lead to enhanced tolerance to drought stress by raising the ROS scavenging enzymes activity and related genes expression levels, particularly <i>PEROXIDASE (MsPOD)</i>. Furthermore, we showed that expression of <i>MsPOD</i> is activated by MsNAC022 in transient assays. Interestingly, <i>Part1</i> (<i>P1</i>) region is the key region for the positive regulation of <i>MsPOD</i> promoter by MsNAC022, and the different <i>POD</i> expression patterns in <i>M. sieversii</i> and <i>M. domestica</i> is attributed to the specific fragments inserted in <i>P1</i> region of <i>M. sieversii</i>. Our findings reveal the function of the miR164g<i>-MsNAC022</i> module in mediating the drought response of <i>M. sieversii</i> and lay a foundation for breeding drought-tolerant apple cultivars.