A phased small interfering RNA-derived pathway mediates lead stress tolerance in maize

Phased small interfering RNAs (phasiRNAs) are a distinct class of endogenous small interfering RNAs, which regulate plant growth, development, and environmental stress response. To determine the effect of phasiRNAs on maize (Zea mays L.) tolerance to lead (Pb) stress, the roots of 305 maize lines under Pb treatment were subjected to generation of individual databases of small RNAs. We identified 55 high-confidence phasiRNAs derived from 13 PHAS genes (genes producing phasiRNAs) in this maize panel, of which 41 derived from 9 PHAS loci were negatively correlated with Pb content in the roots. The potential targets of the 41 phasiRNAs were enriched in ion transport and import. Only the expression of PHAS_1 (ZmTAS3j, Trans-Acting Short Interference RNA3) was regulated by its cis-expression quantitative trait locus and thus affected the Pb content in the roots. Using the Nicotiana benthamiana transient expression system, 5'-rapid amplification of cDNA ends, and Arabidopsis heterologously expressed, we verified that ZmTAS3j was cleaved by zma-miR390 and thus generated tasiRNA targeting ARF genes (tasiARFs), and that the 5' and 3' zma-miR390 target sites of ZmTAS3j were both necessary for efficient biosynthesis and functional integrity of tasiARFs. We validated the involvement of the zma-miR390-ZmTAS3j-tasiARF-ZmARF3-ZmHMA3 pathway in Pb accumulation in maize seedlings using genetic, molecular, and cytological methods. Moreover, the increased Pb tolerance in ZmTAS3j-overexpressed lines was likely attributed to the zma-miR390-ZmTAS3j-tasiARF-ZmARF3-SAURs pathway, which elevated indole acetic acid levels and thus reactive oxygen species-scavenging capacity in maize roots. Our study reveals the importance of the TAS3-derived tasiRNA pathway in plant adaptation to Pb stress.