A long non-coding RNA associated with H3K7me3 methylation negatively regulates OsZIP16 transcription under cadmium stress

Cadmium (Cd) is a toxic environmental pollutant, posing a high risk to crop production and human health. However, the genetic mechanisms for regulation of Cd accumulation in crops are poorly understood. In this study, we functionally identified a novel long non-coding RNA (lncRNA, TCONS_00502780) that repressed a locus encoding an uncharacterized metal transporter ZIP16 (ZRT/IRT-like Protein) in rice. LncRNA-OsZIP16 (L16) is resident in the antisense strand of OsZIP16. Both L16 and OsZIP16 were transcriptionally expressed during the life cycle, but under Cd stress the L16 transcription was repressed, whereas the OsZIP16 expression was upregulated. OsZIP16 is localized to the endoplasmic reticulum. Knocking out OsZIP16 by CRISPR-Cas9 (C16) resulted in Cd sensitivity, manifested by reduced plant growth and intense cellular damage with a slightly higher Cd translocation from roots to shoots, suggesting that OsZIP16 expression is required for rice growth and development under Cd stress. Conversely, OsZIP16 constitutive overexpression (OE16) lines displayed a growth phenotype compatible to the wide-type with lower Cd translocation ratio from roots to shoots. L16 knock-down lines by RNA interference (L16-R) showed a similar phenotype to the OE16 lines, while the L16 overexpression (L16-OE) lines were phenotypically similar to the C16 lines. The OsZIP16 transcription was upregulated in the L16-R lines but downregulated in the L16-OE lines. Using an antibody against the trimethylation of histone H3 lysine 27 (H3K27me3) followed by chromatin immunoprecipitation (ChIP), we found the reduced H3K27me3 methylation marks surrounding the OsZIP16 gene under Cd stress. Further examination of H3K27me3 in the L16-R lines revealed that the methylation levels were also significantly lower than those in WT. Taken together, these data suggest that the L16 could negatively regulate the OsZIP16 transcriptional expression through an epigenetic mechanism for rice adaption to Cd stress.