MicroRNA408 negatively regulates salt tolerance by affecting secondary cell wall development in maize.

Although microRNA408 (miR408) is a highly conserved miRNA, the miR408 response to salt stress differs among plant species. Here, we show that miR408 transcripts are strongly repressed by salt stress and methyl viologen treatment in maize (Zea mays). Application of N, N1-dimethylthiourea partly relieved the NaCl-induced down-regulation of miR408. Transgenic maize overexpressing MIR408b is hypersensitive to salt stress. Overexpression of MIR408b enhanced the rate of net Na+ efflux, caused Na+ to locate in the inter-cellular space, reduced lignin accumulation, and reduced the number of cells in vascular bundles under salt stress. We further demonstrated that miR408 targets ZmLACCASE9 (ZmLAC9). Knockout of MIR408a or MIR408b or overexpression of ZmLAC9 increased the accumulation of lignin, thickened the walls of pavement cells, and improved salt tolerance of maize. Transcriptome profiles of the wild-type and MIR408b-overexpressing transgenic maize with or without salt stress indicated that miR408 negatively regulates the expression of cell wall biogenesis genes under salt conditions. These results indicate that miR408 negatively regulates salt tolerance by regulating secondary cell wall development in maize.

MicroRNA528 Affects Lodging Resistance of Maize by Regulating Lignin Biosynthesis under Nitrogen-Luxury Conditions.

Lodging under nitrogen (N)-luxury conditions substantially reduces crop yield and seed quality. However, the molecular mechanisms of plant lodging resistance remain largely unclear, especially in maize. We report here that the expression of ZmmiR528, a monocot-specific microRNA, is induced by N luxury but reduced by N deficiency. We show by the thioacidolysis and acetyl bromide analysis that N luxury significantly reduces the generation of H, G, and S monomers of the lignin as well as its total content in maize shoots. We further demonstrate that ZmLACCASE3 (ZmLAC3) and ZmLACCASE5 (ZmLAC5), which encode the copper-containing laccases, are the targets of ZmmiR528. In situ hybridization showed that ZmmiR528 is mainly expressed in maize vascular tissues. Knockdown of ZmmiR528 or overexpression of ZmLAC3 significantly increased the lignin content and rind penetrometer resistance of maize stems. In contrast, transgenic maize plants overexpressing ZmmiR528 had reduced lignin content and rind penetrometer resistance and were prone to lodging under N-luxury conditions. RNA-sequencing analysis revealed that ZmPAL7 and ZmPAL8 are upregulated in transgenic maize lines downregulating ZmmiR528. Under N-luxury conditions, the expression levels of ZmPALs were much higher in ZmmiR528-knockdown lines than in the wild type and transgenic maize lines overexpressing ZmmiR528. Taken together, these results indicate that, by regulating the expression of ZmLAC3 and ZmLAC5, ZmmiR528 affects maize lodging resistance under N-luxury conditions.