Brassinosteroid-signaling kinase 4 activates mitogen-activated protein kinase 4 to enhance cold stress tolerance in maize

Abstract Cold stress limits the growth, development, and yield of maize (Zea mays L.). Mitogen-activated protein kinases (MAPKs) play important roles in response to cold stress. However, besides the canonical MAPK cascades, it is unclear whether other kinases directly activate MAPKs under cold stress. Here, we identified brassinosteroid-signaling kinase 4 (ZmBSK4) as an upstream kinase of ZmMAPK4 in regulating cold tolerance. Functional analysis demonstrated that ZmMAPK4 and ZmBSK4 positively regulate cold tolerance in maize. ZmBSK4 directly interacts with and phosphorylates ZmMAPK4 at Ser-171. This Ser-171 phosphorylation augments ZmMAPK4 kinase activity and improves maize cold tolerance. Furthermore, we identified 2 ZmMAPK4-interacting substrates: the 2 basic helix-loop-helix (bHLH) transcription factors ZmbHLH111 and ZmbHLH181. ZmMAPK4 phosphorylates ZmbHLH111 and ZmbHLH181. Ser-171 phosphorylation enhances ZmMAPK4-mediated phosphorylation of ZmbHLH111 and ZmbHLH181, which promotes their transcriptional activity. Then, ZmbHLH111 and ZmbHLH181 induce the expression of the cold-responsive genes Zea mays dehydration response element binding protein1.2/1.4/1.9/1.10 (ZmDREB1.2/1.4/1.9/1.10), thereby enhancing cold tolerance in maize. Taken together, ZmBSK4 phosphorylating ZmMAPK4 at Ser-171 enhances ZmMAPK4-mediated phosphorylation of ZmbHLH111 and ZmbHLH181, which promotes their activity, ultimately triggering the expression of the cold-responsive ZmDREB1 genes and enhancing maize cold tolerance. Our results reveal a noncanonical MAPK regulatory mechanism for enhancing cold tolerance in maize.