Molecular Mechanism Of Arabidopsis MAPK Cascade Involved In The Regulation Of Red-light-induced Cotyledon Opening

Light signal plays important roles in regulating growth and development of plants. The red/far-red-light-absorbing photoreceptor phytochrome regulates multiple developmental processes including the photomorphogenesis. Phytochrome interacting Factors (PIFs), a small subfamily of bHLH transcription factors, repress photomorphogenesis in darkness. Photoactivation of phytochrome induces rapid phosphorylation and subsequent degradation of PIF protein, resulting in promotion of photomorphogenesis. However, the kinase responsible for PIFs phosphorylation is still unknown. Mitogen-activated protein kinase (MAPK) cascades play crucial roles in plant signal transduction and regulate diverse biological processes including cell division and stress responses. Recent advances have unraveled that red light could activate two MAPKs in Cucumis sativus cotyledons, providing new insights into a positive correlation between MAPK cascades and plant response to light.In this study, we found that MPK6 exhibited rapidly increased kinase activity when the etiolated Arabidopsis seedlings exposed to red light. And the mpk6 null mutants displayed slightly decreased cotyledon angle in red light, implying that MPK6 involved in the regulation of red-light induced cotyledon opening. In order to explore the contribution of the MAPK activity to the photomorphogenesis, we analyzed the phenotypes of all constitutively activated MAPKKs (MKKDDs) transgenic seedlings (except MKK3DD and MKK7DD) grown in darkness, and we found only seedlings expressing MKK10DD exhibited obvious opened cotyledons. Further experiments indicated that the cotyledon of MKK10DD is hypersensitive to red light, and MKK10 kinase activity is essential for these phenotypes. Activation assay showed that MKK10 specifically activated MPK6 in vitro. Then we tested the MAPK activity in etiolated MKK10 mutant transgenic seedlings. This result showed that MKK10 could activate MPK6 in vivo. Knockout of mpk6 in MKK10DD transgenic seedlings can obviously suppress the cotyledon phenotype induced by overexpression of MKK10DD, indicating that MPK6 acts downstream of MKK10DD in the regulation of cotyledon opening.It has been reported that PIF3 is a key regulator in the repression of cotyledon opening. In our experiments we showed that overexpression of PIF3 in MKK10DD could apparently rescue the constitutively photomorphogenic phenotype induced by overexpression of MKK10DD, raising the possibility that PIF3 is a substrate of MPK6. Pull-down and Co-IP results showed that PIF3 interacted directly with MPK6 both in vitro and in vivo. And the in vivo interaction was induced by red light. The phosphorylation assay further showed that PIF3 could be phophorylated by MPK6 in vitro. phyB was reported as the main photoreceptor that acts upstream of PIF3. Interestingly, overexpression of MKK10DD in a phyB background caused fully opened cotyledons. Further experiments indicated that overexpression of MKK10DD decreased PIF3 protein accumulation in an MPK6-dependent manner.In conclusion, our results suggested that MKK10-MPK6 cascade might be involved in the regulation of phy-mediated cotyledon opening by inducing PIF3 phosphorylation.