| Tetrapyrroles extensively exist in organisms.Higher plants produce four kinds of tetrapyrroles:heme,siroheme,phytochromobilin and chlorophyll.Chlorophyll,the most abundant tetrapyrrole,functions as photosynthetic pigment to harvest light energy and then transfer the absorbed energy to the reaction center to trigger charge separation.Light is required for chlorophyll biosynthesis.In etiolated Arabidopsis seedlings,chlorophyll biosynthesis stops at Divinyl-protochlorophyllide(Pchlide)stage.Intermediate products of chlorophyll and other tetrapyrroles excited by light can lead to generation of reaction oxygen species(ROS),and then cause oxidative stress and even cell death of the etiolated seedlings.Therefore,the accumulation of Pchlide in etiolated seedlings needs to be regulated precisely.It has been reported that Pchlide accumulation in etiolated seedlings was regulated through regulation of chlorophyll synthesis related enzymes and hormone signaling pathway.The accumulation of Pchlide in etiolated seedlings represses the activity of Glutamate-RNA reductase(Glu-TR),the rate-limiting enzyme of chlorophyll synthesis,by a protein called FLU.Transcription factors,e.g.PIF1,PIF3,FHY3 and HY5,affect Pchlide accumulation by regulating the expression of chlorophyll synthetic genes.Some plant hormones(e.g.GA and ethylene)signaling pathway also affect Pchlide accumulation in etiolated seedlings.Though many regulation factors have been discovered,the mechanism in maintaining appropriated level of Pchlide in etiolated seedlings remains to be uncovered.MAPK cascades are conserved signal modules in eukaryotes and involved in the regulation of Arabidopsis growth,development and response to environment stimulus.To date function of MAPK cascades in regulating Pchlide accumulation has not been reported.Our data showed that mkk5 etiolated seedlings accumulated more Pchlide than wild type among nine MKK null mutants.When transferred to light,mkk5 etiolated seedlings exhibited higher level of ROS accumulation and lower greening rate than wild type.These phenotypes could be recovered in complemented mkk5 etiolated seedlings.MKK5DD(constitutive active fonm of MKK5)over-expressing seedlings exhibited decreased Pchlide.These results suggest that MKK5 negatively regulate Pchlide accumulation in etiolated seedlings.Furthermore,we detennined whether MPK3 and MPK6,two well-known downstream MAPK,were involved in this process.We found that mpk6,but not mpk3,etiolated seedlings accumulated more Pchlide than wild type.Induction of MPK6 expression in mpk6 can rescue this phenotype.mkk5/mpk6 and MKK5DD/mpk6 double mutants showed similar Pchlide accumulation phenotype as mkk5 and mpk6,respectively.And MKK5 could activate MPK6 in etiolated seedlings.These results suggest that the MKK5 and MPK6 form a MAPK cascade negatively regulating Pchlide accumulation in etiolated seedlings.To explore the upstream MAPKKK of MKK5-MPK6 in regulating Pchlide accumulation in etiolated seedlings,we screened some MAPKKK T-DNA insertion mutants by detecting Pchlide accumulation in etiolated seedlings.And we found a MAPKKK null mutant,raf35,accumulated more Pchlide in etiolated seedlings,which can be recovered by expressing RAF35.Pchlide of raf35/mkk5 and raf35/mpk6 double mutants remained in the same level as mkk5 and mpk6 mutants,respectively.The interaction between RAF35 and MKK5 was detected using Y2H and pull-down assay.The kinase domain of RAF35,?RAF35,possessed kinase activity and can activate MKK5-MPK6 in vitro.Deficiency of RAF35 decreased MPK6 kinase activity in etiolated seedlings.These results showed that RAF35-MKK5-MPK6 is a MAPK cascade involved in regulating Pchlide accumulation.In conclusion,we demonstrated a novel MAPK cascade,RAF35-MKK5-MPK6,negatively regulating Pchlide accumulation in Arabidopsis etiolated seedlings.This study provides new evidences for understanding the regulatory mechanism of Pchlide biosynthesis and metabolism in etiolated seedlings. |
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