The Molecular Mechanism Of Brassinosteroids In Regulating Greening Of Etiolated Arabidopsis Seedlings

After germination in the soil,seedlings are characterized by fast growth of the hypocotyl and formation of closed apical hook and yellow cotyledons,which is termed skotomorphogenesis.Instead of mature chloroplasts and chlorophyll,etioplasts and protochlorophyllide(Pchlide),the phototoxic chlorophyll synthesis precursor,are accumulated in yellow cotyledons.The nutrients needed by plant seedlings before emergence through the soil come from storage organs such as endosperm,which is known as heterotrophic growth.Once seedlings break through the soil and are exposed to the light,the Pchlide is promptly transformed into chlorophyllide,which allows seedlings to become photosynthetically active and capable of autotrophic growth.Nonetheless,if there is so excessive Pchlide in etiolated cotyledons that protochlorophyllide oxidoreductase(POR)could not catalyze all of the Pchlide to chlorophyllide under light,the free Pchlide will produce large amounts of reactive oxygen species(ROS),which lead seedlings to suffer from photo-oxidative damage.Under this circumstance,cotyledon bleaching or even seedling death occurs.As a result,the greening process during seedlings emerging from the ground to receive light is a very fragile but critical stage in the whole life cycle of terrestrial plants,which determines whether plants can survive successfully.Thus,plants have evolved complex and sophisticated mechanisms to regulate the biosynthesis of Pchlide to regulate the greening process of etiolated seedlings.Brassinosteroids(BRs),a kind of phytohormone,play crucial roles in seed germination,skotomorphogenesis,cell elongation,stress response and other biological processes.However,the roles of BRs in the transition from skotomorphogenesis to photomorphogenesis of seedlings remain unclear.In this study,we carried out experiments centering on the key scientific question that whether the BRs are involved in the green process of etiolated seedlings.At the first,we analyzed the greening process of BRs-related mutants after dark treatment.The results showed that the abilities to turn green of etiolated seedlings with increased BRs or enhanced BRs signals were stronger than that of wild-type Col-0,while seedlings with BRs deficiency or attenuated BRs signals showed decreased greening rate when compared with Col-0.At the same time,treatment with brassinolide(BL)increased the greening rates of Col-0 and rescued the greening defect of det2-1.Moreover,dominant gain-of-function mutant bzrl-1D can restore the abilities to turn green of det2-1 and bril-301.In addition,the Pchlide content of these mutants was detected,and the results showed that the seedlings defected in turning green accumulated excessive Pchlide.Together,these results demonstrate that BRs and activated BZR1 repress excessive accumulation of Pchlide in the dark to facilitate greening of etiolated seedling upon light irradiation.Previous studies revealed that PIFs are key regulators in this process.Genetic analysis showed that PIFs were required for BZR1 regulation on Pchlide biosynthesis and greening process.To further explore the molecular mechanism by which BZR1 and PIFs promote greening of etiolated seedlings,we analyzed in depth the downstream genes jointly regulated by BZR1 and PIFs and found GRFs are regulated both by BZR1 and PIFs.Further studies showed that GRF7 and GRF8 were induced by BRs through BZR1,but inhibited by light through PIFs.Meanwhile,ChIP-qPCR assays demonstrated that BZR1 and PIF4 both bound to promoters of GRF7 and GRF8,indicating both BZR1 and PIF4 could directly induce the expression of GRF7 and GRF8.GRFs are regulated by microRNA396 in post-transcriptional level in plants.To determine whether GRFs are functionally involved in seedling greening during de-etiolation,we emphatically analyzed greening phenotypes of GRFs related mutants and overexpressed plants.The results showed that 35Spro:miR396a,grf3 grf4 grf7(grf t)and grfl grf4 grf7 grf8(grf-q)mutants displayed remarkable reduction of cotyledon greening,while the greening rates of 35Spro:MIM396 and 35Spro:rGRF7 were higher than that of Col-0.Accordingly,the level of Pchlide in grf-t,grf-q and 35Spro:miR396a seedlings was extremely elevated when compared with Col-0.These results indicate that the miR396-GRFs module plays a critical role in greening of etiolated seedlings upon illumination.In order to further elucidate the defective greening phenotype of 35Spro:miR396a seedlings,we carried out RNA-Seq analysis using wild-type Col-0 and 35Spro:miR396a seedlings dark-grown for 4 days.GO enrichment analysis revealed significant enrichment of genes controlling chloroplast development and photosynthesis in miR396a regulated genes.These microarray data provide strong evidence that miR396 and GRFs play key regulatory roles in plant phomorphogenesis.Genetic analysis showed that GRFs depend on BZR1 and PIFs,while BZR1 and PIFs partly depend on GRFs in regulations of cell elongation and greening.Meanwhile,the interactions between GFR7 and BZR1 or PIF4 in vitro and in vivo were observed through ratiometric bimolecular fluorescence complementation(rBiFC)assays,protein-protein pull-down experiment and Co-immunoprecipitation assays.To further understand the importance of interactions between GRFs,BZR1 and PIF4 on greening of etiolated seedlings,we systematically analyzed the expression of Pchlide biosynthesis genes in GRFs,BZR1 or PIF4 related genetic materials.The results showed that the expression levels of HEMA1,HEMB1,CHLH,GUN4 and CHLD in det2-1 and pifq were significantly increased.The bzrl-1D could recover the high expression of these genes in det2-1,but had no effect on pifq.35Spro:MIM396 can partially inhibit the high expression of some genes in det2-1,but had no effect on pifq.These results indicated that BZR1,GRFs and PIFs could secretly inhibit the expression of Pchlide biosynthesis genes to regulate the content of Pchlide in the dark,but PIFs play more dominant roles.This study reveals the essential roles of phytohormone BRs in transition from skotomorphogenesis to photomorphogenesis of etiolated Arabidopsis seedlings,and clarify the molecular mechanism of BRs to opimize greening during de-etiolation through inhibiting excessive synthesis of Pchlide and ROS,providing fundamental basis for increasing the survival of crop seedlings using BRs during the initial emergence of seedlings from subterranean darkness into sunlight.Secondly,a new key factor miR396 was found in this paper.Overexpressing miR396 or weakening function of its target genes GRFs will lead to the over-accumulation of Pchlide and ROS and failure of etiolated seedlings to green upon exposure to light,while reducing miR396 activity will cause adverse situation.This study expands understanding not only on greening process of etiolated seedlings,but also on microRNA functions in plants.In addition,GRFs mediate the interaction between BRs and light to regulate plant growth and development processes,enhancing comprehension on the overlap regulations of BRs and light on plant growth and development processes.