The miR396-GRFs Module Mediates Brassinosteroid-Prevented Photooxidative 1 Damage during Seedling De-etiolation in Arabidopsis

The developmental switch from skotomorphogenesis to photomorphogenesis is critical for the survival and growth of plants, but its regulatory mechanism remains unclear. Here, we report that the steroid hormone brassinosteroids (BRs) play crucial roles in the transition from skotomorphogenesis to photomorphogenesis by regulating chlorophyll biosynthesis to promote the greening of etiolated seedlings upon light exposure. Seedlings of BR-deficient mutant det2-1 accumulated excess protochlorophyllide when grown in darkness, resulting in photo-oxidative damage upon exposure to light. Conversely, the gain-of-function mutant bzr1-1D suppressed the protochlorophyllide-accumulated phenotype of det2-1, thereby promoting greening of etiolated seedlings. Genetic analysis indicated that phytochrome-interacting factors (PIFs) were required for BZR1-promoted seedlings greening. Furthermore, we revealed that the GROWTH REGULATING FACTOR 7 (GRF7) and GRF8 were induced by BZR1 and PIF4 to repress the chlorophyll biosynthesis and promote seedling greening. Suppression the functions of GRFs by overexpressing microRNA396a (miR396a) caused the high-accumulated photochlorophyllide in darkness and more serious photobleach upon light exposure. Additionally, BZR1, PIF4 and GRF7 interact with each other and precisely regulate the expression of chlorophyll biosynthetic genes. Our findings revealed an essential role of brassinosteroid in promoting seedling development and survival during the critical initial emergence of seedlings from subterranean darkness to sunlight. The seedlings of wild type Col-0 and 35S:miR396a were grown on ½ MS medium containing 1% sucrose for 4 days in the dark, and treated with or without light for 3 hrs. Total RNA was extracted with Trizaol RNA extraction kit (Transgene), and the mRNA sequencing libraries construction and sequencing on the BGISEQ-500 platform were performed at Beijing Geonomics Institute. The sequence reads were mapped to theArabidopsis genome using HISAT and Bowtie2 software, and differential geneexpression was analyzed using Noiseq software. Differentially expressed genes were defined by a 2-fold expression difference with a possibility >0.8.