GSE106223
The COP9 Signalosome regulates seed germination by facilitating protein degradation of RGL2 and ABI5
The control of seed germination and seed dormancy are critical for the successful propagation of plant species, and are important agricultural traits. Seed germination is tightly controlled by the balance of gibberellin (GA) and abscisic acid (ABA), and is influenced by environmental factors. The COP9 Signalosome (CSN) is a conserved multi-subunit protein complex that is best known as a regulator of the Cullin-RING family of ubiquitin E3 ligases (CRLs). Multiple viable mutants of the CSN showed poor germination, except for csn5b-1. Detailed analyses showed that csn1-10 has a stronger seed dormancy, while csn5a-1 mutants exhibit retarded seed germination in addition to hyperdormancy. Both csn5a-1 and csn1-10 plants show defects in the timely removal of the germination inhibitors: RGL2, a repressor of GA signaling, and ABI5, an effector of ABA responses. We provide genetic evidence to demonstrate that the germination phenotype of csn1-10 is caused by over-accumulation of RGL2, a substrate of the SCF (CRL1) ubiquitin E3 ligase, while the csn5a-1 phenotype is caused by over-accumulation of RGL2 as well as ABI5. The genetic data are consistent with the hypothesis that CSN5A regulates ABI5 by a mechanism that may not involve CSN1. Transcriptome analyses suggest that CSN1 has a more prominent role than CSN5A during seed maturation, but CSN5A plays a more important role than CSN1 during seed germination, further supporting the functional distinction of these two CSN genes. Our study delineates the molecular targets of the CSN complex in seed germination, and reveals that CSN5 has additional functions in regulating ABI5, thus the ABA signaling pathway. Overall design: Col-0, csn1-10, and csn5a-1 seeds were collected from plants grown side-by-side in the growth room. Seeds for Col-0 and csn1-10 were 1.5wks in storage, and csn5a-1 seeds were 4wk in storage. Approximately 100 microliter of settled seeds were used for RNA extraction for each sample. The 2-day imbibed seeds, prepared with the same volume of dry seeds as above, were sterilized, washed, and incubate on cell culture wells with 1ml of 0.5X liquid MS medium at 22oC under constant light for 2 days. Each sample points had three biological repeats. Seeds were centrifuged to remove the liquid, and were frozen in liquid nitrogen. The frozen seeds were ground to a fine powder using mortar and pestle in the presence of liquid nitrogen and small quantity of sterile quartz powder. RNA extraction was performed according to a published procedure [73]. High-throughput RNA-seq was carried out at Yale Center for Genome Analysis. The Single-End RNA-sequencing was carried out with Illumina Hi-seq 2000 platform (Genome Center, Yale West Campus). Specifically, libraries were analyzed with a Bioanalyzer 2100 instrument (Agilent, Santa Clara, CA), quantified by Qubit fluorometer (Life Technologies, Carlsbad, CA). The Arabidopsis thaliana genome obtained from TAIR10 (https://www.arabidopsis.org) was used as the genome reference. After adaptor trimming and contaminate sequence removing by fastqc (www.bioinformatics.babraham.ac.uk/projects/fastqc/) and fastx-toolkits (hannonlab.cshl.edu/fastx_toolkit/), Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) was used for genome mapping and followed by the tophat (https://ccb.jhu.edu/software/tophat/index.shtml) transcript assembling. Gene differentially expression profiling was accomplished by cufflink and cuffdiff software package (cole-trapnell-lab.github.io/cufflinks/cuffdiff/) with default parameters and cutoffs, fold change cutoff was set to 2.