Abstract
In Arabidopsis (Arabidopsis thaliana), the F-box protein F-BOX-LIKE17 (FBL17) was previously identified as an important cell-cycle regulatory protein. FBL17 is required for cell division during pollen development and for normal cell-cycle progression and endoreplication during the diploid sporophyte phase. FBL17 was reported to control the stability of the CYCLIN-DEPENDENT KINASE inhibitor KIP-RELATED PROTEIN (KRP), which may underlie the drastic reduction in cell division activity in both shoot and root apical meristems observed in fbl17 loss-of-function mutants. However, whether FBL17 has other substrates and functions besides degrading KRPs remains poorly understood. Here we show that mutation of FBL17 leads not only to misregulation of cell cycle genes, but also to a strong upregulation of genes involved in DNA damage and repair processes. This phenotype is associated with a higher frequency of DNA lesions in fbl17 and increased cell death in the root meristem, even in the absence of genotoxic stress. Notably, the constitutive activation of DNA damage response genes is largely SOG1-independent in fbl17 In addition, through analyses of root elongation, accumulation of cell death, and occurrence of γH2AX foci, we found that fbl17 mutants are hypersensitive to DNA double-strand break-induced genotoxic stress. Notably, we observed that the FBL17 protein is recruited at nuclear foci upon double-strand break induction and colocalizes with γH2AX, but only in the presence of RETINOBLASTOMA RELATED1. Altogether, our results highlight a role for FBL17 in DNA damage response, likely by ubiquitylating proteins involved in DNA-damage signaling or repair.
Highlights
In all eukaryotes, the cell cycle is composed of four phases: in S phase DNA replication occurs and in mitosis (M) phase, chromosomes segregate into two nuclei, followed by cytokinesis, allowing cells to be divided into two daughter cells (Nurse, 2000)
Considering all differentially expressed (DE) genes in fbl17, a Gene Ontology (GO) term enrichment analysis, based on Biological Process functional categories of the ShinyGo software, revealed that genes which expression is widely altered in the mutant are involved in primary metabolic pathways such as the photosynthesis, and other cellular responses, most of them being related to stress conditions (Supplemental Table 2), in line with the severe global phenotypic alterations of the mutant plants
By filtering DE genes based on Fold Change, the comparative RNA sequencing (RNA-seq) analysis revealed that still more than 1400 genes are DE in fbl17 mutant and their GO term enrichment analysis highlighted their implication in cell cycle progression, DNA replication mechanisms, chromosome dynamics and, in an unexpectedly extended manner, DNA repair and stress response
Summary
The cell cycle is composed of four phases: in S phase DNA replication occurs and in mitosis (M) phase, chromosomes segregate into two nuclei, followed by cytokinesis, allowing cells to be divided into two daughter cells (Nurse, 2000). Several CDKs are inactivated by cyclin-dependent kinase inhibitors CKIs (Denicourt and Dowdy, 2004) and both in fungi and metazoans, it has been established that CKI degradation at the G1to-S transition releases CDK activity, which in turn is required to enter S phase. In budding yeast, this is achieved by the ubiquitin E3 ligase complex SCFCdc (Skp, Cdc53/CULLIN, and Cdc, a WD40-type F-box protein), which ubiquitylates the CKI Sic protein leading to its proteolysis shortly before S phase (Schwob et al, 1994)(Feldman et al, 1997). The human SCFSKP2 E3 targets several other essential regulators of S-phase progression as well as other regulatory proteins
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