SUMMARY

The coordination of chloroplast and nuclear genome status is critical for plant cell function. Here, we report that Arabidopsis CHLOROPLAST AND NUCLEUS DUAL-LOCALIZED PROTEIN 1 (CND1) maintains genome stability in the chloroplast and the nucleus. CND1 localizes to both compartments, and complete loss of CND1 results in embryo lethality. Partial loss of CND1 disturbs nuclear cell-cycle progression and photosynthetic activity. CND1 binds to nuclear pre-replication complexes and DNA replication origins and regulates nuclear genome stability. In chloroplasts, CND1 interacts with and facilitates binding of the regulator of chloroplast genome stability WHY1 to chloroplast DNA. The defects in nuclear cell-cycle progression and photosynthesis of cnd1 mutants are respectively rescued by compartment-restricted CND1 localization. Light promotes the association of CND1 with HSP90 and its import into chloroplasts. This study provides a paradigm of the convergence of genome status across organelles to coordinately regulate cell cycle to control plant growth and development.

INTRODUCTION

Chloroplasts in plants and algae have an endosymbiotic origin: over 2 billion years ago, a photosynthetic cyanobacterium-like organism was stably engulfed by a heterotrophic unicellular eukaryote.1 This initial event was followed over the course of evolution by massive gene transfer from the green prokaryote genome to the host-cell nucleus, leading to the complete dependency of the endosymbiont on its surrounding cells and the generation of a novel type of organism, photosynthetic eukaryotes.2 The development of higher plants requires the tight coordination of chloroplast development from undifferentiated proplastids in meristematic cells or from etioplasts, which differentiate from proplastids when seeds germinate in darkness. In addition to being the site of photosynthesis in leaf mesophyll cells, chloroplasts house essential metabolic pathways and contribute to storage and pigmentation in specialized cells.3

Eukaryote genomes are organized into multiple chromosomes whose replication initiates from numerous origins and is strictly regulated.4 To maintain genome integrity and stability, genomic DNA must be replicated completely and accurately, but also only once during the S phase of the cell cycle. The formation of a pre-replication complex (pre-RC) is required for the subsequent ordered assembly of replication factors such as the Origin Recognition Complex (Orc), Cell Division Cycle6 (Cdc6) in budding yeast (Saccharomyces cerevisiae) or its ortholog CDC18 in fission yeast (Schizosaccharomyces pombe), chromatin licensing and DNA replication factor 1 (CDT1), and Mini Chromosome Maintenance (MCM) proteins (MCM2–۷).۵ MCM proteins are essential replication initiation factors that were originally identified as proteins required for minichromosome maintenance, plasmid replication, or cell-cycle progression in yeast.6 Indeed, cell-cycle checkpoints are crucial for genome integrity, as they stop cell-cycle progression upon DNA damage until repairs have been completed. Such mechanisms are particularly important in plants, which endure daily exposure to DNA-damaging agents such as light and reactive oxygen species (ROS).7

RESULTS

CND1 is a nucleus and chloroplast dual-localized protein
To understand the functional coordination between the chloroplast and nucleus, we systematically screened proteins with predicted chloroplast and nucleus dual localization by combining bioinformatics and proteomics analyses. We focus here on one such putative chloroplast and nucleus dual-localized protein, encoded by the nuclear gene At1g32730 that we named CHLOROPLAST AND NUCLEUS DUAL-LOCALIZED PROTEIN 1 (CND1). CND1 contains four predicted functional domains: two targeting domains, consisting of an N-terminal chloroplast transit peptide (cTP) (amino acids [aa] 1–۶۵) based on TargetP prediction27 and a nuclear localization sequence (NLS) containing the amino acid motif KRKK28; a zinc finger domain (ZF); and the domain of unknown function DUF702 (Figure 1A ).