Cullin4-Ring ligase-mediated filamenting temperature-sensitive Z 2 homeostasis affects plastid level and fruit...

Plants have evolved intricate regulatory mechanisms to balance growth and defense. In particular, the UV-damaged DNA-binding protein 1 (DDB1), which can form an E3 ubiquitin ligase with CUL4 (Cullin4-Ring Ligase, CRL4), is widely involved in plant growth and response to adversity. The tomato spontaneous SlDDB1-defective high pigment 1 (hp1) mutant manifests significantly increased plastid level and pigments accumulation, but the underlying molecular mechanism remains unclear. Here we report the isolation and characterization of two novel SlDDB1-interacting proteins, SlFtsZ2-1 and SlFtsZ2-2, identified by a yeast two-hybrid assay. They showed constitutive expression patterns and chloroplast localizations. CRISPR/Cas9 knockout uncovered a functional redundancy between SlFtsZ2-1 and SlFtsZ2-2 since only their double knockout mutant displayed significantly decreased plastid level and fruit nutrient accumulation. The interactions between the SlDDB1/SlCUL4 and SlFtsZ2-1/SlFtsZ2-2 within chloroplasts were subsequently validated through co-immunoprecipitation and fluorescence-based assays. Furthermore, biochemical and molecular analyses demonstrated that both SlFtsZ2-1/SlFtsZ2-2 proteins are targeted for ubiquitination and degradation by the CRL4 E3 ligase complex, uncovering a previously unknown role of CRL4 in plastid proteostasis. Collectively, our findings elucidate a novel regulatory module, SlCUL4-SlDDB1-SlFtsZ2, which is distinct from the existing chloroplast-associated protein degradation (CHLORAD) pathway. This module plays a pivotal role in the precise control of SlFtsZ2 protein homeostasis, thereby influencing the plastid level and fruit quality in tomato. This study provides a mechanistic foundation for improving crop nutrient content through ubiquitination pathway manipulation and indicates potential agricultural applications in fruit quality regulation.