Simultaneous defects in chloroplast development and division cause leaf variegation in Arabidopsis

Chloroplast biogenesis is essential not only for photosynthesis but also for the synthesis of many metabolites critical for plant growth and human nutrition. Leaf variegation provides a powerful model for dissecting the process of chloroplast biogenesis, which encompasses both chloroplast development and division. Here, we show that the Arabidopsis thaliana leaf variegation mutant var2, defective in the thylakoid protease FtsH2, exhibits severe defects in chloroplast biogenesis. Confocal and ultrastructural analyses revealed that chloroplast development is delayed yet prolonged in the green sectors of var2, leading to increased cellular heterogeneity in chloroplast number and size. Strikingly, plastid-free cells were observed in white sectors, indicating impaired chloroplast division. Consistent with this, loss of the chloroplast division factors Paralog of Accumulation and Replication of Chloroplasts 6 (PARC6) or Plastid Division 1 (PDV1) exacerbated var2 variegation, whereas overexpression of PDV1 or PDV2 suppressed it. Similarly, chloroplast division was compromised in the variegated mutant immutans, whereas the virescent mutant clpr4, which exhibits increased chloroplast number, rescued leaf variegation phenotype of var2. Furthermore, VAR2-regulated chloroplast development and division are mediated by Constitutively Photomorphogenic 1 (COP1) and autophagy-related ATG8a, respectively. Collectively, our findings demonstrate that leaf variegation arises from simultaneous defects in chloroplast development and division, unveiling a coordinated regulatory mechanism that maintains chloroplast homeostasis.