PeFtsH1 and PeFtsH2 form a heteromeric hexamer and play conserved roles in chloroplast development and leaf variegation in Phalaenopsis equestris

Breeding new elite variegated cultivars of Phalaenopsis equestris remains a significant challenge due to the scarcity of variegated germplasm resources, despite their high ornamental and commercial value. Here, we investigate the roles of PeFtsH1 and PeFtsH2 in chloroplast development and leaf variegation formation in Phalaenopsis equestris. Phylogenetic analysis showed that thylakoid membrane-localized PeFtsH protease is composed of PeFtsH1 and PeFtsH2 subunits, which are homologs of Arabidopsis VAR1/FtsH5 (type A) and VAR2/FtsH2 (type B), respectively. Genetic complementation assays showed that both PeFtsH1 and PeFtsH2 can rescue the leaf variegation phenotype of var1 and var2 mutants, respectively. Consistently, reduced expression of PeFtsH1 or PeFtsH2 via virus-induced gene silencing (VIGS) resulted in variegated leaves, reduced photosynthetic efficiency, and defective thylakoid development. Furthermore, we found that PeFtsH1 and PeFtsH2 form a heteromeric hexamer with a subunit stoichiometry of approximately 2:1. These findings suggest an evolutionary adaptation of P. equestris to low-light and high-humidity environments. Taken together, our findings reveal a conserved role of PeFtsH1 and PeFtsH2 in chloroplast development through the formation of a 2:1 heterohexameric complex, providing key molecular insights into the mechanism of chloroplast development and leaf variegation in Phalaenopsis equestris. These insights open an avenue for improving ornamental traits in orchids through targeted breeding.