Chloroplast biogenesis is critical for crop biomass and economic yield. However, chloroplast development is a very complicated process coordinated by cross-communication between the nucleus and plastids, and the underlying mechanisms have not been fully revealed. To explore the regulatory machinery for chloroplast biogenesis, we conducted map-based cloning of the Grandpa 1 (Gpa1) gene regulating chloroplast development in barley. The spontaneous mutation gpa1.a caused a variegation phenotype of the leaf, dwarfed growth, reduced grain yield, and increased tiller number. Genetic mapping anchored the Gpa1 gene onto 2H within a gene cluster functionally related to photosynthesis or chloroplast differentiation. One gene (HORVU.MOREX.r3.2HG0213170) in the delimited region encodes a putative plastid terminal oxidase (PTOX) in thylakoid membranes, which is homologous to IMMUTANS (IM) of Arabidopsis. The IM gene is required for chloroplast biogenesis and maintenance of functional thylakoids in Arabidopsis. Using CRISPR technology and gene transformation, we functionally validated that the PTOX-encoding gene, HORVU.MOREX.r3.2HG0213170, is the causal gene of Gpa1. Gene expression and chemical analysis revealed that the carotenoid biosynthesis pathway is suppressed by the gpa1 mutation, rendering mutants vulnerable to photobleaching. Our results showed that the overtillering associated with the gpa1 mutation was caused by the lower accumulation of carotenoid-derived strigolactones (SLs) in the mutant. The cloning of Gpa1 not only improves our understanding of the molecular mechanisms underlying chloroplast biosynthesis but also indicates that the PTOX activity is conserved between monocots and dicots for the establishment of the photosynthesis factory.
Keywords: CRISPR; Gpa1 cloning; barley; chloroplast biogenesis; plastid terminal oxidase.
Published 2023. This article is a U.S. Government work and is in the public domain in the USA.