Screening and Functional Validation of the Chlorophyll Synthesis-Related Gene StaHemF in Sinobambusa tootsik f. albostriata

Abstract

Leaf color variation is an important ornamental trait in bamboo and is primarily determined by chlorophyll accumulation. However, the molecular mechanisms underlying leaf color differentiation in Sinobambusa tootsik f. albostriata remain largely unclear. In this study, fully green (WG) and fully white (WW) leaf buds at three developmental stages (S1–S3) were analyzed to investigate the regulatory mechanisms associated with chlorophyll accumulation and identify key functional genes. Chlorophyll content determination, coproporphyrinogen III oxidase (CPOX) activity assays, and comparative transcriptome analysis were integrated to identify candidate genes involved in leaf color formation. Six StaHemF family members were identified, and StaHemF5 was selected as the primary candidate based on phylogenetic relationships and expression patterns. Compared with WG leaf buds, WW leaf buds exhibited significantly reduced chlorophyll contents and CPOX activities throughout development, indicating impaired chlorophyll biosynthesis. StaHemF5 encodes a chloroplast-localized protein and displays consistent expression trends between transcriptome analysis and qRT-PCR validation. Functional characterization revealed that transient overexpression of StaHemF5 in Nicotiana benthamiana significantly enhanced CPOX activity and chlorophyll accumulation, suggesting a positive role of StaHemF5 in chlorophyll biosynthesis. Collectively, these results demonstrate that StaHemF5 is a conserved HemF family member involved in chlorophyll accumulation and contributes to leaf color differentiation in S. tootsik f. albostriata. This study provides new insights into the molecular mechanisms underlying bamboo leaf color variation and identifies a potential candidate gene for the genetic improvement of ornamental bamboo.