Integrated physiological and transcriptomic analyses reveal insight into leaf coloration variation in cucumber

• Cucumber mutant shows yellow leaves with less chlorophyll and abnormal chloroplasts. • Identified 1702 potential genes affecting photosynthesis and chloroplast development. • Two bZIP transcription factors control chlorophyll and leaf color. • Chlorophyll b reakdown gene CsPTC52 potentially regulated by MYB/bHLH factors. • New model explains how leaf yellowing occurs in mutants. Leaf color mutations contribute to the diverse coloration observed in higher plants and serve as optimal materials for studying pigment metabolism, chloroplast development, and photosynthesis. Here, we identified a novel dominant chlorophyll (Chl) - deficient mutant with yellow-green leaves ( Ygl3 ), which exhibited lower Chl content and a higher Chl a/b ratio than its WT type. Observation of micro- and ultra- structural features revealed that sponge cells were loosely arranged, and that the chloroplasts were abnormal in the Ygl3 leaves, which was further supported by a reduction in light-capture capability. Transcriptome analysis identified 1702 differentially expressed genes, comprising 726 upregulated and 976 downregulated genes. Notably, genes involved in photosynthesis ( Lhcb1 s, PsbA , and petF ) and chloroplast development ( HSP s, SIG2 s, CYP707A2 / A4, H3K36, HY5 s, and PIF4 ) were significantly differentially expressed in the Ygl3 mutant. Also, the expression of transcription factor (TF) family genes, including AP2-ERF, MYB, bHLH, WRKY, and bZIP was abundant; among them, two homologous bZIP s (CsaV3_2G031960 and CsaV3_3G046530) emerged as hub genes in the protein-protein interaction network, elucidating the connection between TFs and physiological characteristics of Ygl3 . By integrating current knowledge and prediction of transcription factor binding sites, we preliminarily established a transcriptional model suggesting that the expression of the CsPTC52 gene, which encodes protochlorophyllide oxidoreductase translocon protein, may be directly regulated by MYB and/or bHLH, thereby activating Chl metabolism. This regulation affected Chl content, chloroplast development, and light harvesting ability, ultimately contributing to leaf yellowing. Our findings provide valuable information regarding the cucumber Ygl3 mutant at phenotypic, physiological, cytological, and transcriptional levels.