Chlorophyll, the pigment in plant leaves, is crucial for capturing light during photosynthesis. Mutations that disrupt chlorophyll production or chloroplast development frequently lead to changes in leaf color. Here, we report the identification and characterization of a chlorotic mutant in cucumber (Cucumis sativus L.), named tnyl3, which exhibits chlorotic cotyledons and seedling lethality. Map-based cloning revealed that the tnyl3 mutation results from a Tnt1 retrotransposon insertion in a gene encoding nuclear factor Y subunit B3 (NF-YB3), a transcription factor subunit. Compared with the wild type, the mutant exhibited dramatic decreases in chlorophyll a and b levels and a significantly lower net photosynthetic rate. Ultrastructural analysis revealed that the chloroplasts in the tnyl3 mutants are structurally abnormal and characterized by underdeveloped thylakoid membranes. Gene expression analyses revealed that CsNF-YB3 is highly expressed in young seedling tissues and is upregulated by light. CRISPR/Cas9 analyses subsequently confirmed that the tnyl3 phenotype is caused by the loss of CsNF-YB3. Yeast two-hybrid and split-LUC assays demonstrated that CsNF-YB3 interacts directly with the cucumber NF-YC2 protein and promotes CsTIC21 transcription, suggesting that it functions as part of an NF-Y transcriptional complex. Transcriptome profiling of the mutant revealed extensive downregulation of photosynthesis-related genes, which is consistent with its impaired chloroplast function. Our findings establish CsNF-YB3 as a crucial genetic factor for chloroplast development and pigment synthesis in cucumber. This work provides new insight into the NF-Y–mediated regulatory network controlling chloroplast biogenesis and offers a potential genetic target for improving plant photosynthetic performance and vigor.

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