CsNF-YB3 acts as a key switch for cucumber greening
image: A model chloroplast development in the WT and tnyl3 mutants. In the WT, CsNF-YB3 interacts with the light-responsive subunit CsNF-YC2 to form a nuclear NF-YB/YC complex, which binds CCAAT-box motifs to activate CsTIC21 genes crucial for chloroplast biogenesis. However, in tnyl3 mutants, the loss of CsNF-YB3 causes chlorosis and chloroplast development defects in cucumber.
Credit: Horticulture Research
A pale cucumber seedling can reveal what keeps a plant alive. Researchers have identified a nuclear gene that acts as a central regulator of chloroplast formation, chlorophyll accumulation, and early photosynthetic growth in cucumber. When the cucumber Nuclear Factor Y subunit B3 gene (CsNF-YB3) was disrupted, seedlings produced yellow cotyledons, malformed chloroplasts, and only a fraction of normal chlorophyll, leaving them unable to sustain ordinary photoautotrophic development. The study further shows that the CsNF-YB3 protein works with the CsNF-YC2 protein to activate a chloroplast-development gene, connecting light-responsive nuclear regulation with the cellular machinery that builds functional chloroplasts. The findings reveal a potential genetic entry point for improving seedling vigor and photosynthetic performance.
Chloroplasts convert light into chemical energy, making their formation essential for crop establishment and productivity. Leaf-color mutants have helped scientists uncover enzymes, transporters, ribosomal factors, and structural proteins involved in chlorophyll synthesis and plastid development. Yet many cucumber mutants studied so far affect chloroplast components themselves, while the upstream nuclear regulators that coordinate chloroplast biogenesis remain less clearly defined. Nuclear Factor Y (NF-Y) transcription factors are known to influence light signaling and photosynthesis in several plant species, but the roles of individual NF-Y subunits can differ by tissue, developmental stage, and protein partner. Because of these challenges, deeper investigation is needed into how specific nuclear transcriptional modules control chloroplast development and early seedling greening in cucumber.
Researchers from Shenyang Agricultural University, Shanghai Jiao Tong University, the Liaoning Academy of Agricultural Sciences, and the Shanghai Pudong Agrotechnology Extension Center reported (DOI: 10.1093/hr/uhag138) the findings in Horticulture Research on May 8, 2026. The study identifies cucumber Nuclear Factor Y subunit B3 gene (CsNF-YB3) as the gene responsible for the seedling-lethal chlorotic mutant tnyl3 in cucumber (Cucumis sativus). By combining genetic mapping, genome editing, cell imaging, protein-interaction tests, promoter analysis, and transcriptome profiling, the team reconstructed a nuclear regulatory pathway that links light-responsive NF-Y activity to chloroplast biogenesis and photosynthetic competence.
The team first traced the tnyl3 phenotype to a 5.3-kilobase Tnt1 retrotransposon insertion that disrupted an exon of CsNF-YB3. Expanded genotyping across 288 second-generation seedlings showed that this insertion consistently cosegregated with chlorosis, supporting single-gene recessive inheritance. In mutant cotyledons, chlorophyll a and b fell to roughly 2%–3% of wild-type (WT) levels, while net photosynthesis dropped below one-third of WT performance. Transmission electron microscopy (TEM) showed irregular chloroplasts with fragmented thylakoid membranes and poorly developed grana. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) knockout lines reproduced the yellow, stunted phenotype, confirming causality. Expression analysis showed that CsNF-YB3 is most active in cotyledons and rises about 2.5-fold under light. Yeast two-hybrid and split-luciferase complementation assays then revealed a selective interaction between the CsNF-YB3 and CsNF-YC2 proteins. Together, they strengthened activation of CsTIC21, which encodes a component of the chloroplast protein-import machinery. Ribonucleic acid sequencing (RNA-seq) identified 5,682 differentially expressed genes: 2,721 were downregulated and 2,961 upregulated. Photosystem, light-harvesting, chlorophyll-biosynthesis, and plastid-ribosome genes were broadly suppressed, while stress- and hormone-responsive programs increased, explaining both the loss of photosynthetic capacity and the mutant’s severe growth failure.
The authors said the results place CsNF-YB3 near the top of a regulatory chain that determines whether young cucumber tissues can build working chloroplasts and begin independent growth. They said the striking seedling lethality makes the gene especially informative: rather than producing only a cosmetic leaf-color change, its loss collapses the photosynthetic program across cellular structure, pigment production, protein import, and nuclear gene expression. The authors added that the selective partnership between CsNF-YB3 and CsNF-YC2 helps explain why closely related NF-Y family members cannot simply compensate for the missing regulator.
The findings provide a molecular framework for breeding or engineering cucurbits with stronger early growth, improved light-use efficiency, and greater photosynthetic resilience. However, because complete loss of CsNF-YB3 is lethal, practical strategies would need to avoid blunt knockout or unrestricted manipulation. More realistic routes may include identifying favorable natural alleles, tuning expression levels, or using tissue- and stage-specific regulation to enhance performance without disrupting chloroplast development. The study also gives researchers a candidate module for examining how light, hormone, and stress signals converge on chloroplast biogenesis. Before crop applications can be considered, direct genomic targets of the CsNF-YB3/CsNF-YC2 complex should be mapped, and complementation, field-performance, and cross-species studies will be needed.
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References
DOI
Original Source URL
https://doi.org/10.1093/hr/uhag138
Funding information
This work was supported by Shanghai Agricultural Science and Technology Innovation 586 Project (T2024305).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.