Kocacinar, F.Ceylan, FaikCömertpay, S.2025-11-202025-11-202025https://doi.org/10.1038/s41598-025-20388-whttps://hdl.handle.net/20.500.12684/22159C4 plants had evolved from C3 ancestors in at least more than 60 independent lineages of angiosperms, most probably in response to decreasing levels of atmospheric CO2 and environmental conditions promoting photorespiration. Salsola, Petrosimonia, and Cyathobasis genera of Salsoloideae subfamily contain some species with C3 cotyledons followed by C4 leaves. The aim of this study was to determine and compare the biochemical and structural differences between C3 cotyledons and subsequent C4 leaves in these genera. The results showed that there were isopalisade C3 cotyledons in Salsola grandis and isolateral C3 cotyledons in Cyathobasis fruticulosa, while salsoloid type C4 Kranz anatomy was present in subsequent leaves. Petrosimonia nigdeensis had dorsiventral C3 cotyledons and salsoloid-type C4 leaves. The proteins of C4 isoform phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) enzymes were absent or very sparse in C3 cotyledons of these species, whereas they were abundant in their C4 leaves. The proteins of photorespiratory isoform glycolate oxidase (GOX) and glycine decarboxylase-H subunit (GDC-H) were generally higher in cotyledons than leaves. The protein level of photosynthetic isoform ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) enzyme was lower in C4 leaves compared to C3 cotyledons. Transcript levels of these enzymes were generally consistent with their protein content except for GOX in S. grandis and S. tragus and glycine decarboxylase complex (GDC) in S. tragus. As a result, we demonstrated that not only the protein and transcript levels of the enzymes required for C4 pathway increased, but also those levels of C3 and photorespiratory enzymes decreased during the transition from C3 cotyledons to C4 leaves. These results are considered significant in terms of shedding light on the understanding of evolutionary transition from C3 to C4 biochemical pathway in a single plant and contributing to C4 engineering.en10.1038/s41598-025-20388-winfo:eu-repo/semantics/closedAccessC3 cotyledons and C4 leavesSalsoloideaeLeaf anatomyQuantitative real time-polymerase chain reaction (qRT-PCR)Western blotPhotosynthetic enzymesC4 evolutionArticle1152-s2.0-105019114105WOS:001597098400022Q1Q1