Biochemical and structural differences between C3 cotyledons and C4 leaves in species of Salsoloideae (Chenopodiaceae)

C₄ 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.