C4 photosynthesis and transition of Kranz anatomy in cotyledons and leaves of Tetraena simplex

Premise of the Study: Tetraena simplex is an independently evolved C-4 species in the Zygophylloideae (Zygophyllaceae) and a characteristic forb of saline flats in hot and sandy desert habitats. During early ontogeny, the species had a morphological shift from planar cotyledons (dorsiventral symmetry) to terete, succulent leaves (radial symmetry). We tested whether this shift had a corresponding change in internal Kranz anatomy and tissue patterning. Methods: For a comprehensive characterization of C-4 photosynthesis across early ontogeny in T. simplex, structural and ultrastructural anatomical properties and localization patterns, activities, and immunoblotting of key C-4 photosynthetic enzymes were compared in mesophyll and bundle sheath tissues in cotyledons and leaves. Key Results: Cotyledons and leaves possessed different types of Kranz anatomy (atriplicoid type and a "Tetraena" variant of the kochioid type, respectively), reflecting the change in leaf morphology. In bundle sheath cells, key differences in ultrastructural features included increased organelle numbers and chloroplast thylakoid stacking. C-4 enzymes had strict tissue-specific localization patterns within bundle sheath and mesophyll cells in both cotyledons and leaves. The decarboxylase NAD-ME maintained the highest activity, increasing from cotyledons to leaves. This classified T. simplex as fully C-4 across ontogeny and a strictly NAD-ME biochemical subtype. Conclusions: Tetraena simplex cotyledons and leaves showed differences in Kranz type, with associated progression in ultrastructural features, and differing activities/expression levels of C-4 enzymes. Furthermore, leaves characterized a new "Tetraena" variation of the kochioid Kranz anatomy.