Full Assessment of C4 Photosynthesis in Blepharis attenuata Napper (Acanthaceae) from Jordan: Evidence from Leaf Anatomy and Key C4 Photosynthetic Enzymes

Blepharis constitutes an important element of the desert vegetation in the Middle East, inhabiting extreme desert habitats of the Saharo-Arabian and Sudanian regions. It is the only genus in the Acanthaceae that includes C4 species; however, comprehensive studies for these species are generally absent. This study presents a thorough assessment of C4 biochemical subtype in B. attenuata Napper through investigating leaf ultrastructural features and activities of C4 acid decarboxylating enzymes. Leaf anatomy of B. attenuata revealed a typical atriplicoid Kranz type, with prominent chlorenchymatous bundle sheath and mesophyll tissues forming concentric rings around leaf veins. Transmission electron microscopy showed characteristic features of NADP-malic enzyme subtype, with bundle sheath chloroplasts having grana with reduced thylakoid appression compared to mesophyll counterparts. Immunolocalizations of key C4 pathway enzymes showed a strict compartmentalization patterns between bundle sheath and mesophyll, with Rubisco localized in the bundle sheath and both PEP-carboxylase and pyruvate, orthophosphate dikinase were confined to the mesophyll. Measurements of enzyme activities showed that NADP-malic enzyme demonstrated the highest activity, suggesting that B. attenuata would best be classified as a NADP-malic enzyme subtype C4 species. This study reveals that Blepharis encompasses at least two biochemical subtypes of C4 photosynthesis within the genus.