Tribulus (Zygophyllaceae) as a case study for the evolution of C2 and C4 photosynthesis.

C2 photosynthesis is a photosynthetic pathway in which photorespiratory CO2 release and refixation are enhanced in leaf bundle sheath (BS) tissues. The evolution of C2 photosynthesis has been hypothesized to be a major step in the origin of C4 photosynthesis, highlighting the importance of studying C2 evolution. In this study, physiological, anatomical, ultrastructural, and immunohistochemical properties of leaf photosynthetic tissues were investigated in six non‐C4Tribulus species and four C4Tribulus species. At 42°C, T. cristatus exhibited a photosynthetic CO2 compensation point in the absence of respiration (C*) of 21 µmol mol−1, below the C3 mean C* of 73 µmol mol−1. Tribulus astrocarpus had a C* value at 42°C of 55 µmol mol−1, intermediate between the C3 species and the C2T. cristatus. Glycine decarboxylase (GDC) allocation to BS tissues was associated with lower C*. Tribulus cristatus and T. astrocarpus allocated 86% and 30% of their GDC to the BS tissues, respectively, well above the C3 mean of 11%. Tribulus astrocarpus thus exhibits a weaker C2 (termed sub‐C2) phenotype. Increased allocation of mitochondria to the BS and decreased length‐to‐width ratios of BS cells, were present in non‐C4 species, indicating a potential role in C2 and C4 evolution. Summary statement: Physiological, structural, and immunohistochemical analyses of leaf tissues indicate sub‐C2 and C2 character states occurring in Tribulus (Zygophyllaceae). These data reveal novel insights into the mechanisms by which photorespiratory CO2 refixation facilitates the evolution of C4 photosynthesis. [ABSTRACT FROM AUTHOR]