Tribulus (Zygophyllaceae) as a case study for the evolution of C 2 and C 4 photosynthesis.

C 2 photosynthesis is a photosynthetic pathway in which photorespiratory CO 2 release and refixation are enhanced in leaf bundle sheath (BS) tissues. The evolution of C 2 photosynthesis has been hypothesized to be a major step in the origin of C 4 photosynthesis, highlighting the importance of studying C 2 evolution. In this study, physiological, anatomical, ultrastructural, and immunohistochemical properties of leaf photosynthetic tissues were investigated in six non-C 4 Tribulus species and four C 4 Tribulus species. At 42°C, T. cristatus exhibited a photosynthetic CO 2 compensation point in the absence of respiration (C * ) of 21 µmol mol -1 , below the C 3 mean C * of 73 µmol mol -1 . Tribulus astrocarpus had a C * value at 42°C of 55 µmol mol -1 , intermediate between the C 3 species and the C 2 T. 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 C 3 mean of 11%. Tribulus astrocarpus thus exhibits a weaker C 2 (termed sub-C 2 ) phenotype. Increased allocation of mitochondria to the BS and decreased length-to-width ratios of BS cells, were present in non-C 4 species, indicating a potential role in C 2 and C 4 evolution.