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-C₄ Tribulus species and four C₄ Tribulus 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 C₂ 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 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.