Nonstomatal limitations are responsible for drought-induced photosynthetic inhibition in four C₄ grasses

•   Here, the contribution of stomatal and nonstomatal factors to photosynthetic inhibition under water stress in four tropical C₄ grasses was investigated (Panicum coloratum, Bothriochloa bladhii, Cenchrus ciliaris and Astrebla lappacea). •   Plants were grown in well watered soil, and then the effects of soil drying were measured on leaf gas exchange, chlorophyll a fluorescence and water relations. •   During the drying cycle, leaf water potential (Ψ_leaf ) and relative water content (RWC) decreased from c. -0.4 to -2.8 MPa and 100-40%, respectively. The CO₂ assimilation rates (A) and quantum yield of PSII (Φ_PSII ) of all four grasses decreased rapidly with declining RWC. High CO₂ concentration (2500 µl l^-1 ) had no effect on A or Φ_PSII at any stage of the drying cycle. Electron transport capacity and dark respiration rates were unaltered by drought. The CO₂ compensation concentrations of P. coloratum and C. ciliaris rose sharply when leaf RWC fell below 70%. In P. coloratum, 5% CO₂ did not prevent the decline of O₂ evolution rates under water stress. •   We conclude that inhibition of photosynthesis in the four C₄ grasses under water stress is dependent mainly on biochemical limitations.