At around the same time as the nature of photorespiration was becoming clearer Hatch and Slack (1966) demonstrated that in tropical grasses (initially sugar cane) the first-formed products of photosynthetic CO₂ fixation were the four-carbon acids oxalacetate, malate and aspartate, rather than the 3-PGA formed in the PCR cycle. Furthermore, the carboxylation reaction involved PEP carboxylase and carbon was subsequently transferred to PCR cycle intermediates. As noted earlier (Section 2.2) C₄ plants show no apparent CO₂ release in light. The explanation lies in their anatomy and multiple carboxylation reactions rather than in the absence of the pathway of photorespiration. Bundle sheath cells are equipped with a CO₂-concentrating mechanism that favours carboxylation over oxygenation reactions due to increased partial pressure of CO₂, while photorespiratory release of CO₂ is further prevented through the activity of PEP carboxylase which refixes any respired CO₂ formed from the oxygenase function of Rubisco.

Unicellular green algae also posed a problem for the simple extrapolation of early models for photorespiratory metabolism in C₃ leaves. Although organisms such as Chlorella had been used to establish the PCR cycle, and indeed provided much early evidence for effects of O₂ on photosynthesis and formation of glycolate in light, they also appeared to lack CO₂ evolution in light (Lloyd et al. 1977). In this case the explanation lies in a CO₂-concentrating mechanism which effectively increases the internal pool of inorganic carbon (CO₂ and HCO₃^–) thereby favouring the carboxylase function of Rubisco over its oxygenase function.