SummaryAt glacial CO2, NAD-ME grasses have higher photosynthetic water use efficiency than NADP-ME and PCK counterparts. Photosynthetic carboxylases rather than decarboxylases modulate the response of C4 photosynthesis to glacial CO2

C4 photosynthesis evolved multiple times in diverse lineages. Most physiological studies comparing C4 plants were not conducted at the low atmospheric CO2 prevailing during their evolution. Here, 24 C4 grasses belonging to three biochemical subtypes [nicotinamide adenine dinucleotide malic enzyme (NAD-ME), phosphoenolpyruvate carboxykinase (PCK) and nicotinamide adenine dinucleotide phosphate malic enzyme (NADP-ME)] and six major evolutionary lineages were grown under ambient (400 μL L(-1) ) and inter-glacial (280 μL L(-1) ) CO2 . We hypothesized that nitrogen-related and water-related physiological traits are associated with subtypes and lineages, respectively. Photosynthetic rate and stomatal conductance were constrained by the shared lineage, while variation in leaf mass per area (LMA), leaf N per area, plant dry mass and plant water use efficiency were influenced by the subtype. Subtype and lineage were equally important for explaining variations in photosynthetic nitrogen use efficiency (PNUE) and photosynthetic water use efficiency (PWUE). CO2 treatment impacted most parameters. Overall, higher LMA and leaf N distinguished the Chloridoideae/NAD-ME group, while NADP-ME and PCK grasses were distinguished by higher PNUE regardless of lineage. Plants were characterized by high photosynthesis and PWUE when grown at ambient CO2 and by high conductance at inter-glacial CO2 . In conclusion, the evolutionary and biochemical diversity among C4 grasses was aligned with discernible leaf physiology, but it remains unknown whether these traits represent ecophysiological adaptation.

The physiological implications of C(3)-C(4) photosynthesis were investigated using closely related Panicum species exposed to industrial-age climate change. Panicum bisulcatum (C(3)), P. milioides (C(3)-C(4)), and P. coloratum (C(4)) were grown in a glasshouse at three CO(2) concentrations ([CO(2)]: 280, 400, and 650 μl l(-1)) and two air temperatures [ambient (27/19 °C day/night) and ambient + 4 °C] for 12 weeks. Under current ambient [CO(2)] and temperature, the C(3)-C(4) species had higher photosynthetic rates and lower stomatal limitation and electron cost of photosynthesis relative to the C(3) species. These photosynthetic advantages did not improve leaf- or plant-level water (WUE) or nitrogen (NUE) use efficiencies of the C(3)-C(4) relative to the C(3) Panicum species. In contrast, the C(4) species had higher photosynthetic rates and WUE but similar NUE to the C(3) species. Increasing [CO(2)] mainly stimulated photosynthesis of the C(3) and C(3)-C(4) species, while high temperature had no or negative effects on photosynthesis of the Panicum species. Under ambient temperature, increasing [CO(2)] enhanced the biomass of the C(3) species only. Under high temperature, increasing [CO(2)] enhanced the biomass of the C(3) and C(3)-C(4) species to the same extent, indicating increased CO(2) limitation in the C(3)-C(4) intermediate at high temperature. Growth [CO(2)] and temperature had complex interactive effects, but did not alter the ranking of key physiological parameters amongst the Panicum species. In conclusion, the ability of C(3)-C(4) intermediate species partially to recycle photorespired CO(2) did not improve WUE or NUE relative to congeneric C(3) or C(4) species grown under varying [CO(2)] and temperature conditions.