Variations in nitrogen use efficiency reflect the biochemical subtype while variations in water use efficiency reflect the evolutionary lineage of C4 grasses at inter-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.

Photosynthesis of C3, C3-C4, and C4 grasses at glacial CO2.

Most physiology comparisons of C3 and C4 plants are made under current or elevated concentrations of atmospheric CO2 which do not reflect the low CO2 environment under which C4 photosynthesis has evolved. Accordingly, photosynthetic nitrogen (PNUE) and water (PWUE) use efficiency, and the activity of the photosynthetic carboxylases [Rubisco and phosphoenolpyruvate carboxylase (PEPC)] and decarboxylases [NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PEP-CK)] were compared in eight C4 grasses with NAD-ME, PCK, and NADP-ME subtypes, one C3 grass, and one C3-C4 grass grown under ambient (400 µl l(-1)) and glacial (180 µl l(-1)) CO2. Glacial CO2 caused a smaller reduction of photosynthesis and a greater increase of stomatal conductance in C4 relative to C3 and C3-C4 species. Panicum bisulcatum (C3) acclimated to glacial [CO2] by doubling Rubisco activity, while Rubisco was unchanged in Panicum milioides (C3-C4), possibly due to its high leaf N and Rubisco contents. Glacial CO2 up-regulated Rubisco and PEPC activities in concert for several C4 grasses, while NADP-ME and PEP-CK activities were unchanged, reflecting the high control exerted by the carboxylases relative to the decarboxylases on the efficiency of C4 metabolism. Despite having larger stomatal conductance at glacial CO2, C4 species maintained greater PWUE and PNUE relative to C3-C4 and C3 species due to higher photosynthetic rates. Relative to other C4 subtypes, NAD-ME and PEP-CK grasses had the highest PWUE and PNUE, respectively; relative to C3, the C3-C4 grass had higher PWUE and similar PNUE at glacial CO2. Biomass accumulation was reduced by glacial CO2 in the C3 grass relative to the C3-C4 grass, while biomass was less reduced in NAD-ME grasses compared with NADP-ME and PCK grasses. Under glacial CO2, high resource use efficiency offers a key evolutionary advantage for the transition from C3 to C4 photosynthesis in water- and nutrient-limited environments.

Panicum milioides (C(3)-C(4)) does not have improved water or nitrogen economies relative to C(3) and C(4) congeners exposed to industrial-age climate change.

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.