Background/Question/Methods The C₄ photosynthetic pathway mostly suppresses RuBP oxygenation and subsequent photorespiration, which is regarded as a wasteful reaction of Rubisco.  In contrast, C₃-C₄ intermediate photosynthesis does not suppress photorespiration, but employs a mechanism that permits these species to recapture some of the CO₂ that would be lost to photorespiration in C₃ leaves.  This pathway is believed to be most important in hot, dry environments where levels of RuBP oxygenation are high and C₃ carbon assimilation is consequently very inefficient.  C₄ photosynthetic species derive corollary ecological advantages from suppression of RuBP oxygenation such as increased water-use and nitrogen-use efficiencies, but it is unclear whether C₃-C₄ species do so as well.  Results from such studies are inconsistent and dependent upon measurements of a few (3-5) individuals.  In this study, we test the hypothesis that water-use efficiency (WUE) and nitrogen-use efficiency (NUE) in intermediates will increase with C₄-cycle activity.
We included fourteen species of Flaveria (Asteraceae): two C₃ species, three C₄ species, and nine C₃-C₄ species with a gradient of C₄-cycle activities (3-95% CO₂ fixation via the C₄ pathway).  Plants were fertilized using one of five nitrogen concentrations (1, 2, 4, 8, 16 mmol) to provide a gradient of photosynthetic capacities.  Reduced photosynthetic capacity results in reduced stomatal conductance (g_s), and at least fifteen individuals of each species were measured to determine the response of g_s to light-saturated CO₂ assimilation rate (A_max), an index of WUE.  Leaves were sampled for Rubisco, chlorophyll, and total nitrogen contents to assess NUE.
Results/Conclusions We found that C₃-C₄ species with <80% C₄-cycle activity exhibited WUE values that were not significantly different from the C₃ species, while the more C₄-like intermediates exhibited WUE values that were not significantly different from the C₄ species.  The response of A_max to leaf Rubisco content showed a similar pattern, with a reduction in Rubisco content to C₄ levels occurring in species with >80% C₄-cycle activity.  Responses of A_max to chlorophyll content and total leaf nitrogen were also similar; an increase in NUE to C₄ levels occurred when C₄-cycle activity was greater than 80%.
These results demonstrate that substantial C₄-cycle activity is required to provide increased WUE and NUE in C₃-C₄ species.  This also implies that WUE and NUE were probably not important factors in selecting for C₃-C₄ photosynthesis.  This points to more efficient CO₂ assimilation through refixation of photorespired CO₂ as the primary, and perhaps only, ecological advantage of C₃-C₄ photosynthesis in Flaveria.