δ13C of bulk leaf matter and cellulose reveal post-photosynthetic fractionation during ontogeny in C4 grass leaves

The 13C isotope composition (δ13C) of leaf dry matter is widely employed as an index of physiological characteristics. δ13C of leaf carries integrated signatures of 12C/13C discrimination occurring during and after photosynthesis. The former is well-understood, and models have been developed to infer physiological processes and key parameters in C3 and C4 photosynthesis. However, much less is known about the downstream post-photosynthetic fractionation (∆post) processes. Several mechanisms have been hypothesized, such as isotope fractionation during respiration and the export of photosynthetic products. ∆post could cause the isotopic difference between newly fixed carbon and leaf biomass and thus complicates the interpretation of physiological responses based on isotopic records. We investigated the effects of ∆post on δ13C of mature leaves of Cleistogenes squarrosa, a perennial C4 grass, in controlled experiments with different levels of vapour pressure deficit and nitrogen supply. We measured the 12C/13C fractionation of leaf organic matter relative to the δ13C of atmosphere CO2 (ΔDM) and that of cellulose (Δcel) along leaf age category. With the increase of leaf age classes, ΔDM increased while Δcel was almost constant. Also, ΔDM of young leaves and Δcel had similar responses to vapour pressure deficit and nitrogen treatments. The divergence between ΔDM and Δcel increased with leaf age classes with a maximum value of 1.6‰, indicating the accumulation post-photosynthetic fractionation. Applying a new mass balance model that accounts for respiration and export of photosynthates, we found an apparent 12C/13C fractionation associated with respiration of –0.7 to –1.1‰ and carbon export of –0.5 to –1.0‰. Furthermore, different 12C/13C fractionation among leaves, pseudostems, daughter tillers and roots indicate that ∆post happens at the whole-plant level. In summary, our study confirmed that leaf became increasingly 13C-depleted during ontogeny and respiration and carbon export are the driving mechanisms. Compared with ΔDM of old leaves, ΔDM of young leaves and Δcel are more reliable proxies for predicting physiological parameters due to the smaller sensitivity to post-photosynthetic fractionation and the similar sensitivity in responses to environmental changes.