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 (Δ_DM) and that of cellulose (Δ_cel) along leaf age category. With the increase of leaf age classes, Δ_DM increased while Δ_cel was almost constant. Also, Δ_DM of young leaves and Δ_cel had similar responses to vapour pressure deficit and nitrogen treatments. The divergence between Δ_DM and Δ_celincreased 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 Δ_DM of old leaves, Δ_DM of young leaves and Δ_cel 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.