Photosynthetic carbon isotope discrimination is a non-destructive tool for investigating C4 metabolism. Tuneable diode laser absorption spectroscopy provides new opportunities for making rapid, concurrent measurements of carbon isotope discrimination and CO2 assimilation over a range of environmental conditions, and this has facilitated the use of carbon isotope discrimination as a probe of C4 metabolism. In spite of the significant progress made in recent years, understanding how photosynthetic carbon isotope discrimination measured concurrently with gas exchange relates to carbon isotope composition of leaf and plant dry matter remains a challenge that requires resolution if this technique is to be successfully applied as a screening tool in crop breeding and phylogenetic research. In this review, we update our understanding of the factors and assumptions that underlie variations in photosynthetic carbon isotope discrimination in C4 leaves. Closing the main gaps in our understanding of carbon isotope discrimination during C4 photosynthesis may help advance research aimed at developing higher productivity and efficiency in key C4 food, feed, and biofuel crops.
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