Grass species displaying the different C₄ biochemical subtypes (NAD-ME, NADP-ME and PCK) show potential for maximizing growth under limited water. Carbon (d¹³C) and oxygen (d¹⁸O) isotopic analysis provide a means of assessing the effects of water status on transpiration and photosynthesis parameters.  The purpose of this study was to (a) determine the effects of water stress on d¹³C and d¹⁸O isotope ratios and bundle sheath leakiness in two subtypes of C₄ grasses; and (b) determine the efficacy of using leaf cellulose d¹⁸O as a proxy for stomatal behavior in C₄ grasses.  The species examined included two NAD-ME grasses: switchgrass ‘Alamo' and ‘Cave-in-Rock' and two NADP-ME: big bluestem ‘Pawnee' and indiangrass ‘Nebraska 54' grasses.

There were significant species effects on leaf d¹³C.  Leaf tissue was 0.81‰ more enriched with ¹³C in the two NAD-ME grasses, relative to the NADP-ME grasses.    The NADP-ME grasses exhibited more stable bundle sheath leakiness (Φ) values with increasing water deficits compared to the NAD-ME grasses.  Differences in bulk leaf water d¹⁸O in control plants between big bluestem and the other grass species were significant (P<0.05).   

Water stress significantly reduced the leaf d¹³C of all the C₄ grasses by an average of 2.45‰.  Water stress significantly (P<0.05) reduced bulk leaf water d¹⁸O in all the grass species.  Water stress did not significantly (P<0.05) affect either the overall or the basal and apical leaf cellulose d¹⁸O signatures in the four grass species. 

The stability of bundle sheath leakiness under conditions of changing water availability suggests that variations in p_i/p_a accounted for most of the variation in leaf carbon isotope ratios and D (carbon isotope discrimination) (r² = 0.89).   Leaf cellulose d¹⁸O signatures cannot be reliably used as a measure of stomatal behavior under water stress in these species.