APPENDIX A

The term is the weighted fractionation across the boundary layer and stomata:

S. Eqn (1)

where ab (2.9 ‰) and as (4.4 ‰) are the fractionation across the boundary layer and stomata, respectively. CL is CO2 partial pressure at the leaf surface. The terms b3 and b4 are defined as (Farquhar, 1983):

S. Eqn (2) S. Eqn (3)

where Rd is the non-photorespiratory CO2 release in the light (derived from light response curve); Rm is mesophyll cell dark respiration rate, which is assumed to be half of the Rd (von Caemmerer, 2000); vc, vo and vp are Rubisco carboxylation rate, Rubisco oxygenation rate and PEP carboxylation rate, respectively. Rates of vc, vo and vp were estimated using the C4 model of photosynthesis (von Caemmerer, 2000) as described by Ubierna et al. (2011) and Sun et al. (2014). Below 1500 mmol quanta m-2 s-1 the light limited model was used and at 1500 mmol quanta m-2 s-1 and above the enzyme limited model was used (Ubierna et al., 2011). The is the fractionation by Rubisco, 30‰ (Roeske and O'Leary, 1984); represents the net affect of CO2 dissolution, hydration and PEPC activity, which at 25 °C has a value of -5.7 ‰ (Farquhar, 1983; Henderson et al., 1992); f is fractionation during photorespiration, 11.6 ‰ (Lanigan et al., 2008); is fractionation associated with dark respiration, which is calculated as (Wingate et al., 2007):

S. Eqn (4)

where e is fractionation during decarboxylation, assumed to equal -6 ‰ (Ghashghaie et al., 2001; Sun et al., 2010); δ13Cmeasurement (-31 ‰) is the carbon isotope signature of CO2 used for the online discrimination measurement, which was measured by the TDLAS; and δ13Cgrowth is the carbon isotope composition of CO2 of the growth conditions, which is assumed to be -8 ‰.

The ternary affect term t is defined as (Farquhar and Cernusak, 2012):

S. Eqn (5)

where E is the transpiration rate, gac is the conductance to diffusion of air in CO2, aac (=1+) is the fractionation factor for the isotopologues of CO2 diffusing in air.

References

Farquhar GD (1983) On the nature of carbon isotope discrimination in C4 species. Australian Journal of Plant Physiology 10: 205-226

Farquhar GD, Cernusak LA (2012) Ternary effects on the gas exchange of isotopologues of carbon dioxide. Plant, Cell and Environment 35: 1221-1231

Ghashghaie J, Duranceau M, Badeck F-W (2001) δ13C of CO2 respired in the dark in relation to δ13C of leaf metabolites: comparison between Nicotiana sylvestris and Helianthus annuus under drought. Plant, Cell and Environment 24: 505-515

Henderson SA, von Caemmerer S, Farquhar GD (1992) Short-Term Measurements of Carbon Isotope Discrimination in Several C4 Species. Australian Journal of Plant Physiology 19: 263-285

Lanigan GJ, Betson N, Griffiths H, Seibt U (2008) Carbon isotope fractionation during photorespiration and carboxylation in Senecio. Plant Physiology 148: 2013-2020

Roeske CA, O'Leary MH (1984) Carbon isotope effects on the enzyme-catalyzed carboxylation of ribulose bisphosphate. Biochemistry 23: 6275-6284

Sun W, Resco V, Williams DG (2010) Nocturnal and seasonal patterns of carbon isotope composition of leaf dark-respired carbon dioxide differ among dominant plant species in a semiarid savanna. Oecologia 164: 297-310

Sun W, Ubierna N, Ma J-Y, Cousins AB (2012) The influence of light quality on C4 photosynthesis under steady-state conditions in Zea mays and Miscanthus × giganteus: changes in rates of photosynthesis but not the efficiency of the CO2 concentrating mechanism. Plant, Cell and Environment 35: 982-993

Sun W, Ubierna N, Ma J-Y, Walker B, Kramer D, Cousins AB (2014) The coordination of C4 photosynthesis and the CO2 concentrating mechanism in Zea mays and Miscanthus× giganteus in response to transient changes in light quality. Plant Physiology 164:1283-92

Ubierna N, Sun W, Cousins AB (2011) The efficiency of C4 photosynthesis under low light conditions: assumptions and calculations with CO2 isotope discrimination. Journal of Experimental Botany 61: 3119-3134

Ubierna N, Sun W, Cousins AB (2013) The efficiency of C4 photosynthesis under low light conditions in Zea mays, Miscanthus x giganteus and Flaveria bidentis. Plant Cell and Environment 36:365-381

von Caemmerer S (2000) Biochemical models of leaf photosynthesis. CSIRO Publishing, Victoria

Wingate L, Seibt U, Moncrieff JB, Jarvis PG, Lloyd J (2007) Variations in 13C discrimination during CO2 exchange by Picea sitchensis branches in the field. Plant, Cell and Environment 30: 600-616

APPENDIX B

The mesophyll surface area exposed to IS per unit leaf area (Smes) was calculated from measurements of total length of mesophyll cell walls exposed to IS and width of section analysed using equation from Evans et al. (1994) with curvature correction factor as 1.34 (Evans et al., 1994):

S. Eqn (6)

For calculation of bundle-sheath surface area per unit leaf area (Sbs) the length of BS cell walls exposed to mesophyll (bundle-sheath perimeter) was measured within the interveinal distance, and the equation from Pengelly et al. (2010) was applied:

S. Eqn (7)

The percentages of the bundle-sheath perimeter exposed to IS (% BS CW to IS) and percentages of bundle-sheath perimeter that is not covered by chloroplasts (% BS CW w/o chloroplast) were calculated by measuring the length of bundle-sheath cell walls exposed to mesophyll that is exposed to IS and that is missing of chloroplasts were calculated as

S. Eqn (8)

S. Eqn (9).