V. Photochemical Energy Supply Colimits Photosynthesis at Low Values of Intercellular Co2 Concentration

Although there is now some agreement with the view that the supply of photochemical energy may influence photosynthetic rate (P) at high CO2 pressures, it is less clear whether this limitation extends to P at low CO2. This was investigated by measuring P per area as a function of the intercellular CO2 concentration (C,) at different levels of photochemical energy supply. Changes in the latter were obtained experimentally by varying the level of irradiance to normal (Fe-sufficient) leaves of Beta vulgaris L. cv F58-554H1, and by varying photosynthetic electron transport capacity using leaves from Fe-deficient and Fe-sufficient plants. P and C, were determined for attached sugar beet leaves using open flow ps exchange. The results suggest that P/area was colimited by the supply of photochemical energy at very low as well as high values of Cl. Using the procedure developed by Perchorowicz et al. (Plant Physiol 1982 69:1165-1168), we investigated the effect of irradiance on ribulose bisphosphate carboxylase (RuBPCase) activation. The ratio of initial extractable activity to total inducible RuBPCase activity increased from 0.25 to 0.90 as leaf irradiance increased from 100 to 1500 microeinsteins photosynthetically active radiation per square meter per second. These data suggest that colimitation by photochemical energy supply at low C, may be mediated via effects on RuBPCase activation. Several researchers have used the relationship between CO2 assimilation and CO2 concentration to elucidate the factors controlling photosynthetic rate. Laing et al. (20) observed that P' was linearly related to the ambient CO2 concentration from r to CO2 saturation and concluded that P was limited by the concentrations of CO2 and 02, and by the kinetic parameters of RuBPCase. Other researchers have studied the relationship between P and intercellular CO2 concentration (C1) (9, 10, 12, 17, 18, 30). Farquhar et al. (13) obtained P/Ci curves under specific environmental conditions and interpreted these data in terms of the characteristics of biochemical systems. They concluded that there are two domains of photosynthetic limitation: the first relates to the initial slope ofthe P/Ci curve where P is dominated by the kinetic parameters of RuBPCase and the concentrations ofCO2 and 02; the second relates to the plateau region ofthe PI Ci curve where P increases relatively less with increase in C1 and represents a condition which is proposed to be controlled by the supply of photochemical energy (e.g. ATP, NADPH). An alternative view to the two-domain theory, one domain ' Abbreviations: P, rate of photosynthetic CO2 uptake; Ci, intercellular CO2 concentration; r, CO2 compensation point; RuBP, ribulose 1,5bisphosphate; RuBPCase, ribulose 1,5-bisphosphate carboxylase; P,,,,,m, maximal rate of photosynthesis. operating at low CO2 and the other at high C02, is that photochemical energy supply may colimit P over the entire range of Ci values. The most recent report in this series (28) indicated that photochemical energy supply may colimit photosynthesis at ambient CO2 concentrations of 300 Ml 1-' or more; however, it was not clear whether this colimitation extended to very low CO2 concentrations as well (28). In the present work, we explored this question further by (a) measuring P/Ci curves for leaves exposed to different levels of irradiance, and (b) determining P/Ci curves for leaves with different photosynthetic electron transport capacities using Fe-sufficient and Fe-deficient plants. Since we found that decreasing the supply of photochemical energy by either procedure led to a reduction in the initial slope ofthe P/Ct curve, we also explored the possibility that the effect of photochemical energy supply may be mediated via the level of activation of RuBPCase as suggested by Perchorowicz et al. (23). MATERIALS AND METHODS Plant Culture. Sugar beet plants (Beta vulgaris L. cv F58554H1) were grown hydroponically in growth chambers and Fe deficiency was induced by transferring plants to culture solution without Fe as described previously (27). Leaf Gas Exchange. P/area, leaf conductance, and Ci of individual attached leaves were determined over a range of ambient CO2 concentrations using open flow gas exchange as described previously (see 28). The measurements were made by exposing the leaf initially to an ambient CO2 concentration ofabout 1000 Al 1-' for 1.5 h at 21% 02, then for 30 min at 1% 02; subsequently, the ambient CO2 concentration was lowered to successive levels with 30-min periods at 21% and 1% 02 at each ambient C02 level. Leaftemperature was maintained at 30 ± 0.5C. Irradiance was held either at a constant level (Fig. 2) or increased gradually to achieve light saturation at each CO2 level (Fig. 4). Photosynthetic Electron Transport. Chloroplasts were isolated from chilled leaves by a brief (5 s) homogenization in a Waring Blendor with an extraction solution of 50 mM Tricine (pH 7.8), 400 mM sorbitol, 10 mM NaCl, 5 mM MgCl2, 1% (w/v) PVP-40, 0.2% (w/v) Na ascorbate, and 0.1% (w/v) BSA, at 4°C. The suspension was filtered through two layers of Miracloth, and the chloroplasts pelleted by centrifugation at 400g for 2 min. The chloroplasts were washed by resuspension in the extraction solution and repelleted. The thylakoids were isolated by rupturing the chloroplasts in extraction solution containing only 100 mM sorbitol; the osmotic strength was then adjusted to 400 mM sorbitol to approximate conditions in vivo, and the thylakoids pelleted by centrifugation at 3000g for 3 min. The isolated thylakoids were tested for whole chain electron transport activity as described previously (21). 02 evolution was measured polarographically with a Rank 02 electrode at 25°C with 1000 ME PAR m 2 s-' in the presence of 2.5 mM NH4C1, 10 mM glyceraldehyde, 1.5 mm K3Fe(CN)6, 400 mm sorbitol, 30 mM