S. B. Ku

Oxygen inhibition of photosynthesis

The effect of leaf temperature, O2 and calculated O2/CO2 solubility ratio in the leaf on the quantum yield of photosynthesis was studied for the C4 species, Zea mays L., and the C3 species, Triticum aestivum L. Over a range of leaf temperatures of 16 to 35° C, the quantum yield of Z. mays was relatively constant and was similar under 1.5 and 21% O2, being ca. 0.059 mol CO2 mol-1 quanta absorbed. Under 1.5% O2 and atmospheric levels of CO2, the quantum yield of T. aestivum was relatively constant (0.083 mol CO2 mol-1 quanta absorbed) at leaf temperatures from 15 to 35° C. Atmospheric levels of O2 (21%) reduced the quantum yield of photosynthesis in T. aestivum and as leaf temperature increased, the quantum yield decreased from 0.062 at 15°C to 0.046 mol CO2 mol-1 quanta absorbed at 35°C. Increasing temperature decreases the solubility of CO2 relatively more than the solubility of O2, resulting in an increased solubility ratio of O2/CO2. Experimentally manipulating the atmospheric levels of O2 or CO2 to maintain a near-constant solubility ratio of O2/CO2 at varying leaf temperatures largely prevented the temperature-dependent decrease in quantum yield in t. aestivum. Thus, the decrease in quantum yield with increasing leaf temperature in C3 species may be largely caused by a temperaturedependent change in the solubility ratio of O2/CO2.

Localization of the C4 and C3 pathways of photosynthesis in the leaves of Pennisetum purpureum and other C4 species. Insignificance of phenol oxidase

SummaryMesophyll protoplasts and bundle-sheath cells of Pennisetum purpureum Schum., a C4 plant with low phenol-oxidase activity, were enzymatically separated according to methods recently developed with sugarcane (Saccharum officinarum L.), maize (Zea mays L.), and sorghum (Sorghum bicolor L.). The phosphoenolpyruvate carboxylase and NADP-malic dehydrogenase of the C4 pathway were found to be localized in the mesophyll protoplasts while ribulose-1,5-diphosphate (RuDP) carboxylase, phosphoribulokinase and NADP-malic enzyme were localized in the bundle-sheath cells. The levels of these enzyme activities in the leaf extracts and in certain cellular preparations of P. purpureum are sufficient to account for the rate of photosynthesis in the leaf. These results on the activities and distribution of photosynthetic enzymes with P. purpureum preparations are consistent with our previous evidence for cellular separation of the C4 and the reductive pentose-phosphate pathways in C4 species.With chlorogenic acid as the substrate, P. purpureum, Setaria lutescens (Weigel) Hubb. and Panicum texanum Buckl. have relatively low phenol-oxidase activity, similar to that found in spinach (Spinacia oleracea L.); while sorghum, sugarcane, maize, Panicum capillare L. and P. miliaceum L. have relatively high phenoloxidase activity, similar to that in tobacco (Nicotiana tabacum L.). C4 species having high phenol-oxidase activity have substantial activity of the enzyme in both mesophyll and bundle-sheath extracts. Since phenol oxidase is found in both cell types it is not logical to expect preferential inhibition of RuDP carboxylase or other photosynthetic enzymes through phenol oxidation in mesophyll extracts, as has been previously suggested. When dithiothreitol and polyvinylpyrrolidone were included in the enzyme extraction medium, the activity of RuDP carboxylase increased 10% in P. purpureum and 59% in sugarcane leaf extracts.

Oxygen Inhibition of Photosynthesis in the C4 Species Amaranthus graecizans L.

In the C4 plant, Amaranthus graecizans, increasing [O2] from 2% up to 100% inhibited photosynthesis, quantum yield, and the carboxylation efficiency, and increased the CO2 compensation point (Γ) from 2 to about 12 μl/l. The O2 inhibition of photosynthesis was fully reversible. When changing from 2.5 to 40% O2 and vice versa, about 1 h was required for full equilibration with an O2 inhibition of 18%; whereas in wheat, a C3 species, inhibition of photosynthesis and its reversal occurs within minutes after changing [O2], resulting in 63% inhibition of photosynthesis by 45% O2. These differences in O2 inhibition between a C4 and C3 species can be explained by high diffusive resistance across bundle-sheath cells of C4 plants and the increased CO2/O2 ratio in bundle-sheath cells which is the consequence of the C4 cycle. In A. graecizans, Γ increased with increasing [O2] but tended to reach a maximum at relatively high O2 levels. The lack of a linear increase in Γ as previously observed for C3 species indicates that a considerable amount of photorespired CO2 may be re-fixed with increasing levels of O2. In comparison to previous reports with other C4 species, photosynthesis of A. graecizans shows greater sensitivity to O2, with a noticeable inhibition occurring with shifts from 2 to 21% O2. A. graecizans has characteristics of other C4 species with respect to Kranz anatomy, localization of PEP carboxylase in mesophyll cells and RuBP carboxylase in bundle-sheath cells, and little fractionation among carbon isotopes during CO2 fixation. The basis for the higher sensitivity of photosynthesis of A. graecizans to O2 may be based upon a lower diffusive resistance of gases across bundle-sheath cells than in some other C4 species.

Protoplast Isolation from Leaves of C3, C4 and CAM Plants and Biochemical Activities

Leaf protoplasts have been isolated from a number of plants by enzymatic digestion with 2% cellulase (Onozuka R-10, All Japan Biochem. Co.) and 0.1% pectinase (1). Leaf segments less than lmm wide were incubated in the isolation medium for 3 hours at 30°C. The protoplasts released were purified in a liquid-liquid two-phase system using dextran T20 and polyethylene glycol 6000. Intact mesophyll protoplasts were collected at the interphase. Bundle sheath strands from C4 plants were collected by filtration techniques through nylon sieves of different porosity. For microscopy observations isolated protoplasts and bundle sheath cells were suspended in 0.5% Evans Blue. The exclusion of the dye was used as an indication of the intactness of the preparation. Comparative studies on the photosynthetic activities by mesophyll protoplasts from C3 and Crassulcean Acie Metabolism plants further showed the viability of the isolated cells. C4 plants, such as maize, sugarcane and Panicum miliaceum, are characterized as having two types of chlorophyllous cells, the mesophyll and the bundle sheath cells, and two carboxylating enzymes, PEP carboxylase of the C4-dicarboxylic acid pathway and RuDP carboxylase of the ribulose pentose phosphate pathway. PEP carboxylase and other enzymes of the C4 pathway were almost exclusively localized in mesophyll protoplasts while RuDP carboxylase and phosphoribulose kinase were localized in bundle sheath cells. Enzyme distribution, photochemical activities and CO2 fixation have been studies in relation to the compartmentation of photosynthetic carbon metabolism (2,3).