Two C4 species, Amaranthus cruentus and Zea mays, were studied to evaluate the mechanism of inhibition of photosynthesis due to water stress. The net rate of carbon dioxide fixation (A) and transpiration (E) were measured by gas exchange, and stomatal conductance (gs) and intercellular CO2 (Ci) calculated, while the true rate of oxygen evolution (JO2) was calculated from chlorophyll fluorescence analysis. Following the withholding of water there was a progressive decrease in gs and E during the stress cycle. The results clearly indicate that, initially, Ci decreased with little effect on A (indicating the CO2 pump is providing sufficient CO2 for carbon assimilation), and that the eventual inhibition of photosynthesis by water stress was caused by a limited supply of CO2 to Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase). As A decreased during water stress, the photosystem II activity per CO2 fixed increased, a phenomenon also observed when well watered plants were provided with very low atmospheric levels of CO2, which is indicative of a decreased supply of CO2 to Rubisco. At the same time the RuBP pool/RuBP binding site on Rubisco increased, and the ratio of initial extractable activity of Rubisco to A increased, which suggests that neither RuBP regeneration nor Rubisco capacity is limiting photosynthesis. When plants were rewatered after photosynthesis had dropped to 5-10% of the original rate, both species showed near full recovery in 2-4 days.

l h e temperature dependence of quantum yields of electron transport from photosystem II (PSII) ($11, determined from chlorophyll a fluorescence) and COz assimilation (Ocoz, apparent quantum yield for CO, assimilation) were determined simultaneously in vivo. With C4 species representing NADP-malic enzyme, NADmalic enzyme, and phosphoenolpyruvate carboxykinase subgroups, the ratio of Oll/OcOZ was constant over the temperature range from 15 to 40'C at high light intensity (1100 pmol quanta m-' s-'1. A similar response was obtained at low light intensity (300 fimol quanta m-' s-'), except the ratio of 011/9c02 increased at high temperature. When the true quantum yield for COz fixation (Ocoz*) was calculated by correcting for respiration in the light (estimated from temperature dependence of dark respiration), the ratio of Q,,/QCo2* remained constant with varying temperature and under both light intensities in all C4 species examined. Because the Qll/Qcoz* ratio was the same in C, monocots representing the three subgroups, the ratio was not affected by differences in the biochemical mechanism of concentrating COz in the bundle sheath cells. l h e results suggest that Psll activity is closely linked to the true rate of CO, fixation in C4 plants. The dose relationship between QII and QcOz" in C, species under varying temperature and light intensity conditions is apparently due to a common low leve1 of photorespiration and a primary requirement for reductive power in the C3 pathway. In contrast, in a C) plant the O 1 l / O~~z * ratio is higher under normal atmospheric conditions than under nonphotorespiratory conditions and it increases with rising temperature. lhis decrease in efficiency in utilizing energy derived from PSll for COz fixation is dueto an increase in photorespiration.In both the C3 and C4 species, photochemistry is limited under low temperature, and thus excess energy must be dissipated by nonphotochemical means.The photochemical efficiency of PSII is regulated such that the capacity of noncyclic electron transport matches the energy-requiring processes that act as sinks for the products of thylakoid photochemistry (ATP and NADPH) (Weis and Berry, 1987; Foyer et al., 1990). Through measurements of Chl a fluorescence emitted from PSII using modulated light, it iipossible to distinguish between the utilization of absorbed energy in photochemical versus nonphotochemical processes and to determine GIl (Genty et al., 1989). Through simultaneous determination of @ll and C02 fixation or Oz evolution by fluorescence and gas exchange, and calculating &o2 or the efficiency in utilizing the photochemically derived energy for carbon fixation can be analyzed. Determination of Supported by U.S. Department of Agriculture Competitive Grant NO. 90-37280-5706.* Corresponding author; fax 1-509-335-3517.507 from fluorescence analysis is based on a model that depends upon nonphotochemical quenching being a process that reduces photochemical yield in direct proportion to the steady-state efficiency in trapping energy (varia...

Internal CO2 and O2 concentrations in Sedum praealtum DC. were determined by gas chromatography of 200-�l gas samples. Day-night monitoring showed that internal CO2 varied from a high of approximately 4000 �l/l during periods of daytime stomatal closure to a low of 270-280 �l/l during the dark period (stomata open). Internal O2 concentrations varied from a high of approximately 26 % at midday to a low of 20.8 % during the dark period. The calculated internal O2/CO2 ratio varied about 12-15-fold from 50-60 near midday to approximately 750 during the dark period (ratio in normal air is roughly 600). Day-night patterns of CO2 exchange and malic acid concentration were typical for a plant with crassulacean acid metabolism (CAM). Influx of CO2 during the late light period was inhibited by O2, but dark CO2 influx was O2-insensitive. Gas samples taken near midday from several CAM plants all showed elevated internal CO2 and O2 concentrations. Ratios of O2/CO2 in these plants ranged from 81 in Sedum praealtum to 285 in Hoya carnosa. The highest internal O2 concentration observed was 41.5% in Kalanchoe gastonis-bonnieri. The high CO2 concentration in leaves of CAM plants during daytime stomatal closure should provide a near- saturating level of this substrate for photosynthesis. In comparison to C3 plants, the relatively low O2/CO2 ratio in the CAM leaf during malic acid decarboxylation should be favourable for photosynthesis and unfavourable for O2 inhibition of photosynthesis.

The quantum yields of non-cyclic electron transport from photosystem II (determined from chlorophyll a fluorescence) and carbon dioxide assimilation were measured in vivo in representative species of the three subgroups of C4 plants (NADP-malic enzyme, NAD-malic enzyme and PEP-carboxykinase) over a series of intercellular CO2 concentrations (CI) at both 21% and 2% O2. The CO2 assimilation rate was independent of O2 concentration over the entire range of Ci (up to 500 μbar) in all three C4 subgroups. The quantum yield of PS II electron transport was similar, or only slightly greater, in 21% v. 2% O2 at all Ci values. In contrast, in the C3 species wheat there was a large O2 dependent increase in PS II quantum yield at low CO2, which reflects a high level of photorespiration. In the C4 plants, the relationship of the quantum yield of PS II electron transport to the quantum yield of CO2 fixation is linear suggesting that photochemical use of energy absorbed by PS II is tightly linked to CO2 fixation in C4 plants. This relationship is nearly identical in all three subgroups and may allow estimates of photosynthetic rates of C4 plants based on measurements of PS II photochemical efficiency. The results suggest that in C4 plants both the photoreduction of O2 and photorespiration are low, even at very limiting CO2 concentrations.

Enzymatic digestion of leaf segments with 2 % cellulase, in combination with a pectinase in some species, yields intact protoplasts mixed with epidermal tissue, vascular tissue, broken protoplasts, and chloroplasts. Epidermal and vascular tissue are removed with sieves of various porosity. Intact protoplasts in the filtrate are separated from other components by an aqueous two-phase system which consists of dextranpolyethylene glycol, with sorbitol and sodium phosphate. Intact protoplasts partition at the interphase, while chloroplasts and broken protoplasts partition in the lower phase when the separation is facilitated by low speed centrifugation. The optimum conditions for purification of maize mesophyll protoplasts with high yields are centrifugation of the two-phase system at 300g for 6 minutes at 2 C with a mixture including 0.46 M sorbitol, 10 mM sodium phosphate, 5.5% polyethylene glycol 6000, and 10% dextran of average molecular weight of 20,000 to 40,000. The collection of protoplasts at the interphase was proportional to the amount of chlorophyll added over a wide range of concentrations regardless of the initial contamination of the preparation by other cellular debris. The two-phase system is applicable for protoplast purification from a wide variety of species, including C3, C4, and Crassulacean acid metabolism plants, regardless of protoplast size.Over the past 12 years there has been a rapid development in the enzymatic isolation and utilization of plant protoplasts for various research purposes (4, 6). Factors broken protoplasts, chloroplasts, and other organelles. Recently, we have isolated mesophyll protoplasts and bundle sheath strands from leaves of many C4 plants4 for comparative biochemical studies on the role of the two cell types in C4 photosynthesis (13,14). It was necessary to purify the isolated protoplasts as well as to obtain suitable yields for metabolic studies. Purification of mesophyll protoplasts of tobacco and cereal leaves, tomato fruit, and onion roots by floating the protoplasts on sucrose solutions has been reported (9, 11, 17, 18), although in most studies yield data have not been given.Attempts to collect the protoplasts of maize, a Co plant, by floating on sucrose has not provided adequate quantitative preparations for our investigations. This led to the development of another purification method with a new principle. We have found that an aqueous dextran-polyethylene glycol twophase system is applicable for purifying leaf protoplasts from a wide variety of plants including C3, C4, and CAM plants. Details of the characteristics of the two-phase system for protoplast purification are described in this report. The viability of the protoplasts was discussed in relation to their photosynthetic capacity. MATERIALS AND METHODSPlants were grown in a growth chamber under 16 hr of light and 8 hr of dark with a day temperature of 30 C and a night temperature of 20 C. Light was provided by a combination of incandescent and fluorescent lamps giving a quantum flux density ...