Effects of the Phosphoenolpyruvate Carboxylase Inhibitor

The effect of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl)propenoate (DCDP), an analog of phosphoenolpyruvate (PEP), on PEP carboxylase activity in crude leaf extracts and on photosynthesis of excised leaves was examined. DCDP is an effective inhibitor of PEP carboxylase from Zea mays or Pancum miliaceum; 50% inhibition was obtained at 70 or 350 micromolar, respectively, in the presence of I millimolar PEP and 1 millimolar HCO3-. When fed to leaf sections via the transpiration stream, DCDP at I millimolar strongly inhibited photosynthesis in C4 species (79-98% inhibition for a range of seven C4 species), but only moderately in C3 species (12-46% for four Cs species), suggesting different mechanisms of inhibition for each photosynthetic type. The response of P. miliaceum (C4) net photosynthesis to intercellular pCO2 showed that carboxylation efficiency, as well as the CO2 saturated rate, are lowered in the presence of DCDP and supported the view that carboxylation efficiency in C4 species is directiy related to PEP carboxylase activity. A fivefold increase in intercellular pCO2 over that occurring in P. millaceum under normal photosynthesis conditions only increased net photosynthesis rate in the presence of 1 millimolar DCDP from zero to about 5% of the maximal uninhibited rate. Therefore, it seems unlikely that direct fixation of atmospheric CO2 by the bundle sheath cells makes any significant contribution to photosynthetic CO2 assimilation in C4 species. The results support the concept that C-selective herbicides may be developed based on inhibitors of C4 pathway reactions. C4 plants dominate in lists of the world's worst weeds (4). Inhibitors of the C4 pathway should prevent photosynthesis and may have potential as herbicides selective to C4 weeds (7), but as yet no good C4-selective inhibitors ofphotosynthesis have been described. We recently reported that DCDP' is a potent, selective inhibitor of PEP carboxylase from leaves of both C4 and C3 plants (1 1). In examining the feasibility that 'Abbreviations: DCDP, 3,3-dichloro-2-dihydroxyphosphinoylmethyl)propenoate; PEP, phosphoenolpyruvate; CE, carboxylation efficiency; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase; G6P, glucose 6-phosphate; RuBP, ribulose 1,5-bisphosphate; pCO2, partial carbon dioxide pressure. 1231 C4-selective herbicides may be designed based on inhibitors of the C4 pathway enzymes, it was of interest to test this compound on photosynthesis in C3 and C4 species. A specific inhibitor of the C4 pathway may also be useful in studies on other aspects of C4 photosynthesis, particularly the CE, and the CO2 diffusibility of the bundle sheath-mesophyll interface of C4 plants. C4 plants reach maximum rates of photosynthesis at lower intercellular [CO2] than C3 plants, and have higher CE (i.e. carbon gain per unit increase in [CO2J at subsaturating [CO2], determined from the slope of the assimilation response to intercellular [CO2]). These gasexchange features have been attributed to the operation ofthe C4 pathway concentrating CO2 in bundle sheath cells at the site of Rubisco, thereby preventing 02 inhibition of carboxylation, and to the involvement of the different primary carboxylases in the C4 and C3 photosynthetic types (4, 8). In normal air and at high light intensity CE in C3 plants is correlated with the amount of Rubisco in the leaf (18). This has been demonstrated by varying the amount of Rubisco by differing levels of nitrogen nutrition or light intensity during growth (18). In contrast, in C4 species CE has been related to the high maximum velocity of PEP carboxylase (generally severalfold higher than maximum photosynthesis rate) and its high effective affinity for the inorganic carbon substrate (while HCO3is the substrate for PEP carboxylase, in C4 mesophyll cells in the presence of carbonic anhydrase HCO3would be in rapid equilibrium with a relatively low concentration ofCO2). Thus, the carboxylation rate at low [CO2] can be high relative to maximum photosynthesis capacity. This involvement of PEP carboxylase in CE is not easily demonstrated experimentally, however, as it is difficult to manipulate C4 pathway activity while keeping constant the activity of the C3 photosynthetic carbon reduction cycle (see, e.g. Refs. 16 and 17). A critical feature for the effective functioning of the C4 pathway is that the interface between the bundle sheath and mesophyll cells in C4 species has a low diffusibility to CO2 to allow generation of high CO2 concentrations in bundle sheath cells. The rate of CO2 leakage from bundle sheath cells is unknown, though it is probably less than 20% of the photosynthesis rate (6). The extent to which this CO2 barrier restricts www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 1989 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 89,1989 entry and direct fixation of atmospheric CO2 by Rubisco in bundle sheath cells under normal conditions is uncertain; although short-term '4CO2-labeling experiments have shown 100% fixation into C4 acids when extrapolated to zero time, indicating no direct fixation by Rubisco (7), it has been suggested elsewhere, based on experiments with leaf slices, that 10 to 15% of carbon assimilation may be due to direct fixation in bundle sheath cells (2, 14, 15). As well as under normal conditions of photosynthesis, it is also of interest to know in relation to the feasibility of C4-specific herbicides whether C4 plants can continue to assimilate atmospheric CO2 in the absence of a functional C4 pathway, by direct fixation of CO2 in the bundle sheath cells. In this report it is shown that DCDP is an effective inhibitor of PEP carboxylase in crude leaf extracts under various conditions, and of photosynthesis with some degree of selectivity toward C4 species. The responses of photosynthesis in a C4 species to variations in intercellular [CO2] and incident light intensity in the presence of this inhibitor have also been examined. MATERIALS AND METHODS