Glyphosate Effects on Carbon Assimilation and Gas Exchange in Sugar Beet Leaves

Abstract

ABSTRACI The mechanism responsible for the inhibition of net carbon exchange (NCE) which was reported previously (DR Geiger et aL 1986 Plant Physiol 82: 468472) was investigated by applying glyphosate [N-(phos-phonomethyl)glycinel to exporting leaves of sugar beet (Beta vulgaris L.). Leaf internal CO2 concentration (Ci) remained constant despite decreases in stomatal conductance and NCE following glyphosate treatment , indicating that the cause of the inhibition was a slowing of carbon assimilation rather than decreased conductance of CO2. Throughout a range of CO2 concentrations, NCE rate at a given Ci declined gradually, with the time-series of response curves remaining parallel. Gas exchange measurements revealed that disruption of chloroplast carbon metabolism was an early and important factor in mediating these glyphosate effects, perhaps by slowing the rate of ribulose bisphosphate regeneration. An increase in the CO2 compensation point accompanied the decrease in NCE and this increase was hastened by stepwise lowering of the ambient CO2 concentration. Eventually the CO2 compensation point approached the CO2 level of air and the difference between internal and external CO2 concentrations decreased. In control and in glyphosate-treated plants, both carbon assimilation and photorespiration at atmospheric CO2 level were inhibited to a similar extent of air level of 02-Maintaining leaves in low 02 concentration did not prevent the decline in NCE rate. The herbicide GLP2, [(N-phosphonomethyl)glycine], inhibited NCE (5, 6, 12, 13) and starch accumulation (5, 6, 12) within 4 h after its application to exporting sugar beet leaves. The primary effect ofGLP on green tissue ofplants is thought to be inhibition of 5-enolpyruvylshikimate 3-phosphate synthase that results in accumulation of SHK and SHK 3-P (1, 10). Stoppage of starch accumulation when NCE slowed and the resumption of starch accumulation after NCE rate was restored by raising CO2 concentration (5) point to disruption of photosynthetic carbon metabolism as a likely cause ofthese GLP-induced effects. It remains to be shown whether these effects on NCE and starch accumulation are related to the primary effect of GLP on the SHK pathway. Determination of carbon pools by steady-state labeling and levels of soluble carbohydrates and SHK revealed that insufficient carbon was diverted to these pools to account directly for ' shikimate; WUE, water use efficiency (NCE/E); WVCD, water vapor concentration difference. the lesser amount of carbon being accumulated in starch (5). The decrease in NCE and its subsequent restoration upon raising CO2 level were totally sufficient to account for the stoppage and later resumption of starch accumulation. The effects of GLP on rates of NCE and starch accumulation include altered allocation of newly fixed carbon among products of photosynthesis. The diversion of carbon and possibly of phosphate to intermediates of the SHK pathway seems to affect the regulation of allocation of newly-fixed carbon. The present study and one that follows (12) were undertaken to determine the mechanism responsible for the GLP-induced effects on inhibition of NCE and, in particular, on the allocation of newly-fixed carbon between starch and sucrose (5). The basis for the inhibition of NCE by GLP, whether mediated by disruption of gas exchange, photosynthetic light reactions or Calvin cycle, is an important question. Munoz-Rueda et al. (8) found that GLP concentrations as low as 0.15 mm affected photosyn-thetic pigments, stomatal response and photosynthetic electron transport 1 d after treatment. With the exception of Geiger et al. (5), few studies have investigated the role that GLP might have on photosynthetic carbon metabolism, a logical site of action. Shaner and Lyons (13) concluded that an early effect of GLP was reduction ofgs and cycling of stomatal aperture which could result in lowered Ci and NCE. Evidence presented here indicates that the decrease in gs may be an effect rather than a cause of inhibition, because the decline in conductance occurs simultaneously with a decrease in the photosynthetic capacity of the mesophyll. These data support the view that g, responds to rather than controls carbon fixation rate (2, 16). In addition to its use as a herbicide, GLP may have application as a tool for understanding photosynthetic carbon metabolism. In this study we have inhibited NCE with GLP and used gas exchange measurements to examine the effect of GLP on g,, internal C02, and the response to CO2 and 02 to identify the nature of the inhibition. MATERIALS AND METHODS Plant Material. Sugar beet plants (Beta vulgaris L., Klein E type multigerm) were grown in 1.5-L containers in a mixture of Jiffy Mix (Ball Jiffy Co.):sand (1: 1, v/v) and watered three times daily with a nutrient solution described by Snyder and Carlson (14). The plants were maintained under a 14-h photoperiod at 25°C, 60% RH day and 17°C, 75% RH night. Photon flux density was 0.75 mmol m-2 s-' at leaf blade level during growth and during experiments. Typically, leaves of plastochrons 4 and 5, having an area of 1.0 to 1.5 dM2, were used from 4 week old plants. Leaf 6 was used when a third leaf on the same plant was needed for study. GLP Application. Solutions of analytical grade GLP, 99% (w/w) purity (Monsanto Agricultural Products Co.), were prepared in 0.01% (v/v) Tween-20 to give a final concentration of 365 www.plant.org on March 4, 2016-Published by www.plantphysiol.org Downloaded from