Effect of trifluoroacetate, a persistent degradation product of halogenated hydrocarbons, on photosynthesis of C3 and C4 crop plants

Abstract

Hydrofluorocarbons (HFCs) and hydrochlorofluorocarbons (HCFCs) have been the main substitutes for CFCs since the signing of the Montreal Protocol in 1987. According to the literature HFCs can degrade in the troposphere by the action of OH, NO and O2 radicals, amongst others to trifluoroacetic acid (TFA). Studies have also shown that thermolysis of fluoropolymers, such as Teflon, can lead to the formation of HFCs. Water bodies with little or no outflow and high evaporation rate may have the potential to accumulate TFA, and can potentially achieve levels as high as 100 μg.l-1 in as little as 30 years. The occurrence of TFA has been detected in water and air samples from many geographical areas and studies have confirmed the fact that TFA is a ubiquitous contaminant of the hydrosphere with levels as high as 41 μg.l-1. Although information is available for the analogous compound trichloroacetic acid (TCA), little is known about the phytotoxic effects of TFA. The prediction of higher estimated future concentrations of TFA, highlights the necessity for research on the phytotoxic relevance of TFA. Both P. vulgaris and Z. mays, representative crop plants of the C3 and C4 photosynthetic pathways, have been used as test plants in this study. Initial experiments were carried out over a 3 day NaTFA treatment period with plants grown and treated in sand culture. In order to obtain better control over treatment levels, later experiments were carried out over a 14 day treatment period with plants grown and treated in water culture. In both experiments in vivo photosynthetic gas exchange and fast phase chlorophyll a fluorescence measurement were routinely measured during the treatment period. In the case of the water culture treatments additional measurements with respect to chlorophyll content and plant development were carried out. At the end of the water culture experiment freeze clamp samples were taken for in vitro Rubisco analysis, and dry weight of roots and shoots were measured. In a separate experiment the effect of trifluoroacetate (NaTFA) and trichloroacetate (NaTCA) on photosynthetic electron transport was studied by measuring oxygen evolution of isolated thylakoids. Motivated by observations of NaTFA treatment on plant growth, a further simple experiment was done to determine the effect of NaTFA and NaTCA on cell growth by means of a bioassay. The results of this study have shown that trifluoroacetate affects not only photosynthesis, but also the utilisation of photosynthates. Photosynthetic gas exchange measurements indicated that stomatal conductance were initially (day 4 and 8 of treatment) increased by low NaTFA concentrations (0.625 mg.l-1) and then subsequently reduced by higher concentrations (40- 160 mg.l-1) for both P. vulgaris and Z. mays. Photosynthetic gas exchange data indicated increasing reduction of Rubisco andlor PEP-case activity with increasing NaTFA concentration. The reduction in Rubisco activity was also confirmed by Rubisco activity assays, which displayed decreases with increasing NaTFA treatment levels (0.625-160 mg.l-1), for both P. vulgaris and Z. mays. The photosynthetic gas exchange data also showed a decline in Jmax which suggested that RuBP regeneration became limiting with increasing NaTFA concentrations, especially in the case of Z. mays. This finding was corroborated by the chlorophyll a fluorescence data, indicating that the formation of reducing equivalents (μR0) as reduced by NaTFA treatment. In vitro measurements of oxygen evolution on isolated thylakoid membranes revealed that trifluoroacetate partly inhibited the photosynthetic electron transport between QA and QB, as well as on the electron acceptor side of PSII (OEC), similarly to inhibition induced by trichloroacetate. These last two mentioned sites of inhibition were however shown not to be the primary point of inhibition of photosynthesis by chlorophyll a fluorescence measurements, and that FNR was also inhibited. Photosynthesis in Z. mays was more severely affected, possibly due to the complex nature of the C, photosynthetic pathway. The accumulation of starch in chloroplasts of treated plants (observed by TEM, in Z. mays) and the observation that all NaTFA concentrations severely reduced root growth, suggested that the inhibition of root growth (more severe for Z. mays) and photosynthate utilisation was an additional cause of the phytotoxic effects of trifluoroacetate. Furthermore the fact that it has been shown that trifluoroacetate affects plant growth and development, in both species, may be due to induced changes in auxin activity. The combination of subtle effects of root growth inhibition and simultaneous stimulation of stomatal conductance suggest that trifluoroacetate may have marked effects on crops and natural vegetation, even at low environmental relevant concentrations, if an additional stress such as drought is added. The fact that this study has shown some phytotoxic effect of trifluoroacetate at much lower concentration than previously reported (0.625 mg NaTFA.l-1) and the fact that environmental concentrations are predicted to rise by orders of magnitude, point out the relevance of this work in the understanding of this pollutant's phytotoxicity.