Paraquat resistance in Chlamydomonas reinhardtii

Abstract

In this research project, the resistance mechanisms to the herbicide “paraquat” in unicellular green alga chlamydomonas reinhardtii were investigated by using resistant cell lines constructed from the corresponding wild type cell (137c). PPQ-10/3 strain was obtained from stepwise selection under the herbicide pressure. UPQ-S1 strain was from chloroplast DNA directed mutagenesis by 5-fluorodeoxyuridine. The degree of paraquat resistance, which was determined by the establishing lethal dose test, was found to be 6.48 and 3.07 µM for PPQ-10/3 and UPQ-S1 respectively in comparison with the value of 0.36 µM for 137c. Maximum growth yield of both resistant strains were significantly lower than that of the wild type. Absence of parquat in the culture medium caused abnormal cell division which from TEM and SEM evidences indicated the alteration in the deposition of the outermost wall layer. Chlorophyll a content of UPQ-S1was not distinct from that found in the wild type which was concomitant with the similar organization of chloroplast discs into very long grana. Whereas in PPQ-10/3 about 2-3 folds higher in the chlorophyll a content was detected accompanying with the highly stacking degree of grana. Extensive large and size of vacuoles were observed in parquat resistant strains together with the enlargement of cells. Activity of UPQ-S1 photosystem I was 2 folds over that of PPQ-10/3 and 137c. Paraquat treatment at sublethal dose caused reduction in photosystem I activity in all call types. Paraquat uptake in C. reinhardtii was illustrated to be facilitated via a membrane carrier-mediated system which was partially inhibited by dinitrophenol, the specific uncoupling agent for oxidative phosphorylation. Paraquat treatment in advance somewhat stimulated paraquat uptake by 2 folds in PPQ-10/3 and UPQ-S1 but not in 137c strain. The Km value of paraquat transport across cell membrane of PPQ-10/3 was about seven times of UPQ-S1and the wild type 137c. No significant difference in the amount of paraquat uptake was observed in paraquat resistant and wild type cells. The amount of the herbicide influx into the chloroplasts was decreased (60%) in the resistant cells when comparing to 137c. The enzymes of the superoxide detoxification pathway, superoxide dismutase, catalase and ascorbate peroxidase, were found to be at constitutively elevated levels in UPQ-S1 and especially PPQ-10/3 strains. Distinct isozyme patterns of superoxide dismutase were observed in UPQ-S1 with two novel copper/zinc-enzymes and two manganese-enzymes. Paraquat treatment unexpectedly reduced the cellular superoxide dismutase content (20-50%) and caused diminution of some isozymes in the three algal strains, whereas, markedly increased activity of ascorbate peroxidase was observed. The data conclusively revealed that sequestration of paraquat from chloroplasts and the ability to prevent peroxidative damage by oxygen radicals were the biochemical bases of paraquat resistance in C. reinhardtiiPPQ-10/3 and UPQ-S1 call lines. Moreover, analyses of chloroplast DNA of UPQ-S1 demonstrated interestingly altered restriction pattern comparing to that of the wild type when digested with the same Pst I endonuclease.