Increased glutathione S-transferase activity in 35 S ( CaMV )-Zmgstf 4 transgenic Arabidopsis thaliana

Clones of 35S-Zmgstf4 transgenic Arabidopsis thaliana expressing the glutathione S-transferase F4 gene of Zea mays, were tested for stress-inductive GST (glutathione S-transferase) activity following treatments with the heavy metals Zn (150 and 1500 μM), Cd (20 and 30 μM) and chloroacetanilide herbicide metolachlor (2000 μM). The overexpression of Zmgstf4 gene in Arabidopsis resulted in an extreme resistance to all treatments. The GST activity of the transgenic plants was almost the double compared to the wild type plant in the untreated samples. After Cd (20 and 30 μM), and Zn (150 and 1500 μM) exposure the stress response activity of GSTs increased in both wild type and transgenic plants, however with significantly higher levels in transgenic plants with extreme level at 20 μM CdSO4 treatment (0.24 in transgenic and 0.13 in wild-type). To compare GST responsivity, Zn treatments was less inductive compared to Cd. Metolachlor (200 μM) was totally tolerated by transgenic plants, compared to wild type plants, which died in 11 days.


Introduction
Plant GSTs (glutathione S-transferases) (EC 2.5.1.18)are a large and diverse stress-protective enzyme family, which catalyze the conjugation of the tripeptide glutathione (gamma-L-glutamyl-L-cysteinylglycine, GSH) with a wide variety of harmful electrophilic xenobiotics.E.g. plant resistance to chloroacetanilide herbicides is mainly caused by the functions of GSTs in maize and soybean, which, in case of atrazine, results in GSH-atrazine a complex (Cummins et al. 2011;Dixon, Skipsey, and Edwards 2010;Labrou et al. 2015).Recent results revealed that GSTs are also involved in heavy metal stress defence mechanisms in plants (Lyubenova and Schröder 2011;Schröder 2001).Genes encoding for GSTs are grouped in diverse gene families of A, B, D, K, M, O, F, T, U, Z, L, M, S. In Arabidopsis, 51 AtGST isoenzyme genes were cloned belonging to F (Phi), T (Theta), U (Tau) and Z (Zeta) families (Fig. 1).In maize 42 ZmGSTs (12 F, 28 T, and 2 Z), and in soybean 25 GmGSTs (20 T, 4 F, and 1 Z) are known.
Here we report a study of GST enzyme activity of wild type and 35S(CaMV)-ZmgstF4 transgenic Arabidopsis following exposure to Zn, Cd and metolachlor.
Seeds of transgenic 35S(CaMV)-ZmgstF4 and wild type Arabidopsis thaliana (ecotype: Col-5) were germinated in vitro on aseptic hormone free agar media.First, seeds were stratified in dark for 72 hour at 4°C to break seed dormancy.Seeds were surface sterilized with 70 % ethanol (2 min), followed by 0.5% NaOCl (3 min) and rinsing with ddH 2 O, sown on ½ MS media, solidified with 7% agar, and supplemented with different chemicals.After germination, seedlings were illuminated with Osram Fluora fluorescent lamps for 16/8 photoperiod (16-27 μmol m -2 s -1 ).Metolachlor was applied to plants as irrigation at rosette stage grown on Jiffy peat.

Biochemical analysis
The aerial parts of plants were harvested, frozen in liquid nitrogen, and stored at -80ºC prior to enzyme extractions (Schröder et al. 2003).The GST enzyme activities were measured according to Lyubenova and Schröder (2009) with CDNB model substrate at 22°C by using a Shimadzu UV-1601 Spectrophotometer at 22°C.

Statistics
At least three independent parallel experiments were carried out in each case.The significant differences between mean values were evaluated by Student's t-test at P=0.05.

Symptoms
Wild type plants treated with Zn and Cd showed typical heavy metal toxicity symptoms: loss of chlorophyll and leaf turgor.The symptoms of metolachlor (200 μM) were also significant (Fig. 2); apparently, transgenic plants were unaffected by metolachlor compared with wild type plants, which died after 11 days (Fig. 2).The root development of wild type plants was also seriously inhibited in wild type.

Enzyme activities
The GST activity of the transgenic plants is 75% higher than in the wild type plant (Fig. 3).After Cd (20 and 30 μM), but not Zn (150 and 1500 μM) exposure, increased GST activities were measured in both wild-type and transgenic plants, but the induction in transgenic plants was significantly higher (Fig. 3).

Discussion
Genetic engineering is a powerful tool to study plant metabolic pathways.Overexpression of specific genes helps to clarify their physiological roles in the metabolism grown under different stress conditions (Bittsánszky et al. 2015).Deeper understanding of the biochemical pathways contributing to the processes of uptake, translocation and accumulation of heavy metals, and tolerance of phytotoxic chemicals will greatly help the improvement of phytoremediation potential of plants.Glutathione S-transferases seem to be valuable targets for these purposes.
Our results has indicated that overexpression of GST enzymes can play important roles in the detoxification of heavy metals, and tolerance to herbicides.The overexpression of Zmgstf4 gene was also found to increase resistance against chloroacetanilide herbicides (Milligan et al. 2001).
In conclusion, Zmgstf4 transgenic Arabidopsis plant investigated in this study provided new data on the understanding of plant GST functions with indications in their use in phytoremediation.Results also show the applicability of the Zmgstf4 gene in molecular plant breeding for phytoremediation purposes.