Characterization of Heavy Metal Tolerance and Accumulation in Indian Mustard Overexpressing Bacterial ECS Gene Janice Lee Abstract The tolerance and accumulation of multiple metals by Indian mustard Brassica juncea plants overexpressing the glutamylcystein synthetase ECS gene was examined in order to study heavy metal tolerance in plants Seedling growth was tested on four different metals including Mn Mo Pb and Ni The ECS seedlings had significantly longer root lengths and higher tolerance than wildtypes for all metals tested However it was likely that the ECS plants did not hyperaccmulate metals as expected since the fresh weights of the transgenic plants did not differ much from the wildtypes Nevertheless the increased root length tolerance in transgenic plants still offers a promising strategy for the production of plants with superior heavy metal phytoremediation capacity Introduction Heavy metal pollution of soils and waters caused by the mining and burning of fossil fuels is a major environmental problem and exposure to these metals can be toxic to living cells Qian et al 1999 Unlike organic pollutants heavy metals cannot be chemically degraded or biodegraded by microorganisms One alternative biological approach to deal with this problem is phytoremediation the use of trees and plants to detoxify chemical waste sites Compared with other technologies phytoremediation is less expensive Cunningham and Ow 1996 and is particularly suitable for treatment of large volumes of substrate with low concentrations of heavy metals Heavy metals and metalloids can be removed from polluted sites by phytoextraction which is a method of phytoremediation and involves the accumulation of pollutants in plant biomass Zayed et al 1998 As a result hyperaccumulators plant species that accumulate extremely high concentrations of heavy metals in their shoots become particularly useful In addition one can genetically engineer these species to improve their metal tolerance and metal accumulating capacity A suitable target species for this strategy is Indian mustard Brassica juncea which has a large biomass production and a relatively high trace element accumulation capacity Most importantly it can easily be genetically engineered Zhu et al 1999b Generally plants have evolved a number of mechanisms to cope with heavy metal stress One example is the production of gluthathione GSH GSH serves as an antioxidant directly detoxify metals by conjugating them forming a non toxic complex through glutathione Stransferase catalyzed reaction Coleman et al 1997 Fig 1 In addition GSH is the precursor to the heavy metal binding peptides phytochelations PCs which are involved in heavy metal tolerance and sequestration Steffens 1990 It is proposed that the rate limiting step for GSH synthesis in the absence of heavy metals is believed to be the reaction catalyzed by the ECS and GS enzymes Noctor et al 1996 Therefore manipulating the expression of such enzymes involved in GSH and PC synthesis may be a good approach to genetically engineer the plants to enhance their heavy metal tolerance and accumulation Figure 1 Regulation of GSH PC Biosynthesis in plants The basis for increased production of GSH and PCs during metal stress was addressed through numerous past studies In one experiment conducted by Zhu et al 1999a the overexpression of GS in Indian mustard plants showed an increased concentration of GSH and PCs when the plants were treated with Cadium Cd Moreover the transgenic plants in the experiment exhibited higher Cd tolerance and accumulation The transgenic seedlings adapted better under metal stress since they had longer root length and higher fresh weight than wildtype plants As for the adult Indian mustards they also had a higher relative growth than wildtype plants Similar results were obtained for Indian mustards overpressing ECS in the cytosol Transgenic seedlings tolerated Cd better than wildtype and had significantly higher levels of GSH Zhu 1999 Mature transgenic ECS plants grew better and accumulated more Cd than wildtype as well Thus overexpression of enzymes GS and ECS are positively correlated to improvements in heavy metal stress response However the majority of previous studies with transgenic plants have focused on the change in tolerance to a single metal primarily Cd In natural environments where phytoremediation techniques are applied a variety of different metals are found Therefore further examination of these transgenic plants tolerance to other heavy metals should be conducted In this research I would like to characterize the tolerance and accumulation of seedling transgenic Indian mustard overexpressing bacteria gene ECS to a variety of metals The metals to be tested include Manganese Mn Molybdenum Mo Nickle Ni and Lead Pb which are all commonly found at heavy metal polluted sites Therefore the objective of this study is to determine if transgenic plants are successful at tolerating other metals besides Cd I predict that the transgenic plants will have longer root lengths than wildtypes since they can adapt better the modified GSH levels help the plants to detoxify metals better Likewise I also hypothesize that the transgenic plants will be heavier than wildtype plants since they will be able to accumulate more metals Methods The objective of this study is to test the hypothesis that transgenic ECS seedlings have superior heavy metal tolerance and accmulation as compared to wildtypes The experiment can be divided into two main parts The focus of the first part is to determine the appropriate level of metal concentration to be used in each treatment solution This is essential as metal concentration levels cannot be too high or otherwise all the wildtype plants will be killed and no data will be available to act as comparison to the results of the transgenic plants Similarly the levels cannot be too low or the wildtype plants will grow just as well as the transgenic ones and no tolerance difference can be observed between them The goal is to find the right metal concentration levels so that the wildtype plants will be significantly affected but not die This means that the metal treated wildtype plants should have root lengths and fresh weights that are about 30 to 40 percent lower than those of the control The second part of the experiment involves growing both the transgenic and wildtype seeds simultaneously at the concentration levels determined in part I The untransformed wildtypes serve
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