Zinc hyperaccumulation in Thlaspi caerulescensas a chemical defence against herbivoryABSTRACTThlaspi caerulescens is one of several plant species known toaccumulate heavy metals in excess of 2% of their above ground plantbiomass. The reasons for hyperaccumulation are unknown, butseveral studies conclude that it may be a plant chemical defence. This has been of interest to biologists because these metals areusually toxic. The accumulation of these metals may serve as amodel for coevolution. We examined the effects of zinchyperaccumulation in Thlaspi on Xanthomonas campestris and foundthat the plants containing zinc thrived when inoculated with thisbacteria, while plants not containing zinc showed signs ofdeterioration. INTRODUCTIONThere are several wild plant species that have the ability toaccumulate high quantities of heavy metals in their above groundbiomass, up to three percent or more. Many of these plants arefound in the Brassicaceae family throughout Europe and the BritishIsles. These plants thrive on mineral outcrops with calamine andserpentine soils rich with high levels of zinc, cadmium, and nickel(Baker et al, 1994). Several theories have been advanced on thereasons for this hyperaccumulation. Boyd and Martens propose thatit could be a form of drought resistance, inadvertent uptake,interference, tolerance or disposal of metal from the plant, or achemical defence against herbivory or pathogens. Several studies have supported the chemical defencehypothesis. Martens and Boyd (1994 and Boyd and Martens, 1994)showed that nickel hyperaccumulation is an effective defenceagainst insect herbivores in two different feeding experiments. Boyd et at (1994) also demonstrated that nickel hyperaccumulatingplants resisted pathogens including Xanthomonas campestris. Thlaspi caerulescens J. and C. Presl (Brassicaceae) is ahyperaccumulating plant found in the British Isles. It has beenshown to accumulate 10,000 ppm (>1%) of its biomass in zinc (Bakeret at, 1994), and Pollard and Baker (1997) suggest that this is aneffective defence against herbivory for this species. This paperexplores the effects of zinc hyperaccumulation in Thlaspi as adefence against Xanthomonas campestris. MATERIALS AND METHODSThlaspi caerulescens seeds were collected in Cloughwood, U. K. These seeds germinated on polyester beads supported in expandedpolystyrene rafts floating on one-tenth strength Rorison’s solution(Hewitt, 1966). These containers were placed in a Conviron E-15environmental growth chamber at the following settings: 20 C, 90%RH, 16 hr day, and 8 hr night. After three weeks, twenty seedlingswere transferred to 4 rafts composed of expanded styrene onpolyethylene, each supporting five plants individually. Tenplants floated on one-tenth strength Rorison’s, and ten plantsfloated on a solution containing Rorison’s and 10ppm zinc, as ZnSOThe solutions were freshened every four days to inhibit anypossible algal growth. After twenty days, each plant was transferred to an individualbeaker containing 25ml of solution. The ten Rorison’s plantsretained the same solution as did the zinc plants. Parafilm heldthe plants in place. The plants were then inoculated with threedifferent strains of Xanthomonas campestris, a bacteria known toharm plants. Each plant had each strain inoculated on threedifferent leaves. The plants grew in the growth chamber for oneweek and then were examined. The plants were analyzed with a ranking scale based on
appearance by three people who did not know which plants containedzinc. RESULTSScale:1= Healthy, green leaf with no brown, small puncture hole2= Longer puncture hole with some white spots3= Some leaf discoloration, expanded hole, some shriveling leaves4= Shriveling of several leaves, whole plant not thriving5= Many leaves dead, small shriveled plantsPlant Solution Average Rank 1 Non-zinc 5.00 2 Zinc 1.67 3 Zinc 2.00 4 Non-zinc 4.675 Non-zinc 2.67 6 Non-zinc 3.67 7 Zinc 2.67 8 Non-zinc 2.00 9Non-zinc 4.00 10 Zinc 1.33 11 Non-zinc 1.67 12 Non-zinc 5.00 13Zinc 2.33 14 Zinc 2.00 15 Zinc 1.00 16 Zinc 1.33 17 Zinc2.00 18 Non-zinc 1.67 19 Zinc 1.67 20 Non-zinc 1.00The rankings showed that the zinc plants were on averagehealthier than were the non-zinc plants after being inoculated.However, the non-zinc plants did show a variety of rankings, butthey were statistically unhealthier than the zinc plants. TheMann-Whitney U Test showed a one-tailed probability of 0.031. Weare 97% confident that the results were significant. The zincplants showed a healthier response to the bacteria than did thenon-zinc plants. DISCUSSIONThese results demonstrate that zinc hyperaccumulation inThlaspi caerulescens is an effective defence against the pathogenXanthonomas campestris. Since the experiment was a double blindinvestigation, the results were not biased. Our results were consistent with other studies in this area.Boyd et al. found that nickel accumulation in S. polygaloides wasan effective defence against pathogens such as Xanthomonas. Of theplants inoculated with this bacteria, the nickel accumulatingplants inhibited the growth of a powdery mildew. Growth of thefungus Alternaria brasssicola was also inhibited by nickelconcentration in the plants. Martens and Boyd (1993) showed in feeding experiments thatnickel accumulation is an effective defence against insectherbivory. The insects fed non nickel bearing leaves survived orshowed weight gain while the insects fed nickel bearing leaves didnot. The nickel accumulation is effective because of broadtoxicity, low cost, and high lethality. Pollard and Baker (1997) conducted studies showing preferencesof locusts (Schistocerca gregaria), slugs (Deroceras caruanae), andcaterpillars (Pieris brassicea) to Thlaspi caerulescens grown inlow zinc and zinc amended solutions. They all showed preferentialfeeding on plants with low zinc concentrations. This is an important finding because zinc in these quantitiesis normally lethal to a plant. Boyd and Martens (1993) suggestthat it is reasonable to assume that the hyperaccumulated metals,especially nickel, might also be toxic to pathogens and herbivoressince they are widely used in fungicides and bactericides. Studiesshow that hyperaccumulators are more susceptible to fungi whengrown on non-serpentine soil. Studies by Martens and Boyd (1993)suggest that metal accumulation may provide a useful example of thecoevolution of defence mechanisms because of the increased fitnessit allows for these plants. Herbivores were given a choice betweenaccumulating and non-accumulating S. polygaloides, and the fitnessof non-hyperaccumulating plants was 0.42 of that of thehyperaccumulators. Selective pressures could favor the evolutionof these plants. All of these findings suggest that plants accumulating heavymetals may be utilizing an effective defence against herbivory andpathogens. They may also be good examples of the methods ofcoevolution.