Nickel hyperaccumulation in Alyssum murale as a protective mechanism against herbivory from Pieris rapae: the elemental defense hypothesis

Adrian Chan Bio 2290G Section 7 TM Gray 2012 03 27

Abstract Nickel hyperaccumulation in plants is not essential for survival, so why has this trait evolved in some plant species? The elemental defense hypothesis proposes that plants hyperaccumulate Ni in their leaves to induce Ni toxicity in their predators, thus increasing the plants' evolutionary fitness. The objective of this study was to test this hypothesis using Alyssum murale as the Ni hyperaccumulator and Pieris rapae as the insect predator. Plants of A. murale were grown in three types of soil: unamended, serpentine, and Ni-amended soil. Plant leaves from these three groups were fed to early instar larvae of P. rapae, and the number of surviving larvae from each group was counted at two day intervals. Larvae fed leaves from the serpentine and Ni-amended plants experienced a higher mortality rate than larvae fed leaves from the unamended plants. Ni analysis of the plant leaves revealed that the Ni-amended and serpentine leaves contained significantly more Ni than the unamended plant leaves. These results suggest that Ni hyperaccumulation acts as a self-defense strategy against insect predators, coinciding with the elemental defense hypothesis.

Introduction Nickel pollution in soil and water is prevalent in Asia, Europe, and North America, and is due to human activities such as mining, fossil fuel burning, and disposing industrial and urban waste (Chen et al. 2009). Contaminated soil and surface water possess about 20 to 30 times more Ni than their uncontaminated counterparts (Chen et al. 2009). As a result, human and plant exposure to Ni is increased and can be dire. Human exposure of Ni is mainly through food, water, dust, soil, and air, where food and water

consumption are the most common pathways (De Brouwere et al. 2012). Excessive oral exposure can exuberate existing dermatitis in Ni-sensitive individuals while excessive inhalation exposure can cause lung cancer and lung inflammation (De Brouwere et al. 2012). In plants, depending on their species, growth stage, and environmental conditions, high concentrations of Ni in the soil can inhibit growth, reduce germination, and decrease fruit yield and quality (Chen et al. 2009). Therefore, farmland that has been contaminated with Ni are incapable of crop cultivation (Chen et al. 2009). One proposed solution to rely on plant species that can sequester unusually high amounts of Ni from the soil (Chen et al. 2009). These plants are called Ni hyperaccumulators, which contain at least 1000 mg Ni kg-1 dry weight (Brooks et al. 1977). In contrast, Ni accumulators contain between 100-1000 mg Ni kg-1 dry weight and non-Ni accumulators contain less than 10 mg Ni kg-1 dry weight (Reeves and Baker 2000). After root absorption of Ni, it is transported to the plant's shoots and leaves (Chen et al. 2009). Taxonomically, over 310 plant species have been classified as Ni hyperaccumulators, and are distributed across at least 22 plant families, of which 80 species belong to Brassicaceae (Martens and Boyd 1994; Chen et al. 2009). Many Ni hyperaccumulators are endemic to serpentine soils that are deficient in essential plant nutrients but abundant in heavy metals (Martens and Boyd 1994; Brady et al. 2005). However, it is unknown why this trait has evolved in some plant species because Ni hyperaccumulators are able to grow on non-Ni-bearing soils as well (Martens and Boyd 1994). Boyd and Martens (1992) have postulated five hypotheses to explain the evolution of Ni hyperaccumulation: 1) metal tolerance/disposal, 2) drought resistance, 3) interference with neighboring plants, 4) inadvertent uptake, and 5) elemental defense.

The elemental defense hypothesis suggests that Ni hyperaccumulation may have evolved as a defensive mechanism against herbivores and pathogens. Predators that feed or infect Ni hyperaccumulators may exhibit Ni toxicity, and, as a result, the Ni hyperaccumulator experiences reduced damage. Understanding this role of Ni hyperaccumulation in plants will help authorities contemplate the ecological impact of using Ni hyperaccumulators for phytoremediation in foreign environments. The objective of this study is to test the elemental defense hypothesis that Ni hyperaccumulation in Alyssum murale, a member of Brassicaceae, provides defense against herbivory from the insect herbivore Pieris rapae. To test this hypothesis, larvae of P. rapae were fed plant leaves from plants grown in unamended soil, serpentine soil, and Ni-amended soil, and the number of surviving larvae was counted and compared. It is predicted that there will be a lower number of surviving larvae in the serpentine and Niamended treatment group than in the unamended group.

Methods Serpentine soil was collected from Newfoundland at the foot of the Blow Me Down Range (494' N, 5815' W) located 16 km west of Corner Brook, NL. The soil had a concentration of approximately 300 mg Ni kg-1. Ni-amended soil was prepared by mixing Miracle-Gro Organic Choice potting mix with nickel chloride to a concentration of approximately 1000 mg NiCl2 kg-1. The unamended (control) soil was simply the Miracle-Gro Organic Choice potting mix, which contained 2.35 ppm Ni. Each treatment group (unamended, serpentine, and Ni-amended) consisted of eight replicate pots filled to 10 cm of each soil type. A layer of perlite was placed over all pots.

Seeds of Alyssum murale Waldstein and Kitamura, purchased from the North Central Regional Plant Introduction Station (Ames, IA), were planted into each pot. Pots were thinned to one plant per pot. Plants grew in a greenhouse under a 16/8 hours of light/dark photoperiod and 30/22C thermoperiod for 16 weeks. X-ray absorption spectroscopy was conducted on 3 plants and three 50 g soil samples from each treatment group for their Ni profiles. Six leaves of each plant from each treatment group were analyzed. Larvae of Pieris rapae L. (the cabbage white butterfly) were collected after females oviposited on young cabbage plants kept separately in the greenhouse. Each early instar larva was randomly placed into a 50 mm Petri dish containing 3 to 4 plant leaves from 1 of the 3 soil treatments (18 replicates per treatment). Petri dishes were stored at room temperature, and water and plant leaves were replenished as needed. Surviving larvae were counted in two day intervals for ten days. Larvae were fed a synthetic diet from Bio-Serv Inc. (Frenchtown, NJ), which was amended with NiCl2 to concentrations of 0, 100, 500, and 1000 ppm Ni dry weight. Eight replicates per Ni concentration were conducted. A one-way analysis of variance (ANOVA) was conducted to compare means among the three treatments, and this analysis was followed by Tukey's HSD test to determine differences among means (PASW 2010).

Results In the Ni-amended and serpentine treatment groups, all the larvae died on the eighth and tenth day, respectively, while in the unamended treatment group, no larvae

died (Fig. 1). Larvae fed the Ni-amended synthetic diet at concentrations above 500 ppm died after 6 days (data not shown). Ni concentration in the Ni-amended and serpentine leaves were significantly greater (F(2,51) = 10868.921, P<0.001) than the unamended leaves (Table 1). The Ni-amended and serpentine soil in which the plants grew also had a greater Ni concentration than the unamended soil (Table 2).
Unamended 18 16 Serpentine Ni-amended

Number of Larvae

14 12 10 8 6 4 2 0 0 2 4 6 8 10

Number of Days

Fig. 1. Number of Pieris rapae larvae at each two day interval after fed unamended, serpentine, and Ni-amended plant leaves of Alyssum murale containing various Ni levels. Table 1. Mean Ni (ppm SE) in plant leaves of Alyssum murale grown in each soil type. Soil type Ni level (ppm) Unamended 2.18 0.22a Serpentine 291.61 4.45b Ni-amended 934.06 6.55c Note: Means followed by the same letter are not significantly different (P<0.05) according to Tukey's HSD test. Table 2. Mean Ni concentration (mg kg-1 SD) in each soil type. Soil type Ni concentration (mg kg-1) Unamended 0.0024 0.0009 Serpentine 326 37.9 Ni-amended 977 15

Discussion The elemental defense hypothesis was tested by feeding P. rapae larvae plant leaves of A. murale hyperaccumulated with Ni. As predicted, more larvae survived in the unamended group than in the serpentine and Ni-amended group; in fact, mortality of the larvae occurred only in those that were fed Ni hyperaccumulated leaves from the serpentine and Ni-amended plants. Moreover, larvae fed a Ni-amended synthetic diet died at concentrations above 500 ppm, demonstrating that Ni toxicity alone was responsible for the mortality. These results support the hypothesis that Ni hyperaccumulation protects the plant from herbivory by poisoning the herbivore. This study confirms the results of previous studies that Ni hyperaccumulation in plant leaves is toxic to insect herbivores (Boyd and Martens 1994; Martens and Boyd 1994). Martens and Boyd (1994) showed that larvae of P. rapae fed leaves of Streptanthus polygaloides containing 7000 mg Ni kg-1and 7400 mg Ni kg-1 died by 10 days while the unamended leaves containing 180 mg Ni kg-1 caused no mortality. They also conducted an elemental analysis of the plant leaves, and argued that the large difference in Ni levels between the unamended and Ni-containing plant leaves must be responsible for larvae mortality. Another study by Boyd and Martens (1994) found similar results: larvae of P. rapae fed Ni-amended leaves of Thlaspi montanum var. montanum containing 3000 ppm Ni died by 12 days. In contrast, only 20% of the larvae fed the unamended plant leaves died by 12 days. The high-Ni leaves contained a significant 167-fold more Ni than the low Ni-leaves, thus supporting the conclusion that Ni hyperaccumulation provides defense against insect herbivores.

However, while this study and previous studies (Boyd and Martens 1994; Martens and Boyd 1994) demonstrated that Ni toxicity in P. rapae larvae occurs at Ni concentrations above 500 ppm, the serpentine plant leaves caused a similar mortality pattern to the Ni-amended leaves, even though the Ni-amended leaves contained significantly three times more Ni than the serpentine leaves. This similarity poses the question: is there another mechanism that augments the toxicity of Ni in A. murale? The joint effects hypothesis states that elemental defenses (e.g. Ni hyperaccumulation and Ni accumulation) may work in concert with other plant defenses to produce additive, synergistic, or antagonistic effects (Boyd 2007). This concept suggests that the concentration necessary for elemental defense can be lowered when combined additively or synergistically with organic plant defenses. For example, Jhee et al. (2006) found additive effects between Ni and tannic acid, atropine, and nicotine, which are all organic defense chemicals. In addition, Coleman et al. (2005) showed that Ni toxicity in the herbivore Plutella xylostella occurred at only 20 mg Ni kg-1 when fed amended artificial diet. Therefore, the joint effects hypothesis may explain why the A. murale plants grown in the serpentine soil, although possessing Ni levels in the accumulator range in their leaves, can still defend themselves from herbivory. In addition, since the plants from the serpentine group were grown in a native soil rather than a potting mix, this could inadvertently supply the plants with additional elements that were not accounted for. For example, zinc has been shown to co-accumulate with Ni and provide additive effects (Coleman et al. 2005). This co-accumulation could be responsible for killing more larvae than expected, so a complete elemental profile must be obtained, rather than a Ni profile, to clear this possibility.

The elemental defense mechanism may provide Ni hyperaccumulators (and possibly accumulators) selective advantage over non-Ni accumulating plants in terms of survival against herbivory, in addition to defense. For example, Martens and Boyd (1994) demonstrated that Ni-amended S. polygaloides plants (7400 mg Ni kg-1) had significantly greater dry weight and survival rate after 21 days of herbivory from P. rapae females than the unamended plants (180 mg Ni kg-1). Besides reducing herbivory, Palomino et al. (2007) showed that Ni hyperaccumulation increases a plant's tolerance to herbivory. Using stem diameter as a measure of plant flower number and growth, they showed that T. montanum plants grown in Ni-amended soil (600 mg Ni kg-1) had a bigger stem diameter than those that grew in unamended soil (0 mg Ni kg-1) when artificially damaged (using scissors) at 0%, 10%, and 50% intensity. Martens and Boyd (1994) also suggested that there is no apparent cost to Ni hyperaccumulation, as they found aboveground yield of S. polygaloides grown in Ni-amended (7400 mg Ni kg-1) and unamended (180 mg Ni kg-1) soil had no significant difference in biomass. These findings from Martens and Boyd (1994) and Palomino et al. (2007) are consistent with this study's findings and conclusionNi hyperaccumulation provides defense against herbivory, and in doing so, increases the survival rate and the fitness of the plant. However, when tested in the field, Martens and Boyd (2002) failed to support the elemental defense hypothesis. Ni hyperaccumulated plants suffered extensive damage from herbivory, except from small insect herbivores, which is consistent with previous laboratory studies. Therefore, more research needs to be done in field conditions to expand the scope of the elemental defense hypothesis to include non-insect herbivores.

As suggested by the elemental defense hypothesis, postulated by Boyd and Martens (1992), Ni hyperaccumulation may have evolved in plants to protect themselves from herbivory. By poisoning their predators with Ni, they avoid prolonged damage compared to their non-Ni hyperaccumulator counterparts, thus increasing their fitness. This study has shown that Ni hyperaccumulation (and possibly Ni accumulation) in plant leaves of A. murale causes mortality in larvae of P. rapae while normal Ni levels failed to cause mortality. This result provides evidence for the elemental defense hypothesis, thus fulfilling the objective of this study. However, although many studies have supported the elemental defense hypothesis, most have only focused on Ni hyperaccumulation (not accumulation), members of Brassicaceae, and laboratory conditions (Boyd 2007). In order to bring more meaningful evidence to the elemental defense hypothesis, different experimental approaches are needed. Once these challenges are met with consistent results, we can better understand what ecological impact Ni hyperaccumulators may have on foreign environments when used for phytoremediation. Word Count: 2239

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