Hyperaccumulation the facultative hyperaccumulator Alyssum serpyllifolium. Plants were grown

 Hyperaccumulation is a complex trait that describes a plant’sability to concentrate heavy metals up to 100 to 1000-fold more than typicalconcentrations within their shoot.

Over 500 species are knownhyperaccumulators, of which 73% are nickel hyperaccumulators. Its enormouspotential for commercial application such as for bioremediation of pollutedsites requires a deeper understanding of its mechanism. The influence of Nickelon the uptake of essential nutrients, Calcium, Iron, Magnesium, and Potassium,is investigated in two serpentine populations (S) of the facultativehyperaccumulator Alyssum serpyllifolium.

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Plants were grown in hydroponic solutions supplemented with 0, 30, 100 or 300µM of nickel. Two non-serpentine populations (NS) of A. serpyllifolium and a NS population of a related species of Clypeola jonthlaspi werealso grown to compare foliar elemental concentration (Ca, Fe, Mg and K) under noNi exposure. Elemental concentrations were determined using an atomicabsorption spectrophotometry. When exposed to increased concentrations of Ni,lower Fe, and higher K in both S populations, and lower Mg in one S populationwere recorded.

The two S populations had lower Mg than NS population, and one Shad higher Ca than one NS. The potential contribution of K to physiologicalresponses involved in Ni hyperaccumulation was revealed along with thedemonstration of the antagonistic effect of Ni on Fe. This is important to knowthe mechanism of a hyperaccumulator to tolerate the increased concentration ofheavy metals, for a better understanding to eventually being able to use it.    IntroductionFirstdiscovered in 1948, the phenomenon of metal hyperaccumulation has now beenrecorded in over 500 plant species across the globe (Van der Ent et al., 2012; Anjum, 2013). Hyperaccumulation, a term coined by Jaffré et al. (1976), is the ability of plantsto accumulate metals and metalloids to exceptionally high concentrations intheir above-ground tissues (de la Fuente etal., 2007).

The metals include cadmium, copper, cobalt, chromium, leadmanganese, nickel, thallium, and metalloids such as arsenic. Hyperaccumulatorshave an enormous potential for commercial application, notably for the clean-upof polluted sites. Methods including phytoextraction (extracting soil elementsinto the plant shoots) and phytostabilization (transforming soil metals intononbioavailable form) would effectively liberate sites for various purposes(e.g. agriculture). However, currently known species are not well suited tocommercial use, as they have a low biomass associated with their tolerance toharsh environments and typically only accumulate one type of metal.

Extensiveresearch on the genetics and mechanism of this complex trait is thereforerequired to gain sufficient understanding to unleash its potential. Thisproject attempts to add to the current knowledge by investigating the influenceof nickel (Ni) on the accumulation of four essential macronutrients withinhyperaccumulators, focusing on the nickel hyperaccumulator species Alyssum serpyllifolium, found growingnaturally on serpentine soils. Serpentine soilsSerpentinesoils are metal-rich soils distributed across the world in countries such as inAlbania, Greece, Spain, France, Ural Mountains, United States and Indonesia(Whittaker, 1954). They derive from the weathering of ultramafic rocks,consisting of at least 70% of mafic materials, which originate from themetamorphosis of peridotite (Kruckeberg, 2002; Kristy et al., 2005).

Peridotite principally consists of olivine, (MgFe)2SiO4,and other components such as pyroxenes and amphiboles (Whittaker, 1954). Theproportions of the compounds vary, which is reflected in the colour ofserpentine soils being red, green, blue or black. Serpentine soils sharecharacteristics of high degrees of species endemism, poor plant productivityand distinct vegetation from its neighbouring areas (Whittaker, 1954). Theseare consequences of the “serpentine syndrome”, the cumulative effect a plantexperiences due to the soils’ unusual chemical and physical properties (Kristy et al.

, Jenny 1980). Thedistinctive chemical properties of serpentine soils include their highconcentration of magnesium, low Ca/Mg ratio, low bioavailability of majornutrients, and elevated concentrations of heavy metals. The magnesium (Mg)concentration in serpentine soils is very high due to the parent rocks beingrich in Mg, such as olivine, but these soils are correspondingly poor inbioavailable calcium (Ca).

The ratio of Ca/Mg is therefore much lower than the ratioof unity originally determined for optimal plant growth by Loew and May (1901).Excess Mg also induces localized necrosis at the root tip by actingantagonistically on the uptake of Ca in the root, leading to Ca deficiency(Marschner, 2002; Baker et al.,2013). Serpentine plants have developed physiological mechanisms to toleratethis nutrient imbalance, which include selective uptake of Ca over Mg,requiring higher concentrations of Mg for normal functioning, or the ability tosequester excessive Mg in non-toxic forms that are not harmful to metabolism(Baker et al., 2008). Elevatedconcentrations of potentially toxic metals such as nickel, iron, chromium,cobalt and manganese is also a stress experienced by plants living onserpentine soils. Typical soils have a Ni concentration of 7 to 50 mg kg?1,whereas serpentine soils have 700 to 5000 mg kg?1, i.

e. 100-foldmore Ni (Reeves & Adigüzel, 2008).Thephysical conditions of serpentine soils are also relatively unfavourable forplant growth. Serpentine areas are often found in rocky environments that lackcomplete vegetation cover and therefore suffer from soil erosion (Kruckeberg,2002; Kristy et al.

, 2005). Thisresults in shallow soils with minimal silt and clay content, which reduces soilmoisture levels (Yusuf et al., 2010).Some areas also suffer from droughts as they are characteristic of theMediterranean bioclimatic regions (Rivas-Martinez et al., 2002). Serpentine-tolerant plants share some morphologicaltraits as adaptations to these conditions, such as a smaller height and biomassthan relatives on non-serpentine soils, and xeromorphic foliage with reducedleaf size and sclerophylly (Kristy et al.

,2005).Distributionof metal-hyperaccumulator plants                                                                                                                      Mostexamples of metal-enriched soils are those enriched with nickel, which isthought to explain why nickel hyperaccumulator plants make up more 73% of theknown hyperaccumulator, with 532 out of 721 species. (Brooks, 1998; Reeves andBaker, 2000; Krämer, 2010; Reeves et al.,2017). Out of the 52 families of nickel-hyperaccumulator, Brassicaceae is themost represented with 83 species with the majority being in the genus Alyssum (de la Fuente et al., 2007; Reeves et al., 2017).

Of the 188 species ofAlyssum distributed across the Mediterranean and South West Asian region, 51can hyperaccumulate nickel and are found on serpentine soils (Burge and Barker,2010). The nickel-hyperaccumulating species are either obligate, found on onlyserpentine soil, or facultative, found on both serpentine and limestone-derivedsoils. Alyssum serpyllifolium is a facultativehyperaccumulator, therefore making it a suitable candidate to studyintraspecific variation in nickel hyperaccumulation (Baker et al., 2013).

 Nickel as an essential and toxicelementHyperaccumulatorplants have an unusually high Ni-tolerance threshold, an indispensable propertyto allow them to withstand Ni foliar concentrations greatly exceeding typicalrequirements. Nickel is an essential nutrient for plants that is required invery low amounts, typically 1–10 mg kg?1 of dry biomass, for theenzyme urease, which prevents accumulation of urea produced by metabolicprocesses (Yusuf et al., 2011; Witte,2011). Ni is mainly absorbed in its ionic form Ni2+ which mayinvolve low-affinity transporters from the ZIP family.

Ni exposure has beenshown to stimulate gene expression of the Fe2+ transporter ITR1 andZn transporter ZIP10, both of which are members of the ZIP family (Halimma et al., 2014). Intracellularly, themetals ions are chelated by organic ligands, such as amino acids, to reducetheir toxicity.  Levels of histidine havebeen shown to increase 36-fold in the hyperaccumulator Alyssum lesbiacum when exposed to Ni, as opposed to remaining atconstant levels in non-hyperaccumulator A.montanum (Krämer et al., 1996).

Histidinechelation with Ni is essential for the plant hypertolerance to Ni and for itshigh root-to-shoot Ni flux in the xylem (Krämer, 2010).Oncetaken up by the root system, nickel is then transported through the xylem tothe shoot, driven mainly by transpiration. Ultimately, Ni is transferred to thevacuole and chelated by carboxylic acids (Smart et al., 2010; Van der Ent etal., 2017). The process is tightly regulated to prevent nickelphytotoxicity, which would interfere with numerous aspects of plant metabolismsuch as growth, mitotic and enzymatic activities, photosynthesis, seedgermination, and transport of nutrients. Nickel can also induce chlorosis,narcosis and wilting (Bhalerao et al.,2015).

More specifically, Ni can reduce photosynthetic pigments, restrictswater movement from the roots to the shoots by decreasing transpiration rate,conductance, leaf water potential and total moisture content, and induceschanges in morphology such as decreasing mesophyll thickness and width ofepidermal cells (Bishnoi et al., 1993; Seregin and Kozhevnikova, 2006a; Dubey et al., 2011). Lessresearch has been conducted on the effect of Ni on the concentrations of otherfoliar elements compared to the effect of Ni on plant physiology. The mostwell-known effect of Ni is on foliar iron (Fe), which has been reported torespond antagonistically in non-hyperaccumulators (e.g. maize) as well as inhyperaccumulators such as Alyssum inflatum (Maksimovi? et al., 2007; Ghasemi et al.

, 2009). This is partly due toiron-regulated transporter 1 (ITR1), which is the primary transport system forFe uptake into roots, but which has a broad substrate specificity and cantransport divalent metals such as Ni (Nishida et al., 2011). In rice, wheat and maize seedlings, exposure to Nisignificantly reduces Ca, Mg and potassium (K) content in the aerial part ofthe plants (Rubio et al., 1994;Matraszek, 2016; Pavlovkin et al.,2017). The effects of Ni on elements inhyperaccumulators have been even less studied, although this is essential for aproper understanding of hyperaccumulation.

Recently, however, a study byQuintela- Sabarís et al. (2017) on Alyssum serpyllifolium analysed theinfluence of Ni on foliar concentrations of four nutrients (Ca, K, P and Mg) inplants of populations found on serpentine and limestone-derived soils grown inexperimental soils supplemented with Ni (Quintela-Sabarís et al., 2017). It is not clear, however, from this experiment howincreased Ni in particular, as opposed to other serpentine factors in general,affected the accumulation on the other foliar elements.

Experiments by Brooks et al. (1981) began to elucidate thisinfluence by demonstrating a decrease in Ca and an increase in Mg in aserpentine population of A.serpyllifolium exposed to Ni. The current project therefore attempts toexplore this influence more systematically by studying the effect of nickel ata range of concentrations on the accumulation of four elements in Alyssum serpyllifolium. To do so, theexperimental plants were grown in hydroponic solutions, which gave more precisecontrol over the concentration of Ni to which plants were exposed, andelemental concentrations were measured in plant extracts using atomicabsorption spectrophotometry (AAS).

Objectives The interactions between Ni and thenutrients, Ca, Fe, Mg and K are investigated in two serpentine populations of Alyssum serpyllifolium. Nickel exposureis expected to reduce the uptake of Fe as a result of ions Ni2+competing with Fe2+ for the transporter, to reduce the uptake of Caand increase the uptake of Mg (as demonstrated in a previous study) and as theeffect of K is unknown in the literature, it is suggested to decrease, as withnon-hyperaccumulators.  The variation in concentrations ofnutrients in the shoots between two serpentines and two non-serpentinepopulations of Alyssum serpyllifoliumand one non-serpentine population of Clypeolajonthlaspi was also investigated under no Ni exposure.

The two serpentinespopulation are expected to have a higher concentration of Ca and a lowerconcentration of Mg due to their tolerance of serpentine soils, which isthought to include selective uptake of Ca over Mg and limiting uptake of Mg.The two other macronutrients are not expected to differ significantly betweenthe populations.