The nickel hyperaccumulating plants of genus Odontarrhena (Brassicaceae): novel insights from molecular, physiological and biochemical analyses

Due to the high nickel concentrations, serpentine soils provide a very restrictive and selective environment for plant life. Some plants, termed “Ni-hyperaccumulators”, are adapted to live on these heavy-metal-enriched soils without toxicity symptoms. Ni-hyperaccumulators are increasingly important for research on metal tolerance, homeostasis and biotechnological applications. This project aims to investigate nickel accumulation in taxa and populations of Odontarrhena, a genus of tribe Alysseae (Brassicaceae) that includes over 85 species many of which are Ni-hyperaccumulators. Based on a previous systematic study conducted on poorly-known populations of Odontarrhena native to Albania we performed a molecular study to characterize taxa and populations of this genus. To this purpose we used DNA sequencing and the AFLP-fingerprint technique to reconstruct the species phylogenetic relationships and the population differentiation patterns in relation to their distribution, ploidy level, intensity of anthropic site disturbance, altitude, soil type and metal concentration population (Ni, Cr, Co, Ca, Mg). We found significant population differentiation, dominance of within-population variation, no isolation by geographic distance and existence of six genetic groups variously represented across the six taxa possibly due to hybridization especially in disturbed sites. Next, we compared metal concentrations in native Odontarrhena populations from Albania in relation to their soil of origin. We determined the concentration of the most important trace metals (Ni, Co, Cr, Mg, Ca, K, Fe and Mn) in soil, plant roots and shoots of five taxa from 20 different outcrops. We found large differences in mineral element concentrations in soils and also between the plants; shoot Ni concentrations in Albanian Odontarrhena taxa depend on soil Ni concentrations but not on species identity. For O. chalcidica, the most widely distributed species, this “environmental fingerprint” was found not only for Ni, but also for Ca and Mg. After these investigations on native populations from the natural environment, we designed an experimental study in controlled conditions. Plant seedlings of seven taxa and 11 populations of Odontarrhena from serpentine and non-serpentine sites of the Balkan peninsula and Italy were cultivated in hydroponics with increasing NiSO4 concentrations to determine plant growth and Ni accumulation. These plantlets were analyzed to test inter- and intra-specific differences in nickel tolerance and accumulation, in relation to Ni levels in the soils and in wild plants. We found a metal stimulatory effect on growth that was present in the low-dose zone and significantly fitted the Brain-Cousens hormetic model. Taxa showed broad variation in tolerance, with the most tolerant plants requiring the highest Ni concentration for optimal growth. Our data suggested that tolerance is associated with hyperaccumulation ability. Among the obligate and facultative serpentinophytic species of Odontarrhena that have been investigated we found a notable exception, O. sibirica, a facultative serpentinophyte in which accumulation ability was enigmatic from previous studies. We addressed this issue using observational and experimental methods as in our previous researches. We found that Ni-concentrations in the native populations sampled on serpentine soils in Greece were always much lower than the hyperaccumulation threshold. When cultivated together with other Ni-accumulating Odontarrhena species on the same natural ultramafic soil, O. sibirica was the only one unable to accumulate the metal. When grown in hydroponics at different NiSO4 levels Ni-accumulation occurred only at higher concentrations which, however, had a toxic effect. This peculiar combination of Ni-response traits could be the result of a partial evolutionary loss of ability with respect to all other Ni-accumulating congeneric species. For its unique characteristics, O. sibirica could therefore represent a unique model system for further studies on the evolutionary dynamics, physiological mechanisms and genetic control of metal accumulation and homeostasis. In a parallel study, we investigated photosynthesis responses of the same plants using an experimental approach. In non-hyperaccumulator plants, toxicity symptoms to above 10 μg g-1 DW nickel concentrations in soils can include inhibition of photosynthesis, impaired nitrogen assimilation and disturbed enzyme activity. However, there is a complete lack of information about how Ni-hyperaccumulators reconcile that extraordinary amount of metal in their shoots with an efficient photosynthetic activity, or at least on which photosynthetic parameters the excess of Ni impacts less in these plants. We measured Ni effects on growth, root and shoot metal accumulation and several photosynthetic parameters, such as gas exchange, chlorophyll fluorescence analyses and pigments content in three Odontarrhena taxa (two hyperaccumulators, one not) grown in hydroponics and exposed to three NiSO4 treatments. We found that Ni-hyperaccumulators species are photosynthetically more efficient under Ni excess in respect to the non-accumulating species. In fact, Ni treatment in O. chalcidica increased not only the photochemical efficiency of PSII and the CO2 assimilation rate, but also the stomatal conductance. Finally, this project focused on the determination of the activity of the enzyme urease, the only Nimetalloenzyme known so far in plants, in selected Odontarrhena taxa. The hypothesis to test was whether the high basal requirement for this micronutrient in these plants could be linked to a depletion of the Ni cytosolic pool at low external metal concentration, due to hyperaccumulation mechanism and impairing urease activity. To this purpose, enzyme activity and Ni shoot concentration were determined in plants of accumulating and non-accumulating taxa of Odontarrhena cultivated on Ni-rich serpentine soil and on garden soil, as well as in samples of O. bertolonii cultivated in hydroponics at increasing Ni concentrations. Odontarrhena hyperaccumulators showed similar urease activity when grown on both kinds of soils, with no relation between the enzyme activity and the leaf Ni accumulation. Contrarily, clear indications came from the experiment in controlled conditions, where the presence of Ni determined a progressive stimulation, in respect to control samples, of the activity of the enzyme, associated with an increase in shoot metal concentration. A significant relationship was found between the levels of urease activity and the amount of Ni accumulated in the leaves. Therefore, the already known Ni-stimulated growth of O. bertolonii at increasing metal concentrations in the low-dose zone could be explained by a Ni-induced activity of urease, associable to an enhanced nitrogen metabolism, unless other still unknown physiological functions of Ni in hyperaccumulating plants.