The nickel hyperaccumulating plants of genus Odontarrhena (Brassicaceae): novel insights from molecular, physiological and biochemical analyses
Creators: name: BETTARINI, ISABELLA
Publisher: name: Universit� degli Studi di Siena
Year: 2021

description: 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 ?g 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.