Discussion competition for ITR1 is the up-regulation of

Discussion

In
this project, the influence of Ni exposure on the accumulation of four
essential nutrient (Ca, Fe, Mg and K) in two serpentine population of Alyssum serpyllifolium was investigated.
A significant reduction in Fe and an increase of K in both populations, and
reduction in Mg in Carratraca were detected. The variation in foliar elements
between two serpentines, two non-serpentine populations of Alyssum serpyllifolium and one non-serpentine population of Clypeola jonthlaspi was also
investigated. The two serpentine populations had significantly lower Mg than
one or two non-serpentine population, and one serpentine population (Bragança)
had significantly higher Ca than one serpentine population (Morata). 

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Iron
was significantly reduced overall in both population. Plants
of Bragança exposed to 300 µM showed signs of chlorosis (figure 4), a visual
symptom of Fe-deficiency (Lešková et al.,2017).
However, the leaves recovered within the successive week and did not show
further signs of chlorosis for the remaining of the experiment. The
reduced Fe in the shoots would partly be due to reduced Fe uptake as the ion Ni2+
competes with Fe2+ for the IRT1 in the roots (Nishida et al., 2011). A physiological response
to this competition for ITR1 is the up-regulation of IRT1 (Lešková et al.,2017). This could explain the
increase in Fe observed in Carratraca at 30 µM Ni as the Ni in the
concentration may be dilute enough to not saturate the IRT1. However, a
consequence of this upregulation is the increased uptake of Ni which results in
less Fe uptake, explaining the overall significant decrease in Fe. The
reduction of Fe is also observed in non-serpentine populations such as A. inflatum with a 17.4% and 12.4%
decrease in Fe when exposed to 100 and 300 µM Ni (Ghasemi et al., 2009).

The
Fe of the four population of Alyssum serpyllifolium differed significantly
with the closely related species Clypeola
jonthlaspi but not between each other. Their concentrations of Fe are
within the critical Fe deficiency range 0.005 to 0.015 (w/w) %, whereas
Clypeola Fe is higher (Marschner, 2011). This suggests that A. serpyllifolium may be tolerant to
lower foliar Fe even without the exposure to Ni. This could be in support of
the “incremental advantage” hypothesis, outlined by Pollard et al (2014) to explain facultative
hyperaccumulation. The hypothesis suggests that maintaining physiological mechanism
associated with hyperaccumulation confers a selective advantage to plants on non-serpentine
soils. These include tolerance to drought, allelopathic interference with neighbouring
plants, and defence against herbivores and pathogens (). In this example, requiring
a lower Fe for optimal growth would be the selective advantage over neighbouring
plants. Although Fe is the second most abundant metal in the earth crust, its bioavailability
in aerobic and alkaline soil is low as Fe is in its insoluble form, ferric oxide
(Morrissey et al., 2009; Marschner,
2011).

 

The
K in the leaves of population Bragança and Carratraca significantly increase
when exposed to Ni. Unlike Fe, the uptake of K is highly selective thus Ni2+
would not be interfering (Marschner, 2011). K is found in high concentration in
the cytosol of plant cells which contributes to maintaining an optimum pH of 7
to 8 by balancing the charges of soluble and insoluble ions, such as organic
acids (Marschner, 2011). Within the leaf cells of Alyssum, Ni is in a complex with carboxylic acids such as malic and
malonic acids to reduce its toxicity (Van der Ent et al., 2017). Therefore, the increase in K could be a
physiological response to the increase in carboxylic acids being accumulated
within the cells as the plants accumulate Ni. K is also involved in in
regulating the osmotic potential within vacuoles (Krämer et al., 2000). As Ni is concentrated mainly in the vacuoles, the
increase in K could also be a response to the change in its osmotic potential
(Smart et al., 2010). The four
populations of A. serpyllifolium do
not have significantly different K with no Ni exposure. However, serpentine
population of A. serpyllifolium have
been shown to have significantly higher K than non-serpentine populations when
grown on serpentine soils (Quintela-Sabarís et
al., 2017). This would support the suggestion that an increase in K is a
result of the accumulation of Ni within the cells. 

 Mg
significantly decrease within shoots of Carratraca as higher concentration of
Ni was accumulated. The transporters of Mg facilitate the transport of Mg2+
along the gradient in electro-chemical potential. The uptake has been shown to
be repressed by other cations such as K+ and Ca2+ which
could suggest that Ni2+ could also interfere with the uptake (Marshner,
2011).  Studies on Saccharomyces cerevisiae revealed
that the cells could be rescued from toxicity of nickel by supplementing higher
concentration of Mg. This suggesting that Ni is taken up into the cells via
presumed Mg2+ transport system located in the plasma membrane
(Nishimura et al., 1998). The reduced
Mg in Carratraca would support that Ni interferes with Mg although Bragança
does not reveal the same relationship. However, a study on serpentine
subspecies Alyssum malacitanum, grown
on 100, 1000 and 10000 µg/g Ni, demonstrated an increase in Mg (Brooks et al., 1981). Mg2+ is
involved in regulating the pH and cation-anion balance thus similarly to K,
could be increased as a response to higher concentration of carboxylic acid-Ni
complexes (Marschner, 2011). Brooks et al.,
(1981) used higher concentration of
Ni exposure which could explain the difference in relationships demonstrated
although Bragança was also shown to increase, albeit not statistically justified.
This suggests that the interaction between Ni and Mg needs to be explored
further in larger number of populations.

Within
Alyssum serpyllifolium, the
serpentine populations had significantly lower Mg than one or two non-serpentine
populations. This reflects their tolerance to serpentine soils as hypothesised
by Kruckerberg (1954), that plants limit their uptake of Mg to tolerate high Mg
concentrations of serpentine soils. Alternatively, the lower Mg could be due to
serpentine populations requiring higher Mg bioavailability than in the standard
nutrient solutions to uptake sufficient levels of Mg. This has been shown in
serpentine endemic Poa curtifolia and
serpentine races of Agrostis stolonifera which
needed a higher Mg soil concentration (Main, 1981; Marrs and Proctor,
1976). 

As
the plants were grown in identical environment, this physiological difference is
suggested to be due to local adaptation as opposed to phenotypic plasticity, trait
is inherited and not due an interaction with the environment (Kawecki and Ebert, 2004).
Reciprocal transplant experiments could be conducted to compare the populations
fitness when grown on the other type of soils to investigate this idea. Also,
it would be interesting to investigate if this difference is sustained through
generations when grown on the same environment. 

Ni
did not influence the uptake of Ca in either populations. With no Ni exposure,
one serpentine population (Bragança) had significantly higher Ca than a
non-serpentine population (Morata). Bragança also had the lowest Mg which
illustrates another hypothesis of serpentine tolerance, which is the selective
uptake of Ca over Mg (Baker et al.,
2008). Serpentine populations of A.
serpyllifolium had significantly higher Ca and lower Mg than non-serpentine
populations when grown on serpentine soils (Quintela-Sabarís et al., 2017). Their Ca/Mg molar ratio
were > 4 compared to 1 in non-serpentine population, further supporting the
hypothesis of serpentine plants having greater absorption of Ca over Mg.

It
is important to note that the plants of both serpentine populations remained
visually healthy throughout the experiment, with one exception mentioned
earlier. This suggests that although Ni may influence the uptake of nutrient,
plants have altered physiology and/or mechanisms to cope with this influence.

The
influence of Ni need to be investigated in more populations as the results obtained
would be more reliable with more data supporting it, especially as some of the
results are not consistent between the two serpentine populations. It is difficult
to suggests whether this is due to variation between serpentine populations,
potentially due to variation in the chemical composition of the soil of origin,
or if the variation is due biological variation within the study. It is being
debated whether Ni hyperaccumulation and tolerance are population specific or
species wide traits, although it is suggested that the answer is more nuanced
(Pollard et al., 2014). For example,
studies on Thlaspi caerulescens
revealed that the uptake, root to shoot translocation and tolerance of heavy
metals are uncorrelated within serpentine and non-serpentine populations (Assunção
et al., 2003). The intraspecific
variation in these three traits could explain differences between Bragança and
Carratraca, notably its Mg response to Ni. Studies on the offspring of
non-serpentine and serpentine populations, presuming the ability of producing
viable seeds, would provide insights on the heritability and genetics which
could help in the debate illustrated above.

Further work should also integrate
the effect of Ni on the root cells with the effect on shoot cells. Hyperaccumulators
have physiological mechanisms that allows them to concentrate heavy metals in
their leaves as opposed to non-accumulators that concentrate in their root (Karmer,
2010). For example, Ni sequestration in root vacuoles have been found to be
repressed by Ni2+chelation with histidine as higher concentrations
of histidine has been found in roots of Ni hyperaccumulators Alyssum lesbiacum than in
non-hyperaccumulator Brassica juncea
(Kerkeb and Krämer, 2003; Kozhevnikova et al., 2014). This results in Ni
shoot: root ratio to be higher than in non-hyperaccumulators. Comparison
between elements shoot: root ratio and effect of Ni could provide insight in
the transport mechanism of Ni through the stems and within the leaves. This
would reveal the plants altered physiology to remain healthy and tolerate high
concentration of Ni.  

 

Conclusion

In
conclusion, the negative interaction between Ni and Fe and positive interaction
between Ni and K in the shoots were revealed in the two serpentine populations
of Alyssum serpyllifolium. Serpentine
populations were shown to have a lower uptake of Ca and higher uptake of Mg, consistent
with hypothesis of serpentine tolerance. Further works should be focused on  shoots and roots of a larger set of populations
in more species of facultative hyperaccumulators to provide a more extensive
and complete data on the influence of Ni on foliar nutrients. This is fundamental
in understanding plants ability to tolerate and accumulate heavy metals, crucial
for any development of future commercial application.