4.1.1 – Chemically-assisted phytoextraction experiments

C

4. D

4.1 – Phytoextraction experiments

4.1.1 – Chemically-assisted phytoextraction experiments

The degree of germination of seeds in the presence of toxic metals, which is to some extent a measure of the tolerance to the element concerned (Baker and Brooks, 1989), allowed a preliminary screening of the starting nine species (Table 3.1). The species which showed the highest germinability (B. carinata, S. cereale, T. alexandrinum, H.

annus and Z. mays) were further screened for selecting the species that showed the highest tolerance index (B. carinata, S. cereale and T. alexandrinum) (Table 3.2). Since one of the main damaging effects of metals is the reduction of the root length due to limited cell division and elongation (Macnair et al., 2000), it is not desirable to use plants that would have a limited below-ground development.

The hydroponic study using each metal separately was aimed to compare the effectiveness of their uptake among the plants under investigation without chelate side- effects and interferences by other metals, and thus to select the species which shows overall the highest potential for multiple metal extraction. In selecting plants for effective phytoextraction the use of metal concentrations on a dry weight basis may lead to wrong choices because data should be considered on a plant basis so as to take into account the large variability showed in biomass among species. It must be considered that the potential biomass yield under field conditions is generally higher compared to greenhouse one. However, this could be true for all species screened in the present study. The higher dry matter production of B. carinata in comparison with T.

alexandrinum compensated for the lower metal concentrations (Tables 3.3 and 3.4). For several plant species it has been shown that photosynthesis is inhibited by metals at different target sites with a consequent reduction of growth (Haag-Kerwer et al., 1999).

Thus, to maintain biomass production under altered environmental conditions, plants

require efficient and different cellular detoxification systems (Hall, 2002). This capacity

to activate defence mechanisms might account, at least in part, for the slight reduction of B. carinata dry biomass and root length in the presence of redox (As, Cu) and non- redox metals (Cd, Pb, Zn) (Table 3.3).

The hydroponic experiment with all the metals together was carried out to evaluate the effects of chelators on the overall ability of B. carinata to take up metals and accumulate them in the shoots. Although in the presence of both chelators free metal concentrations in the solutions were negligible, since about the 99% of metals, except for As, was bound to the chelating agents, B. carinata grown in presence of five metals and one chelator (NTA or EDDS) showed a huge ability to accumulate metals in roots and shoots, with EDDS being more effective than NTA in enhancing their translocation (Figs. 3.1a and 3.1b). That capacity appeared to be more evident when the amounts of metals accumulated and translocated from the multiple-contaminated solutions (Figs.

3.1a and 3.1b) were compared with those accumulated from the single-contaminated solutions (Table 3.4). Therefore, the presence of chelators seemed to favour the accumulation of metals in plant tissues instead of making it more difficult due to their high capacity to bind metals in solution (Table 3.6). The dramatic enhancement of metal uptake by the chelators can be explained by their influence on metal speciation in the nutrient solutions. Due to its high content of carboxylic groups, the apoplasm acts as an effective cation exchanger. Therefore, cations tend to be bound to the intercellular space of roots, whereas anions are not. In the chelant treatments, the formation of negatively charged complexes prevented free metals from binding to the cation exchange sites in the cell walls of the roots and allowed them to enter into the cells by a non-selective apoplastic mechanism (Wenger et al., 2003, 2005). It has been hypothesised that chelates may enter the roots at breaks in the root endodermis and the Casparian strip, and be rapidly transported to the shoots (Nowack et al., 2006). With a high dissolved metal concentration, the non-selective uptake in the presence of chelants would exceed selective uptake along the symplastic pathway (Nowack et al., 2006). The detection of EDDS in roots, shoots and xylem sap of sunflower grown either in soil or in hydroponics (Tandy et al., 2006a, b) and of EDDS and NTA in roots and shoots of B.

carinata (Fig. 3.22) is an indication that metal complexes or free chelators are taken up

metal-EDDS chelates were more mobile within the plant apoplastic system and, therefore, more accumulated in the shoots (Figs. 3.1a, 3.21 and 3.22b). According to these results, more EDDS, compared to NTA, was found in roots and shoots of B.

carinata (Fig. 3.22). It can be suggested that the interactions of the metal-NTA complexes with the root matrix were stronger than those with EDDS and/or that available uncoordinated free EDDS (HEDDS

and H

EDDS

) destroyed the physiological barriers in roots by removing Fe

, Ca

and other divalent cations from the plasma membrane, which is thought to play a major role in the root selectivity properties (Luo et al., 2005). On the other hand, NTA alone was not found to be phytotoxic compared to other chelators (Schuman et al., 1991). To reach a high accumulation of Pb-EDTA in shoots, a threshold concentration of free protonated EDTA was required in the solution (Vassil et al., 1998). It is suggested that in the present case EDDS, but not NTA, may have exceeded such a limit and a higher concentration of metal-EDDS complexes and free EDDS reached the stele and the xylem. As chelating agents are thought to protect cells from the oxidative damaging effects of metals inactivating and minimizing the negative impact of free metal ions (Ruley et al., 2006), the presence of high concentrations of EDDS in its free form might account in part for the reduced growth of B. carinata shoots (Fig. 3.1c). The higher toxicity of EDDS, compared to NTA, is confirmed by the fact that in presence of NTA a similarly huge amount of metals was accumulated in roots and shoots of B. carinata (Figs. 3.1a and 3.1b) without any significant growth reduction (Fig. 3.1c).

In agreement with comparative experiments (Tandy et al., 2004; Meers et al., 2005), the ability of EDDS to desorb metals from the soil was higher in comparison to NTA (Fig.

3.3b) due to its higher affinity constants for Cu, Pb and Zn (Table 3.6). Even though

total Zn was present in the mixed soil at a higher concentration compared to Pb (Table

3.5), and Zn-NTA or Zn-EDDS complexes have higher or similar logK values than Pb

ones, the capacity of both chelators to desorb Zn from the soil was quite low as a

consequence of its adsorption to soil particles (Fig. 3.3a). For the same reason and due

to the low stability constant values of the complexes (logK 10.5 and 11.4 for NTA and

EDDS, respectively), Cd was not available for plant uptake following chelators

addition. Arsenic does not form complexes with the chelating agents; this can explain the low soluble As amounts found in the amended soil (Fig. 3.3b).

Since a sufficient fertilization was used, the low biomass yields of pot-grown plants (Fig. 3.4) might be attributed to the fact that the number of plants per pot was relatively high and/or that peat addition did not improve soil structure and characteristics enough for plant optimal growth. Differently from what was observed in hydroponics, in the pot experiment none of the treatments significantly affected biomass production of B.

carinata (Fig. 3.4). This may be attributed to the shorter time span between treatments and harvest (one week) compared to that of hydroponical experiments (4 weeks). The longer the time period between treatments and harvest, the greater the growth depression caused by metal/chelator toxicity. In addition, the higher complexity of the soil system compared to the hydroponic culture and the higher biomass of Brassica plants at the moment of amendment must be also taken into consideration. However, both amendments caused acute symptoms on leaves which showed large necrotic areas and curling of the edges (Fig. 3.5) due to the rapid intake of metal complexes and/or chelants which overcame cellular antioxidative defence system (Navari-Izzo and Quartacci, 2001). It has been found that application of free chelators resulted in the removal of essential metal nutrients from soil leading to deficiencies in the plants (Geebelen et al., 2002), even if in the present case it is likely that the short time between chelator application and plant harvest did not cause this effect. Moreover, chelators have a higher affinity for transition metals than for alkaline and alkaline earth elements.

In agreement with the available metal amounts detected in the soil, NTA and EDDS applications enhanced the capacity of B. carinata shoots to accumulate Cu, Pb and Zn (Fig. 3.6a). A two-step process for assisted phytoextraction was suggested (Ensley et al., 1999). Plants first accumulate free metals in their roots in the time period before chelator application and then, through the application of a chelating agent, metals are complexed within the roots and translocated as metal chelates. Alternatively, free metals could have been taken up by a selective active transport mechanism and then complexed by free chelators in the plant (Tandy et al., 2006b).

The 1.4- to 2-fold (NTA) and the 2- to 4-fold (EDDS) increases in Cu, Pb and Zn

considered suitable for planning a realistic and short-term phytoremediation program of the pyrite ashes-polluted area. The relatively high bioavailability of metals in the multiple metal-contaminated soil severely limited B. carinata growth. On the other hand, a high biomass at the moment of chelant addition is important to provide a high water flow for chelate uptake and to store metal complexes (Tandy et al., 2006b). Only moderately contaminated soils or soils with low bioavailability of the contaminating metals could be cleaned-up by phytoextraction techniques using B. carinata. However, B. carinata demonstrated the ability to survive and tolerate more metals supplied together, and in the presence of multiple contaminations the simultaneous uptake of more metals cannot be discarded.

In the present study, only a very limited fraction of metals (< 0.2% of total metal

content) was effectively absorbed and translocated by the plants. This fact confirms that

amendments and their effects should be short-lived in order to allow the remaining

portion of available metals to be re-stabilised in the soil by (bio)degradation of the

chelate, and to reduce the risk of percolation (Meers et al., 2005). One week after

chelant amendments, the amounts of available metals in the soil treated with EDDS

were lower than in the NTA treatment (Fig. 3.6b) in spite of the higher capacity of

EDDS to desorb metals. The higher accumulation of B. carinata following EDDS

amendment (Fig. 3.6a) could explain this pattern or, alternatively, another hypothesis

regarding the higher biodegradability of EDDS, compared to NTA, could come into

play. To confirm the latter hypothesis, the variable plant was eliminated and the pattern

of extractable amount of metals in soil was analysed over time (Fig. 3.7). The reduction

of DTPA-extractable metals in EDDS-amended soil, not observed in NTA-amended

soil, could be due to the faster degradation in the soil system of metal-EDDS complexes

compared to NTA ones. In the mixed soil condition, the half-live of 7 days for EDDS

(Table 3.7) further confirmed that the reduction of DTPA-extractable metals was due to

a degradation of the chelating agent and a subsequent re-association of metals with the

soil particles. Those results are in agreement with those of other authors. In fact, it has

been found that half-lives for EDDS ranged between 3.8 and 7.5 days depending on the

applied dose and soil characteristics, and that metal availability following EDDS

application was not different from that in the control group 28 days after chelator

addition (Meers et al., 2005; Luo et al., 2006). As a consequence, in the present phytoextraction program the risk of metal leaching to the surrounding environments can be considered potentially low, especially when EDDS was used. The biodegradability of metal chelates strongly depends on the type of metal involved and is not related to the stability constant of the complex (Vandevivere et al., 2001). Pb-EDDS biodegrades much more readily than Zn-EDDS, although both complexes have very similar stability constants (logK = 13.45 and 14.20 for Pb and Zn, respectively). This may account for the similar Pb and Zn concentrations in the soil one week after amendments notwithstanding a higher Pb concentration was initially desorbed by EDDS (Fig. 3.3b).

4.1.2 – Wild species-assisted phytoextraction experiments

Although EDDS was found to be the most biodegradable among the strong transition metal chelators, the data of Meers et al. (2005) suggest that under less optimal conditions ligand or ligand effect persistence may increase dramatically. These results caution against the use of any amendment, biodegradable or otherwise, without proper investigation of its effects and longevity.

Although in the conditions used in the present experiment EDDS appeared to be a non- persistent chelating agent, the search of new methods, more environmentally friendly than the present technologies, for enhancing metal bioavailability is still necessary.

The idea that some plants with specific characteristics could improve the extraction capacity of other plants is new and interesting, because it could improve phytoextraction in an environmentally safe way.

The evidence that non-accumulating plants exude more organic compounds than

hyperaccumulator ones (Salt et al., 2000; Zhao et al., 2001) suggests the possibility of

using these species for producing natural chelators for assisted phytoextraction

programs. If the roots exudates remain stable in soils for sufficient time, they can

improve the phytoextraction capacities of high biomass crops grown in succession to

the excluder plants. To date, this is the first study addressing this topic.

In order to investigate if a exudates-assisted phytoextraction could be possible, three excluders plants (Plantago lanceolata, Pinus pinaster and Silene paradoxa) had been grown in the mixed soil immediately before B. carinata (a high biomass crop species) was planted .

The amount of DTPA-extractable metals, analysed immediately after the harvest of the three excluders species, showed that their cultivation had a positive effect on metals mobilisation in spite of the strong association of those metals to the soil particles (Figs.

3.3a and 3.8) with the available metal values approaching or exceeding, as in the cases of Cd and Cu, the values obtained following chelators application (Figs. 3.7 and 3.8).

Although the pattern of mobilising metals was strictly dependent on the wild species used, it appeared that Pinus pinaster and Silene paradoxa showed the same capacity of mobilising metals from the mixed soil and that their potential was significantly higher than that of Plantago lanceolata (Fig. 3.8). In agreement with previous findings (Figs.

3.3a, 3.6b and 3.7), the extractable fraction of Cu was particularly enhanced in soil in which wild species had been grown (Fig. 3.8).

The mobilisation due to wild species cultivation was probably due to the decrease of soil pH. A confirmation of this hypothesis lies in the fact that the DTPA-extractable amount of arsenic was not influenced by the growth of the excluders plants. In fact, it is widely known that the availability of arsenic does not increase under acidic soil conditions.

An efflux of organic anions substances cannot occur without being balanced by the

release of cations because the membrane potential would otherwise collapse to the

equilibrium potential and rapidly inhibit any transport (Ryan et al., 2001). Jones (1998)

proposed that the efflux of organic substances could be driven by the electric charge

gradient that exists across the plasma membrane (ca. –180 mV) due to the operation of

ATP-driven proton pumps (H

-ATPases). While the H

expelled into the apoplast by

these H

-ATPases creates a charge gradient to facilitate the uptake of cations from the

soil, it also tends to draw anions (e.g. citrate

) out of the cells and into the external soil

solution. Organic acids typically flow across the lipid bilayer at a slow rate in response

to this electrochemical gradient (baseline exudation); however, efflux can be increased

greatly, for example in stress conditions, by the opening of channels embedded in the

lipid bilayer (Dennis et al., 1997). It has been shown that also K

, Ca

and Mg

can serve as counter-ions to maintain electroneutrality when metal-activated efflux of organic anions occurs (Qin et al., 2007; Osawa and Matsumoto, 2002). The activation of the ATP-driven proton pumps located in the plasma membrane due to the necessity of exuding organic compounds from roots could explain the decrease in soil pH observed following the cultivation of the three wild species. However, the fact that the decrease in the pH was similar for the three excluders while the capacity of mobilising metals was different among each other and was also metal specific (Fig. 3.8) allows to consider that the decrease of pH values only partly account for interspecific differences in metal mobilisation among the three wild species considered. The exudation of specific organic ligands or the amount of organic ligands exuded by roots could account for the rest of the differences found in the present study.

For this reason, the analysis of the organic compounds, in terms of flavonoids, organic and phenolic acids, exuded by Pinus pinaster, Plantago lanceolata and Silene vulgaris as response to heavy metals toxicity was performed in solution culture. It is known that the hydroponic culture technique suffers from a series of severe drawbacks among which the most serious criticism is that roots grown under these conditions may be morphologically and physiologically very different from those growing in a real soil. In addition, the aeration, microbial and nutritional status of these hydroponic cultures is often very different from those of a typical soil environment. However, despite all these methodological limitations, hydroponical experiments are the most useful practices for studying the organic exudates of plants. In addition, when hydroponical experiments are used in comparative studies (control vs treatment solutions), as it was in the present work, they become a strong instruments in investigating the role of organic exudates in plant responses to many environmental stresses.

Since organic acids with only one carboxylic group, such as lactate, formate and acetate, have very little metal-complexing ability, only dicarboxylic and tricarboxylic acids, potentially strong metal chelators (Martell and Smith, 1977), have been looked for.

Several different organic acids, principally oxalic (Ma et al., 1997a; Ahonen-Jonnarth et

Pellet et al., 1997) acid were found to be exuded in increased amounts by roots exposed to heavy metals, with aluminium being the most widely studied. Accordingly the present study identified oxalic and malic acid (probably exuded as oxalate and malate) as the most induced organic acids in response to multiple heavy metal stress (As, Cd, Cu, Pb and Zn) (Table 3.8). In addition to oxalic and malic acid, other low molecular weight organic acids were detected. However, these compounds did not show a consistent pattern, being species-specific, and their concentrations, although in many cases significantly different between control and treatment, were much lower than those of oxalic and malic acid (Table 3.8). Although citric acid is generally considered to play an important role in heavy metal tolerance, especially in Al and Zn tolerance (Godbold et al., 1984; Ma et al., 1997b; Nguyen et al., 2003), it was actually released only by Plantago lanceolata and in a very small amount (Table 3.8). The five heavy metals stimulated the excretion of fumaric and tartaric acid from roots of Pinus and Plantago, respectively, allowing to propose their release as a part of the exclusion mechanism of this two wild-species. To date, this study is the first report identifying fumaric and tartaric acids in root exudates of excluder wild species.

Although organic acids are doubtlessly the major components of Pinus, Plantago and Silene exudates, other organic compounds were exuded.

Several different phenolic acids and three flavonoids were detected both under control

and treatment conditions, though their release was two or three orders of magnitude

lower than that of organic acids. While phenolic compounds showed a species-variable

pattern, in terms of kind and amount of phenols detected (Fig. 3.10), the three

flavonoids exhibited a consistent pattern since all of them were found to be produced in

increased amounts by roots exposed to heavy metals (Fig. 3.11). As total phenolics and

flavonoids are only present at micromolar levels, the low concentrations of those

substances do not suggest their participation in increasing metal exclusion in Pinus,

Plantago and Silene although both classes of compounds can form stable complexes

with heavy metals (Martell and Smith, 1977). Those results are in agreement with those

found for Norway spruce (Heim et al., 2000). Also in that case, the exudation of

phenolic complexators was not the major Al tolerance mechanism. Therefore, the

exudation of phenols and flavonoids could be better explained if discussed in relation to

the possible functions of those organic substances as chemical signals in root-soil- microbe interactions exuded to enhance plant adaptation to particular environmental conditions.

As hoped, the organic exudates of Pinus, Plantago and Silene remained stable in soil and improved phytoextraction capacity of B. carinata. Since organic compounds excreted by plants grown in soil are likely to be rapidly degraded by microorganisms, a continuous excretion from roots during the growth of the three wild species should by hypothesised in the present experiment.

In agreement with the available metal amount in soil, the growth of wild species enhanced B. carinata capacity to accumulate and translocate Cd, Cu, Pb and Zn (Fig.

3.9). The pattern of accumulation in shoot of B. carinata, although very similar to the pattern of extractable metals in soil, is difficult to explain as it depends on several factors. The multiple-contamination of the soil and the differences in the amount of organic exudates and in their affinity for the various metals present are doubtlessly the most important factors. Further studies aimed, for example, to investigate the metal binding properties of each exuded organic compound, are necessary in order to assess the role that those organic substances have in the excluding capacity of Pinus, Plantago and Silene and in the accumulating capacity of B. carinata.

4.1.3 – Genetic engineering approach: AtMT2b-transformed tobacco plants

The biomass production and the root development under As(III) stress are doubtlessly two parameters to measure the tolerance to the element. The capacity of the wild type of Nicotiana tabacum SR1 to maintain a relatively high biomass also following the 12 µM As(III) treatment (data not shown) and the slight reduction of its root development under the arsenite stress (Fig. 3.32) suggest that the wild type was more able than the AtMT2b-trasformed tobacco plants to face up to the As(III) toxicity.

However, the higher resistance of the wild type to As(III) is certainly not due to a

reduction of the net As(III) content in the plants. In fact, the wild type, if compared to

the AtMT2b-transformed plants, showed the highest values of As(III) accumulation especially at high external As(III) concentrations (Figs. 3.33, 3.34 and 3.35).

Therefore, conversely with what observed for Cd and Cu (Evans et al., 1992; Lee et al., 2004; Zhigang et al., 2006 for example), the insertion of AtMT2b gene in tobacco plants didn’t increase As(III) accumulation and tolerance although MTs proteins, thanks to their high affinity for As(III) (Garrett et al., 2001; Merrifield et al., 2004; Zhang et al., 2005; Zimeri et al., 2005), could be involved in intracellular As(III) sequestration.

The results obtained can be explained in other terms. The overexpression of the AtMT2b gene leads to an enhanced production of MTs proteins. As a result of this overproduction, the transformed plants can suffer from a depletion in their glutathione and phytochelatins content leading to a reduced capacity of those plants to bind As(III) and sequester it in the vacuolar cellular compartment as As(III)-GS

or As(III)-PCs (Salt and Rauser, 1995; Bleeker et al., 2006). Investigations about the glutathione and phytochelatin contents in wild type and AtMT2b-transformed Nicotiana tabacum SR1 plants may confirm this hypothesis.

Unless regulatory genes are identified that simultaneously induce many metal-related genes, it is feasible that more than one gene will need to be upregulated in order to substantially enhance metal phytoremediation capacity. Since metal tolerance, accumulation and plant productivity are largely independent properties; they should be all engineered to get a plant with high metal accumulation and tolerance as well as high productivity. This would be the ideal plant suitable for metal phytoextraction.

Encouraging for transgenic approaches, is that classic genetic studies indicate that there are usually very few genes responsible for metal tolerance (MacNair et al., 2000).

Some previous results (for example those of Yeargan et al., 1992) demonstrated that

trace element uptake observed on non-soil substrates under glasshouse or growth

chamber conditions cannot be extrapolated to predict the performances of transgenic

plants on soil substrates or under field conditions. Therefore, the next step for the

geneticists is to test the transgenic plants on more realistic contaminated substrates,

collected from the environment.

4.2 – Arsenic

Time course studies on B. carinata showed the presence of linear phases both over the first 90 min of arsenate uptake and over 8 h of arsenite uptake (Fig. 3.12).

In time course studies it has frequently been observed that a rapid initial uptake phase is associated with a passive uptake process involving diffusion of ions into the apparent free space (AFS) of the roots.

In order to avoid this problem, it would be better to perform experiments in the “steady- state” period. However, in the present case, it was not possible to perform the uptake experiments on the concentration-dependent kinetics in the “steady-state” periods because of the long incubation times required for arsenate (about 90 min) and the absence of a real “steady-state” phase for arsenite (Fig. 3.12).

The error due to the passively absorbed components can be avoid using a suitable desorption solution. In fact, the passively adsorbed component of ion uptake is readily reversible by placing the roots in an opportune desorption solution for 20-30 min (Asher and Reay, 1979). Therefore, it is very important to look for the best desorbing solution.

As little work has been done on arsenic, preliminary experiments were conducted with Brassica carinata roots allowing the selection of deionised water as the best desorbing solution. According to other authors (see for example Abedin et al., 2002), the duration of the desorption process was fixed at 30 min.

In agreement with Asher and Reay (1979), the initial uptake steps at room temperature were greater than the likely free space uptake at the concentration employed (0.25 mM) and were in any case detected after desorbing the arsenic taken up in the free space (Fig.

3.12). In addition, the linear phases of arsenate and arsenite uptakes were markedly affected by temperature (Fig. 3.13). These large effects of temperature observed on the initial phases of arsenate and arsenite uptake confirmed that these phases have to be linked with root metabolism although the true nature of this mechanism is still in doubt.

Finally, the observation that phosphate is a strong inhibitor of arsenate uptake also after

30 min of incubation (Fig. 3.16) confirmed, at least in the case of As(V), the metabolic

nature of As(V) uptake.

When arsenate and arsenite were supplied at the same concentration (0.25 mM), both the initial and the “steady-state” phases showed that arsenate was taken up at a higher rate of uptake than arsenite (Fig. 3.12). However, despite the lower uptake rate of arsenite relative to arsenate, studies on several plant species have shown that, at equal substrate concentrations, arsenite, reacting with sulphydryl groups (-SH) of enzymes and proteins, is the most toxic inorganic arsenic form (Meharg and Hartley-Whitaker, 2002). In addition, As(III) is, generally, the predominant form of arsenic present in plants since arsenate is promptly reduced to arsenite and stored as As(III)-tris-thiolate (Pickering et al., 2000; Zhang et al., 2002).

Investigations on the mechanisms involved in the uptake of arsenite, together with studies on the better known uptake of arsenate, could be essential for a better understanding plant uptake mechanisms and for the development of an efficient phytoextraction strategy for this metalloid.

The kinetic pattern obtained for arsenate uptake as a function of external concentration showed a typical “enzyme saturation” curve (Fig. 3.14). The observed hyperbolic kinetics curve up to 1 mM external As(V) concentration (Fig. 3.14) is in agreement with that reported by several authors (Asher and Reay, 1979; Meharg and Macnair 1990, 1992a,b; Abedin et al., 2002; Wang et al., 2002) and suggests that the uptake of this inorganic form of arsenic is an active process which requires both an energy supply and the presence of selective binding sites.

However, uptake curves for toxic elements such as arsenate must be interpreted with caution because of the possible inhibitory effects that harmful substances could have on the uptake process itself.

Uptake of 0.25 mM arsenate was strongly inhibited by the presence of different

concentrations of phosphate (0 - 2.5 mM) (Fig. 3.16) confirming the active uptake of

As(V) via P binding sites and, therefore, the competition between arsenate and

phosphate for the same transporters. This result is in full agreement with previous

studies on barley (Asher and Reay, 1979), Holcus lanatus (Meharg and Macnair 1990,

1992a,b), Deschampsia cespitosa (Meharg and Macnair, 1994) and rice (Abeding et al.,

2002). In addition, in the arsenic hyperaccumulator Pteris vittata P starvation for eight

days was found to increase V

for arsenate 2.5-fold suggesting an increased density of

phosphate/arsenate transporters on the plasma membranes in root cells (Wang et al., 2002).

The change in the pattern of uptake observed when arsenate concentration was higher than 1 mM (Fig. 3.14) might be explained in terms of a dual uptake mechanism, consisting of a high-affinity uptake system dominating at lower substrate concentrations and a low-affinity uptake system progressively taking over at higher substrate concentrations. Although other mechanisms have been invoked to explain ion uptake isotherms, such as the multiphasic uptake mechanism (Nissen and Nissen, 1983), the existence of two distinctive uptake systems in the case of arsenate/phosphate was provided by the presence of mutants containing only one system. The apparent absence of a high-affinity P uptake mechanism in arsenate-tolerant Holcus lanatus was the first example of such mutants in higher plants (Meharg and Macnair, 1990).

In the present case, the take over of the low-affinity (passive) uptake mechanism could be explained by the loss of membrane integrity when the As(V) concentration was higher than 1 mM (Table 3.9).

A linear mono-phasic concentration-dependent influx was evident for arsenite (Fig.

3.15). This mechanism being also present after the desorbing procedure, the data allow to consider an osmoregulated uptake of arsenite by B. carinata roots. Loss of membrane integrity detected also at very low arsenite concentrations (Table 3.9) confirmed this hypothesis being, thus, the damage of the plasmalemma responsible of the osmoregulated arsenite uptake. Although this hypothesis is in contrast with the results of Abedin and colleagues (2002) who found an active transport involved in the As(III) uptake by rice roots, it is in agreement with the results of Sharples et al. (2000). These authors found that the rate of H

AsO

uptake by Hymenoscyphus ericae isolated from heathland and from mine sites increased linearly in response to the increase of H

AsO

external concentrations. In addition, it has been shown that arsenite was transported

across the plasma membrane of Saccharomyces cerevisiae via the glycerol channel

protein Fps1p. Deletion of FPS1 gene (fps1∆ mutant) improved tolerance to arsenite

decreasing the rate of uptake, whereas cells expressing a constitutively open form of the

Fps1p channel were highly sensitive to this trivalent metalloid. Yeast cells appeared to

gene was repressed upon As(III) addition (Wysocki et al., 2001). Fps1p is a member of the ubiquitous MIP family of water and solute transporters found in bacteria, fungi and animals that have a role in osmoregulation. In fact, under high osmolarity conditions, when the Fps1p channel is closed, wild type cells of S. cerevisiae showed the same degree of As(III) uptake as the fps1∆ mutant (Wysocki et al., 2001).

This family of genes being ubiquitous also in plants, it is not unreasonable to suppose

its involvement in the uptake of arsenite by B. carinata roots. This could explain the

linear As(III) isotherm influx obtained in the present study (Fig. 3.15). However, further

studies are necessary to investigate uptake of this trivalent metalloid in plants and to

clarify the discrepancies regarding the mechanisms involved.

4.3 – Copper

4.3.1 – Mechanisms of free and complexed copper uptake

Time course studies on uptake of copper in B. carinata showed the presence of a linear phase over the first 60 min followed by a “steady-state” period (Fig. 3.17). In time course studies it has frequently been observed that a rapid initial uptake phase is associated with a passive uptake process involving diffusion of ions into the apparent free space (AFS) of the roots. Therefore, in order to avoid this problem, it would be better to perform experiments in the “steady-state” period. However, in the present study, it was not possible to perform the experiments on the concentration-dependent uptake in the “steady-state” period because of the long incubation times required for copper uptake. Those long times of incubation, apart from being experimentally unfeasible, can determine significant errors in studying cation nutrients transport across the plasma membrane. In fact, the trace metal uptake has to be measured over periods sufficiently short to reflect unidirectional influx. The experiments of Reid and Smith (1992) showed that efflux of Ca

could be up to 50% of influx and, unless influx measurements were made within 30 min, significant errors were introduced. Being Ca

a divalent cation, it is reasonable to suppose that the same could happen also for other divalent cations such as Cu

. This was the reason for which an incubation time of 30 min was chosen for concentration-dependent influx of Cu

. The error due to the passively adsorbed components can be avoided not by increasing the time of incubation but using a suitable desorption solution. In fact, the passively adsorbed component of ion uptake can be readily reversible by placing the roots in the suitable desorption solution for 20-30 min (Asher and Reay, 1979).

Although few studies have been conducted on the uptake of micronutrients, with copper

in particular, investigations of the mechanisms involved in the influx of copper could be

essential for better understanding plant uptake mechanisms and for the development of

an efficient phytoextraction strategy for this heavy metal.

The observed hyperbolic kinetics curve up to 60 µM external Cu

(Fig. 3.18) confirms some general features about divalent metal transport into cells of yeasts, mosses and plants. First, uptake of most of the first series transition metals is energy-dependent and requires selective binding sites. In fact, the existence of an active copper uptake was previously demonstrated for yeasts (Lin and Kosmann, 1990 and papers reported therein), mosses (Couto et al., 2004 and papers reported therein) and plants (Veltrup, 1977; Marquenie-van der Werff and Ernst, 1979; Lidon and Henriques, 1992). Second, the Michaelis-Menten constants for uptake of these first transition series metals fall into a range of values between a few and a few hundreds micromolar. The K

value for Cu

uptake reported here, 1.41 µM, is very similar to K

values determined for copper uptake in Saccharomyces cerevisiae (4.4 µM) by Lin and Kosmann (1990), in barley roots (9.3 µM) by Veltrup (1977) and in Elodea nuttalii (0.4-5.6 µM) by Marquenie-van der Werff and Ernst (1979). The V

value for Cu

uptake reported here, 0.95 µmol Cu

g

fw h

, is similar to maximal uptake velocities determined in the above- mentioned studies, as well.

The change in the pattern of uptake observed when the external copper concentration

was higher than 60 µM (Fig. 3.18) might be explained in terms of a dual uptake

mechanism, consisting of a high-affinity uptake system dominating at lower substrate

concentrations and a low-affinity uptake system progressively taking over at higher

substrate concentrations. The existence of high-affinity and low-affinity transport

systems is well established for many of the macronutrients (Forde, 2000; Schachtman

2000; Smith et al., 2000) or for molecules, such as arsenate, that are structural

homologues of macronutrients (see paragraph 4.2) and the same appears to apply for

many of the micronutrients. Biphasic uptake behaviour has been attributed to a

combination of cells surface adsorption and intracellular accumulation. In fact, one of

the main important problems in studying trace metals, especially divalent cations,

uptake is the often extensive binding to the plant cell walls that blurs the

characterisation of flux kinetics. In practice, the measurement of the degree to which

residual binding in cell walls contributed to the observed fluxes is a difficult task

because many micronutrients occur in a variety of apoplasmic pools with widely

varying exchange times. The only way to avoid this problem will be to make

measurements of micronutrient fluxes across membranes. However, the low internal requirements for these cations means that uptake rates will be very small compared with the macronutrients, and this, as hypothesised by Kochian (1991) may require techniques more sophisticated than the conventional patch-clamp procedures. The application of a desorption with a suitable solution for a sufficiently long period still remains the most common method to avoid cell wall bound components in the uptake kinetic studies. In the present study, however, experiments directed towards the measurements of the relative leakage ratio of membranes have demonstrated that the take over of the low- affinity uptake mechanism could be explained by the loss of membrane integrities when the external copper concentration was higher than 60 µM (Table 3.10).

It is believed that a competition among trace metals for common carriers is existent.

Therefore, defining the kinetic characteristics for a specific transporter against the background of non-specific transporters presents a scientific challenge and could introduce significant errors. However, one distinguishing feature of the copper transporter lies in the fact that, at least in a certain range of concentrations, it shows a relative selectivity. For example, Veltrup (1977) concluded that the uptake of copper by intact barley roots is mediated by a mechanism which is not influenced by the presence of Zn. Subsequently, Lin and Kosmann (1990) demonstrated that Cu

uptake by the yeast Saccharomyces cerevisiae was relatively selective for Cu

in competition with Zn

and Ni

. More recently, a putative copper-specific transporter was definitively identified in Arabidopsis thaliana (Kampfenkel et al., 1995).

The majority of previous studies on copper uptake by plants has been made using inorganic copper, applied as a solution of a simple copper (II) salt in which the chemical form of the applied copper is well defined as the hexaquocopper (II) cation, [Cu(OH

)

]

. Such experiments have clearly established, as discussed above, that copper uptake is biphasic for dilute and more concentrated applications of copper solutions to excised roots (Fig. 3.18).

However, in soils or copper-rich sewage sludge, copper may be less readily available

for uptake, and total copper content is not necessarily an accurate indication of the

biologically available copper. Copper may bind to organic matter or to the aluminium-

study, a huge amount of total copper (1846 mg kg

soil) was found in the contaminated soil (Table 3.5), but only the 3.2% of total amount (59.3 mg kg

soil) was present in the exchangeable form (Fig. 3.3a). Therefore, in order to enhance bioavailability of this metal, biodegradable chelators (NTA and EDDS) were added to the soil. As complexing of copper ions will alter not only their reactivity but also, possibly, their effective charge (depending on the nature of the chelator), it is not unreasonable to assume that complexing of soil copper may affect copper uptake by plants.

NTA and [S,S]-EDDS were actually found to enhance the capacity of B. carinata to accumulate Cu (Fig. 3.6a) with none of the amendants significantly affecting biomass production (Fig. 3.4). Majumder and Dunn (1959) presented similar evidences finding that the presence of EDTA reduced copper toxicity in Zea mays grown in solution culture. In addition, Hadi Khadr and Wallace (1966) showed that the addition of ethylenediaminedi-(O-hydroxyphenylacetate)] (EDDHA) to solutions containing potentially phytotoxic copper levels prevented the development of copper toxicity in bush beans.

The effect of NTA and EDDS in enhancing the capacity of plants to take up and translocate metals was already studied for various plant species by other authors and for B. carinata in the present study; however, to date, no studies have been performed in order to clarify the mechanisms involved in the uptake of copper present as complexed ion. The experiments reported here were, therefore, designed to investigate the effects of NTA-Cu or EDDS-Cu forms on the subsequent uptake of copper by excised B.

carinata roots. Both chelators were added at sufficiently high concentrations in order to create chelator-buffered solutions (Chaney et al., 1989) in which the free-metal ion activity doesn’t decrease during plant uptake of metal because the metal activity is buffered.

In the presence of both chelators, the free copper concentration was negligible and all copper was bound to the chelating agents in the form of Cu-NTA

and Cu-EDDS

(Gustafsson, 2006).

The kinetic patterns obtained for Cu-NTA

and Cu-EDDS

complexes, as a function of

external copper concentration, showed typical “enzyme saturation” curves (Fig. 3.19)

with the anionic complexes accumulated at a slower rates than the positive charged

hexaquocopper (II) cation (Fig. 3.18). During uptake, copper is normally bound to negatively charged sites on the root surface or within the free space before being absorbed, while copper in the negatively charged forms of Cu-NTA

and Cu-EDDS

cannot occupy such sites. This, obviously, get the absorption of the complexes more difficult and could explain the reason because the Cu-NTA

and Cu-EDDS

complexes were accumulated poorly and with kinetic patterns completely different from that of free copper cation.

It has been reported that synthetic chelators destroy the physiological barriers in roots that normally function to control uptake of solutes and that, consequently, the negatively charged complexes metal-chelator may enter the root through via a non- selective apoplastic pathway (Vassil et al., 1998; Wenger et al., 2003; Luo et al., 2005;

Nowack et al., 2006). However, the observed Michaelis-Menten patterns allow to think that active mechanisms and specific binding sites should be involved in uptake of Cu- NTA

and Cu-EDDS

. This hypothesis may come into play only if Cu-NTA

and Cu- EDDS

complexes are considered as anionic nutrients. In fact, the anionic nutrients, such as Cl

or MoO4

, are electrically repelled from the cytoplasm and almost certainly their uptake occurs via active mechanisms involving symport with protons (Reid, 2001).

However, hypothesising the absorption of Cu-NTA

and Cu-EDDS

, although plausible

in the opinion of Coombes et al. (1977) and Welch (1995), clashes with the common

opinion according to which only the free copper ion can be absorbed by roots. The Cu-

complexes are thought to be destabilised at the plasma membrane surface by the

standard reductase systems that cause the release of copper from its complexes (Holden

et al., 1995; Cohen et al., 1997). Looking at the results from the point of view that only

free Cu

ion is absorbed by roots, the differences among the kinetic patterns of Cu

,

Cu-NTA

and Cu-EDDS

could be explained in terms of an inhibition of the uptake due

to the presence of the two chelators in the treatment solutions. That inhibition is then a

direct function of the stability constants of the three species studied. In fact, since Cu-

NTA

and Cu-EDDS

have much higher stability constants than [Cu(OH

)

]

, copper

could be much more readily displaced from the latter complex and therefore much more

easily absorbed. In addition, also the charge carried by the copper complex is thought to

much faster than the negative ones (Coombes et al., 1977). The hypothesis of a displacement from chelators before absorption could be confirmed by the kinetic parameters obtained by fitting the data to additive Michaelis-Menten functions. In fact, the K

values for Cu-NTA

and [Cu(OH

)

]

(0.98 ± 0.37 and 1.41 ± 0.09 µM, respectively) were not significantly different, strengthening the idea that the same substrate was absorbed in both cases, while the V

value of Cu-NTA

(0.43 ± 0.10 µmol g

fw h

) was halved in comparison with the maximal velocity of the hexaquocopper ion absorption (0.96 ± 0.06 µmol g

fw h

), indicating that an inhibition could come into play when NTA is present in solution. Also the V

value for the Cu- EDDS

complex (0.31 ± 0.01 µmol g

fw h

) appeared to be halved if compared to that of free copper ion but, in this case, the K

value, that arise from three different experiments (0.20 ± 0.05 µM), is significantly different from that of [Cu(OH

)

]

, although it was only one order of magnitude smaller than that of Cu

. Since in literature the K

values are considered different when two or more orders of magnitude occur (see for example the studies of Meharg), the small difference, although significant, between the values reported here allows to think that also in the case of Cu- EDDS

complex, an inhibition due to the presence of EDDS in solution is more likely to occur than the absorption of the negative complex.

Damage to the root plasma membrane integrity is probably not involved in the uptake of Cu-NTA

and Cu-EDDS

(Fig. 3.19). However, to explain the results obtained from the hydroponical experiments designed for testing the capacity of B. carinata to remediate the contaminated site (Fig. 3.1), it was suggested that EDDS, but not NTA, exceeded the limit required to damage the root membranes (Vassil et al., 1998) and a higher concentration of metal-EDDS complexes or free EDDS reached the stele and the xylem.

Since the concentration of EDDS was the same in both experiments, the differences in the chelator effect on the plants may be likely attributed to the shorter incubation period (only 30 min) used for the uptake kinetic experiments.

To test the effect of both chelators in the uptake and translocation of copper during time, long-term experiments of accumulation of Cu, NTA and EDDS were performed.

In this case, synthetic chelators have likely induced plasma membrane destabilization as

a huge amount of copper was accumulated in shoots of B. carinata after 12 h of

incubation (Fig 3.21) although an inhibition of copper uptake into B. carinata roots by EDDS and NTA was observed (Fig. 3.20). This pattern of metal distribution tallies with the hypothesised membrane damages since the destabilization of the plasma membranes leads to a rapid equilibration of hydroponic or soil solution with the xylem sap. Once in the xylem, solutes, such as Cu-NTA

and Cu-EDDS

, would follow the transpiration stream and accumulate to a high concentration in shoots.

The results reported here, although in contrast with Tandy et al. (2006a), who found that the concentrations of the essential metals Cu and Zn were decreased in sunflower shoots in the presence of EDDS, are in agreement with other authors (Wenger et al. 2003). In addition, the same effect of the two chelators on the translocation of metals, with EDDS more effective than NTA, was found both in the hydroponic and soil experiments (Figs.

3.1a and 3.6a).

Direct measurements of EDDS and NTA in roots and shoots of B. carinata incubated for different period with Cu-NTA

and Cu-EDDS

confirmed the absorption and translocation of both chelating agents (Fig. 3.22) demonstrating that their presence is the reason of the enhanced copper translocation. The two complexing agents were actually present in the shoots in similar amounts (Fig. 3.22b), while the EDDS content in roots was much higher that the NTA one (Fig. 3.22a). The high correlation between chelators and copper amounts in shoots of B. carinata (Figs. 3.23b and 3.24) (R

= 0.95 and 0.90 for EDDS and NTA, respectively) further corroborates the involvement of chelators in copper translocation although the content of the two chelators was always much higher than that of copper. Preliminary experiments designed to detect NTA and EDDS accumulation in roots and shoots of B. carinata showed that both chelators are accumulated also in their free forms (data not shown) explaining their higher accumulation compared to copper.

In contrast to NTA, EDDS showed a high interdependence with copper also in roots

(Fig. 3.23a). This, together with the K

value of the Cu-EDDS

complex significantly

different from that of [Cu(OH

)

]

, contradicts the previous hypothesis about the nature

of the absorbed compound again. In fact, this direct relationship could be due to the

higher toxicity of EDDS that allows the entrance of the chelator in high amount also

after short period of incubation or it could be due to a real absorption of the Cu-EDDS

complex.

The data presented here on the accumulation and transport of chelated metals in plants do not only advance the understanding of the role of metal chelators in plant nutrition, but also point to potential areas of improvement for the development of chelate-assisted phytoextraction. However, the relative importance of the form of copper (free or complexed) in governing the process of uptake is still an open question. Till now, no direct evidence showing the uptake of Cu as a complexed form has been reported.

Further researches, eventually designed for studying the absorption at plasma membrane level, are therefore needed to address this problem.

4.3.2 – Mechanism of copper translocation

Mechanisms involved in translocation of copper from its absorption sites in the roots to different organs of the shoots are only partly understood. Because metals move acropetally via the xylem fluid, improved understanding of translocation requires studies within that system. Because heavy metals are highly reactive as such, long- distance movement involves relatively non-reactive or chelated metals. Numerous studies have been conducted to identify the most important chelating agent of copper in the xylem sap. Simulation models (White et al., 1981a, b; Mullins et al., 1986) and, more recently, in vivo studies (Herbik et al., 1996; Pich and Scholz, 1996; Liao et al., 2000; Takahashi et al., 2003; Kim et al., 2005) agree in affirming that the most important long-distance Cu transporters seem to be amino acidic compounds.

Although B. carinata showed a great capacity to accumulate copper and other heavy

metals when grown in a multiple-contaminated site, to date, no study has been

performed in order to investigate its translocation mechanisms. This is a big omission

considering that the understanding of metal translocation pathways is the first step in

order to improve the phytoextraction capacity of this high biomass plant that already

exhibits a natural high metal accumulation capacity.

Since no previous studies have been conducted on B. carinata, it is not possible to compare the amino acids concentrations detected in the present work (Table 3.11) with those found earlier. However, as the values are of the same order of magnitude or one order less than those found previously for other plant species (White et al., 1981b;

Senden et al., 1992; Krjiger et al., 1999; Liao et al., 2000), one might conclude that the xylem amino acidic content of plants grown in optimal condition doesn’t vary significantly. Conversely, the amino acidic composition seems to be species-dependent although in most cases histidine and nicotianamine (if analysed) are two of the most abundant amino acids in xylem sap. In the present experiments, nicotianamine was the major amino acid present in B. carinata xylem sap followed by lysine, alanine, leucine, histidine and arginine (Table 3.11). Nicotianamine, a phytosiderophore, was detected, recently, in tomato and chicory xylem saps (Liao et al., 2000) and previously in other studies (Noma et al., 1971; Noma and Noguchi, 1976; Budêšínský et al., 1980; Herbik et al., 1996; Pich and Scholz, 1996) confirming the widespread occurrence of this amino acid in higher plants. To date, the occurrence of nicotianamine in B. carinata has not been reported.

When exposed to high Cu concentrations in the nutrient solutions, relative increases in the concentration of certain amino acids were greater than others. Notably, histidine, which can form complexes with Cu with high association constant (logK

= 17.5), was the amino acid with the greatest relative increase (Table 3.11 and Fig. 3.25) followed by other amino acids with lower stability constants (May et al., 1977). This findings are in agreement with Krämer et al. (1996) who detected increased free histidine concentrations in the xylem sap of the nickel hyperaccumulator Alyssum lesbiacum in response to Ni treatments. They subsequently identified histidine as the major Ni chelator. Surprisingly, an almost constant content of nicotianamine, the amino acid with the highest stability constant (logK

= 18.6), was detected during both copper excess treatments (Table 3.11 and Fig. 3.25). This is in contrast with Liao et al. (2000) who found that both nicotianamine and histidine were the amino acids with the greatest increments when tomato or chicory plants were exposed to high copper concentrations.

However, it is not unreasonable to consider the plant response to copper stress as

Under copper starvation, methionine, nicotianamine, glutamine and threonine were detected as the amino acids with the highest relative increases (Table 3.11 and Fig.

3.26). Since the production of free amino acids in the xylem of plants as a response to copper starvation has not been previously reported, it is not possible to confirm the data obtained by comparing them with others. It seems that the response to copper stress is not only species-specific, as previously affirmed, but also stress-specific since the same plant, B. carinata, faced up to different stress conditions stimulating the accumulation of different free amino acids in the xylem sap.

The pH sensitive Cu-binding signature curves of Brassica carinata xylem sap and simulated saps containing single amino acids (Fig. 3.28) showed that histidine and proline are likely to be the most important copper ligands in xylem exudates of B.

carinata treated with excess of copper. The titration of simulated saps containing combinations of amino acids (Fig. 3.29), confirmed the role of histidine as the major copper chelator although not the only one. The structure of Cu-His

complex is shown in Fig. 4.1.

In the absence of histidine, proline would play a very important role in Cu binding (Fig.

3.29 and Table 3.12). The role of threonine (21.75 µM), glutamine (7.43 µM), glycine (22.92 µM) and methionine (12.5 µM) were negligible in the presence of 0.14 mM histidine and 6.33 µM proline (Fig. 3.28 and Table 3.12). These results are in contrast

Fig. 4.1. Molecular structure of the Cu(II)-Histidine complex. Manikandan et al., 2001.

with White et al. (1981a) who indicated, using a chemical speciation model, that Cu would be bound primarily to asparagine, glutamine and histidine.

Cations, especially the heavy metals Co, Fe, Ni and Zn, may affect Cu speciation because of a competition of those metals with copper for the same ligands. However, no other heavy metals, except zinc, were present in B. carinata xylem saps since these plants were grown in control conditions and then treated with excess copper only.

Although the xylem concentrations of Zn were not determined in the present study because of the limited xylem sap volume, xylem sap Zn concentrations in Brassica plants receiving normal Zn treatment were likely too low to have an effect on Cu complexation by histidine and proline because of the lower association constants with histidine and proline compared to Cu (May et al., 1977). Additional analyses of xylem sap from B. carinata incubated for three days in copper excess treatments (2.5 and 5 µM) showed consistent increases in histidine and proline as solution copper concentration increased (Figs. 3.27 and 3.30). In particular, the histidine concentration increased in direct proportion with the external Cu concentration. However, sap histidine and proline concentrations were always high enough to account for all the complexed Cu in xylem sap.

Although in the case of copper starvation conditions the pH sensitive Cu-binding signature curves were performed on xylem saps to which exogenous copper amounts were added (Fig. 3.31), it is not unreasonable to suggest that the same trends would exist in native xylem saps.

The pH sensitive Cu-binding signature curves of Brassica carinata xylem sap and simulated saps containing single amino acids (Fig. 3.31) showed that nicotianamine is likely the most important copper ligand in xylem exudates of B. carinata in condition of copper starvation.

Nicotianamine was already identified and quantified in plant xylem sap and suggested

as a possible copper translocator (Pich et al., 1994; Herbik et al., 1996; Pich and Scholz,

1996; Liao et al., 2000). Herbik et al. (1996) showed that the nicotianamine-deficient

mutant chloronerva of tomato suffers from Cu deficiency in the shoots. These results

were confirmed by Liao et al. (2000). They reported that NA is likely to be the most

in the regulation of the level of nicotianamine is the nicotianamine aminotransferase (NAAT) that catalyses the amino group transfer of NA in the biosynthetic pathway of phytosiderophores. The gene that encodes NAAT from barley was introduced into the non-graminaeous plant tobacco. Transgenic tobacco plants (naat tobacco) showed a lower concentration of Cu, Mn, Fe and Zn in both young leaves and flower than the wild-type. This decrease, attributable to the depletion of endogenous NA, is a further confirmation of the role of NA in metals transport (Takahashi et al., 2003).

The detected increase of nicotinamine in copper starvation conditions but not in copper excess conditions could be explained when the biosynthetic pattern of nicotianamine is taken into consideration. Nicotianamine is an intermediate in mucigenic-acid (MAs) biosynthesis formed via the nicotianamine synthase-catalised trimerisation of S- adenosyl-L-methionine. Mucigenic acids (MAs) are naturally secreted from graminaceous plants in iron starvation conditions in order to solubilize Fe in the soil.

Unlike MAs, nicotianamine is not secreted from roots but it is not unreasonable to think

it is involved in internal copper transport when the plant is in metal deficiency rather

than in metal excess.

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