C 3. R 3.1 – Phytoextraction experiments

C

3.

R

3.1 – Phytoextraction experiments

3.1.1 – Selection of the species

Germination and tolerance index (TI) tests were used to select the species for the phytoextraction program. The germination test, performed on the starting nine species (Brassica carinata, Helianthus annuus, Lotus corniculatus, Phleum pratense, Secale

cereale, Sorghum vulgare, Triticum aestivum, Trifolium alexandrinum, Zea mays), indicated that B. carinata, T. alexandrinum, S. cereale, H. annuus and Z. mays showed the highest germinability after 48 h in presence of metals (Table 3.1). The five species mentioned above showed a decrease in the percentage of germination not higher than 20% in comparison with the control (Table 3.1).

Table 3.1. Percentage of germination of nine species germinated for 48 h in 50 µM Na2AsO4 or 20 µM CdSO4 or 75 µM CuSO4 or 200 µM Pb(NO3)2 or 750 µM

Zn(NO3)2 or demineralised water (control).

Control As 50 µM Cd 20 µM Cu 75 µM Pb 200 µM Zn 750 µM B.carinata 92 96 96 84 100 74 T. alexandrinum 96 96 96 100 80 88 S. cereale 80 92 84 72 100 88 H. annuus 88 92 88 96 100 76 Z. mays 84 84 100 56 92 84 T. aestivum 88 76 100 56 72 56 P. pratense 84 52 68 56 88 72 L. corniculatus 60 52 68 52 56 36 S. vulgare 70 52 68 72 56 58

Results are the means* of five replicates of three independent experiments (n = 15). The standard deviation* from the mean was always smaller than 10%. *The means and the standard deviations were calculated after transformation of percentages as arcsin√P.

The five species selected by the germination test were grown for five days in presence of As, Cd, Cu, Pb and Zn added separately at the same concentration used for the germination test. At the end of the fifth day, the root length was measured and the tolerance index was calculated (Table 3.2) allowing to reduce the number of suitable species to three crops (B. carinata, T. alexandrinum, S. cereale).In fact, in the presence of metals, the selected three crops showed the highest tolerance indexes (Table 3.2). B.

carinata and T. alexandrinum were the most resistant plants (average tolerance index of 76% and 63%, respectively) while H. annuus appeared to be the most sensitive to metals (average tolerance index of 44%) (Table 3.2).

A further screening was performed growing the three species for four weeks in nutrient solutions containing As, Cr, Cu, Pb and Zn separately (Tables 3.3 and 3.4). Chemical speciation modelling was carried out to give an insight into the speciation of the hydroponic solutions. In the treatments with only one metal, 96% As (H2AsO4

-), 93% Cd, 89% Cu, 85% Pb, and 94% Zn were calculated to be in the free form. The remaining metals were present as complexes with sulphate (Cd 4.5%, Cu 4.2%, Pb 8.5%, Zn 5%), phosphate (Cd 1.3%, Cu 3.3%) and tartrate (Cu, Pb 1.6%).

Shoot dry biomass of B. carinata was significantly reduced only in the Zn treatment (-34%), whereas root length showed on average a 21% reduction in comparison with the control (Table 3.3). S. cereale proved to be the most sensitive to metals with an average

Table 3.2. Tolerance index (TI) of the five species grown for five days in 50 µM Na2AsO4 or 20 µM CdSO4 or 75 µM CuSO4 or 200 µM Pb(NO3)2 or 750

µM ZnSO4. As 50 µM Cd 20 µM Cu 75 µM Pb 200 µM Zn 750 µM B.carinata 73.2 74.6 70.7 78.3 81.7 T. alexandrinum 89.2 54.7 45.2 81.3 44.3 S. cereale 56.6 56.7 68.4 72.7 44.4 H. annuus 49.3 45.9 39.3 40.7 46.6 Z. mays 51.4 56.6 54.8 41.5 47.9

The results are the means* of 30 replicates (n = 30). The standard deviation* was always ≤ 10%. *Means and standard deviations were calculated after transformation of percentages as arcsin√P.

Table 3.3. Shoot dry biomass (mg plant-1

) and root length (cm) of B. carinata, T.

alexandrinum and S. cereale grown for 4 weeks in hydroponics with solutions containing 50 µM Na2AsO4 or 20 µM CdSO4 or 75 µM CuSO4 or 200 µM Pb(NO3)2

or 750 µM Zn(NO3)2. Nutrient solution was used as a control.

Control As 50 µM Cd 20 µM Cu 75 µM Pb 200 µM Zn 750 µM Shoot biomass B.carinata 35.8±2.0 34.4±1.7 32.9±2.1 31.9±1.8 32.9±2.2 23.6±2.2 T. alexandrinum 7.6±0.7 5.6±0.5 6.1±0.4 5.8±0.3 6.2±0.5 5.7±0.7 S. cereale 35.5±2.4 15.6±0.8 14.8±1.4 21.0±1.6 24.6±1.9 14.7±1.7 Root length B.carinata 5.7±0.6 4.4±0.4 4.4±0.4 4.4±0.5 4.8±0.5 4.6±0.6 T. alexandrinum 6.6±0.5 5.9±0.7 3.5±0.5 3.0±0.8 5.5±0.6 2.7±0.3 S. cereale 21.6±1.9 13.9±0.7 12.0±1.1 14.4±1.0 15.2±1.2 9.2±0.8 Results are the means ± SD of 30 replicates (n = 30).

On a shoot dry matter basis, Cu, Pb and Zn concentrations were higher in T.

alexandrinum, but when calculated on a plant basis (content), B. carinata showed the highest values for As, Cd, Cu and Pb (1.1, 2.0, 1.9 and 9.2 µg plant-1, respectively) due to its higher biomass (Tables 3.3 and 3.4). Zn accumulated more in S. cereale (8.3 µg plant-1) (Table 3.4).

Table 3.4. Metal concentrations (mg kg-1

dw) in shoots of B. carinata, T.

alexandrinum and S. cereale grown for 4 weeks in hydroponics with solutions containing 50 µM Na2AsO4 or 20 µM CdSO4 or 75 µM CuSO4 or 200 µM Pb(NO3)2 or

750 µM Zn(NO3)2. Nutrient solution was used as a control.

As Cd Cu Pb Zn

50 µM 20 µM 0.12 µM 75 µM 200 µM 0.3 µM 750 µM

B.carinata 31±3 60±5 12±1 60±4 280±18 167±10 333±16

T. alexandrinum 35±4 67±3 14±2 96±8 773±39 180±17 800±32

S. cereale 24±2 40±3 11±2 46±4 253±19 240±20 566±19

Root length, biomass and shoot metal concentrations identified B. carinata as the most metal tolerant crop.

3.1.2 – Brassica carinata hydroponical assay

To assess the potential of B. carinata to accumulate the five metals when present together, the nutrient solutions were amended with 10 mM NTA or 5 mM [S,S]-EDDS in order to avoid salt precipitation. The concentration of NTA was two-fold higher compared to EDDS because of the lower capacity of the former to keep metals in solution. In the presence of both chelants, free metal concentrations in the solutions were negligible as almost all the metals (> 99%) were bound to the chelating agents, with the exception of As that was for more than 95% in the free H2AsO4- form. In the

metal-NTA solution 81% of the NTA was in the HNTA2- form, whereas in the EDDS treatment 49% of it was present as anionic HEDDS3- and H2EDDS

forms. When bound to metals, NTA was as metal-NTA- _(Cd-NTA- _{0.2%, Cu-NTA}- _{0.7%, Pb-NTA}

-1.0% and Zn-NTA- 7.5% of total, respectively), whereas EDDS was as metal-EDDS 2-complex (Cd-EDDS2- 0.4%, Cu-EDDS2- 1.5%, Pb-EDDS2- 2.0% and Zn-EDDS2- 14.9% of total, respectively).

The metal concentrations of shoots (Fig. 3.1a) indicate that metals were present in higher amount following EDDS addition (1.8-fold on average). In contrast, in B.

carinata roots (Fig. 3.1b) NTA was more effective than EDDS in increasing the concentrations of Cu, Pb and Zn, whereas EDDS induced a higher root accumulation for Cd (about 1.9-fold). While NTA did not affect root length and shoot:root mass ratio of

B. carinata, EDDS significantly lowered both parameters in comparison with the control (Fig. 3.1c). In comparison with the plants grown without metals in excess, NTA treatment did not change shoot dry weight (35.8 and 32.3 mg plant-1, respectively) unlike EDDS that significantly reduced biomass to 15.1 mg plant-1 (Fig. 3.2).

Fig. 3.1. Concentrations of metals in shoots (a) and roots (b), root length and shoot to root mass ratio (c) of B. carinata plants grown in hydroponics for 4 weeks in the presence of the five metals and 10 mM NTA or 5 mM EDDS. Values are means ± SD (n = 3 and n = 30 for metal determination and growth parameters, respectively).

3.1.3 – Pot experiments

Physical and chemical characteristics of the mixed soil are reported in Table 3.5.

Table 3.5. Physical and chemical characteristics of the contaminated soil mixed with peat in a 2:1 ratio by volume.

Parameters Total metals

Sand (%) 79 As (mg kg-1) 639 Silt (%) 14 Cd (mg kg-1) 61 Clay (%) 7 Cu (mg kg-1) 1846 pH (H2O) 7.3 Pb (mg kg -1 ) 246 Organic matter (%) 6.8 Zn (mg kg-1) 1143 CEC (cmol(+) kg -1 ) 4.4 Total S (%) 0.01 Total N (g kg-1) 0.7 Exchangeable P (mg kg-1) 11.7 Exchangeable K (mg kg-1) 170.8

Results are the means of five replicates, analysed in triplicate (n = 5). The standard deviation was always ≤ 10%. In the case of percentages, the means and the SDs were calculated after transformation of the data as arcsin√P.

Fig. 3.2. Brassica

carinata grown for 4 weeks in nutrient solution (on the left) and in nutrient solution added with As (50 µM), Cd (20 µM), Cu (75 µM), Pb (200 µM), Zn (750 µM) and EDDS 5 mM (on the right).

The results of the sequential extraction of metals from the mixed soil (Fig. 3.3a) indicate that As and Cd were equally distributed over the three binding forms, exchangeable, bound to iron and manganese oxides, and bound to organic matter and sulphides, respectively. The extractable amounts of these metals were low compared to their total contents in the soil (2 and 13%, respectively). The most abundant metal was Cu, of which about 60% of the total extractable amount was bound to organic matter and sulphides. For extractable Pb, the most represented binding fraction was the reducible one (26% of the total soil content). In contrast, extractable Zn showed the highest association (6% of the total soil content) with the weakly-bound acid-soluble fraction.

Fig. 3.3. Concentrations of metals in the different soil fractions following sequential extraction (a) and effects of NTA and EDDS applied to the soil at 5 mmol kg-1 _before

sowing on the

solubilisation of metals (b). 1st step, acid-soluble phase; 2nd step, reducible phase; 3rd step, oxidisable phase. Values are means ± SD (n = 5).

The ability of NTA and EDDS to desorb As, Cd, Cu, Pb and Zn from the multiple metal-contaminated soil is illustrated in Fig. 3.3b. Both complexing agents used were not able to extract significant amounts of As and Cd from the soil. The extraction of Cu, Pb and Zn from the polluted soil by 5 mmol kg-1 EDDS was on average about 2.4-fold higher than that by NTA, with Cu showing the greatest difference between the two chelators (3.2-fold). Pb was the more effective metal extracted by EDDS (23% of soil total metal content) followed by Cu (about 14% desorption).

The calculated stability constants (logK) of each chelate were higher for EDDS complexes than for NTA ones (Table 3.6).

In the pot experiment, dry weights of B. carinata shoots were not reduced following EDDS and NTA soil amendments (Fig. 3.4).

Table 3.6. Estimated stability constants (logK) of metal complexes in the soil following chelator addition.

Cd-NTA- Cu-NTA- Pb-NTA- Zn-NTA- Cd-EDDS2- Cu-EDDS2- Pb-EDDS2- Zn-EDDS

2-logK 10.54 13.79 12.27 11.35 11.42 19.42 13.45 14.20

Fig. 3.4. Effect of NTA and EDDS applied to the soil at 5 mmol kg-1 one week before harvest on shoot dry weight of B.

carinata. NA, untreated pots. Values are means ± SD (n = 30).

However, both amendments caused acute necrosis and curling of the edges of the leaves (Fig. 3.5).

Shoots of NA plants did not accumulate metals during the week between chelator additions and harvest (Fig. 3.6a). In comparison with NA plants, the two complexing agents did not cause any significant change in As and Cd uptake. In comparison with the control (NA), EDDS yielded an about 4-fold increase of Cu and Pb and doubled the Zn content. Further, the NTA treatment enhanced Cu, Pb and Zn concentrations in shoots in comparison with NA plants, though at a lower extent than EDDS (Fig. 3.6a). Compared to NTA, the amendment with EDDS almost doubled Cu and Pb concentrations (157 and 122 mg kg-1 dry weight, respectively), whereas Zn accumulation was only slightly increased (+ 19%). Since shoot dry biomass of B.

carinata did not change due to the amendments (Fig. 3.4), the metal concentrations expressed on a plant basis maintained the same behaviour showing the highest values in the EDDS-treated plants (23.6, 18.3 and 19.3 µg plant-1 for Cu, Pb and Zn, respectively). In Figure 3.6b the amounts of DTPA-extractable metals desorbed after the harvest from the unamended (NA) and amended soils are shown. Also in this case, As and Cd were always extracted in trace amounts according to their tight association with soil particles (Figs. 3.3a and 3.3b). In comparison to the untreated soil, both chelating agents enhanced DTPA-extractable Cu, Pb and Zn concentrations by about 2-, 7- and 5-fold, respectively. However, NTA-treated soil showed slight but significantly higher

Fig. 3.5. Non-amended, NTA-amended and EDDS-amended B. carinata (from left to right) in pot experiments.

values of these metal concentrations if compared to EDDS-treated soil. Neither of the complexing agents, nor B. carinata growth changed the soil pH value (data not shown).

Fig. 3.6. Effect of NTA and EDDS applied to the soil at 5 mmol kg-1 one week before

harvest on metal uptake by shoots of B. carinata (a) and DTPA-extractable metals in the soil at the harvest (b). NA, untreated pots after harvest; BA, untreated pots before amendments. Values are means ± S.D. (n = 3 and n = 5 for metal uptake and extractable metals in the soil, respectively). With the exception of As and Cd, the differences between the two chelators were significant at P ≤ 0.01.

3.1.4 – Extractable metals during time

The amount of DTPA-extractable metals present in unamended (NA) and in 5 mmol kg-1 soil NTA- or EDDS-amended soils were measured 2 and 7 days after the amendments. The amount of DTPA-extractable metals of NTA-amended soil remained almost constant with time, except for Zn that showed a 24% decrease (Fig. 3.7a).

The amount of DTPA-extractable Cu, Pb and Zn in EDDS-amended soil significantly decreased with time. In comparison with the values found the second day, the DTPA-extractable amounts of Cu, Pb and Zn measured the 7th day decreased by about 23, 21

Fig. 3.7. DTPA- extractable metals in NTA-(a) and EDDS-amended (b) soils 2 days and 7 days after the amendments. 0 days represents the amount of DTPA-extractable metals in soil immediately before amendments. One-way ANOVA was used to evaluate the differences with increasing time. Different letters indicate significantly different values at P≤0.01. n = 5.

a

and 25%, respectively (Fig. 3.7b). No significant differences were found for DTPA-extractable amounts of arsenic and cadmium both after NTA and EDDS amendments (Fig. 3.7).

3.1.5 – Biodegradation of EDDS and NTA in soil

The amount of extractable EDDS and NTA was measured immediately (4 h), and 2 and 7 days after the amendment. EDDS content showed a significant and continuous decrease with time. Its amount was halved in 7 days (Table 3.7). No significant differences were detected within 7 days for NTA (Table 3.7).

3.1.6 – Wild species – assisted phytoextraction by B. carinata

The cultivation of the wild species had a positive effect on metal solubilization. In comparison with the control (soil in which no wild species had been grown), the presence of three wild species caused an enhancement of DTPA-exchangeable metals by about three fold, except for Cd that showed higher rates of increase (3, 7 and 22 fold for Plantago, Silene and Pinus, respectively) (Fig. 3.8). In particular, Silene paradoxa was the most effective wild species in increasing Cu and Zn extractable amounts while

Table 3.7. Extractable EDDS and NTA (mmol kg-1 soil) 4 h, 2 and 7 days after chelates addition (5 mmol kg-1 soil).

4h 2d 7d

EDDS 3.821 ± 0.10 a 3.089 ± 0.10 b 1.905 ± 0.11 c

NTA 4.005 ± 0.22 a 3.967 ± 0.10 a 3.665 ± 0.19 a

Results are the means ± SD of three replicates, analysed in triplicate, of three independent experiments (n = 9). One-way ANOVA was used to evaluate the differences.

Cd and Pb extractable amounts were increased more by the presence of Pinus pinaster (Fig. 3.8).

The growth of the wild species caused a significant decrease in soil pH from 7.3 ± 0.3 for the control to 5.4 ± 0.2 for the soil in which Pinus Pinaster and Silene paradoxa had been grown and to 5.6 ± 0.5 for the soil in which Plantago lanceolata had been grown.

The accumulation in the shoot of Brassica carinata of Cd, Cu and Pb was favoured, in comparison with the control by Pinus pinaster whereas Silene paradoxa was the most effective in increasing shoot Zn concentration. No significant differences between the control and the treatments were detected for arsenic (Fig. 3.9). The values of metal

a d c b a a a a a a a b b b b c c c c d

Fig. 3.8. DTPA – extractable metals of the soils in which Pinus pinaster, Plantago lanceolata and Silene paradoxa had been grown immediately before planting B. carinata seeds. The control is represented by the soil in which no wild species had been grown. Values are the means of 5 replicates, analysed in triplicates (n = 5). One-way ANOVA was used to evaluate the differences between the control and the treatments. Different letters indicate significantly different values at P≤ 0.01.

concentrations in shoots of B. carinata grown in succession to the wild species (Fig. 3.9) approached or exceeded (as in the case of Cd) the values detected after EDDS or NTA applications (Fig. 3.6a).

3.1.7 – Wild species root exudates

Phenolic acids were detected in root exudates of Silene paradoxa, Plantago lanceolata and Pinus pinaster, the amount and kind being strictly dependent on the species analysed (Fig. 3.10). Silene paradoxa, with a total phenolic acids concentration of 78.25

a

a

a

a

a

a

a

a

b

b

b

b

c

c

c

c

d

d

d

d

Fig. 3.9. Metal concentration in shoots of B. carinata grown to succession to Pinus

pinaster, Plantago lanceolata and Silene paradoxa. B. carinata plants grown in soil in which no wild species had previously been grown represent the control. The results are the means of three replicates, analysed in triplicate (n = 3). One-way ANOVA was used to evaluate the differences between the control and the treatments. Different letters indicate significantly different values at P ≤ 0.01.

followed by Plantago lanceolata (23.56 µg phenolic acid g-1 root dw) and Pinus

pinaster (13.61 µg phenolic acid g-1 root dw). The phenolic acids present in the exudates of Silene paradoxa grown in nutrient solution were protocatechuic (Pro; 36% of total concentration), syringic (Syr; 31% of total concentration), p-OH-benzoic (Ben; 23% of total concentration) and vanillic acid (Van; 10% of total concentration). Ferulic (Fer; 31% of total concentration), syringic (29% of total concentration), p-cumaric (Cum; 11% of total concentration) and caffeic acid (Caf; 11% of total concentration) were the main constituents of the phenolic exudates of Plantago lanceolata grown in control conditions while only protocatechuic (30% of total concentration), p-OH-benzoic (37% of total concentration) and syringic acid (33% of total concentration) were exuded by Pinus pinaster.

The incubation of the wild species in metal contaminated solutions caused changes in the composition of the exuded phenolic compounds (Fig. 3.10). Also in this case, the changes in phenolic composition varied among the species. In comparison with the control, 2-, 3- and 6-fold increases of vanillic, gallic (Gal) and chlorogenic (Chl) acid were found in root exudates of Silene paradoxa, an about 3- and 8-fold increase of p-OH-benzoic and vanillic acid was detected in root exudates of Plantago lanceolata, and a 3- and 7- fold increase of vanillic and chlorogenic acid was measured in root exudates of Pinus pinaster (Fig. 3.10).

Among flavonoids, quercetin, kaempferol and naringenin were found in exudates of the three wild species although their amounts were very small. Plantago lanceolata and

Silene paradoxa exuded a total flavonoid concentration higher than Pinus pinaster both in the control and in the treatment (Fig. 3.11). Plantago was the species that showed the greatest increase in flavonoid exudates following the treatment (from 1.90 µg flavonoids g-1 root dw in the control to 9.55 µg flavonoids g-1 root dw in the treatment) followed by Silene (from 3.08 µg flavonoids g-1 _{root dw to 6.55 µg flavonoids g}-1

root dw) and Pinus (from 0.41 µg flavonoids g-1 _{root dw to 0.86 µg flavonoids g}-1

Fig. 3.10. Phenolic acids (µg g-1

root dw) in Silene (a), Plantago (b) and Pinus (c) for the control (□) and the metal treatment (■). Data are the means of three replicates, analysed in triplicate (n = 3). One-way ANOVA was used to evaluate the differences between the control and the treatment for each compound. Only caffeic acid in Plantago showed no significant differences between control and treatment. µ g g -1 r o o t d w 0 2 4 6 8 10 12 14 F er B en V a n C h l C a f S y r C u m b µ g g -1 r o ot d w 0 5 10 15 20 25 30 P ro B en G a l V a n C h l S y r a 0 1 2 3 4 5 6 7 P ro B en V a n C h l C a f S y r C u m µ g g -1 r o o t d w c

In all of the three species naringenin was the flavonoid whose exudation was the most stimulated by the metal stress. It increased by about 18-, 7- and 3-fold in Pinus,

Plantago and Silene exudates, respectively (Fig. 3.11).

The main components of exudates were doubtlessly organic acids. Four different organic acids were detected in wild species exudates, their composition and amount being strictly species-dependent. Oxalic and malic acid were detected in all of the three species while tartaric and malonic were acid typical of Pinus, citric and fumaric acid of

Plantago and malonic and fumaric acid of Silene. Oxalic and malic acids were the main representative organic acids both in the control and in the treatment exudates (Table 3.8). The organic acid composition present in root exudates remained unchanged under

µ g g -1 r o o t d w 0 1 2 3 4 5 6 Q u e K a e N a r

a

b

c

root dw) in Plantago (a),

Silene (b) and Pinus (c) for the control (□) and the metal treatment (■). Data are the means of three replicates, analysed in triplicate (n = 3). One-way ANOVA was used to evaluate the differences between the control and the treatment for each compound. Significant differences were always found between control and treatment.

treatment conditions although the rate of their increase was dependent on the species analysed (Table 3.8). Under treatment, the highest increase in Pinus exudates was seen for oxalic acid (173-fold) followed by malic acid (93-fold) (Table 3.8).

Table 3.8. Organic acids (mg g-1 root dry weight) in exudates of 15-d-old Pinus, Plantago and Silene incubated for 24 h in nutrient solution (control) and in nutrient solution added with five heavy metals (As, Cd, Cu, Pb and Zn).

Pinus pinaster Control Treatment Oxalic 2.15 ± 0.17 373.22 ± 46.29 Tartaric 0.35 ± 0.09 11.60 ± 0.07 Malic 0.51 ± 0.08 47.45 ± 2.90 Malonic 0.10 ± 0.01 5.89 ± 0.58 Plantago lanceolata Control Treatment Oxalic 7.19 ± 1.23 68.76 ± 3.20 Malic 1.55 ± 0.34 12.86 ± 1.37 Citric 0.10 ± 0.02 0.41 ± 0.01 Fumaric 0.04 ± 0.004 1.34 ± 0.08 Silene paradoxa Control Treatment Oxalic 7.08 ± 0.71 556.72 ± 18.77 Malic 0.86 ± 0.09 123.52 ± 10.58 Malonic 1.17 ± 0.18 18.82 ± 2.32 Fumaric 0.01 ± 0.002 0.01 ± 0.002

Results are the means ± SD of three replicates, analysed in triplicate (n = 3). Except for fumaric acid in Silene, the treatment values were always significantly different from the control values at P≤0.01.

Similar huge increases in response to metal treatment were also found in Silene

paradoxa of which oxalic and malic acid contents increased about 78- and 143-fold, respectively. Plantago lanceolata was the wild species that showed the lowest rates of increase. Its fumaric acid content increased about 25 fold followed by oxalic acid which

3.2 – Arsenic

3.2.1 – Time-dependent kinetics of As(III) and As(V) uptake

Absorption of inorganic arsenic species (nmol Asabs g-1 fw) by excised B. carinata roots

was monitored over 8 h (Fig. 3.12). The net uptake at 0.25 mM arsenate, expressed on a root fresh weight basis, appeared to display saturation kinetics (Fig. 3.12). Uptake was found to consist of a rapid initial phase over the first 90 min (R2 = 0.99) (Insert of Fig. 3.12), followed by a “steady-state” phase in which the rate of uptake decreased (Fig. 3.12). Mono-phasic time-dependent influx was evident at 0.25 mM arsenite. The uptake appeared to be linear over 8 h (R2 = 0.98) (Fig. 3.12). When supplied both at 0.25 mM, the uptake of arsenate was 1.5 to 5 times higher than that of arsenite (Fig. 3.12).

The initial phases (up to 90 min) of arsenate and arsenite uptake were markedly affected by temperature. A temperature reduction of 20°C caused an about 10-fold reduction of the net uptake of As(V) and As(III) (Fig. 3.13).

Fig. 3.12. Time-dependent kinetics for arsenite (■) and arsenate (●) influxes into

B. carinata roots. Insert depicts the time-dependent influxes of arsenite (■) and arsenate (●) for the first 90 min. Each point is the mean ± SD of three replicates, analysed in triplicate, of three independent experiments (n = 9).

R2 _{= 0.98}

R2 _{= 0.97}

R

2

3.2.2 – Concentration-dependent kinetics of As(III) and As(V) uptake

The excised roots of B. carinata showed biphasic uptake kinetics for arsenate (Fig. 3.14). The concentration-dependent influx isotherm for arsenate showed an active pattern at lower substrate concentrations ([As(V)] ≤ 1 mM) that fitted very well (R2 = 0.95) to a Michaelis-Menten function and a passive absorption mechanism progressively taking over (R2 = 0.98) at increasing As(V) concentrations ([As(V)] ≥ 1 mM). The transition occurred when the external arsenate concentration was about 1 mM (Fig. 3.13). The high-affinity Vmax and KM values were 51.28 ± 2.07 nmolAs(V)abs g-1fw

h-1 and 2.05 ± 0.21 nM, respectively.

For arsenite a mono-phasic concentration-dependent influx was found. The isotherm data obtained fitted better to a linear model (R2 = 0.99) rather than to a non-linear one (R2 = 0.80), also at very low concentrations (Insert of Fig. 3.15).

Fig. 3.13. Effect of temperature of the test solution on the first 90 min of the time course of arsenite (a) and arsenate (b) uptake by B. carinata roots. (___{) 25°C; (--) 5°C.}

Each point is the average ± SD of three replicates, analysed in triplicate, of three independent experiments (n = 9).

Fig. 3.14. Concentration-dependent kinetics for high- and low- affinity arsenate influx into B. carinata roots. Insert depicts the kinetics uptake of arsenate at low substrate concentrations (0 – 1 mM). Each point is the average ± SD of three replicates, analysed in triplicate, of three independent experiments (n = 9).

Fig. 3.15. Concentration-dependent kinetics for arsenite influx into B. carinata roots. Insert depicts the uptake kinetics of arsenite at low substrate concentrations (0 – 0.1 mM). Each point is the average ± SD of three replicates, analysed in triplicate, of three independent experiments (n = 9).

3.2.3 – Measurement of membrane integrity

No significant differences (P ≤ 0.01) in the relative leakage ratio, in comparison with the control, were detected up to 1 mM As(V) (Table 3.9). When the roots of B. carinata were treated with Na2HAsO4.7H2O concentrations higher than 1 mM, the relative

leakage ratio increased linearly with the external As(V) concentration (Table 3.9). In the case of arsenite, a continuous increase of the relative leakage ratio of B. carinata roots was found with increasing external NaAsO2 concentration (Table 3.9).

Table 3.9. Relative leakage ratio (RLR) of B. carinata roots treated with different concentrations of Na2AsO4.7H2O or NaAsO2 (mM).

As(V) mM 0 0.1 0.25 0.5 1 1.5 2.5

RLR 3.4±0.3a 3.7±0.2a 3.4±0.5a 3.8±0.3a 3.4±0.3a 5.9±0.2b 6.7±0.4c

As(III) mM 0 0.1 0.25 0.5 1 1.5 2.5

RLR 3.3±0.2a 4.3±0.4b 5.1±0.2c 6.1±0.3d 6.7±0.3e 7.6±0.2f 8.3±0.3g Results are the means ± SD of three replicates, analysed in triplicate, of three independent experiments (n = 9). One-way ANOVA was used to evaluate the differences between the controls and the treatments. Different letters indicate significantly different values at P ≤ 0.01.

3.2.4 – As(III) and As(V) influxes at different phosphate concentrations

The uptake rate of 0.25 mM arsenate decreased significantly with increasing phosphate concentration in the incubating solution (Fig. 3.16). Without phosphate in the uptake medium, the net uptake rate of arsenate was 55.00 nmolAs(V)abs g

-1

fw h-1. The rate of arsenate accumulation decreased with increasing phosphate supply till 38 nmol As(V)abs

g-1 fw h-1 when phosphate was supplied at 0.4 mM (Fig. 3.16).

On the contrary, the rate of arsenite accumulation by B. carinata roots was not influenced by the phosphate concentration in the medium (Fig. 3.16). The arsenite concentrations in roots incubated with 0.25 mM NaAsO2 remained almost constant (~

Fig. 3.16. Uptake of arsenite (■) and arsenate (●), supplied at 0.25 mM, by B. carinata roots at different concentrations of phosphate (0 – 2.5 mM). Inserts depict the uptake kinetics of arsenite (■) and arsenate (●) at low phosphate concentrations (0 – 0.25 mM). Each point is the mean ± SD of three replicates, analysed in triplicate, of three independent experiments (n = 9).

3.3 – Copper

3.3.1 – Time-dependent kinetics of Cu(II) uptake

Uptake of copper sulphate (µmolCuabs g-1 fw) by excised B.carinata roots was

monitored over 8 h. The net uptake of 50 µM CuSO4, expressed on a root fresh weight

basis, appeared to display saturation kinetics (R2 = 0.96) (Fig. 3.17). Uptake was found to consist of a rapid initial phase over the first 60 min (R2 = 0.90) (Insert of Fig. 3.17) followed by a “steady-state” phase in which the rate of uptake decreased (Fig. 3.17).

3.3.2 – Concentration-dependent kinetics of Cu(II) uptake

Excised roots of B. carinata showed two concentration-dependent uptake systems for Cu+2 (Fig. 3.18). The high-affinity uptake system dominated at low external substrate

Fig. 3.17. Time-dependent kinetics for CuSO4 influx into B. carinata roots. Insert depicts

the time-dependent influx of CuSO4 for the first 60 min. Each point is the mean ± SD of

three replicates, analysed in triplicate, of three independent experiments (n = 9).

R2 _{= 0.90}

R2

took over at increasing external substrate concentrations ([Cu+2] ≥ 60 µM) (Fig. 3.18). The concentration-dependent influx isotherm for Cu+2 _{fitted well (R}2 _{= 0.92) to a}

Michaelis-Menten equation at external substrate concentrations up to 60 µM CuSO4.

This produced a Vmax of 0.95 ± 0.06 µmolCu+2 g-1fw h-1 and KM of 1.41 ± 0.09 µM. At

external copper sulphate concentrations higher than 60 µM, the influx data fitted better to a linear model (R2 = 0.95) rather than a nonlinear one (R2 = 0.77) (Fig. 3.18).

3.3.3 – Measurement of membrane integrity

No significant differences (P ≤ 0.01) in the relative leakage ratio, in comparison with the control, were detected up to 50 µM CuSO4 (Table 3.10). When the roots of B.

carinata were treated with CuSO4 concentrations higher than 50 µM, the relative

leakage ratio increased with the increasing of the external copper concentration (Table 3.10).

Fig. 3.18. Concentration-dependent kinetics for high- and low-affinity copper influx into B. carinata roots. Insert depicts the uptake of copper at low external substrate concentration (0.12 – 60 µM). Each point is the mean ± SD of three replicates, analysed in triplicate, of three independent experiments (n = 9).

R2

= 0.92

R2

Table 3.10. Relative leakage ratio (RLR) of B. carinata roots treated with different concentrations of CuSO4 (µM)

Cu(II) µM 0.12 10 25 50 100 150 300

RLR 0.14±0.03a 0.17±0.01a 0.15±0.01a 0.18±0.03a 0.24±0.01b 0.30±0.03c 0.83±0.01d Results are the means ± SD of three replicates, analysed in triplicate, of three independent experiments (n = 9). One-way ANOVA was used to evaluate the differences between the control and the treatments. Different letters indicate significantly different values at P≤0.01.

3.3.4 – Concentration-dependent kinetics of [S,S]-EDDS-Cu and NTA-Cu

The presence of chelators in the uptake solution influenced the concentration-dependent copper influx since EDDS and NTA chelated copper changing the pattern of its absorption (Fig. 3.19). In presence of 5 mM [S,S]-EDDS or NTA the above reported biphasic copper uptake mechanism (Fig. 3.18) was replaced by simple Michaelis-Menten functions (R2 = 0.98 for EDDS-Cu complex and R2 = 0.90 for NTA-Cu complex) (Fig. 3.19). Data fitting produced a Vmax of 0.31 ± 0.01 µmolCu g

-1

fw h-1 and a KM of 0.20 ± 0.05 µM for EDDS-Cu complex uptake (Fig. 3.19) and a Vmax of 0.43 ±

0.10 µmolCu g-1

fw h-1 and a KM of 0.98 ± 0.37 µM for NTA-Cu complex uptake (Fig.

3.19).

Further studies were conducted maintaining the same concentration range of CuSO4

(from 0.12 to 300 µM) and changing the amount of chelators that were, anyway, in excess. When EDDS and NTA were added at 2.5 mM to the uptake solutions no significant changes were detected in their Vmax and KM values in comparison with the

3.3.5 – Long-term copper, EDDS and NTA accumulation in plants tissues

The rates of long-term Cu and EDDS or NTA accumulation in roots and shoots of B.

carinata treated with CuSO4 (control), Cu-EDDS and Cu-NTA were monitored over 72

h. Copper uptake by roots of B. carinata showed a saturation pattern with an initial linear phase (first 25 h) followed by a “steady state” phase in which the rate of uptake decreased and copper content remained almost constant (~ 30 µmol g-1

dw) (Fig. 3.20). Cu uptake by roots in the Cu-EDDS or Cu-NTA treatments was about 10-fold lower than in CuSO4 treatment. After 72 h of incubation, the net Cu uptake from the CuSO4

solution was about 30 µmol g-1 dw while the net uptake from Cu-EDDS or Cu-NTA solution was about 3-4 µmol g-1

dw. As shown by the insert of figure 3.20, the pattern of copper uptake by B. carinata roots from Cu-EDDS and Cu-NTA solutions wasn’t dependent on the kind of chelator.

Fig. 3.19. Concentration-dependent kinetics of Cu-NTA (___{) and Cu-EDDS (- -) influxes into}

B. carinata roots. Insert depicts the uptake of the complexes at low external substrate concentrations (0.12 – 25 µM). Each point is the mean ± SD of three replicates, analysed in triplicate, of three independent experiments (n = 9).

R2 _{= 0.90}

The translocation of Cu showed an opposite behaviour with the shoot content in the Cu-EDDS and Cu-NTA treatments much higher than that in the CuSO4 treatment. After 72

h of incubation, the net shoot accumulation in the CuSO4 treatment was about 2 µmol g -1

dw while the net accumulation in the Cu/NTA or Cu/EDDS treatments were about 13 and 26 µmol g-1 _{dw, respectively (Fig. 3.21). A significant difference was detected}

between the two chelators with EDDS being two times more effective than NTA in increasing Cu translocation (Fig. 3.21).

The presence of EDDS and NTA had not only an influence on the amount of copper translocated from roots to shoots but also on the time required for its translocation. When Brassica plants were incubated in CuSO4 solution, no translocation was observed

before 36 h whereas when the seedlings were incubated in Cu-NTA or Cu-EDDS solutions a significant increase in translocated copper was detected already after 12 h (Fig. 3.21).

Fig. 3.20. Copper concentration in roots of Brassica carinata treated for different times (from 20 min up to 72 h) with 300 µM CuSO4 (■) or with 300 µM CuSO4 + 5 mM NTA (□)

or with 300 µM CuSO4 + 5 mM EDDS (●). Results are the means of three replicates,

analysed in triplicate (n = 3). Insert depicts copper content in roots of B. carinata treated with Cu-EDDS (●_{) and Cu-NTA (}□_{). The size of error bars doesn’t exceed that of symbols.}

Time µ m o lC u (I I) g -1 d w 0 0,5 1 1,5 2 2,5 3 3,5 4 0 20 40 60 80 0 5 10 15 20 25 30 35 40 0 10 20 30 40 50 60 70 80 ■ CuSO4 □ Cu-NTA ● Cu-EDDS

Measurements of EDDS and NTA content in roots and shoots of B. carinata were performed as well. EDDS was observed in roots already after 20 min of incubation, thereafter its content continued to increase with a pattern that fitted better a saturation model (R2 = 0.92) rather than a linear one (R2 = 0.80). About 350 µmol EDDS g-1 dw were detected in roots after 72 h of incubation in Cu-EDDS solution (Fig. 3.22a). NTA was undetectable in roots before 36 h. Thereafter its content increased linearly with increasing time reaching a maximum root accumulation of 7.48 µmol NTA g-1

dw after 72 h of incubation (Insert of Fig. 3.22a). The EDDS concentration was higher than the NTA concentration also in shoots, although the difference was no so pronounced as in roots. In this case, both chelators showed a linear pattern (R2 = 0.88 and 0.90 for EDDS and NTA, respectively) with their concentration linearly increasing with time of incubation (Fig. 3.22b).

Fig. 3.21. Copper concentration in shoots of B. carinata treated for different times (from 20 min up to 72 h) with 300 µM CuSO4 (■) or with 300 µM CuSO4 + 5 mM NTA

(□_{) or with 300 µM CuSO4 + 5 mM EDDS (}●_{). Results are the means ± SD of three} replicates, analysed in triplicate (n = 3). In some cases, the size of error bars doesn’t exceed that of symbols.

0 5 10 15 20 25 30 0 10 20 30 40 50 60 70 80 Time (h) µm o lC u (I I) g -1 d w ■ CuSO4 □ Cu-NTA ● Cu-EDDS

A positive correlation was found between EDDS and Cu both in roots and shoots of B.

carinata (R2 = 0.92 and 0.95 in roots and shoots, respectively) (Fig. 3.23).

Fig. 3.22. NTA (□) and EDDS (●) concentration in roots (a) and shoots (b) of Brassica

carinata treated for different times (from 20 min up to 72 h) with 300 µM CuSO4 + 5 mM

NTA (□) or with 300 µM CuSO4 + 5 mM EDDS (●).

The results are the means ± SD of three replicates, analysed in triplicate (n = 3). Insert depicts the root NTA content. µm o lC h el a to r g -1 d w Time (h) a b 0 100 200 300 400 500 0 20 40 60 80 0 4 8 0 50 100 0 100 200 300 400 500 □ Cu-NTA ● Cu-EDDS 0 100 200 300 400 500 0 1 2 3 4 µ m o lE D D S g -1 d w µmolCu(II) g-1 dw _{µmolCu(II) g}-1 dw 0 10 0 20 0 30 0 40 0 50 0 0 10 20 30 µ m o lE D D S g -1 d w b a

Fig. 3.23. Correlation between EDDS and Cu concentration in roots (a) and shoots (b) of

B. carinata. Plants were treated for different times (from 20 min up to 72 h) with 300 µM

CuSO4 and 5 mM EDDS.

20 min

20 min 72 h

No relationship was found between Cu and NTA in roots while in shoots they were linearly related although the correlation factor (R2 = 0.90) was slightly lower than the one of EDDS (Fig. 3.24).

3.3.6 – Xylem sap analysis

The xylem pH of Brassica carinata wasn’t dependent on the amount of copper present in culture solutions. Its value was always around 5.8.

The cytosolic marker enzymes (malate dehydrogenase, c-mdh, EC 5.3.1.9; hexose phosphate isomerase, c-hpi, EC 1.1.1.37) were always ≤ 1% of those found in total plant tissue homogenates indicating that no appreciable cytosolic contamination was present in B. carinata xylem sap.

3.3.7 – Amino acids in B. carinata xylem sap

Xylem saps of Brassica carinata plants grown in nutrient solution (0.12 µM CuSO4;

control) or incubated for three days in treatment solutions (0 µM, 2.5 µM and 5 µM CuSO4) were collected.

Fig. 3.24. Correlation between NTA and Cu concentration in shoots of B. carinata. Plants were treated for different times (from 20 min up to 72 h) with 300 µM CuSO4 and 5 mM NTA.

0 40 80 120 160 200 0 5 10 15 µmolCu(II) g-1 _dw µ m o lN T A g -1 d w 72 h 20 min

Under control conditions, nicotianamine (NA) was the major amino acid present in

Brassica carinata xylem sap (19.60 µg ml-1 xylem) followed by lysine (Lys) (7.56 µg ml-1 xylem), alanine (Ala) (3.89 µg ml-1 xylem), leucine (Leu) (2.91 µg ml-1 xylem), histidine (His) (2.89 µg ml-1 xylem) and arginine (Arg) (2.59 µg ml-1 xylem) (Table 3.11).

Table 3.11. Amino acids concentrations (µg ml-1 xylem) in xylem saps of Brassica

carinata exposed for three days to 0, 0.12 (control), 2.5 and 5 µM CuSO4.

CuSO4µµµµM 0.12 µµµµM 2.5 µM µµµ 5 µµµM µ 0 µµµµM

Aspartic acid (Asp) 0.38±0.04 0.63±0.21 0.48±0.22 0.38±0.16

Glutamic acid (Glu) 1.15±0.09 1.38±0.17 1.04±0.08 1.30±0.50

Asparagine (Asn) 0.14±0.04 0.10±0.001 0.16±0.08 0.15±0.08

Glutamine (Gln) 0.34±0.08 0.74±0.06 1.19±0.10 1.42±0.32

Serine (Ser) 1.26±0.40 0.81±0.07 0.55±0.05 0.85±0.31

Glicine (Gly) 0.91±0.11 1.66±0.11 1.72±0.28 0.56±0.02

γ-aminobutyric acid (Gaba) 0.18±0.06 0.22±0.004 0.21±0.03 0.16±0.01

Threonine (Thr) 0.52±0.08 2.04±0.11 2.59±0.35 2.09±0.06 Histidine (His) 2.89±0.50 15.96±1.34 21.41±2.55 2.03±0.65 Arginine (Arg) 2.59±0.61 2.64±0.43 2.47±0.52 2.25±0.28 Alanine (Ala) 3.89±0.38 1.41±0.11 2.34±0.56 4.28±0.60 Proline (Pro) 0.31±0.07 0.67±0.02 0.73±0.09 0.41±0.07 Tyrosine (Tyr) 1.19±0.19 1.49±0.20 1.59±0.33 1.68±0.41 Valine (Val) 1.94±0.30 1.55±0.48 1.32±0.37 1.27±0.14 Methionine (Met) 0.90±0.07 1.64±0.02 1.87±0.31 4.41±0.11 Cysteine (Cys) 0.68±0.06 0.92±0.15 0.67±0.03 ND Isoleucine (Ile) 1.46±0.43 1.61±0.42 1.77±0.29 1.91±0.40 Leucine (Leu) 2.91±0.08 1.83±0.39 1.69±0.17 2.23±0.50 Phenylalanine (Phe) 1.15±0.14 1.44±0.17 1.33±0.04 0.96±0.33 Lysine (Lys) 7.56±0.48 8.33±0.76 8.53±0.17 9.27±1.17 Nicotianamine (NA) 19.60±4.08 23.12±1.93 17.73±1.91 82.38±11.33 Results are the means ± SD of three replicates, analysed in triplicate, of three independent experiments (n = 9).

One-way ANOVA was used to evaluate the differences between the control and the treatments. The results

of statistical analysis is reported in Figs. 3.25 and 3.26.

Compared to the control (CuSO4 0.12 µM), the xylem amino acids concentrations

The amino acids that showed the greatest relative increases in concentration when B.

carinata was exposed for three days to 2.5 and 5 µM CuSO4 were: histidine (+ 452%

and + 641%, respectively), threonine (+ 292% and + 398%, respectively), glutamine (+ 118% and + 250%, respectively), proline (+ 116% and + 135%, respectively), methionine (+ 82% and +108%, respectively) and glycine (+ 82% and + 89%, respectively) (Table 3.11 and Fig. 3.25).

Compared to the control (CuSO4 0.12 µM), the xylem amino acid concentrations

showed some significant changes when B. carinata plants were grown for three days under copper deficiency (0 µM CuSO4) (Table 3.11 and Fig. 3.26).

Fig. 3.25. Changes (%) in xylem amino acid concentrations of B. carinata plants exposed for three days to 2.5 µM (□) and 5 µM (■) CuSO4 relative to amino acid concentration in the

xylem sap of control plants (0.12 µM CuSO4). The control value of each amino acid is denoted

0. The asterisks indicate those treatment values that are significantly different from the control ones. The analysis of variance was performed before the conversion in percentage. Each data is the mean of three replicates, analysed in triplicate, of three independent experiments (n = 9). Amino acids are listed from left to right in the order of their chromatographic retention times.

A sp G lu A sn G ln S e r G ly G a b a T h r H is A r g A la P r o T y r V a l M e t C y s Il e L e u P h e L y s N A R e la ti v e am in o a ci d s v ar ia ti o n s (% ) □ 2.5 µM CuSO4 ■ 5 µM CuSO4 -100 0 100 200 300 400 500 600 700 * * * * * * * * * * * * * _{* *} * *

The amino acids that showed significant relative increases in concentration when B.

carinata was grown in copper starvation were: methionine (+ 390%), nicotianamine (+ 320%), glutamine (+ 318%) and threonine (+ 302%).

3.3.8 – Cu in xylem sap

Total Cu concentration in Brassica carinata xylem sap increased with increasing Cu concentration in the nutrient solution (Fig. 3.27). When Cu in culture solutions increased from 0 to 5 µM, Cu concentration in Brassica xylem sap increased 6 fold Fig. 3.26. Changes (%) in xylem amino acid concentrations of B. carinata plants grown for three days under copper starvation (■ 0 µM CuSO4) relative to amino acid concentration in the

xylem sap of control plants (0.12 µM CuSO4). The control value of each amino acid is denoted

0. The asterisks indicate those treatment values that are significantly different from the control ones. The analysis of variance was performed before the conversion in percentage. Each data is the mean of three replicates, analysed in triplicate, of three independent experiments (n = 9). Amino acids are listed from left to right in the order of their chromatographic retention times. ND = non-detectable. * A sp G lu A sn G ln S er G ly G a b a T h r H is A rg A la P ro T y r V al M et C ys Il e L eu P h e L y s N A ■ 0 µM CuSO4 -50 0 50 100 150 200 250 300 350 400 . R e la ti v e am in o a ci d s v ar ia ti o n s (% ) * * * * * ND

3.3.9 –Brassica carinata xylem sap and simulated saps for excess of copper

Increased Cu concentration in the nutrient solution induced selective accumulation of certain amino acids in the xylem sap. The plant response, in terms of amino acid accumulation, was directly correlated with the copper concentration. In fact, the 2.5 and 5 µM Cu treatments induced the accumulation of the same amino acids although at different degrees. For this reason the free Cu2+concentration vs pH titration signatures were conducted only for plants treated with 5 µM CuSO4 (Fig. 3.28).

The free Cu2+ concentrations vs pH titration curves of Brassica carinata xylem sap and key amino acids detected in xylem sap are presented in Fig. 3.28. For a single complexation agent, the pattern of titration curve of 0.14 mM histidine (His) with 0.94 µM Cu (Fig. 3.28a) was the most similar, closely followed by that of 6.33 µM proline (Pro) (Fig. 3.28e), to the curve of Brassica carinata xylem sap.

Fig. 3.27. Effect of CuSO4 concentration in nutrient solution on copper concentration in

xylem sap of Brassica carinata. Data are the means of three replicates, analysed in triplicate, of three independent experiments (n = 9). One-way ANOVA was used to evaluate the differences between control (0.12 µM) and treatments (0, 2.5 and 5 µM). Different letters indicate significantly different values at P ≤ 0.01.

a

b

c

In order to investigate the relative importance of histidine and proline on Cu binding in the presence of other amino acids, titration experiments were conducted on the following solutions: simulated xylem sap (His + Thr + Gln + Gly + Pro + Met) (Fig.

0 10 2 0 3 0 4 0 50 6 0 70 3 4 5 6 7 0 10 20 30 40 50 60 70 3 4 5 6 7 0 10 20 30 40 50 60 70 3 4 5 6 7 0 10 20 30 40 50 60 70 3 4 5 6 7 0 10 2 0 3 0 4 0 50 6 0 70 3 4 5 6 7 F re e C u 2 + ( µg l -1 ) pH pH pH pH pH F re e C u 2 + ( µ g l -1 ) F re e C u 2 + ( µg l -1 ) F re e C u 2 + ( µ g l -1 ) F re e C u 2 + ( µ g l -1 ) ■ Brassica xylem ∆ Thr ■ Brassica xylem ∆ His ■ Brassica xylem

∆ Gln ■ Brassica xylem ∆ Gly

■ Brassica xylem ∆ Pro 0 10 2 0 3 0 4 0 50 6 0 70 3 4 5 6 7 ■ Brassica xylem ∆ Met pH F re e C u 2 + ( µ g l -1 )

Fig. 3.28. Free Cu2+ concentration vs pH titration curves of Brassica carinata xylem sap and

simulated saps containing single amino acids. Each point is the mean of three replicates, analysed in triplicate, of three independent experiments (n = 9). The standard deviation was always smaller than 10%.

b a

c d

3.29b); simulated xylem sap without proline (His + Thr + Gln + Gly + Met) (Fig. 3.29c); Brassica carinata xylem sap (Fig. 3.29).

The pattern of Cu2+ release from Cu-complexes of the simulated xylem sap without proline was the most similar to the pattern of Brassica carinata xylem sap, followed by the solution that didn’t contain histidine (Fig. 3.29). Therefore, histidine proved to be the most important copper chelator in xylem sap of B. carinata grown for three days in copper excess (5 µM CuSO4).

In the xylem sap of Brassica carinata treated with copper excess (5 µM CuSO4), 0.14

mM histidine (His), 21.75 µM threonine (Thr), 7.42 µM glutamine (Gln), 22.92 µM glycine (Gly), 6.331 µM proline (Pro) and 12.51 µM methionine (Met) could account, at pH = 5.8 ± 0.6 (Brassica carinata xylem sap pH), for 67.4%, 41.6%, 45.3%, 52.1%, 59.5% and 32.7% complexation of 0.94 µM Cu, respectively (Fig. 3.28 and Table 3.12).

F re e C u 2 + ( µ g l -1 ) F re e C u 2 + ( µ g l -1 ) 0 10 20 30 40 50 60 70 3 4 5 6 7 0 10 20 30 40 50 60 70 3 4 5 6 7 0 10 20 30 40 50 60 70 3 4 5 6 7 F re e C u 2 + ( µ g l -1 ) pH pH pH ■ Brassica xylem

∆ His + Thr + Gln + Gly + Pro + Met

Fig. 3.29. Free Cu2+ concentration vs pH

titration curves of Brassica carinata xylem sap and simulated saps containing combinations of amino acids. Each point is the mean of three replicates, analysed in triplicate, of three independent experiments (n = 9). The standard deviation was always ≤ 10%.

■ Brassica xylem

∆ Thr + Gln + Gly + Pro + Met

■ Brassica xylem

∆ His + Thr + Gln + Gly + Met

a b

Combination of 6 key amino acids (His + Thr + Gln + Gly + Pro + Met) could account for 69.9% complexation of 0.94 µM Cu, which is very similar to the measured level of Cu complexation in Brassica carinata xylem sap (72.7%) (Fig. 3.29 and Table 3.12). Simulated xylem sap without histidine (Thr + Gln + Gly + Pro + Met) and simulated xylem sap without proline (His + Thr + Gln + Gly + Met) could account for 59.2% and 68.4% complexation of 0.94 µM Cu, respectively (Fig. 3.29 and Table 3.12).

3.3.10 – Effect of Cu excess treatments on xylem sap histidine and proline

The histidine content increased with increasing external Cu concentration. Regression analyses showed that the concentration of histidine in B. carinata xylem saps was linearly related with the external copper concentration (y = 7.07x where y = [His]xyl and

x = [Cu] ; R2 _{= 0.88) and xylematic Cu concentration (y = 463x – 5.1 where y =}

Table 3.12. Free Cu2+ concentration in simulated xylem sap containing single amino acids or a combination of them and Brassica carinata xylem sap from high Cu treatment (5 µM CuSO4) at pH = 5.8 (xylem sap pH).

Titration solution Total Cu (µg l-1 xylem) Free Cu (µg l-1 xylem) Free Cu as % of total Cu 0.14 mM His 60 19.59 32.6 21.75 µM Thr 60 35.01 58.4 7.43 µM Gln 60 32.85 54.7 22.92 µM Gly 60 28.76 47.9 6.33 µM Pro 60 24.31 40.5 12.51 µM Met 60 40.38 67.3

His + Thr + Gln + Gly + Pro + Met 60 18.06 30.1

Thr + Gln + Gly + Pro + Met 60 24.45 40.8

His + Thr + Gln + Gly + Met 60 18.93 31.6

Brassica xylem sap 60 16.40 27.3

Amino acid concentrations simulate those found in Brassica carinata xylem sap. Data are the means of three replicates, analysed in triplicate, of three independent experiments ( n = 9).

For the percentages, the means were calculated after transformation of the data as arcsin√P.

increase only at the 2.5 µM CuSO4 treatment since no significant differences were

detected between the contents of proline at the 2.5 and 5 µM CuSO4 treatments (Fig.

3.30). However, both the histidine and proline concentrations in Brassica carinata xylem sap increased upon treatment with excess copper (Fig. 3.30), indicating that the accumulation of histidine and proline in xylem saps was induced by Cu in growing media.

3.3.11 – Brassica carinata xylem sap and simulated saps for copper starvation

Copper starvation induced a selective accumulation of certain amino acids in the xylem sap. In order to investigate the relative importance of those amino acids in Cu xylematic transport, Cu selective titrations were conducted. Since the free copper concentration in xylem of Brassica carinata grown for three days in copper deficiency was too low (0.01 ppm total Cu) to be detected by the cupric electrode, the titrations were performed using the amino acids concentrations found in xylem of Brassica incubated at 0 µM CuSO4

but adding an opportune amount of copper needed to reach a final total Cu concentration of 0.06 ppm (the copper concentration found in 5 µM CuSO4 treatment)

(Fig. 3.31). Good performances of the electrode were found in the previous experiments using that amount of copper.

Fig. 3.30. Effect of CuSO4 concentration in nutrient solution on Brassica carinata

xylem sap histidine and proline concentrations. Data shown are the means of three replicates, analysed in triplicate, of three independent experiments (n = 9). The standard deviations are reported in Table 3.11.

Pro His CuSO4 (µM) CuSO4 (µM) µg H is m l -1 x y le m µg P ro m l -1 x y le m 0 0,5 1 0.12 2.5 5 0 5 10 15 20 25 0.12 2.5 5 a a b b b c

The free Cu2+ concentrations vs pH titration curves of Brassica carinata xylem sap and key amino acids detected in xylem sap are presented in Fig. 3.31.

When singly applied, glutamine (Gln), methionine (Met) and threonine (Thr) showed titration patterns completely different from that of the xylem fluid of B. carinata (Figs. 3.31a,c,d). Conversely, the 0.27 mM nicotianamine (NA) pattern appeared to be very similar to the curve of Brassica carinata xylem sap (Fig. 3.31b), indicating that, among the amino acids analysed, it is likely to be the most important copper chelator in the xylem sap of B. carinata under copper starvation conditions (Fig. 3.30).

0 10 20 30 40 50 60 70 3 4 5 6 7 ■ Brassica xylem ∆ NA pH F re e C u 2 + (m g l -1 ) 0 10 20 30 40 50 60 70 3 4 5 6 7 ■ Brassica xylem ∆ Gln pH F re e C u 2 +(m g l -1) 0 10 20 30 40 50 60 70 3 4 5 6 7 ■ Brassica xylem ∆ Met pH F re e C u 2 +(m g l -1) 0 10 20 30 40 50 60 70 3 4 5 6 7 ■ Brassica xylem ∆ Thr pH F re e C u 2 + (m g l -1 )

Fig. 3.31. Free Cu2+ _{concentration vs pH titration curves of Brassica carinata xylem sap}

and simulated saps containing single amino acids. Each point is the mean of three replicates, analysed in triplicate, of three independent experiments (n = 9). The standard deviation was always ≤ 10%.

a

3.4 – AtMT2b-transformed tobacco

3.4.1 – As(III) resistance and accumulation tests

Two AtMT2b-transformed lines, M9 and M10, and the wild type of Nicotiana tabacum SR1 were subjected to detailed testing for As(III) resistance and accumulation using a hydroponic system.

To assess As(III) tolerance of AtMT2b-transformed tobacco plants, the two T1 lines were tested against the wild type for root growth under exposure to different supply rates of As(III) from 0 (control) up to 12 µM (Fig. 3.32).

As inferred from the root-growth response (Fig. 3.32), wild type and transformants didn’t show any significant As(III) sensitivity up to 6 µM concentration of external As(III). A 12 µM concentration of external As(III) inhibited root growth of wild type and both transformants severely (Fig. 3.32). At this concentration, M9 and M10 lines didn’t exhibit significant difference between each other in root development. Conversely, the wild type showed a different sensitivity to 12 µM As(III) being more resistant than both transformed lines (Fig. 3.32). At a 12 µM concentration of external

R o o t le n g th ( c m ) As(III) µµµµM Plant type ** As Conc ns Interaction ***

Fig. 3.32. Nicotiana tabacum SR1 root growth during 5 days of exposure to As(III) in hydroponic culture. ● Wild type; □ M9; ■ M10. Data point are the means of 12 plants. Vertical bars represent ± SE. Two ways ANOVA was used at P ≤ 0.05.

As(III), the difference between wild type and the other lines in terms of root length was highly significant (P ≤ 0.001).

Wild type and AtMT2b-transformed tobacco plants showed a very similar pattern of As(III) accumulation in roots. The root As(III) content (µmol As(III) g-1

root dw) increased almost linearly with increasing external concentration of As(III) for all plants tested (Fig. 3.33).

At 3 µM external As(III) there were no significant differences among the tobacco lines but, at higher concentrations, the wild type showed a significantly (P ≤ 0.01) higher As(III) content than both transformed lines, M9 and M10. M9 appeared to be more efficient than M10 in accumulating As(III) in roots (Fig. 3.33).

No significant differences (P ≤ 0.05) were detected in shoot As(III) concentration among the different lines analysed up to 6 µM external As(III) concentration (Fig. 3.34). At the highest concentration, however, M10 showed a significantly different pattern if compared to M9 and wild type. At 12 µM external concentration of As(III), the difference between M10 and the other lines (M9 and wild type) in term of shoot As(III) content was highly significant (Fig. 3.34).

µµµµ m o l A s( II I) g -1 r o o t d w As(III) µµµµM Fig. 3.33. Nicotiana tabacum SR1 As(III) root content after 5 days of exposure to As(III) in hydroponic culture. ● Wild type; □ M9; ■ M10. Data point are the means of 4 plants. Vertical bars represent ± SE. Two ways ANOVA was used at P ≤ 0.01.

Plant type *** As Conc ns Interaction *

The higher dry biomass of wild type and M9 lines in comparison with M10 line (data not shown) balanced their lower metal concentrations (Fig. 3.34). So, when calculated on a plant basis, the wild type showed the highest values of As(III) accumulation due to its higher biomass especially at the highest external As(III) concentrations (Fig. 3.35).

µµµµ m o l A s( II I) g -1 s h o o t d w As(III) µµµµM Fig. 3.34. Nicotiana tabacum SR1 As(III) shoot content after 5 days of exposure to As(III) in hydroponic culture. ● Wild type; □ M9; ■ M10. Data point are the means of 4 plants. Vertical bars represent ± SE. Two ways ANOVA was used at P ≤ 0.05.

Plant type ** As Conc ns Interaction ** n m o l A s( II I) p la n t -1 As(III) µµµµM Fig. 3.35. Nicotiana tabacum SR1 As(III) plant content after 5 days of exposure to As(III) in hydroponic culture. ● Wild type; □ M9; ■ M10. Data point are the means of 4 plants. Vertical bars represent ± SE. Two ways ANOVA was used at P ≤ 0.05.

Plant type ** As Conc ns Interaction ns