**4. Discussion**

### **4.1 Growth, physiological and biochemical parameters**

Zinc is an essential trace element for normal plant growth. There are important enzymes that contain zinc, such as the enzyme alcohol dehydrogenase, carbonic anhydrase, ribonucleic acid (RNA) polymerase, and superoxide dismutase, a key enzyme in protection against oxidative stress. Zinc activates different enzymes responsible for the synthesis of certain proteins. It is involved in the formation of chlorophyll and some carbohydrates. It is essential in the formation of auxins, which help regulate stem development and elongation, in addition to being the precursor of tryptophan [14]. Copper also plays a key function in normal plant growth. For example, it participates in CO2 assimilation and adenosine triphosphate (ATP) production [15]. It is the main constituent of diverse proteins such as plastocyanin of the photosynthetic system and cytochrome oxidase of the electron transport chain [16]. It plays a significant function in cell wall metabolism, signaling to the transcription protein trafficking apparatus, oxidative phosphorylation, iron armament, and biogenesis of

molybdenum cofactor [17]. Both are essential micronutrients necessary for the correct growth and development of plants; however, in high concentrations, they turn out to be phytotoxic, generating various negative metabolism modifications.

The results of our experiment indicate that some physiological and biochemical parameters of *C. indica* were significantly different at high Zn(II) and Cu(II) concentrations (**Figures 1-6**). The biomass decreased (both aerial part and root) for both metals (**Figure 1**), but only Cu(II) treatments showed a decline in the content of chlorophyll and carotenes (**Figure 2**). Root-relative conductivity (RC) increased with the Zn(II) and Cu(II) increasing concentrations (**Figure 3**), and the same occurred for the malondialdehyde (MDA) content in shoots with both metals, whereas, in roots, only Cu(II) treatments showed an increase (**Figure 4**). The soluble proteins content increased in the roots of the plants treated with Zn(II) but decreased in shoots of Cu (II)-treated plants. (**Figure 5**). For proline shoot content, a decline was shown in the lowest concentrations of both metals but increased at the highest concentrations while, in roots, increased only in the lowest concentration of Zn(II) but then decreased again to the levels of control treatment, showing no significant difference (**Figure 6**).

The decrease observed in the biomass of *C. indica* is highly reported in this and other species for zinc [18–20] and copper [21, 22] toxicity as one of the most obvious symptoms of plants growing in these conditions.

The biomass reduction related to Zn(II) toxicity is a consequence of mitosis inhibition that causes growth alterations product of the inhibition of deoxyribonucleic acid (DNA) synthesis [23]. Also could be the result of the alteration in macronutrient absorption [24] or the micronutrient distribution in different parts of the plant [25] such as lower uptake of Fe+2 and Fe+3; modification of the metabolic activity [26], inhibition of cellular division in the meristematic region, lengthening of root cells [27], reduction of cell viability, and death in the root tips [28].

Additionally, copper excess generates reactive oxygen species, which causes oxidative stress [29] that disrupts numerous metabolic pathways and modifies essential macromolecules [30]. Also, high copper concentrations cause negative modifications to DNA, photosynthesis, cell membrane integrity, enzyme activity, and respiration leading to general growth reduction [31]. Excess of copper in the roots can trigger alterations in the root system design that causes growth reduction, bronzing, necrosis, and nutritional inequities [32, 33].

#### **Figure 1.**

*Shoot and root dry weight (mg) of* Canna indica *plants in Zn(II) (A) and Cu(II) (B) systems. Columns represent the mean (*n *= 5), and vertical bars show the standard deviation (S.D.). Means followed by different letters (a-b-c) represent statistically significant differences (*p *< 0.05), e.g., "a" is statistically different from "b" and "c", but not from "ab".*

*Phytoextraction of Zn(II) and Cu(II) by* Canna indica*: Related Physiological Effects DOI: http://dx.doi.org/10.5772/intechopen.102450*

#### **Figure 2.**

*Chlorophyll A, B, total and carotenes content of* Canna indica *plant in Zn(II) (A) and Cu(II) (B) systems. Columns represent the mean (*n *= 5), and vertical bars show the standard deviation (S.D.). Means followed by different letters (a-b) represent statistically significant differences (*p *< 0.05), e.g., "a" is statistically different from "b", but not from "ab".*

#### **Figure 3.**

*Relative conductivity (RC) percentage (%) in roots and leaves biomass of* Canna indica *plants in Zn(II) (A) and Cu(II) (B) systems. Columns represent the mean (n = 5), and vertical bars show the standard deviation (S.D.). Means followed by different letters (a-b-c) represent statistically significant differences (*p *< 0.05), e.g., "a" is statistically different from "b" and "c", but not from "ab".*

Zinc helps to maintain membrane integrity, preserving the structural orientation of macromolecules and protecting the transportation systems [18], but in high concentrations, triggers reactions that promote oxidative stress and the breakdown of membrane integrity [24]. Similar behavior happens with copper excess, causing the

#### **Figure 4.**

*Malondialdehyde (MDA) content in the roots and leaves of* Canna indica *plant in Zn(II) (A) and Cu(II) (B) systems. Columns represent the mean (n=5), and vertical bars show the standard deviation (S.D.). Means followed by different letters (a-b) represent statistically significant differences (*p *< 0.05), e.g., "a" is statistically different from "b", but not from "ab".*

#### **Figure 5.**

*Soluble protein content in the roots and leaves of* Canna indica *plant in Zn(II) (A) and Cu(II) (B) systems. Columns represent the mean (*n *= 5), and vertical bars show the standard deviation (S.D.). Means followed by different letters (a-b) represent statistically significant differences (p < 0.05), e.g., "a" is statistically different from "b", but not from "ab".*

#### **Figure 6.**

*Proline content in the roots and leaves of* Canna indica *plants in Zn(II) (A) and Cu(II) (B) systems. Columns represent the mean (*n *= 5), and vertical bars show the standard deviation (S.D.). Means followed by different letters (a-b-c) represent statistically significant differences (*p *< 0.05), e.g., "b" is statistically different from "a" and "c", but not from "ab" and "bc".*

disruption of cell wall integrity and deposition of electron-dense material in the cytoplasmic membranes [34]. An increase in the relative conductivity (RC) of cellular membranes would indicate damage at the membrane level; higher values than 30%

*Phytoextraction of Zn(II) and Cu(II) by* Canna indica*: Related Physiological Effects DOI: http://dx.doi.org/10.5772/intechopen.102450*

#### **Figure 7.**

*(A) Zn(II) and (B) Cu(II) bioaccumulation in shoot, root, and total biomass of* Canna indica *plants and heavy metal accumulation in substrate. Columns represent the mean (*n *= 4), and vertical bars show the standard deviation (S.D.). Means followed by different letters (a-b-c-d) represent statistically significant differences (*p *< 0.05), e.g., "a" is statistically different from "b", "c" and "d".*

indicate damage [35]. In this work, results show that RC significantly increased only in roots for both metals. However, the values obtained were relatively low, showing damage only in the highest concentrations. The degree of peroxidation of lipids and the degree of membrane damage are related and can be analyzed from the malondialdehyde (MDA) concentration and RC [36]. Increased levels of reactive oxygen species (ROS) caused by heavy metal stress could develop in damage to lipid membranes, proteins, pigments, and nucleic acids [37]. The malondialdehyde is a product of the lipid peroxidation of polyunsaturated fatty acids in cell membranes caused by oxidative stress and the production of ROS [35]. In this work, shoot MDA levels increased in the maximum concentration, in comparison to the control, for both metals, while in roots only copper treatments showed an increase in the maximum concentration. Also, this suggests that the antioxidant enzymes present in the roots of zinc treatments could have compensated the damage caused by ROS [38]. Similar results were found in different species such as *Salix fragilis* and *Salix aurita*, which showed an increase in the electrolytic leakage (similar parameter associated to relative conductivity) related to heavy metal concentrations [39], or *Canna orchioides*, which also showed an increase in the relative conductivity and MDA accumulation associated to this type of stress [40]. Metal-induced stress induces reactive oxygen species (ROS) generation, which can lead to lipid peroxidation, protein impairment, enzyme inactivation, and DNA damage [23]. Membrane disruption and lipid peroxidation are generally contemplated as dependable biomarkers of oxidative status in plants [24].

Another distinctive heavy metal toxicity symptom in plants is a reduction of the content of photosynthetic pigments [41]. They are directly related to photosynthesis and plant growth so, a decrease of the content of these pigments or damage done to chloroplasts results in lower CO2 assimilation and a biomass decrease [42]. Carotenoids participate in antioxidant defense systems and impart a significant role in ROS sequestration [43], preventing the peroxidation of lipid membranes. [42]. Chloroplasts, mitochondria, and cellular membranes are some of the main sites that generate ROS. They are interconnected to the electron transport system, so when oxidative stress occurs, these sites are the first to be affected [44]. The decline in chlorophyll content in plants exposed to heavy metals stress is related to the inhibition of important enzymes, such as 6-aminolevulinic acid dehydratase (ALA-dehydratase) and protochlorophyllide reductase associated with chlorophyll biosynthesis, and the reduction of Mg+2 and Fe+2 supply. Zinc in phytotoxic concentrations may be equivalent to magnesium, causing processes of substitution of the central ion of the

tetrapyrrolic chlorophyll ring, inhibiting its function and decreasing its concentration [45]. Similar effects are caused by excessive copper concentrations. Photosynthetic pigments decrease might be the result of displacement of magnesium required for chlorophyll biosynthesis or ultra-structural alteration of chloroplast under metal toxicity [46]. Also, this reduction might be due to the inhibited activities of various enzymes associated with chlorophyll biosynthesis [47]. A similar effect was observed in the present work but only with statistical significance in copper-treated *C. indica* plants where a decrease in chlorophyll and carotene contents was observed with the increment of this metal. This can be associated with the smaller biomass and the increment of oxidative stress indicated by the increase of MDA contents found in the highest concentrations of copper. Similar diminution in chlorophyll and carotenes caused by copper excess was found in different species such as *Citrus aurantium* [48], *Phragmites australis* [49], *Lemna minor* [50], and *Camellia sinensis* [51].

Shoot-soluble protein content of *C. indica* plants decreased with the increase of copper concentrations concerning the control, whereas the opposite was found in the roots of the lowest zinc treatment. Similar results were found in *L. minor* [52] and *Hordeum vulgare* [53] treated with high concentrations of heavy metals. The decrease in the level of soluble proteins is another symptom characteristic of the stress caused by metals [54]. Proteins not only can act as metal chelators; they can also act in the movement toward the interior of the cell, for compartmentalization in vacuoles, as well as the exterior by an ion flow [55]. Therefore, the increase of the protein content observed in the zinc-treated *C. indica* roots might be due to a nutritional boost caused by the lowest zinc concentration. Also, biosynthesis of various biomolecules is another way to tolerate zinc excess; this process includes the induction of metallochaperones, proteins of low molecular weight, or chelators such as nicotianamine, putrescine, spermine, mugineic acid, organic acids, glutathione, phytochelatins, and specific metallothioneins, such as proline and histidine [56]. A similar increment was found in different poplar clones [57] and was associated with antioxidant enzymes synthesis during oxidative stress induced by heavy metals. On the contrary, in this work, shootsoluble protein content decreased in copper-treated *C. indica* plants. A similar reduction was found in *Brassica napus* growing on copper excess [58]. This decrease may be due to ROS generation. ROS are likely to target proteins that contain sulfur-containing amino acids and thiol groups [59]. Proteins can also be damaged in oxidative conditions by their reactions with lipid peroxidation products [60], and it can result in the deleterious effect of the normal protein form by disrupting the pathways and protein synthesis [61].

Proline is an amino acid that helps in activating many physiological and molecular responses in stress conditions. Its accumulation is a widespread response to heavy metal stress [62]. Shoot proline content con *C. indica* in this work showed a tendency to increase with the increment of both metal concentrations, whereas for roots only an increment in the first concentration of zinc treatment was observed. Proline accumulation increases the tolerance to heavy metals through several mechanisms, such as osmoregulation, stabilization of protein synthesis, and enzyme protection against denaturation [63]. It is suggested that proline accumulation is triggered by ROS, which allows their direct detoxification without the intervention of antioxidant enzymes [64]. Oxidative stress can lead to lipid peroxidation that produces a disruption at the cellular level, especially plasma membrane and leaking potassium from the plant cell; exogenous proline applications suppress the heavy metal induces [65]. Several authors found an increment in proline content in different species growing in excessive zinc [66–68] and copper [69–71] concentrations.
