**4.3 Correlation between physiological and biochemical parameters and Zn(II) and Cu(II) bioaccumulation: indicators for different applications**

Some associations between physiological and biochemical parameters and the exposition of metals can be estimated by Pearson's correlation coefficient (r). In this work, *C. indica* plants showed a significant negative correlation for shoot (*r* = 0.74) and root dry weight (r = 0.8) in Zn(II) treatments and shoot dry weight (r = 0.67), chlorophyll (*r* = 0.61) and protein (*r* = 0.58) content in Cu(II) treatments showing that when the concentration of this metals increases, these parameters are affected negatively. The opposite occurred for shoot MDA (*r* = 0.53) and proline (*r* = 0.6) content and root-relative conductivity (*r* = 0.63) in Zn(II) treatments and shoot proline content (*r* = 0.66) and roots-relative conductivity (*r* = 0.93) in Cu(II) treatments. Proline accumulation in shoots, relative conductivity increment in roots, and

the diminution of dry weight could be useful indicators of the strategies of this plant to overcome heavy metal stress and could be used to monitor the phytoremediation process.

The analysis of the correlation between metal accumulation and physiological parameters could be useful in different areas, such as variety selection, genetic improvement, environmental monitoring, or index construction as an indirect indicator of the phytoremediation process [88]. Various studies have demonstrated the correlation between metal accumulation and the antioxidant system. Antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), show an increased production to protect the plant from the damage caused by reactive oxygen species (ROS) under metals exposure [89]. Also, malondialdehyde (MDA) could act as an indicator of lipid peroxidation and is usually related to assessing oxidative damage [28]. Lipid peroxidation and oxidative damage cause alterations in metabolic processes [90] such as photosynthesis or protein productions leading to a decrease of photosynthetic pigments, less CO2 assimilation, and diminution of biomass [91]. On the other hand, the accumulation of metabolites is another mechanism that plants use for stress tolerance. Proline is an amino acid that is involved in different stress mechanisms; it performs functions such as osmoregulation, stabilization of protein, and enzyme synthesis or even can chelate metal ions to help in the vacuolar sequestration [92]. These correlations are another way to demonstrate the tolerance mechanisms, and it helps to create comparations between species from the same genus or different cultivars to select the best for specific phytoremediation techniques becoming these, indicators of phytoremediation efficiency parallel to heavy metal accumulation [93].

Another use of these correlations is the construction of biomarkers. These represent the biological response to environmental disturbances or contamination, and they allow the detection of pollution at different contamination levels corresponding to concentrations difficult to achieve or when yield is not easy to form an integrative sample. There are three types of biomarkers: biomarkers of exposure: such as DNA breaks, stress proteins, and phytochelatins; biomarkers of effects such as morphological and physiological parameters; and biomarkers of susceptibility such as genetic mutations [94]. The use of such tools is currently increasing in the field of biomonitoring and bioremediation. Some biomarkers that have already been reported in plants are the following: oxidative stress by the production of reactive oxygen species [95], the reduction of macromorphological parameters such as plant height, stem diameter, and the number of leaves and negative modifications in chloroplasts with implications in photosynthesis [96]. These have been useful biomarkers for showing the adverse effects of metal exposition on the development, growth, and physiology of different plants exposed to this type of stress [97, 98].
