## 4 Discussion
In fact, this particular wavelength has been shown to be the most responsive to different kinds of stress in diverse plant species [37]. Besides this specific wavelength, we also found that reflectance values based on wavelengths around 400 nm are also very selective, indicating a possible relation with water stress in particular. Albeit other spectroscopic techniques, such as Raman spectroscopy, are able to provide detailed information about chemical structure of materials, due to its simplicity, reflectance spectroscopy is generally use in biological materials [38]. Reflectance depends on leaf surface properties and internal structure and the concentration and distribution of biochemical components, and, in the visible domain, reflectance depends mainly on the presence of photosynthetic pigments and defense chromophores [38]. Water also deeply influences the reflectance spectra of leaves, but mostly in the shortwave infrared region of the electromagnetic spectrum ([39, 40] and references therein). Several studies indicate that leaf reflectance alteration by stress, at the 400-750 nm wavelength range, is related to changes in chlorophyll and anthocyanin concentrations [41, 42]. Photosynthetic pigments composition (chlorophyll and carotenoids) showed significant changes in drought stressed _Arabidopsis thaliana_ (Figure 2c). Also the absorption spectra of photosynthetic pigments and anthocyanins presented differences between S0 and S2 plants (Figure 2d). Therefore, in this work, the different reflectance bands that presented significant discriminant power may be attributed to different biological chromophores. The increase of reflectance at wavelengths around 670 nm and higher is probably due to the sharp decrease of of chlorophyll a and b absorbance in that spectral band (Figure 2c). [PERSON] et al. [43] reported maximum absorption peaks of Chl. a dissolved in diethyl ether of 660.8 and 429.0 nm (Soret), and 643.0 nm and 454.0 nm (Soret) for Chl.b. [PERSON] [28] reported maximum absorption peaks of Chl. a dissolved in ethanol (95%) at 664 and 431 nm (Soret) and 648 and 463 nm (Soret) for Ch. b. In our work, photosynthetic pigments were also dissolved in ethanol 95% (v/v) and the absorption peaks of total pigments extract were at 437 nm (Soret) and 665 nm (Figure 2d). However, in vivo chlorophylls are bound to an ensemble of different proteins to form the structurally complex photosystems, and the spectral characteristics of protein-bound chlorophyll molecules are modulated by interaction with their environment [44], presenting shifts on absorption peaks that may explain the effect we observed at 670 nm. Albeit in a previous work, with a similar experimental setup, we did not find a significant decrease in the chlorophyll content of drought stressed _Arabidopsis_ plants [27], in this work, where we extended the drought stress for 5 additional days, significant differences were found between S0 and S2 for both Chl. a and Chl. b (Figure 2c). Moreover, significant differences were also observed on the Chl.a/Chl.b and on the Chl.a+b/Cx+c ratios (Figure 2c) contributing to changes on the photosynthetic pigments' absorption spectra and, most probably, on the leaf reflectance spectra. The significant changes observed in the anthocyanins' absorption spectrum (Figure 2d) is in line with the massive increase of anthocyanins content previously reported [27]. Anthocyanins are a class of different non-photosynthetic water-soluble pigments derived from flavonoids via the shikimic acid pathway [17]. Their biosynthesis is elicited by a number of abiotic stresses, including drought stress [45]. They may function as a screen to increase photoproduction and as a pool of antioxidant metabolites [46]. In vivo, anthocyanins present strong absorption between 450 and 600 nm [47], suggesting that they may be responsible for the high frequency observed between 500 and 600 nm in Figure 7. In fact, our results show a significant increase on the anthocyanins absorption spectra, from S0 to S2, on that wavelength range (Figure 2d).