*2.1. Spectral Data Measurements*

To record the spectral response of each plant, considering the different treatments, a darkroom was prepared to avoid light interference from other materials. The spectral response of the lettuce was measured using a Fieldspec HandHeld ASD spectroradiometer, operating at a spectral range of 325–1075 nm, in 512 channels with a spectral resolution of 1.6 nm and a 1◦ field of view. The equipment was carefully placed close to the leaves, at a 45◦ inclination, in relation to the height of the plant so that its field of view (FOV) did not exceed the area of the plant and register the spectral response of the substrate. A halogenic lamp was also placed at 45◦ on the other side. Before each measurement, the equipment was calibrated with a Lambertian (Spectralon® plate) surface plate.

The spectroradiometer registered 10 spectral curves during the same measurement for different leaves. This resulted in 360 spectral signatures for each measured day. These signatures represent the radiance from the leaves along the electromagnetic wavelength. Because the spectroradiometer records the radiance that reaches the equipment, we needed to transform it into the reflectance factor. For that, the leaf radiance was divided by a reference radiance, which corresponds with the Lambertian plate measured previously. The spectroradiometer also has a known calibration factor (K), which must be multiplied by the values of the described operation. This factor, together with the radiance values of the reference plate of the Lambertian surface, was used to estimate the bidirectional reflectance factor (BRF), as shown in Equation (1) [32].

$$BRF(\boldsymbol{\omega}\_{l}\boldsymbol{\omega}\_{r}) = \frac{dL\ (\boldsymbol{\theta}\_{r\prime}\boldsymbol{\Phi}\_{r})\ (\text{target})}{dL\ (\boldsymbol{\theta}\_{r\prime}\boldsymbol{\Phi}\_{r})\ (\text{reference})}\ \text{K}\ (\boldsymbol{\theta}\_{l\prime}\boldsymbol{\Phi}\_{l\prime}\boldsymbol{\theta}\_{r\prime}\boldsymbol{\Phi}\_{r})\tag{1}$$

where *dL* is the spectral radiance, ω is the solid angle, θ and Φ are in order, the zenith and azimuth angles, respectively; *i* is the incident flux, and *r* is the reflected energy flux. As mentioned, the *K* value is the correction factor from the equipment manufacturer. The BRF represents the spectral signature of the recorded radiometric target, also called the spectral response of the selected target.

To remove regions with a low signal-to-noise ratio, the spectral range from 380 to 1020 nm was selected to compose the spectral data, removing everything outside this range. The spectral curves were evaluated in terms of reflectance and absorbance values. Following Beer–Lambert's law, which shows that a concentration of an absorbent is proportional to the absorbance, the spectral reflectance values were converted using Equation (2).

$$A = \log\left(\frac{1}{R}\right) \tag{2}$$

where *A* corresponds to absorbance and *R* corresponds to the spectral reflectance obtained with the Fieldspec HandHeld ASD spectroradiometer.

## *2.2. Biophysical Data Measurements*

The leaf chlorophyll content (α + β) was recorded using a portable chlorophyllometer (Clorofilog Falker). The measurements were taken in the leaves of the apical part, median part, and basal part of each lettuce plant. This device operates in three spectral regions, in which the first two are in the red and red-edge regions and the third one in the near-infrared region [33]. The diameter of the leaves of each plant was also measured using a millimetric tape. The plants were then detached and weighed using a digital balance. At this stage, the aerial part (leaf and stem) was removed from the root and weighed separately, obtaining the fresh mass (g). The material was then left to dry in the open air for 48 h and weighed again to find its dry mass (g).
