Plant Measurements

All measured plant traits along with abbreviations and the device used for measuring are shown in Table 1.

Plants were scanned using a PlantEye F500 multispectral 3D scanner (Phenospex, Heerlen, Netherlands). PlantEye measures the spectral reflectance in Red (peak wavelength 620–645 nm), Green (peak wavelength 530–540 nm), Blue (peak wavelength 460–485 nm), Near-Infrared (peak wavelength 820–850 nm), and the 3D laser (940 nm) of the plant. Resolution of the PlantEye was set up as follows: Z-range (the distance measured from the scanner down) 40 cm, Y-resolution (Vscan = 50 mm s^–1) 1 mm, X-resolution 0.19 mm, and Z-resolution < 0.1 mm. Calculation of vegetation indices and morphological parameters starts from the 3D point cloud from which the 3D plant model is built by integrated Phena software (Phenospex, Heerlen, Netherlands). All points that belong to the same sector are triangulated. Triangles were created by connecting adjacent points. Different vegetation indices and morphological parameters were calculated using HortControl software (Phenospex, Heerlen, Netherlands).

Calculated vegetation indices from 3D plant model were: HUE_3D, calculated in the same way as described above, but using 3D plant model, Greenness index (GI) (2 × R_Green – R_Red – R_Blue)/(R_Green + R_Red + R_Blue), normalized difference vegetation index (NDVI) (R_NIR – R_Red)/(R_NIR + R_Red) (Rouse et al., 1974), normalized pigments chlorophyll ratio index (NPCI) (R_Red – R_Blue)/(R_Red + R_Blue) (Peñuelas et al., 1995), and plant senescence reflectance index (PSRI) PSRI = (R_Red - R_Green)/(R_NIR) (Merzlyak et al., 1999).

Calculated morphological parameters from 3D plant model were: plant height (PH; mm) calculated as distribution of elementary triangles along the z-axis; leaf area projected (LAP; cm²) calculated as an area of the projection of all elementary triangles on X–Y plane; total leaf area (TLA; cm²) calculated as the sum of all triangle domains, where each domain represents a group of triangles that form a uniform surface; digital biomass (DB; cm³) calculated as the product of the height and 3D leaf area; leaf area index (LAI, mm² mm^–2) calculated as TLA/sector size; leaf inclination (LINC; mm² mm^–2) which describes how leaves on the plant are erected and calculated as TLA/LAP; leaf angle [LANG; degree (°)]; and light penetration depth (LPD; mm) measured by the deepest point in which the laser can penetrate the canopy along the z-axis.

Chlorophyll fluorescence and multispectral imaging were performed using the CropReporter^TM (PhenoVation B.V., Wageningen, Netherlands). The CropReporter^TM consists of a cabinet with a camera system that houses controller computer, charge-coupled device (CCD) camera with optical filter wheel and focusing unit, integrated high-intensity red light-emitting diodes (LEDs) for excitation of the photosynthesis, LEDs at six spectral bands [broadband white (3000 K), far-red (730 nm), red (660 nm), green (520 nm), blue (460 nm), and UV/blue (405 nm)], controllable in intensity (0–780 μmol m^–2 s^–1), and spectrum for spectral imaging. All images are captured with the same lens (10 Mp lens, 200 Lp mm^–1 resolution, 400–1000 nm spectral range) and CCD camera (1.3 Mp, 1296 × 966 pixels), with real 14-bit signal resolution. Plants were imaged at 80 cm distance from the camera. The output is 16-bit RAW format, and automatic analysis of chlorophyll fluorescence, color, and multispectral images was performed by DA^TM software (PhenoVation B.V., Wageningen, Netherlands).

Plants were imaged with the optimized quenching protocol or dark-to-light slow fluorescence induction (Brestic and Zivcak, 2013), which includes dark adaptation, measurement of the induction curve of the dark-adapted plant followed by actinic light switching on for light adaptation, and measurement of induction curve of light-adapted plants.

For chlorophyll fluorescence measurements of dark-adapted plants (overnight dark adaptation), saturating light pulse (4500 μmol m^–2 s^–1 for an 800 ms) was used. Minimum chlorophyll fluorescence (F₀) was measured after 20 μs, and maximum chlorophyll fluorescence (F_m) was measured after saturation. Four dark frames were captured and averaged to one single frame during the time red LEDs were off; 20 frames were captured for the induction curve during 800 ms; integration time for capturing the chlorophyll fluorescence images was 200 μs.

Following the measurement of dark-adapted plants, plants were relaxed in the dark for 15 s, and then actinic lights (300 μmol m^–2 s^–1) were switched on enabling plants to adapt to light for 5 min. Steady-state fluorescence yield (F_s’) was measured at the onset of the saturating pulse, and maximum chlorophyll fluorescence (F_m’) of light-adapted plants was measured at saturation, using the saturating pulse intensity (4500 μmol m^–2 s^–1). Again, four dark frames were captured and averaged to one single frame during the time red LEDs were off; 20 frames were captured for the induction curve during 800 ms; integration time for capturing the chlorophyll fluorescence images was 200 μs.

Measured F₀, F_m, F_m’, and F_s’ were used for calculation of the following fluorescence parameters:

The maximum quantum yield of PSII (F_v/F_m): F_v/F_m = (F_m − F₀)/F_m (Genty et al., 1989)

Effective quantum yield of PSII (F_q’/F_m’): F_q’/F_m’ = (F_m’ − F_s’)/F_m’ (Genty et al., 1989)

Electron transport rate (ETR) = F_q’/F_m’ × PPFD × (0.5) (Genty et al., 1989)

Non-photochemical quenching (NPQ) = (F_m − F_m’)/F_m’ (Bilger and Björkman, 1990).

After chlorophyll fluorescence imaging, color and spectral reflectance (R) images were captured at 300 μmol m^–2 s^–1 produced by broadband white LEDs. Reflectance images were captured at R_Red—640 nm, R_Green—550 nm, R_Blue—475 nm, R_Chlorophyll (R_Chl)—730 nm, R_Anthocyanin (R_Anth)—540 nm, R_NIR—769 nm, and R_FarRed—710 nm. During imaging, spectral ratio (R_Anth: R_FarRed: R_NIR) and color ratio (R_Red: R_Green: R_Blue) were kept constant.

From reflectance images, chlorophyll index (CHI) and anthocyanin index (ARI) were calculated using the following equations: CHI = (R_Chl)^–1 − (R_NIR)^–1 (Gitelson et al., 2003), and ARI = (R_Anth)^–1 − (R_FarRed)^–1 (Gitelson et al., 2001). Hue, saturation, and value were calculated after converting R_Red, R_Green, and R_Blue into values between 0 and 1.

Hue (0–360°) was calculated as follows:

HUE = 60 × [0 + (R_Green − R_Blue)/(max − min)], if max = R_Red;

HUE = 60 × [2 + (R_Blue − R_Red)/(max − min)], if max = R_Green;

HUE = 60 × [4 + (R_Red − R_Green)/(max − min)], if max = R_Blue.

360 was added in case of HUE < 0.

Value (0–1) was calculated as: VAL = (max + min)/2, while max and min were selected from the R_Red, R_Green, R_Blue. Saturation (0–1) was calculated as: SAT = (max – min)/(max + min) if VAL > 0.5, or SAT = (max – min)/(2.0 – max – min) if VAL < 0.5, while max and min were selected from the R_Red, R_Green, R_Blue.