*4.3. Plant Analysis*

Phenological analysis [35] was performed through the recording of different developmental stages for a period of 100 days after sowing, considered as day 0 (Table 1). A total of 10 plants were monitored for each growth condition.

### **Table 1.** *A. thaliana* growth stages recorded in this work.


For morphological measurements, a total of 10 *A. thaliana* plants for each growth condition were sampled after 33 days from sowing. The leaves (complete rosette) of the plants were scanned at 800 dpi with the Epson Expression 12000XL instrument and then oven-dried at 70 ◦C until constant weight. Plant roots were freed from soil media by carefully washing them under running water and subsequently oven-dried until constant weight. The dry organs were then weighed on an analytical balance (Orma AL220S) and the shoot-to-root ratio (S/R) was calculated. The scanned images were processed with WinRhizo (Regent Instrument) to measure the projected rosette area (PRA) and with ImageJ (NIH, USA) to measure the rosette diameter (RD) as well as lamina and petiole length of three leaves for each plant. The lamina-to-petiole ratio (L/P) was then calculated.

Physiological measurements were conducted as follows on 10 different plants for each growth condition.

(A) Chlorophyll fluorescence analysis was performed with a modulated chlorophyll fluorometer (OS1-FL; Opti-Sciences) after 35 days from sowing. The maximum quantum efficiency of photosystem II (PSII) photochemistry (*Fv*/*Fm*) was measured pre-dawn, while the PSII operating efficiency (ΦPSII) was measured after at least 3 h of plant exposure to light. Non-photochemical quenching (NPQ) was subsequently calculated using Fm and Fm' values [33,36].

(B) The leaf pigment content was measured on the upper face of completely expanded young leaves with the Dualex Scientific Instrument (Force-A) 36 days after sowing. The concentrations of chlorophyll, anthocyanins, and flavonoids are reported by the instrument in µg/cm<sup>2</sup> .

(C) The Ciras 2 instrument (PP Systems) was used to measure the net photosynthetic rate (Pn), the stomatal conductance (Gs), and the evapotranspiration rate (ET) 48 days after sowing. The single leaf gas exchange measurements were taken under ambient light with the 25 × 7 mm leaf cuvette oriented perpendicularly to the light source. At least three measurements for each leaf were taken on completely expanded young leaves. For leaves smaller than 25 × 7 mm, digital pictures were made to determine the projected leaf area inside the cuvette and properly scale the measurement [37].
