Plant growth and morpho-physiological assays
The experiments were carried out on Arabidopsis thaliana plants (cv Columbia 0) following the protocol proposed by Rizhsky et al. (2004) with some modifications. A professional potting soil (Orticole alveolo TecnoGrow, Tercomposti, Calvisano BS, Italy) was sterilised before the experiment (120 °C for 20 min). Before sowing, seed germination was synchronised by soaking the seeds in sterile water for 76 hours at 4 °C in dark conditions. Ten seeds were then sown per pot, and after germination, they were thinned, leaving one seedling per pot. A total of 24 pots (5 x 5 x 5 cm) were prepared for each treatment and replicate.
Seedlings were grown in a growth chamber under controlled conditions: 12 h light / 12 h dark photoperiod (long day), 21±1 °C, 100 µmol m-2 s-1, and relative humidity of 60-70%. Seedlings were fertilised every other day through sub-irrigation using a half-strength Hoagland solution and grown for 22 days [Arabidopsis growth stage 3.50, Rosette is ~ 50% final size (Boyes et al. 2001).
The following treatments were applied: C (Control watered plants), D (Drought stress), H (heat stress), and D+H (Drought + Heat stress). Drought treatment was achieved by blocking plant irrigation until they reached a relative water content (RWC) of 65 % to 70 % (typically 5–6 d). In contrast, heat stress was applied by raising the temperature gradually (~ 4 °C per hour) to avoid heat shock, in the growth chamber to 35 °C, and then keeping the plants at this temperature for 12 h.
At the end of the experiments, 1 g of the rhizosphere and root samples were collected for the microbiome analysis. Roots were carefully washed in sterilised distilled water and immediately snap-frozen in liquid nitrogen. Both soil and powdered roots were stored at -80 °C (Thijset al. 2017).
During the experiments, morphological and physiological parameters were monitored. In particular, the changes in biomass were evaluated at the end of the experiments on the fully developed rosette, measuring the changes in fresh weight (FW), dry weight (DW) and the DW/FW ratio. Another indicator of plant stress was the chlorophyll content, carried out at the end of the experiment using a chlorophyll meter SPAD (MC100, Qingdao Tlead International Co. Ltd). Finally, to have an indicator of plant water status and the plant’s ability to preserve it, the temperature of the leaves was monitored using a thermocamera (FLIR), and the leaf relative water content (% RWC) as previously reported by Araniti et al. , 2020.