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.