2.2 Experimental and treatment conditions
The preculture as well as the drought, heat, and combined drought and
heat treatments, were carried out in three artificial climate chambers
(AI000, Canada).
- Preculture (June 5–12, 2019): The plants were acclimated to
the growth chamber for seven days before being subjected to the
treatments. Plant samples collected in the field were transplanted
into an experimental pot with an inner diameter of 8 cm and a height
of 10 cm. Then, the samples were placed into an artificial
intelligence climate box, where the daytime and night temperatures
were 18.5 ℃ and 4 ℃, respectively, and the air humidity was 60%; they
were watered once every 2 d, with the water quantity being 100% of
the moisture evaporation (ET) (the necessary water quantity was
weighed). The preculture lasted 7 d; then, in order to ensure a
survival rate, dead plants were removed, and 27 basins with plants of
the same size and similar growth rate were selected for each of the
four species, totaling 108 basins, to be used in the treatment
experiment.
- Stress treatment (June 13–July 13, 2019): Two drought
gradients (D1, D2), two heat gradients (H1, H2), two drought and heat
compound treatment gradients (D1H1, D2H2), and a control (CK)
treatment were established in the experiment(Table 1). Each treatment
was performed in triplicate.
CK, D1, D2: The day and night temperature were set at 18.5 °C
and 4 °C, respectively. The plants were watered every two days for 100%
ET. D1 and D2 were treated at the same temperature as CK and watered
once every two days (at 8:00 a.m. every day). The watering amount was
reduced to 50% ET for D1 and 20% ET for D2. A total of 36 samples (3
treatments × 4 species × 3 replicates) were placed in the same
incubator.H1, D1H1: The day and night temperatures were set at 21.5 °C
and 7 °C, respectively, in H1. The plants were watered every two days
for 100% ET. A total of 24 samples (2 treatments × 4 species × 3
replicates) were placed in the same incubator at the same time D1 was
processed.H2, D2H2: The day and night temperatures were set at 24.5 °C
and 10 °C, respectively, in H2. The plants were watered every two days
for 100% ET. A total of 24 samples (2 treatments × 4 species × 3
replicates) were placed in the same incubator at the same time D2 was
processed.