Plant treatment
The plants used for the cadmium stress experiments were grown in the
greenhouse of Shandong University, Qingdao, China (36°23′10′′ N,
120°36′44′′ E). The greenhouse equipped with glass ceilings receives
natural light, with light intensity similar with natural conditions and
a photoperiod of 12-14 hours of daylight from May to October. The
average daily temperature was maintained at 30.2℃ and the air humidity
around 85% by ventilating fans. The same greenhouse settings were
maintained throughout the experimental period to guarantee controlled
and constant growth conditions. First of all, rhizomes of two
genotypes Heze and Panjin of P. australis were collected from the
common garden in May, 2022. After one week in the water, the rhizomes
were cut into pieces of similar lengths with buds and placed in the
pots. Each rhizome piece was planted in a pot with mixed vermiculite and
nutrient soil (in a volumn ratio of 2:1) as the substrate. The pots had
a top diameter of 16.0 cm, a bottom diameter of 13.0 cm, and a height of
17.5 cm. The water saturated soil weight was about 1.25 kg in each pot,
and the dry weight was about 0.35 kg. Three clones grown from the
rhizomes of the same genotype were used as independent biological
replicates (in separate pots) for each treatment. The concentration of
the Cd solution was made of 1.61g CdCl2 per liter of
water, i.e., 1mg mL-1 Cd. A survey of spatial and
temporal distribution of Cd pollution in farmland soil showed that the
farmland soil Cd concentrations ranged from 0.012 to 23.33
mg·kg-1 in China, with the geometric mean of 0.473
mg·kg-1 (J. Wang, Wei, & Pan, 2023). According to the
control standards of pollutants in sludge for agricultural use in China
issued in 2018 (GB4284-2018), the total cadmium content below 3 mg
kg-1 can be classified as Class A pollutants, while
the total cadmium content below 15 mg kg-1 can be
classified as Class B pollutants. Class A sludge products can be used
for farmland, gardens, and pasture, whereas Class B sludge products can
be used for gardens, pasture, and non-food crop farmland. Therefore, we
set three Cd treatment levels in this study: 0mg
kg-1(the control, C), 4mg
kg-1 (the low Cd treatment, L), and 20mg
kg-1(the high Cd treatment, H). After two-month
cultivation of the buds, we will treat these plants with
CdCl2 solution. After one-month treatment, the fresh
leaves of each individual were collected for RNA extraction, and the
cultivars were used for functional traits measurement.