Seedling growth experiment

In June 2013, the germinated seeds were sown in plug trays (five seeds in each plug tray) and cultivated in an artificial climate chamber. After 22 days’ cultivation, the plug tray was changed to nutrition bag. One healthy seedling in each plug tray was transferred into 0.5 L pots with a substrate of 10:1 peat soil:perlite. During the seedling cultivation period, the nutrient solution was supplemented once every 10 d. The environment of the growth chamber was set as follows: temperature, 25/20 °C (12 h day/12 h night, the same below); humidity, 80%; and PPFD, 200 μmol· m⁻²·s^−1 35,36.

After five months of seedling cultivation, 20 C. lanceolata seedlings of similar size were selected and placed in four artificial climate chambers, corresponding to the four different day/night temperature gradients used in the seed germination experiment.

Eight seedlings from each population were randomly selected before the temperature treatments and subjected to testing to determine the total biomass of each plant and its distribution as well as the dry weight (W1). One month later, eight healthy seedlings were selected from each treatment and each population. Each of these seedlings was removed whole from the substrate and cleaned. Then, each seedling was divided into three parts: roots, stems and leaves. The fresh weight of each part was determined. The plant materials were dried at 80 °C until there was no further weight loss. The dry weight of each tissue was determined. Based on the aforementioned measurements, the total dry weight (W2) and its allocation (proportions of roots, stems and leaves of the total biomass of the plant), the root-shoot ratio, and the relative growth rate (RGR) were calculated.

The RGR is calculated using the following equation:

where W1 and W2 represent the biomass at the first sampling time and the second sampling time, respectively, and Δt represents the time interval between the first sampling time and the second sampling time. The RGR unit is in g·g⁻¹·d⁻¹. In addition to biomass, we also determined the chlorophyll content of the leaves, the photosynthetic efficiency of the leaves, and the chemical composition of the leaves. The chlorophyll in the leaves was extracted using the ethanol extraction method³⁷. The indexes for evaluating the photosynthetic efficiency of the leaves mainly include the maximum photosystem II (PS II) light energy conversion efficiency (variable fluorescence (Fv)/maximum fluorescence (Fm)), PS II electron transport (Fv/initial fluorescence (F₀)), and actual PS II photosynthetic efficiency (Y(II)). The required parameters were measured using a PAM-2500 portable pulse-amplitude modulation fluorometer (Walz, Germany). The indices for determining the chemical composition of the leaves include non-structural carbohydrates (i.e., soluble sugars and starch) and the C, N and P contents of the leaves. The soluble sugar and starch contents of each organ of each plant were determined using the anthrone-sulfuric acid colorimetric method³⁸. The C content of the leaves was determined using the potassium dichromate-concentrated sulfuric acid oxidation method with external heating³⁹. Prior to the determination of the N and P contents of the leaves, the leaves were subjected to a sulfuric acid-hydrogen peroxide digestion process. Afterward, the N content of the leaves was determined using a Kjeltec N analyzer (KDN-102F, Shanghai Xianjian Instruments CO., LTD), and the P content of the leaves was determined using an ultraviolet spectrophotometer³⁹.