Carotenoids

We used a semi-quantitative FFQ of 101 food items to assess the usual daily intake of foods and nutrients (available at: http://bibliodieta.umh.es/files/2011/07/CFA101.pdf). The FFQ was a modified version from a previous FFQ based on the Harvard questionnaire [15 (link)], which we developed and validated using four 1-week dietary records in an adult population in Valencia. The validity correlation coefficients (adjusted for energy intake) ranged from 0.27 for folate intake to 0.67 for calcium intake (average 0.47), and the reproducibility correlation coefficient s ranged from 0.30 for carotene intake to 0.65 for calcium intake (average 0.40) [16 ,17 (link)]; this is a similar range to other established diet questionnaires [3 ,4 (link)]. For the dietary assessment of pregnant women in the INMA cohort study, we added additional food items in the FFQ in order to capture the major sources of the most relevant nutrients, including specific carotenoids.
Participants in the study were asked twice during pregnancy how often, on average, they had consumed each food item over two periods of several months. The first period covered the time from the last menstruation to the first prenatal visit that occurred between the 10–13 weeks of pregnancy; the second period was the time between the first visit and the second one between weeks 28–32 of gestation. Serving sizes were specified for each food item in the FFQ. The questionnaire had nine possible responses, ranging from ‘never or less than once per month’ to ‘six or more per day’. Additionally, we asked whether study participants followed special diets.
Nutrient values were primarily obtained from the food composition tables of the US Department of Agriculture publications as well as other published sources for Spanish foods and portion sizes [18 ,19 ]. In order to obtain average daily nutrient intakes from diet for each individual, we multiplied the frequency of use for each food by the nutrient composition of the portion/serving size specified on the FFQ and added the results across all foods. For those nutrients often used in supplements during pregnancy such as folate, vitamin C and vitamin B12, the total daily nutrient intake was estimated by adding the average daily intake from supplements and the usual daily nutrient intake from the FFQ. In order to convert folic acid intake from supplements to dietary folate, we used the equivalence of 1 mcg of folate in the diet equals to 0.6 mcg of folic acid from supplements [20 (link)]. We estimated the mean daily consumption for 17 foods and food groups by grouping the intake of specific foods in the FFQ (Table 1).

The contents of chlorophyll, carotenoid, and anthocyanin of the QHP, ZSY, and L2025 leaves were determined based on a previously described method (Tian et al., 2021b (link)). Fresh leaves were used to measure total chlorophyll content based on the method described by Wu et al. (2020) (link). Leaf tissue (0.1 g) was ground into powder and extracted in 5 mL of 95% ethanol at 50°C for 2 h. The mixture was vortexed and centrifuged at 5,000 rpm for 5 min. The absorbance of the supernatant was measured at 470, 649, and 665 nm using an ultraviolet–visible spectrophotometer (Wu et al., 2020 (link); Zhuang et al., 2021b (link)). Fresh leaves were used to determine the carotenoid content in accordance with the method used by (Gao et al., 2021 (link)). Approximately 100 mg of fresh leaf tissue were cut into pieces with scissors, and extracted in 10 mL of 1% (v/v) HCl–ethanol at 60°C for 30 min. The mixture was vortexed and centrifuged at 13,000 × g for 5 min. The absorbance of the supernatant was measured at 530, 620, and 650 nm using an ultraviolet-visible spectrophotometer (Gao et al., 2021 (link)).

QHP with bright purple leaves, L2025 with green leaves, and ZSY with bright purple and green leaves were planted in the experimental field of Nanjing Botanical Garden, Memorial Sun Yat-Sen (32°3′N, 118°49′E). The fourth and fifth fully expanded mature leaves were collected from two-year-old seedlings of QHP (P) and L2025 (G), respectively. Fully expanded purple leaves (F_P) and green leaves (F_G) from two-year-old ZSY seedlings were also harvested. The leaf samples were immediately frozen in liquid nitrogen and stored at −80°C until use for the determination of chlorophyll, carotenoid, and anthocyanin contents, metabolite detection, RNA-seq, and qRT-PCR analyses. Three independent biological replicates were used for each experiment.

The well-stirred egg yolk (0.5 g) was added to acetone (10 ml), vortexed for 15 min and then centrifuged at 4000 rpm for 15 min. The absorbance value of the supernatant at 475 nm was determined by UV–visible spectrophotometer. Carotenoids content was calculated according to the following formula:
where A is absorbance value at 475 nm; V is the volume of acetone being added, ml; 0.16 is molar extinction coefficient of carotene; W is weight of the egg yolk used in the measurement process, g.