Litchi chinensis

Total RNA from the pericarps of litchi and tobacco was extracted and first strand cDNA was synthesized as described above. The transcription levels of both the litchi and tobacco anthocyanin biosynthetic genes were analyzed using quantitative real-time PCR (qRT-PCR),) with THUNDERBIRD qPCR Mix (TOYOBO, Japan) and ABI 7500 Real-Time PCR Systems (Applied Biosystems, USA) according to the manufacturers' instructions. The primers are shown in Table S1. Each reaction (20 μL final volume,) contained 9.96 μL 2×SYBR® qPCR Mix (TOYOBO), 0.04 μL 50×ROX reference dye, 1.0 μL of each the forward and reverse primers (0.25 μM), 2.0 μL of the cDNA template (corresponding to 50.0 ng of total RNA), and 7.0 μL of RNase-free water. The reaction mixtures were heated to 95°C for 30 s, followed by 40 cycles at 95°C for 10 s, 56°C for 15 s, and 72°C for 35 s. A melting curve was generated for each sample at the end of each run to ensure the purity of the amplified products. The specific qRT-PCR primers were designed using a Primer 5.0 program (PREMIER Biosoft International, Canada) (Table 2). Using these gene-specific primers, each assay amplified a single product of the correct size and demonstrated an acceptable PCR efficiency (approximately 90%). qRT-PCR reactions were normalized to the Ct values for LcActin (HQ615689) and NtACT (GQ281246) in litchi and tobacco, respectively. The relative expression levels of the target genes were calculated using the formula 2^−ΔΔCT[61] (link). All biological replicates were measured in triplicate.

Longitudinal face-to-face interviews were conducted by trained interviewers three times during the follow-up period: during the first trimester (≤12 weeks), the second trimester (13 to 27 weeks) and the last trimester (≥28 weeks) of gestation. Each subject was asked to complete a 3-day food record (2 weekdays and 1 weekend day), which was reported in a face-to-face interview on the first day and by telephone interview 2 days later. The participants were required to provide information on the types of food they had eaten each day and how much of each type they had eaten. A commonly used portion size was specified for each food type (e.g. a glass, a slice or a unit such as one apple or banana). To reduce measurement error, photographs of normal portions were provided to help the subjects to estimate and record their food consumption in the face-to-face interviews. The types and amounts of food were classified and coded based on the Chinese Food Composition Table40 . The average daily consumption of all fresh fruit items reported by each participant was summed to calculate the total consumption of fresh fruit during the second trimester of pregnancy.
Individual fruits were categorised into three groups based on their GI values: low was defined as GI ≤ 55, moderate as GI > 55 & <70 and high as GI ≥ 70. Because the consumption of fruit in the moderate- and high-GI groups was too small to be analysed separately, fruits with moderate and high GI values were merged into a single group, defined as GI > 5541 . Of the fruit consumed frequently by the participants, apples, pears, oranges, tangerines, grapefruits, peaches/nectarines, apricots, plums and strawberries were defined as low-GI fruit, and bananas, cantaloupes, melons, watermelons, pineapples and lychees were defined as moderate- and high-GI fruit41 . In addition, following Lai et al., the above fruit types were divided into six subgroups according to their polyphenol content using the Phenol-Explorer database. These subgroups comprised pome fruit, citrus fruit, berries, drupe fruit, gourd fruit and tropical fruit (Table 5)17 (link)32 (link). Tropical fruit was defined as fruit cultivated in the tropics, such as bananas, mangoes and persimmons.

After being air-dried, the serial litchi seed tissue sections were used for MALDI matrix coating (Figure 1C). A 2-MBT matrix solution was prepared at an optimal concentration of 15 mg/ml and dissolved in methanol/water/TFA (80:20:0.2, v/v/v). Air-dried tissue sections were coated with the 2-MBT matrix solution by a GET-Sprayer (III) (HIT Co., Ltd, Beijing, China). Briefly, the 2-MBT matrix solution 15 cycles (5 s spray, 10 s incubation, and 20 s drying time) was sprayed on the surface of the tissue sections to pre-seed a thin layer of the 2-MBT matrix. After the tissue sections were completely air-dried in a vented fume hood, the matrix solution was evenly sprayed for 50 more of the same cycles.

Fresh litchi fruit was collected from the Yongfuda litchi orchard (Haikou, Hainan, China) in June 2022. Haikou is located on Hainan Island in China. It has a typical tropical marine climate and annual sunshine duration of over 2,000 h. The climate is humid, the temperature rises fast, and the average annual precipitation is approximately 260 mm. The Yongfuda litchi orchard is located in a volcanic rock soil planting area. Once harvested, the peel and flesh of the litchi were immediately removed, and the litchi seeds were flash-frozen with liquid nitrogen by slow immersion to prevent seed shattering and endogenous compound changes. The commonly used MALDI matrix, 2-mercaptobenzothiazole (2-MBT), was obtained from Sigma-Aldrich (St. Louis, MO, USA). Amino acid and oligopeptide standards, including His, Gly-Gly-Leu (tripeptide), Ala-His-Lys (tripeptide), Leu-Leu-Tyr (tripeptide), and Arg-Gly-Asp-dTyr-Lys (pentapeptide), were purchased from Bankpeptide Biological Technology Co., Ltd. (Hefei, Anhui, China). Trifluoroacetic acid (TFA) and liquid chromatography–mass spectrometry (LC-MS)-grade methanol and ethanol were obtained from Merck & Co., Inc. (Darmstadt, Germany). Ultrapure water in the whole process of the experiments was prepared using a Millipore Milli-Q system (Bedford, MA, USA). All other reagents and chemicals were purchased from Merck, unless otherwise noted.