Transcriptomic analysis reveals key genes regulating organic acid synthesis and accumulation in the pulp of Litchi chinensis Sonn. cv. Feizixiao✰
Introduction
Litchi (Litchi chinensis Sonn.) is an evergreen tree belonging to the Sapindaceae family, bearing delicious fruit and known as the fruit king of Lingnan (Lora et al., 2018). Among litchi cultivars, L. Chinensis Sonn. CV. Feizixiao (FZX) is one of the main cultivars in Chinese litchi-producing areas, including Hainan Province (Wang et al., 2017). Litchi's flavor mainly depends on the composition and concentration of sugar and acid in pulp (Liu et al., 2016). Organic acids are an important component of fruit flavor quality, and they affect the overall fruit quality by influencing fruit flavor, taste, nutrition and health care value (Lama et al., 2020). Therefore, clarifying the mechanism of accumulation and metabolism of organic acids could regulate the flavor quality of litchi fruits.
In recent years, the accumulation and metabolism of organic acids during the growth and development of different fruit species have been largely reported. It has been proven that organic acid components in litchi fruits mainly include malic acid, succinic acid, tartaric acid, citric acid (Wang et al., 2006a), lactic acid, acetic acid, oxalic acid, fumaric acid and pyruvate acid (Huang, 2005), and malic acid and citric acid are the most abundant organic acids in litchi fruits (Chen et al., 2005). However, succinic acid and malic acid are dominant in litchi pulp (Paull et al., 1984). Furthermore, based on the mass fraction of monomeric organic acids, tartaric acid and malic acid are the main organic acids in the fruits of litchi varieties (Wang et al., 2005). There have been no consistent conclusions on the composition of organic acids in litchi pulp.
Organic acid metabolism enzymes have a strong relationship with organic acid content in fruits. Enzymes related to organic acid metabolism include phosphoenolpyruvate carboxylase (PEPC) (Berüter, 2004), malate dehydrogenase (MDH) (Crecelius et al., 2003), malic enzyme (ME) (Maldonado et al., 2004), malate synthetase (Shukla et al., 2020), citrate synthase (CS) (Park et al., 2021), aconitase (ACO) (Jiang et al., 2014), isocitrate dehydrogenase (IDH) (Jiang et al., 2014) and isocitrate lyase (ICL) (Krieger et al., 2012). PEPC is a key enzyme in the synthesis of organic acids in litchi, while NAD-MDH and NADP-ME also affect the content of organic acids (Wen, 2012). In loquat fruits, the activity of NAD-MDH, NADP-ME and PEPC may play important roles in the biosynthesis and degradation of malic acid (Chen et al., 2009). In citrus, the main organic acid is citric acid (Dinari and Nabiyan, 2016), and PEPC, CS, ACO, and IDH are key enzymes for citric acid metabolism (Saradhuldhat, 2005). MS and ICL are key enzymes in the glyoxalic acid cycle (Mclaughlin and Smith, 1994), which is a complementary pathway of the tricarboxylic acid cycle and plays an important role in the accumulation of citric acid and malic acid.
In addition, a large number of genes encoding enzymes related to organic acids have been reported to affect the acidity of fruit. Genes encoding phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate carboxy kinase (PEPCK), NAD-malate dehydrogenase (NAD-MDH), NAD-isocitrate dehydrogenase (NAD-IDH), glutamine synthetase (GS), and fructose-1,6-bisphosphatase (FBPase) play an important role in the acid synthesis and degradation of ‘Huapi’ kumquat (Wei et al., 2021). Glutamate decarboxylase (GAD)-related genes affect the metabolism of citric acid (Liu et al., 2014). The NAD-MDH1 gene controls the synthesis of malic acid, while pyruvate dehydrogenase kinase (PDK), pyruvate kinase (PK) and alcohol dehydrogenase (ADH) affect the synthesis of citric acid through the pyruvate-to-acetyl-CoA-to-citrate pathway (Zheng et al., 2020). Moreover, several genes related to vacuolar organic acid transport were found to play a crucial role in determining the acidity of fruit. For example, SL-ALMT9, which encodes an aluminum-activated malic acid transporter, controlled tomato acidity (Ye et al., 2017). In apples, malic acid accumulation is regulated by MdBT2-MdMYB73 (Zhang et al., al.,2020), and MdMYB73 regulates malic acid accumulation by directly binding to the promoters of MdVHA-A, MdVHP1 and MdALMT9 (Hu et al., 2017). Ma1, one of two aluminum (Al)-activated malic acid transporter (ALMT) genes, was correlated with malic acid concentration (Bai et al., 2015). Previous researchers also identified a gene belonging to the ALMT family that specifically contributed to the release of malic acid (Zhou et al., 2020).
In general, organic acid compositions vary considerably between different fruit species, even between cultivars of the same species, and the key enzymes affecting the accumulation of organic acids are also diverse. At present, studies on the molecular and physiological mechanisms of organic acids in FZX are still being conducted. In the present study, investigations that used the fruit of FZX from Hainan Province as experimental material were conducted to determine the composition of organic acids and perform transcriptome analysis. The aim of this study was to discover key genes regulating the synthesis and accumulation of different organic acids in the pulp of FZX and to provide a necessary theoretical basis to explore measures to regulate the acid content in fruit.
Section snippets
Plant materials
The experimental site was located in Litchi Orchard No. 5 at Team of Jinpai Farm, Lingao County, Hainan Province, with terrain of the Qiongbei platform and a tropical monsoon climate. The annual average temperature was 23–24 °C, and it had an annual average sunshine duration of 2175 h and an annual average rainfall of 1100–1800 mm; moreover, rain and heat belong to the same season. The soil was fertile latosol. Five 16-year-old Feizixiao litchi trees having the same growth potential and without
Change in total acid content
As shown in Fig. 1, the content of total acid in 2019 increased from 35 d to 43 d after anthesis, which may be the process of carbohydrate transformation into organic acid accumulation. The change trend from 43 d to 77 d after anthesis was consistent with that in 2020, which decreased first and then tended to be gentle. The peak value in 2019 appeared at 43 d, and the content range was 0.23–5.79%. The highest value in 2020 was 42 d, and the content range was 0.64–4.43%. The highest and lowest
Compositions and content changes of organic acids in FZX litchi pulp
Organic acid compositions and contents are important factors of fruit quality in FZX litchi pulp and play an important role in the nutritional quality and metabolism of fruit (Zhang et al., 2021). Wu et al. reported that total acid showed a decreasing trend from the beginning of rapid growth of litchi to full maturity (Wu et al., 2016). Previous studies have reported that the main components of organic acids in FZX pulp were malic acid and tartaric acid, and the former was 2.6 times that of the
Conclusion
In summary, tartaric acid and malic acid were the major organic acids in FZX litchi pulp, in which the malic acid content showed a 'downward gentle' trend and that of tartaric acid showed an 'upward and then downward' trend. The expressed transcripts of organic acid metabolism were significantly enriched in correlated pathways that were glycolysis and gluconeogenesis, citric acid cycle (TCA cycle), ascorbate and aldarate metabolism, glyoxylate and dicarboxylate metabolism, and carbon fixation
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Foundation items: National Natural Science Foundation of China (NSFC) (No. 31,960,570); World First-class Discipline Construction Fund of Hainan University (No. RZZX201906).