Elsevier

Scientia Horticulturae

Volume 303, 20 September 2022, 111220
Scientia Horticulturae

Transcriptomic analysis reveals key genes regulating organic acid synthesis and accumulation in the pulp of Litchi chinensis Sonn. cv. Feizixiao

https://doi.org/10.1016/j.scienta.2022.111220Get rights and content

Highlights

  • Determining the kinds of the organic acids in the pulp of Feizixiao litchi and observing the dynamic change characteristics of the different organic acids content.

  • Identifing the key biochemical pathways for the metabolism of the organic acids in the pulp of Feizixiao litchi through RNA-seq.

  • Confirming the key genes regulating the organic acids accumulation in the pulp of Feizixiao litchi and their expressions characteristics through RNA-seq.

  • Verifing the expression of the above key genes through real-time fluorescence quantitative PCR.

Abstract

To investigate the gene expression characteristics and patterns of organic acid synthesis and accumulation in the pulp of Litchi chinensis Sonn. cv. Feizixiao (FZX), 16-year-old Feizixiao litchi trees were used as the experimental material, and the dynamic changes in the main organic acids in the pulp were determined for two years. RNA sequencing was carried out when the total acids in the pulp changed drastically 35, 50 and 73 d after anthesis in 2020 to discover the molecular mechanism of the synthesis and accumulation of different organic acids in pulp. The results indicate that tartaric acid and malic acid were the main organic acids in pulp. The RNA-seq contained 165,886 transcripts, and 12 176, 19 835 and 5 048 DEGs were observed in the 35 d vs. 50 d, 35 d vs. 73 d and 50 d vs. 73 d groups, respectively. These expressed transcripts of organic acid metabolism were significantly enriched in correlated pathways, including glycolysis and gluconeogenesis, citric acid cycle (TCA cycle), and ascorbate and aldarate metabolism. In this paper, 75 genes of the enzymes related to organic acid metabolic pathways were screened, and these enzymes included PEPC, MDH, ME, CS, IDH and ACO. Ten unigenes were selected to confirm by real-time PCR, and the linear regression between the RNA-Seq results and real-time PCR data was significant (r = 0.8637). In addition, the expression of PEPC and ACO genes was consistent with the content of citric acid, the expression of the CS gene increased with time, and the change in expression of the MDH gene was similar to that of the malic acid content, while the expression of the ME gene showed an opposite trend. Therefore, it was possible to regulate acid accumulation in pulp by regulating the expression of key genes related to pathways such as the TCA cycle, and then the comprehensive fruit quality was regulated.

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.

References (51)

  • P. Saradhuldhat et al.

    Pineapple organic acid metabolism and accumulation during fruit development

    Scientia Horticulturae

    (2007)
  • C. Sweetman et al.

    Regulation of malate metabolism in grape berry and other developing fruits

    Phytochemistry

    (2009)
  • Z.C. Wu et al.

    Methyl-inositol, γ-aminobutyric acid and other health benefit compounds in the aril of Litchi

    Int. J. Food Sci. Nutr.

    (2016)
  • Y. Bai et al.

    A Co-expression gene network associated with developmental regulation of apple fruit acidity

    Molecular Genetics & Genomics

    (2015)
  • F.X. Chen et al.

    Advances in research on organic acid metabolism in fruits

    J. Fruit Ence

    (2005)
  • M. Chen et al.

    Effect of hot air treatment on organic acid- and sugar-metabolism in Ponkan (Citrus Reticulata) fruit

    Scientia Horticulturae

    (2012)
  • T.H. Claus et al.

    Glucagon and Gluconeogenesis; Glucagon and Gluconeogenesis

    (1983)
  • F. Crecelius et al.

    Malate metabolism and reactions of oxidoreduction in cold-hardened winter rye (Secale Cereale L.) Leaves

    J. Exp. Bot.

    (2003)
  • Dinari, M.; Nabiyan, A. Citric acid-modified layered double hydroxides as a green reinforcing agent for improving...
  • D.-.G. Hu et al.

    The R2R3-MYB transcription factor MdMYB73 is involved in malate accumulation and vacuolar acidification in apple

    The Plant J.

    (2017)
  • Huang, H. Fruit set, development and maturation: A. Litchi. 2005....
  • T. Ishikawa et al.

    Progress in manipulating ascorbic acid biosynthesis and accumulation in plants

    Physiologia Plantarum

    (2006)
  • Kou, X.; Wang, S.; Zhang, Y.; Guo, R.; Wu, M.; Chen, Q.; Xue, Z. Effects of chitosan and calcium chloride treatments on...
  • B. Lai et al.

    Characterization of a novel litchi R2R3-MYB transcription factor that involves in anthocyanin biosynthesis and tissue acidification

    BMC Plant Biol.

    (2019)
  • K. Lama et al.

    Tissue-specific organic acid metabolism in reproductive and non-reproductive parts of the fig fruit is partially induced by pollination

    Physiologia Plantarum

    (2020)
  • Foundation items: National Natural Science Foundation of China (NSFC) (No. 31,960,570); World First-class Discipline Construction Fund of Hainan University (No. RZZX201906).

    View full text