Integrated Metabolomic and Transcriptomic Analysis of Nitraria Berries Indicate the Role of Flavonoids in Adaptation to High Altitude

Abstract

Background: Plants of Nitraria, belonging to the Zygophyllaceae family, are not only widely distributed at an altitude of about 1000 m but also at an altitude of about 3000 m, which is a rare phenomenon. However, little is known about the effect of altitude on the accumulation of metabolites in plants of Nitraria. Furthermore, the mechanism of the high-altitude adaptation of Nitraria has yet to be fully elucidated. Methods: In this study, metabolomics and transcriptomics were used to investigate the differential accumulation of metabolites of Nitraria berries and the regulatory mechanisms in different altitudes. Results: As a result, the biosynthesis of flavonoids is the most significant metabolic pathway in the process of adaptation to high altitude, and 5 Cyanidins, 1 Pelargonidin, 3 Petunidins, 1 Peonidin, and 4 Delphinidins are highly accumulated in high-altitude Nitraria. The results of transcriptomics showed that the structural genes C4H (2), F3H, 4CL (2), DFR (2), UFGT (2), and FLS (2) were highly expressed in high-altitude Nitraria. A network metabolism map of flavonoids was constructed, and the accumulation of differential metabolites and the expression of structural genes were analyzed for correlation. Conclusions: In summary, this study preliminarily offers a new understanding of metabolic differences and regulation mechanisms in plants of Nitraria from different altitudes.

Keywords: Nitraria berries; altitude; flavonoids; metabolomic; transcriptomic.

Figure 1

The PCA score plot of metabolites in HNT, LNT, HNS, and LNS (a). The amount of up−regulated and down–regulated DAMs; (b) the results of DAM pathway analysis of Nitraria berries; and (c) the results of DAM pathway analysis of Nitraria berries at different altitudes. The color from yellow to red indicates that the smaller the p value, the larger the diameter of the circle, indicating that the number of metabolites enriched in the pathway is more. (a: Anthocyanin biosynthesis; b: Flavone and flavonol biosynthesis; c: Valine; leucine and isoleucine biosynthesis).

Figure 2

Content of 6 metabolites in NT and NS at different altitudes (* p < 0.05, ** p < 0.01): (a) HNT vs. LNT; (b) HNS vs. LNS.

Figure 3

The length distribution of de novo assembled unigenes: (a) Venn diagram of unigene annotation results from four databases; (b) Principal component analysis; and (c) volcano plots to filter the DEGs of berries in different altitudes groups of HNS vs. LNS (d) and HNT vs. LNT (e).

Figure 4

KEGG enrichment analysis and functional classification of DEGs in different altitudes groups: (A) KEGG primary classification results of DEGs; (B) secondary classification results of DEGs; and (C) tertiary classification results of DEGs enriched into pathways.

Figure 5

Flavonoid biosynthetic structural genes of DEGs in different altitudes groups. The log₁₀FC–transformed values of DEGs are indicated from blue to red (low to high). PAL: phenylalaninammo–ialyase; C4H: cinnamic acid–4–hydroxylase; 4CL: 4–Coumaric acid coenzyme ligase; CHS: chalcone synthase; CHI: chalcone isomerase; F3H: flavanone–3–hydroxylase; FLS: flavonol Synthase; F’3′5′H: flavonoid 3′; 5′–hydroxylase; FLS: flavonol Synthase; DFR: dihydroflavonol 4–reductase; F3′H: fla–vonoid 3′–monooxygenase; ANS: anthocyanidin synthase; FG2: flavonol–3–O–glucoside L–rhamnosyltransferase; UFGT: UDP–glycose flavonoid glycosyltransferase.

Figure 6

The number and species of differentially expressed transcription factors in HNS vs. LNS (a) and HNT vs. LNT (b). (c,d) Protein–Protein interaction analysis of regulatory factors with structural genes in HNS vs. LNS and in HNT vs. LNT; (e) correlation analysis of flavonoids and related transcription factors in HNS vs. LNS and (f) in HNT vs. LNT.