Transcriptome analysis associated with polysaccharide synthesis and their antioxidant activity in Cyclocarya paliurus leaves of different developmental stages

Abstract

Background: Cyclocarya paliurus (Batal.) Iljinskaja is a common endemic tree species and used as a Chinese medicine. The main active components in the leaves of this plant are polysaccharides. However, the temporal patterns of gene expression underlying the synthesis of polysaccharides in C. paliurus at different leaf developmental stages and its relationship with the polysaccharide content and antioxidant activities has not been reported to date.

Methods: RNA-seq was used to investigate the biosynthesis pathway of polysaccharides at the four developmental stages of C. paliurus leaves. The content and the antioxidant activities of polysaccharides were measured with typical biochemical methods and the identified correlations were statistically evaluated.

Results: Sixty-nine differentially expressed genes were found in the leaves during different developmental stages of C. paliurus. These are associated with glycosyltransferases and belong to 18 families. During different developmental stages of C. paliurus, the polysaccharide content first increased and then decreased, and the UDP-glucose 4-epimerase gene was found to be significantly positively correlated with the polysaccharide content. The clearance rates of DPPH radicals, superoxide anion radicals, hydroxyl radicals, and the reducing power of polysaccharides in the leaves of C. paliurus at different developmental stages showed a dose-dependent relationship with the concentration of polysaccharides.

Conclusions: The smallest fully expanded leaves are suitable for high-quality tea, and leaves with sizes below the largest fully expanded leaves are suitable for industrial production of polysaccharides.

Keywords: Antioxidant activity; Cyclocarya paliurus; Industrial production; Polysaccharide biosynthesis.

Figure 1. Classification and number of carbohydrate-active enzyme families in Cyclocarya paliurus unigenes.

GT, glycosyltransferase; GH, glycoside hydrolase; CE, carbohydrate esterase; CBM, carbohydrate-binding module; PL, polysaccharide lyase.

Figure 2. Heat map of differentially expressed glycosyltransferase genes at four different developmental stages (i.e., F1, F2, F3, and F4) of Cyclocarya paliurus leaves.

The relative expression levels of genes are highlighted with different colors from blue (low) to red (high) across the F1, F2, F3, and F4 developmental stages.

Figure 3. Real-time PCR validation of candidate unigenes involved in Cyclocarya paliurus polysaccharide biosynthesis by RNA-seq.

(A–J) Respectively indicate real-time PCR validation of different genes. The histograms show the relative gene expression levels obtained via real-time PCR. The transcripts per million (TPM) of each million mapped fragment of the transcriptome are represented by line graphs. The right y-axis indicates gene expression levels calculated as TPM. The left y-axis indicates relative gene expression levels obtained by real time PCR.

Figure 4. Putative biosynthesis pathway map and unigene expression pattern of Cyclocarya paliurus polysaccharide.

(A) The pathway of C. paliurus polysaccharide biosynthesis was constructed based on KEGG analysis. The full names of enzymes (with EC number) are provided in Table S1. The red font represents activated monosaccharides. The text formatted in blue font represents detected metabolites. (B)–(C) The bar graph shows the contents of these two metabolites across the F1, F2, F3, and F4 developmental stages. (D) The average expression level of the enzyme-encoding unigenes at different leaf stages is indicated with a heat map. The relative expression levels of genes are highlighted with different colors from blue (low) to red (high) across the F1, F2, F3, and F4 developmental stages.

Figure 5. Polysaccharide content in leaves of Cyclocarya paliurus at four developmental stages (F1, F2, F3, and F4).

Figure 6. Antioxidant activities of Cyclocarya paliurus polysaccharides in vitro.

(A) Scavenging activity of DPPH radicals; (B) scavenging activity of superoxide radicals; (C) scavenging activity of hydroxyl radicals; (D) reducing power.