Metabolome and transcriptome analyses reveal chlorophyll and anthocyanin metabolism pathway associated with cucumber fruit skin color

Abstract

Background: Fruit skin color play important role in commercial value of cucumber, which is mainly determined by the content and composition of chlorophyll and anthocyanins. Therefore, understanding the related genes and metabolomics involved in composition of fruit skin color is essential for cucumber quality and commodity value.

Results: The results showed that chlorophyll a, chlorophyll b and carotenoid content in fruit skin were higher in Lv (dark green skin) than Bai (light green skin) on fruit skin. Cytological observation showed more chloroplast existed in fruit skin cells of Lv. A total of 162 significantly different metabolites were found between the fruit skin of the two genotypes by metabolome analysis, including 40 flavones, 9 flavanones, 8 flavonols, 6 anthocyanins, and other compounds. Crucial anthocyanins and flavonols for fruit skin color, were detected significantly decreased in fruit skin of Bai compared with Lv. By RNA-seq assay, 4516 differentially expressed genes (DEGs) were identified between two cultivars. Further analyses suggested that low expression level of chlorophyll biosynthetic genes, such as chlM, por and NOL caused less chlorophylls or chloroplast in fruit skin of Bai. Meanwhile, a predicted regulatory network of anthocyanin biosynthesis was established to illustrate involving many DEGs, especially 4CL, CHS and UFGT.

Conclusions: This study uncovered significant differences between two cucumber genotypes with different fruit color using metabolome and RNA-seq analysis. We lay a foundation to understand molecular regulation mechanism on formation of cucumber skin color, by exploring valuable genes, which is helpful for cucumber breeding and improvement on fruit skin color.

Keywords: Anthocyanin; Chlorophyll; Cucumis sativus L.; Metabolome; RNA-Seq.

Fig. 1

Phenotype of Bai and Lv about chlorophyll in fruit skins. a Fruit external characteristic of Lv and Bai. b Crosscutting observation of fruit from Lv and Bai. c Measurement of chlorophyll and carotenoid content of fruit skins from Lv and Bai. Scar bar in (a) 3 cm, (b) 2 cm. Data is presented as the mean ± standard deviation (n = 9). *0.01 ≤ P ≤ 0.05, **P ≤ 0.01, Student’s t test

Fig. 2

Transmission electron microscopy observation of Bai and Lv fruit skins. a-c Transmission electron microscopic photos of cells from Lv. d-f Transmission electron microscopic photos of cells from Bai. “T” in the figure represents thylakoid. Scar bar in (a), (c), (d) and (f) 20 μm, (b) 2 μm, (e) 1.0 μm

Fig. 3

Epidermal cells from Bai showed larger single cell area and perimeter. a, b Observation of paraffin section of fruit skins from Lv (a) and Bai (b). c Single cell area of epidermal cells from Lv and Bai fruit skins. d Single cell perimeter of epidermal cells from Lv and Bai fruit skin. e, f SEM observation of fruit skin from Lv (e) and Bai (f). Scar bar in (a, b): 150 μm. Data is presented as the mean ± standard deviation (n = 9). *0.01 ≤ P ≤ 0.05, **P ≤ 0.01, Student’s t test

Fig. 4

Comparison and KEGG analysis of different metabolites in fruit skin between Lv and Bai. a Different metabolites in fruit skins of Lv and Bai. Red, green and black correspond to up-regulated, down-regulated, and unchanged content of metabolites, respectively. b KEGG enrichment of annotated metabolites from Lv and Bai. The y-axis indicates the KEGG pathway and the x-axis indicates the enrichment factor

Fig. 5

Comparison and KEGG analysis of DEGs in fruit skin between Lv and Bai. a Analysis of DEGs in fruit skin of Lv and Bai. Red, green and blue correspond to up-regulated, down-regulated, and normal content of metabolites, respectively. b Histogram of GO terms assigned to DEGs in fruit skin of Lv and Bai. All GO terms are grouped into three ontologies: green for biological process, orange for cellular component, and purple for molecular function

Fig. 6

The detailed information on DEGs involved in the pathway of chlorophyll metabolism. HemA, glutamyl-tRNA reductase; HemL, glutamate-1-semialdehyde 2,1-aminomutase; HemB, porphobilinogen synthase; HemC, hydroxymethylbilane synthase; HemD, uroporphyrinogen-III synthase; HemE, uroporphyrinogen decarboxylase; HemF, coproporphyrinogen III oxidase; chlH, magnesium chelatase subunit H; chlM, magnesium-protoporphyrin O-methyltransferase; chlE, magnesium-protoporphyrin IX monomethyl ester; por, protochlorophyllide reductase; DAR, divinyl chlorophyllide a 8-vinyl-reductase; CAO, chlorophyllide a oxygenase; chlG, chlorophyll/bacteriochlorophyll a synthase; NOL, chlorophyll(ide) b reductase; HCAR, 7-hydroxymethyl chlorophyll a reductase; CLH, chlorophyllase

Fig. 7

Regulatory network of predicted flavonoid biosynthesis in Lv and Bai. PAL, phenylalanine ammonia-lyase; C4H, trans-cinnamate 4-hydroxylase; 4CL, 4-coumarate: CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3’H, flavonoid 3′-hydroxylase; DFR, dihydroflavonol 4-reductase; FLS, flavonol synthesis; LDOX, leucoanthocyanidin dioxygenase; UFGT, UDP glucose-flavonoid 3-O-glcosyl-transferase; LAR, leucocyanidin reductase; ANR, anthocyanidin reductase