Integrated Transcriptomic and Metabolomic Analyses Shed Light on the Regulation of Aromatic Amino Acid Biosynthesis in a Novel Albino Tea (Camellia sinensis) Mutation

Abstract

Off-white or yellowish shoots are common in tea plants (Camellia sinensis L.), and such albino variations are often accompanied by metabolic reprogramming, including increased contents of amino acids and lower levels of polyphenols. Nonetheless, the molecular mechanisms that underlie these albino variations remain to be fully clarified. Here, we examined the ultrastructural characteristics of novel, naturally occurring, yellowish mutated tea leaves and performed metabolomic analyses on green and albino leaves and stems. Then, transcriptomic analyses were also conducted on green and albino leaves to investigate the mechanistic basis of the albino variation. As expected, the cells of albino tea leaves contained fewer and smaller chloroplasts with disorganized thylakoids and smaller starch granules. Widely targeted metabolomics analysis revealed 561 differentially abundant metabolites between green and albino leaves and stems, but there was little difference between green and albino stems. Then, RNA sequencing of green and albino leaves revealed downregulation of genes associated with light harvesting and photosynthesis, and integration of the metabolomic and transcriptomic results indicated that biosynthesis of aromatic amino acids (AAAs) was strongly upregulated in albino leaves. To gain additional insight into the molecular basis of the increased AAA levels, Oxford Nanopore long-read sequencing was performed on green and albino leaves, which enabled us to identify differences in long non-coding RNAs (lncRNAs) and alternatively spliced transcripts between green and albino leaves. Interestingly, the amino acid biosynthesis genes arogenate dehydratase/prephenate dehydratase (ADT) and serine hydroxymethyltransferase (SHMT) were highlighted in the lncRNA and alternative splicing analyses, and the transcription factor genes PLATZ, B3 Os04g0386900, and LRR RLK At1g56140 showed significant changes in both expression and alternative splicing in albino leaves. Together, our data suggest that biosynthesis of AAAs might be crucial for albino mutations in tea plants and could be coordinated with the regulation of lncRNAs and alternative splicing. This is a complex regulatory network, and further exploration of the extensive metabolic reprogramming of albino tea leaves will be beneficial.

Keywords: Camellia sinensis; albino mutation; alternative splicing; aromatic amino acid; lncRNAs.

Figure 1

Phenotypes and ultrastructures of green and albino leaves. (A) Tea leaf phenotypes. An albino shoot is visible near the center of the image. (B) The ultrastructure of a third green leaf. (C) The chloroplast ultrastructure of a third green leaf. (D) The ultrastructure of a third albino leaf. (E) The chloroplast ultrastructure of a third albino leaf. The bars indicate 5 μm in (B,D) and 2 μm in (C,E).

Figure 2

PCA (A) and clustering analysis (B) of differential metabolites in albino and normal leaves (GLs and YLs) and stems (GSs and YSs).

Figure 3

KEGG enrichment analyses of differentially abundant metabolites. (A) Normal leaves (GLs) versus albino leaves (YLs). (B) Albino leaves (YLs) versus albino stems (YSs). The top twenty enriched pathways for each comparison are shown.

Figure 4

Hierarchical clustering and GO enrichment analysis of DEGs from normal and albino leaves. (A) Heatmap of DEGs in normal leaves (GLs) versus albino leaves (YLs). (B) GO enrichment analysis of DEGs upregulated in YLs versus GLs. (C) GO enrichment analysis of DEGs downregulated in YLs versus GLs.

Figure 5

Integrated analysis of differentially abundant metabolites and DEGs in normal leaves (GLs) and albino leaves (YLs). (A) Integrated KEGG enrichment analysis, with yellow boxes highlighting two KEGG pathways that were significantly enriched in both the differentially abundant metabolites and DEGs. The blue and yellow dotted lines represent the significance of enrichment (p < 0.05 and p < 0.01, respectively). (B) Integrated analysis of key genes and metabolites associated with the enriched KEGG pathway “biosynthesis of amino acids”. Heatmaps show the abundance of key metabolites in this pathway, and the red, green, and blue fonts indicate genes that were upregulated, downregulated, and both up- and downregulated in YLs versus GLs, respectively. (C) The relative expression of key genes in the “biosynthesis of amino acids” pathway as measured by qRT–PCR. Each heatmap contains 6 cells, representing three biological samples from GLs and YLs. BCAT2: branched-chain amino acid aminotransferase; leuC: 3-isopropylmalate dehydratase large subunit; DHAD: dihydroxy-acid dehydratase; PHGDH1: D-3-phosphoglycerate dehydrogenase 1; PSPH: phosphoserine phosphatase; SHMT: serine hydroxymethyltransferase; L-TA: low-specificity L-threonine aldolase 1; ASA1: anthranilate synthase alpha subunit 1; trpB1: tryptophan synthase beta chain 1; ADT1: arogenate dehydratase/prephenate dehydratase 1; ADT1 X1: arogenate dehydratase/prephenate dehydratase 1 isoform X1; ADH1: arogenate dehydrogenase 1.

Figure 6

Analysis of lncRNAs and their predicted DEG targets associated with the KEGG pathway “biosynthesis of amino acids”. (A) The numbers of different types of lncRNA obtained in C. sinensis leaves. (B) lncRNAs and their predicted DEG targets associated with the “biosynthesis of amino acids” KEGG pathway. (C) The relative expression of ADT2 X1, ADT1, SHMT, and their predicted cognate lncRNAs as measured by qRT–PCR.

Figure 7

Analysis of alternatively spliced transcription factor genes. (A) Top twenty enriched KEGG pathways of alternatively spliced transcription factor genes. (B) Expression heatmap of alternatively spliced transcription factor genes.

Figure 8

The expression levels and alternative sequences of three transcription factors that exhibited alternative splicing. (A,B) The relative expression levels of PLATZ (A) and LRR RLK At1g56140 (B) as measured by qRT–PCR. (C) The relative expression levels of B3 Os04g0386900 and the lncRNA ONT.12712.2. (D) The alternatively spliced sequence of B3 Os04g0386900 in the YLs.