Integrated Metabolomics and Proteomics Analyses of the Grain-Filling Process and Differences in the Quality of Tibetan Hulless Barleys

Abstract

Tibetan hulless barley (qingke) grains are becoming more popular because of their high nutritional benefits. Comparative metabolomics and proteomics analyses of qingke grains (at 16, 20, 36, and 42 days after flowering) were conducted to explore the metabolic dynamics during grain filling and compare the differences in quality among three different varieties, Dulihuang, Kunlun 14, and Heilaoya. A total of 728 metabolites and 4864 proteins were identified. We first found that both the metabolite and protein profiles were more closely associated with the grain developmental stage in each cultivar than across different stages in a single cultivar. Next, we focused on the energy metabolism and biosynthesis pathways of key quality components, such as flavonoids, starch, and β-glucans in qingke grains. Quantitative analysis revealed significant variation in the abundance of cellulose synthase-like enzyme (CslF) among the three cultivars. Notably, Heilaoya displayed substantially lower CslF6 levels at 36 and 42 DAF than Kunlun 14 and Dulihuang did. These observed differences in CslF6 abundance may represent a key regulatory mechanism underlying the distinct β-glucan biosynthesis patterns among the three cultivars. Collectively, our results enhance the understanding of metabolic networks involved in qingke grain development and serve as a foundation for advancing breeding studies.

Keywords: Tibetan hulless barley; difference in quality; metabolomics; proteomics.

Figure 1

Qingke seed morphology and metabolite analysis at four key developmental stages (I, II, III, and IV): (A) Grain phenotype. (B) PCA of metabolomic data from four developmental stages and 36 qingke samples. (C) Hierarchical clustering analysis of metabolite distribution profiles of Dulihuang, Kunlun 14, and Heilaoya at four developmental stages. The color scale 0−1 represents Spearman’s correlation coefficient. (D) Numbers and classification of the identified and annotated metabolites. (E) Statistics of differentially accumulated metabolites, including upregulated and downregulated metabolites, in each comparison group.

Figure 2

Cluster analysis of the accumulation patterns of the shared DAMs in Dulihuang, Kunlun 14, and Heilaoya: (A) Venn diagram of the DAMs in each of the three cultivars. (B) Distribution patterns of 293 metabolites at four seed developmental stages.

Figure 3

Protein identification and analysis: (A) PCA of proteomic data from four developmental stages and 36 qingke samples. (B) hierarchical clustering analysis of the protein expression profiles of Dulihuang, Kunlun 14, and Heilaoya at four developmental stages. The color scale 0−1 represents Spearman’s correlation coefficient. (C) statistics of the differentially expressed proteins, including upregulated and downregulated proteins, in each comparison group. (D) Top 10 GO terms of each category for the recognized proteins in the proteome. (E) KEGG pathway enrichment of the identified proteins. The enrichment factor is the percentage of members out of the total number detected. The bubble size represents the number of members detected in the KEGG pathway, and the color of the bubble represents the −log 10 (p value).

Figure 4

PPI networks of the DEPs: (A) Dulihuang vs. Kunlun. (B) Dulihuang vs. Heilaoya. (C) Kunlun 14 vs. Heilaoya. The circles represent the DEPs in each comparison, and darker colors indicate greater connectivity.

Figure 5

Cluster analysis and functional enrichment of proteins with shared expression patterns in Dulihuang, Kunlun 14, and Heilaoya: (A) Major clusters (Clusters 1~3) are shown with light blue trends for proteins with the same trend in Dulihuang, Kunlun 14, and Heilaoya. The average expression levels of genes in each cluster are shown by blue lines. (B) Functional category enrichments among the three major clusters are shown in a heatmap. Red, significant enrichment; white, nonsignificant; gray, not detected.

Figure 6

Visualization of the metabolites and proteins in biochemical pathway maps related to carbohydrate metabolism and energy metabolism in developing qingke seeds. Z score fold change values are shown on a color scale that is proportional to the abundance of each metabolite and protein. 3.2.1.20, maltase-glucoamylase; 3.2.1.26, beta-fructofuranosidase; 4.1.2.13, fructose-bisphosphate aldolase; 1.2.1.12, glyceraldehyde 3-phosphate dehydrogenase; 2.7.2.3, phosphoglycerate kinase; 5.4.2.12, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase; 4.2.1.11, phosphopyruvate hydratase; 2.7.1.40, pyruvate kinase; 4.2.1.3, aconitate hydratase; 1.1.1.42, isocitrate dehydrogenase; 6.2.1.4, 6.2.1.5, succinyl-CoA synthetase alpha subunit; 1.3.5.1, succinate dehydrogenase; 4.2.1.2, fumarate hydratase; 1.1.5.4, malate dehydrogenase; 2.7.1.1, hexokinase; PGM, phosphoglucomutase; AGPase, ADP-glucose pyrophosphorylase; 2.7.7.9, UDP-glucose pyrophosphorylase; CslF6, cellulose synthase-like F6; CslF10, cellulose synthase-like F10; SS, starch synthase; SBE, starch branching enzyme.

Figure 7

Visualization of the metabolites and proteins in biochemical pathway maps related to flavonoid biosynthesis in developing qingke seeds. The heatmap was plotted using normalized expression values of metabolites and proteins with Z scores. Total anthocyanin content in the three cultivars at 42 DAF. PAL, phenylalanine ammonia-lyase; C4H, 4-hydroxylase; 4CL, CoA ligase; 1.2.1.44, cinnamoyl-CoA reductase; 1.1.1.195, cinnamyl-alcohol dehydrogenase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; DFR, dihydroflavonol-4-reductase; LAR, leucoanthocyanidin reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; 1.14.13.-, p-coumaroyl-CoA: caffeoyl-CoA 3-hydroxylase; 2.1.1.68, caffeic acid 3-O-methyltransferase; F5H, flavanone 5- hydroxylase; 2.1.1.104, caffeoyl-CoA O-methyltransferase; 1.14.20.5, flavone synthase I; 1.14.19.76, flavone synthase II; 1.14.14.81, flavonoid 3′,5′-hydroxylase.