De novo transcriptome and tissue-specific gene expression analysis in three types of barley following exploring the mechanism of quercetin content in purple barley (Hordeum vulgare)

Overview

This investigation examined the transcriptional regulation of quercetin biosynthesis and accumulation across three barley phenotypes at distinct developmental stages. Quercetin, a flavonol with documented bioactive properties, exhibits type-dependent accumulation patterns in barley grains, with detection restricted to purple barley varieties compared to yellow and black cultivars. The study employed comparative transcriptome sequencing to identify molecular mechanisms governing this differential quercetin content and its developmental dynamics during grain maturation.

Methods and approach

Comparative transcriptome analysis was conducted on yellow, black, and purple barley grains sampled at both grain-filling and grain maturation stages. RNA sequencing was performed followed by de novo assembly and structural optimization. Functional annotation was applied to identified genes, and differential expression analysis was executed to compare transcript abundance across barley types. Alternative splicing events and single-nucleotide polymorphisms were systematically catalogued. Gene ontology enrichment analysis and KEGG pathway mapping were employed to contextualize differentially expressed genes within established metabolic frameworks, with protein-protein interaction network construction performed for identified candidates.

Key Findings

De novo transcriptome assembly resulted in structural optimization of 6,393 genes, with 4,986 achieving functional annotation. Analysis identified 12 alternative splicing event categories and 561,743 putative single-nucleotide polymorphisms across the three barley types. Differential expression profiling revealed 4,540 differentially expressed genes, with purple barley demonstrating the highest proportion of upregulated transcripts. Gene ontology enrichment and network analysis indicated dense protein-protein interaction patterns among differentially expressed genes distributed across biological process, cellular component, and molecular function categories. KEGG pathway analysis demonstrated significant enrichment of phenylpropanoid and flavonoid biosynthesis pathways specifically in purple barley. Critical quercetin biosynthesis genes, including F3ʹH, F3ʹ5ʹH, FLS1, and FLS2, exhibited substantially elevated expression during grain maturation relative to grain-filling stages, suggesting developmental coordination of quercetin accumulation with seed maturity.

Implications

This transcriptome-level characterization establishes the first systematic comparison of quercetin-associated gene regulation among barley phenotypes and identifies a coordinated transcriptional framework governing quercetin biosynthesis in purple varieties. The developmental expression pattern of key biosynthetic enzymes suggests that grain maturation represents a critical window for quercetin accumulation, implicating both transcriptional and post-transcriptional regulatory mechanisms in this process. These findings delineate molecular targets for investigating quercetin biosynthetic regulation through functional genomic approaches. The identified genes and regulatory pathways constitute genetic resources applicable to marker development and allele discovery for selective breeding objectives. Future functional validation of candidate genes and promoter characterization will enable rational engineering strategies to enhance quercetin content in purple barley cultivars for agricultural and nutritional applications.