Transcriptome Screening of Long Noncoding RNAs and Their Target Protein-Coding Genes Unmasks a Dynamic Portrait of Seed Coat Coloration Associated with Anthocyanins in Tibetan Hulless Barley

Abstract

Many plants have the capability to accumulate anthocyanins for coloration, and anthocyanins are advantageous to human health. In the case of hulless barley (Hordeum vulgare L. var. nudum), investigation into the mechanism of anthocyanin formation is limited to the level of protein-coding genes (PCGs). Here, we conducted a comprehensive bioinformatics analysis to identify a total of 9414 long noncoding RNAs (lncRNAs) in the seed coats of purple and white hulless barley along a developmental gradient. Transcriptome-wide profiles of lncRNAs documented several properties, including GC content fluctuation, uneven length, a diverse range of exon numbers, and a wide variety of transcript classifications. We found that certain lncRNAs in hulless barley possess detectable sequence conservation with Hordeum vulgare and other monocots. Furthermore, both differentially expressed lncRNAs (DElncRNAs) and PCGs (DEPCGs) were concentrated in the later seed development stages. On the one hand, DElncRNAs could potentially cis-regulate DEPCGs associated with multiple metabolic pathways, including flavonoid and anthocyanin biosynthesis in the late milk and soft dough stages. On the other hand, there was an opportunity for trans-regulated lncRNAs in the color-forming module to affect seed coat color by upregulating PCGs in the anthocyanin pathway. In addition, the interweaving of hulless barley lncRNAs and diverse TFs may function in seed coat coloration. Notably, we depicted a dynamic portrait of the anthocyanin synthesis pathway containing hulless barley lncRNAs. Therefore, this work provides valuable gene resources and more insights into the molecular mechanisms underlying anthocyanin accumulation in hulless barley from the perspective of lncRNAs, which facilitate the development of molecular design breeding in crops.

Keywords: anthocyanin formation; hulless barley; long noncoding RNA; protein-coding gene; seed coat coloration.

Figure 1

Transcriptome-wide identification and characterization of long noncoding RNAs (lncRNAs) in Tibetan hulless barley seed coats. (a) Schematic computational pipeline for the identification of lncRNAs in hulless barley seed coats. Kunlun 10 (white) and Nierumuzha (purple) Tibetan hulless barley grains were divided into three developmental stages: early milk (PC1 and WC1), late milk (PC2 and WC2), and soft dough (PC3 and WC3). (b) Transcriptome-wide characterization of hulless barley seed coat lncRNAs. The gray curve in the middle of the circos plot shows the collinearity of PCGs in the hulless barley genome. (c) Length distribution in hulless barley seed coat lncRNAs. (d) Distribution of exon numbers in hulless barley seed coat lncRNAs. (e) Classification of hulless barley seed coat lncRNAs based on the relative position of lncRNAs to annotated genes in the reference assembly. The yellow arrow represents the reverse transcription direction.

Figure 2

Evolutionary sequence conservation and expression pattern dynamics of lncRNAs in Tibetan hulless barley seed coats. (a) Sequence conservation of lncRNAs in seed coats of hulless barley and 39 other species. Information on the taxonomic category of plants is provided in the evolutionary tree. The numbers represent the number of homologues. Green areas indicate monocotyledonous plant taxa. (b) Unique and shared lncRNAs among six different samples. (c) Expression heatmap and hierarchical clustering of white and purple hulless barley lncRNAs along the developmental gradient (log scale: base = 2; logwith = 1; col scale: normalized).

Figure 3

Defining DElncRNAs and DEPCGs between white and purple Tibetan hulless barley seed coats among three developmental stages. (a) The number of DElncRNAs and DEPCGs in Nierumuzha at three different developmental stages compared to Kunlun 10 (|log₂FC| values ≥ 1; p value ≤ 0.01; q value ≤ 0.05). (b–d) DElncRNAs (upregulated, downregulated, and total) between purple and white hulless barley along the developmental gradient. (e–g) DEPCGs (upregulated, downregulated, and total) between purple and white hulless barley along the developmental gradient.

Figure 4

Prediction and metabolic function of potential cis-regulated target DEPCGs and their DElncRNAs in Tibetan hulless barley seed coats. (a–c) In three developmental stages (early milk, late milk, and soft dough) of hulless barley, expression pattern comparisons of cis-regulated target DEPCGs and their DElncRNAs. (d) Heatmap of cis-regulated DEPCGs of DElncRNAs associated with metabolic processes along the developmental gradient.

Figure 5

Co-expression network of trans-regulated lncRNAs and their PCGs in Tibetan hulless barley seed coats. (a) Investigation of module-trait correlations. Each row shows a module, and each column represents different developmental stages of hulless barley. Red represents a positive correlation, and green represents a negative correlation. The module marked by black arrow is potential color-forming modules. (b) The eigengene expression heatmap for the color-forming module (“yellow” module). (c) Metabolic function enrichment of PCGs in PC3 of the “yellow” module. (d) The correlation network of trans-regulated hub-lncRNAs and their hub-PCGs involved in phenylpropanoid and flavonoid biosynthesis (kME = 0.8; GS = 0.8).

Figure 6

Transcription factors associated with cis- and trans-regulated lncRNAs. (a) Cis-regulated DETFs and their DElncRNAs in the early milk stage. (b) Cis-regulated DETFs and their DElncRNAs in the soft dough stage. (c) Possible trans-regulated lncRNA-TF interaction correlation network of the color-forming module. The different color points represent different transcription factor families.

Figure 7

Dynamic molecular mechanism portrait of the anthocyanin synthesis pathway illustrating the color formation of Tibetan hulless barley seed coats. The red line represents the higher PCG expression level at the soft dough stage in purple Nierumuzha relative to white Kunlun 10. The blue line represents a much lower level of expression. Both cis- and trans-regulated lncRNAs that are consistent with the expression pattern of their target PCGs are written in red font, and those that are not consistent with the expression pattern of their target PCGs are written in blue.