Identification and functional analysis of long non-coding RNA (lncRNA) and metabolites response to mowing in hulless barley (Hordeum vulgare L. var. nudum hook. f.)

Abstract

Background: Hulless barley (Hordeum vulgare L. var. nudum Hook. f.) is a significant cereal crop and a substantial source of forage for livestock. Long non-coding RNAs (lncRNAs) and metabolites play crucial roles in the nutrient accumulation and regeneration of hulless barley plants following mowing. The study aimed to identify differentially expressed lncRNAs and metabolites in hulless barley plants by analyzing transcriptomic and metabolomic datasets at 2 h, 24 h, and 72 h following mowing.

Results: The study revealed that 190, 90, and 438 lncRNA genes were differentially expressed at the 2 h, 24 h, and 72 h time points compared to the non-mowing control. We identified 14 lncRNA genes-11 downregulated and 3 upregulated-showing consistently significant differential expression across all time points after mowing. These differentially expressed lncRNAs target genes involved in critical processes such as cytokinin signaling, cell wall degradation, storage protein accumulation, and biomass increase. In addition, we identified ten differentially expressed metabolites targeting diverse metabolic pathways, including plant hormones, alkaloids, and flavonoids, before and after mowing at various time points. Endogenous hormone analysis revealed that cytokinin most likely played a crucial role in the regeneration of hulless barley after mowing.

Conclusions: This study created a comprehensive dataset of lncRNAs, metabolites, and hormones in hulless barley after mowing, revealing valuable insights into the functional characteristics of lncRNAs, metabolites, and hormones in regulating plant regeneration. The results indicated that cytokinin plays a significant role in facilitating the regeneration process of hulless barley after mowing. This comprehensive dataset is an invaluable resource for better understanding the complex mechanisms that underlie plant regeneration, with significant implications for crop improvement.

Keywords: Cytokinin; Hulless barley; Metabolome; Mowing; Plant hormones; lncRNA.

Fig. 1

The identification and classification of lncRNAs in hulless barley.  A Various analytical methods used to identify lncRNAs. B Category of lncRNAs. C Classification of lncRNAs

Fig. 2

Comparison of lncRNA and mRNAs characteristics in hulless barley. A Differentially expressed lncRNA gene number, (B-D) Volcano plots of differentially expressed lncRNA genes after mowing at 2 h, 24 h, and 72 h, (E) transcript length, (F) exon number, (G) ORF length, (H) expression level, I intron length, (J) intron number, CK, control check; M1, mowing after 2 h; M2, mowing after 24 h; M3, mowing after 72 h

Fig. 3

Differentially expressed mRNA and lncRNAs in hulless barley following mowing. A DEGs of mRNA. B DEGs of lncRNAs. C Cluster heat map of differentially expressed mRNAs. D Cluster heat map of differentially expressed lncRNAs. E Venn diagram showing differentially expressed mRNAs. F Venn diagram showing differentially expressed lncRNAs

Fig. 4

KEGG enrichment analysis of differentially expressed mRNA and lncRNA. A PCA analysis of mRNA. B PCA analysis of lncRNA. C-E KEGG analysis of differential cis-target genes of mRNA in the M1 vs CK, M2 vs CK and M3 vs CK groups, respectively. F–H KEGG analysis of differentially expressed trans-target genes of lncRNA in the M1 vs CK, M2 vs CK and M3 vs CK groups, respectively

Fig. 5

Analysis of mRNA target genes by qRT-PCR. The expression pattern of mRNA target genes in response to mowing is displayed here. The shown data are the mean ± SD of three replicates. 18S rRNA was used as the reference gene

Fig. 6

Differential metabolites analysis. A PCA analysis. B-D Network visualization of differential metabolite molecular pairs in M1 vs CK, M2 vs CK, and M3 vs CK groups, with correlation coefficient |r|> 0.8 and p < 0.05. E–G The top 20 most significant differential metabolites in M1 vs CK, M2 vs CK, and M3 vs CK groups, respectively

Fig. 7

KEGG analysis of differential metabolites. A-C KEGG analysis of differential metabolites in the M1 vs CK, M2 vs CK, and M3 vs CK groups, respectively. D Venn diagram of differential metabolites after mowing

Fig. 8

Hormones content in hulless barley after mowing at 0 h, 2 h, 24 h, and 72 h. A TZR content, (B) tZT content, (C) iPA content, (D) IAA content, (E) ABA content. Values are means ± SD (n = 3) (*P ≤ 0.05, **P ≤ 0.01, **P ≤ 0.001, Student’s t-test)