Unveiling the mysteries of HvANS: a study on anthocyanin biosynthesis in qingke (hordeum vulgare L. var. Nudum hook. f.) seeds

Abstract

Background: Based on our previous research, a full-length cDNA sequence of HvANS gene was isolated from purple and white Qingke. The open reading frame (ORF) in the purple variety Nierumuzha was 1320 base pairs (bp), encoding 439 amino acids, while the ORF in the white variety Kunlun 10 was 1197 bp, encoding 398 amino acids. A nonsynonymous mutation was found at the position of 1195 bp (T/C) in the coding sequence (CDS) of the HvANS gene. We carried out a series of studies to further clarify the relationship between the HvANS gene and anthocyanin synthesis in Qingke.

Results: The conservative structural domain prediction results showed that the encoded protein belonged to the PLN03178 superfamily. Multiple comparisons showed that this protein had the highest homology with Hordeum vulgare, at 88.61%. The approximately 2000 bp promoter sequence of the HvANS gene was identical in both varieties. The real-time fluorescence PCR (qRT-PCR) results revealed that HvANS expression was either absent or very low in the roots, stems, leaves, and awns of Nierumuzha. In contrast, the HvANS expression was high in the seed coats and seeds of Nierumuzha. Likewise, in Kunlun 10, HvANS expression was either absent or very low, indicating a tissue-specific and variety-specific pattern for HvANS expression. The subcellular localization results indicated that HvANS was in the cell membrane. Metabolomic results indicated that the HvANS gene is closely related to the synthesis of three anthocyanin substances (Idaein chloride, Kinetin 9-riboside, and Cyanidin O-syringic acid). Yeast single hybridization experiments showed that the HvANS promoter interacted with HvANT1, which is the key anthocyanin regulatory protein. In a yeast two-hybrid experiment, we obtained two significantly different proteins (ZWY2020 and POMGNT2-like) and verified the results by qRT-PCR.

Conclusions: These results provide a basis for further studies on the regulatory mechanism of HvANS in the synthesis of anthocyanins in Qingke purple grains.

Keywords: HvANS gene; Anthocyanin; Expression pattern; Functions and mechanisms; Qingke.

Fig. 1

Bioinformatics analysis of the HvANS gene CDS. (A) Prediction of conserved structural domains of HvANS proteins. (B) Prediction of hydrophilicity and hydrophobicity of HvANS proteins. (C) Secondary structure prediction of the HvANS protein. (D) Tertiary structure prediction of the HvANS protein. 1 is Nierumuzha, 2 is Kunlun 10

Fig. 2

Homology comparison and phylogenetic analysis of ANS proteins. (A) Homologous comparison of ANS proteins. (B) Phylogenetic analysis of ANS proteins. (C) Validation of base mutation loci for 28 Qingke materials. N-HvANS, Hordeum vulgare L. var. nudum Hook. f. ‘Nierumuzha’; K-HvANS, Hordeum vulgare L. var. nudum Hook. f. ‘Kunlun 10’; Hv, Hordeum vulgare (Ensembl: HORVU.MOREX.r3.5HG0509790); Td, Triticum dicoccoides (GeneBank: XP_037444903.1); At, Aegilops tauschii (GeneBank: XP_020171118.1); Ih, Indosasa hispida (GeneBank: AHC07955.1); Os, Oryza sativa Japonica Group (GeneBank: CAA69252.1); Ta, Triticum aestivum (GeneBank: AXM42875.1); Tu, Triticum Urartu (GeneBank: XP_048537655.1); Tt, Triticum turgidum subsp. Durum (GeneBank: VAI52396.1); Lr, Lolium rigidum (GeneBank: XP_047057945.1)

Fig. 3

Functional studies of the HvANS gene. (A) Spatial and temporal expression patterns of HvANS in grain color formation of barley varieties with different grain colors. (B) Subcellular localization of the HvANS protein. (C) Positive transgenic Arabidopsis thaliana seedlings and seeds. (D)Y1H assay showing the interaction between HvANT1 and the HvANS promoter. p53AbAi x pGADT7-Rec53 was used as a positive control, pANS-AbAi x pGADT7 was used as a negative control, and pANS-AbAi x рGADT7-ANT1 was used as an experimental group. Capital letters in graph A represent highly significant differences. GFP, green fluorescence; Chlorophy II, Chloroplast; Bright, bright field; Merge, Superimposed. A, B, C, D. statistically significant (p < 0.01). SD-Leu, yeast-deficient medium (without leucine); AbA, gold tamoxifen

Fig. 4

Interactions of the HvANS protein with eight proteins revealed by yeast two-hybrid experiments

Fig. 5

(A) Expression of two differentially expressed proteins in two varieties. (B) DNA pattern of ANS-interacting proteins in purple Qingke. A, B, C, D. statistically significant (p < 0.01)