Table 1

qRT-PCR primers for the ScbHLH13 gene and the synthetic genes of anthocyanin branch pathway"

Fig. 1

Characteristic of ScbHLH13 nucleotide and its coding amino acid sequence A: the alignment of nucleic acid sequences from sorghum bHLH13 and ScbHLH13, the un-conserved sites was indicated with green; B: the conserved domain prediction of ScbHLH13 protein; C: the prediction of the transmembrane structure of ScbHLH13 protein."

Fig. 2

Multiple alignments of the amino acid sequences encoded by the ScbHLH13 and the bHLH13 from other homologous species"

Fig. 3

Tertiary structure analysis of bHLH13 protein based on AlphaFold modeling The tertiary structure of bHLH13 proteins from Saccharum spp. hybrids (GenBank accession No.: USN24528.1), Sorghum bicolor (GenBank accession No.: XP_002440844.1), Setaria italic (GenBank accession No.: XP_004960675.1), Panicum miliaceum (GenBank accession No.: RLN28435.1), Hordeum vulgare (GenBank accession No.: KAE8807589.1), and Aegilops tauschii (GenBank accession No.: XP_020150590.1) were achieved from AlphaFold database (https://alphafold.com/). The blue dashed line indicates the difference in the red β-sheet region, and the black arrow indicates the difference in the random coil or α-helix region."

Fig. 4

Phylogenetic tree of bHLH proteins from different plant species Red font indicates ScbHLH proteins. According to the previous reports about classification of bHLH family[31], 18 sequence, specifically belonging to different subfamily, from Arabidopsis and rice were used to generate the phylogenetic tree[21,32]. Different color represent the different bHLH subfamily including VII (a+b), VIIIb, III (a+c), IIIb, III (d+e), and IIIf."

Fig. 5

ScbHLH13 gene expression in different transcriptomes A: the transcriptomic data from sugarcane varieties Badila and ROC22 under low nitrogen stress; B: the transcriptomic data from sugarcane variety ROC22 under Sorghum mosaic virus stress; C: the transcriptomic data from sugarcane variety YC05-179 and ROC22 under Sporisorium scitamineum stress."

Fig. 6

Subcellular localization analyses of ScbHLH13 protein in the epidermis cell of Nicotiana benthamiana leaf The same field on the leaf was pictured through bright and yellow fluorescence, and merge channels. 35S::YFP was the result of injection of agrobacterium solution with empty pEarlegate101-YFP. 35S::ScbHLH13::YFP was the result of infusion of agrobacterium with pEarlegate101-ScbHLH13-YFP."

Fig. 7

Subcellular localization analyses of ScbHLH13 in sugarcane sheath protoplasts YFP is localized inside the cell. The fluorescence microscope selectes the blue fluorescence channel, and the excitation wavelength is 358-360 nm, and the emission wavelength 460-461 nm. Bar: 10 μm."

Fig. 8

Relative expression profile of anthocyanin synthesis genes in sugarcane protoplast bHLH represents the sugarcane ScbHLH13 gene. F3’H, A1, CHI, F3H, CHS, and ANS are the members of the anthocyanin branch pathway. bHLH is the experimental group and YFP is the control group. Different lowercase letters above the bars represent significant differences at P < 0.05."