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Alternative Splicing of the Basic Helix-Loop-Helix Transcription Factor Gene CmbHLH2 Affects Anthocyanin Biosynthesis in Ray Florets of Chrysanthemum (Chrysanthemum morifolium).

ABSTRACT: Chrysanthemum is an important ornamental crop worldwide. Some white-flowered chrysanthemum cultivars produce red ray florets under natural cultivation conditions, but little is known about how this occurs. We compared the expression of anthocyanin biosynthetic and transcription factor genes between white ray florets and those that turned red based on cultivation conditions to comprehend the underlying mechanism. Significant differences in the expression of CmbHLH2 were detected between the florets of different colors. CmbHLH2 generated two alternatively spliced transcripts, designated CmbHLH2^Full and CmbHLH2^Short . Compared with CmbHLH2^Full , CmbHLH2^Short encoded a truncated protein with only a partial MYB-interaction region and no other domains normally present in the full-length protein. Unlike the full-length form, the splicing variant protein CmbHLH2^Short localized to the cytoplasm and the nucleus and could not interact with CmMYB6. Additionally, CmbHLH2^Short failed to activate anthocyanin biosynthetic genes and induce pigment accumulation in transiently transfected tobacco leaves, whereas CmbHLH2^Full promoted both processes when simultaneously expressed with CmMYB6. Co-expressing CmbHLH2^Full and CmMYB6 also enhanced the promoter activities of CmCHS and CmDFR. Notably, the Arabidopsis tt8-1 mutant, which lacks red pigmentation in the leaves and seeds, could be complemented by the heterologous expression of CmbHLH2^Full, which restored red pigmentation and resulted in red pigmentation in high anthocyanin and proanthocyanidin contents in the leaves and seeds, respectively, whereas expression of CmbHLH2^Short did not. Together, these results indicate that CmbHLH2 and CmMYB6 interaction plays a key role in the anthocyanin pigmentation changes of ray florets in chrysanthemum. Our findings highlight alternative splicing as a potential approach to modulate anthocyanin biosynthesis in specific tissues.

SUBMITTER: Lim SH

PROVIDER: S-EPMC8222801 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundChrysanthemum morifolium is one of the most important global cut flower and pot plants, and has been cultivated worldwide. However, limited genomic resources are available and the molecular mechanisms involved in the two morphologically distinct floret developmental cycles in chrysanthemum remain unclear.ResultsThe transcriptomes of chrysanthemum ray florets, disc florets and leaves were sequenced using Illumina paired-end sequencing technology. In total, 16.9 G reads were assembled into 93,138 unigenes with an average length of 738 bp, of which 44,364 unigenes showed similarity to known proteins in the Swissprot or NCBI non-redundant protein databases. Additionally, 26,320, 22,304 and 13,949 unigenes were assigned to 54 gene ontology (GO) categories, 25 EuKaryotic Orthologous Groups (KOG) categories, and 280 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. A total of 1863 differentially expressed genes (DEGs) (1210 up-regulated and 653 down-regulated) were identified between ray florets and disc florets, including genes encoding transcription factors and protein kinases. GO and KEGG pathway enrichment analyses were performed on the DEGs to identify differences in the biological processes and pathways between ray florets and disc florets. The important regulatory genes controlling flower development and flower organ determination, as well as important functional genes in the anthocyanin biosynthetic pathway, were identified, of which two leucoanthocyanidin dioxygenase-encoding genes showed specific expression in ray florets. Lastly, reverse transcription quantitative PCR was conducted to validate the DEGs identified in our study.ConclusionsComparative transcriptome analysis revealed significant differences in patterns of gene expression and signaling pathways between ray florets and disc florets in Chrysanthemum morifolium. This study provided the first step to understanding the molecular mechanism of the differential development of ray florets and disc florets in chrysanthemum, and also provided valuable genomic resources for candidate genes applicable for the breeding of novel varieties in chrysanthemum.

Project description:The generation of chrysanthemum (Chrysanthemum × morifolium) flower color is mainly attributed to the accumulation of anthocyanins. Light is one of the key environmental factors that affect the anthocyanin biosynthesis, but the deep molecular mechanism remains elusive. In our previous study, a series of light-induced structural and regulatory genes involved in the anthocyanin biosynthetic pathway in the chrysanthemum were identified using RNA sequencing. In the present study, differentially expressed proteins that are in response to light with the capitulum development of the chrysanthemum 'Purple Reagan' were further identified using isobaric tags for relative and absolute quantification (iTRAQ) technique, and correlation between the proteomic and the transcriptomic libraries was analyzed. In general, 5106 raw proteins were assembled based on six proteomic libraries (three capitulum developmental stages × two light treatments). As many as 160 proteins were differentially expressed between the light and the dark libraries with 45 upregulated and 115 downregulated proteins in response to shading. Comparative analysis between the pathway enrichment and the gene expression patterns indicated that most of the proteins involved in the anthocyanin biosynthetic pathway were downregulated after shading, which was consistent with the expression patterns of corresponding encoding genes; while five light-harvesting chlorophyll a/b-binding proteins were initially downregulated after shading, and their expressions were enhanced with the capitulum development thereafter. As revealed by correlation analysis between the proteomic and the transcriptomic libraries, GDSL esterase APG might also play an important role in light signal transduction. Finally, a putative mechanism of light-induced anthocyanin biosynthesis in the chrysanthemum was proposed. This study will help us to clearly identify light-induced proteins associated with flower color in the chrysanthemum and to enrich the complex mechanism of anthocyanin biosynthesis for use in cultivar breeding.

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Alternative Splicing of the Basic Helix-Loop-Helix Transcription Factor Gene CmbHLH2 Affects Anthocyanin Biosynthesis in Ray Florets of Chrysanthemum (Chrysanthemum morifolium).

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