Overexpression of ThMYC4E Enhances Anthocyanin Biosynthesis in Common Wheat.

The basic helix-loop helix (bHLH) transcription factor has been inferred to play an important role in blue and purple grain traits in common wheat, but to date, its overexpression has not been reported. In this study, the bHLH transcription factor ThMYC4E, the candidate gene controlling the blue grain trait from Th. Ponticum, was transferred to the common wheat JW1. The positive transgenic lines displayed higher levels of purple anthocyanin pigments in their grains, leaves and glumes. Stripping the glumes (light treatment) caused white grains to become purple in transgenic lines. RNA-Seq and qRT-PCR analysis demonstrated that the transcript levels of structural genes associated with anthocyanin biosynthesis were higher in transgenic wheat than the wild-type (WT), which indicated that ThMYC4E activated anthocyanin biosynthesis in the transgenic lines. Correspondingly, the anthocyanin contents in grains, roots, stems, leaves and glumes of transgenic lines were higher than those in the WT. Metabolome analysis demonstrated that the anthocyanins were composed of cyanidin and delphinidin in the grains of the transgenic lines. Moreover, the transgenic lines showed higher antioxidant activity, in terms of scavenging DPPH radicals, in the ethanol extracts of their grains. The overexpression of ThMYC4E sheds light on the traits related to anthocyanin biosynthesis in common wheat and provide a new way to improve anthocyanin content.

A bHLH transcription factor TsMYC2 is associated with the blue grain character in triticale (Triticum × Secale).

KEY MESSAGE: RNA-Seq was employed to compare the transcriptome differences between the triticale lines and to identify the key gene responsible for the blue aleurone trait. The accumulation of anthocyanins in the aleurone of triticale results in the formation of the blue-grained trait, but the identity of the genes associated with anthocyanin biosynthesis in the aleurone has not yet been reported. In this manuscript, RNA-Seq was employed to compare the transcriptome differences between the triticale lines HM13 (blue aleurone) and HM5 (white aleurone), and to identify the key genes responsible for the blue aleurone trait. There were 32,406 differentially expressed genes between HM13 and HM5. Seventy-three unigenes were homologous to the structural genes related to anthocyanin biosynthesis, and the average transcript level of the structural genes was higher in HM13 than in HM5, so that quantitative differences between the two lines in transcription rates could be the cause of the blue aleurone. The MYB and bHLH transcription factors had two homologous unigenes, but contained only one differentially expressed unigene each. The relative transcript level of bHLH Unigene5672_All (TsMYC2) in HM13 was 42.71 times that in HM5, while the relative transcript level of the MYB transcription factor Unigene12228_All in HM13 was 2.20 times that in HM5. qPCR experiments determined the relative transcript level of TsMYC2 in developing grain, with the expression of TsMYC2 in grain being the highest compared with that in root, stem or leaf tissue. TsMYC2 was homologous to the bHLH transcription factor regulating anthocyanin biosynthesis and contained three entire functional domains: bHLH-MYC_N, HLH and ACT-like, which were important for exercising regulation of anthocyanin biosynthesis as a bHLH transcription factor. Transient expression of ZmC1 and TsMYC2 could induce anthocyanin biosynthesis in white wheat coleoptile cells, demonstrating that TsMYC2 was a functional bHLH transcription factor. These results indicated that TsMYC2 was associated with the blue aleurone trait and could prove to be a valuable gene with which to breed new triticale cultivars with the blue aleurone trait.

Transcriptome Analysis Identifies Key Genes Responsible for Red Coleoptiles in Triticum Monococcum.

Red coleoptiles can help crops to cope with adversity and the key genes that are responsible for this trait have previously been isolated from Triticum aestivum, Triticum urartu, and Aegilops tauschii. This report describes the use of transcriptome analysis to determine the candidate gene that controls the trait for white coleoptiles in T. monococcum by screening three cultivars with white coleoptiles and two with red coleoptiles. Fifteen structural genes and two transcription factors that are involved in anthocyanin biosynthesis were identified from the assembled UniGene database through BLAST analysis and their transcript levels were then compared in white and red coleoptiles. The majority of the structural genes reflected lower transcript levels in the white than in the red coleoptiles, which implied that transcription factors related to anthocyanin biosynthesis could be candidate genes. The transcript levels of MYC transcription factor TmMYC-A1 were not significantly different between the white and red coleoptiles and all of the TmMYC-A1s contained complete functional domains. The deduced amino acid sequence of the MYB transcription factor TmMYB-A1 in red coleoptiles was homologous to TuMYB-A1, TaMYB-A1, TaMYB-B1, and TaMYB-D1, which control coleoptile color in corresponding species and contained the complete R2R3 MYB domain and the transactivation domain. TmMYB-a1 lost its two functional domains in white coleoptiles due to a single nucleotide deletion that caused premature termination at 13 bp after the initiation codon. Therefore, TmMYB-A1 is likely to be the candidate gene for the control of the red coleoptile trait, and its loss-of-function mutation leads to the white phenotype in T. monococcum.

Transcriptome Analysis Clarified Genes Involved in Betalain Biosynthesis in the Fruit of Red Pitayas (Hylocereus costaricensis).

The red flesh trait gives red pitayas more healthful components and a higher price, while the genetic mechanism behind this trait is unknown. In this manuscript, transcriptome analysis was employed to discover the genetic differences between white and red flesh in pitayas. A total of 27.99 Gb clean data were obtained for four samples. Unigenes, 79,049 in number, were generated with an average length of 1333 bp, and 52,618 Unigenes were annotated. Compared with white flesh, the expression of 10,215 Unigenes was up-regulated, and 4853 Unigenes were down-regulated in red flesh. The metabolic pathways accounted for 64.6% of all differentially expressed Unigenes in KEGG pathways. The group with high betalain content in red flesh and all structural genes, related to betalain biosynthesis, had a higher expression in red flesh than white flesh. The expression of the key gene, tyrosine hydroxylase CYP76AD1, was up-regulated 245.08 times, while 4,5-DOPA dioxygenase DODA was up-regulated 6.46 times. Moreover, the special isomers CYP76AD1α and DODAα were only expressed in red flesh. The competitive anthocyanin biosynthesis pathway had a lower expression in red flesh. Two MYB transcription factors were of the same branch as BvMYB1, regulating betalain biosynthesis in beet, and those transcription factors had expression differences in two kinds of pitayas, which indicated that they should be candidate genes controlling betalain accumulation in red pitayas. This research would benefit from identifying the major gene controlling red flesh trait and breed new cultivars with the red flesh trait. Future research should aim to prove the role of each candidate gene in betalain biosynthesis in red pitayas.