The Paeonia qiui R2R3-MYB Transcription Factor PqMYB113 Positively Regulates Anthocyanin Accumulation in Arabidopsis thaliana and Tobacco

Paeonia qiui is a wild species of tree peony native to China. Its leaves are purplish red from the bud germination to the flowering stage, and anthocyanin is the main pigment in purplish red leaves. However, the anthocyanin synthesis regulation mechanism in tree peony leaves remains unclear. In this study, an R2R3-MYB, PqMYB113 was identified from the leaves of P. qiui. Phylogenetic analysis revealed that PqMYB113 clustered with Liquidambar LfMYB113 and grape VvMYBA6. Subcellular location analysis showed that PqMYB113 was located in the cell nucleus. The transient reporter assay suggested that PqMYB113 was a transcriptional activator. The overexpression of PqMYB113 in Arabidopsis thaliana and tobacco (Nicotiana tabacum) resulted in increased anthocyanin accumulation and the upregulation of CHS, F3H, F3’H, DFR, and ANS. The dual luciferase reporter assay showed that PqMYB113 could activate the promoters of PqDFR and PqANS. Bimolecular fluorescence complementation assays and yeast two-hybrid assays suggested that PqMYB113 could form a ternary MBW complex with PqbHLH1 and PqWD40 cofactors. These results provide insight into the regulation of anthocyanin biosynthesis in tree peony leaves.

Triticum Aestivum L. cv. Guizi 1 ANS-6D Positively Regulates Leaf Senescence Through the Abscisic Acid Mediated Chlorophyll Degradation in Tobacco

Anthocyanidin synthase (ANS) is involved in the synthesis of anthocyanins, which are important phytonutrients because of their beneficial effects on human health. Here, we identified ANS-6D of purple-colored Triticum aestivum L. cv. Guizi 1 (Gz) that is involved in leaf senescence through the abscisic acid (ABA) mediated chlorophyll degradation pathway in tobacco. After characterizing the leaf-senescence phenotype in GzANS-6D overexpression (OxGzANS-6D) lines, we found that the increased anthocyanin accumulation and decreased chlorophyll content in OxGzANS-6D lines were closely correlated with the expression levels of anthocyanin synthesis-related structural genes and senescence marker genes, as well as the accumulation of reactive oxygen species. The endogenous ABA content increased and ethylene content decreased in OxGzANS-6D transgenic lines compared with wild type. Additionally, the levels of the abscisic acid-responsive transcription factors ABF1 and ABF2, as well as those of chlorophyll degradation-related genes (PAO, NYC, SGR and CHL), were significantly higher in OxGzANS-6D transgenic lines than in wild type. Furthermore, we found that GzABF1 and NtABF1 binds to the promoter of GzANS-6D, and NtABF2 binds to the promoter of NtSGR. Thus, GzANS-6D participated in leaf senescence through ABA-mediated chlorophyll degradation, and ABF1/2 play important role in GzANS-6D functions.

Changes in the Polyphenol Content of Red Raspberry Fruits during Ripening

Berry fruits that contain large amounts of polyphenol compounds are expected to exhibit health and anti-aging effects due to the antioxidant activities of these components. Among the various polyphenols, flavan-3-ol derivatives are known to have a particularly high functionality. In this study, the maturity of red raspberry fruits is classified into eight stages based on the polyphenol content at each stage. Quantification of the various compounds and investigation of the DPPH and ABTS radical scavenging activities were carried out. The total polyphenol content, including that of the flavan-3-ol derivatives, was the highest in immature fruits, gradually decreasing during fruit maturation, during which the radical scavenging activity also decreased. Based on our quantitative results, it was considered that the decrease in the flavan-3-ol derivative content due to fruit ripening was largely related to the increase in the amount of anthocyanin derivatives. Considering that the decreased contents of these compounds were related to the expression levels of polyphenol biosynthetic enzymes, quantification was performed using the semi-quantitative polymerase chain reaction, but the only change observed was the increased expression of the enzyme that synthesizes anthocyanins during maturation. Therefore, it was suggested that it is necessary to inhibit anthocyanin synthesis to increase the contents of highly functional flavan-3-ol derivatives in the mature fruit.

VvMYB15 and VvWRKY40 Positively Co-regulated Anthocyanin Biosynthesis in Grape Berries in Response to Root Restriction

In most grapevine planting regions, especially in south of China, plenty of rainfall and high water level underground are the characteristic of the area, a series of problem during fruit ripening easily caused poor color quality. Thereby affecting fruit quality, yield and economic benefits. The accumulation of anthocyanin is regulated by transcriptional regulatory factor and a series of cultivation measures, root restriction can make plants in the environment of stress and stress relief, root restriction induced the higher expression of VvMYB15 and VvWRKY40, and consistent with anthocyanin accumulation. Whether and how root restriction-inducible VvMYB15 and VvWRKY40 transcription factor regulate anthocyanin synthesis in grape berry is still unclear. In this study, we identified that the transient overexpression of VvMYB15 and VvWRKY40 alone or both in strawberry fruits and grape berries can promote anthocyanin accumulation and increase the expression level of anthocyanin biosynthetic genes, indicating VvMYB15 and VvWRKY40 play a positive regulator of anthocyanin biosynthesis. Furthermore, we confirmed that both VvMYB15 and VvWRKY40 specifically bind to the promoter region of VvF3′5′H and VvUFGT, and the expression of VvF3′5′H and VvUFGT is further activated through the heterodimer formation between VvMYB15 and VvWRKY40. Finally, we confirmed that VvMYB15 promoted anthocyanin accumulation by interacting with VvWRKY40 in grape berries, our findings provide insights into a mechanism involving the synergistic regulation of root restriction-dependent coloration and biosynthesis via a VvMYB15 and VvWRKY40 alone or both in grape berries.

Regulation of MYB Transcription Factors of Anthocyanin Synthesis in Lily Flowers

Flower color is the decisive factor that affects the commercial value of ornamental flowers. Therefore, it is important to study the regulation of flower color formation in lily to discover the positive and negative factors that regulate this important trait. In this study, MYB transcription factors (TFs) were characterized to understand the regulatory mechanism of anthocyanin biosynthesis in lily. Two R2R3-MYB TFs, LvMYB5, and LvMYB1, were found to regulate anthocyanin biosynthesis in lily flowers. LvMYB5, which has an activation motif, belongs to the SG6 MYB protein subgroup of Arabidopsis thaliana. Transient expression of LvMYB5 indicated that LvMYB5 can promote coloration in Nicotiana benthamiana leaves, and that expression of LvMYB5 increases the expression levels of NbCHS, NbDFR, and NbANS. VIGS experiments in lily petals showed that the accumulation of anthocyanins was reduced when LvMYB5 was silenced. Luciferase assays showed that LvMYB5 can promote anthocyanin synthesis by activating the ANS gene promoter. Therefore, LvMYB5 plays an important role in flower coloration in lily. In addition, the transient expression experiment provided preliminary evidence that LvMYB1 (an R2R3-MYB TF) inhibits anthocyanin synthesis in lily flowers. The discovery of activating and inhibitory factors related to anthocyanin biosynthesis in lily provides a theoretical basis for improving flower color through genetic engineering. The results of our study provide a new direction for the further study of the mechanisms of flower color formation in lilies.

The evidence for anthocyanins in the betalain-pigmented genus Hylocereus is weak

Here we respond to Zhou et al., 2020 'Combined Transcriptome and Metabolome analysis of Pitaya fruit unveiled the mechanisms underlying Peel and pulp color formation' published in BMC Genomics. Given the evolutionary conserved anthocyanin biosynthesis pathway in betalain-pigmented species, we are open to the idea that species with both anthocyanins and betalains might exist. However, in absence of LC-MS/MS spectra, apparent lack of biological replicates, and no comparison to authentic standards, the findings of Zhou et al., 2020 are not a strong basis to propose the presence of anthocyanins in betalain-pigmented pitaya. In addition, our re-analysis of the datasets indicates the misidentification of important genes and the omission of key anthocyanin synthesis genes ANS and DFR. Finally, our re-analysis of the RNA-Seq dataset reveals no correlation between anthocyanin biosynthesis gene expression and pigment status.

Phosphate (Pi) Starvation Up-Regulated GmCSN5A/B Participates in Anthocyanin Synthesis in Soybean (Glycine max) Dependent on Pi Availability

Phosphorus (P) is an essential macronutrient for plant growth and development. Among adaptive strategies of plants to P deficiency, increased anthocyanin accumulation is widely observed in plants, which is tightly regulated by a set of genes at transcription levels. However, it remains unclear whether other key regulators might control anthocyanin synthesis through protein modification under P-deficient conditions. In the study, phosphate (Pi) starvation led to anthocyanin accumulations in soybean (Glycine max) leaves, accompanied with increased transcripts of a group of genes involved in anthocyanin synthesis. Meanwhile, transcripts of GmCSN5A/B, two members of the COP9 signalosome subunit 5 (CSN5) family, were up-regulated in both young and old soybean leaves by Pi starvation. Furthermore, overexpressing GmCSN5A and GmCSN5B in Arabidopsis thaliana significantly resulted in anthocyanin accumulations in shoots, accompanied with increased transcripts of gene functions in anthocyanin synthesis including AtPAL, AtCHS, AtF3H, AtF3′H, AtDFR, AtANS, and AtUF3GT only under P-deficient conditions. Taken together, these results strongly suggest that P deficiency leads to increased anthocyanin synthesis through enhancing expression levels of genes involved in anthocyanin synthesis, which could be regulated by GmCSN5A and GmCSN5B.

Transcriptome Analysis Revealed the Mechanism by Which Exogenous ABA Increases Anthocyanins in Blueberry Fruit During Veraison

Blueberry (Vaccinium spp.) is a popular healthy fruit worldwide. The health value of blueberry is mainly because the fruit is rich in anthocyanins, which have a strong antioxidant capacity. However, because blueberry is a non-model plant, little is known about the structural and regulatory genes involved in anthocyanin synthesis in blueberries. Previous studies have found that spraying 1,000 mg/L abscisic acid at the late green stage of “Jersey” highbush blueberry fruits can increase the content of anthocyanins. In this experiment, the previous results were verified in “Brightwell” rabbiteye blueberry fruits. Based on the previous results, the anthocyanin accumulation process in blueberry can be divided into six stages from the late green stage to the mature stage, and the transcriptome was used to systematically analyze the blueberry anthocyanin synthesis process. Combined with data from previous studies on important transcription factors regulating anthocyanin synthesis in plants, phylogenetic trees were constructed to explore the key transcription factors during blueberry fruit ripening. The results showed that ABA increased the anthocyanin content of blueberry fruits during veraison. All structural genes and transcription factors (MYB, bHLH, and WD40) involved in the anthocyanin pathway were identified, and their spatiotemporal expression patterns were analyzed. The expression of CHS, CHI, DFR, and LDOX/ANS in ABA-treated fruits was higher in the last two stages of maturity, which was consistent with the change in the anthocyanin contents in fruits. In general, six MYB transcription factors, one bHLH transcription factor and four WD40 transcription factors were found to change significantly under treatment during fruit ripening. Among them, VcMYBA plays a major role in the regulation of anthocyanin synthesis in ABA signaling. This result preliminarily explained the mechanism by which ABA increases the anthocyanin content and improves the efficiency of the industrial use of blueberry anthocyanins.