Anthocyanin concentration is an important determinant of the colour of many fruits. In apple (Malus x domestica), centuries of breeding have produced numerous varieties in which levels of anthocyanin pigment vary widely and change in response to environmental and developmental stimuli. The apple fruit cortex is usually colourless, although germplasm does exist where the cortex is highly pigmented due to the accumulation of either anthocyanins or carotenoids. From studies in a diverse array of plant species, it is apparent that anthocyanin biosynthesis is controlled at the level of transcription. Here we report the transcript levels of the anthocyanin biosynthetic genes in a red-fleshed apple compared with a white-fleshed cultivar. We also describe an apple MYB transcription factor, MdMYB10, that is similar in sequence to known anthocyanin regulators in other species. We further show that this transcription factor can induce anthocyanin accumulation in both heterologous and homologous systems, generating pigmented patches in transient assays in tobacco leaves and highly pigmented apple plants following stable transformation with constitutively expressed MdMYB10. Efficient induction of anthocyanin biosynthesis in transient assays by MdMYB10 was dependent on the co-expression of two distinct bHLH proteins from apple, MdbHLH3 and MdbHLH33. The strong correlation between the expression of MdMYB10 and apple anthocyanin levels during fruit development suggests that this transcription factor is responsible for controlling anthocyanin biosynthesis in apple fruit; in the red-fleshed cultivar and in the skin of other varieties, there is an induction of MdMYB10 expression concurrent with colour formation during development. Characterization of MdMYB10 has implications for the development of new varieties through classical breeding or a biotechnological approach.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2006.02964.x

Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10

The expansion of gene families encoding regulatory proteins is typically associated with the increase in complexity characteristic of multi-cellular organisms. The MYB and basic helix-loop-helix (bHLH) families provide excellent examples of how gene duplication and divergence within particular groups of transcription factors are associated with, if not driven by, the morphological and metabolic diversity that characterize the higher plants. These gene families expanded dramatically in higher plants; for example, there are approximately 339 and 162 MYB and bHLH genes, respectively, in Arabidopsis, and approximately 230 and 111, respectively, in rice. In contrast, the Chlamydomonas genome has only 38 MYB genes and eight bHLH genes. In this review, we compare the MYB and bHLH gene families from structural, evolutionary and functional perspectives. The knowledge acquired on the role of many of these factors in Arabidopsis provides an excellent reference to explore sequence-function relationships in crops and other plants. The physical interaction and regulatory synergy between particular sub-classes of MYB and bHLH factors is perhaps one of the best examples of combinatorial plant gene regulation. However, members of the MYB and bHLH families also interact with a number of other regulatory proteins, forming complexes that either activate or repress the expression of sets of target genes that are increasingly being identified through a diversity of high-throughput genomic approaches. The next few years are likely to witness an increasing understanding of the extent to which conserved transcription factors participate at similar positions in gene regulatory networks across plant species.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2010.04459.x

Evolutionary and comparative analysis of MYB and bHLH plant transcription factors

Flavonoids are secondary metabolites involved in several aspects of plant development and defence. They colour fruits and flowers, favouring seed and pollen dispersal, and contribute to plant adaptation to environmental conditions such as cold or UV stresses, and pathogen attacks. Because they affect the quality of flowers (for horticulture), fruits and vegetables, and their derivatives (colour, aroma, stringency, etc.), flavonoids have a high economic value. Furthermore, these compounds possess pharmaceutical properties extremely attractive for human health. Thanks to easily detectable mutant phenotypes, such as modification of petal pigmentation and seeds exhibiting transparent testa, the enzymes involved in the flavonoid biosynthetic pathway have been characterized in several plant species. Conserved features as well as specific differences have been described. Regulation of structural gene expression appears tightly organized in a spatial and temporal way during plant development, and is orchestrated by a ternary complex involving transcription factors from the R2R3-MYB, basic helix-loop-helix (bHLH), and WD40 classes. This MYB-bHLH-WD40 (MBW) complex regulates the genes that encode enzymes specifically involved in the late steps of the pathway leading to the biosynthesis of anthocyanins and condensed tannins. Although several genes encoding transcription factors from these three families have been identified, many gaps remain in our understanding of the regulation of this biosynthetic pathway, especially about the respective roles of bHLH and WD40 proteins. A better knowledge of the regulatory mechanisms of the flavonoid pathway is likely to favour the development of new biotechnological tools for the generation of value-added plants with optimized flavonoid content.

/pdf/recent-advances-in-the-transcriptional-regulation-of-the-38kdsk8jcj.pdf

Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway

Plant phenolics: recent advances on their biosynthesis, genetics, and ecophysiology.

https://munin.uit.no/bitstream/handle/10037/5893/article.pdf?sequence=1

New insights into the regulation of anthocyanin biosynthesis in fruits

We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes. We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs. In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

/pdf/transient-expression-vectors-for-functional-genomics-4s6jcdwc3w.pdf

Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species. We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA) are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins. This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

/pdf/an-r2r3-myb-transcription-factor-associated-with-regulation-3eochh883k.pdf

An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae

Background

The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species.

Results

We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA) are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins.

Conclusions

This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

Karryn Grafton

Papers

Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae

An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae