A Mutation in the MYBL2-1 Gene Is Associated with Purple Pigmentation in Brassica oleracea.

Anthocyanins are well-known antioxidants that are beneficial for plants and consumers. Dihydroflavonol-4-reductase (DFR) is a key gene of anthocyanin biosynthesis, controlled by multiple transcription factors. Its expression can be enhanced by mutations in the negative regulator of anthocyanin biosynthesis myeloblastosis family transcription factor-like 2 (MYBL2). The expression profiles of the DFR gene were examined in 43 purple and green varieties of Brassica oleracea L., Brassica napus L., Brassica juncea L., and Brassica rapa L. MYBL2 gene expression was significantly reduced in purple varieties of B. oleracea, and green varieties of B. juncea. The MYBL2 gene sequences were screened for mutations that can affect pigmentation. Expression of the DFR gene was cultivar-specific, but in general it correlated with anthocyanin content and was higher in purple plants. Two single nucleotide polymorphysms (SNPs) were found at the beginning of the DNA-binding domain of MYBL2 gene in all purple varieties of B. oleracea. This mutation, leading to an amino acid substitution and the formation of a mononucleotide repeat (A)8, significantly affects RNA structure. No other noteworthy mutations were found in the MYBL2 gene in green varieties of B. oleracea and other studied species. These results bring new insights into the regulation of anthocyanin biosynthesis in genus Brassica and provide opportunities for generation of new purple varieties with precise mutations introduced via genetic engineering and CRISPR/Cas.

The Role of the GSTF11 Gene in Resistance to Powdery Mildew Infection and Cold Stress.

Oilseed rape (Brassica napus) is an economically important crop. In a temperate climate, powdery mildew Erysiphe crucifertaum can drastically reduce its yield. Nevertheless, cultivars resistant to this fungal disease have not yet been selected. Glutathione S-transferase GSTF11 is involved in glucosinolate (GSL) biosynthesis and response to stress, including fungal deceases. However, the impact of exogenous GSTF11 gene expression on resistance to powdery mildew has not yet been confirmed and requires further investigation. Transgenic B. napus was generated for this purpose. It demonstrated increased GST activity and a higher GSH:GSSG ratio under normal conditions. Powdery mildew Erysiphe crucifertaum caused 50% mortality in wild type (WT) plants. In most of transgenic plants, mycelium growth was inhibited. The infection contributed to higher GSTF11 expression and increased levels of glutathione (GSH) and oxidized glutathione (GSSG) in both transgenic and WT plants. In contrast, GSTF11 mRNA content, GST activity and GSSG level were lower only in WT plants. In transgenic plants, increased resistance to powdery mildew correlated with a lower GSH:GSSG ratio, indicating a higher content of neutralized toxic molecules. GSTF11 expression was also affected by cold stress, but not drought. At -1 °C, the expression level increased only in transgenic plants. Therefore, GSTF11 appears to be nonspecific and is able to protect plants under several types of stress. This gene could be used as a target in the production of stress tolerant cultivars.

Anthocyanin Biosynthesis Genes as Model Genes for Genome Editing in Plants.

CRISPR/Cas, one of the most rapidly developing technologies in the world, has been applied successfully in plant science. To test new nucleases, gRNA expression systems and other inventions in this field, several plant genes with visible phenotypic effects have been constantly used as targets. Anthocyanin pigmentation is one of the most easily identified traits, that does not require any additional treatment. It is also associated with stress resistance, therefore plants with edited anthocyanin genes might be of interest for agriculture. Phenotypic effect of CRISPR/Cas editing of PAP1 and its homologs, DFR, F3H and F3'H genes have been confirmed in several distinct plant species. DFR appears to be a key structural gene of anthocyanin biosynthesis, controlled by various transcription factors. There are still many promising potential model genes that have not been edited yet. Some of them, such as Delila, MYB60, HAT1, UGT79B2, UGT79B3 and miR156, have been shown to regulate drought tolerance in addition to anthocyanin biosynthesis. Genes, also involved in trichome development, such as TTG1, GLABRA2, MYBL2 and CPC, can provide increased visibility. In this review successful events of CRISPR/Cas editing of anthocyanin genes are summarized, and new model genes are proposed. It can be useful for molecular biologists and genetic engineers, crop scientists, plant genetics and physiologists.