Prospects for Knockout of MYB60, a Transcriptional Repressor of Anthocyanin Biosynthesis, in Brassicaceae Plants by Genome Editing

The Brassicaceae plant family contains many economically important crops such as Brassica napus L., Brassica rapa L., Brassica oleracea L., Brassica juncea L., Eruca sativa Mill., Camelina sativa L. and Raphanus sativus L. Insufficient data on the genetic regulation of agronomic traits in these species complicates the editing of their genomes. In recent years, the attention of the academic community has been drawn to anthocyanin hyperaccumulation. This trait is not only beneficial for human health, but can also increase plant resistance to stress. MYB transcription factors are the main regulators of flavonoid biosynthesis in plants. Some of them are well studied in Arabidopsis thaliana. The AtMYB60 gene is a transcriptional repressor of anthocyanin biosynthesis, and it also negatively impacts plant responses to drought stress. Myb60 is one of the least studied transcription factors with similar functions in Brassicaceae. There is a high degree of homology between predicted MYB60 genes of A. thaliana and related plant species. However, functions of these homologous genes have never been studied. Gene knockout by CRISPR/Cas technology remains the easiest way to perform genome editing in order to discover the role of individual plant genes. Disruption of genes acting as negative regulators of anthocyanin biosynthesis could result in color staining of plant tissues and an increase in stress tolerance. In the present study, we investigated the AtMYB60 gene and its homologs in Brassicaceae plants and suggested universal gRNAs to knockout these genes. Keywords: CRISPR, Brassicaceae, MYB60, knockout, anthocyanin

Genome-wide Identification, Evolutionary and Functional Analyses of Kfb Family Members in Potato

Background: Kelch repeat F-box (KFB) proteins play vital roles in the regulation of multitudinous biochemical and physiological processes in plants, including growth and development, stress response and secondary metabolism. Multiple KFBs have been characterized in various plant species, but this family members have not been systematically identified and analyzed in potato. Results: Genome and transcriptome analyses of StKFB gene family were conducted to dissect the structure, evolution and function of the KFBs in Solanum tuberosum L. Totally, 44 StKFB members were identified and were classified into 5 groups according to their structural and phylogenetic features. The chromosomal localization analysis showed that the 44 StKFB genes were located on 12 chromosomes. Among these genes, two pairs of genes (StKFB15/16 and StKFB40/41) were predicted to be tandemly duplicated genes, and one pair of genes (StKFB15/29) was segmentally duplicated genes. The syntenic analysis showed that the KFBs in potato were closely related to the KFBs in tomato and pepper. Expression profiles of StKFBs in 13 different tissues and in potato plants with different treatments uncovered distinct spatial expression patterns of these genes and their potential roles in response to various stresses. Transcriptomic and qRT-PCR analyses of StKFBs deciphered that multiple StKFB genes were differentially expressed in three colored potato tubers. Genes that were highly expressed in yellow fleshed tubers (Jin-16) and were lowly expressed in the red- (Red Rose-2) or purple-fleshed (Xisen-8) tubers, such as StKFB07, StKFB15, StKFB23, StKFB29 and StKFB44, may negatively regulate anthocyanin biosynthesis.Conclusions: This study reports the structure, evolution and expression characteristics of the KFB family in potato. These findings set the stage for further study of functional mechanisms of StKFBs, and also provide candidate genes for potato genetic improvement.

Dynamic Changes in Anthocyanin Accumulation and Cellular Antioxidant Activities in Two Varieties of Grape Berries during Fruit Maturation under Different Climates

As popularly consumed fruit berries, grapes are widely planted and processed into products, such as raisins and wine. In order to identify the influences of different climatic conditions on grape coloring and quality formation, we selected two common varieties of grape berries, ‘Red Globe’ and ‘Xin Yu’, for investigation. Grapes were separately grown in different climates, such as a temperate continental arid climate and a temperate continental desert climate, in Urumqi and Turpan, China, for five developmental stages. As measured, the average daily temperature and light intensity were lower in Urumqi. Urumqi grape berries had a lower brightness value (L*) and a higher red-green value (a*) when compared to Turpan’s. A RT-qPCR analysis revealed higher transcriptions of key genes related to anthocyanin biosynthesis in Urumqi grape berries, which was consistent with the more abundant phenolic substances, especially anthocyanins. The maximum antioxidant activity in vitro and cellular antioxidant activity of grape berries were also observed in Urumqi grape berries. These findings enclosed the influence of climate on anthocyanin accumulation and the antioxidant capacity of grapes, which might enlarge our knowledge on the quality formation of grape berries and might also be helpful for cultivating grapes with higher nutritional value.

Genome-Wide Identification and Co-Expression Analysis of ARF and IAA Family Genes in Euscaphis konishii: Potential Regulators of Triterpenoids and Anthocyanin Biosynthesis

Euscaphis konishii is an evergreen plant that is widely planted as an industrial crop in Southern China. It produces red fruits with abundant secondary metabolites, giving E. konishii high medicinal and ornamental value. Auxin signaling mediated by members of the AUXIN RESPONSE FACTOR (ARF) and auxin/indole-3-acetic acid (Aux/IAA) protein families plays important roles during plant growth and development. Aux/IAA and ARF genes have been described in many plants but have not yet been described in E. konishii. In this study, we identified 34 EkIAA and 29 EkARF proteins encoded by the E. konishii genome through database searching using HMMER. We also performed a bioinformatic characterization of EkIAA and EkARF genes, including their phylogenetic relationships, gene structures, chromosomal distribution, and cis-element analysis, as well as conserved motifs in the proteins. Our results suggest that EkIAA and EkARF genes have been relatively conserved over evolutionary history. Furthermore, we conducted expression and co-expression analyses of EkIAA and EkARF genes in leaves, branches, and fruits, which identified a subset of seven EkARF genes as potential regulators of triterpenoids and anthocyanin biosynthesis. RT-qPCR, yeast one-hybrid, and transient expression analyses showed that EkARF5.1 can directly interact with auxin response elements and regulate downstream gene expression. Our results may pave the way to elucidating the function of EkIAA and EkARF gene families in E. konishii, laying a foundation for further research on high-yielding industrial products and E. konishii breeding.

Novel Insights into Anthocyanin Metabolism and Molecular Characterization of Associated Genes in Sugarcane Rinds Using the Metabolome and Transcriptome

Saccharum officinarum (sugarcane) is the fifth major cultivated crop around the world. Sugarcane rind is a promising source for anthocyanin pigments; however, limited information is available on the anthocyanin and its biosynthesis in sugarcane rinds. In this study, we have quantified 49 compounds including 6 flavonoids and 43 anthocyanins in the rind of 6 sugarcane cultivars by using LCMS/MS approach. Thirty of them were quantified for the first time in sugarcane. The 43 anthocyanins included 10 cyanidin (Cya), 11 pelargonidin (Pel), 9 peonidin (Peo), 5 malvidin (Mal), 4 delphinidin (Del), and 4 petunidin (Pet) metabolites. High contents of Cya derivatives were observed in the rind of YT71/210 (dark purple rind), such as cya-3-O-(6-O-malonyl)-glu 1283.3 µg/g and cya-3-O-glu 482.67 µg/g followed by ROC22 (red rind) 821.3 µg/g and 409 µg/g, respectively, whereas the YT93/159 (green rind) showed a minimum level of these compounds. Among six cultivars, ROC22 rind has high levels of Peo derivatives such as peo-3-O-glu (197 µg/g), peo-3-O-(6-O-malonyl)-glu (69 µg/g) and peo-3-O-(6-O-p-coumaryl)-glu (55.17 µg/g). The gene expression analysis revealed that some genes, including a MYB(t) gene, were highly associated with the color phenotype. Thus, we cloned and overexpressed the gene in Arabidopsis and found the pinkish brown color in the hypocotyl of all transgenic lines compared with the wild type. Hence, we have quantified a wide range of anthocyanins in major sugarcane cultivars, reported many new anthocyanins for the first time, and concluded that Cya and Peo derivatives are the major contributing factor of dissimilar colors in sugarcane. The finding and the verification of a novel MYB gene involved in anthocyanin biosynthesis have demonstrated that our study was very valuable for gene discovery and genetic improvement of sugarcane cultivars to harvest high anthocyanin contents.

Integrated Metabolomic and Transcriptomic Analysis of the Anthocyanin and Proanthocyanin Regulatory Networks in Red Walnut Natural Hybrid Progeny Leaves

BackgroundWalnuts are one of the most important dry fruit crops worldwide, typically exhibiting green leaves and yellow–brown or gray–yellow seed coats. A specific walnut type, red walnut ‘RW-1’, with red leaves and seed coats was selected as the plant material because of its higher anthocyanin and proanthocyanin (PA) contents. Anthocyanins and PAs coprise important secondary defense methods for plants to respond to biotic and abiotic stresses. However, few studies have focused on the molecular mechanism of anthocyanin biosynthesis in walnuts.ResultsFrom the results of widely targeted metabolome and anthocyanidin detection analysis, 395 substances, including 4 PAs and 26 anthocyanins, were identified from the red-leaf walnuts of RW-1 natural hybrid progenies (SR) and the green-leaf walnuts of RW-1 natural hybrid progenies (SG). Among these, all anthocyanin types in SR were significantly upregulated compared with SG. Additionally, delphinidin 3-O-galactoside, cyanidin 3-O-galactoside, delphinidin 3-O-glucoside and cyanidin 3-O-glucoside were identified as the primary components of anthocyanidins because of their higher contents. Nine anthocyanidins, malvidin 3-O-galactoside, malvidin 3-O-arabinoside, cyanidin 3-O-(6-O-malonyl-beta-D-glucoside), delphinidin 3-O-glucoside, delphinidin 3,5-O-diglucoside (Delphin), peonidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-arabinoside and pelargonidin 3-O-(6-O-malonyl-beta-D-glucoside), were detected only in the SR walnuts. For PAs, proanthocyanin C1 was upregulated in SR compared with SG, while proanthocyanin B1 and proanthocyanin B3 were upregulated in SR-1 and SR-3 but downregulated in SR-2 compared with the controls. Furthermore, transcriptome analysis demonstrated that the expression of structural genes (C4H, F3H, F3’5’H, UFGTs, LAR and ANR), four MYBs and six WD40s in the anthocyanin and PA biosynthetic pathways were significantly higher in the SR walnut.ConclusionsOur results provide valuable information on anthocyanin and PA metabolites and candidate genes in anthocyanin and PA biosynthesis, which provides new insights into anthocyanin and PA biosynthesis in walnuts.

Characterisation of chalcone isomerase gene isolated from Pueraria montana var.lobata plant

Chalcone isomerase (CHI) is well-known as an important enzyme in the biosynthetic pathways such as flavonoid, isoflavonoid, and anthocyanin biosynthesis. The enzyme was investigated in some kinds of plants in Fabaceae but no research was conducted about the CHI gene of Pueraria montana var. lobata (P. lobata) in Vietnam. In order to provide more information and characterisation of the gene, our study isolated the CHI gene by RT-PCR and Sangersequencing. The sequence of the CHIgene was analysed with nucleotide and deduced amino acid sequences to find the main domains. A full-length CDS of CHI gene from P. lobata is 672 bp encoded 224 amino acids. By using bioinformatic tools to compare, the isolated gene shared 99.7% homology with the same species reference (code D63577.1). Two different nucleotides in the gene were altered the amino acids in the protein, but the differences have not happened in active sites. Additionally, the conserved amino acids related to active catalysis of a hydrogen bond network also appeared in the P. lobataCHI gene. SWISS-MODEL was used to build the complete protein modeling showing that P. lobataCHI protein was the most similar with CHI of Medicago sativa - was defined structure in which all alpha-helix and beta-helix were completelyhomologies.