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

Combined application of zinc and iron-lysine and its effects on morpho-physiological traits, antioxidant capacity and chromium uptake in rapeseed (Brassica napus L.)

Environmental contamination of chromium (Cr) has gained substantial consideration worldwide because of its high levels in the water and soil. A pot experiment using oil seed crop (rapeseed (Brassica napus L.)) grown under different levels of tannery wastewater (0, 33, 66 and 100%) in the soil using the foliar application of zinc (Zn) and iron (Fe)–lysine (lys) has been conducted. Results revealed that a considerable decline in the plant growth and biomass elevates with the addition of concentrations of tannery wastewater. Maximum decline in plant height, number of leaves, root length, fresh and dry biomass of root and leaves were recorded at the maximum level of tannery wastewater application (100%) compared to the plants grown without the addition of tannery wastewater (0%) in the soil. Similarly, contents of carotenoid and chlorophyll, gas exchange parameters and activities of various antioxidants (superoxidase dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX)) were also reduced significantly (P < 0.05) with the increasing concentration of tannery wastewater (33, 66 and 100%) in the soil. In addition, a combined application of Zn and Fe-lys reduced the accumulation and uptake of toxic Cr, while boosting the uptake of essential micronutrients such as Zn and Fe in different tissues of the plants. Results concluded that exogenous application of micronutrients chelated with amino acid successfully mitigate Cr stress in B. napus. Under field conditions, supplementation with these micronutrient-chelated amino acids may be an effective method for alleviating metal stress in other essential seed crops.

Antioxidant Response and Calcium-Dependent Protein Kinases Involvement in Canola (Brassica napus L.) Tolerance to Drought

Canola is an important temperate oil crop that can be severely affected by drought. Understanding the physiological and molecular mechanisms involved in canola tolerance to water deficit is essential to obtain drought-tolerant productive cultivars. To investigate the role of antioxidant response and the possible involvement of calcium-dependent protein kinases (CDPKs) in canola tolerance to drought, we analyzed four genotypes with different sensitivity to water stress. Leaf relative water content, canopy temperature, PSII efficiency, electrolyte leakage index and lipid peroxidation were used as indicators to classify the cultivars as drought-tolerant or drought-sensitive. Antioxidant enzymes superoxide dismutase, guaiacol peroxidase and catalase displayed significantly higher activities in drought-tolerant than in drought-sensitive cultivars subjected to water deficit, suggesting that the efficiency of the antioxidant response is essential in canola drought tolerance. The increased expression of genes BnaCDPK6 and BnaCDPK14 under drought conditions, their differential expression in drought-tolerant and drought-sensitive genotypes, and the presence of multiple cis-acting stress-related elements in their promoter regions suggest that CDPKs are part of the signaling pathways that regulate drought response in canola. We propose the BnaCDPK genes and their regulator elements as potential molecular targets to obtain drought-tolerant productive canola cultivars through breeding or genetic transformation.

Evaluation of expression changes, proteins interaction network, and microRNAs targeting catalase and superoxide dismutase genes under cold stress in rapeseed (Brassica napus L.)

Rapeseed is the third-largest source of plant oil and one of the essential oil plants worldwide. Cold stress is one of the critical factors that affect plant yield. Therefore, improving cold stress tolerance is necessary for yield increase. The present study investigated BnCAT1 and BnCSD1 genes’ expression behavior in a tolerant and sensitive cultivar under cold stress (4 °C). Besides, protein-protein interaction networks of CATs and CSDs enzymes, and their association with other antioxidant enzymes were analyzed. Moreover, the microRNAs targeting BnCAT1 and BnCSD1 genes were predicted. This study indicated many direct and indirect interactions and the association between the components of the plant antioxidant system. However, not only did the CATs and CSDs enzymes have a relationship with each other, but they also interacted directly with ascorbate peroxidase and glutathione reductase enzymes. Also, 23 and 35 effective microRNAs were predicted for BnCAT1 and BnCSD1 genes, respectively. The gene expression results indicated an elevated expression of BnCAT1 and BnCSD1 in both tolerant and sensitive cultivars. However, this increase was more noticeable in the tolerant cultivar. Thus, the BnCSD1 gene had the highest expression in the early hour of cold stress, especially in the 12th h, and the BnCAT1 gene showed the highest expression in the 48th h. This result may indicate a functional relationship between these enzymes.

Chemical Differentiation and Antimicrobial Potential of Four Brassica napus L Seed Oils

The conducted study compares the phytochemical and the antimicrobial potential of four varieties of Brassica napus seed oils. The plant seeds were cultivated during the winter growing season. Soxhlet extractor and Gas Chromatography-Mass Spectrometer (GC-MS) were used for essential oil analysis. The micro broth dilutionassay was applied to test the antimicrobial potential (MIC: Minimum inhibitory concentration, MBC: Minimum bactericidal concentration) of the extracted essential oils against different bacterial strains. A total of 56 phytochemicals were found, including 23 and 25 compounds in the oils of Pactol and Rapifera seed varieties,respectively, and 21 compounds in each of Bacara and Rally seed oils. Oleic acid constituted about 35.79 %, 15.62%, 7%, and 2.41 % for Rally, Bacara, Rapifera, and Pactol seed oils, respectively. Gram-positive bacteria, Streptococcus pyogenes and Streptococcus agalactiae, showed lower resistance potentials (MIC= 0.78%, 3.125%respectively) (MBC=1.36%, 6.25% respectively) to the essential oils compared with Staphylococcus aureus. Escherichia coli showed higher sensitivity (6.25% and 12.5% for MIC and MBC, respectively) than Klebsiella pneumonia and Pseudomonas aeruginosa to the B. napus seed oils. Gram-positive bacteria weremore sensitive to the tested essential oils than Gram-negative bacteria. Overall, four different seed varieties have important chemicals and fatty acids. Oleic acid was the most common carboxylic acid (fatty acid) and 2,4-decadienal with hexanal were the most prevalent aldehydes in four seed oils. Tested B. napus seed essential oilsshowed antimicrobial activities against various Gram-positive and negative bacteria and Candida albicans, with Pactol seed oils exerting the highest activity.

Yield of Winter Oilseed Rape (Brassica napus L. var. napus) in a Short-Term Monoculture and the Macronutrient Accumulation in Relation to the Dose and Method of Sulphur Application

The objective of this study was to assess the yield efficiency of sulphur-enhanced fertilisers, depending on the dose and application method, in a short-lived (three-year) monoculture of winter oilseed rape under the climate and soil conditions of south-eastern Poland. The experiment was carried out between 2010 and 2013 on winter oilseed rape (Brassica napus L. var. napus) of the Orlando variety, fertilised with different sulphur doses—0, 20, 40 or 60 kg S ha−1 applied in different method—soil application sowing, foliar application in the spring, and soil application sowing + foliar application in the spring (combined application). Following the harvest, seed and straw yields and the content of macroelements (N, S, P, K, Ca and Mg) in the seed and straw samples were determined. The harvest indices were also established for each of these elements. The impact of sulphur on winter oilseed rape yield depended significantly on both the dose and the application method. Even at the lowest dose (20 kg·ha−1), sulphur materially increased seed yield, regardless of the application method. With autumn soil application and foliar application, differences between the lowest dose and the higher doses (40 and 60 kg·ha−1) were not significant. However, with combined application, the highest dose (60 kg·ha−1) significantly increased yield compared to the lower doses. In general, all the fertilisation approaches significantly increased the N, P, K, Ca and Mg contents compared to the control sample, but the differences between them were not substantial. Each of the sulphur application approaches decreased the harvest index for sulphur. The foliar application of each of the doses decreased the harvest indices for N, P, K and Ca. The soil application of 20 kg·ha−1, and the mixed application of 40 and 60 kg·ha−1, all increased the harvest indices for P, K and Ca.