Canola (Brassica napus) enhances sodium chloride and sodium ion tolerance by maintaining ion homeostasis, higher antioxidant enzyme activity and photosynthetic capacity fluorescence parameters.

Under salt stress, plants are forced to take up and accumulate large amounts of sodium (Na+ ) and chloride (Cl- ). Although most studies have focused on the toxic effects of Na+ on plants, Cl- stress is also very important. This study aimed to clarify physiological mechanisms underpinning growth contrasts in canola varieties with different salt tolerance. In hydroponic experiments, 150mM Na+ , Cl- and NaCl were applied to salt-tolerant and sensitive canola varieties. Both NaCl and Na+ treatments inhibited seedling growth. NaCl caused the strongest damage to both canola varieties, and stress damage was more severe at high concentrations of Na+ than Cl- . High Cl- promoted the uptake of ions (potassium K+ , calcium Ca2+ ) and induced antioxidant defence. Salt-tolerant varieties were able to mitigate ion toxicity by maintaining lower Na+ content in the root system for a short period of time, and elevating magnesium Mg2+ content, Mg2+ /Na+ ratio, and antioxidant enzyme activity to improve photosynthetic capacity. They subsequently re-established new K+ /Na+ and Ca2+ /Na+ balances to improve their salt tolerance. High concentrations of Cl salts caused less damage to seedlings than NaCl and Na salts, and Cl- also had a positive role in inducing oxidative stress and responsive antioxidant defence in the short term.

Identification and expression analysis of the Xyloglucan transglycosylase/hydrolase (XTH) gene family under abiotic stress in oilseed (Brassica napus L.).

XTH genes are key genes that regulate the hydrolysis and recombination of XG components and plays role in the structure and composition of plant cell walls. Therefore, clarifying the changes that occur in XTHs during plant defense against abiotic stresses is informative for the study of the plant stress regulatory mechanism mediated by plant cell wall signals. XTH proteins in Arabidopsis thaliana was selected as the seed sequences in combination with its protein structural domains, 80 members of the BnXTH gene family were jointly identified from the whole genome of the Brassica napus ZS11, and analyzed for their encoded protein physicochemical properties, phylogenetic relationships, covariance relationships, and interoperating miRNAs. Based on the transcriptome data, the expression patterns of BnXTHs were analyzed in response to different abiotic stress treatments. The relative expression levels of some BnXTH genes under Al, alkali, salt, and drought treatments after 0, 6, 12 and 24 h were analyzed by using qRT-PCR to explore their roles in abiotic stress tolerance in B. napus. BnXTHs showed different expression patterns in response to different abiotic stress signals, indicating that the response mechanisms of oilseed rape against different abiotic stresses are also different. This paper provides a theoretical basis for clarifying the function and molecular genetic mechanism of the BnXTH gene family in abiotic stress tolerance in rapeseed.

Knockout of stigmatic ascorbate peroxidase 1 (APX1) delays pollen rehydration and germination by mediating ROS homeostasis in Brassica napus L.

The successful interaction between pollen and stigma is a critical process for plant sexual reproduction, involving a series of intricate molecular and physiological events. After self-compatible pollination, a significant reduction in reactive oxygen species (ROS) production has been observed in stigmas, which is essential for pollen grain rehydration and subsequent pollen tube growth. Several scavenging enzymes tightly regulate ROS homeostasis. However, the potential role of these ROS-scavenging enzymes in the pollen-stigma interaction in Brassica napus remains unclear. Here, we showed that the activity of ascorbate peroxidase (APX), an enzyme that plays a crucial role in the detoxification of hydrogen peroxide (H2O2), was modulated depending on the compatibility of pollination in B. napus. We then identified stigma-expressed APX1s and generated pentuple mutants of APX1s using CRISPR/Cas9 technology. After compatible pollination, the BnaAPX1 pentuple mutants accumulated higher levels of H2O2 in the stigma, while the overexpression of BnaA09.APX1 resulted in lower levels of H2O2. Furthermore, the knockout of BnaAPX1 delayed the compatible response-mediated pollen rehydration and germination, which was consistent with the effects of a specific APX inhibitor, ρ-Aminophenol, on compatible pollination. In contrast, the overexpression of BnaA09.APX1 accelerated pollen rehydration and germination after both compatible and incompatible pollinations. However, delaying and promoting pollen rehydration and germination did not affect the seed set after compatible and incompatible pollination in APX1 pentuple mutants and overexpression lines, respectively. Our results demonstrate the fundamental role of BnaAPX1 in pollen rehydration and germination by regulating ROS homeostasis during the pollen-stigma interaction in B. napus.

BnaABF3 and BnaMYB44 regulate the transcription of zeaxanthin epoxidase genes in carotenoid and abscisic acid biosynthesis.

Zeaxanthin epoxidase (ZEP) is a key enzyme that catalyzes the conversion of zeaxanthin to violaxanthin in the carotenoid and abscisic acid (ABA) biosynthesis pathways. The rapeseed (Brassica napus) genome has 4 ZEP (BnaZEP) copies that are suspected to have undergone subfunctionalization, yet the 4 genes' underlying regulatory mechanisms remain unknown. Here, we genetically confirmed the functional divergence of the gene pairs BnaA09.ZEP/BnaC09.ZEP and BnaA07.ZEP/BnaC07.ZEP, which encode enzymes with tissue-specific roles in carotenoid and ABA biosynthesis in flowers and leaves, respectively. Molecular and transgenic experiments demonstrated that each BnaZEP pair is transcriptionally regulated via ABA-responsive element-binding factor 3 s (BnaABF3s) and BnaMYB44s as common and specific regulators, respectively. BnaABF3s directly bound to the promoters of all 4 BnaZEPs and activated their transcription, with overexpression of individual BnaABF3s inducing BnaZEP expression and ABA accumulation under drought stress. Conversely, loss of BnaABF3s function resulted in lower expression of several genes functioning in carotenoid and ABA metabolism and compromised drought tolerance. BnaMYB44s specifically targeted and repressed the expression of BnaA09.ZEP/BnaC09.ZEP but not BnaA07.ZEP/BnaC07.ZEP. Overexpression of BnaA07.MYB44 resulted in increased carotenoid content and an altered carotenoid profile in petals. Additionally, RNA-seq analysis indicated that BnaMYB44s functions as a repressor in phenylpropanoid and flavonoid biosynthesis. These findings provide clear evidence for the subfunctionalization of duplicated genes and contribute to our understanding of the complex regulatory network involved in carotenoid and ABA biosynthesis in B. napus.

Efficient preparation of alginate oligosaccharides by using alginate lyases and evaluation of the development promoting effects on Brassica napus L. in saline-alkali environment.

Enzymatic degradation of alginate for the preparation of alginate oligosaccharides (AOS) is currently receiving significant attention in the field. AOS has been shown to promote crop growth and improve plant resistance to abiotic stresses. In this study, two PL6 family alginate lyases, AlyRmA and AlyRmB, were expressed and characterized. These enzymes demonstrate exceptional activity and stable thermophilicity compared to other known alginate lyases. AlyRmA (8855.34 U/mg) and AlyRmB (7879.44 U/mg) exhibited excellent degradation activity towards sodium alginate even at high temperatures (70 °C). The AlyRmA and AlyRmB were characterized and utilized to efficiently produce AOS. The study investigated the promotional effect of AOS on the growth of Brassica napus L. seedlings in a saline-alkaline environment. The results of this study demonstrate the high activity and thermal stability of AlyRmA and AlyRmB, highlighting their potential in the preparation of AOS. Moreover, the application of AOS prepared by AlyRmB could enhance the resistance of Brassica napus L. to saline-alkali environments, thereby broadening the potential applications of AOS.

Identification and characterization of the plasma membrane H+-ATPase genes in Brassica napus and functional analysis of BnHA9 in salt tolerance.

As a primary proton pump, plasma membrane (PM) H+-ATPase plays critical roles in regulating plant growth, development, and stress responses. PM H+-ATPases have been well characterized in many plant species. However, no comprehensive study of PM H+-ATPase genes has been performed in Brassica napus (rapeseed). In this study, we identified 32 PM H+-ATPase genes (BnHAs) in the rapeseed genome, and they were distributed on 16 chromosomes. Phylogenetical and gene duplication analyses showed that the BnHA genes were classified into five subfamilies, and the segmental duplication mainly contributed to the expansion of the rapeseed PM H+-ATPase gene family. The conserved domain and subcellular analyses indicated that BnHAs encoded canonical PM H+-ATPase proteins with 14 highly conserved domains and localized on PM. Cis-acting regulatory element and expression pattern analyses indicated that the expression of BnHAs possessed tissue developmental stage specificity. The 25 upstream open reading frames with the canonical initiation codon ATG were predicted in the 5' untranslated regions of 11 BnHA genes and could be used as potential target sites for improving rapeseed traits. Protein interaction analysis showed that BnBRI1.c associated with BnHA2 and BnHA17, indicating that the conserved activity regulation mechanism of BnHAs may be present in rapeseed. BnHA9 overexpression in Arabidopsis enhanced the salt tolerance of the transgenic plants. Thus, our results lay a foundation for further research exploring the biological functions of PM H+-ATPases in rapeseed.

Systematic characterization of Brassica napus UBC13 genes involved in DNA-damage response and K63-linked polyubiquitination.

BACKGROUND: Ubc13 is the only known ubiquitin conjugating enzyme (Ubc/E2) dedicated to promoting Lys (K)63-linked polyubiquitination, and this process requires a Ubc/E2 variant (UEV). Unlike conventional K48-linked polyubiquitination that targets proteins for degradation, K63-linked polyubiquitination, which is involved in several cellular processes, does not target proteins for degradation but alter their activities. RESULTS: In this study we report the identification and functional characterization of 12 Brassica napus UBC13 genes. All the cloned UBC13 gene products were able to physically interact with AtUev1D, an Arabidopsis UEV, to form stable complexes that are capable of catalyzing K63-linked polyubiquitination in vitro. Furthermore, BnUBC13 genes functionally complemented the yeast ubc13 null mutant defects in spontaneous mutagenesis and DNA-damage responses, suggesting that BnUBC13s can replace yeast UBC13 in mediating K63-linked polyubiquitination and error-free DNA-damage tolerance. CONCLUSION: Collectively, this study provides convincing data to support notions that B. napus Ubc13s promote K63-linked polyubiquitination and are probably required for abiotic stress response. Since plant Ubc13-UEV are also implicated in other developmental and stress responses, this systematic study sets a milestone in exploring roles of K63-linked polyubiquitination in this agriculturally important crop.

Evolution and functional diversity of lipoxygenase (LOX) genes in allotetraploid rapeseed (Brassica napus L.).

Lipoxygenase (LOX, EC 1.13.11.12) is a non-haeme iron-containing dioxygenase family that catalyzes the oxygenation of polyunsaturated fatty acids into bio-functionally fatty acid diverse (oxylipins) and plays vital role in plant growth and development and responses to abiotic and biotic stresses. Though LOX genes have been studied in many plant species, their roles in Brassicaceae species are still unknown. Here, a set of 14, 18, and 33 putative LOX genes were identified in Brassica rapa, Brassica oleracea and Brassica napus (allotetraploid rapeseed), respectively, which could be divided into 9-LOX (LOX1/5), 13-LOX type I (LOX3/4/6), and type II (LOX2) subgroups. There was an expansion of LOX2 orthologous genes in Brassicaceae. Most of the LOX genes are intron rich and conserved in gene structure, and the LOX proteins all have the conserved lipoxygenase and PLAT/LH2 domain. Ka/Ks ratio revealed that the majority of LOXs underwent purifying selection in Brassicaceae. The light-, ABA-, MeJA-related cis-elements and MYB-binding sites in the promoters of BnaLOXs were the most abundant. BnaLOXs displayed different spatiotemporal expression patterns and various abiotic/biotic stress responsive expression patterns. BnaLOX1/5 were slightly or no response to phytohormones and abiotic stresses. BnaLOX3/4/6 predominantly express in roots and were strongly up-regulated by salinity and PEG treatments, and BnaLOX3/4 were the methyl jasmonate (MeJA) and salicylic acid (SA) early response genes and strongly induced by infection of Sclerotinia sclerotiorum; while the BnaLOX2 members predominantly express in stamens, were MeJA and SA continuous response genes and strongly repressed by cold, heat and waterlogging treatments in leaves. Our results are useful for understanding the biological functions of the BnaLOX genes in allotetraploid rapeseed.

Oilseed rape (Brassica napus): the importance of aminopeptidases in germination under normal and heavy metals stress conditions.

BACKGROUND: Oilseed rape is one of the most important oilseed crops worldwide, crucial in the food and feed industries. Different environment and climatic conditions can influence its sustainable cultivation and crop yield. Aminopeptidases are crucial enzymes in many physiological processes in all organisms, including humans, so it is important to learn their behavior in food and feed sources. This study presents, for the first time, a detailed discussion on the importance of aminopeptidases, during the oilseed rape germination process, under standard and stress conditions. RESULTS: During the germination of oilseed rape under standard conditions, a significant increase in aminopeptidases activity toward N-terminal amino acids - phenylalanine (Phe), alanine (Ala), glycine (Gly), leucine (Leu), proline (Pro), methionine (Met) - was observed. The change was substrate specific, with the highest increase being observed for Gly (3.2-fold), followed by Ala (2.9-fold), Pro (2.5-fold), Met (1.5-fold), and Phe (1.3-fold). Generally, N-terminal Phe was preferentially cleaved. Germination under stress conditions, caused by several heavy metal ions (e.g. divalent copper, zinc, cadmium, and lead ions), negatively influenced the plants' growth and quality, but significantly enhanced the expression of genes encoding aminopeptidases (or potentially activated aminopeptidases precursors), which was related to the dramatic increase of their activity. CONCLUSIONS: The activity/concentration of aminopeptidases in plants is adjusted to the needs at each stage of development and stress factors occurrence. The most significant increase of activity toward N-terminal Gly and Pro proved the key role of aminopeptidases in the defense mechanisms, by supplying the plants with osmoprotectants and organic nitrogen. The results provide new concepts of oilseed rape growth and cultivation under different conditions. © 2021 Society of Chemical Industry.

Exogenous 3,3'-Diindolylmethane Improves Vanadium Stress Tolerance in Brassica napus Seedling Shoots by Modulating Antioxidant Enzyme Activities.

3,3'-diindolylmethane (DIM) belongs to a family of indole glucosinolate compounds that have been shown to improve Brassica napus growth through the modulation of reactive oxygen species when applied exogenously. The B. napus cultivar AV Garnet was previously identified as a vanadium-sensitive cultivar. Therefore, in this study we investigated whether exogenous DIM could improve the vanadium tolerance of AV Garnet. We performed the following experiments: seed germination assessment, dry weight assessment, cell viability assay, chlorophyll content assay, malondialdehyde (MDA) assay, conjugated diene (CD) content assay, hydrogen peroxide (H2O2) content assay, superoxide (O2-) content determination, methylglyoxal (MG) content determination, hydroxyl radical (·OH) concentration determination, ascorbate peroxidase (APX) activity assay, superoxide dismutase (SOD) activity assay, glyoxalase I (Gly I) activity assay, glutathione S-transferase (GST) activity assay and inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis for vanadium content determination. Under vanadium stress, exogenous DIM increased the seed germination percentage, shoot dry weight, cell viability and chlorophyll content. Exogenous DIM also led to a decrease in MDA, CD, H2O2, O2-, MG and ·OH, under vanadium stress in the shoots. Furthermore, DIM application led to an increase in the enzymatic activities of APX, SOD, Gly I and GST under vanadium stress. Interestingly, under vanadium stress, DIM treatment did not alter vanadium content in B. napus shoots. Our results indicate that exogenous application of DIM can improve B. napus seedling shoot growth and biomass under vanadium stress by priming the antioxidant enzymes via reactive oxygen species (ROS) signaling.

Acylation of non-specific phospholipase C4 determines its function in plant response to phosphate deficiency.

Non-specific phospholipase C (NPC) is involved in plant growth, development and stress responses. To elucidate the mechanism by which NPCs mediate cellular functions, here we show that NPC4 is S-acylated at the C terminus and that acylation determines its plasma membrane (PM) association and function. The acylation of NPC4 was detected using NPC4 isolated from Arabidopsis and reconstituted in vitro. The C-terminal Cys-533 was identified as the S-acylation residue, and the mutation of Cys-533 to Ala-533 in NPC4 (NPC4C533A ) led to the loss of S-acylation and membrane association of NPC4. The knockout of NPC4 impeded the phosphate deficiency-induced decrease of the phosphosphingolipid glycosyl inositol phosphoryl ceramide (GIPC), but introducing NPC4C533A to npc4-1 failed to complement this defect, thereby supporting the hypothesis that the non-acylated NPC4C533A fails to hydrolyze GIPC during phosphate deprivation. Moreover, NPC4C533A failed to complement the primary root growth in npc4-1 under stress. In addition, NPC4 in Brassica napus was S-acylated and mutation of the S-acylating cysteine residue of BnaC01.NPC4 led to the loss of S-acylation and its membrane association. Together, our results reveal that S-acylation of NPC4 in the C terminus is conserved and required for its membrane association, phosphosphingolipid hydrolysis and function in plant stress responses.

Membrane-bound Δ12 fatty acid desaturase (FAD12); From Brassica napus to E. coli expression system.

Fatty acid desaturase catalyzes the desaturation reactions by insertion of double bonds into the fatty acyl chain, producing unsaturated fatty acids. Though soluble fatty acid desaturases have been studied widely in advanced organisms, there are very limited studies of membrane fatty acid desaturases due to the difficulty of generating recombinant desaturase. Brassica napus is a rapeseed, which possesses a range of different membrane-bound desaturases capable of producing fatty acids including Δ3, Δ4, Δ8, Δ9, Δ12, and Δ15 fatty acids. The 1155 bp open reading frame of Δ12 fatty acid desaturase (FAD12) from Brassica napus codes for 383 amino acid residues with a molecular weight of 44 kDa. It was expressed in Escherichia coli at 37 °C in soluble and insoluble forms when induced with 0.5 mM IPTG. Soluble FAD12 has been purified using Ni2+-Sepharose affinity chromatography with a total protein yield of 0.728 mg/mL. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that desaturase activity of FAD12 could produce linoleic acid from oleic acid at a retention time of 17.6 with a conversion rate of 47%. Characterization of purified FAD12 revealed the optimal temperature of FAD12 was 50 °C with 2 mM preferred substrate concentration of oleic acid. Analysis of circular dichroism (CD) showed FAD12 was made up of 47.3% and 0.9% of alpha-helix and ß-sheet secondary structures. The predicted Tm value was 50.2 °C.

Down-regulation of MANNANASE7 gene in Brassica napus L. enhances silique dehiscence-resistance.

KEY MESSAGE: MANNANASE7 gene in Brassica napus L. encodes a hemicellulose which located at cell wall or extracellular space and dehiscence-resistance can be manipulated by altering the expression of MANNANASE7. Silique dehiscence is an important physiological process in plant reproductive development, but causes heavy yield loss in crops. The lack of dehiscence-resistant germplasm limits the application of mechanized harvesting and greatly restricts the rapeseed (Brassica napus L.) production. Hemicellulases, together with cellulases and pectinases, play important roles in fruit development and maturation. The hemicellulase gene MANNANASE7 (MAN7) was previously shown to be involved in the development and dehiscence of Arabidopsis (Arabidopsis thaliana) siliques. Here, we cloned BnaA07g12590D (BnMAN7A07), an AtMAN7 homolog from rapeseed, and demonstrate its function in the dehiscence of rapeseed siliques. We found that BnMAN7A07 was expressed in both vegetative and reproductive organs and significantly highly expressed in leaves, flowers and siliques where the abscission or dehiscence process occurs. Subcellular localization experiment showed that BnMAN7A07 was localized in the cell wall. The biological activity of the BnMAN7A07 protein isolated and purified through prokaryotic expression system was verified to catalyse the decomposition of xylan into xylose. Phenotypic studies of RNA interference (RNAi) lines revealed that down-regulation of BnMAN7A07 in rapeseed could significantly enhance silique dehiscence-resistance. In addition, the expression of upstream silique development regulators is altered in BnMAN7A07-RNAi plants, suggesting that a possible feedback regulation mechanism exists in the regulation network of silique dehiscence. Our results demonstrate that dehiscence-resistance can be manipulated by altering the expression of hemicellulase gene BnMAN7A07, which could provide an available genetic resource for breeding practice in rapeseed which is beneficial to mechanized harvest.

Interaction between Brassica napus polygalacturonase inhibition proteins and Sclerotinia sclerotiorum polygalacturonase: implications for rapeseed resistance to fungal infection.

MAIN CONCLUSION: BnPGIPs interacted with Sclerotinia sclerotiorum PGs to improve rapeseed SSR resistance at different levels; the BnPGIP-overexpression lines did not affect plant morphology or seed quality traits. Plant polygalacturonase-inhibiting proteins (PGIPs) play a crucial role in plant defence against phytopathogenic fungi by inhibiting fungal polygalacturonase (PG) activity. We overexpressed BnPGIP2, BnPGIP5, and BnPGIP10 genes in an inbred line 7492 of rapeseed (Brassica napus). Compared with 7492WT, the overexpression of BnPGIP2 lines significantly increased Sclerotinia sclerotiorum resistance in both seedlings and adult plants. BnPGIP5 overexpression lines exhibited decreased S. sclerotiorum disease symptoms in seedlings only, whereas BnPGIP10 overexpression lines did not improve Sclerotinia resistance for seedlings or adult plants. Quantitative real-time PCR analysis of S. sclerotiorum PG1, SsPG3, SsPG5, and SsPG6 genes in overexpressing BnPGIP lines showed that these pathogenic genes in the Sclerotinia resistance transgenic lines exhibited low expression in stem tissues. Split-luciferase complementation experiments confirmed the following: BnPGIP2 interacts with SsPG1 and SsPG6 but not with SsPG3 or SsPG5; BnPGIP5 interacts with SsPG3 and SsPG6 but not with SsPG1 or SsPG5; and BnPGIP10 interacts with SsPG1 but not SsPG3, SsPG5, or SsPG6. Leaf crude protein extracts from BnPGIP2 and BnPGIP5 transgenic lines displayed high inhibitory activity against the SsPG crude protein. BnPGIP-overexpression lines with Sclerotinia resistance displayed a lower accumulation of H2O2 and higher expression of the H2O2-removing gene BnAPX (ascorbate peroxidase) than 7492WT, as well as elevated expression of defence response genes including jasmonic acid/ethylene and salicylic acid pathways after S. sclerotiorum infection. The plants overexpressing BnPGIP exhibited no difference in either agronomic traits or grain yield from 7492WT. This study provides potential target genes for developing S. sclerotiorum resistance in rapeseed.

Analyses of Lysin-motif Receptor-like Kinase (LysM-RLK) Gene Family in Allotetraploid Brassica napus L. and Its Progenitor Species: An In Silico Study.

The LysM receptor-like kinases (LysM-RLKs) play a crucial role in plant symbiosis and response to environmental stresses. Brassica napus, B. rapa, and B. oleracea are utilized as valuable vegetables. Different biotic and abiotic stressors affect these crops, resulting in yield losses. Therefore, genome-wide analysis of the LysM-RLK gene family was conducted. From the genome of the examined species, 33 LysM-RLK have been found. The conserved domains of Brassica LysM-RLKs were divided into three groups: LYK, LYP, and LysMn. In the BrassicaLysM-RLK gene family, only segmental duplication has occurred. The Ka/Ks ratio for the duplicated pair of genes was less than one indicating that the genes' function had not changed over time. The BrassicaLysM-RLKs contain 70 cis-elements, indicating that they are involved in stress response. 39 miRNA molecules were responsible for the post-transcriptional regulation of 12 Brassica LysM-RLKs. A total of 22 SSR loci were discovered in 16 Brassica LysM-RLKs. According to RNA-seq data, the highest expression in response to biotic stresses was related to BnLYP6. According to the docking simulations, several residues in the active sites of BnLYP6 are in direct contact with the docked chitin and could be useful in future studies to develop pathogen-resistant B. napus. This research reveals comprehensive information that could lead to the identification of potential genes for Brassica species genetic manipulation.

A predicted transmembrane region in plant diacylglycerol acyltransferase 2 regulates specificity toward very-long-chain acyl-CoAs.

Triacylglycerols are the main constituent of seed oil. The specific fatty acid composition of this oil is strongly impacted by the substrate specificities of acyltransferases involved in lipid synthesis, such as the integral membrane enzyme diacylglycerol acyltransferase (DGAT). Two forms of DGAT, DGAT1 and DGAT2, are thought to contribute to the formation of seed oil, and previous characterizations of various DGAT2 enzymes indicate that these often are associated with the incorporation of unusual fatty acids. However, the basis of DGAT2's acyl-donor specificity is not known because of the inherent challenges of predicting structural features of integral membrane enzymes. The recent characterization of DGAT2 enzymes from Brassica napus reveals that DGAT2 enzymes with similar amino acid sequences exhibit starkly contrasting acyl-donor specificities. Here we have designed and biochemically tested a range of chimeric enzymes, substituting parts of these B. napus DGAT2 enzymes with each other, allowing us to pinpoint a region that dramatically affects the specificity toward 22:1-CoA. It may thus be possible to redesign the acyl-donor specificity of DGAT2 enzymes, potentially altering the fatty acid composition of seed oil. Further, the characterization of a DGAT2 chimera between Arabidopsis and B. napus demonstrates that the specificity regulated by this region is transferrable across species. The identified region contains two predicted transmembrane helices that appear to reoccur in a wide range of plant DGAT2 orthologues, suggesting that it is a general feature of plant DGAT2 enzymes.

Cleaved amplified polymorphic sequences (CAPS) marker for identification of two mutant alleles of the rapeseed BnaA.FAD2 gene.

Two mutants of winter rapeseed (Brassica napus L. var. oleifera) with an increased amount of oleic acid in seeds were created by chemical mutagenesis (HOR3-M10453 and HOR4-M10464). The overall performance of the mutated plants was much lower than that of wild-type cultivars. Multiple rounds of crossing with high-yielding double-low ("00") cultivars and breeding lines having valuable agronomic traits, followed by selection of high oleic acid genotypes is then needed to obtain new "00" varieties of rapeseed having high oleic acid content in seeds. To perform such selection, the specific codominant cleaved amplified polymorphic sequences (CAPS) marker was used. This marker was designed to detect the presence of two relevant point mutations in the desaturase gene BnaA.FAD2, and it was previously described and patented. The specific polymerase chain reaction product (732 bp) was digested using FspBI restriction enzyme that recognizes the 5'-C↓TAG-3' sequence which is common to both mutated alleles, thereby yielding band patterns specific for those alleles. The method proposed in the patent was redesigned, adjusted to specific laboratory conditions, and thoroughly tested. Different DNA extraction protocols were tested to optimize the procedure. Two variants of the CAPS method (with and without purification of amplified product) were considered to choose the best option. In addition, the ability of the studied marker to detect heterozygosity in the BnaA.FAD2 locus was also tested. Finally, we also presented some examples for the use of the new CAPS marker in the marker-assisted selection (MAS) during our breeding programs. The standard CTAB method of DNA extraction and the simplified, two-step (amplification/digestion) procedure for the CAPS marker are recommended. The marker was found to be useful for the detection of two mutated alleles of the studied BnaA.FAD2 desaturase gene and can potentially assure the breeders of the purity of their HOLL lines. However, it was also shown that it could not detect any other alleles or genes that were revealed to play a role in the regulation of oleic acid level.

Genome-wide identification and expression profiling of the carotenoid cleavage dioxygenase (CCD) gene family in Brassica napus L.

Carotenoid cleavage dioxygenase (CCD), a key enzyme in carotenoid metabolism, cleaves carotenoids to form apo-carotenoids, which play a major role in plant growth and stress responses. CCD genes had not previously been systematically characterized in Brassica napus (rapeseed), an important oil crop worldwide. In this study, we identified 30 BnCCD genes and classified them into nine subgroups based on a phylogenetic analysis. We identified the chromosomal locations, gene structures, and cis-promoter elements of each of these genes and performed a selection pressure analysis to identify residues under selection. Furthermore, we determined the subcellular localization, physicochemical properties, and conserved protein motifs of the encoded proteins. All the CCD proteins contained a retinal pigment epithelial membrane protein (RPE65) domain. qRT-PCR analysis of expression of 20 representative BnCCD genes in 16 tissues of the B. napus cultivar Zhong Shuang 11 ('ZS11') revealed that members of the BnCCD gene family possess a broad range of expression patterns. This work lays the foundation for functional studies of the BnCCD gene family.

Gene silencing of BnaA09.ZEP and BnaC09.ZEP confers orange color in Brassica napus flowers.

Brassica napus is currently cultivated as an important ornamental crop in China. Flower color has attracted much attention in rapeseed genetics and breeding. Here, we characterize an orange-flowered mutant of B. napus that exhibits an altered carotenoid profile in its petals. As revealed by map-based cloning, the change in color from yellow to orange is attributed to the loss of BnaC09.ZEP (zeaxanthin epoxidase) and a 1695-bp deletion in BnaA09.ZEP. HPLC analysis, genetic complementation and CRISPR/Cas9 experiments demonstrated that BnaA09.ZEP and BnaC09.ZEP have similar functions, and the abolishment of both genes led to a substantial increase in lutein content and a sharp decline in violaxanthin content in petals but not leaves. BnaA09.ZEP and BnaC09.ZEP are predominantly expressed in floral tissues, whereas their homologs, BnaA07.ZEP and BnaC07.ZEP, mainly function in leaves, indicating redundancy and tissue-specific diversification of BnaZEP function. Transcriptome analysis in petals revealed differences in the expression of carotenoid and flavonoid biosynthesis-related genes between the mutant and its complementary lines. Flavonoid profiles in the petals of complementary lines were greatly altered compared to the mutant, indicating potential cross-talk between the regulatory networks underlying the carotenoid and flavonoid pathways. Additionally, our results indicate that there is functional compensation by BnaA07.ZEP and BnaC07.ZEP in the absence of BnaA09.ZEP and BnaC09.ZEP. Cloning and characterization of BnaZEPs provide insights into the molecular mechanisms underlying flower pigmentation in B. napus and would facilitate breeding of B. napus varieties with higher ornamental value.

Synergistic mutations of two rapeseed AHAS genes confer high resistance to sulfonylurea herbicides for weed control.

KEY MESSAGE: A double mutant 5N of rapeseed was obtained with a synergistic effect of high resistance to sulfonylurea herbicide. Excellent weed control was observed in Ning R201 created by 5N resources. Sulfonylurea herbicides, which inhibit acetohydroxyacid synthase (AHAS), have become the most widely used herbicides worldwide. However, weed control in rapeseed crop production remains challenging in China due to the shortage of available herbicide-resistant cultivars. In this study, we developed a rapeseed line (PN19) with sulfonylurea herbicide resistance through seed mutagenesis. Molecular analysis revealed a Trp-574-Leu mutation in BnAHAS1-2R of PN19 according to the sequence of Arabidopsis thaliana, and an allele-specific cleaved amplified polymorphic sequence marker was developed to target the point mutation. A double mutant (5N) with very high sulfonylurea resistance was then created through pyramiding two mutant genes of PN19 and M342 by molecular marker-assisted selection. Herbicide resistance identification, toxicology testing, and an in vitro enzyme activity assay of AHAS in 5N indicated that each mutant was four and eight times more resistant to sulfonylurea than M342 and PN19, respectively. Protein structure analysis of AHAS1 demonstrated that the leucine of mutant Trp-574-Leu destroyed the original π-plane stacking effect of the local region for tribenuron-methyl binding, leading to herbicide tolerance. Isobole graph analysis showed a significant synergistic effect of the combination of two mutant genes in 5N for improved tolerance to sulfonylurea herbicides. Finally, we bred rapeseed variety Ning R201 using 5N herbicide resistance resources, and observed excellent weed control performance. Together, these results demonstrate the practical value of 5N application for optimizing and simplifying rapeseed cultivation in China.