Overexpression of BnaA10.WRKY75 Decreases Cadmium and Salt Tolerance via Increasing ROS Accumulation in Arabidopsis and Brassica napus L.

Soil is indispensable for agricultural production but has been seriously polluted by cadmium and salt in recent years. Many crops are suffering from this, including rapeseed, the third largest global oilseed crop. However, genes simultaneously related to both cadmium and salt stress have not been extensively reported yet. In this study, BnaA10.WRKY75 was screened from previous RNA-seq data related to cadmium and salt stress and further analyses including sequence comparison, GUS staining, transformation and qRT-PCR were conducted to confirm its function. GUS staining and qRT-PCR results indicated BnaA10.WRKY75 was induced by CdCl2 and NaCl treatment. Sequence analysis suggested BnaA10.WRKY75 belongs to Group IIc of the WRKY gene family and transient expression assay showed it was a nuclear localized transcription factor. BnaA10.WRKY75-overexpressing Arabidopsis and rapeseed plants accumulated more H2O2 and O2- and were more sensitive to CdCl2 and NaCl treatment compared with untransformed plants, which may be caused by the downregulation of BnaC03.CAT2. Our study reported that BnaA10.WRKY75 increases sensitivity to cadmium and salt stress by disrupting the balance of reactive oxygen species both in Arabidopsis and rapeseed. The results support the further understanding of the mechanisms underlying cadmium and salt tolerance and provide BnaA10.WRKY75 as a valuable gene for rapeseed abiotic stress breeding.

Genetic mapping and physiological analysis of chlorophyll-deficient mutant in Brassica napus L.

BACKGROUND: Leaf color mutants have reduced photosynthetic efficiency, which has severely negative impacts on crop growth and economic product yield. There are different chlorophyll mutants in Arabidopsis and crops that can be used for genetic control and molecular mechanism studies of chlorophyll biosynthesis, chloroplast development and photoefficiency. Chlorophyll mutants in Brassica napus are mostly used for mapping and location research but are rarely used for physiological research. The chlorophyll-deficient mutant in this experiment were both genetically mapped and physiologically analyzed. RESULTS: In this study, yellow leaf mutant of Brassica napus L. mutated by ethyl methyl sulfone (EMS) had significantly lower chlorophyll a, b and carotenoid contents than the wild type, and the net photosynthetic efficiency, stomatal conductance and transpiration rate were all significantly reduced. The mutant had sparse chloroplast distribution and weak autofluorescence. The granule stacks were reduced, and the shape was extremely irregular, with more broken stromal lamella. Transcriptome data analysis enriched the differentially expressed genes mainly in phenylpropane and sugar metabolism. The mutant was mapped to a 2.72 Mb region on A01 by using BSA-Seq, and the region was validated by SSR markers. CONCLUSIONS: The mutant chlorophyll content and photosynthetic efficiency were significantly reduced compared with those of the wild type. Abnormal chloroplasts and thylakoids less connected to the stroma lamella appeared in the mutant. This work on the mutant will facilitate the process of cloning the BnaA01.cd gene and provide more genetic and physiological information concerning chloroplast development in Brassica napus.

Integrated genetic mapping and transcriptome analysis reveal the BnaA03.IAA7 protein regulates plant architecture and gibberellin signaling in Brassica napus L.

KEY MESSAGE: A novel mutation in the BnaA03.IAA7 protein reduces plant height and enhances gibberellin signaling in Brassica napus L. Rapeseed (Brassica napus) is an excellent and important source for vegetable oil production, but its production is severely affected by lodging. Lodging hinders mechanization and decreases yield, and an ideal solution is semidwarf breeding. Limited by germplasm resources, semidwarf breeding developed slowly in rapeseed. In the current study, a mutant called sdA03 was isolated from EMS-mutagenized lines of Zhongshuang 11 (ZS11). The inheritance analysis showed that phenotypes of sdA03 were controlled by a single semidominant gene. Genetic mapping, RNA-seq and candidate gene analysis identified BnaA03.IAA7 as a candidate gene, and a function test confirmed that the mutated BnaA03.iaa7 regulates plant architecture in a dose-dependent manner. Yeast two-hybrid and transient expression experiments illustrated the P87L substitution in the GWPPV/I degron motif of BnaA03.iaa7 impaired the interaction between BnaA03.IAA7 and TIR1 proteins, and BnaA03.iaa7 prevented ARF from activating the auxin signaling pathway.The gibberellin (GA) content was higher in sdA03 hypocotyls than in those of ZS11. Further expression analysis showed more active gibberellin signaling in hypocotyl and richer expression of GA synthetic genes in root and cotyledon of sdA03 seedlings. Finally, a marker was developed based on the SNP found in BnaA03.iaa7 and used in molecular breeding. The study enriched our understanding of the architectural regulation of rapeseed and provided germplasm resources for breeding.

Genome-wide exploration and characterization of miR172/euAP2 genes in Brassica napus L. for likely role in flower organ development.

BACKGROUND: APETALA2-like genes encode plant-specific transcription factors, some of which possess one microRNA172 (miR172) binding site. The miR172 and its target euAP2 genes are involved in the process of phase transformation and flower organ development in many plants. However, the roles of miR172 and its target AP2 genes remain largely unknown in Brassica napus (B. napus). RESULTS: In this study, 19 euAP2 and four miR172 genes were identified in the B. napus genome. A sequence analysis suggested that 17 euAP2 genes were targeted by Bna-miR172 in the 3' coding region. EuAP2s were classified into five major groups in B.napus. This classification was consistent with the exon-intron structure and motif organization. An analysis of the nonsynonymous and synonymous substitution rates revealed that the euAP2 genes had gone through purifying selection. Whole genome duplication (WGD) or segmental duplication events played a major role in the expansion of the euAP2 gene family. A cis-regulatory element (CRE) analysis suggested that the euAP2s were involved in the response to light, hormones, stress, and developmental processes including circadian control, endosperm and meristem expression. Expression analysis of the miR172-targeted euAP2s in nine different tissues showed diverse spatiotemporal expression patterns. Most euAP2 genes were highly expressed in the floral organs, suggesting their specific functions in flower development. BnaAP2-1, BnaAP2-5 and BnaTOE1-2 had higher expression levels in late-flowering material than early-flowering material based on RNA-seq and qRT-PCR, indicating that they may act as floral suppressors. CONCLUSIONS: Overall, analyses of the evolution, structure, tissue specificity and expression of the euAP2 genes were peformed in B.napus. Based on the RNA-seq and experimental data, euAP2 may be involved in flower development. Three euAP2 genes (BnaAP2-1, BnaAP2-5 and BnaTOE1-2) might be regarded as floral suppressors. The results of this study provide insights for further functional characterization of the miR172 /euAP2 module in B.napus.

Genome-Wide Identification of the LAC Gene Family and Its Expression Analysis Under Stress in Brassica napus.

Lignin is an important biological polymer in plants that is necessary for plant secondary cell wall ontogenesis. The laccase (LAC) gene family catalyzes lignification and has been suggested to play a vital role in the plant kingdom. In this study, we identified 45 LAC genes from the Brassica napus genome (BnLACs), 25 LAC genes from the Brassica rapa genome (BrLACs) and 8 LAC genes from the Brassica oleracea genome (BoLACs). These LAC genes could be divided into five groups in a cladogram and members in same group had similar structures and conserved motifs. All BnLACs contained hormone- and stress- related elements determined by cis-element analysis. The expression of BnLACs was relatively higher in the root, seed coat and stem than in other tissues. Furthermore, BnLAC4 and its predicted downstream genes showed earlier expression in the silique pericarps of short silique lines than long silique lines. Three miRNAs (miR397a, miR397b and miR6034) target 11 BnLACs were also predicted. The expression changes of BnLACs under series of stresses were further investigated by RNA sequencing (RNA-seq) and quantitative real-time polymerase chain reaction (qRT-PCR). The study will give a deeper understanding of the LAC gene family evolution and functions in B. napus.