Sclerotinia sclerotiorum causes a devastating disease in oilseed rape (Brassica napus) resulting in a tremendous yield loss worldwide. Studies on various host-pathogen interactions have shown that plant WRKY transcription factors are essential for defence. For the B. napus-S. sclerotiorum interaction, little direct evidence has been found with regard to the biological roles of specific WRKY genes in host resistance. In this study, we isolated a B. napus WRKY gene, BnWRKY33, and found that the gene is highly responsive to S. sclerotiorum infection. Transgenic B. napus plants overexpressing BnWRKY33 showed markedly enhanced resistance to S. sclerotiorum, constitutive activation of the expression of BnPR1 and BnPDF1.2, and inhibition of H2 O2 accumulation in response to pathogen infection. Further, we isolated a mitogen-activated protein (MAP) kinase substrate gene, BnMKS1, and found that not only can BnWRKY33 interact with BnMKS1, which can also interact with BnMPK4, using the yeast two-hybrid assay, consistent with their collective nuclear localization, but also BnWRKY33, BnMKS1 and BnMPK4 are substantially and synergistically expressed in response to S. sclerotiorum infection. In contrast, the three genes showed differential expression in response to phytohormone treatments. Together, these results suggest that BnWRKY33 plays an important role in B. napus defence to S. sclerotiorum, which is most probably associated with the activation of the salicylic acid (SA)- and jasmonic acid (JA)-mediated defence response and inhibition of H2 O2 accumulation, and we propose a potential mechanism in which BnMPK4-BnMKS1-BnWRKY33 exist in a nuclear localized complex to regulate resistance to S. sclerotiorum in oilseed rape.
Background: Triacylglycerols (TAGs) are the main composition of plant seed oil. Long-chain acyl-coenzyme A synthetases (LACSs) catalyze the synthesis of long-chain acyl-coenzyme A, which is one of the primary substrates for TAG synthesis. In Arabidopsis, the LACS gene family contains nine members, among which LACS1 and LACS9 have overlapping functions in TAG biosynthesis. However, functional characterization of LACS proteins in rapeseed have been rarely reported. Results: An orthologue of the Arabidopsis LACS2 gene (BnLACS2) that is highly expressed in developing seeds was identified in rapeseed (Brassica napus). The BnLACS2-GFP fusion protein was mainly localized to the endoplasmic reticulum, where TAG biosynthesis occurs. Interestingly, overexpression of the BnLACS2 gene resulted in significantly higher oil contents in transgenic rapeseed plants compared to wild type, while BnLACS2-RNAi transgenic rapeseed plants had decreased oil contents. Furthermore, quantitative real-time PCR expression data revealed that the expression of several genes involved in glycolysis, as well as fatty acid (FA) and lipid biosynthesis, was also affected in transgenic plants. Conclusions: A long chain acyl-CoA synthetase, BnLACS2, located in the endoplasmic reticulum was identified in B. napus. Overexpression of BnLACS2 in yeast and rapeseed could increase oil content, while BnLACS2-RNAi transgenic rapeseed plants exhibited decreased oil content. Furthermore, BnLACS2 transcription increased the expression of genes involved in glycolysis, and FA and lipid synthesis in developing seeds. These results suggested that BnLACS2 is an important factor for seed oil production in B. napus.
Seed storage oil, in the form of triacylglycerol (TAG), is degraded to provide carbon and energy during germination and early seedling growth by the fatty acid β-oxidation in the peroxisome. Although the pathways for lipid degradation have been uncovered, understanding of the exact involved enzymes in soybean is still limited. Long-chain acyl-CoA synthetase (ACSL) is a critical enzyme that activates free fatty acid released from TAG to form the fatty acyl-CoA. Recent studies have shown the importance of ACSL in lipid degradation and synthesis, but few studies were focused on soybean. In this work, we cloned a ACSL gene from soybean and designated it as GmACSL2. Sequence analysis revealed that GmACSL2 encodes a protein of 733 amino acid residues, which is highly homologous to the ones in other higher plants. Complementation test showed that GmACSL2 could restore the growth of an ACS-deficient yeast strain (YB525). Co-expression assay in Nicotiana benthamiana indicated that GmACSL2 is located at peroxisome. Expression pattern analysis showed that GmACSL2 is highly expressed in germinating seedling and strongly induced 1 day after imbibition, which indicate that GmACSL2 may take part in the seed germination. GmACSL2 overexpression in yeast and soybean hairy root severely reduces the contents of the lipids and fatty acids, compared with controls in both cells, and enhances the β-oxidation efficiency in yeast. All these results suggest that GmACSL2 may take part in fatty acid and lipid degradation. In conclusion, peroxisomal GmACSL2 from Glycine max probably be involved in the lipid degradation during seed germination.
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