Overexpression of Cinnamoyl-CoA Reductase 2 in Brassica napus Increases Resistance to Sclerotinia sclerotiorum by Affecting Lignin Biosynthesis

Sclerotinia sclerotiorum causes severe yield and economic losses for many crop and vegetable species, especially Brassica napus. To date, no immune B. napus germplasm has been identified, giving rise to a major challenge in the breeding of Sclerotinia resistance. In the present study, we found that, compared with a Sclerotinia-susceptible line (J902), a Sclerotinia-resistant line (J964) exhibited better xylem development and a higher lignin content in the stems, which may limit the invasion and spread of S. sclerotiorum during the early infection period. In addition, genes involved in lignin biosynthesis were induced under S. sclerotiorum infection in both lines, indicating that lignin was deposited proactively in infected tissues. We then overexpressed BnaC.CCR2.b, which encodes the first rate-limiting enzyme (cinnamoyl-CoA reductase) that catalyzes the reaction of lignin-specific pathways, and found that overexpression of BnaC.CCR2.b increased the lignin content in the stems of B. napus by 2.28–2.76% under normal growth conditions. We further evaluated the Sclerotinia resistance of BnaC.CCR2.b overexpression lines at the flower-termination stage and found that the disease lesions on the stems of plants in the T2 and T3 generations decreased by 12.2–33.7% and 32.5–37.3% compared to non-transgenic control plants, respectively, at 7days post-inoculation (dpi). The above results indicate that overexpression of BnaC.CCR2.b leads to an increase in lignin content in the stems, which subsequently leads to increased resistance to S. sclerotiorum. Our findings demonstrate that increasing the lignin content in the stems of B. napus is an important strategy for controlling Sclerotinia.

The rice SCFOsFBK1 E3 ligase mediates jasmonic acid induced degradation of a RING-H2 protein and the cinnamoyl-CoA reductase, OsCCR14

We had previously shown the rice F-box, OsFBK1, plays a role in anther development by mediating the turnover of OsCCR14, a cinnamoyl CoA-reductase regulating lignification. Another substrate identified in the same Y2H library screening was OsATL53, a member of the ATL family of RING-H2 proteins that is primarily localized to the cytoplasm. We found OsATL53 to be a component and substrate of SCFOsFBK1 by immunoprecipitation and cell-free studies. Incidentally, OsATL53 was found to interact with OsCCR14 in the cytoplasm and form a stable complex in cell-free experiments and bimolecular fluorescence complementation assays. Biochemically, OsATL53 was found to influence the enzymatic activity of OsCCR14 by decreasing its efficiency. Degradation studies have shown OsFBK1 mediates turnover of OsCCR14 in the nucleus, while OsATL53 is degraded in both cytoplasm and nucleus. The degradation of ATLs by F-box proteins has not been reported before. In presence of jasmonic acid (JA), which plays a role in anther dehiscence, OsATL53 has been found to gather around the nucleus, and this property enables the translocation of the OsATL53-OsCCR14 complex from a cytoplasmic localization to accumulate around the nuclear periphery. FLIM analyses revealed OsCCR14-OsATL53 complex undergoing conformational changes in presence of JA and this triggers OsFBK1 to mediate the targeted degradation of OsATL53 in the cytoplasm, thereby dissociating the cytoplasmic OsCCR14-OsATL53 complex and enabling OsCCR14 to enter the nucleus and eventually get degraded by SCFOsFBK1 E3 ligase. We have thus studied the signalling mechanism of a variant JA-induced E3 ligase-mediated substrate turnover in plants at the molecular level.

Cinnamoyl-CoA Reductase 1 (CCR1) and CCR2 Function Divergently in Tissue Lignification, Flux Control and Cross-talk with Glucosinolate Pathway in Brassica napus

Cinnamoyl-CoA reductase (CCR) is the entry point of lignin pathway and a crucial locus in dissection and manipulation of associated traits. Brassica crops have worldwide importance, but their CCR-related metabolisms and traits are largely uncharacterized. Here, 16 CCR genes are identified from B. napus and its parental species B. rapa and B. oleracea. They are divided into CCR1 subfamily and CCR2 subfamily, which differ from each other in organ-specificity, participation in yellow-seed trait and responses to various stresses. BnCCR1 is preferentially involved in biosynthesis of G and H lignins and development of vascular system, while BnCCR2 is preferentially involved in biosynthesis of S lignin and development of interfascicular fibers. BnCCR1 has stronger effects on lignification-related development, lodging resistance, flux control and seed color, whereas BnCCR2 has stronger effect on sinapates biosynthesis. BnCCR1 overexpressing plants show a delay in bolting and flowering, while BnCCR2 overexpressing plants have less developed vascular system in leaf due to suppressed G lignin accumulation. Unexpectedly, both BnCCR1 and BnCCR2 overexpressors show no improvement in resistance to UV-B and S. sclerotiorum. Besides, their glucosinolate profiles are greatly and almost oppositely remodeled through pathway crosstalk. These results provide systemic dissection on Brassica CCRs and CCR1-CCR2 divergence in Brassicaceae.