Sufficient coumarin accumulation improves apple resistance toCytospora maliunder high-potassium status

Abstract

Cytospora canker, caused by Cytospora mali, is the most destructive disease in production of apples (Malus domestica). Adding potassium (K) to apple trees can effectively control this disease. However, the underlying mechanisms of apple resistance to C. mali under high-K status remain unknown. Here, we found that high K (HK, 9.30 g/kg) apple tissues exhibited high disease resistance. The resistance was impeded when blocking K channels, leading to susceptibility even under HK conditions. We detected a suite of resistance events in HK apple tissues, including upregulation of resistance genes, callose deposition, and formation of ligno-suberized tissues. Further multi-omics revealed that the phenylpropanoid pathway was reprogrammed by increasing K content from low K (LK, 4.30 g/kg) status, leading to increases of 18 antifungal chemicals. Among them, the physiological concentration of coumarin (1,2-benzopyrone) became sufficient to inhibit C. mali growth in HK tissues, and exogenous application could improve the C. mali resistance of LK apple branches. Transgenic apple calli overexpressing beta-glucosidase 40 (MdBGLU40), which encodes enzyme for coumarin synthesis, contained higher levels of coumarin and exhibited high resistance to C. mali even under LK conditions. Conversely, suppression of MdBGLU40 through RNAi reduced coumarin content and resistance in HK apple calli, supporting the importance of coumarin accumulation in vivo for apple resistance. Moreover, we found that the upregulation of transcription factor MdMYB1r1 directly activated MdBGLU40, and the binding affinity of MdMYB1r1 to the MdBGLU40 promoter increased in HK apple tissue, leading to high levels of coumarin and resistance in HK apple. Overall, we found that accumulation of defensive metabolites strengthened resistance in apple when raising K from insufficient to optimal status, and these results highlight the optimization of K content in fertilization practices as a disease management strategy.

Major quality regulation network of flavonoid synthesis governing the bioactivity of black wolfberry

“…The minimum number of samples necessary in one group was set as 3 for its validation. Identification of metabolites was performed (Du et al ., 2023) based on MS/MS and retention time information of each compound by software P eak V iew and xcms online (https://xcmsonline.scripps.edu). An in‐house MS/MS database (BiotreeDB) based on standard metabolites from Shanghai Biotree Biomedical Technology (Shanghai, China) was applied in metabolite annotation.…”

“…Typically, the flavonoids that exhibited antioxidant activity were further identified and aligned by standard metabolites according to its MS1 information (ppm < 10) and retention time. Further metabolomic analysis was performed via M etaboanalyst (Du et al ., 2023). Differential metabolites were screened based on the threshold P < 0.05 and fold change > 2.0.…”

“…The full coding sequence of LrMYB94 / LrWRKY32 was amplified with a primer pair containing BamHI site (Dataset S2) and ligated to pBIN‐eGFP vector as described (Du et al ., 2023). The pBIN:LrMYB94‐eGFP and pBIN:LrWRKY32‐eGFP plasmids were introduced into Agrobacterium strain LBA4404 and transformed into black wolfberry calli.…”

“…For the MS conditions, the capillary temperature and source voltage were 550°C and 3.5 kV, respectively. Both positive and negative modes were analyzed following Du et al (2023).…”

“…Subsequently, Agrobacterium strains harboring LrCYP71B1-Pro:LUC, pSK:LrWRKY32 together with 35S:LrMYB94 (no GFP) were co-transformed into N. benthamiana leaves. Then, the luciferase activity assay was performed using the Firefly Luciferase Reporter Gene Assay Kit (Beyotime, Shanghai, China) following Du et al (2023). To investigate the effects of environmental factors in affecting gene expression, the recombinant Agrobacterium GV3101 containing LrFLS/LrCYP75B1/LrCHS/LrF3HPro: LUC vector was transformed into black wolfberry leaves (Huang et al, 2024).…”