Characterisation of Polyamines and Their Biosynthetic Pathways Contributing to Postharvest Anthracnose Resistance in Mango ( <scp> <i>Mangifera indica</i> </scp> L.)

Polyamines and their derivatives, such as phenolamides, play essential roles in plant defence systems, especially under biotic stress conditions (Gerlin et al. 2021). Anthracnose, caused by Colletotrichum gloeosporioides, is a major postharvest disease affecting mango (Mangifera indica L.), leading to significant economic losses during storage and transportation. Sustainable approaches that leverage mango's intrinsic defence mechanisms offer promising alternatives for managing postharvest diseases. This study identifies p-coumaroyl-feruloyl-putrescine (CFP), a phenolamide derived from polyamines, as a critical metabolite in mango's defence against anthracnose, and investigates its biosynthesis, regulation, and antifungal efficacy. To identify key metabolites involved in mango's response to anthracnose, we conducted a time-course metabolomic analysis of infected (T1d to T3d, 1–3 days post-inoculation) and control (CK0d to CK3d) fruit samples, with each sample comprising three biological replicates. Using UPLC–MS, we identified 175 distinct alkaloids, including phenolamides (Table S1). Metabolite values were normalised using Z-scores before performing orthogonal partial least squares discriminant analysis (OPLS-DA). The OPLS-DA model revealed distinct metabolic profiles between infected and control samples, highlighting infection-driven metabolic reprogramming (Figure 1a). Among the metabolites, CFP emerged as the most significant, exhibiting the highest score and a pronounced accumulation during the early stages of infection (Figure 1a–c; Tables S2 and S3). These findings suggest that CFP plays a central role as a rapidly induced defence metabolite during the initial response to anthracnose. To elucidate the biosynthetic pathways underlying CFP production, we conducted a transcriptomic analysis to identify genes associated with CFP production (Tables S4 and S5). k-means clustering was performed using the Euclidean distance metric. Metabolites and genes were grouped into five distinct clusters (Figure 1d). Cluster 3, containing CFP and seven other metabolites, displayed a sharp increase at 1 and 2 dpi, followed by a decline at 3 dpi, reflecting an early and transient defence response. The genes in cluster 3 showed expression profiles closely mirroring the temporal patterns observed in metabolomic cluster 3, confirming a coordinated activation of metabolic and transcriptional defences. In this cluster, a series of genes involved in phenylpropanoid and polyamine pathways were identified. Co-expression analysis further linked these genes to CFP biosynthesis (Figure 1e; Table S6), suggesting that phenylpropanoid and polyamine pathway genes are temporally and spatially regulated to regulate CFP biosynthesis. Then we proposed the biosynthetic pathways leading to CFP synthesis by integrating both the phenylpropanoid and polyamine pathways (Figure 1f; Table S7). Enzymes in the phenylpropanoid pathway, including phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), and caffeic acid O-methyltransferase (OMT), were significantly upregulated during infection. These enzymes synthesise the acyl donors p-coumaroyl-CoA and feruloyl-CoA (Wang et al. 2023). Similarly, arginine decarboxylase (ADC), a critical enzyme in the polyamine pathway, showed elevated expression, producing putrescine as the amine donor for CFP synthesis. The final steps of CFP biosynthesis involve the conjugation of p-coumaroyl-CoA and feruloyl-CoA with putrescine, catalysed by acyltransferase (AT) and berberine bridge enzyme-like (BBL) proteins (Bai et al. 2022). To identify regulatory elements involved in CFP biosynthesis, we focused on MYB14 and MYB15, two Jasmonic acid (JA) responsive transcription factors (He et al. 2020). These regulators showed strong co-expression with CFP biosynthetic genes (Figure 1e), indicating their central role in activating CFP production. Functional validation of CFP biosynthetic genes was performed using transient overexpression in Nicotiana benthamiana leaves (Figure 1g; Table S8). Overexpression of individual biosynthetic genes did not significantly increase CFP accumulation. However, the expression of MYB14 or MYB15 led to substantial production of CFP and its precursor, feruloyl-putrescine (FP), reinforcing the regulatory role of these transcription factors in orchestrating the phenylpropanoid and polyamine pathways. Additionally, exogenous JA treatments significantly enhanced the expression of MYB14, MYB15, and CFP-related genes, further supporting the role of JA as a key hormonal signal driving phenolamide-mediated defence. In contrast, salicylic acid (SA) treatments had minimal effects on CFP biosynthetic gene expression (Figure 1h). To assess the antifungal efficacy of CFP, its precursors, putrescine and FP, were selected due to the unavailability of commercially standardised CFP compounds. We conducted in vitro and in vivo assays (Figure S1). In vitro antifungal activity was evaluated by incorporating putrescine and FP into potato dextrose agar (PDA) medium and measured fungal colony growth to assess inhibitory effects. For in vivo assays, mango and apple fruits were inoculated with C. gloeosporioides conidia, followed by the application of putrescine or FP to the infection site. In vitro tests revealed that neither putrescine nor FP inhibited fungal growth on PDA plates, indicating limited direct antifungal activity under these conditions. However, in vivo assays demonstrated significant reductions in anthracnose lesion sizes on mango and apple fruits treated with putrescine or FP. FP exhibited greater protective effects than putrescine, reducing lesion sizes by approximately 30% compared to untreated controls. This context-specific activity suggests that CFP and its precursors function by modulating host defences and fortifying structural barriers, rather than directly inhibiting fungal growth (Zeiss et al. 2021). In conclusion, CFP plays a pivotal role in mango's defence against anthracnose, synthesised through the coordinated action of phenylpropanoid and polyamine pathways and regulated by MYB14 and MYB15 under JA signalling (Figure 1i). B.A. and B.Z. designed the project. B.Z., L.H., Q.X., and Q.W. performed the experiments. B.Z., L.H. and Q.W. analysed the data. B.A., B.Z., Q.W. and H.L. wrote the manuscript. This research was funded by the Central Public-interest Scientific Institution Basal Research Fund for CATAS (1630032022007); National Natural Science Foundation of China (32160594, 32260042). The data that support the findings of this study are available in the Supporting Information of this article. Figure S1. Tables S1–S8. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.