Graphene oxide enhances aphid resistance in sorghum via the miR319-SbTCP7-SbLOX3 Pathway

Abstract

The aphid (Melanaphis sacchari) has emerged as a formidable pest, devastating sorghum plants and highlighting the need for sustainable management strategies. Graphene oxide (GO), as a novel material, has garnered attention for its use in crop cultivation and management, but its effects on biotic stresses remain elusive. Here, we used 10 mg/L GO to spray aphid-stressed sorghum seedlings four times in total. GO exposure reduced 50% H₂O₂ from the reactive oxygen species (ROS) burst induced by the aphid. Further analysis revealed that GO within the cells acts as a nanozyme, mimicking and enhancing the catalytic activity of the ROS-scavenging system to maintain ROS homeostasis, protecting normal plant growth and development under aphid stress. Moreover, the moderate increase in H₂O₂ in GO-treated, aphid-infected seedlings blocked the biogenesis of miR319, leading to the induction of its target gene SbTCP7, which in turn activated the transcription of SbLOX3, a rate-limiting enzyme in jasmonic acid (JA) biosynthesis. Subsequent molecular and genetic assays confirmed that the miR319-SbTCP7 module enhances JA metabolism, promoting the accumulation of JA and its active derivative jasmonic acid-isoleucine (JA-Ile) to combat aphids. Our results suggest that GO, as a potential nanozyme, enhances the aphid resistance of sorghum through the miR319-SbTCP7 module to regulate JA synthesis, indicating a novel cultivation strategy for improving pest management via nanomaterials. This frontier research has opened new avenues for crop protection against invasive pests like aphids.

Keywords: aphid; graphene oxide; jasmonic acid (JA); miR319; sorghum.

Figure 1

Graphene oxide mimics ROS‐scavenging enzyme activity in sorghum cells to enhance aphid resistance (a) Characteristics of GO nanoparticles (I) Scanning electron microscope image and the Raman spectra of the used GO. D and G represented the disordered structure of graphene caused by oxidation and π bonds formed by sp2 hybridization, respectively. (II–IV) Raman spectra from leaves, stems and roots of GO‐treated sorghum seedlings. (b) Phenotypes of sorghum seedlings with different treatments at 10 dpi. Scale bars, 10 cm. (c–e) Plant height, fresh weight and aphid number of sorghum seedlings under different treatments at 10 dpi, mean ± SD, n = 10. Different letters indicate significant differences at P < 0.05 based on one‐way analysis of variance (ANOVA). (f) Antioxidant enzyme activities and contents of H₂O₂ in sorghum seedlings under different treatments. SOD, POD, CAT and APX activity (U min⁻¹ g⁻¹ FW) and H₂O₂ content (μmol g⁻¹ FW), mean ± SD, n = 3. Different letters indicate significant differences at P < 0.05 based on one‐way ANOVA. Heatmap of key antioxidant gene expression from RNA‐seq data. Gene expression level is indicated by log₂ and converted to a colour scale, triplicates were normalized between 0 and 1. (g) Michaelis–Menten kinetic assay of graphene oxide with 5, 10, 50, 100, 150, 200 and 250 mM H₂O₂, mean ± SD, n = 3. (h and i) 0.1 mg POD and CAT activity combined with 0, 2, 5, 10, 20, 50 μg GO in vitro, mean ± SD, n = 3. Different letters indicate significant differences at P < 0.05 based on one‐way ANOVA.

Figure 2

GO modulates JA biosynthesis under aphid stress. (a) Venn diagram of the DEGs among the four comparisons. (b) K‐mean trend cluster analysis and (c) KEGG enrichment of the DEGs. (d) Heatmap of genes involved in JA biosynthesis. Gene expression level is indicated by log₂ and converted to a colour scale; triplicates were normalized between 0 and 1. (e and f) Extracted ion chromatograms (EIC) of JA (m/z: 209.1/58.8) and jasmonoyl‐isoleucine (JA‐Ile, m/z: 322.4/122.7) in treated leaves. (g) Phenotypes image of SbLOX3 VIGS seedlings 10 days after aphid inoculation. Scale bars, 10 cm. (h–j) Aphid count, fresh weight and survival rate of VIGS seedlings in (g), mean ± SD, n = 6, different letters indicate significant differences at P < 0.05 based on one‐way ANOVA. (k and l) JA and JA‐Ile contents in VIGS seedlings shown in (g), mean ± SD, n = 6, different letters indicate significant differences at P < 0.05 based on one‐way ANOVA.

Figure 3

SbTCP7 activates SbLOX3 transcription to upregulate JA level. (a) Differential expression transcription factors from RNA‐seq. (b) Relative expression level of SbTCP7 under GO, aphid and GO+aphid treatments. (c) Prediction of SbTCP7 binding to SbLOX3 promoter by AlphaFold3. (d) Yeast one‐hybrid (Y1H) assay and (e) electrophoretic mobility shift assay (EMSA) of SbTCP7 protein and SbLOX3 promoter. (f) Schematic diagram of the SbLOX3 promoter (−500 to −1500 bp). Yellow ellipses represent the predicted TCP binding site (GGCCC), mSbLOX3 represents the mutated promoter from GGCCC to CAGTT. (g) Diagrams of the effectors and reporters used in the dual‐luciferase reporter assays. (h) Image of LUC activities with the combination shown in (g). (i) Quantitative LUC activities from (h), mean ± SD, n = 3, different letters indicate significant differences at P < 0.05 based on one‐way ANOVA. (j) Phenotypes image of SbTCP7 VIGS seedlings 10 days after aphid inoculation. Scale bars, 10 cm. (k–m) Aphid count, fresh weight and survival rate of VIGS seedlings in (j), mean ± SD, n = 6, different letters indicate significant differences at P < 0.05 based on one‐way ANOVA. (k and l) JA and JA‐Ile contents in VIGS seedlings shown in (j), mean ± SD, n = 6, different letters indicate significant differences at P < 0.05 based on one‐way ANOVA.

Figure 4

Optimal H₂O₂ levels under aphid stress inhibit miR319 biogenesis and activate JA synthesis. (a and b) Relative expression of (a) SbTCP7 and (b) miR319 after H₂O₂ and glutathione (GSH) treatment. The level of the control was set to 1, means ± SD, n = 3; Different letters indicate significant differences at P < 0.05 based on one‐way ANOVA. (c) The analysis for the 5′ RACE. The red numbers represent the gene positions, while the black arrow indicates the cleavage site. Out of 25 reads, 22 were successfully sequenced at this site. (d) Schematic diagrams showing effector and reporter constructs used in dual‐luciferase assays. (e) Quantitative LUC activities, mean ± SD, n = 3. **P < 0.01, Student's t‐test. (f) Phenotypes image of miR319 VIGS seedlings 10 days after aphid inoculation. Scale bars, 10 cm. (g and h) Aphid count and survival rate of VIGS seedlings in (f), mean ± SD, n = 6, different letters indicate significant differences at P < 0.05 based on one‐way ANOVA. (i) Relative expression of SbTCP7 and key genes of JA biosynthesis and signalling pathway in VIGS seedlings shown in (f), mean ± SD, n = 3. * P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, Student's t‐test. (j and k) JA and JA‐Ile of in VIGS seedlings shown in (f), mean ± SD, n = 6, different letters indicate significant differences at P < 0.05 based on one‐way ANOVA. (l) Proposed model illustrating how GO acts as a nanozyme to reduce excess H₂O₂ and promote aphid resistance. Under aphid stress, a burst of ROS causes oxidative damage to plant cell, the miR319‐TCP7‐LOX3 module was inactive, leading to plant death. Following GO treatment, GO mimics peroxidase activity to degrade excess H₂O₂, thereby maintaining ROS homeostasis. Concurrently, miR319 biogenesis is inhibited, and the SbTCP7‐SbLOX3 module is activated, leading to the accumulation of JA and enhancing resistance to aphids.