Jasmonate-induced prey response in the carnivorous plant Drosera capensis

Abstract

Drosera capensis is a carnivorous plant native to South Africa. Central to its prey capture and digestive processes is a complex array of biochemical processes triggering the production of both enzymes and small molecules. These processes are in part activated by the release of jasmonic acid, a plant defense hormone repurposed as a prey detection signal. Here, we use RNASeq and untargeted LC-MS metabolomics to study the response of D. capensis to a feeding stimulus. We confirm the expression of digestive proteins predicted in prior genomic work and show up- and downregulation for a number of enzyme classes in response to jasmonic acid. Metabolomics experiments indicate that many small molecules produced during feeding depend on specific nutrient inputs from prey (and not merely a jasmonic acid stimulus). These results shed light on the molecular basis of plant carnivory and the recruitment of existing biochemical pathways to perform specialized functions.

Figure 1:. Differential expression of aspartic and cysteine proteases upon treatment with jasmonic acid.

(A) Jasmonic acid treatment induces upregulation of four aspartic proteases, three nepenthesins (Supplementary Figure S1) and one protease associated with drought resistance (Supplementary Figure S5). (B) Twelve cysteine proteases are upregulated in response to jasmonic acid treatment (Supplementary Figure S9). (C) Although molecular models show that aspartic proteases share a common fold and active site architecture (33) (active site residues shown in bright red), the nepenthesin NEP_DCAP can be distinguished by the presence of the nepthesin-specific insert (NAP, light yellow). The expression level of Droserasin 1 (left), does not significantly change in response to jasmonic acid treatment, which is consistent with its hypothesized role in removal of pro-peptides in vacuoles. NEP_DCAP (right), is one of three nepenthesins upregulated upon treatment with jasmonic acid. (D) Both cysteine proteases (models from (35)) share the same Cys (yellow) -His (purple) - Asn (magenta) cataytic triad. DCAP_6097 (left) is a cysteine protease with a C-terminal granulin domain (light yellow), a structure typical of proteins involved in degradation of storage proteins during seed germination; it is not upregulated in response to jasmonic acid. DCAP_4952, a Dionain 1 homolog, is upregulated upon jasmonic acid treatment.

Figure 2:. Chitinases and esterases.

(A) Five chitinases are upregulated upon treatment with jasmonic acid, three from Family 18 and two from Family 19. (B) Esterases, a diverse category that includes esterases, acylesterases. and GDSL esterase/lipases, have more complex expression patterns. Seven of them are upregulated and eight are downregulated in response to jasmonic acid treatment.

Figure 3:. Differential expression of new enzymes upon treatment with jasmonic acid.

A) Two neprosins are upregulated, while two more show unchanged expression levels. (B) Genes whose assigned EC numbers appear in the KEGG flavonoid biosynthesis pathway are largely downregulated.

Figure 4:. Jasmonic acid derivatives peak at about 6 hours post-treatment.

A time-course analysis of jasmonic acid (JA) derivatives identified in Drosera capensis (N = 5–9 replicates per timepoint) shows a peak at about 6 hours post treatment.

Figure 5:. Certain free amino acids increase in abundunce upon feeding.

A) Experimental design. Leaves were treated with different combinations of jasmonic acid (molecular structure), BSA (protein emoji), and bloodworm homogenate (fly) as indicated by the black and white circles. Time for the leaf to respond to the treatment is indicated by the clock. B) Differential treatment analysis of D. capensis (N = 7 for each group), tracking the abundance of four amino acids: arginine, tyrosine, phenylalanine, and tryptophan. Each cell of the heatmap shows the average relative abundance of an amino acid, relative to the maximum signal of that amino acid across all replicates. The bar plot shows the average relative abundance of all four amino acids for each treatment. Bars show mean relative abundance, calculated from the relative abundance of all replicates in the treatment group across features, ± 95% confidence interval. Untreated leaves contain very low levels of any of the four free amino acids. Arginine and tyrosine both appear in bloodworm homogenate. All amino acids increased in abundance in response to the consumption of food (either bloodworm homogenate or protein). Arginine and tryptophan levels rose much more in response to bloodworm homogenate, while phenylalanine and tyrosine increased more in response to BSA. When jasmonic acid accompanied treatment with bloodworm homogenate, abundances of all amino acids increased relative to being fed bloodworm homogenate without jasmonic acid.

Figure 6:. Production of flavonoids and anthocyanins.

A) A heatmap and bar plot showing the relative abundances of different flavonoids present in D. capensis leaves, in response to various treatments. The color of each cell corresponds to the average replicate abundance (N = 7 for all groups) for D. capensis leaves treated with the corresponding substance(s). The bar plot represents the average relative abundance of all flavonoids for a particular treatment. The data show that flavonoids, on average, increase in abundance, when comparing untreated leaves to any treatment where the plant consumes nutrients (bloodworm homogenate or protein). Interestingly, consumption of protein produced a significantly larger increase in the abundance of flavonoids than the consumption of bloodworm homogenate ± jasmonic acid. Treatment with jasmonic acid alone led to a statistically significant decrease in flavonoid abundance, relative to untreated leaves. The three arrows mark example flavonoids chosen as representatives: their individual plots are shown in Supplementary Figure S22 to highlight their differential abundance in response to various treatments. Bars show mean relative abundance, calculated from the relative abundance of all replicates in the treatment group across features, ± 95% confidence interval. B) Structures of three representative flavonoids observed in the dataset.

Figure 7:. Production of lipids.

A) A heatmap and bar plot showing the relative abundances of different lipids present in D. capensis leaves, in response to various treatments. The color of each cell corresponds to the average replicate abundance (N = 7 for all groups) for leaves that have undergone the corresponding treatment. The bar plot represents the average relative abundance of all lipids for a particular treatment. Only a small fraction of lipids observed in bloodworm homogenate were also found in the plant tissue. We observed a statistically significant decrease in lipid abundance in response to treatment with jasmonic acid, relative to untreated leaves. Interestingly, lipid abundance was reduced even further in response to the consumption of protein, relative to treatment with jasmonic acid. Consumption of bloodworm homogenate, with or without jasmonic acid, resulted in the opposite response – an increase in the abundance of lipids. B) Representative lipid structures illustrating different response patterns to the treatments. Individual bar plots showing the abundance for each structure are shown in Supplementary Figure S23. Bars show mean relative abundance, calculated from the relative abundance of all replicates in the treatment group across features, ± 95% confidence interval.

Figure 8:. Differential expression of enzymes and small molecules in the KEGG flavonoid biosynthesis pathway (–73) upon treatment with jasmonic acid or jasmonic acid and bloodworm homogenate).

bottom: The log₂FC for enzyme expression with jasmonic acid alone are visualized by blue to red colored rectangles. Blue/red coloration represents the average log₂FC of all significant genes assigned the specified EC number. The log₂FC for small molecule expression with the jasmonic acid alone and jasmonic acid + bloodworm homogenate are visualized in cyan to magenta splitcircles; five small molecules were identified in this pathway: galangin, kaempferol, quercetin, luteolin, and myricetin. EC numbers in grey were found in the transcriptome, but were not assigned to any significant genes. EC numbers in white were not found in the transcriptome. top: Structures of the five identified molecules are shown along with close-ups of their positions in the pathway, highlighting the split circles illustrating the differential abundance of each metabolite depending on whether the plant was treated with jasmonic acid alone or with bloodworm homogenate.