Improved pokeweed genome assembly and early gene expression changes in response to jasmonic acid

Abstract

Background: Jasmonic acid (JA) is a phytohormone involved in regulating responses to biotic and abiotic stress. Although the JA pathway is well characterized in model plants such as Arabidopsis thaliana, less is known about many non-model plants. Phytolacca americana (pokeweed) is native to eastern North Americana and is resilient to environmental stress. The goal of this study was to produce a publicly available pokeweed genome assembly and annotations and use this resource to determine how early response to JA changes gene expression, with particular focus on genes involved in defense.

Results: We assembled the pokeweed genome de novo from approximately 30 Gb of PacBio Hifi long reads and achieved an NG50 of ~ 13.2 Mb and a minimum 93.9% complete BUSCO score for gene annotations. With this reference, we investigated the early changes in pokeweed gene expression following JA treatment. Approximately 5,100 genes were differentially expressed during the 0-6 h time course with almost equal number of genes with increased and decreased transcript levels. Cluster and gene ontology analyses indicated the downregulation of genes associated with photosynthesis and upregulation of genes involved in hormone signaling and defense. We identified orthologues of key transcription factors and constructed the first JA gene response network integrated with our transcriptomic data from orthologues of Arabidopsis genes. We discovered that pokeweed did not use leaf senescence as a means of reallocating resources during stress; rather, most secondary metabolite synthesis genes were constitutively expressed, suggesting that pokeweed directs its resources for survival over the long term. In addition, pokeweed synthesizes several RNA N-glycosylases hypothesized to function in defense, each with unique expression profiles in response to JA.

Conclusions: Our investigation of the early response of pokeweed to JA illustrates patterns of gene expression involved in defence and stress tolerance. Pokeweed provides insight into the defense mechanisms of plants beyond those observed in research models and crops, and further study may yield novel approaches to improving the resilience of plants to environmental changes. Our assembled pokeweed genome is the first within the taxonomic family Phytolaccaceae to be publicly available for continued research.

Keywords: Phytolacca americana; Gene regulatory network; Genome assembly; Jasmonic acid; Pokeweed antiviral protein; RNA-seq; Stress response.

Fig. 1

Cluster analysis and subsequent GO analysis of differentially expressed genes in pokeweed during the JA treatment time-course. Hierarchical Euclidean clusters, with four clusters as the target, were generated with the R package dtwclust. Each cluster is represented by a colour identified in the legend. (A) Top significantly differentially enriched GO terms per cluster (p value less than 0.01 using the ‘elim’, ‘weight’, and classic methods among the top 25 differentially expressed GO terms). X-axis represents the percentage of each GO term present per cluster compared to all instances of that term, and the y-axis indicates each enriched GO term. (B) Bar graph specifying the number of genes represented per cluster. (C) Ribbon plot of the mean logFC of each cluster, flanked by the interquartile range. Genes were selected as differentially expressed if they had an FDR < 0.01 in any contrast

Fig. 2

Upset plot of the number of genes in various combinations of differential expression groups over time (top 20 largest). All time points are specified in hours (h). “Decreased” refers to gene transcript levels that have a FC < -1.5 and an FDR < 0.01 for the time point specified. “Increased” refers to gene transcript levels that have a FC > 1.5 and an FDR < 0.01 for the time point specified. “Not_DE” refers to genes that are not differentially expressed (have an FDR > = 0.01) for the time point specified. Genes not differentially expressed (FDR < 0.01) in all four contrasts were not included.

Fig. 3

The expression pattern of each differentially expressed gene identified as a ribosome inactivating protein by InterProScan5. The y-axis represents the logFC of the contrast between JA and Et at each of the four time points in comparison to time 0, the x-axis denotes the time points in hours, and the coloured lines represent the transcripts of genes encoding RIPs

Fig. 4

Number of pokeweed genes involved in secondary metabolite production and their expression patterns throughout the JA time course. GO terms relating to secondary metabolite biosynthesis were identified by keyword searches from the dataset of GO identifiers and their associated terms from the GO.db package, and then all pokeweed genes containing at least one of these terms were separated into another table for plotting. (A) Bar graph of the number of secondary metabolite biosynthesis-related genes, separated into columns based on whether they were differentially expressed or not in pokeweed, and colour-coded based on their product. (B) Line graphs of the logFC pattern of each gene involved in secondary metabolite production; the line colour represents genes with transcript levels that are only decreased (red), only increased (blue), or both decreased and increased (purple) during the time course

Fig. 5

Known gene regulatory network of Arabidopsis in response to JA with the differential expression patterns of identified pokeweed orthologues. The Araport11 Arabidopsis amino acid sequences for the genes shown were downloaded from TAIR (arabidopsis.org; March 1, 2023) and used as the query sequences in a blastp search against the translated gene sequences from the pokeweed annotations. Genes were considered orthologues if they had an e-value less than 0.01 and percent identity greater than 50. Coloured bars represent the logFC values of each orthologue at the time points 1, 2, 4, and 6 h (left to right)