The Pokeweed Leaf mRNA Transcriptome and Its Regulation by Jasmonic Acid

Abstract

The American pokeweed plant, Phytolacca americana, is recognized for synthesizing pokeweed antiviral protein (PAP), a ribosome inactivating protein (RIP) that inhibits the replication of several plant and animal viruses. The plant is also a heavy metal accumulator with applications in soil remediation. However, little is known about pokeweed stress responses, as large-scale sequencing projects have not been performed for this species. Here, we sequenced the mRNA transcriptome of pokeweed in the presence and absence of jasmonic acid (JA), a hormone mediating plant defense. Trinity-based de novo assembly of mRNA from leaf tissue and BLASTx homology searches against public sequence databases resulted in the annotation of 59 096 transcripts. Differential expression analysis identified JA-responsive genes that may be involved in defense against pathogen infection and herbivory. We confirmed the existence of several PAP isoforms and cloned a potentially novel isoform of PAP. Expression analysis indicated that PAP isoforms are differentially responsive to JA, perhaps indicating specialized roles within the plant. Finally, we identified 52 305 natural antisense transcript pairs, four of which comprised PAP isoforms, suggesting a novel form of RIP gene regulation. This transcriptome-wide study of a Phytolaccaceae family member provides a source of new genes that may be involved in stress tolerance in this plant. The sequences generated in our study have been deposited in the SRA database under project # SRP069141.

Keywords: Phytolacca americana; RNA-seq; jasmonic acid; natural antisense transcript; pokeweed; pokeweed antiviral protein; ribosome inactivating protein; transcriptome.

FIGURE 1

Length distributions of pokeweed mRNA transcriptomes.

FIGURE 2

Identification of JA-responsive genes in pokeweed. (A) Heat map of expression values (log₂FPKM, median-centered) of the top differentially expressed transcripts (FDR < 0.001, FC ≥ 4). (B) Venn diagram depicting treatment-specific expression patterns of transcripts in (A).

FIGURE 3

Validation of RNA-seq differential expression results. The correlation of JA-induced expression changes obtained from RNA-seq and qRT-PCR is shown for 10 transcripts, eight of which were randomly selected. Results for qRT-PCR are from three independent biological replicates for each transcript.

FIGURE 4

Functional analysis of NATs in pokeweed. GO enrichment analysis was conducted on all NATs (Test Set) against the raw pokeweed transcriptome assembly (Reference Set). Enriched terms (FDR < 0.001) are listed in order of increasing significance (bottom to top).

FIGURE 5

Expression of PAP sense and antisense transcripts. The correlation of JA-induced expression changes obtained by RNA-seq is shown for each PAP isoform and its corresponding NAT.