The American pokeweed plant, Phytolacca americana, displays broad-spectrum resistance to plant viruses and is a heavy metal hyperaccumulator. However, little is known about the regulation of biotic and abiotic stress responses in this non-model plant. To investigate the control of miRNAs in gene expression, we sequenced the small RNA transcriptome of pokeweed treated with jasmonic acid (JA), a hormone that mediates pathogen defense and stress tolerance. We predicted 145 miRNAs responsive to JA, most of which were unique to pokeweed. These miRNAs were low in abundance and condition-specific, with discrete expression change. Integration of paired mRNA-Seq expression data enabled us to identify correlated, novel JA-responsive targets that mediate hormone biosynthesis, signal transduction, and pathogen defense. The expression of approximately half the pairs was positively correlated, an uncommon finding that we functionally validated by mRNA cleavage. Importantly, we report that a pokeweed-specific miRNA targets the transcript of OPR3, novel evidence that a miRNA regulates a JA biosynthesis enzyme. This first large-scale small RNA study of a Phytolaccaceae family member shows that miRNA-mediated control is a significant component of the JA response, associated with widespread changes in expression of genes required for stress adaptation.

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.

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Ribosome-inactivating proteins (RIPs) are RNA glycosidases thought to function in defense against pathogens. These enzymes remove purine bases from RNAs, including rRNA; the latter activity decreases protein synthesis in vitro , which is hypothesized to limit pathogen proliferation by causing host cell death. Pokeweed antiviral protein (PAP) is a RIP synthesized by the American pokeweed plant ( Phytolacca americana ). PAP inhibits virus infection when expressed in crop plants, yet little is known about the function of PAP in pokeweed due to a lack of genomic tools for this non-model species. In this work, we de novo assembled the pokeweed genome and annotated protein-coding genes. Sequencing comprised paired-end reads from a short-insert library of 83X coverage, and our draft assembly (N50 = 42.5 Kb) accounted for 74% of the measured pokeweed genome size of 1.3 Gb. We obtained 29,773 genes, 73% of which contained known protein domains, and identified several PAP isoforms. Within the gene models of each PAP isoform, a long 5′ UTR intron was discovered, which was validated by RT-PCR and sequencing. Presence of the intron stimulated reporter gene expression in tobacco. To gain further understanding of PAP regulation, we complemented this genomic resource with expression profiles of pokeweed plants subjected to stress treatments [jasmonic acid (JA), salicylic acid, polyethylene glycol, and wounding]. Cluster analysis of the top differentially expressed genes indicated that some PAP isoforms shared expression patterns with genes involved in terpenoid biosynthesis, JA-mediated signaling, and metabolism of amino acids and carbohydrates. The newly sequenced promoters of all PAP isoforms contained cis -regulatory elements associated with diverse biotic and abiotic stresses. These elements mediated response to JA in tobacco, based on reporter constructs containing promoter truncations of PAP-I, the most abundant isoform. Taken together, this first genomic resource for the Phytolaccaceae plant family provides new insight into the regulation and function of PAP in pokeweed.

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