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

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.

Computational curation and analysis of publicly available protein sequence data from a single protein family.

The wealth of sequence data available on public databases is increasing at an exponential rate, and while tremendous efforts are being made to make access to these resources easier, these data can be challenging for researchers to reuse because submissions are made from numerous laboratories with different biological objectives, resulting in inconsistent naming conventions and sequence content. Researchers can manually inspect each sequence and curate a dataset by hand but automating some of these steps will reduce this burden. This paper is a step-by-step guide describing how to identify all proteins containing a specific domain with the Conserved Protein Domain Architecture Retrieval Tool, download all associated amino acid sequences from NCBI Entrez, tabulate, and clean the data. I will also describe how to extract the full taxonomic information and computationally predict some physicochemical properties of the proteins based on amino acid sequence. The resulting data are applicable to a wide range of bioinformatic analyses where publicly available data are utilized. • Step-by-step guide to gathering, cleaning, and parsing data from publicly available databases for computational analysis, plus supplementation of taxonomic data and physicochemical characteristics from sequence data. • This strategy allows for reuse of existing large-scale publicly available data for different downstream applications to answer novel biological questions.

Phylogeny and domain architecture of plant ribosome inactivating proteins.

Ribosome inactivating proteins (RIPs) are rRNA N-glycosylases (EC 3.2.2.22) best known for hydrolyzing an adenine base from the conserved sarcin/ricin loop of ribosomal RNA. Protein translation is inhibited by ribosome depurination; therefore, RIPs are generally considered toxic to cells. The expression of some RIPs is upregulated by biotic and abiotic stress, though the connection between RNA depurination and defense response is not well understood. Despite their prevalence in approximately one-third of flowering plant orders, our knowledge of RIPs stems primarily from biochemical analyses of individuals or genomics-scale analyses of small datasets from a limited number of species. Here, we performed an unbiased search for proteins with RIP domains and identified several-fold more RIPs than previously known - more than 800 from 120 species, many with novel associated domains and physicochemical characteristics. Based on protein domain configuration, we established 15 distinct groups, suggesting diverse functionality. Surprisingly, most of these RIPs lacked a signal peptide, indicating they may be localized to the nucleocytoplasm of cells, raising questions regarding their toxicity against conspecific ribosomes. Our phylogenetic analysis significantly extends previous models for RIP evolution in plants, predicting an original single-domain RIP that later evolved to acquire a signal peptide and different protein domains. We show that RIPs are distributed throughout 21 plant orders with many species maintaining genes for more than one RIP group. Our analyses provide the foundation for further characterization of these new RIP types, to understand how these enzymes function in plants.

Pistil Mating Type and Morphology Are Mediated by the Brassinosteroid Inactivating Activity of the S-Locus Gene BAHD in Heterostylous Turnera Species.

Heterostyly is a breeding system that promotes outbreeding through a combination of morphological and physiological floral traits. In Turnera these traits are governed by a single, hemizygous S-locus containing just three genes. We report that the S-locus gene, BAHD, is mutated and encodes a severely truncated protein in a self-compatible long homostyle species. Further, a self-compatible long homostyle mutant possesses a T. krapovickasii BAHD allele with a point mutation in a highly conserved domain of BAHD acyl transferases. Wild type and mutant TkBAHD alleles were expressed in Arabidopsis to assay for brassinosteroid (BR) inactivating activity. The wild type but not mutant allele caused dwarfism, consistent with the wild type possessing, but the mutant allele having lost, BR inactivating activity. To investigate whether BRs act directly in self-incompatibility, BRs were added to in vitro pollen cultures of the two mating types. A small morph specific stimulatory effect on pollen tube growth was found with 5 µM brassinolide, but no genotype specific inhibition was observed. These results suggest that BAHD acts pleiotropically to mediate pistil length and physiological mating type through BR inactivation, and that in regard to self-incompatibility, BR acts by differentially regulating gene expression in pistils, rather than directly on pollen.