Hybrid de novo genome assembly of red gromwell (Lithospermum erythrorhizon) reveals evolutionary insight into shikonin biosynthesis

Abstract

Lithospermum erythrorhizon (red gromwell; zicao) is a medicinal and economically valuable plant belonging to the Boraginaceae family. Roots from L. erythrorhizon have been used for centuries based on the antiviral and woundhealing properties produced from the bioactive compound shikonin and its derivatives. More recently, shikonin, its enantiomer alkannin, and several other shikonin/alkannin derivatives have collectively emerged as valuable natural colorants and as novel drug scaffolds. Despite several transcriptomes and proteomes having been generated from L. erythrorhizon, a reference genome is still unavailable. This has limited investigations into elucidating the shikonin/ alkannin pathway and understanding its evolutionary and ecological significance. In this study, we obtained a de novo genome assembly for L. erythrorhizon using a combination of Oxford Nanopore long-read and Illumina short-read sequencing technologies. The resulting genome is ∼367.41 Mb long, with a contig N50 size of 314.31 kb and 27,720 predicted protein-coding genes. Using the L. erythrorhizon genome, we identified several additional phydroxybenzoate:geranyltransferase (PGT) homologs and provide insight into their evolutionary history. Phylogenetic analysis of prenyltransferases suggests that PGTs originated in a common ancestor of modern shikonin/alkanninproducing Boraginaceous species, likely from a retrotransposition-derived duplication event of an ancestral prenyltransferase gene. Furthermore, knocking down expression of LePGT1 in L. erythrorhizon hairy root lines revealed that LePGT1 is predominantly responsible for shikonin production early in culture establishment. Taken together, the reference genome reported in this study and the provided analysis on the evolutionary origin of shikonin/alkannin biosynthesis will guide elucidation of the remainder of the pathway.

Convergent evolution of plant specialized 1,4-naphthoquinones: metabolism, trafficking, and resistance to their allelopathic effects

“…Examples of 1,4-naphthoquinones observed in ferns, magnoliids, monocots, and dicots are shown. Shaded arrows next to orders indicate experimentally supported metabolic origins ( Widhalm and Rhodes, 2016 ; Rajniak et al , 2018 ; Auber et al , 2020 ). Arrow colors depicting metabolic routes are the same as those in Fig.…”

“…1B ). Since then, several studies have further corroborated the existence of different 1,4-naphthoquinone pathways across plants and provided insight into their metabolic evolution ( McCoy et al , 2018 ; Rajniak et al , 2018 ; S. Wang et al , 2019 ; Auber et al , 2020 ; Song et al , 2020 ; Ueoka et al , 2020 ) ( Box 1 ). Moreover, genome assemblies from 1,4-naphthoquinone-producing species also revealed potential glimpses into the metabolic innovation that facilitated 1,4-naphthoquinone pathway evolution.…”

“…Moreover, genome assemblies from 1,4-naphthoquinone-producing species also revealed potential glimpses into the metabolic innovation that facilitated 1,4-naphthoquinone pathway evolution. De novo assembly of the red gromwell ( Lithospermum erythrorhizon ) and Echium plantagineum genomes indicated that retrotransposition-derived duplication ( Auber et al , 2020 ) and whole-genome duplication ( Tang et al , 2020 ) within the Boraginales contributed to establishing the shikonin/alkannin pathway. Reported genome assemblies from English walnut ( Juglans regia ) ( Martínez-García et al , 2016 ), J. regia × J. microcarpa hybrid ( Zhu et al , 2019 ), and several other Juglans species ( Stevens et al , 2018 ) underscored the importance of whole-genome duplication ( Luo et al , 2015 ) in driving metabolic diversification in the Juglandaceae.…”

“…De novo assembly of the red gromwell ( Lithospermum erythrorhizon ) genome and phylogenetic analysis by Auber et al (2020) showed that PGT genes arose in a common ancestor of modern shikonin/alkannin-producing Boraginaceae species via a retrotransposition-derived duplication event and subsequent neofunctionalization of an ancestral prenyltransferase gene.…”

“…floral volatiles; Dudareva et al , 2013 ). With recent progress in understanding plant 1,4-naphthoquinone distribution, metabolism, and function, it is becoming evident that the ability to synthesize these compounds has independently evolved several times and was facilitated by extraordinary events of metabolic innovation and/or metabolic links to respiratory and photosynthetic quinone metabolism ( McCoy et al , 2018 ; Auber et al , 2020 ; Ueoka et al , 2020 ). In this review, we highlight advances in understanding specialized 1,4-naphthoquinone biosynthesis, trafficking, and resistance strategies with emphasis on how these discoveries have shed light on the convergent evolution and diversification of this class of compounds in plants.…”