Haplotype-phased genome unveils the butylphthalide biosynthesis and homoploid hybrid origin of Ligusticum chuanxiong.

Butylphthalide is one of the first-line drugs for ischemic stroke therapy, while no biosynthetic enzyme for butylphthalide has been reported. Here, we present a haplotype-resolved genome of Ligusticum chuanxiong, a long-cultivated and phthalide-rich medicinal plant in Apiaceae. On the basis of comprehensive screening, four Fe(II)- and 2-oxoglutarate-dependent dioxygenases and two CYPs were mined and further biochemically verified as phthalide C-4/C-5 desaturases (P4,5Ds) that effectively promoted the forming of (S)-3-n-butylphthalide and butylidenephthalide. The substrate promiscuity and functional redundancy featured for P4,5Ds may contribute to the high phthalide diversity in L. chuanxiong. Notably, comparative genomic evidence supported L. chuanxiong as a homoploid hybrid with Ligusticum sinense as a potential parent. The two haplotypes demonstrated exceptional structure variance and diverged around 3.42 million years ago. Our study is an icebreaker for the dissection of phthalide biosynthetic pathway and reveals the hybrid origin of L. chuanxiong, which will facilitate the metabolic engineering for (S)-3-n-butylphthalide production and breeding for L. chuanxiong.

Evolution of a plant gene cluster in Solanaceae and emergence of metabolic diversity.

Plants produce phylogenetically and spatially restricted, as well as structurally diverse specialized metabolites via multistep metabolic pathways. Hallmarks of specialized metabolic evolution include enzymatic promiscuity and recruitment of primary metabolic enzymes and examples of genomic clustering of pathway genes. Solanaceae glandular trichomes produce defensive acylsugars, with sidechains that vary in length across the family. We describe a tomato gene cluster on chromosome 7 involved in medium chain acylsugar accumulation due to trichome specific acyl-CoA synthetase and enoyl-CoA hydratase genes. This cluster co-localizes with a tomato steroidal alkaloid gene cluster and is syntenic to a chromosome 12 region containing another acylsugar pathway gene. We reconstructed the evolutionary events leading to this gene cluster and found that its phylogenetic distribution correlates with medium chain acylsugar accumulation across the Solanaceae. This work reveals insights into the dynamics behind gene cluster evolution and cell-type specific metabolite diversity.

Plants produce a vast variety of different molecules known as secondary or specialized metabolites to attract pollinating insects, such as bees, or protect themselves against herbivores and pests. The secondary metabolites are made from simple building blocks that are readily available in plants, including amino acids, fatty acids and sugars. Different species of plant, and even different parts of the same plant, produce their own sets of secondary metabolites. For example, the hairs on the surface of tomatoes and other members of the nightshade family of plants make metabolites known as acylsugars. These chemicals deter herbivores and pests from damaging the plants. To make acylsugars, the plants attach long chains known as fatty acyl groups to molecules of sugar, such as sucrose. Some members of the nightshade family produce acylsugars with longer chains than others. In particular, acylsugars with long chains are only found in tomatoes and other closely-related species. It remained unclear how the nightshade family evolved to produce acylsugars with chains of different lengths. To address this question, Fan et al. used genetic and biochemical approaches to study tomato plants and other members of the nightshade family. The experiments identified two genes known as AACS and AECH in tomatoes that produce acylsugars with long chains. These two genes originated from the genes of older enzymes that metabolize fatty acids ­ the building blocks of fats ­ in plant cells. Unlike the older genes, AACS and AECH were only active at the tips of the hairs on the plant's surface. Fan et al. then investigated the evolutionary relationship between 11 members of the nightshade family and two other plant species. This revealed that AACS and AECH emerged in the nightshade family around the same time that longer chains of acylsugars started appearing. These findings provide insights into how plants evolved to be able to produce a variety of secondary metabolites that may protect them from a broader range of pests. The gene cluster identified in this work could be used to engineer other species of crop plants to start producing acylsugars as natural pesticides.

Tip of the trichome: evolution of acylsugar metabolic diversity in Solanaceae.

Acylsugars are insecticidal plant specialized metabolites produced in the Solanaceae (nightshade family). Despite having simple constituents, these compounds are unusually structurally diverse. Their structural variations in phylogenetically closely related species enable comparative biochemical approaches to understand acylsugar biosynthesis and pathway diversification. Thus far, varied enzyme classes contributing to their synthesis were characterized in cultivated and wild tomatoes, including from core metabolism - isopropylmalate synthase (Leu) and invertase (carbon) - and a group of evolutionarily related BAHD acyltransferases known as acylsucrose acyltransferases. Gene duplication and neofunctionalization of these enzymes drove acylsugar diversification both within and beyond tomato. The broad set of evolutionary mechanisms underlying acylsugar diversity in Solanaceae make this metabolic network an exemplar for detailed understanding of the evolution of metabolic form and function.

Functionally divergent alleles and duplicated Loci encoding an acyltransferase contribute to acylsugar metabolite diversity in Solanum trichomes.

Glandular trichomes from tomato (Solanum lycopersicum) and other species in the Solanaceae produce and secrete a mixture of O-acylsugars (aliphatic esters of sucrose and glucose) that contribute to insect defense. Despite their phylogenetic distribution and diversity, relatively little is known about how these specialized metabolites are synthesized. Mass spectrometric profiling of acylsugars in the S. lycopersicum x Solanum pennellii introgression lines identified a chromosome 11 locus containing a cluster of BAHD acyltransferases with one gene (named Sl-ASAT3) expressed in tip cells of type I trichomes where acylsugars are made. Sl-ASAT3 was shown to encode an acyl-CoA-dependent acyltransferase that catalyzes the transfer of short (four to five carbons) branched acyl chains to the furanose ring of di-acylsucrose acceptors to produce tri-acylsucroses, which can be further acetylated by Sl-ASAT4 (previously Sl-AT2). Among the wild tomatoes, diversity in furanose ring acyl chains on acylsucroses was most striking in Solanum habrochaites. S. habrochaites accessions from Ecuador and northern Peru produced acylsucroses with short (≤C5) or no acyl chains on the furanose ring. Accessions from central and southern Peru had the ability to add short or long (up to C12) acyl chains to the furanose ring. Multiple ASAT3-like sequences were found in most accessions, and their in vitro activities correlated with observed geographical diversity in acylsugar profiles.