Salicylic acid (SA) is a ubiquitous plant hormone with a long history in human civilization^1,2. Because of the central role of SA in orchestrating plant pathogen defence, understanding SA biosynthesis is fundamental to plant immunity research and crop improvement. Isochorismate-derived SA biosynthesis has been well defined in Arabidopsis. However, increasing evidence suggests a crucial function for phenylalanine-derived SA biosynthesis in many other plant species¹. Here we reveal the phenylalanine-derived SA biosynthetic pathway in rice by identifying three dedicated enzymes - peroxisomal benzoyl-CoA:benzyl alcohol benzoyltransferase (BEBT), the endoplasmic reticulum-associated cytochrome P450 enzyme benzylbenzoate hydroxylase (BBH), and cytosolic benzylsalicylate esterase (BSE) that sequentially convert benzoyl-CoA to benzylbenzoate, benzylsalicylate and SA. The pathogen-induced gene expression pattern and SA biosynthetic functions of this triple-enzyme module are conserved in diverse plants. This work fills a major knowledge gap in the biosynthesis of a key plant defence hormone, establishing a foundation for new strategies to create disease-resistant crops.

Background: The morning glories (Convolvulaceae) are distributed worldwide and produce economically important crops, medicinal herbs, and ornamentals. Members of this family are diverse in morphological characteristics and trophic modes, including the leafless parasitic Cuscuta (dodders). Organelle genomes were generally used for studying plant phylogeny and genomic variations. Notably, plastomes in parasitic plants always show non-canonical features, such as reduced size and accelerated rates. However, few organelle genomes of this group have been sequenced, hindering our understanding of their evolution, and dodder mitogenome in particular. Results: We assembled 22 new mitogenomes and 12 new plastomes in Convolvulaceae. Alongside previously known ones, we totally analyzed organelle genomes of 23 species in the family. Our sampling includes 16 leafy autotrophic species and 7 leafless parasitic dodders, covering 8 of the 12 tribes. Both the plastid and mitochondrial genomes of these plants have encountered variations that were rarely observed in other angiosperms. All of the plastomes possessed atypical IR boundaries. Besides the gene and IR losses in dodders, some leafy species also showed gene and intron losses, duplications, structural variations, and insertions of foreign DNAs. The phylogeny reconstructed by plastid protein coding sequences confirmed the previous relationship of the tribes. However, the monophyly of ‘Merremieae’ and the sister group of Cuscuta remained uncertain. The mitogenome was significantly inflated in Cuscuta japonica, which has exceeded over 800 kb and integrated massive DNAs from other species. In other dodders, mitogenomes were maintained in small size, revealing divergent evolutionary strategies. Mutations unique to plants were detected in the mitochondrial gene ccmFc, which has broken into three fragments through gene fission and splicing shift. The unusual changes likely initially happened to the common ancestor of the family and were caused by a foreign insertion from rosids followed by double-strand breaks and imprecise DNA repairs. The coding regions of ccmFc expanded at both sides after the fission, which may have altered the protein structure. Conclusions: Our family-scale analyses uncovered unusual scenarios for both organelle genomes in Convolvulaceae, especially in parasitic plants. The data provided valuable genetic resources for studying the evolution of Convolvulaceae and plant parasitism.

The mystery of genomic alternations in heterotrophic plants is among the most intriguing in evolutionary biology. Compared to plastid genomes (plastomes) with parallel size reduction and gene loss, mitochondrial genome (mitogenome) variation in heterotrophic plants remains underexplored in many aspects. To further unravel the evolutionary outcomes of heterotrophy, we present a comparative mitogenomic study with 13 de novo assemblies of Gastrodia (Orchidaceae), one of the largest fully mycoheterotrophic plant genera, and its relatives. Analyzed Gastrodia mitogenomes range from 0.56 to 2.1 Mb, each consisting of numerous, unequally abundant chromosomes or contigs. Size variation might have evolved through chromosome rearrangements followed by stochastic loss of “dispensable” chromosomes, with deletion-biased mutations. The discovery of a hyper-abundant (∼15 times intragenomic average) chromosome in two assemblies represents the hitherto most extreme copy number variation in any mitogenomes, with similar architectures discovered in two metazoan lineages. Transferred sequence contents highlight asymmetric evolutionary consequences of heterotrophy: despite drastically reduced intracellular plastome transfers convergent across heterotrophic plants, their rarity of horizontally acquired sequences sharply contrasts parasitic plants, where massive transfers from their hosts prevail. Rates of sequence evolution are markedly elevated but not explained by copy number variation, extending prior findings of accelerated molecular evolution from parasitic to heterotrophic plants. Putative evolutionary scenarios for these mitogenomic convergence and divergence fit well with the common (e.g. plastome contraction) and specific (e.g. host identity) aspects of the two heterotrophic types. These idiosyncratic mycoheterotrophs expand known architectural variability of plant mitogenomes and provide mechanistic insights into their content and size variation.

Rehmannia chingii is an important medicinal plant with immense value in scientific research. However, its mitochondrial genome (mitogenome) has not yet been characterized. Herein, based on whole-genome Illumina short reads and PacBio HiFi reads, we obtained the complete mitogenome of R. chingii through a de novo assembly strategy. We carried out comparative genomic analyses and found that, in comparison with the plastid genome (plastome) showing a high degree of structural conservation, the R. chingii mitogenome structure is relatively complex, showing an intricate ring structure with 16 connections, owing to five repetitive sequences. The R. chingii mitogenome was 783,161 bp with a GC content of 44.8% and contained 77 genes, comprising 47 protein-coding genes (CDS), 27 tRNA genes, and 3 rRNA genes. We counted 579 RNA editing events in 47 CDS and 12,828 codons in all CDSs of the R. chingii mitogenome. Furthermore, 24 unique sequence transfer fragments were found between the mitogenome and plastome, comprising 8 mitogenome CDS genes and 16 plastome CDS genes, corresponding to 2.39% of the R. chingii mitogenome. Mitogenomes had shorter but more collinear regions, evidenced by a comparison of the organelles of non-parasitic R. chingii, hemiparasitic Pedicularis chinensis, and holoparasitic Aeginetia indica in the Orobanchaceae family. Moreover, from non-parasitic to holoparasitic species, the genome size in the mitogenomes of Orobanchaceae species did not decrease gradually. Instead, the smallest mitogenome was found in the hemiparasitic species P. chinensis, with a size of 225,612 bp. The findings fill the gap in the mitogenome research of the medicinal plant R. chingii, promote the progress of the organelle genome research of the Orobanchaceae family, and provide clues for molecular breeding.

Fagales are an order of woody plants and comprise more than 1,100 species, most of which produce economically important timbers, nuts, and fruits. Their nuclear and plastid genomes are well-sequenced and provided valuable resources to study their phylogeny, breeding, resistance, etc. However, little is known about the mitochondrial genomes (mitogenomes), which hinder a full understanding of their genome evolution. In this study, we assembled complete mitogenomes of 23 species, covering five of the seven families of Fagales. These mitogenomes had similar gene sets but varied 2.4 times in size. The mitochondrial genes were highly conserved, and their capacity in phylogeny was challenging. The mitogenomic structure was extremely dynamic, and synteny among species was poor. Further analyses of the Fagales mitogenomes revealed extremely mosaic characteristics, with horizontal transfer (HGT)-like sequences from almost all seed plant taxa and even mitoviruses. The largest mitogenome, Carpinus cordata, did not have large amounts of specific sequences but instead contained a high proportion of sequences homologous to other Fagales. Independent and unequal transfers of third-party DNA, including nuclear genome and other resources, may partially account for the HGT-like fragments and unbalanced size expansions observed in Fagales mitogenomes. Supporting this, a mitochondrial plasmid-like of nuclear origin was found in Carpinus. Overall, we deciphered the last genetic materials of Fagales, and our large-scale analyses provide new insights into plant mitogenome evolution and size variation.