Phylogenomic Analysis and Functional Characterization of the APETALA2/Ethylene-Responsive Factor Transcription Factor Across Solanaceae.

The AP2/ERF family constitutes one of the largest groups of transcription factors in the Solanaceae. AP2/ERF contributes to various plant biological processes, including growth, development, and responses to various stresses. The origins and functional diversification of AP2/ERF within the Solanaceae family remain poorly understood, primarily because of the complex interactions between whole-genome duplications (WGDs) and tandem duplications. In this study, a total of 1282 AP2/ERF proteins are identified from 7 Solanaceae genomes. The amplification of AP2/ERF genes was driven not only by WGDs but also by the presence of clusters of tandem duplicated genes. The conservation of synteny across different chromosomes provides compelling evidence for the impact of the WGD event on the distribution pattern of AP2/ERF genes. Distinct expression patterns suggest that the multiple copies of AP2/ERF genes evolved in different functional directions, catalyzing the diversification of roles among the duplicated genes, which was of great significance for the adaptability of Solanaceae. Gene silencing and overexpression assays suggest that ERF-1 members' role in regulating the timing of floral initiation in C. annuum. Our findings provide insights into the genomic origins, duplication events, and function divergence of the Solanaceae AP2/ERF.

Deciphering the evolutionary development of the "Chinese lantern" within Solanaceae.

MAIN CONCLUSION: The key genetic variation underlying the evo-devo of ICS in Solanaceae may be further pinpointed using an integrated strategy of forward and reverse genetics studies under the framework of phylogeny. The calyx of Physalis remains persistent throughout fruit development. Post-flowering, the fruiting calyx is inflated rapidly to encapsulate the berry, giving rise to a "Chinese lantern" structure called inflated calyx syndrome (ICS). It is unclear how this novelty arises. Over the past 2 decades, the role of MADS-box genes in the evolutionary development (evo-devo) of ICS has mainly been investigated within Solanaceae. In this review, we analyze the main achievements, challenges, and new progress. ICS acts as a source for fruit development, provides a microenvironment to protect fruit development, and assists in long-distance fruit dispersal. ICS is a typical post-floral trait, and the onset of its development is triggered by specific developmental signals that coincide with fertilization. These signals can be replaced by exogenous gibberellin and cytokinin application. MPF2-like heterotopic expression and MBP21-like loss have been proposed to be two essential evolutionary events for ICS origin, and manipulating the related MADS-box genes has been shown to affect the ICS size, sepal organ identity, and/or male fertility, but not completely disrupt ICS. Therefore, the core genes or key links in the ICS biosynthesis pathways may have undergone secondary mutations during evolution, or they have not yet been pinpointed. Recently, we have made some encouraging progress in acquiring lantern mutants in Physalis floridana. In addition to technological innovation, we propose an integrated strategy to further analyze the evo-devo mechanisms of ICS in Solanaceae using forward and reverse genetics studies under the framework of phylogeny.

De novo domestication in the Solanaceae: advances and challenges.

The advent of highly efficient genome editing (GE) tools, coupled with high-throughput genome sequencing, has paved the way for the accelerated domestication of crop wild relatives. New crops could thus be rapidly created that are well adapted to cope with drought, flooding, soil salinity, or insect damage. De novo domestication avoids the complexity of transferring polygenic stress resistance from wild species to crops. Instead, new crops can be created by manipulating major genes in stress-resistant wild species. However, the genetic basis of certain relevant domestication-related traits often involve epistasis and pleiotropy. Furthermore, pan-genome analyses show that structural variation driving gene expression changes has been selected during domestication. A growing body of work suggests that the Solanaceae family, which includes crop species such as tomatoes, potatoes, eggplants, peppers, and tobacco, is a suitable model group to dissect these phenomena and operate changes in wild relatives to improve agronomic traits rapidly with GE. We briefly discuss the prospects of this exciting novel field in the interface between fundamental and applied plant biology and its potential impact in the coming years.

The evolution of tropane alkaloids: Coca does it differently.

It is undeniable that tropane alkaloids (TAs) have been both beneficial and detrimental to human health in the modern era. Understanding their biosynthesis is vital for using synthetic biology to engineer organisms for pharmaceutical production. The most parsimonious approaches to pathway elucidation are traditionally homology-based methods. However, this approach has largely failed for TA biosynthesis in angiosperms. In the recent decade, significant progress has been made in elucidating the TA synthesis pathway in Erythroxylum coca, highlighting the parallel development of TAs in both the Solanaceae and Erythroxylaceae families. This separate evolutionary path has uncovered substantial divergence in the TAs formed by E. coca and distinct enzymatic reactions that differ from the traditional TA biosynthetic pathway found in TA-producing nightshade plants.

Family ties: Root-root communication within Solanaceae.

Root-root communication effects on several physiological and metabolic aspects among Solanaceae relatives were studied. We examined cherry (C) and field (F) tomato (Solanum lycopersicum) and bell pepper (B) (Capsicum annuum), comprising three degrees of relatedness (DOR): high (H-DOR; CC, FF and BB), medium (M-DOR; CF) and low (L-DOR; CB and FB). Plants were grown in pairs of similar or different plants on a paper-based and non-destructive root growth system, namely, rhizoslides. Root growth, including the proliferation of fine roots, and respiration increased as the DOR decreased and were highest in paired L-DOR plants, as was shown for root respiration that increased by 63, 110 and 88 % for C, F, and B when grown with B, B and F, respectively. On the other hand, root exudates of L-DOR plants had significantly lower levels of total organic carbon and protein than those of H-DOR plants, indicating different root-root communication between individuals with different DOR. Our findings indicate, for the first time, that carbon allocation to root growth, exudation and respiration depends on the degree of genetic relatedness, and that the degree of relatedness between individual plants plays a key role in the root-root communication within Solanaceae.

Pathogen elicitor peptide (pep), systemin, and their receptors in tomato: sequence analysis sheds light on standing disagreements about biotic stress signaling components.

Peps are endogenous damage-associated polypeptides that evoke defense responses in plants. Like other damage-associated molecular patterns, Pep signals are transduced by receptors. PEPRs are the receptors that transduce Pep danger signals. This paper identifies new putative Peps in the Solanaceae (including Solanum spp., Nicotiana spp., and Petunia spp.) and Coffea and explores their properties. Using these newly identified Peps we derive sequence logos that present a refinement of the current understanding of the importance of specific residues in the Pep signaling molecules in Solanaceae, including several arginines, prolines that restrict peptide's conformations, and C-terminal asparagine. We examine the degree of disorder in Pep, which is likely important to the mechanism of Pep perception. This work also calls into question some of the evolutionary relationships between Peps in Solanaceae and specific Arabidopsis Peps published in previous literature, culminating in a conclusion that SlPep should not be named SlPep6 due to the lack of conservation of protein sequences in AtPROPEP6 and SlPROPEP, and that SlPep probably does not have two receptors in tomato, based on phylogenetic analysis. Our analyses advance understanding of the Pep signaling system in Solanaceae.

Incomplete lineage sorting and hybridization underlie tree discordance in Petunia and related genera (Petunieae, Solanaceae).

Despite the overarching history of species divergence, phylogenetic studies often reveal distinct topologies across regions of the genome. The sources of these gene tree discordances are variable, but incomplete lineage sorting (ILS) and hybridization are among those with the most biological importance. Petunia serves as a classic system for studying hybridization in the wild. While field studies suggest that hybridization is frequent, the extent of reticulation within Petunia and its closely related genera has never been examined from a phylogenetic perspective. In this study, we used transcriptomic data from 11 Petunia, 16 Calibrachoa, and 10 Fabiana species to illuminate the relationships between these species and investigate whether hybridization played a significant role in the diversification of the clade. We inferred that gene tree discordance within genera is linked to hybridization events along with high levels of ILS due to their rapid diversification. Moreover, network analyses estimated deeper hybridization events between Petunia and Calibrachoa, genera that have different chromosome numbers. Although these genera cannot hybridize at the present time, ancestral hybridization could have played a role in their parallel radiations, as they share the same habitat and life history.

Molecular mechanisms of self-incompatibility in Brassicaceae and Solanaceae.

Self-incompatibility (SI) is a mechanism for preventing self-fertilization in flowering plants. SI is controlled by a single S-locus with multiple haplotypes (S-haplotypes). When the pistil and pollen share the same S-haplotype, the pollen is recognized as self and rejected by the pistil. This review introduces our research on Brassicaceae and Solanaceae SI systems to identify the S-determinants encoded at the S-locus and uncover the mechanisms of self/nonself-discrimination and pollen rejection. The recognition mechanisms of SI systems differ between these families. A self-recognition system is adopted by Brassicaceae, whereas a collaborative nonself-recognition system is used by Solanaceae. Work by our group and subsequent studies indicate that plants have evolved diverse SI systems.

The phylogeographic journey of a plant species from lowland to highlands during the Pleistocene.

Phylogeographic history refers to how species evolve and diversify in response to historical, ecological, and demographic factors. The climate fluctuation during the Pleistocene period marked a crucial time in shaping many species' distribution and genetic structure, particularly those from southern South American grasslands. This work investigated the phylogeographic history of a highland grassland, Petunia altiplana T. Ando & Hashim. (Solanaceae), its diversity, and geographic distribution using a population genomic approach based on RAD-seq data. Our results indicated that, during the Pleistocene, when the grasslands expanded to highlands, the lowland populations of P. altiplana reached the higher open fields, enlarging their geographic distribution. We found that the P. altiplana genetic diversity followed the geographic division into eastern (E) and western (WE) population groups, with a subtle division in the E group regarding the Pelotas River headwater. The results also showed that isolation by distance was the main divergence pattern, with elevation playing a pivotal role in shaping WE and E groups. Our findings indicated that lowland-adapted populations quickly colonized highlands during the late Pleistocene.

Chromosome level genome assembly of endangered medicinal plant Anisodus tanguticus.

Anisodus tanguticus is a medicinal herb that belongs to the Anisodus genus of the Solanaceae family. This endangered herb is mainly distributed in Qinghai-Tibet Plateau. In this study, we combined the Illumina short-read, Nanopore long-read and high-throughput chromosome conformation capture (Hi-C) sequencing technologies to de novo assemble the A. tanguticus genome. A high-quality chromosomal-level genome assembly was obtained with a genome size of 1.26 Gb and a contig N50 of 25.07 Mb. Of the draft genome sequences, 97.47% were anchored to 24 pseudochromosomes with a scaffold N50 of 51.28 Mb. In addition, 842.14 Mb of transposable elements occupying 66.70% of the genome assembly were identified and 44,252 protein-coding genes were predicted. The genome assembly of A. tanguticus will provide genetic repertoire to understand the adaptation strategy of Anisodus species in the plateau, which will further promote the conservation of endangered A. tanguticus resources.

A chromosome-level genome assembly of anesthetic drug-producing Anisodus acutangulus provides insights into its evolution and the biosynthesis of tropane alkaloids.

Tropane alkaloids (TAs), which are anticholinergic agents, are an essential class of natural compounds, and there is a growing demand for TAs with anesthetic, analgesic, and spasmolytic effects. Anisodus acutangulus (Solanaceae) is a TA-producing plant that was used as an anesthetic in ancient China. In this study, we assembled a high-quality, chromosome-scale genome of A. acutangulus with a contig N50 of 7.4 Mb. A recent whole-genome duplication occurred in A. acutangulus after its divergence from other Solanaceae species, which resulted in the duplication of ADC1 and UGT genes involved in TA biosynthesis. The catalytic activities of H6H enzymes were determined for three Solanaceae plants. On the basis of evolution and co-expressed genes, AaWRKY11 was selected for further analyses, which revealed that its encoded transcription factor promotes TA biosynthesis by activating AaH6H1 expression. These findings provide useful insights into genome evolution related to TA biosynthesis and have potential implications for genetic manipulation of TA-producing plants.

An evolutionary view of vanillylamine synthase pAMT, a key enzyme of capsaicinoid biosynthesis pathway in chili pepper.

Pungent capsaicinoid is synthesized only in chili pepper (Capsicum spp.). The production of vanillylamine from vanillin is a unique reaction in the capsaicinoid biosynthesis pathway. Although putative aminotransferase (pAMT) has been isolated as the vanillylamine synthase gene, it is unclear how Capsicum acquired pAMT. Here, we present a phylogenetic overview of pAMT and its homologs. The Capsicum genome contained 5 homologs, including pAMT, CaGABA-T1, CaGABA-T3, and two pseudogenes. Phylogenetic analysis indicated that pAMT is a member of the Solanaceae cytoplasmic GABA-Ts. Comparative genome analysis found that multiple copies of GABA-T exist in a specific Solanaceae genomic region, and the cytoplasmic GABA-Ts other than pAMT are located in the region. The cytoplasmic GABA-T was phylogenetically close to pseudo-GABA-T harboring a plastid transit peptide (pseudo-GABA-T3). This suggested that Solanaceae cytoplasmic GABA-Ts occurred via duplication of a chloroplastic GABA-T ancestor and subsequent loss of the plastid transit signal. The cytoplasmic GABA-T may have been translocated from the specific Solanaceae genomic region during Capsicum divergence, resulting in the current pAMT locus. A recombinant protein assay demonstrated that pAMT had higher vanillylamine synthase activity than those of other plant GABA-Ts. pAMT was expressed exclusively in the placental septum of mature green fruit, whereas tomato orthologs SlGABA-T2/4 exhibit a ubiquitous expression pattern in plants. These findings suggested that both the increased catalytic efficiency and transcriptional changes in pAMT may have contributed to establish vanillylamine synthesis in the capsaicinoid biosynthesis pathway. This study provides insights into the establishment of pungency in the evolution of chili peppers.

Multiple independent losses of the biosynthetic pathway for two tropane alkaloids in the Solanaceae family.

Hyoscyamine and scopolamine (HS), two valuable tropane alkaloids of significant medicinal importance, are found in multiple distantly related lineages within the Solanaceae family. Here we sequence the genomes of three representative species that produce HS from these lineages, and one species that does not produce HS. Our analysis reveals a shared biosynthetic pathway responsible for HS production in the three HS-producing species. We observe a high level of gene collinearity related to HS synthesis across the family in both types of species. By introducing gain-of-function and loss-of-function mutations at key sites, we confirm the reduced/lost or re-activated functions of critical genes involved in HS synthesis in both types of species, respectively. These findings indicate independent and repeated losses of the HS biosynthesis pathway since its origin in the ancestral lineage. Our results hold promise for potential future applications in the artificial engineering of HS biosynthesis in Solanaceae crops.

Genome-Wide Analysis of MBF1 Family Genes in Five Solanaceous Plants and Functional Analysis of SlER24 in Salt Stress.

Multiprotein bridging factor 1 (MBF1) is an ancient family of transcription coactivators that play a crucial role in the response of plants to abiotic stress. In this study, we analyzed the genomic data of five Solanaceae plants and identified a total of 21 MBF1 genes. The expansion of MBF1a and MBF1b subfamilies was attributed to whole-genome duplication (WGD), and the expansion of the MBF1c subfamily occurred through transposed duplication (TRD). Collinearity analysis within Solanaceae species revealed collinearity between members of the MBF1a and MBF1b subfamilies, whereas the MBF1c subfamily showed relative independence. The gene expression of SlER24 was induced by sodium chloride (NaCl), polyethylene glycol (PEG), ABA (abscisic acid), and ethrel treatments, with the highest expression observed under NaCl treatment. The overexpression of SlER24 significantly enhanced the salt tolerance of tomato, and the functional deficiency of SlER24 decreased the tolerance of tomato to salt stress. SlER24 enhanced antioxidant enzyme activity to reduce the accumulation of reactive oxygen species (ROS) and alleviated plasma membrane damage under salt stress. SlER24 upregulated the expression levels of salt stress-related genes to enhance salt tolerance in tomato. In conclusion, this study provides basic information for the study of the MBF1 family of Solanaceae under abiotic stress, as well as a reference for the study of other plants.

Flower Development in the Solanaceae.

Flower development is the process leading from a reproductive meristem to a mature flower with fully developed floral organs. This multi-step process is complex and involves thousands of genes in intertwined regulatory pathways; navigating through the FLOR-ID website will give an impression of this complexity and of the astonishing amount of work that has been carried on the topic (Bouché et al., Nucleic Acids Res 44:D1167-D1171, 2016). Our understanding of flower development mostly comes from the model species Arabidopsis thaliana, but numerous other studies outside of Brassicaceae have helped apprehend the conservation of these mechanisms in a large evolutionary context (Moyroud and Glover, Curr Biol 27:R941-R951, 2017; Smyth, New Phytol 220:70-86, 2018; Soltis et al., Ann Bot 100:155-163, 2007). Integrating additional species and families to the research on this topic can only advance our understanding of flower development and its evolution.In this chapter, we review the contribution that the Solanaceae family has made to the comprehension of flower development. While many of the general features of flower development (i.e., the key molecular players involved in flower meristem identity, inflorescence architecture or floral organ development) are similar to Arabidopsis, our main objective in this chapter is to highlight the points of divergence and emphasize specificities of the Solanaceae. We will not discuss the large topics of flowering time regulation, inflorescence architecture and fruit development, and we will restrict ourselves to the mechanisms included in a time window after the floral transition and before the fertilization. Moreover, this review will not be exhaustive of the large amount of work carried on the topic, and the choices that we made to describe in large details some stories from the literature are based on the soundness of the functional work performed, and surely as well on our own preferences and expertise.First, we will give a brief overview of the Solanaceae family and some of its specificities. Then, our focus will be on the molecular mechanisms controlling floral organ identity, for which extended functional work in petunia led to substantial revisions to the famous ABC model. Finally, after reviewing some studies on floral organ initiation and growth, we will discuss floral organ maturation, using the examples of the inflated calyx of the Chinese lantern Physalis and petunia petal pigmentation.

Abundance of distal repetitive DNA sequences in Capsicum L. (Solanaceae) chromosomes.

Chili peppers (Solanaceae family) have great commercial value. They are commercialized in natura and used as spices and for ornamental and medicinal purposes. Although three whole genomes have been published, limited information about satellite DNA sequences, their composition, and genomic distribution has been provided. Here, we exploited the noncoding repetitive fraction, represented by satellite sequences, that tends to accumulate in blocks along chromosomes, especially near the chromosome ends of peppers. Two satellite DNA sequences were identified (CDR-1 and CDR-2), characterized and mapped in silico in three Capsicum genomes (C. annuum, C. chinense, and C. baccatum) using data from the published high-coverage sequencing and repeats finding bioinformatic tools. Localization using FISH in the chromosomes of these species and in two others (C. frutescens and C. chacoense), totaling five species, showed signals adjacent to the rDNA sites. A sequence comparison with existing Solanaceae repeats showed that CDR-1 and CDR-2 have different origins but without homology to rDNA sequences. Satellites occupied subterminal chromosomal regions, sometimes collocated with or adjacent to 35S rDNA sequences. Our results expand knowledge about the diversity of subterminal regions of Capsicum chromosomes, showing different amounts and distributions within and between karyotypes. In addition, these sequences may be useful for future phylogenetic studies.

Evolution and Expression of the Meprin and TRAF Homology Domain-Containing Gene Family in Solanaceae.

Meprin and TRAF homology (MATH)-domain-containing proteins are pivotal in modulating plant development and environmental stress responses. To date, members of the MATH gene family have been identified only in a few plant species, including Arabidopsis thaliana, Brassica rapa, maize, and rice, and the functions of this gene family in other economically important crops, especially the Solanaceae family, remain unclear. The present study identified and analyzed 58 MATH genes from three Solanaceae species, including tomato (Solanum lycopersicum), potato (Solanum tuberosum), and pepper (Capsicum annuum). Phylogenetic analysis and domain organization classified these MATH genes into four groups, consistent with those based on motif organization and gene structure. Synteny analysis found that segmental and tandem duplication might have contributed to MATH gene expansion in the tomato and the potato, respectively. Collinearity analysis revealed high conservation among Solanaceae MATH genes. Further cis-regulatory element prediction and gene expression analysis showed that Solanaceae MATH genes play essential roles during development and stress response. These findings provide a theoretical basis for other functional studies on Solanaceae MATH genes.

Anatomical and biophysical basis for graft incompatibility within the Solanaceae.

Interspecies grafting is an economically relevant technique that allows beneficial shoot and root combinations from separate species to be combined. One hypothesis for the basis of graft compatibility revolves around taxonomic relatedness. To test how phylogenetic distance affects interspecific graft compatibility within the economically important Solanaceae subfamily, Solanoideae, we characterized the anatomical and biophysical integrity of graft junctions between four species: tomato (Solanum lycopersicum), eggplant (Solanum melongena), pepper (Capsicum annuum), and groundcherry (Physalis pubescens). We analyzed the survival, growth, integrity, and cellular composition of the graft junctions. Utilizing various techniques, we were able to quantitatively assess compatibility among the interspecific grafts. Even though most of our graft combinations could survive, we show that only intrageneric combinations between tomato and eggplant are compatible. Unlike incompatible grafts, the formation of substantial vascular reconnections between tomato and eggplant in the intrageneric heterografts likely contributed to biophysically stable grafts. Furthermore, we identified 10 graft combinations that show delayed incompatibility, providing a useful system to pursue deeper work into graft compatibility. This work provides new evidence that graft compatibility may be limited to intrageneric combinations within the Solanoideae subfamily. Further research amongst additional Solanaceous species can be used to test the extent to which our hypothesis applies to this family.

Genomic and structural basis for evolution of tropane alkaloid biosynthesis.

The tropane alkaloids (TAs) cocaine and hyoscyamine have been used medicinally for thousands of years. To understand the evolutionary origins and trajectories of serial biosynthetic enzymes of TAs and especially the characteristic tropane skeletons, we generated the chromosome-level genome assemblies of cocaine-producing Erythroxylum novogranatense (Erythroxylaceae, rosids clade) and hyoscyamine-producing Anisodus acutangulus (Solanaceae, asterids clade). Comparative genomic and phylogenetic analysis suggested that the lack of spermidine synthase/N-methyltransferase (EnSPMT1) in ancestral asterids species contributed to the divergence of polyamine (spermidine or putrescine) methylation in cocaine and hyoscyamine biosynthesis. Molecular docking analysis and key site mutation experiments suggested that ecgonone synthases CYP81AN15 and CYP82M3 adopt different active-site architectures to biosynthesize the same product ecgonone from the same substrate in Erythroxylaceae and Solanaceae. Further synteny analysis showed different evolutionary origins and trajectories of CYP81AN15 and CYP82M3, particularly the emergence of CYP81AN15 through the neofunctionalization of ancient tandem duplication genes. The combination of structural biology and comparative genomic analysis revealed that ecgonone methyltransferase, which is responsible for the biosynthesis of characteristic 2-substituted carboxymethyl group in cocaine, evolved from the tandem copies of salicylic acid methyltransferase by the mutations of critical E216 and S153 residues. Overall, we provided strong evidence for the independent origins of serial TA biosynthetic enzymes on the genomic and structural level, underlying the chemotypic convergence of TAs in phylogenetically distant species.

Nuclear phylogeny and insights into whole-genome duplications and reproductive development of Solanaceae plants.

Solanaceae, the nightshade family, have ∼2700 species, including the important crops potato and tomato, ornamentals, and medicinal plants. Several sequenced Solanaceae genomes show evidence for whole-genome duplication (WGD), providing an excellent opportunity to investigate WGD and its impacts. Here, we generated 93 transcriptomes/genomes and combined them with 87 public datasets, for a total of 180 Solanaceae species representing all four subfamilies and 14 of 15 tribes. Nearly 1700 nuclear genes from these transcriptomic/genomic datasets were used to reconstruct a highly resolved Solanaceae phylogenetic tree with six major clades. The Solanaceae tree supports four previously recognized subfamilies (Goetzeioideae, Cestroideae, Nicotianoideae, and Solanoideae) and the designation of three other subfamilies (Schizanthoideae, Schwenckioideae, and Petunioideae), with the placement of several previously unassigned genera. We placed a Solanaceae-specific whole-genome triplication (WGT1) at ∼81 million years ago (mya), before the divergence of Schizanthoideae from other Solanaceae subfamilies at ∼73 mya. In addition, we detected two gene duplication bursts (GDBs) supporting proposed WGD events and four other GDBs. An investigation of the evolutionary histories of homologs of carpel and fruit developmental genes in 14 gene (sub)families revealed that 21 gene clades have retained gene duplicates. These were likely generated by the Solanaceae WGT1 and may have promoted fleshy fruit development. This study presents a well-resolved Solanaceae phylogeny and a new perspective on retained gene duplicates and carpel/fruit development, providing an improved understanding of Solanaceae evolution.