Genome sequence for the blue-flowered Andean shrub Iochroma cyaneum reveals extensive discordance across the berry clade of Solanaceae

The tomato family, Solanaceae, is a model clade for a wide range of applied and basic research questions. Currently, reference-quality genomes are available for over 30 species from seven genera, and these include numerous crops as well as wild species (e.g., Jaltomata sinuosa and Nicotiana attenuata). Here we present the genome of the showy-flowered Andean shrub Iochroma cyaneum, a woody lineage from the tomatillo subfamily Physalideae. The assembled size of the genome (2.7Gb) is more similar in size to chilipepper (2.6Gb) than to other sequenced diploid members of the berry clade of Solanaceae (e.g., potato, tomato, and Jaltomata). Our assembly recovers 92% of the conserved orthologous set, suggesting a nearly complete genome for this species. Most of the genomic content is repetitive (69%), with gyspy elements alone accounting for 52% of the genome. Despite the large amount of repetitive content, most of the 12 Iochroma chromosomes are highly syntenic with tomato. Bayesian concordance analysis provides strong support for the berry clade, including Iochroma, but reveals extensive discordance along the backbone, with placement of pepper and Jaltomata being highly variable across gene trees. The Iochroma genome contributes to a growing wealth of genomic resources in Solanaceae and underscores the need for expanded sampling of diverse berry genomes to dissect major morphological transitions.

California TRV-based VIGS vectors mediate gene silencing at elevated temperatures but with greater growth stunting

Background Tobacco rattle virus (TRV) based virus-induced gene silencing (VIGS), a widely used functional genomics tool, requires growth temperatures typically lower than those of the plant’s native environment. Enabling VIGS under native conditions in the field according to applicable safety regulations could be a revolutionary advance for ecological research. Results Here, we report the development of an enhanced thermal tolerant VIGS vector system based on a TRV California isolate. cDNA clones representing the whole viral genome were sequenced and used to construct separate binary plant transformation vectors for functional elements of RNA1 (6765 nt) and RNA2 (3682 nt). VIGS of target genes was induced by transient transformation of the host plant with both vectors or by treating the host plant with sap from already VIGS induced plants. In Nicotiana attenuata the silencing efficiency of the PDS (phytoene desaturase) gene was 90% at 28 °C and 78% at 30 °C. Silencing at these temperatures was more prominent and durable than silencing induced by the widely used TRV PpK20-based pBINTRA6/pTV00 system, but was associated with a viral phenotype. Differences in the suppressor protein and RNA dependent RNA polymerase sequences between the TRV California isolate and PpK20 may be the reason for their different thermal tolerance. Conclusions The new TRV California-based VIGS vectors induce gene silencing in Nicotiana attenuata at higher temperatures than the existing pBINTRA6/pTV00 vector system, but cause greater growth defects. The new vector system opens up an avenue to study genes functions in planta under field conditions.

Tissue-specific systemic responses of the wild tobacco Nicotiana attenuata against stem-boring herbivore attack

Background Plants are able to optimize defense responses induced by various herbivores, which have different feeding strategies. Local and systemic responses within a plant after herbivory are essential to modulate herbivore-specific plant responses. For instance, leaf-chewing herbivores elicit jasmonic acid signaling, which result in the inductions of toxic chemicals in the attacked leaf (tissue-specific responses) and also in the other unattacked parts of the plant (systemic responses). Root herbivory induces toxic metabolites in the attacked root and alters the levels of transcripts and metabolites in the unattacked shoot. However, we have little knowledge of the local and systemic responses against stem-boring herbivores. In this study, we examined the systemic changes in metabolites in the wild tobacco Nicotiana attenuata, when the stem-boring herbivore Trichobaris mucorea attacks. Results To investigate the systemic responses of T. mucorea attacks, we measured the levels of jasmonic acid (JA), JA-dependent secondary metabolites, soluble sugars, and free amino acids in 7 distinct tissues of N. attenuata: leaf lamina with epidermis (LLE), leaf midrib (LM), stem epidermis (SE), stem pith (SP), stem vascular bundle (SV), root cortex with epidermis (RCE), and root vascular bundle (RV). The levels of JA were increased in all root tissues and in LM by T. mucorea attacks. The levels of chlorogenic acids (CGAs) and nicotine were increased in all stem tissues by T. mucorea. However, CGA was systematically induced in LM, and nicotine was systematically induced in LM and RCE. We further tested the resource allocation by measuring soluble sugars and free amino acids in plant tissues. T. mucorea attacks increased the level of free amino acids in all tissues except in LLE. The levels of soluble sugars were significantly decreased in SE and SP, but increased in RV. Conclusions The results reveal that plants have local- and systemic-specific responses in response to attack from a stem-boring herbivore. Interestingly, the level of induced secondary metabolites was not consistent with the systemic inductions of JA. Spatiotemporal resolution of plant defense responses against stem herbivory will be required to understand how a plant copes with attack from herbivores from different feeding guilds.

Moths sense, but do not learn flower odors with their proboscis during flower investigation

Insect pollinators, like the tobacco hawkmoth Manduca sexta, are known for locating flowers and learning floral odors by using their antennae. A recent study revealed, however, that the tobacco hawkmoth additionally possesses olfactory sensilla at the tip of its proboscis. Here, we ask whether this second “nose” of the hawkmoth is similarly involved in odor learning as are the antennae. We first show that Manduca foraging efficiency at Nicotiana attenuata flowers increases with experience. This raises the question whether olfactory learning with the proboscis is playing a role during flower handling. By rewarding the moths at an artificial flower, we show that – while moths learn an odor easily when they perceive it with their antennae – experiencing the odor just with the proboscis is not sufficient for odor learning. Furthermore, experiencing the odor with the antennae during training does not affect the behavior of the moths when they later can detect the learned odor with the proboscis only. Therefore, there seems to be no cross-talk between antennae and proboscis and information learnt by the antennae cannot be retrieved by the proboscis.

Variation in Manduca sexta Pollination-Related Floral Traits and Reproduction in a Wild Tobacco Plant

Most flowering plants depend on animal pollination for successful sexual reproduction. Floral signals such as color, shape, and odor are crucial in establishing this (often mutualistic) interaction. Plant and pollinator phenotypes can vary temporally but also spatially, thus creating mosaic-like patterns of local adaptations. Here, we investigated natural variation in floral morphology, flower volatile emission, and phenology in four accessions of a self-compatible wild tobacco, Nicotiana attenuata, to assess how these traits match the sensory perception of a known pollinator, the hawkmoth Manduca sexta. These accessions differ in floral traits and also in their habitat altitudes. Based on habitat temperatures, the accession occurring at the highest altitude (California) is less likely to be visited by M. sexta, while the others (Arizona, Utah 1, and Utah 2) are known to receive M. sexta pollinations. The accessions varied significantly in flower morphologies, volatile emissions, flower opening, and phenology, traits likely important for M. sexta perception and floral handling. In wind tunnel assays, we assessed the seed set of emasculated flowers after M. sexta visitation and of natural selfed and hand-pollinated selfed flowers. After moth visitations, plants of two accessions (Arizona and Utah 2) produced more capsules than the other two, consistent with predictions that accessions co-occurring with M. sexta would benefit more from the pollination services of this moth. We quantified flower and capsule production in four accessions in a glasshouse assay without pollinators to assess the potential for self-pollination. The two Utah accessions set significantly more seeds after pollen supplementation compared with those of autonomous selfing flowers, suggesting a greater opportunistic benefit from efficient pollinators than the other two. Moreover, emasculated flowers of the accession with the most exposed stigma (Utah 2) produced the greatest seed set after M. sexta visitation. This study reveals intraspecific variation in pollination syndromes that illuminate the potential of a plant species to adapt to local pollinator communities, changing environments, and altered pollination networks.

Single-cell RNA-sequencing of Nicotiana attenuata petal cells reveals the entire biosynthetic pathway of a floral scent

High-throughput single-cell RNA sequencing (scRNA-seq) identifies distinct cell populations based on cell-to-cell heterogeneity in gene expression. By examining the distribution of the density of gene expression profiles, the metabolic features of each cell population can be observed. Here, we employ the scRNA-seq technique to reveal the entire biosynthetic pathway of a flower volatile. The corolla (petals) of the wild tobacco Nicotiana attenuata emits a bouquet of scents that are composed mainly of benzylacetone (BA), a rare floral volatile. Protoplasts from the N. attenuata corolla were isolated at three different time points, and the transcript levels of >16,000 genes were analyzed in 3,756 single cells. We performed unsupervised clustering analysis to determine which cell clusters were involved in BA biosynthesis. The biosynthetic pathway of BA was uncovered by analyzing gene co-expression in scRNA-seq datasets and by silencing candidate genes in the corolla. In conclusion, the high-resolution spatiotemporal atlas of gene expression provided by scRNA-seq reveals the molecular features underlying cell-type-specific metabolism in a plant.