Michael Gutensohn

SUMMARY: Successful fertilization of insect‐pollinated crops hinges on a delicate interplay of olfactory and visual signals of pollinator attraction, the chemical complexity of nectar and pollen rewards, and the physical interaction between insects and flower anatomy for efficient pollen transfer. These traits, which are controlled genetically and exhibit phenotypic variance even within species, present opportunities for breeding technologies to map and select genotypes with floral traits that actively guide pollinator preferences. Recent technological advancements and automation have enabled high‐throughput metabolic phenotyping of floral chemical traits of pollinator attraction and rewards. These measurements, when integrated with computed tomography (CT) scans of flower shape analysis and video tracking of pollinator behavior, can guide the selection of genotypes with enhanced insect visitation rates and effective cross‐pollination. In this perspective article, we highlight the potential of this strategy for blueberry (Vaccinium corymbosum L.), a crop heavily reliant on bee pollination for fruit production and with flowers that display considerable variance of chemical and morphological traits and pollinator visitation rates. Leveraging blueberry's genetic diversity can address pollination issues exacerbated by global warming and declining health of managed bees, thus contributing to a more sustainable agricultural production.

The invasive shrub glossy buckthorn (Frangula alnus) has been progressively colonizing the Northeastern United States and Southeastern Canada for more than a century. To determine the dominant arthropod orders and species associated with F. alnus, field surveys were conducted for two years across 16 plots within the Allegheny National Forest, Pennsylvania, USA. Statistical analyses were employed to assess the impact of seasonal variation on insect order richness and diversity. The comprehensive arthropod collection yielded 2845 insects and arachnids, with hemipterans comprising the majority (39.8%), followed by dipterans (22.3%) and arachnids (15.5%). Notably, 16.2% of the hemipterans collected were in the immature stages, indicating F. alnus as a host for development. The two dominant insect species of F. alnus were Psylla carpinicola (Hemiptera: Psyllidae) and Drosophila suzukii (Diptera: Drosophilidae); D. suzukii utilized F. alnus fruits for reproduction. Species richness and diversity exhibited significant variations depending on the phenology of F. alnus. The profiles of volatile compounds emitted from the leaves and flowers of F. alnus were analyzed to identify factors that potentially contribute to the attraction of herbivores and pollinators. The results of our study will advance the development of novel F. alnus management strategies leveraging the insects associated with this invasive species.

Over the last decade, the Allegheny National Forest (ANF) in the USA has experienced issues with the regeneration of black cherry (Prunus serotina). This study was conducted to investigate the effects of silvicultural treatment on the insect communities that may affect black cherry pollination and regeneration. We conducted a 2-year study to compare the abundance, richness, and diversity of insects in unmanaged, shelterwood seed-tree, and shelterwood clear-cut stands. Using pan traps, we sampled insects at the ground level and in the canopies of flowering mature black cherry trees. The results of this study showed significant increases in the abundance of insects captured in shelterwood seed-tree stands and in species richness and diversity of insects captured in the canopy of black cherry in shelterwood removal stands, indicating that silvicultural treatment affected the insect community significantly. The dominant insect order was Diptera (true flies, 72.91%, n = 12,668), and Anthalia bulbosa (Diptera: Hybotidae) was the dominant species comprising 33% of all insects found in the canopy of flowering black cherry. The findings in this study could help land managers in managing black cherry for its pollination and natural regeneration.

Terpenoids constitute one of the largest and most diverse classes of plant metabolites. While some terpenoids are involved in essential plant processes such as photosynthesis, respiration, growth, and development, others are specialized metabolites playing roles in the interaction of plants with their biotic and abiotic environment. Due to the distinct functions and properties of specific terpenoid compounds, there is a growing interest to introduce or modify their production in plants by metabolic engineering for agricultural, pharmaceutical, or industrial applications. The MVA and MEP pathways and the prenyltransferases providing the general precursors for terpenoid formation, as well as the enzymes of the various downstream metabolic pathways leading to the formation of different groups of terpenoid compounds have been characterized in detail in plants. In contrast, the molecular mechanisms directing the metabolic flux of precursors specifically toward one of several potentially competing terpenoid biosynthetic pathways are still not well understood. The formation of metabolons, multi-protein complexes composed of enzymes catalyzing sequential reactions of a metabolic pathway, provides a promising concept to explain the metabolic channeling that appears to occur in the complex terpenoid biosynthetic network of plants. Here we provide an overview about examples of potential metabolons involved in plant terpenoid metabolism that have been recently characterized and the first attempts to utilize metabolic channeling in terpenoid metabolic engineering. In addition, we discuss the gaps in our current knowledge and in consequence the need for future basic and applied research.

Tomato produces a number of terpenes in their glandular trichomes that contribute to host plant resistance against pests. While glandular trichomes of cultivated tomato Solanum lycopersicum primarily accumulate a blend of monoterpenes, those of the wild tomato species Solanum habrochaites produce various sesquiterpenes. Recently, we have identified two groups of sesquiterpenes in S. habrochaites accessions that negatively affect the performance and choice behavior of the potato aphid (Macrosiphum euphorbiae). Aphids are piercing-sucking herbivores that use their mouthpart to penetrate and probe plant tissues in order to ultimately access vascular tissue and ingest phloem sap. Because secondary metabolites produced in glandular trichomes can affect the initial steps of the aphid feeding behavior, introducing the formation of defensive terpenes into additional plant tissues via metabolic engineering has the potential to reduce tissue penetration by aphids and in consequence virus transmission. Here, we have developed two multicistronic expression constructs based on the two sesquiterpene traits with activity toward M. euphorbiae previously identified in S. habrochaites. Both constructs are composed of sequences encoding a prenyl transferase and a respective S. habrochaites terpene synthase, as well as enhanced green fluorescent protein as a visible marker. All three coding sequences were linked by short nucleotide sequences encoding the foot-and-mouth disease virus 2A self-processing oligopeptide which allows their co-expression under the control of one promoter. Transient expression of both constructs under the epidermis-specific Arabidopsis CER5-promoter in tomato leaves demonstrated that formation of the two sets of defensive sesquiterpenes, β-caryophyllene/α-humulene and (−)-endo-α-bergamotene/(+)-α-santalene/(+)-endo-β-bergamotene, can be introduced into new tissues in tomato. The epidermis-specific transgene expression and terpene formation were verified by fluorescence microscopy and tissue fractionation with subsequent analysis of terpene profiles, respectively. In addition, the longevity and fecundity of M. euphorbiae feeding on these engineered tomato leaves were significantly reduced, demonstrating the efficacy of this novel aphid control strategy.

Black cherry is an ecologically important high-value wood. A decline of its regeneration has been reported in the USA, which could be associated with a lack of pollination. This study was conducted to identify insects visiting black cherry flowers, to determine whether insects captured on the flowers carry black cherry pollen and to identify the volatile organic compounds (VOCs) emitted by flowers of black cherry. A two-year insect survey was conducted before, during and after the black cherry bloom. A total of 9533 insects were captured in traps and Diptera was the most abundant (64.1%). Significantly more insects in Diptera, Lepidoptera and Thysanoptera were captured in the traps installed in the canopy than those on the ground, and Anthalia bulbosa (Diptera: Hybotidae) was the dominant species. Electron microscopy analyses demonstrated that insects captured in the canopy indeed carried black cherry pollen. Black cherry flowers emitted a VOC blend that is composed of 34 compounds and dominated by β-ocimene and several phenylpropanoids/benzenoids. This floral VOC profile is similar to that of other pollinator-dependent Prunus species. This study reports pollinator insects and associated VOCs, for the first time, that could play a significant role in the pollination and regeneration of black cherry.

Terpenoids are a large and diverse class of plant metabolites that also includes volatile mono- and sesquiterpenes which are involved in biotic interactions of plants. Due to the limited natural availability of these terpenes and the tight regulation of their biosynthesis, there is strong interest to introduce or enhance their production in crop plants by metabolic engineering for agricultural, pharmaceutical and industrial applications. While engineering of monoterpenes has been quite successful, expression of sesquiterpene synthases in engineered plants frequently resulted in production of only minor amounts of sesquiterpenes. To identify bottlenecks for sesquiterpene engineering in plants, we have used two nearly identical terpene synthases, snapdragon (Antirrhinum majus) nerolidol/linalool synthase-1 and -2 (AmNES/LIS-1/-2), that are localized in the cytosol and plastids, respectively. Since these two bifunctional terpene synthases have very similar catalytic properties with geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), their expression in target tissues allows indirect determination of the availability of these substrates in both subcellular compartments. Both terpene synthases were expressed under control of the ripening specific PG promoter in tomato fruits, which are characterized by a highly active terpenoid metabolism providing precursors for carotenoid biosynthesis. As AmNES/LIS-2 fruits produced the monoterpene linalool, AmNES/LIS-1 fruits were found to exclusively produce the sesquiterpene nerolidol. While nerolidol emission in AmNES/LIS-1 fruits was 60- to 584-fold lower compared to linalool emission in AmNES/LIS-2 fruits, accumulation of nerolidol-glucosides in AmNES/LIS-1 fruits was 4- to 14-fold lower than that of linalool-glucosides in AmNES/LIS-2 fruits. These results suggest that only a relatively small pool of FPP is available for sesquiterpene formation in the cytosol. To potentially overcome limitations in sesquiterpene production, we transiently co-expressed the key pathway-enzymes hydroxymethylglutaryl-CoA reductase (HMGR) and 1-deoxy-D-xylulose 5-phosphate synthase (DXS), as well as the regulator isopentenyl phosphate kinase (IPK). While HMGR and IPK expression increased metabolic flux toward nerolidol formation 5.7- and 2.9-fold, respectively, DXS expression only resulted in a 2.5-fold increase.

Secondary metabolites produced in glandular trichomes of tomato are involved in interactions with herbivores. In cultivated tomato (Solanum lycopersicum) glandular trichomes accumulate a blend of abundant monoterpenes and smaller amounts of a few sesquiterpenes. These mono- and sesquiterpenes are synthesized by three terpene synthases, TPS20 as well as TPS9 and TPS12, respectively. To study effects of these terpenes on performance and choice behavior of potato aphid (Macrosiphum euphorbiae), we utilized two tomato trichome mutants, hairless and odorless-2, that are differently affected in mono- and sesquiterpene production. Non-choice assays demonstrated that longevity and fecundity of M. euphorbiae were increased when kept on the trichome mutants. A principal component analysis of these aphid performance parameters and terpene production in the trichome mutants indicated that longevity and fecundity of M. euphorbiae were negatively correlated with production of the TPS12-derived sesquiterpenes β-caryophyllene and α-humulene. While we had previously shown that addition of pure β-caryophyllene/α-humulene to an artificial feeding diet affected M. euphorbiae apterae survivorship and feeding behavior, no such effects were observed here upon addition of a mixture of pure TPS20-derived monoterpenes. In olfactometer assays M. euphorbiae alates displayed differential choice behaviors towards the hairless and odorless-2 mutants suggesting a role of TPS20-derived monoterpenes in aphid attraction, which was further confirmed using a mixture of pure monoterpenes. Our analyses revealed contrasting roles of glandular trichome-derived terpenes in S. lycopersicum. While TPS12-derived sesquiterpenes contribute to host plant resistance against M. euphorbiae, TPS20-derived monoterpenes appear to be exploited as cue for host plant orientation by aphids.

Significance: As the largest class of natural products found in all living organisms, terpenoids play essential roles in plant growth, development, respiration, photosynthesis, and environmental interactions. Geranylgeranyl diphosphate (GGPP), a precursor for several terpenoid metabolic branches including chlorophyll, carotenoid, and gibberellin biosynthesis, is produced by GGPP synthase (GGPPS) in plastids. We discovered that rice GGPPS recruiting protein (GRP), which forms a heterodimer with the only plastidic GGPPS, controls GGPPS dimerization state and enhances its catalytic properties. By interacting with GGPPS, GRP determines its allocation from stroma to thylakoid membranes, where the heterodimer exists in a complex with chlorophyll biosynthetic proteins. GGPPS recruitment to thylakoids by GRP represents a mechanism directing metabolic flux toward a specific product in the terpenoid metabolic network.