Plant glandular secreting trichomes are epidermal protuberances that produce structurally diverse specialized metabolites, including medically important compounds. Trichomes of many plants in the nightshade family (Solanaceae) produce O-acylsugars, and in cultivated and wild tomatoes these are mixtures of aliphatic esters of sucrose and glucose of varying structures and quantities documented to contribute to insect defense. We characterized the first two enzymes of acylsucrose biosynthesis in the cultivated tomato Solanum lycopersicum. These are type I/IV trichome-expressed BAHD acyltransferases encoded by Solyc12g006330─or S. lycopersicum acylsucrose acyltransferase 1 (Sl-ASAT1)─and Solyc04g012020 (Sl-ASAT2). These enzymes were used-in concert with two previously identified BAHD acyltransferases-to reconstruct the entire cultivated tomato acylsucrose biosynthetic pathway in vitro using sucrose and acyl-CoA substrates.Comparative genomics and biochemical analysis of ASAT enzymes were combined with in vitro mutagenesis to identify amino acids that influence CoA ester substrate specificity and contribute to differences in types of acylsucroses that accumulate in cultivated and wild tomato species. This work demonstrates the feasibility of the metabolic engineering of these insecticidal metabolites in plants and microbes.Solanum | glandular trichomes | acylsugar | specialized metabolism | genotype to phenotype P lants are masters of metabolism, producing hundreds of thousands of small molecules known as specialized metabolites, which vary widely in structure, abundance, and physical and biological properties. These metabolites tend to be produced by enzymes that evolve faster than those that produce "central" metabolites such as amino acids, nucleotides, sugars, and cofactors (1-3), and the pathways and metabolic intermediates involved in biosynthesis of many specialized metabolites remain mysterious. Despite the growing availability of genomic DNA sequences, understanding the genetic and biochemical mechanisms that contribute to this phenotypic diversity and plasticity presents enduring and major challenges in plant biochemistry. It is of great interest to understand and manipulate the biosynthesis of these biologically active molecules.Specialized metabolites typically are produced in a cell-or tissue-specific manner and are generally limited in their taxonomic distribution. Glandular secreting trichomes provide an example of such a differentiated structure; these epidermal "hairs" produce a variety of metabolites of importance to humans, including aromatic flavor components (e.g., in hops for beer and Mediterranean herbs for cooking), psychoactive cannabinoids in Cannabis, and the antimalarial drug artemisinin in Artemisia annua (4, 5).Some trichome-produced metabolites have documented direct and indirect antiherbivore activities (4, 6-8). For example, acylsugars are a group of structurally related specialized metabolites produced in plants of the nightshade family-the Solanaceae (9, 10). Characterized examples ...

Plants produce hundreds of thousands of structurally diverse specialized metabolites via multistep biosynthetic networks, including compounds of ecological and therapeutic importance. These pathways are restricted to specific plant groups, and are excellent systems for understanding metabolic evolution. Tomato and other plants in the nightshade family synthesize protective acylated sugars in the tip cells of glandular trichomes on stems and leaves. We describe a metabolic innovation in wild tomato species that contributes to acylsucrose structural diversity. A small number of amino acid changes in two acylsucrose acyltransferases alter their acyl acceptor preferences, resulting in reversal of their order of reaction and increased product diversity. This study demonstrates how small numbers of amino acid changes in multiple pathway enzymes can lead to diversification of specialized metabolites in plants. It also highlights the power of a combined genetic, genomic and in vitro biochemical approach to identify the evolutionary mechanisms leading to metabolic novelty.

Polarized growth requires the integration of polarity pathways with the delivery of exocytic vesicles for cell expansion and counterbalancing endocytic uptake. In budding yeast, the myosin-V Myo2 is aided by the kinesin-related protein Smy1 in carrying out the essential Sec4-dependent transport of secretory vesicles to sites of polarized growth. Over-expression suppressors of a conditional myo2 smy1 mutant identified a novel F-BAR-containing RhoGAP, Rgd3, that has activity primarily on Rho3, but also Cdc42. Internally tagged Rho3 is restricted to the plasma membrane in a gradient corresponding to cell polarity that is altered upon Rgd3 over-expression. Rgd3 itself is localized to dynamic polarized vesicles that, while distinct from constitutive secretory vesicles, are dependent on actin and Myo2 function. In vitro Rgd3 associates with liposomes in a PIP2-enhanced manner. Further, the Rgd3 C-terminal region contains several phosphorylatable residues within a reported SH3-binding motif. An unphosphorylated mimetic construct is active and highly polarized, while the phospho-mimetic form is not. Rgd3 is capable of activating Myo2, dependent on its phospho-state and Rgd3 overexpression rescues aberrant Rho3 localization and cell morphologies seen at the restrictive temperature in the myo2 smy1 mutant. We propose a model where Rgd3 functions to modulate and maintain Rho3 polarity during growth. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Interactive programs in BASIC for the leaf energy-budget equation are described. The programs are available on disc for the Apple II microcomputer and feature text output, screen graphics, multiple curves, exploration of curves by roving dots, entries for adaxial and abaxial cuticular and stomatal resistance, the Swinbank approximation, and Grashof–Reynolds numbers.

In cultivated (Solanum lycopersicum) and wild tomatoes, glandular trichomes produce structurally diverse acylsugars. These insect protective metabolites are mixtures of aliphatic esters of sucrose and glucose, with acylchains varying in length from C2 to C11. We are analyzing acylsugar biosynthesis in the cultivated tomato, along with the biochemical and evolutionary mechanisms that generate acylsugar diversity across wild tomatoes (1, 2). Previous studies identified four BAHD family Acylsucrose Acyltransferase (ASAT) enzymes. These catalyze consecutive reactions from sucrose and acyl‐CoA substrates to produce the full set of cultivated tomato acylsucroses in vitro (3). Recently we identified a modified ‘upside down’ acylsugar pathway in the wild tomatoes S. pennellii and S. habrochaites that produces unusual types of acylsucroses, namely triacylsucroses with all three chains on the pyranose ring (P‐type) (4). This is in contrast with the furanose ring acylated acylsucroses (F‐type) produced in cultivated tomatoes and a variety of wild tomato species. Biochemical analysis demonstrates that the acyl acceptor substrate specificities were changed in two S. pennellii ASAT orthologous enzymes, which reversed their enzymatic activity order, leading to the ‘flipped pathway’. A phylogeny‐based structure‐function analysis of the ASAT2 and ASAT3 enzymes led to identification of a small number of amino acid changes that switch their activities between the F‐ and P‐ acylsucrose pathways (4, 5). Investigation of these pathways in other tomato species allowed inference of the evolutionary events giving rise to divergence of F‐ and P‐type acylsucroses across tomato species. This work demonstrates multiple evolutionary mechanisms in biochemical pathway evolution, including gene duplication and loss, amino acid substitution, and how the emergence of enzyme promiscuity restructured a specialized metabolic pathway and led to metabolic product innovation.Support or Funding InformationThis work was funded by National Science Foundation grants IOS‐1025636 and IOS‐PGRP‐1546617 to A.D.J. and R.L.L. Abigail Miller was supported by NSF REU grant DBI‐1358474 in the summer of 2014 and an American Society of Plant Biologists Summer Undergraduate Research Award during the summer of 2015. A.D.J. acknowledges support from the USDA National Institute of Food and Agriculture, Hatch project MICL‐02143.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Polarized growth requires the integration of polarity pathways with the delivery of exocytic vesicles for cell expansion and counterbalancing endocytic uptake. In budding yeast, the myosin-V Myo2 is aided by the kinesin-related protein Smy1 in carrying out the essential Sec4dependent transport of secretory vesicles to sites of polarized growth. Over-expression suppressors of a conditional myo2 smy1 mutant identified a novel F-BAR-containing RhoGAP, Rgd3, that has activity primarily on Rho3, but also Cdc42. Internally tagged Rho3 is restricted to the plasma membrane in a gradient corresponding to cell polarity that is altered upon Rgd3 overexpression. Rgd3 itself is localized to dynamic polarized vesicles that, while distinct from constitutive secretory vesicles, are dependent on actin and Myo2 function. In vitro Rgd3 associates with liposomes in a PIP2-enhanced manner. Further, the Rgd3 C-terminal region contains several phosphorylatable residues within a reported SH3-binding motif. An unphosphorylated mimetic construct is active and highly polarized, while the phospho-mimetic form is not. Rgd3 is capable of activating Myo2, dependent on its phospho-state and Rgd3 overexpression rescues aberrant Rho3 localization and cell morphologies seen at the restrictive temperature in the myo2 smy1 mutant. We propose a model where Rgd3 functions to modulate and maintain Rho3 polarity during growth.