More Like this

1. Development of a relative quantification method for infrared matrix‐assisted laser desorption electrospray ionization mass spectrometry imaging of Arabidopsis seedlings

   https://doi.org/10.1002/rcm.8616  

   Bagley, M. Caleb ; Stepanova, Anna N. ; Ekelöf, Måns ; Alonso, Jose M. ; Muddiman, David C. ( February 2020 , Rapid Communications in Mass Spectrometry)

   Mass spectrometry imaging of young seedlings is an invaluable tool in understanding how mutations affect metabolite accumulation in plant development. However, due to numerous biological considerations, established methods for the relative quantification of analytes using infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI) mass spectrometry imaging are not viable options. In this study, we report a method for the quantification of auxin‐related compounds using stable‐isotope‐labelled (SIL) indole‐3‐acetic acid (IAA) doped into agarose substrate.

   Wild‐typeArabidopsis thalianaseedlings,sur2andwei8 tar2loss‐of‐function mutants, andYUC1gain‐of‐function line were grown for 3 days in the dark in standard growth medium. SIL‐IAA was doped into a 1% low‐melting‐point agarose gel and seedlings were gently laid on top for IR‐MALDESI imaging with Orbitrap mass spectrometry analysis. Relative quantification was performed post‐acquisition by normalization of auxin‐related compounds to SIL‐IAA in the agarose. Amounts of auxin‐related compounds were compared between genotypes to distinguish the effects of the mutations on the accumulation of indolic metabolites of interest.

   IAA added to agarose was found to remain stable, with repeatability and abundance features of IAA comparable with those of other compounds used in other methods for relative quantification in IR‐MALDESI analyses. Indole‐3‐acetaldoxime was increased insur2mutants compared with wild‐type and other mutants. Other auxin‐related metabolites were either below the limits of quantification or successfully quantified but showing little difference among mutants.

   Agarose was shown to be an appropriate sampling surface for IR‐MALDESI mass spectrometry imaging ofArabidopsisseedlings. SIL‐IAA doping of agarose was demonstrated as a viable technique for relative quantification of metabolites in live seedlings or tissues with similar biological considerations.

    

   more » « less
2. Complete Biosynthesis of Anthocyanins Using E. coli Polycultures

   https://doi.org/10.1128/mBio.00621-17  

   Jones, J. Andrew ; Vernacchio, Victoria R. ; Collins, Shannon M. ; Shirke, Abhijit N. ; Xiu, Yu ; Englaender, Jacob A. ; Cress, Brady F. ; McCutcheon, Catherine C. ; Linhardt, Robert J. ; Gross, Richard A. ; et al ( June 2017 , mBio)

   ABSTRACT Fermentation-based chemical production strategies provide a feasible route for the rapid, safe, and sustainable production of a wide variety of important chemical products, ranging from fuels to pharmaceuticals. These strategies have yet to find wide industrial utilization due to their inability to economically compete with traditional extraction and chemical production methods. Here, we engineer for the first time the complex microbial biosynthesis of an anthocyanin plant natural product, starting from sugar. This was accomplished through the development of a synthetic, 4-strain Escherichia coli polyculture collectively expressing 15 exogenous or modified pathway enzymes from diverse plants and other microbes. This synthetic consortium-based approach enables the functional expression and connection of lengthy pathways while effectively managing the accompanying metabolic burden. The de novo production of specific anthocyanin molecules, such as calistephin, has been an elusive metabolic engineering target for over a decade. The utilization of our polyculture strategy affords milligram-per-liter production titers. This study also lays the groundwork for significant advances in strain and process design toward the development of cost-competitive biochemical production hosts through nontraditional methodologies. IMPORTANCE To efficiently express active extensive recombinant pathways with high flux in microbial hosts requires careful balance and allocation of metabolic resources such as ATP, reducing equivalents, and malonyl coenzyme A (malonyl-CoA), as well as various other pathway-dependent cofactors and precursors. To address this issue, we report the design, characterization, and implementation of the first synthetic 4-strain polyculture. Division of the overexpression of 15 enzymes and transcription factors over 4 independent strain modules allowed for the division of metabolic burden and for independent strain optimization for module-specific metabolite needs. This study represents the most complex synthetic consortia constructed to date for metabolic engineering applications and provides a new paradigm in metabolic engineering for the reconstitution of extensive metabolic pathways in nonnative hosts. 

   more » « less
3. A combination of conserved and diverged responses underlies Theobroma cacao’s defense response to Phytophthora palmivora

   https://doi.org/10.1186/s12915-024-01831-2  

   Winters, Noah P. ; Wafula, Eric K. ; Knollenberg, Benjamin J. ; Hämälä, Tuomas ; Timilsena, Prakash R. ; Perryman, Melanie ; Zhang, Dapeng ; Sheaffer, Lena L. ; Praul, Craig A. ; Ralph, Paula E. ; et al ( February 2024 , BMC Biology)

   Plants have complex and dynamic immune systems that have evolved to resist pathogens. Humans have worked to enhance these defenses in crops through breeding. However, many crops harbor only a fraction of the genetic diversity present in wild relatives. Increased utilization of diverse germplasm to search for desirable traits, such as disease resistance, is therefore a valuable step towards breeding crops that are adapted to both current and emerging threats. Here, we examine diversity of defense responses across four populations of the long-generation tree cropTheobroma cacaoL., as well as four non-cacaoTheobromaspecies, with the goal of identifying genetic elements essential for protection against the oomycete pathogenPhytophthora palmivora.

   We began by creating a new, highly contiguous genome assembly for theP. palmivora-resistant genotype SCA 6 (Additional file 1: Tables S1-S5), deposited in GenBank under accessions CP139290-CP139299. We then used this high-quality assembly to combine RNA and whole-genome sequencing data to discover several genes and pathways associated with resistance. Many of these are unique, i.e., differentially regulated in only one of the four populations (diverged 40 k–900 k generations). Among the pathways shared across all populations is phenylpropanoid biosynthesis, a metabolic pathway with well-documented roles in plant defense. One gene in this pathway, caffeoyl shikimate esterase (CSE), was upregulated across all four populations following pathogen treatment, indicating its broad importance for cacao’s defense response. Further experimental evidence suggests this gene hydrolyzes caffeoyl shikimate to create caffeic acid, an antimicrobial compound and known inhibitor ofPhytophthora spp.

   Our results indicate most expression variation associated with resistance is unique to populations. Moreover, our findings demonstrate the value of using a broad sample of evolutionarily diverged populations for revealing the genetic bases of cacao resistance toP. palmivora. This approach has promise for further revealing and harnessing valuable genetic resources in this and other long-generation plants.

    

   more » « less
4. Occurrence, Function, and Biosynthesis of the Natural Auxin Phenylacetic Acid (PAA) in Plants

   https://doi.org/10.3390/plants12020266  

   Perez, Veronica C. ; Zhao, Haohao ; Lin, Makou ; Kim, Jeongim ( January 2023 , Plants)

   Auxins are a class of plant hormones playing crucial roles in a plant’s growth, development, and stress responses. Phenylacetic acid (PAA) is a phenylalanine-derived natural auxin found widely in plants. Although the auxin activity of PAA in plants was identified several decades ago, PAA homeostasis and its function remain poorly understood, whereas indole-3-acetic acid (IAA), the most potent auxin, has been used for most auxin studies. Recent studies have revealed unique features of PAA distinctive from IAA, and the enzymes and intermediates of the PAA biosynthesis pathway have been identified. Here, we summarize the occurrence and function of PAA in plants and highlight the recent progress made in PAA homeostasis, emphasizing PAA biosynthesis and crosstalk between IAA and PAA homeostasis. 

   more » « less
5. Diverse MarR bacterial regulators of auxin catabolism in the plant microbiome

   https://doi.org/10.1038/s41564-022-01244-3  

   Conway, Jonathan M. ; Walton, William G. ; Salas-González, Isai ; Law, Theresa F. ; Lindberg, Chloe A. ; Crook, Laura E. ; Kosina, Suzanne M. ; Fitzpatrick, Connor R. ; Lietzan, Adam D. ; Northen, Trent R. ; et al ( October 2022 , Nature Microbiology)

   Chemical signalling in the plant microbiome can have drastic effects on microbial community structure, and on host growth and development. Previously, we demonstrated that the auxin metabolic signal interference performed by the bacterial genusVariovoraxvia an auxin degradation locus was essential for maintaining stereotypic root development in an ecologically relevant bacterial synthetic community. Here, we dissect theVariovoraxauxin degradation locus to define the genesiadDEas necessary and sufficient for indole-3-acetic acid (IAA) degradation and signal interference. We determine the crystal structures and binding properties of the operon’s MarR-family repressor with IAA and other auxins. Auxin degradation operons were identified across the bacterial tree of life and we define two distinct types on the basis of gene content and metabolic products:iac-like andiad-like. The structures of MarRs from representatives of each auxin degradation operon type establish that each has distinct IAA-binding pockets. Comparison of representative IAA-degrading strains from diverse bacterial genera colonizingArabidopsisplants show that while all degrade IAA, only strains containingiad-like auxin-degrading operons interfere with auxin signalling in a complex synthetic community context. This suggests thatiad-like operon-containing bacterial strains, includingVariovoraxspecies, play a key ecological role in modulating auxins in the plant microbiome.

    

   more » « less