Source: PURDUE UNIVERSITY submitted to
MOLECULAR AND BIOCHEMICAL DETERMINANTS OF FLORAL SCENT PRODUCTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1016140
Grant No.
(N/A)
Project No.
IND065039
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2018
Project End Date
Sep 30, 2023
Grant Year
(N/A)
Project Director
Dudareva, NA.
Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Biochemistry
Non Technical Summary
Progress by our groups (Adebesin et al., 2017) and others (Bird et al., 2007; McFarlane et al., 2010; Zhu et al., 2017c, 2017b) have already begun to illuminate how hydrophobic metabolites cross the plasma membrane, but this work will uncover the biological processes involved in shuttling plant metabolites across the cytosol, cell wall, and cuticle. This study will piece together the complex puzzle linking metabolite biosynthesis with their passage through cellular barriers into the environment. As volatiles were previously believed to passively diffuse out of cells, this work will be transformative, as it conceptually changes our way of understanding volatile emission. Moreover, because metabolites produced in different plant organs/tissues have to cross the same cellular barriers, this work will illuminate general biological mechanisms for hydrophobic compounds beyond volatiles and root allelochemicals, including waxes and lignin precursors. This work will uncover new targets for metabolic engineering to either accumulate compounds of interest or accelerate their release from the plant. Such strategies may improve agronomic traits (e.g. pollination, defense), biofuel production (e.g. phenylethanol, terpenoids), palatability of fresh fruits and vegetables (e.g. aroma, flavors), commercial value of ornamentals (e.g. floral scent), and provide platforms to produce and deploy natural product-based herbicides. It will also provide a foundation for development of more accurate atmospheric volatile emission models as plant volatiles significantly contribute to our environment. Beyond plants, chemical communication plays a very important role in the lives of insects and interactions between animals. How metabolites are released from these organisms is a similar biological question to that in plants (minus the cell wall and in some cases the cuticle). Thus, the information gained in this research may enhance understanding of the molecular release mechanisms used by other eukaryotes.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2062121100020%
2062121104015%
2062121102015%
2062499100020%
2062499104015%
2062499102015%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
2121 - Cut flowers, foliage, and greens; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology; 1000 - Biochemistry and biophysics; 1020 - Physiology;
Goals / Objectives
Over the last several years, our labs have concentrated on elucidating the biosynthetic pathways for plant volatiles and allelochemicals, the latter of which are specialized metabolites negatively impacting the growth and development of neighboring plants. We have not only isolated genes responsible for the formation of some of these compounds, but we have also begun to understand the regulation of metabolic fluxes through these pathways using stable isotope-labeled precursors and metabolic modeling. Next, we will investigate how these compounds, primarily lipophilic low-molecular-weight molecules (~100-250 Da), are released to facilitate plant communication. To reach the environment, volatiles and root allelochemicals have to move from their site of synthesis, through cytosol, then cross the plasma membrane, hydrophilic cell wall, and often an outer waxy cuticle in aerial tissues or a hydrophobic suberin layer in roots. To study these processes, we will use Petunia hybrida flowers, which emit high levels of benzenoid/phenylpropanoid volatiles, and hairy roots of Lithospermum erythrorhizon, which secrete large amounts of the hydrophobic allelochemical shikonin and shikonin derivatives (Zhu et al., 2017a). Both systems have available genomic (Bombarely et al., 2016; https://www.ncbi.nlm.nih.gov/bioproject/386534) and transcriptomic (Widhalm et al., 2015a; Widhalm laboratory, unpublished) resources and are amenable to genetic manipulations (Boatright, 2004; Verdonk et al., 2003; Zhu et al., 2017b, 2017c).In this collaborative project, we will use our combined expertise to study different aspects of metabolite trafficking out of cells. We identified three key areas to fill important gaps in our knowledge about the molecular process(es) involved in the release of plant metabolites from their sites of biosynthesis to the environment:Determine the role of vesicle trafficking in shuttling lipophilic specialized metabolites through the cytosol to the plasma membrane.Elucidate the mechanisms for carrying lipophilic specialized metabolites across the cell wall to the outer layer of the cell.Determine the effect of cuticle chemical composition on volatile emission.
Project Methods
Objective 1: Determine the role of vesicle trafficking in shuttling lipophilic specialized metabolites through the cytosol to the plasma membrane.Before lipophilic metabolites cross the plasma membrane, they must traverse the cytosol. However, instead of directly reaching the plasma membrane after being synthesized, we hypothesize that hydrophobic metabolites partition into subcellular membranes and are directionally moved to the plasma membrane via vesicle trafficking (Widhalm et al., 2015b). Our inhibitory study in petunia and published reports in L. erythrorhizon hairy roots (Tatsumi et al., 2016) with the vesicle-trafficking inhibitor Brefeldin A (BFA) implicate the involvement of vesicle trafficking in hydrophobic metabolite release from cells. These results indicate that (i) metabolites may be directly trafficked to the plasma membrane via the secretory pathway and/or (ii) that proteins involved in transporting hydrophobic compounds across the plasma membrane or shuttling them through the cell wall are delivered via vesicle transport. To determine if vesicles directly transport metabolites to the plasma membrane we will metabolically profile endomembranes prepared from petunia flowers and L. erythrorhizon hairy roots. To determine if vesicles deliver known ABC transporters established to export volatiles and shikonin across plasma membranes in petunia (Adebesin et al., 2017) and L. erythrorhizon (Zhu et al., 2017b, 2017c), respectively, we will examine if these GFP-tagged proteins are mislocalized after BFA treatment.By combining biochemistry with reverse genetics and systems biology approaches we will screen for additional molecular players involved in metabolite release in our two model systems. We have already uncovered a novel petunia protein, synaptic vesicle protein 2 (SV2), that is homologous to one of the hallmark players in vesicle-mediated trafficking of neurotransmitters in mammals. Our preliminary data indicate that downregulation of SV2 in petunia flowers reduces volatile emission with concomitant accumulation of volatiles in the flower. Moreover, the SV2-downregulation phenotype can be complemented with mouse SV2, suggesting that the plant homolog is involved in vesicle trafficking similar to its mammalian counterpart. Next, we will analyze SV2 subcellular localization within the secretory network using GFP-tagged proteins and confocal microscopy in the presence and absence of vesicle trafficking inhibitors. To determine what protein cargo resides in PhSV2 vesicles we will subject SV2 affinity-purified vesicles to proteomic analysis (Drakakaki et al., 2012) and metabolic profiling. Similar experiments will also be performed using L. erythrorhizon hairy roots to determine if this is a conserved mechanism for hydrophobic metabolite trafficking in plants.Objective 2. Elucidate the mechanisms for carrying lipophilic specialized metabolites across the cell wall to the outer layer of the cell.Recent studies suggest that plant small carrier proteins, such as extracellular lipid transfer proteins (LTPs), function in the export of hydrophobic wax lipids and diterpenes from the plasma membrane, across the cell wall, to the cuticle (Cameron et al., 2006; Debono et al., 2009). We hypothesize that LTPs, or other carrier proteins, shuttle volatiles and hydrophobic root allelochemicals from the plasma membrane across the aqueous cell wall space to the outer cell layer. We have already identified several LTP candidates from RNA-sequencing datasets from petunia flowers and L. erythrorhizon hairy roots generated by our laboratories. To determine if these LTP candidates are involved in passage of volatiles and shikonins across cell walls, we will use an RNA interference (RNAi) strategy in our respective model systems. Those candidates showing reduced metabolite release upon downregulation will be cloned and subjected to GFP fusion experiments to confirm their localization in the cell wall and to biochemical characterization.Objective 3. Determine the effect of cuticle chemical composition on volatile emission.The final interface between aerial plant tissues and the atmosphere is the cuticle. Based on the structure and chemical properties of the cuticle there are likely no active biological mechanisms in place for shuttling volatiles to the atmosphere. Thus, we hypothesize that passage of compounds across the cuticle is solely diffusive and is dependent on the physicochemical properties of the compound itself and the composition of the cuticle. Due to high volatile cuticle partition coefficients (Kc/w), cuticle thickness is unlikely to significantly affect emission (Riederer and Schreiber, 2001) in contrast to the cuticle composition. To test this, we have designed three strategies to modify the cuticle composition in petunia flowers: (i) by overexpressing transcription factor, Arabidopsis thaliana AtMYB106, known to regulate expression of genes involved both in cutin and wax biosynthesis; (ii) by reducing export of cuticle components via RNAi downregulation of two petunia orthologues of A. thaliana ABCG11 and ABCG12, known to be involved in wax and cutin component transport; and (iii) by changing the expression of genes involved in wax/cutin biosynthesis. Overexpression of A. thaliana CER1 and CER3 proteins known to be essential for the synthesis of alkanes, as well as three A. thaliana genes GPAT6, CYP77A6 and CYP86A4 known to be involved in petal-specific cutin synthesis and conical epidermal cell nanoridge formation will be used to modify wax/cutin biosynthesis. All constructs will be generated under InMYBpro promoter from Japanese morning Glory showing specific expression in flower buds.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Research provides a basic knowledge about biosynthesis of plant-produced volatile compounds and their emission into the atmosphere and release of natural products released into the rhizosphere. The work is important for plant biologists, biochemists, atmospheric chemists, and scientists involved in plant metabolic engineering and investigation of plant-plant and plant insect interactions. This research is also of interest for scientists working in floriculture, flavor, cosmetic and fragrance industries, as well as those working to develop novel herbicides and other agricultural products. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This work provided strong multidisciplinary training in plant biochemistry, molecular biology, genetics, in vivo isotopic labeling, GC-MS and LC-MS-based metabolic profiling and integrative modeling for undergraduate and graduate students, as well as post-doctoral scientists. Dr. Joseph Lynch - Research Assistant (Dudareva): Analysis of transgenic petunia plants with overexpression of PhCM2 and biochemical interface between Phe and auxin biosynthesis. Pan Liao - Post-doc (Dudareva): Analysis of PhABCG12 downregulation on wax composition and volatile emission. Itay Maoz - Post-doc (Dudareva): Analysis of PhLTP(s) downregulation on volatile emission. Xing-Qi (David) Huang - Post-doc (Dudareva): Identification of transporter involved in shikimate transport out of plastids. Elucidation of benzaldehyde biosynthesis in plants. Shannon Sterling - Graduate Student (Dudareva) Investigation of molecular mechanisms of natural fumigation Mithila Shukla - Graduate Student (Dudareva) Investigating flux through the shikimate pathway Ji Hee Lee - Graduate Student (Dudareva) Investigating the role of vesicle trafficking in VOC emission Dr. Manoj Ghaste - Post-doc (Widhalm): Dicamba volatility research. Rachel McCoy - Ph.D. candidate (Widhalm): Investigation of plant quinone metabolism. Thiti Suttiyut - Ph.D. student (Widhalm): Elucidation of cytoplasmic geranyl diphosphate formation in Lithospermum erythrorhizon George Meyer - Ph.D. student (Widhalm): Investigation of juglone resistance mechanisms in plants Awards: Dr. Postdoc Joseph Lynch received 2020 Linda Siersema Staff Excellence Award, Purdue University, Biochemistry Department, May, 2020. Graduate student Shannon Stirling received a Bird Stair Graduate Research Fellowship ($5,000) for her project "KAI2 acts as a receptor for germacrene D and is essential for stigma development", Spring 2020. Graduate student Rachel McCoy received the "Outstanding Graduate Research Award, Ph.D." in the Department of Horticulture and Landscape Architecture How have the results been disseminated to communities of interest?Invited seminars and invited speaker at meetings: 2019 Ball Horticultural Company, West Chicago, IL, November 22. (Dudareva) 2019 University Jean Monnet Saint-Etienne, University de Lyon, Saint-Etienne, France, October 14. (Dudareva) 2019 Biochemistry club (for undergraduate students), Department of Biochemistry, Purdue University, October 1. (Dudareva) 2020 Plant Biology 2020 ASPB Annual Meeting, Washington, DC, July 27 -31, 2020. (Dudareva) 2020 Biochemistry Horizons Symposium "From Organelles to Metabolites and Beyond", Purdue University, West Lafayette, IN, March 6. (Dudareva) 2020 ABC2020 Keynote Plenary Lecture, FEBS Special Meeting 2020 ATP-Binding Cassette (ABC) Proteins: From Multidrug Resistance to Genetic Disease, Innsbruck, Austria, March 1-7. (Dudareva) 2019 "Small Molecules in Plant Research: Chemistry and Biology Come Together Symposium", Valencia, Spain, December 10-11. (Dudareva) 2019 2019 International Plant Biology Workshop at UCSD "Plant Synthetic Biology and Genome Engineering, University of California San Diego, San Diego, CA, November 4. (Dudareva) 2020 Investigating the metabolic origins of specialized plant quinones. Horticulture Research Symposium. Nanjing, China. Note: seminar given online due to COVID-19. (Widhalm) 2020 Investigating the metabolic origins and pathways of specialized plant 1,4-naphthoquinones. University of Illinois at Urbana-Champaign, PMPB seminar series. Champaign, IL. (Widhalm) What do you plan to do during the next reporting period to accomplish the goals?We will continue to investigate the functions, biosynthesis and mode of trafficking of secondary metabolites into the environment using multidisciplinary approaches. We will specifically study the role of vesicles and LTPs in VOC emission from flowers and release of allelochemicals from roots.

Impacts
What was accomplished under these goals? Plants synthesize an amazing diversity of volatile organic compounds (VOCs) that are important for reproduction and defense, serve as practical products for humans, and influence atmospheric chemistry and climate. To date, the chemistry of plant volatiles is well understood, however, little is known about the biosynthesis of this diverse group of compounds and their release from the cell to the atmosphere. Phenylpropanoid compounds constitute the second largest class of plant volatiles, which originate from the aromatic amino acid phenylalanine (Phe). In plants Phe is synthesized predominantly via the arogenate pathway in plastids. However, we have recently demonstrated that Phe is also synthesized in the cytosol via the microbial-like phenylpyruvate pathway, which branches from the known plastidial arogenate pathway at chorismate. In contrast to the arogenate pathway, the phenylpyruvate route is subject to less stringent feedback regulation, because cytosolic chorismate mutase 2 (CM2), which catalyzes the initial step in this pathway, is insensitive to allosteric regulation by aromatic amino acids. Thus, we investigated the metabolic consequences of altering carbon flux towards the cytosolic phenylpyruvate pathway in Petunia hybrida flowers, which typically produce high levels of Phe and Phe-derived benzenoid/phenylpropanoid volatiles. Overexpression of petunia chorismate mutase 2 (PhCM2) increased flux in cytosolic phenylalanine biosynthesis, but paradoxically decreased the levels of phenylalanine and phenylalanine-derived volatiles. Concomitantly, the levels of auxins, including indole-3-acetic acid and its precursor indole-3-pyruvic acid, were elevated. Biochemical and genetic analyses revealed the existence of metabolic crosstalk between the cytosolic phenylalanine biosynthesis and tryptophan-dependent auxin biosynthesis mediated by an aminotransferase that uses cytosolic phenylalanine biosynthetic pathway metabolites as amino acceptors for auxin formation. Plants synthesize volatile organic compounds (VOCs) to attract pollinators and beneficial microorganisms, to defend themselves against herbivores and pathogens and for plant-plant communication. The cuticle is the final physical barrier for VOCs to cross in most plant cells. Due to their hydrophobicity, the rate of VOC transport should depend on the physicochemical properties of the compound itself (diffusivity and solubility in the cuticle) and the cuticle composition and thickness. Although cuticle structure and composition vary considerably between plants, organs and developmental stages, it is commonly composed of cutin and cuticular wax, the latter of which is embedded in and deposited on the surface of the cutin matrix. The cuticular wax is a semi-crystalline mixture of alkanes, aldehydes, primary and secondary alcohols, ketones and esters, all derived from very-long-chain fatty acids. The cuticle generally acts as a molecular transport barrier, protecting tissues from stresses and preventing organ fusion during plant development. While the role of the cuticle in transpirational water flux is well established and extensive prior research provides knowledge about the interaction of atmospheric VOCs with the cuticle, how the cuticle is involved in the release of hydrophobic volatiles into the atmosphere remains an unresolved question. We used Petunia hybrida flowers with a combination of reverse-genetic and chemical approaches to investigate the role of the cuticle in the emission process. We show that reduction in cuticle thickness not only alters VOC emission, but also leads to redistribution of VOC internal pools and feedback inhibition of VOC biosynthesis. While VOCs also play important roles belowground, so do plant exudates released from roots containing non-volatile compounds. Unlike VOCs, however, the roles that these compounds play in mediating plant-biotic interactions is poorly understood. Moreover, little is known about biosynthesis of these compounds and the mechanisms responsible for their release from the cell into the rhizosphere. Specialized 1,4-naphthoquinones (1,4-NQ) have emerged as a major class of compounds involved in belowground plant-biotic interactions. Plants collectively synthesize hundreds of 1,4-NQs proposed to play roles in various plant-plant, plant-microbe and plant-animal interactions. Based on developments in understanding of plant metabolic networks, reinterpretations of classical tracer studies suggest that 1,4-NQs are synthesized through several routes. We have made major progress on understanding one of these routes, that which leads to shikonin. Shikonin is produced by the medicinal plant Lithospermum erythrorhizon (red gromwell; zicao). Roots from L. erythrorhizon have been used for centuries based on the antiviral and wound- healing properties produced from the bioactive compound shikonin and its derivatives. Shikonin also functions as an allelochemical when released into the soil. More recently, shikonin, its enantiomer alkannin, and several other shikonin/alkannin derivatives have collectively emerged as valuable natural colorants and as novel drug scaffolds. Despite several transcriptomes and proteomes having been generated from L. erythrorhizon, a reference genome is still unavailable. This has limited investigations into elucidating the shikonin/ alkannin pathway and understanding its evolutionary and ecological significance. In this study, we obtained a de novo genome assembly for L. erythrorhizon using a combination of Oxford Nanopore long-read and Illumina short-read sequencing technologies. The resulting genome is ∼367.41 Mb long, with a contig N50 size of 314.31 kb and 27,720 predicted protein-coding genes. Using the L. erythrorhizon genome, we identified several additional p- hydroxybenzoate:geranyltransferase (PGT) homologs and provide insight into their evolutionary history. Phylogenetic analysis of prenyltransferases suggests that PGTs originated in a common ancestor of modern shikonin/alkannin- producing Boraginaceous species, likely from a retrotransposition-derived duplication event of an ancestral prenyltransferase gene. Furthermore, knocking down expression of LePGT1 in L. erythrorhizon hairy root lines revealed that LePGT1 is predominantly responsible for shikonin production early in culture establishment. Taken together, the reference genome reported in this study and the provided analysis on the evolutionary origin of shikonin/alkannin biosynthesis will guide elucidation of the remainder of the pathway.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Bornowski N, Hamilton JP, Liao P, Wood JC, Dudareva N, Buell CB. 2020. Genome sequencing of four culinary herbs reveals terpenoid genes underlying chemodiversity in the Nepetoideae, DNA Research, 27 (3), dsaa016.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Lynch JH and Dudareva N. 2020. Aromatic amino acids: A complex network ripe for future exploration. Trends in Plant Sci., 25: 670-681.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Lynch JH, Qian Y, Guo L, Maoz I, Huang X-Q, Garcia A, Louie G, Bowman ME, Noel JP, Morgan JA, Dudareva N. 2020. Modulation of auxin formation by the cytosolic phenylalanine biosynthetic pathway, Nature Chem. Biol., 16: 850-856.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Lichman BR, Godden GT, Hamilton JP, Palmer L, Kamileen MO, Zhao D, Vaillancourt B, Wood J, Sun M, Kinser TJ, Henry LK, Lopez CR, Dudareva N, Soltis DE, Soltis PS, Buell CR, OConnor SE. 2020. The evolutionary origins of the cat attractant nepetalactone in catnip, Science Advances, 6: eaba0721.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Hivert G, Davidovich-Rikanati R, Bar E, Sitrit Y, Schaffer A, Dudareva N, Lewinsohn E. 2020. Prenyltransferases catalyzing geranyldiphosphate formation in tomato fruit. Plant Sci., 296: 110504
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Boachon B, Burdloff Y, Ruan J-X, Rojo R, Vincent B, Junker RR, Bringel F, Lesot A, Henry L, Bassard J-E, Mathieu S, Allouche L, Kaplan I, Dudareva N, Vuilleumier S, Miesch L, Andr� F, Navrot N, Chen X-Y, Werck-Reichhart D. 2019. A promiscuous CYP706A3 reduces terpene volatile emission from Arabidopsis flowers, with impacts on florivores and floral microbiome. The Plant Cell, 31: 2947-2972.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Islam AKMM and Widhalm JR. (2020) Agricultural uses of juglone: Opportunities and challenges. Agronomy, 10, 1500.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Ghaste M, Hayden N, Osterhold MJ, Young JM, Young BG, and Widhalm JR*. (2020) Evaluation of a stable isotope-based direct quantification method for dicamba analysis from air and water using single-quadrupole LCMS. Molecules, 25(16), 3649.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Auber RP, Suttiyut T, McCoy RM, Ghaste M, Crook JW, Pendleton AL, Widhalm JR*, and Wisecaver JH*. (2020) Hybrid de novo genome assembly of red gromwell (Lithospermum erythrorhizon) reveals evolutionary insights into shikonin biosynthesis. Horticulture Research, 7:82.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: McCoy RM, Meyer GW, Rhodes D, Murray GC, Sors TG, and Widhalm JR*. (2020) Investigation of the foliar incorporation and stability of isotopically labeled amino acids applied to turfgrass. Agronomy, 10, 358.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Li Y, Brooks M, Yeoh-Wang J, McCoy RM, Rock T, Pasquino A, Moon CI, Patrick RM, Tanurdzic M, Ruffel S, Widhalm JR, McCombie WR, and Coruzzi GM. (2020) SDG8-mediated histone methylation and RNA processing function in the response to nitrate signaling. Plant Physiology, 182:215-227.
  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Maoz I, Sun P, Haring MA, Schuurink RC, Dudareva N. 2020. Emission and perception of plant volatiles. In E. Pichersky, N. Dudareva (eds), Biology of Plant Volatiles, CRC Press, Taylor and Francis Group, Chapter 14, pp. 251-267.
  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Lynch JH, Pichersky E, Dudareva N. 2020. Floral scent metabolic pathways and their regulation. In E. Pichersky, N. Dudareva (eds), Biology of Plant Volatiles, CRC Press, Taylor and Francis Group, Chapter 8, pp. 147-164.
  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Plasmeier M, Liao P, Haring MA, Dudareva N, Schuurink RC. 2020. Molecular engineering of plant volatiles: floral scent, flavors, defense. In E. Pichersky, N. Dudareva (eds), Biology of Plant Volatiles, CRC Press, Taylor and Francis Group, Chapter 20, pp. 379-403.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Investigating the subcellular architecture of the phylloquinone pathway. McCoy RM, Sun B, Herrera K, Widhalm JR. Plant Biology 2020. American Society of Plant Biologists annual meeting. Virtual in 2020
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Investigating resistance mechanisms to the natural product-based herbicide juglone. Meyer GW, Naranjo M, House R, Widhalm JR. Plant Biology 2020. American Society of Plant Biologists annual meeting. Virtual in 2020
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Elucidating the metabolic origin of the geranyl diphosphate moiety of shikonin. Suttiyut T, Auber RP, Ghaste M, Wisecaver JH, Widhalm JR. Plant Biology 2020. American Society of Plant Biologists annual meeting. Virtual in 2020
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Evaluation of a stable isotope-based direct quantification method for dicamba analysis from air and water using single quadrupole LC-MS. Ghaste M, Hayden N, Osterhold MJ, Young J, Young BG, Widhalm JR. Plant Biology 2020. American Society of Plant Biologists annual meeting. Virtual in 2020
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Plant natural product biosynthesis and trafficking. Widhalm JR. Chemical Machinery of the Cell Scialog Conference. Tucson, AZ 2019


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Research provides a basic knowledge about biosynthesis of plant-produced volatile compounds and their emission into the atmosphere and release of natural products released into the rhizosphere. The work is important for plant biologists, biochemists, atmospheric chemists, and scientists involved in plant metabolic engineering and investigation of plant-plant and plant insect interactions. This research is also of interest for scientists working in floriculture, flavor, cosmetic and fragrance industries, as well as those working to develop novel herbicides and other agricultural products. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Post-doc Dr. Manoj Ghaste has attended training sessions focused on new developments in mass spectrometer technology hosted by Agilent in Indianapolis Graduate student Rachel McCoy attended professional development workshops at the Botany 2019 conference entitled "Using HHMI Videos and Data Points As Tools For Engaging Students From Molecules to Ecosystems" and "Strategies for successful faculty/undergraduate student collaborative research at PUIs." Graduate student Rachel McCoy attended professional development workshops at the Plant Biology 2019 conference entitled "Primarily Undergraduate Institution Faculty Development Workshop," "Being a Plant Biologist in a Primarily Undergraduate Institution," and "How Machine Learning Can Be Used to Solve Plant Biology Problems." Training: This work provided strong multidisciplinary training in plant biochemistry, molecular biology, genetics, in vivo isotopic labeling, GC-MS and LC-MS-based metabolic profiling and integrative modeling for undergraduate and graduate students, as well as post-doctoral scientists. Dr. Joseph Lynch - Post-doc (Purdue University): Analysis of transgenic petunia plants with overexpression of PhCM2 and biochemical interface between Phe and auxin biosynthesis. Yichun Qian - Ph.D. candidate (Purdue University): Generation and metabolic profiling of transgenic petunia plants with overexpression of PhCM2 Pan Liao - Post-doc (Purdue University): Analysis of PhABCG12 downregulation on wax composition and volatile emission. Itay Maoz - Post-doc (Purdue University): Analysis of PhLTP(s) downregulation on volatile emission. Xing-Qi (David) Huang - Post-doc (Purdue University): Identification of transporter involved in shikimate transport out of plastids. Elucidation of benzaldehyde biosynthesis in plants. Shannon Sterling - Graduate Student (Purdue University) Investigation of molecular mechanisms of natural fumigation Dr. Manoj Ghaste - Post-doc (Purdue University): Dicamba volatility research. Rachel McCoy - Ph.D. candidate (Purdue University): Investigation of plant quinone metabolism. Thiti Suttiyut - Ph.D. student (Purdue University): Elucidation of cytoplasmic geranyl diphosphate formation in Lithospermum erythrorhizon George Meyer - Ph.D. student (Purdue University): Investigation of juglone resistance mechanisms in plants Elena Yakubova - Research technician (Purdue University): Determination of quinone content in Impatiens species nectaries. How have the results been disseminated to communities of interest?Invited seminars and invited speaker at meetings: 2019 University Jean Monnet Saint-Etienne, University de Lyon, Saint-Etienne, France, October 14. (Dudareva) 2019 Biochemistry club (for undergraduate students), Department of Biochemistry, Purdue University, October 1. (Dudareva) 2019 XXIII Meeting of the Spanish Society of Plant Physiology and the XVI Hispano-Portuguese Congress of Plant Physiology, Pamplona, Spain, June 26-28. (Dudareva) 2019 2019 Gordon Research Conference on Plant Metabolic Engineering, Lucca (Barga), Italy, June 16-21. (Dudareva) 2019 Institute for Biochemistry and Biology, University of Potsdam, Potsdam-Golm, Germany, June 3. (Dudareva) 2019 2019 Congress "At the Forefront of Plant Research", Barcelona, Spain, May 6 -8. 2019 2019 Annual Meeting of the American Society for Biochemistry and Molecular Biology (ASBMB), Orlando, FL, April 6-10. (Dudareva) 2019 Friday Seminar at John Innes Center, Norwich, UK, March 15. (Dudareva) 2019 College of Landscape Architecture and Horticulture Sciences, Southwest Forestry University, Kunming, China, February 26. (Dudareva) 2019 School of Landscape Architecture, National Engineering Research Center for Floriculture, Beijing Forestry University, Beijing, China, February 22. (Dudareva) 2019 Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China, February 21. (Dudareva) 2019 Purdue University Botany and Plant Pathology Seminar Series, April 17. (Widhalm) 2018 8th Annual Plant Sciences Symposium, University of Wisconsin - Madison, Madison, WI, (student invited), November 16. (Dudareva) What do you plan to do during the next reporting period to accomplish the goals?We will continue to work on original three objectives of proposed research. Specifically, we will investigate the role of LTPs in volatile emission, and will work on elucidation of molecular mechanisms of natural fumigation.

Impacts
What was accomplished under these goals? Plants synthesize an amazing diversity of volatile organic compounds (VOCs) that are important for reproduction and defense, serve as practical products for humans, and influence atmospheric chemistry and climate. To date, the chemistry of plant volatiles is well understood, however, little is known about the biosynthesis of this diverse group of compounds and their release from the cell to the atmosphere. Phenylpropanoid compounds constitute the second largest class of plant volatiles, which originate from the aromatic amino acid phenylalanine (Phe). In plants Phe is synthesized predominantly via the arogenate pathway in plastids. However, we have recently demonstrated that Phe is also synthesized in the cytosol via the microbial-like phenylpyruvate pathway, which branches from the known plastidial arogenate pathway at chorismate. In contrast to the arogenate pathway, the phenylpyruvate route is subject to less stringent feedback regulation, because cytosolic chorismate mutase 2 (CM2), which catalyzes the initial step in this pathway, is insensitive to allosteric regulation by aromatic amino acids. Thus, we investigated the metabolic consequences of altering carbon flux towards the cytosolic phenylpyruvate pathway in Petunia hybrida flowers, which typically produce high levels of Phe and Phe-derived benzenoid/phenylpropanoid volatiles. Overexpression of PhCM2 in petunia flowers increased flux in cytosolic Phe biosynthesis, but paradoxically decreased the levels of Phe and Phe-derived volatiles. Concomitantly, the levels of auxins, including indole-3-acetic acid and its precursor indole-3-pyruvic acid (IPA), were elevated. A combination of reverse genetics, biochemistry, targeted metabolic profiling, metabolic flux modelling and electron microscopy revealed that (i) overexpression of PhCM2 leads to perturbation of plastid development due to altered auxin metabolism; (ii) this phenotype could be reverted by overexpression of Arabidopsis VAS1, an aminotransferase that converts the auxin IPA back to tryptophan; and (iii) PhTrp-AT, an identified aminotransferase that transaminates tryptophan to auxin, is able to use intermediates/byproducts of the cytosolic Phe biosynthetic pathway as amino acceptors, thereby linking extra-plastidial metabolism of tryptophan and Phe. Overall, our results show the existence of a metabolic crosstalk between the cytosolic Phe pathway and tryptophan-dependent auxin biosynthesis. Despite the relatively low direct contribution of the cytosolic phenylpyruvate pathway to Phe biosynthesis, plants likely use this pathway for rapid production of Phe in response to biotic and abiotic stresses. Thus, the existence of a biochemical interface between Phe and auxin biosynthesis might provide an efficient metabolic mechanism for tissue-specific coordination of stress responses with plant growth and development. Since auxin metabolism intersects through complex networks with the biosynthesis of several other plant hormones, modulation of auxin levels via the cytosolic Phe biosynthetic pathway can have a broader impact on plant growth and development under stress conditions. Plants synthesize volatile organic compounds (VOCs) to attract pollinators and beneficial microorganisms, to defend themselves against herbivores and pathogens and for plant-plant communication. Generally, accumulation and emission of VOCs occur from the tissue of their biosynthesis. However, using biochemical and reverse genetic approaches, we demonstrate a new physiological phenomenon: inter-organ aerial transport of VOCs via natural fumigation. Before petunia flowers open, a tube-specific terpene synthase produces sesquiterpenes, which are released inside the buds and then accumulate in the stigma, potentially defending the developing stigma from pathogens. These VOCs also affect reproductive organ development and seed yield, which is a previously unknown function for terpenoid compounds. Regulation of pistil development by volatile terpenoids from surrounding tube could serve as a mechanism to coordinate the timing of pistil maturation with petal development in order to ensure that the stigma is receptive when the flowers are most likely to attract pollinators. Further studies are required to assess whether natural fumigation is conserved in flowering plants, to uncover the mechanisms involved, and to determine its evolutionary advantage in plant reproduction. While VOCs also play important roles belowground, so do plant exudates released from roots containing non-volatile compounds. Unlike VOCs, however, the roles that these compounds play in mediating plant-biotic interactions is poorly understood. Moreover, little is known about biosynthesis of these compounds and the mechanisms responsible for their release from the cell into the rhizosphere. Specialized 1,4-naphthoquinones (1,4-NQ) have emerged as a major class of compounds involved in belowground plant-biotic interactions. Plants collectively synthesize hundreds of 1,4-NQs proposed to play roles in various plant-plant, plant-microbe and plant-animal interactions. Based on developments in understanding of plant metabolic networks, reinterpretations of classical tracer studies suggest that 1,4-NQs are synthesized through several routes. We hypothesize that these routes rely on precursors from four different primary metabolic sources. Recently, we used comparative transcriptomics and isotopic labeling to demonstrate that the 1,4-NQ moiety of juglone, the 1,4-NQ responsible for the allelopathic effects of black walnut (Juglans nigra) trees, is derived from the pathway responsible for producing the naphthalenoid moiety of phylloquinone. We also showed that juglone can be synthesized de novo in roots without translocation of precursors from the phylloquinone pathway present in aerial tissues. These results demonstrate indeed that the pathway to synthesize juglone, and likely other structurally similar 1,4-NQs produced from black walnut trees and other members of the Juglandaceae family, branches from primary quinone metabolism. This provides direct evidence for the connection between primary and specialized quinone metabolism, which may help guide elucidation of the metabolic origins of other 1,4-NQs in other plants. In addition to belowground roles, the occurrence of specialized 1,4-NQs in aerial tissues suggests that they have additional ecological roles aboveground and/or that the mechanisms for their deployment into the rhizosphere rely on tissues/organs present on the aerial portion of the plant. Therefore, we investigated whether 1,4-NQs present in Impatiens glandulifera flowers play a role in influencing the growth of nectar microbes, a mechanism we hypothesized is analogous to the role of structurally similar compounds in the pitcher fluid of carnivorous Nepenthes species. After systematic profiling of 1,4-NQs in different Impatiens organs, we found that the compounds are present in all parts of the plant, including floral nectaries. We also found that the two major compounds present in nectaries, lawsone and 2-methoxynaphthoquinone, exhibit antimicrobial activity against common nectar microbes. In addition to 1,4-NQs being detected in floral nectaries, they were found to be present in higher abundance in extra-floral nectaries. The nectar excreted by these organs, which are connected to vegetative tissues, is hypothesized to attract beneficial insects and other animal mutalists. Analysis of the nectar itself collected from extra-floral nectaries revealed high concentrations of 1,4-NQs. This leads us to hypothesize that broadly throughout plants, natural products are secreted into the nectar that gets released by extra-floral nectaries. This would then allow extra-floral nectaries to play an additional unsuspected role as a conduit to deploy non-volatile compounds from aerial tissues into the rhizosphere via nectar that is washed into the ground through rainfall.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Allelopathy as an evolutionarily stable strategy. McCoy RM, Widhalm JR, McNickle GG. Botany 2019. July 2019, Tucson AZ.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Using synthetic biology to investigate the subcellular architecture of the phylloquinone pathway. McCoy RM and Widhalm JR. Plant Biology 2019, August 3-7, San Jose CA.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Yahyaa M, Berim A, Nawade B, Ibdah M, Dudareva N, Ibdah M. 2019. Biosynthesis of methyleugenol and methylisoeugenol in Daucus carota leaves: Characterization of eugenol/isoeugenol synthase and O-methyltransferase. Phytochemistry, 159: 179-189.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhao D, Hamilton JP, Bhat WW, Johnson SR, Godden GT, Kinser TJ, Boachon B, Dudareva N, Soltis DE, Soltis PS, Hamberger B, Buell CR. 2019. A chromosomal-scale genome assembly of Tectona grandis reveals the importance of tandem gene duplication and enables discovery of genes in natural product biosynthetic pathways. GigaScience, 8, 1-10.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Johnson SR, Bhat WW, Bibik J, Turmo A, Hamberger B, Evolutionary Mint Genomics Consortium (Dudareva N. et al.), Hamberger B. 2019. A database-driven approach identifies additional diterpene synthase activities in the mint family (Lamiaceae). J. Biol. Chem., 294: 1349-1362.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: McCoy RM, Utturkar S, Crook JW, Thimmapuram J, and Widhalm JR. 2018. The origin and biosynthesis of the naphthalenoid moiety of juglone in black walnut. Horticulture Research, 5:67.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Block AK, Yakubova E, and Widhalm JR. (2019) Specialized naphthoquinones present in Impatiens glandulifera nectaries inhibit the growth of fungal nectar microbes. Plant Direct, 3:1-7.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Smith SD, Angelovici R, Heyduk K, Maeda HA, Moghe GD, Pires JC, Widhalm JR, and Wisecaver JH. (2019) The renaissance of comparative biochemistry. American Journal of Botany, 106(1):1-11.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Epigenetic modifications are essential for the rhythmic control of volatile organic compound emission in Petunia hybrida. Stirling S, Patrick R, Lynch J, Mensah I, Li Y, Dudareva N. 2019 ASPB Midwest Conference, March 16, 2019, Morgantown, West Virginia.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Natural fumigation as a mechanism for volatile transport between flower organs. Lynch JH, Boachon B, Ray S, Yuan J, Morgan JA, Dudareva N. 2019. Gordon Research Conference on Plant Metabolic Engineering, June 16-21, Lucca (Barga), Italy.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Using synthetic biology to investigate the subcellular architecture of the phylloquinone pathway. McCoy RM and Widhalm JR. Plant Synthetic Biology 2019, August 8-10, San Jose CA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: The Effects of Adjuvants and Carrier Water Characteristics on Dicamba Volatilization in a Controlled Environment. Hayden C, Young JM, Ghaste MS, Johnson WG, Widhalm JR, Young BG. North Central Weed Science Society Conference 2019.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Gene co-expression network analysis identifies gene candidates for shikonin biosynthesis pathway in the medicinal plant Lithospermum erythrorhizon. Auber R, Suttiyut T, Crook JW, Widhalm JR, Wisecaver J. SMBE 2019 (Society for Molecular Biology and Evolution). Manchester, UK.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Plant natural product biosynthesis and trafficking. Widhalm JR. 2018 Chemical Machinery of the Cell Scialog Conference. October 2018, Tucson, AZ.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Jantzen F, Lynch JH, Kappel C, H�fflin J, Skaliter O, Wozniak N, Sicard A, Sas C, Adebesin F, Ravid J, Vainstein A, Hilker M, Dudareva N, Lenhard M. 2019. Retracing the molecular basis and evolutionary history of the loss of benzaldehyde emission in the genus Capsella. New Phytologist, 224: 1349-1360.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Boachon B, Lynch JH, Ray S, Yuan J, Caldo KMP, Junker RR, Kessler SA, Morgan JA, Dudareva N. 2019. Natural fumigation as a mechanism for volatile transport between flower organs. Nature Chem. Biol., 15: 583-588 (Cover, highlighted in Research Highlights, Nature Plants 2019, 5: 644).
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Qian Y, Lynch JH, Guo L, Rhodes D, Morgan JA, Dudareva N. 2019. Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants. Nature Communications, 10: 15; doi: 10.1038/s41467-018-07969-2.