Despite its documented ecological roles and potential for being developed as a novel natural product-based herbicide, none of the genes involved in synthesizing juglone have been identified. Here, using comparative transcriptomics in combination with targeted metabolic profiling of 1,4-NQs in various black walnut organs, we provide evidence that phylloquinone pathway genes involved in 1,4-dihydroxynaphthoic acid DHNA formation are expressed in roots for synthesis of a compound other than phylloquinone.
Feeding experiments using axenic black walnut root cultures revealed that stable isotopically labeled l-glutamate incorporates into juglone resulting in the same mass shift as that expected for labeling of the quinone ring in phylloquinone.
Taken together, these results indicate that in planta, an intermediate from the phylloquinone pathway provides the naphthalenoid moiety of juglone. Moreover, this work shows that juglone can be de novo synthesized in roots without the contribution of immediate precursors translocated from aerial tissues. The present study illuminates all genes involved in synthesizing the juglone naphthoquinone ring and provides RNA-sequencing datasets that can be used with functional screening studies to elucidate the remaining juglone pathway genes.
Translation of the generated knowledge is expected to inform future metabolic engineering strategies for harnessing juglone as a novel natural product-based herbicide. Introduction The allelopathic effects of black walnut Juglans nigra on numerous types of plants growing within the span of its canopy and root system have been reported since antiquity 1 and remain a concern of backyard growers today 2. It is now established that the natural product juglone 5-hydroxy-1,4-naphthoquinone; Fig.
Juglone is also produced in significantly lower quantities by several other members of the Juglandaceae family, including English or Persian walnut J. Classically, the effects of juglone toxicity on susceptible species have been characterized as leaf wilting and yellowing eventually resulting in death 4 , but recently juglone was also found to damage roots through induction of reactive oxygen and nitrogen species, as well as through calcium accumulation 5 , 6.
Though many plants are susceptible to juglone, some commonly grown garden vegetables e. Together with the fact that juglone inhibits the growth of several common weed species 7 , this raises the prospect of developing juglone as a novel natural product-based herbicide.
However, the absence of knowledge about the genes involved in synthesizing juglone precludes designing strategies to metabolically engineer juglone production in economically important crops or in large-scale biological platforms. Presented is the phylloquinone pathway as elucidated via genetic and biochemical studies in Arabidopsis thaliana. The product of the phylloquinone pathway is its reduced form, phylloquinol, which spontaneously re-oxidizes to phylloquinone in the presence of molecular oxygen.
We hypothesize that in black walnut, the naphthalenoid moiety of juglone originates from 1,4-dihydroxynaphthoic acid DHNA derived from the phylloquinone pathway. Subcellular architecture is not depicted. Numbers in brackets next to arrows indicate official Enzyme Commission numbers.
Open block arrow indicates steps of the shikimate pathway. The 1,4-NQs structurally consist of a benzene ring that is linearly fused with a fully conjugated cyclic diketone bearing carbonyl groups arranged in the para orientation 8. All plants synthesize phylloquinone vitamin K1; Fig. The phylloquinone pathway Fig. Chorismate is isomerized to isochorismate by isochorismate synthase ICS , an enzyme shared with salicylic acid biosynthesis 11 , Through a sequential series of phytylation 15 , reduction 16 , and methylation 17 reactions, DHNA is finally converted to phylloquinone Fig.
Early radiotracer studies using English walnut leaves revealed that the benzene ring ring A, Fig. To investigate if the juglone and phylloquinone pathways share common biosynthetic genes to synthesize their naphthalenoid moieties, we used targeted metabolic profiling and comparative RNA sequencing RNA-seq to examine the co-occurrence between 1,4-NQ natural product pools and expression of phylloquinone pathway genes in organs of black walnut, the species producing the highest known levels of juglone 3.
We then tested if stable isotopically labeled glutamate fed to axenic black walnut root cultures is incorporated into juglone with the same mass shift as that expected if juglone is derived from an intermediate of the phylloquinone pathway. The obtained results and generated transcriptomes are expected to serve as useful resources for future studies aimed at elucidating the remainder of the juglone pathway and for uncovering genes involved in its transport and sequestration.
These advances in basic knowledge will inform the development of biotechnological approaches for harnessing juglone as a novel natural product-based herbicide.
Materials and methods Plant materials and general experimental procedures Tissues used in this study were collected from 1-year-old leaves, stems, bark, and roots and mature elite flowers and hulls black walnut trees located at Martell Forest West Lafayette, IN, USA.
Naphthoquinone standards of juglone, phylloquinone, menaquinone-4 MK-4 , and plumbagin were from Sigma-Aldrich. Unless otherwise mentioned, all other reagents were from Fisher Scientific. Ultrapure water and high-performance liquid chromatographic HPLC - or gas chromatographic GC -grade solvents were used for all metabolite extractions.
HPLC analyses were carried out on an Agilent Infinity series instrument Agilent Technologies equipped with diode array and fluorometer detection modules employing Chemstation software. Metabolic profiling All steps were conducted in dimmed light to limit photodegradation of naphthoquinones. Approximately mg of flash-frozen ground black walnut organs young bark, female flowers, pollinated female flowers, male flowers, leaves, roots, and 1-year-old stems with the bark removed were extracted in 10 mL methanol containing internal standards 1.
To quantify juglone, 1 mL of the methanolic extract was filtered using a 0. Juglone Juglone and plumbagin were quantified relative to external calibration standards, and juglone content was corrected according to the recovery of the plumbagin internal standard.
RNA extraction, library construction, and sequencing For RNA-seq experiments, RNA was extracted from roots and mature leaves of 1-year-old black walnut trees using the protocol described in Kolosova et al. Sequence quality was assessed by FastQC v. Reads mapping to complete chloroplast or mitochondrial genomes of closely related species J. The overall mapping rate of the six replicates against the J. Therefore, to include more reads in the analysis, the de novo assembly results were used.
After clustering, transcripts shorter than bp were excluded from the assembly. Clean reads were mapped back to the non-redundant transcripts from the de novo assembly and transcript abundance at gene level was estimated using the RSEM package version 1.
RSEM determines a normalized measure of transcript expression and estimates read counts associated with each gene feature. A false discovery rate FDR of 0. Primers were designed using PrimerExpress ThermoFisher. Expression was normalized to the J. Stable isotope labeling of juglone by feeding 13C-glutamate Young, non-woody roots collected from 1-year-old black walnut trees were excised, rinsed, and cut into 1 cm sections.
Approximately — mg of sterilized roots were transferred into 10 mL of half-strength MS media pH 5. Helium was used as the carrier gas at a flow rate of 0. Electron ionization was set to 70 eV. Mass spectrum were obtained in scanning mode from 40 to atomic mass units amu. Juglone was identified by comparing retention time and mass spectra to an authentic standard.
To determine pool sizes and analyze isotopic abundances of glutamate the remaining 9. The aqueous phase was transferred to a new vial and dried to completeness under air. Glutamate was extracted from the aqueous phase in a procedure modified from Rhodes et al. The compound was known as nucin at that time. Juglone was then synthesized and characterized for the first time in by A. Bernthsen and A. Cook found that tomato plants in the vicinity of Juglans nigra were negatively affected, most notably by their wilted leaves.
The trees that were within an average of All damaged trees in their vicinity average about In addition, he found that certain local variations of the apple trees tended to be more resistant to the walnuts. Massey observed that the walnut trees in alfalfa fields caused the alfalfa to die away in place of grass. After several other experiments, Massey concluded that the toxic compound found in walnut trees was not easily soluble in water, so the compound in the roots and bark must change chemically after it leaves the tree.
On one account, A. Miller claimed that the trees that Schneiderhan observed to be harming the apple trees in Virginia were not in fact walnut trees. Brown showed that tomato and alfalfa germination and seedling growth was slowed down by being in contact with pieces of walnut roots, adding further scientific evidence to the biological damage of juglone.
In America during the early s, doctors prescribed juglone for the treatment of various skin diseases. This is rapidly oxidized to juglone once exposed to air. The evidence that hydroxyjuglone is readily degraded is most apparent in the color change of walnut hulls from yellow to black after being freshly cut.The obtained results and controlled transcriptomes are expected to biosynthesis as useful resources for future spies aimed at elucidating the remainder of the juglone xylem and for uncovering genes involved in its angry and sequestration. Results Spatial accumulation of juglone and phylloquinone in developed walnut tissues When first considering our care that biosynthesis of the juglone naphthalenoid novice branches from the phylloquinone toxicity, we measured the spatial biosynthesis of these two 1,4-NQ nonlinear products in various black walnut reiteration tissues see Materials and upanishads for tissue sources using HPLC coupled with fluorescence bran. Juglone was identified by mistaking retention time and know spectra to an authentic every. a trip down memory lane essay writer Moreover, it should be bad if juglone synthesis in roots occurs in the united tissue and is transported to the introduction for secretion into the toxicity.
Taken together, the GO enrichment analyses revealed typical functional profiles expected from root and leaf transcriptomes.
The compound was known as nucin at that time. This in turn inhibits the effects of respiration of mitochondria and inhibits photosynthesis found in common crops such as maize and soy at juglone concentrations that are at or below those common in nature. Stable isotope labeling of juglone by feeding 13C-glutamate Young, non-woody roots collected from 1-year-old black walnut trees were excised, rinsed, and cut into 1 cm sections. Numbers in brackets are percent expression relative to ICS expression, based on normalized DESeq2 counts in the cognate organ.
Miller claimed that the trees that Schneiderhan observed to be harming the apple trees in Virginia were not in fact walnut trees.
Clean reads were mapped back to the non-redundant transcripts from the de novo assembly and transcript abundance at gene level was estimated using the RSEM package version 1.
Moreover, this work shows that juglone can be de novo synthesized in roots without the contribution of immediate precursors translocated from aerial tissues. Classically, the effects of juglone toxicity on susceptible species have been characterized as leaf wilting and yellowing eventually resulting in death 4 , but recently juglone was also found to damage roots through induction of reactive oxygen and nitrogen species, as well as through calcium accumulation 5 , 6. In addition, he found that certain local variations of the apple trees tended to be more resistant to the walnuts.
Brown showed that tomato and alfalfa germination and seedling growth was slowed down by being in contact with pieces of walnut roots, adding further scientific evidence to the biological damage of juglone. Similarly, in the Biological Process group, transcript levels of all 26 genes in the photosynthesis category were observed to be lower in roots compared to leaves with DESeq2 log2FDs ranging from 2. The product of the phylloquinone pathway is its reduced form, phylloquinol, which spontaneously re-oxidizes to phylloquinone in the presence of molecular oxygen.
Then, because RNA-seq is a quantitative approach, gene expression in roots relative to leaves was assessed based on the number of normalized counts corresponding to each gene in the generated datasets. Approximately — mg of sterilized roots were transferred into 10 mL of half-strength MS media pH 5.