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Plant Physiol. (1999) 119: 417-428 Cell-Specific Production and Antimicrobial Activity of Naphthoquinones in Roots of Lithospermum erythrorhizon1
Department of Plant Pathology, The Pennsylvania State University, University Park, Pennsylvania 16802-4507
Pigmented
naphthoquinone derivatives of shikonin are produced at specific times
and in specific cells of Lithospermum erythrorhizon roots.
Normal pigment development is limited to root hairs and root border
cells in hairy roots grown on "noninducing" medium, whereas
induction of additional pigment production by abiotic (CuSO4) or biotic (fungal elicitor) factors increases the
amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When
the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and
Plants communicate with their environment (the soil, climate
conditions, neighboring plants, microorganisms, insects, etc.) by
producing a diverse array of chemicals. These secondary metabolites are
often characteristic of specific plants and plant families. Many
members of the Boraginaceae family produce naphthoquinones in their
roots. Naphthoquinones are colored substances derived from
phenylpropanoid and isoprenoid precursors (Gaisser and Heide, 1996
Because of its value as a pharmaceutical agent, most research to date
has focused on understanding the production and regulation of shikonin
with the goal of increasing production of the compound, but the
biological significance of shikonin in the plant has been virtually
ignored. Because shikonin and its derivatives have biological activity
against microorganisms, it seems likely that these compounds may play a
role in plant defense in the rhizosphere. Naphthoquinones have been
shown to function in allelopathy (juglone; Binder et al., 1989
In view of the observations on the antimicrobial properties reported
for shikonin and its derivatives, the production of numerous derivatives of shikonin in the roots of the plant, and the existing information on regulation of the pathways in common with many other
plant defense responses, we investigated the regulation and biological
activity of shikonin and its derivatives in hairy-root cultures of
Lithospermum erythrorhizon. Because these compounds are
brightly colored, the timing and cellular specificity of their production can be followed with simple equipment such as a dissecting microscope. The suite of compounds can be studied in the context of
understanding the multiple actions of plants in fine tuning their
response to their environment. The objectives of this study were: (a)
to characterize the production of naphthoquinone pigments in L. erythrorhizon hairy-root cultures, (b) to analyze the effect(s) of
shikonin and its derivatives on soil-borne microorganisms, and (c) to
determine if the L. erythrorhizon hairy-root cultures could
respond to microorganisms by modifying the production of shikonin and
its derivatives.
Plant Material
Hairy-Root Production and Growth Conditions Seeds of L. erythrorhizon were sterilized with 10% commercial bleach for 20 min, rinsed in five changes of sterile water, and germinated under sterile conditions on solid Murashige and Skoog medium (Murashige and Skoog, 1962 ). Seedlings were grown in GA7
hydroponic culture vessels (Magenta Corp., Chicago, IL), and stems were
inoculated with Agrobacterium rhizogenes strain 15834 (ATCC)
to produce hairy roots. Root cultures were obtained from 16 independent
transformation events. All root culture lines appeared morphologically
indistinguishable, and experiments described in this paper were
performed on a single root line. Root cultures from the selected line
were grown in the dark at 24°C on M medium (Bécard and Fortin,
1988) or M-9 (Fujita et al., 1981a) medium. Roots grown on solid or
liquid M medium did not produce shikonin in sufficient quantities to be
observable on visual inspection. M-9 served as an induction medium for
shikonin because it contains 2.3 times the amount of copper sulfate as
the M medium. Liquid root cultures were grown in the dark on a rotary
shaker (model G-53, New Brunswick Scientific, Edison, NJ) at 90 rpm at
24°C. Solid root cultures were grown on medium solidified with 0.3% (w/v) Phytagel (Sigma).
Microscopy Roots and fungal-growth plates were viewed and photographed using a Zeiss STEMI SV8 dissecting microscope and Kodak 160T slide film. Root hairs were viewed and photographed on a Zeiss inverted microscope and Kodak 160T slide film.Bacterial and Fungal Inhibition Assays Bacterial Growth Inhibition Bacterial cultures were grown overnight at 24°C with shaking in Luria-Bertani broth (Luria and Burrows, 1957) to an optical density of
0.2 at 600 nm. A 100-µL aliquot of this liquid culture was spread
onto 82-mm plates with solid Luria-Bertani medium. A filter disk
saturated with 50 µg of shikonin or shikonin derivative (dissolved in
chloroform and allowed to air dry) was placed on the bacterial lawn.
The plates were incubated in the dark at 24°C. The clear zone from
the filter disk to the bacteria was measured. Each experiment was
performed three times for each isolate tested. The following bacterial
strains were obtained from a collection of field isolates maintained in
the laboratory of Dr. Leland S. Pierson III (Department of Plant
Pathology, University of Arizona, Tucson): Bacillus subtilis
613R, Bacillus thuringiensis Gnatrol, Clavibacter
michigenensis subsp. nebraskensis CN74-1,
Agrobacterium radiobacter K84, Agrobacterium
tumefaciens C58, Burkholdaria cepacea Deny,
Escherichia coli ESS, Erwinia amylovora,
Erwinia carotovora ATCC 15713, Pseudomonas
aureofaciens 30-84, Pseudomonas fluorescens 2-79, Pseudomonas syringae B, Pseudomonas syringae pv
phaseolicola, Ralstonia solanacearum,
and Serratia marsecens. Erwinia
herbicola was isolated from pea seedling roots (L.A. Brigham,
unpublished data). A. rhizogenes ATCC 15834 was maintained
by Paula Michaels at The Pennsylvania State University (University
Park). Xanthomonas campestris pv pelargonii was
from the collection of Dr. Gary Moorman (The Pennsylvania State
University).
Fungal Growth Inhibition Fungal isolates were maintained on V-8 medium (200 mL of commercial vegetable juice, 2 g of CaCO3, and 1 g of Glc per liter of water) solidified with 2% agar (Difco Laboratories, Detroit, MI) in the dark at 24°C. Inhibition assays were performed by dissolving shikonin in solid M-2 medium (modified Martin's medium: 10 g of Glc, 5 g of peptone, 1 g of KH2PO4 0.5 g of MgSO4, 22 g of agar per liter of double-distilled water) in 35-mm plastic Petri dishes. A 4-mm plug of fungal hyphae was placed on one side of the plate and hyphal length was measured at 24-h intervals. Each isolate was tested on all concentrations in three separate replicates. Fungal isolates Nectria hematococca 34-18 and N. hematococca T488 were from the laboratory cultures of Dr. Hans Vanetten (Department of Plant Pathology, University of Arizona, Tucson). All of the other fungal isolates were from Scott Rasmussen (Department of Plant Pathology, University of Arizona).Elicitation of Shikonin Derivatives in Liquid Root Cultures Fungal elicitors were prepared as described previously (Flores et al., 1988). Mycelium from a 2-week-old fungal culture was inoculated
into a 2-L Erlenmeyer flask containing 1 L of Schenk and Hildebrandt
medium and grown in the dark on a rotary shaker (model G-53, New
Brunswick Scientific) at 90 rpm at 25°C for 3 weeks (Schenk and
Hildebrandt, 1972). Mycelium was filtered off, resuspended in distilled
water, homogenized, centrifuged at 18,000 rpm for 30 min, autoclaved at
121°C for 30 min, and stored at  20°C. A 2.5-mL aliquot of this
filtrate was used per 50 mL of root-culture medium. Total shikonin was
quantified by treatment of derivatives with 2.5% KOH for 10 min
and spectrographic reading at 622 nm (Mizukami et al., 1977).
Identification and Quantification of Compounds TLC Analysis Shikonin derivatives were extracted from the roots and liquid media with chloroform. The chloroform layer was collected and evaporated to dryness. The residue was redissolved in chloroform and spotted onto TLC plates (channeled Kieselgel 60CF254, Merck, Darmstadt, Germany) using chloroform as the mobile phase. Standards were obtained from TCI America (Portland, OR). Specific compounds were extracted from the plates by scraping off and grinding the silica with a mortar and pestle, and dissolving in chloroform. The identification of the compounds was verified by comparison of retention time with known standards by HPLC.HPLC Analysis HPLC was performed on a C18 60-Å 4-µm column (3.9- × 300-mm, Nova-Pak, Waters) fitted to a HPLC device comprising a system controller (model 600E), a Waters Intelligent Sample Processor (model 712), and a photodiode array detector (model 990, all Waters). The isocratic solvent system was CH3CN:H2O:CH3COOH:Et3N (630:370:3:3, v/v) Chromatography was performed at a flow rate of 1.2 mL min 1, a pressure of 30 kg/cm2, and a column temperature of 23°C.
A520 was monitored, and peaks were compared
with known standards (TCI America).
Development of Naphthoquinone Pigments in Pot-Grown Plants and in Hairy-Root Cultures Pigment Development under "Noninducing" Conditions L. erythrorhizon hairy-root cultures grown on solid M medium in the dark at 24°C appeared white (Fig. 2A). Roots grew at a rate of 1 to 2 mm/d. Under magnification pigmentation was apparent in two cell types within these roots: the root hairs (Fig. 2, C and E) and the root border cells (Hawes and Brigham, 1992; Brigham et al., 1995; Hawes et al., 1998)
(Fig. 2, C and K). Pigment granules in the root hairs were seen at the
apex of the root hairs as they emerged (Fig. 2E). Pigmentation was not
apparent before the hair had attained a length of at least 1 mm. As the
root hair elongated, the pigment appeared to remain at the same
distance from the epidermal surface as when it was first formed. The
pigment was limited to a very specific region and appeared to be a
ring, perhaps because the vacuole occurred at this region of the root
hair. In the root-cap region, pigments were confined to the border
cells alone (Fig. 2, K and M). Removing the border cells from the cap
revealed that the pigment was formed in the border cells subsequent to
separation from the cap, because the cap cells themselves were
completely devoid of pigment. Border cells were left behind as the root
grew through the medium and, in some cases, appeared to outline the root (Fig. 2L). When border cells were visualized under light microscopy in M medium, they appeared deep purple, as opposed to the
white color of the underlying cap cells (Fig. 2M). Cells of the cap
take up the vital stain fluorescence diacetate, whereas those of the
detached border cells do not (Fig. 2N).
Induction of Shikonin Derivatives Roots grown on the "inducing" medium (M-9) formed pigments sooner, in greatly increased amounts, and in more cell types than those grown on the noninducing medium (M) described above. L. erythrorhizon hairy-root cultures grown in the dark on M-9 medium at 24°C appeared fully pigmented, and the pigments had diffused into the medium, giving it a pinkish color (Fig. 2B). The root cap appeared to have greatly increased pigmentation and the border cells were not as distinct, probably because of increased mucilage production (Fig. 2D). The root hairs appeared to contain more pigment than the noninduced hairs, and the pigment was distributed throughout the cell (Fig. 2F). The amount of pigment production was increased to the point that it was exuded from the root hair and appeared in droplets on the surface of the hair. Lateral root formation occurred at shorter intervals than in the controls, and clusters of lateral roots were commonly observed emerging from a single site with greatly increased pigment production (Fig. 2H). In contrast to the noninduced state, in which these were the only two cell types that produced pigment, in the induced state pigment was produced in all of the epidermal cells of the root much earlier and before other phenolic products had turned the roots brown (Fig. 2J). The pigments appeared to accumulate in the cell wall and were seen throughout the apoplast.Effects of Shikonin on Bacterial and Fungal Growth Because shikonin has been used as an antimicrobial agent in human applications, and because shikonin and its derivatives are produced exclusively by the root of the plant, we tested whether shikonin was inhibitory to soil-borne microorganisms. Both bacterial and fungal isolates were assayed.Bacterial Growth Inhibition Bacterial isolates from plant roots covering a broad phylogenetic range were tested for inhibition of growth by the filter-disk method with 50 µg of standard shikonin per filter. The concentration was chosen within the range shown to inhibit human pathogens (Tanaka and Odani, 1972). Of the 31 strains tested, 10 were inhibited to some
degree by shikonin. Table III lists a
representative sample of the strains tested. Whereas no definite
patterns emerge, it is clear that not all strains are sensitive to
shikonin and some strains are more sensitive than others. As reported
previously by Papageorgiou (1980), the E. coli strains were
not affected. The two pathogenic strains of Erwinia were not
sensitive, whereas the nonpathogenic E. herbicola was
inhibited. All of the Agrobacterium strains were affected,
but the nonpathogenic A. radiobacter was inhibited to a
greater degree than the pathogenic strains. All gram-positive strains
tested were inhibited. In summary, shikonin shows selective inhibition
to some bacterial strains, including important root pathogens.
Fungal Growth Inhibition and Morphological Changes Hyphal growth inhibition was tested by a linear growth assay. Twelve fungal isolates were tested and showed a wide range of growth responses on medium containing 5, 50, 100, and 200 µg/mL shikonin standard. Growth was measured for each isolate until the hyphae of any treatment reached the edge of the 35-mm plate. The length of time for the control to reach the edge of the plate varied from 1 d (Pythium aphanidermatum) to 9 d (Colletotrichum destructivum). Figure 3 illustrates four different growth patterns. Pythium ultimum showed increasing sensitivity to increasing amounts of shikonin (Fig. 3A). P. aphanidermatum was increasingly inhibited by increasing concentrations of shikonin until the highest concentration (200 µg/mL), at which point it showed a slight increase in growth over the 100 µg/mL concentration (Student's t test, P = 0.06; Fig. 3B). Nectria hematococca 34-18 showed little inhibition of hyphal growth even at the highest shikonin concentration (Fig. 3C). Phytophthora parasitica grew faster than the control at 5 and 50 µg/mL (Student's t test, P = 0.01; Fig. 3D). Comparisons of growth patterns among all of the fungi tested were made by calculation of hyphal growth on each concentration of shikonin as a percentage of the growth of the control (Table IV).
Production of Shikonin Derivatives
Effect of Shikonin Derivatives on Bacterial Growth
In Situ Fungal Inhibition and Plant Response to Fungi To analyze the dynamics of shikonin production in root cultures on fungi, we placed plugs of fungal hyphae on 4-week-old L. erythrorhizon root cultures on solid M and M-9 medium. One-half of them were grown in the light (to inhibit shikonin derivative production) and one-half were grown in the dark. Four isolates were chosen based on growth rates and response to the shikonin concentration assays (Table VI). The amount of shikonin derivatives the fungi were exposed to on the plates was not quantified. Pigment was apparent upon visual inspection in the roots grown on M-9 at this point. P. aphanidermatum was sensitive to both the existing shikonin derivatives on the M-9 plates and to increased shikonin derivative produced in the dark on both the M and M-9 plates. On the light-grown plates, shikonin production was inhibited and hyphal growth was twice that seen on the dark-grown M plates (Table VI; Fig. 5A). R. solani, which is very sensitive to high concentrations of shikonin (Table VI), was inhibited on the M-9 plates, but not significantly on the M plates, even though the fungus did induce pigment formation in these roots. N. hematococca T488, which is sensitive to shikonin at relatively low concentrations, showed marked inhibition on dark-grown plates whether preinduced or not. A. niger, the least-sensitive and slowest-growing isolate, showed no significant difference between light- and dark-grown plates, preinduced or not. This was the only one of the four isolates tested that did not induce localized pigment production in the roots (Table VI). Figure 5B shows an example of a region of hyphal contact with the root. The cells in the region of contact show considerable pigmentation. Areas normally pigmented, such as sites of lateral root emergence, had fewer hyphae than regions in between (Fig. 5C). However, all regions where hyphae made contact eventually produced pigment. Whereas most of the roots on the plate were eventually overcome by hyphal growth, the root tips were always devoid of hyphae. These tips, like those grown on M-9 medium, were moist and very red, showing increased pigment production (Fig. 5D). Whereas root epidermal cells in direct contact with hyphae showed increased pigment production compared with adjacent noncontact cells, the root caps showed increased production without direct hyphal contact.
The transformed roots of L. erythrorhizon appear to be
a useful model in which to study the production and potential
function(s) of a suite of antimicrobial compounds in plant roots
(Rhodes et al., 1997
2 Present address: Department of Plant Pathology, The University of Arizona, Tucson, AZ 85721-0036. * Corresponding author; e-mail lbrigham{at}ag.arizona.edu; fax 1-520-621-9290. Received May 26, 1998;
accepted October 22, 1998.
Abbreviations:
AS, acetyl-shikonin.
DMAS,
We thank Drs. Leland S. Pierson and Derek Wood (University of Arizona, Tucson) for providing the bacterial strains used in these experiments, Dr. Hans Vanetten and Scott Rasmussen (University of Arizona) for providing most of the fungal isolates, Dr. Martha Hawes for providing laboratory space for the experiments performed at the University of Arizona, Tom Orum (University of Arizona) for providing assistance in the statistical analyses, and Anthony Omeis (Biology Greenhouse at The Pennsylvania State University) for propagating the L. erythrorhizon plants and caring for them in the greenhouse. The carrot hairy-root cultures were a gift from Dr. Roger Koide of The Pennsylvania State University. Dr. Gary Moorman graciously provided a critical reading of the manuscript, and Dr. Kazufumi Yazaki (Kyoto University, Japan) provided invaluable background information, discussion, and advice for this research.
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Top
Abstract
Introduction
-hydroxyisovaleryl-shikonin, the two
most abundant derivatives in both Agrobacterium
rhizogenes-transformed "hairy-root" cultures and
greenhouse-grown plant roots, were the most biologically active of the
seven compounds tested. Hyphae of the pathogenic fungi
Rhizoctonia solani, Pythium
aphanidermatum, and Nectria hematococca induced
localized pigment production upon contact with the roots. Challenge by
R. solani crude elicitor increased shikonin derivative
production 30-fold. We have studied the regulation of this suite of
related, differentially produced, differentially active compounds to
understand their role(s) in plant defense at the cellular level in the
rhizosphere.
), 5 µg/mL shikonin (
), 50 µg/mL shikonin (
),
100 µg/mL shikonin (×), or 200 µg/mL shikonin (
). A,
P. ultimum. B, P. aphanidermatum. C,
N. hematococca 34-18. D, P. parasitica.
-methyl-n-butyl-shikonin.