Cell-specific production and antimicrobial activity of naphthoquinones in roots of lithospermum erythrorhizon

Abstract

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 beta-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.

Figure 1

Proposed biochemical pathway for shikonin and shikonin derivative synthesis (after Gaisser and Heide, 1996). Derivatives of shikonin are formed by replacement of the R-group with the various fatty acid chains listed.

Figure 2

L. erythrorhizon hairy-root cultures showing pigment formation in different cells under varying conditions. A, Transformed roots growing on M medium. B, Transformed roots growing on M-9 medium. Pigment diffusion from the roots into the medium was apparent at 3 weeks. C, Root tip grown on M medium showing normal pigment production pattern in border cells and root hairs. D, Root tip grown on M-9 medium showing increased pigmentation in border cells and root hairs. E, Pigment deposition patterns in M-grown roots. Emerging root-hair tips have a cap of pigment, which remains at about that same distance from the epidermis as the root hair grows. F, Root hairs of M-9-grown roots showing exudation of pigment in droplets all over the hairs. G, Lateral root formation in M-grown roots. Pigment is apparent in the region of root emergence. H, Lateral root formation in M-9-grown roots. Numerous roots emerge at a single point and pigmentation is more intense than in M-grown roots. I, Pigment deposition in a few of the cells near lateral root eruption. J, Pigment production in all epidermal cells of M-9-grown root. K, Border cell pigmentation on root cap of M-grown root. Pigments are confined to border cells. L, Border cell deposition along growing root in solid M medium. M, Light micrograph of normal (untransformed) root tip placed in water showing dispersion of border cells from the cap. Border cells are purple, cap is white. N, UV fluorescence of root stained with fluorescein diacetate showing living cells of root. Most border cells do not take up the stain.

Figure 3

Fungal growth on medium containing no shikonin (control, ♦), 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.

Figure 4

Changes in fungal morphology when grown on medium containing shikonin. A, R. solani hyphal growth on control plate showing an even growth pattern. B, R. solani hyphal growth on 100 μg/mL shikonin showing clumping of hyphae. C, R. solani hyphal growth on 200 μg/mL shikonin showing sequestration of shikonin pigment in the hyphal clumps. D, Spores of A. niger grown on 5 μg/mL shikonin. Spores are dark and dumbbell shaped. E, Spores of A. niger grown on 200 μg/mL shikonin. Spores are beige and round. F, Spores of G. intraradices grown on M medium in the presence of L. erythrorhizon hairy roots. Pigment is not apparent in the medium by visual inspection. Pigment is transported through the hyphae and deposited on a small fraction of the spores in any one area.

Figure 5

In situ growth inhibition and stimulation of pigment production of R. solani in the presence of L. erythrorhizon hairy roots. A, Hairy roots grown in the light on M medium (left) and grown in the dark on M medium (right). The fungal plug is seen on the left in each plate. B, Pigment formation in root cells at the points of contact with hyphae. C, Accumulation of shikonin at lateral root eruptions on roots grown in M-9 medium showing inhibition of fungal growth. D, Root tip with greatly increased pigment production is free of hyphae, whereas the surrounding root surfaces are completely overcome.