A novel dark-inducible protein, LeDI-2, and its involvement in root-specific secondary metabolism in Lithospermum erythrorhizon

Abstract

Lithospermum erythrorhizon produces red naphthoquinone pigments that are shikonin derivatives. They are accumulated exclusively in the roots of this plant. The biosynthesis of shikonin is strongly inhibited by light, even though other environmental conditions are optimized. Thus, L. erythrorhizon dark-inducible genes (LeDIs) were isolated to investigate the regulatory mechanism of shikonin biosynthesis. LeDI-2, showing the strict dark-specific expression, was further characterized by use of cell suspension cultures and hairy root cultures as model systems. Its mRNA accumulation showed a similar pattern with that of shikonin. In the intact plants LeDI-2 expression was observed solely in the root, and the longitudinal distribution of its mRNA was also in accordance to that of shikonin. LeDI-2 encoded a very hydrophobic polypeptide of 114 amino acids that shared significant similarities with some root-specific polypeptides such as ZRP3 (maize) and RcC3 (rice). Reduction of LeDI-2 expression by its antisense DNA in hairy roots of L. erythrorhizon decreased the shikonin accumulation, whereas other biosynthetic enzymes, e.g. p-hydroxybenzoic acid:geranyltransferase, which catalyzed a critical biosynthetic step, showed similar activity as the wild-type clone. This is the first report of the gene that is involved in production of secondary metabolites without affecting biosynthetic enzyme activities.

Figure 1

A, Nucleotide sequence of cDNA and deduced amino acid sequence of LeDI-2. Putative polyadenylation signal is underlined. B, Hydrophobicity profile of LeDI-2 polypeptide. Kyte-Doolittle hydrophobicity index appears in the left margin. Positive values indicate hydrophobic regions. Amino acid numbers are shown beneath the plot.

Figure 2

Multiple alignment of amino acid sequences of LEDI-2 and the orthologs. The origins of each molecular species are as follows: RcC2 and RcC3 (rice), ZRP3 (maize), DC2.15 (carrot), SAC51 (oilseed rape), CR14KD (Catharanthus roseus; Hotze et al., 1994), PVR5 (Phaseolus vulgaris), and AIR1 (Arabidopsis). Conserved Cys residues are highlighted with asterisks.

Figure 3

A, Northern-blot hybridization of LeDI-2 in cell suspension cultures of L. erythrorhizon in M9 medium. Left, Gel blots of illuminated cultures, and those of dark-cultured cells are shown on the right. β-ATPS indicates a β-subunit of ATP synthase used as a load control (Boutry and Chua, 1985). B, Time course of shikonin production in those cell cultures agitated under illumination or in the dark. C, Organ-specific expression of LeDI-2 in the intact plant of L. erythrorhizon. L, Leaves; S, stems; MR, main root; LR, lateral roots. Shikonin accumulation monitored with the plant materials are shown beneath the blot: (+), shikonin derivatives are detected; (-), shikonin derivatives are not detectable.

Figure 4

Effects of light on LeDI-2 expression and shikonin production in hairy root cultures of L. erythrorhizon in M9 medium. A, Left panels show the accumulation of LeDI-2 mRNA in hairy roots cultured in the dark, and right ones are those under illumination. B, Cell growth and accumulation of shikonin in hairy roots cultured in the dark (left) or under illumination (right). The culture was started by inoculating 0.5 g of hairy roots that had been cultured in Murashige and Skoog liquid medium in the dark. C, Light-induced up- and down-regulation of LeDI-2 expression during the culture period. Hairy root cultures were transferred from a dark to light (left) or light- to-dark (right) condition 7 d after inoculation.

Figure 5

Longitudinal distribution of LeDI-2 mRNA throughout the primary root of L. erythrorhizon (clone no. 5). Root tissues were cut to three sections of 5 mm in length as shown in A. Northern-blot analysis of RNA isolated from each segment of the hairy root using LeDI-2 as a hybridization probe (B). Shikonin contents in each root segment are shown in C.

Figure 6

A, Binary vector constructs (pBinHygLeDI-2as) for transformation of L. erythrorhizon. A full-length cDNA of LeDI-2 was subcloned under the control of El2 promoter. The orientation of the coding region of LeDI-2 cDNA is shown with an arrow, and the flanking regions of non-coding sequences are indicated as white. El2 has two enhancer sequences in tandem attached upstream of the cauliflower mosaic virus 35S promoter. B, Genomic PCR to detect exogenous LeDI-2 and hpt gene integrated in plant genome. P, Positive control in which plasmid DNA (pBinHygLeDI-2as) was used as the template; N, negative control using genomic DNA of control hairy root (clone 5) as the template; L, DNA ladder as size marker. C, Northern-blot analyses of LeDI-2 mRNA level in transformants. LeDI-2 sense shows the level of sense strand detected by antisense probe, and LeDI-2 antisense shown was probed with the sense probe. Three control hairy root clones that were not transformed with a binary vector were employed as control. The mRNA level of PAL was also detected with a consensus sequence of PAL cDNA fragment (Yazaki et al., 1997).

Figure 7

A, Fresh weight of hairy root clones 14 d after inoculation. Each clone was cultured in 30 mL of medium (inoculum size 0.5g). B, Shikonin productivity of each hairy root clone. Shikonin derivatives were extracted from culture media and from hairy root tissues after 14 d of culture kept in the dark.

Figure 8

A, Biosynthetic pathway of shikonin. When the expression of geranyltransferase is inhibited by light, the excess amount of PHB is accumulated as its glucoside form. PHB-OG, O-glucoside of PHB; GPP, geranylpyrophosphate. Shikonin production and PHB:geranyltransferase activity in control and LeDI-antisense hairy root clones are indicated in B and C, respectively. The accumulation level of PHB-OG in the root tissues of those clones are also monitored in D. Cells were cultured in the dark or under continuous light (80 μE/m⁻² s⁻¹) with fluorescent lamps.