A Trp-574-Leu mutation in the acetolactate synthase (ALS) gene of Lithospermum arvense L. confers broad-spectrum resistance to ALS inhibitors.

Lithospermum arvense is a troublesome dicotyledonous winter annual weed of wheat in China. A L. arvense population (HN01) suspected of being resistant to acetolactate synthase (ALS) inhibitors was found in Henan Province, China. This study aimed to testify the sensitivity of this HN01 population to eight herbicides from 3 different modes of action, and to explore the potential target-site-resistance mechanism to tribenuron-methyl. The whole-plant bioassays indicated that the population was highly resistant to tribenuron-methyl (SU, 350-fold), pyrithiobac sodium (PTB, 151-fold), pyroxsulam (TP, 62.7-fold), florasulam (TP, 80.6-fold), and imazethapyr (IMI, 136-fold), but was sensitive to carfentrazone-ethyl and fluroxypyr-meptyl. ALS gene sequencing revealed that the Trp (TGG) was substituted by Leu (TTG) at codon 574 in resistant plants. In in vitro ALS assays, the concentration of tribenuron-methyl required to inhibit 50% ALS activity (I50) for HN01 was 117-fold greater than that required to inhibit a susceptible population (HN05), indicating that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron-methyl. To the best of our knowledge, this is the first report of ALS gene Trp-574-Leu amino acid mutation confer resistance to tribenuron-methyl in L. arvense.

The shikonin derivatives and pyrrolizidine alkaloids in hairy root cultures of Lithospermum canescens (Michx.) Lehm.

Hairy root cultures of Lithospermum canescens were established using three strains of Agrobacterium rhizogenes: ATCC 15834, LBA 9402 and NCIB 8196. Eight lines resulting from infection with A. rhizogenes ATCC 15834 demonstrated sufficient biomass increase and were submitted to further investigations. The contents of acetylshikonin (ACS) and isobutyrylshikonin (IBS) in transformed hairy roots made up ca. 10% of those observed in natural roots of L. canescens (24.35 and 14.48 mg g(-1) DW, respectively). One line, Lc1-D, produced the largest amounts of ACS (2.72 mg g(-1) DW) and IBS (0.307 mg g(-1) DW). Traces of pyrrolizidine alkaloids (PA), canescine and canescenine, were found in all lines of transformed hairy roots.

Ethylene induced shikonin biosynthesis in shoot culture of Lithospermum erythrorhizon.

Lithospermum erythrorhizon shoots, cultured on phytohormone-free Murashige and Skoog solid medium, produced shikonin derivatives, whereas shoots cultured in well-ventilated petri dishes, produced small amount. Analysis by gas chromatography revealed the presence of ethylene in non-ventilated petri dishes where the shoots, producing shikonin derivatives, were cultured. Therefore, the possible involvement of ethylene in shikonin biosynthesis of shoot cultures was investigated. Treatment of ethylene or the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, resulted in increasing shikonin derivatives contents in cultured shoots. Silver ion, an ethylene-response inhibitor, or aminoethoxyvinylglycine, an ethylene biosynthesis inhibitor, decreased production of shikonin derivatives in cultured shoots. Our results indicate that ethylene is one of the regulatory elements of shikonin biosynthesis in L. erythrorhizon shoot culture.

Characterization of berberine transport into Coptis japonica cells and the involvement of ABC protein.

Cultured Coptis japonica cells are able to take up berberine, a benzylisoquinoline alkaloid, from the medium and transport it exclusively into the vacuoles. Uptake activity depends on the growth phase of the cultured cells whereas the culture medium had no effect on uptake. Treatment with several inhibitors suggested that berberine uptake depended on the ATP level. Some inhibitors of P-glycoprotein, an ABC transporter involved in multiple drug resistance in cancer cells, strongly inhibited berberine uptake, whereas a specific inhibitor for glutathione biosynthesis and vacuolar ATPase, bafilomycin A1, had little effect. Vanadate-induced ATP trap experiments to detect ABC proteins expressed in C. japonica cells showed that three membrane proteins of between 120 and 150 kDa were photolabelled with 8-azido-[alpha-32P] ATP. Two revealed the same photoaffinity-labelling pattern as P-glycoprotein, and the interaction of these proteins with berberine was also demonstrated. These results suggest that ABC proteins of the MDR-type are involved in the uptake of berberine from the medium.

CYP98A6 from Lithospermum erythrorhizon encodes 4-coumaroyl-4'-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis.

Rosmarinic acid is the dominant hydroxycinnamic acid ester accumulated in Boraginaceae and Lamiaceae plants. A cytochrome P450 cDNA was isolated by differential display from cultured cells of Lithospermum erythrorhizon, and the gene product was designated CYP98A6 based on the deduced amino acid sequence. After expression in yeast, the P450 was shown to catalyze the 3-hydroxylation of 4-coumaroyl-4'-hydroxyphenyllactic acid, one of the final two steps leading to rosmarinic acid. The expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitor-induced change in rosmarinic acid production, indicating that CYP98A6 plays an important role in regulation of rosmarinic acid biosynthesis.