Use of an antisense gene to characterize glutathione S-transferase functions in transformed suspension-cultured rice cells and calli

Abstract

Rice glutathione S-transferases (GSTs) conjugate the herbicide pretilachlor with glutathione and this reaction is induced by the safener fenclorim. A cDNA construct of the gene coding for the OsGST III subunit of rice GSTs was inserted in antisense orientation into the vector pBC302 under the control of an enhanced p CaMV35S promoter. The final clone containing the bar gene as marker was used to transform suspension-cultured cells and calli of Lemont rice ( indica× japonica) via the Agrobacterium Ti vector pTOK233. Five lines of suspension-cultured rice cells transformed with the antisense GST gene (α-3, α-5, α-8, α-12, and α-15) were selected and analyzed by Southern and Northern blot analyses. Transformed suspension-culture cells and calli of rice exhibited resistance to the herbicide glufosinate because of the bar gene, but were sensitive to pretilachlor. The α-5 and α-8 rice cell lines contained a single copy of the OsGST III gene, while the α-3 line contained two copies of this gene. OsGST III mRNA levels present in wild type rice cells were induced by fenclorim. OsGST III mRNA levels were undetectable in the transformed cell lines of rice and were not induced by fenclorim treatments, except for line α-5. GST activity utilizing cinnamic acid, CDNB, and pretilachlor as substrates was reduced in all cell lines of transformed rice. SDS–PAGE analysis showed that the band of the OsGST III subunit was very faint in α-3 rice cells. HPLC analysis showed that the levels of cinnamic acid, coumaric acid, and phenylalanine were significantly higher in transformed cells than in wild type cells of rice. These results suggest that the OsGST III gene plays an important role in the detoxification of the herbicide pretilachlor and the metabolism of phenolic compounds in suspension-cultured rice cells and calli.