Metabolic engineering of indole glucosinolates in Chinese cabbage plants by expression of Arabidopsis CYP79B2, CYP79B3, and CYP83B1.
Indole glucosinolates (IG) play important roles in plant defense, plant-insect interactions, and stress responses in plants. In an attempt to metabolically engineer the IG pathway flux in Chinese cabbage, three important Arabidopsis cDNAs, CYP79B2, CYP79B3, and CYP83B1, were introduced into Chinese cabbage by Agrobacterium-mediated transformation. Overexpression of CYP79B3 or CYP83B1 did not affect IG accumulation levels, and overexpression of CYP79B2 or CYP79B3 prevented the transformed callus from being regenerated, displaying the phenotype of indole-3-acetic acid (IAA) overproduction. However, when CYP83B1 was overexpressed together with CYP79B2 and/or CYP79B3, the transformed calli were regenerated into whole plants that accumulated higher levels of glucobrassicin, 4-hydroxy glucobrassicin, and 4-methoxy glu-cobrassicin than wild-type controls. This result suggests that the flux in Chinese cabbage is predominantly channeled into IAA biosynthesis so that coordinate expression of the two consecutive enzymes is needed to divert the flux into IG biosynthesis. With regard to IG accumulation, overexpression of all three cDNAs was no better than overexpression of the two cDNAs. The content of neoglucobrassicin remained unchanged in all transgenic plants. Although glucobrassicin was most directly affected by overexpression of the transgenes, elevated levels of the parent IG, glucobrassicin, were not always accompanied by increases in 4-hydroxy and 4-methoxy glucobrassicin. However, one transgenic line producing about 8-fold increased glucobrassicin also accumulated at least 2.5 fold more 4-hydroxy and 4-methoxy glucobrassicin. This implies that a large glucobrassicin pool exceeding some threshold level drives the flux into the side chain modification pathway. Aliphatic glucosinolate content was not affected in any of the transgenic plants.
Zang
Yun-Xiang
YX
Department of Molecular Biotechnology, Konkuk University, Seoul 143-701, Korea.
Lim
Myung-Ho
MH
Park
Beom-Seok
BS
Hong
Seung-Beom
SB
Kim
Doo Hwan
DH
D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D001937:Brassica / Q000201:enzymology / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004269:DNA, Bacterial; D005961:Glucosinolates / Q000737:chemistry / Q000378:metabolism*; D007210:Indoleacetic Acids / Q000737:chemistry; D010641:Phenotype; D030821:Plants, Genetically Modified; D020133:Reverse Transcriptase Polymerase Chain Reaction
D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D005961:Glucosinolates; D007210:Indoleacetic Acids; C036483:T-DNA; C030737:indoleacetic acid; D003577:Cytochrome P-450 Enzyme System; C530432:cytochrome P-450 79B2 protein, Arabidopsis; C530433:cytochrome P-450 79B3, Arabidopsis; C431937:CYP83B1 protein, Arabidopsis