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. 1999 Oct 26;96(22):12929-34.
doi: 10.1073/pnas.96.22.12929.

"VSports手机版" Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway

I E Burbulis  1 , B Winkel-Shirley
Affiliations

Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway

I E Burbulis et al. Proc Natl Acad Sci U S A. .

"VSports" Abstract

Flavonoids are secondary metabolites derived from phenylalanine and acetate metabolism that perform a variety of essential functions in higher plants. Studies over the past 30 years have supported a model in which flavonoid metabolism is catalyzed by an enzyme complex localized to the endoplasmic reticulum [Hrazdina, G. & Wagner, G. J. (1985) Arch. Biochem. Biophys. 237, 88-100]. To test this model further we assayed for direct interactions between several key flavonoid biosynthetic enzymes in developing Arabidopsis seedlings. Two-hybrid assays indicated that chalcone synthase, chalcone isomerase (CHI), and dihydroflavonol 4-reductase interact in an orientation-dependent manner. Affinity chromatography and immunoprecipitation assays further demonstrated interactions between chalcone synthase, CHI, and flavonol 3-hydroxylase in lysates from Arabidopsis seedlings. These results support the hypothesis that the flavonoid enzymes assemble as a macromolecular complex with contacts between multiple proteins. Evidence was also found for posttranslational modification of CHI VSports手机版. The importance of understanding the subcellular organization of elaborate enzyme systems is discussed in the context of metabolic engineering. .

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Figures

Figure 1
Figure 1
Schematic of flavonoid biosynthesis in Arabidopsis. CHS catalyses the first committed step in this pathway. Three general classes of end product are produced in Arabidopsis: flavonols, anthocyanins, and proanthocyanidins (or condensed tannins). Enzymes are indicated in bold, with the corresponding genetic loci in parentheses. FLS, flavonol synthase; LDOX, leucoanthocyanidin dioxygenase.
Figure 2
Figure 2
Two-hybrid assay for protein–protein interactions among Arabidopsis flavonoid enzymes. Growth of transformed HF7C yeast cells was assayed on his-, 5 mM 3-aminotriazole medium at 30°C for 4 days. The top row illustrates a representative interaction assay. The Gal41–147 DNA-binding domain (DB) fusion is indicated at the top of each plate, and the Gal4768–881 transactivation domain (TA) fusion is indicated in each sector. The bottom row shows the controls—yeast cells transformed with single plasmids carrying TA fusions, as indicated.
Figure 3
Figure 3
Immunoblot analysis of Arabidopsis flavonoid enzymes recovered by affinity chromatography. Recombinant CHS or CHI was covalently attached to Affi-Gel 10 resin and used as an affinity matrix to bind interacting flavonoid enzymes from wild-type Columbia (Co), Landsberg (La), tt4(2YY6) (CHS null), or tt5 (CHI null) lysates. Bound endogenous plant proteins were eluted from resin and identified by using immunoblot analysis. U, unbound; B, bound proteins from lysate; and T, total lysate control.
Figure 4
Figure 4
Coimmunoprecipitation of flavonoid enzymes. Affinity-purified anti-CHI bound to Affi-Gel Hz resin was used to precipitate CHI from crude soluble protein extracts of 3-day-old seedlings. A control experiment was performed by using the corresponding preimmune serum. Immunoblot analysis was used to identify other flavonoid enzymes that coimmunoprecipitated with CHI. T, total lysate; U, unbound; and P, precipitated proteins. Asterisks indicate the positions of the light and heavy chains of the IgY (anti-CHI) or IgG (preimmune serum) that were eluted from the resin together with the immunoprecipitated proteins.
Figure 5
Figure 5
Analysis of the two molecular mass variants of CHI. Samples of plant extracts treated with specific compounds were fractionated by using reducing SDS/PAGE and then assayed for CHI by immunoblot analysis. Time points were taken at 1, 4, or 7 h for plant lysates with and without 2 mM 2-mercaptoethanol (+/− me). Samples were collected after 30 min of incubation with 1 M Tris, pH 7.2 (T); 0.2 M KOH (OH); 1 M hydroxylamine, pH 7.2 (H); or 5 M 2-mercaptoethanol (me). S, plant lysate, used as a size standard, that contains both molecular mass species of CHI after incubation in buffer containing 2-mercaptoethanol for 4 h. The two bands migrate at approximately 31 and 26 kDa relative to molecular mass standards.
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