Biomedical subjects

I E Burbulis

Publications and source records attributed to I E Burbulis.

4 recordsLinked to original sources

PubMed1999

Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway.

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. The importance of understanding the subcellular organization of elaborate enzyme systems is discussed in the context of metabolic engineering.

Arabidopsis↗

PubMed1999

Disruption of specific flavonoid genes enhances the accumulation of flavonoid enzymes and end-products in Arabidopsis seedlings.

Polyclonal antibodies were developed against the flavonoid biosynthetic enzymes, CHS, CHI, F3H, FLS, and LDOX from Arabidopsis thaliana. These antibodies were used to perform the first detailed analysis of coordinate expression of flavonoid metabolism at the protein level. The pattern of flavonoid enzyme expression over the course of seedling development was consistent with previous studies indicating that chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), and flavonol synthase (FLS) are encoded by 'early' genes while leucoanthocyanidin dioxygenase (LDOX) is encoded by a 'late' gene. This sequential expression may underlie the variations in flavonoid end-products produced during this developmental stage, as determined by HPLC analysis, which includes a shift in the ratio of the flavonols, quercetin and kaempferol. Moreover, immunoblot and HPLC analyses revealed that several transparent testa lines blocked at intermediate steps of the flavonoid pathway actually accumulated higher levels of specific flavonoid enzymes and end-products. These results suggest that specific intermediates may act as inducers of flavonoid metabolism.

Acyltransferases↗

PubMed1996

A null mutation in the first enzyme of flavonoid biosynthesis does not affect male fertility in Arabidopsis.

Flavonoids are a major class of secondary metabolites that serves a multitude of functions in higher plants, including a recently discovered role in male fertility. Surprisingly, Arabidopsis plants deficient in flavonoid biosynthesis appear to be fully fertile. Using RNA gel blot analysis and polymerase chain reaction-based assays, we have shown that a mutation at the 3' splice acceptor site in the Arabidopsis chalcone synthase gene completely disrupts synthesis of the active form of the enzyme. We also confirmed that this enzyme, which catalyzes the first step of flavonoid biosynthesis, is encoded by a single-copy gene. HPLC analysis of whole flowers and stamens was used to show that plants homozygous for the splice site mutation are completely devoid of flavonoids. This work provides compelling evidence that despite the high levels of these compounds in the pollen of most plant species, flavonoids are not universally required for fertility. The role of flavonoids in plant reproduction may therefore offer an example of convergent functional evolution in secondary metabolism.

Arabidopsis↗

PubMed1996

Are flavonoids synthesized by a multi-enzyme complex?

An enormous variety of metabolic processes are characterized by enzyme complexes, which are likely to play important roles in directing the efficient operation and specificity of cellular metabolism. In many cases membranes or cytoskeletal elements provide scaffolding for these highly ordered assemblies of enzymes. Biochemical and immunocytochemical studies indicate that the flavonoid biosynthetic pathway of higher plants involves a complex of sequentially-acting enzymes localized at the cytoplasmic face of the endoplasmic reticulum. This paper describes preliminary efforts to define the organization of this putative flavonoid biosynthetic complex and elucidate its role in controlling the synthesis of different flavonoid end-products in the model plant, Arabidopsis thaliana.

Acyltransferases↗