75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

343 A Yeast-2-Hybrid Assay Reveals that Chalcone Synthase and Chalcone Isomerase Selectively
Bind Proteins Encoded by Non-Flavonoid Related Genes
Jonathan Watkinson, Brenda Winkel
Virginia Tech
The biosynthetic enzymes of the flavonoid pathway are generally believed to form a complex or metabolon. This
multi-enzyme complex allows for tight regulation over the synthesis of flavonoid end-products and prevents the buildup
of toxic intermediates by channeling them from one enzyme in the pathway to the next. Originally, the complex was
believed to be located exclusively at the cytoplasmic face of the endoplasmic reticulum with flavonoid 3' hydroxylase as
the membrane anchor. However, there is now evidence that nuclear localization of chalcone synthase (CHS), chalcone
isomerase (CHI) and perhaps other flavonoid enzymes are also targeted to nuclei and that this localization may be
dynamic, changing in response to external stimuli and possibly, developmental cues.
We have undertaken a yeast-2-hybrid screen to search for binding partners of flavonoid enzymes in an effort to
determine whether other factors can affect localization of the metabolon. CHS, CHI, and flavanone 3 hydroxylase (F3H)
were previously cloned into the yeast pBI880 DNA binding domain bait vector. An Arabidopsis, cDNA, activation domain
(prey) library in pBI771 was used to transform HF7c yeast cells harboring one of the three bait vectors. The transformed
cells were plated onto selective synthetic dextrose medium lacking histidine and supplemented with 3-aminotriazole.
Positives were confirmed by plating on medium lacking uracil as well as histidine and by confirming that no activation
of the reporter gene took place in the absence of bait. Plasmids were isolated from these colonies and the sequences of
the inserts determined. From the CHS screen eight positive clones have been isolated. The encoded proteins include
ribosomal proteins, a GA responsive protein, a cytosolic amino peptidase and a putative FYVE domain type protein.
Knockout plants for the cytosolic amino peptidase and putative FYVE domain protein are being analyzed. Interaction
between CHS and these proteins is also being characterized using surface plasmon resonance refractometery. The screen
for CHI generated four positives of which two have been eliminated and 2 are undergoing further analysis. Screening of
F3H did not generate any positive hits in the initial experiment.
344 Closely related Arabidopsis thaliana R2R3-MYB transcription factors act as distinct
flavonol-specific regulators of phenylpropanoid biosynthesis
Ralf Stracke, Frank Mehrtens, Bernd Weisshaar
Bielefeld University
The Arabidopsis thaliana R2R3-MYB transcription factor MYB12 was identified as a flavonol-specific activator
of flavonoid biosynthesis. A high degree of functional similarity between MYB12 and the structurally closely related
factor P from Zea mays was revealed by transient expression in A. thaliana protoplasts. Both displayed similar target
gene specificity, and both activated the target gene promoter only in presence of a functional MYB recognition element
(MRE). The genes encoding the flavonoid biosynthesis enzymes CHS, CFI, F3H and FLS were identified as target genes.
A tight linkage between the expression level of functional MYB12 and the flavonol content of young seedlings was
observed by HPLC analyses of myb12 mutants and MYB12 overexpression plants. qRT-PCR using seedlings of these
mutant plants showed MYB12 to be a transcriptional regulator of CHS and FLS in planta. These enzymes are essential
for flavonol biosynthesis and katalyze key branch point steps in the pathway. Transient expression of the closely related
A. thaliana R2R3-MYB factors MYB11 and MYB111 (together with MYB12 defining the subgroup 7 of the R2R3-MYB
gene family) showed similar target gene specificity. Analyses of myb11 and myb111 mutant plants revealed impact of
the corresponding genes on flavonol biosynthesis.