22]

Characterization of Cytochrome P450 Proteins that Regulate Plant Gravitropism.  J.C. Withers¹ and S.E. Wyatt^{1  1}Dept. of Environmental and Plant Biology, Ohio University, Athens, OH.

   An understanding of gene expression that occurs during the gravity response is of central importance to studying the cellular mechanisms linking the physical perception of gravity to the biochemical activities governing the growth response.  Plants with a mutation in the GRAVITY PERSISTENCE SIGNAL (GPS)1 locus of Arabidopsis thaliana show a “No Response” phenotype during gravistimulation experiments.  Cloning of GPS1 revealed that it encodes CYP705A22 (A22), a cytochrome P450 protein (P450) of unknown function.  Microarray data collected from Arabidopsis root tips indicated that the expression of a closely related family member, CYP705A5 (A5), is increased following a gravity stimulus in roots.   

   An expression profile was generated for A5 using real-time quantitative PCR, and the data indicate that A5 is up-regulated nearly five fold within the first five minutes of gravity stimulation.  Reporter gene constructs that link the A5 gene to the green fluorescent protein (GFP) have shown that A5 is expressed in the root zones of elongation and maturation.  To further investigate the role of P450s in regulating gravitropism, plants containing a T-DNA insertion at the A5 locus were obtained and characterized with respect to their gravity response.  Homozygous mutants showed an attenuated rate of curvature that was able to be rescued by the addition of dihydroquercetin, a flavonol known to be the product of a P450 hydroxylation event.   

   Computer modeling of the catalytic domain and screening of potential substrates has generated a list of 130 compounds that may have the ability to bind to A22 and A5, and nearly 50% of the compounds are derivatives of the phenylpropanoid biosynthetic pathway.  Protein expression constructs were created using the cDNA sequence encoding each of the proteins and expressed in cell culture in order to isolate the proteins and determine the specific biological substrate for each. 

(Supported by NASA: NAG2-1608 and NSF: 0618506 to SEW)