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

327 The Role of Tryptophan Metabolism in Auxin Homeostasis
Anna Hull 2 , Arifa Ahamed 1 , Adedamola Adepoju 1 , Jeanine Ledoux 1 , Jennifer Normanly 1 , John Celenza 3 , Mike Pieck 3 ,
Judith Bender 4
1
Dept. of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, (MA) , 2 Lincoln University, (PA),
3
Boston University, Boston, (MA), 4 Dept. of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg
School of Public Health, Baltimore, (MD)
Plants synthesize a great variety of secondary metabolites derived from amino acids. Some of these compounds serve as growth
regulators, while most function in defense against pathogens. In Arabidopsis thaliana, the amino acid tryptophan (Trp) is a precursor
for the growth regulator, IAA, and for two distinct defense compounds: the anti-microbial compound camalexin and the anti-herbivory
class compounds called indole glucosinolates.
Two Arabidopsis cytochrome P450s, CYP79B2 and CYP79B3 convert Trp to indole-3-acetaldoxime (IAOx). The cloning of and
subsequent genetic analysis of the CYP79B2 and CYP79B3 genes has pointed to (IAOx) being a key metabolite in the production
of IAA, indole glucosinolates and camalexin. CYP79B2-overexpressing plants (CYP79B2-OEX) have elevated levels of free IAA
and indole glucosinolates while the cyp79B2 cyp79B3 double mutant has decreased free IAA and make no indole glucosinolates or
camalexin (Hull et al., 2000; Mikkelsen et al., 2000; Zhao et al., 2002; Glawischnig et al., 2004).
We have also found evidence in Arabidopsis for cross-talk between Trp and IAA primary metabolic pathways and secondary
metabolic pathways. A dominant overexpression allele of the Arabidopsis Myb transcription factor ATR1, atr1D, increases flux of Trp
into indole glucosinolates by increasing expression of CYP79B2, CYP79B3 and CYP83B1 while resulting in only a modest increase
in IAA. In addition, atr1-2 mutants have reduced CYP79B2, CYP79B3, and CYP83B1 transcription and produce approximately
30% reduced indole glucosinolate accumulation in adult leaves compared to WT (Celenza et al., 2005).
Our current goals are to define further the role of IAOx in IAA synthesis by characterizing CYP79B2-mediated IAA synthesis
in tobacco and Arabidopsis and to confirm or negate a role for IAOx in IAA synthesis in non-cruciferous plant families. In addition,
we wish to identify genes in the IAOx to IAA pathway as well as identifying alternate routes for IAA synthesis. A combination of
mutants screens and targeted metabolic profiling is being used to achieve these goals.
Celenza, J.L., Quiel, J.A., Smolen, G.A., Merrikh, H., Silvestro, A.R., Normanly, J., and Bender, J. (2005). The Arabidopsis ATR1 Myb
transcription factor controls indolic glucosinolate homeostasis. Plant Physiol 137, 253-262. Hull, A.K., Vij, R., and Celenza, J.L. (2000).
Acad. Sci.
USA 97, 2379-2384. Zhao, Y., Hull, A.K., Gupta, N.R., Goss, K.A., Alonso, J., Ecker, J.R., Normanly, J., Chory, J., and Celenza, J.L. (2002).
Trp-dependent auxin biosynthesis in Arabidopsis: involvement of cytochrome P450s CYP79B2 and CYP79B3. Genes Dev 16, 3100-3112.
328 Interaction between SHMT and Fd-GOGAT in photorespiration
Aziz Jamai 1 , Patrice Salome 1 , Lars Voll 2 , Andreas P. Weber 2 , C. Robertson McClung 1
1
Dartmouth College, Hanover, (NH) USA, 2 Michigan State University, East Lansing, (MI) USA
In the leaves, Rubisco catalyzes the carboxylation of ribulose-1, 5-bisphosphate (RuBP), but it also catalyzes an
oxygenase reaction. Photorespiration is a complex series of reactions to salvage the C2 product of the oxygenation of
RuBP. Mitochondrial serine hydroxymethyltransferase (SHMT) is one of the key enzymes of this cycle. A mutant (shm1-
1) defective in SHM1 function lacks SHMT activity and is unable to grow at ambient CO2 concentrations but grows
normally at elevated CO2 concentrations (Voll et al., 2006, Plant Physiol. 140:59).
Here we address the molecular basis of a second mutant, glu1-201, that also lacks photorespiratory SHMT activity.
A test of allelism between shm1-1 and glu1-201 indicates that they are two distinct loci. glu1-201 maps to the top of
chromosome V in a region that lacks SHM genes. We have established that the GLU1 gene, which encodes Fd-GOGAT,
is the defective gene responsible for the glu1-201 phenotype. There is a single nucleotide change between wild type and
mutant glu1-201 changing amino acid 1270 from L to F. Introduction of a wild type GLU1 allele under control of either the
35S or the GLU1 promoter into glu1-201 restores wild type levels of SHMT activity and allows growth at ambient CO2
concentrations . However, introduction of the glu1-201 coding sequence driven by the 35S promoter fails to rescue.
Although glu1-201 has a missense mutation in the Fd-GOGAT coding sequence, the mutant is defective in SHMT
activity and exhibits wild-type Fd-GOGAT activity. In contrast, a T-DNA insertion mutant, glu1-202, lacks Fd-GOGAT
activity and shows reduced SHMT activity. The F1 progeny of a cross between glu1-201 and glu1-202 exhibit loss
of SHMT activity and wild-type Fd-GOGAT activity. These data suggest that Fd-GOGAT plays an essential role in
mitochondrial SHMT activity that is independent of Fd-GOGAT activity. To test this, we generated a glu1-203 allele
with several missense mutations in the catalytic region and showed that it fails to restore wild type Fd-GOGAT activity
in the glu1-202 T-DNA mutant, yet fully rescues SHMT activity in the glu1-201 mutant. We obtained similar results
with a glu1-204 transgene missing 2 kb of coding sequence, including the catalytic domain, but retaining the C-terminal
domain containing L1270. Our results demonstrate that mitochondrial SHMT activity requires the expression of the Fd-
GOGAT, although the mechanism by which Fd-GOGAT supports SHMT activity is unknown.