Progress 09/01/02 to 06/30/06
Outputs
1) We have analyzed various mutant combinations between the cyp79B2 cyp79B3 double mutant and mutants with altered tryptophan (Trp), indole glucosinolate and/or IAA metabolism. Below are listed summaries of combinations done with the cyp79B2 cyp79B3 double mutant: a) trp3 - cyp79B2 cyp79B3 partially suppresses the conditional tryptophan auxotroph phenotype b) alf3 - no genetic interactions were observed d) alf1, an allele of superroot1 (superroot1 encodes a C-S lyase central to glucosinolate biosynthesis) - cyp79B2 cyp79B3 suppresses the superroot phenotype. These plants have greatly reduced glucosinolate production and have a bushy morphology similar to that described for CYP79F1. e) yucca - triple mutants fail to produce indolic glucosinolates but maintain the morphological phenotypes of yucca c) cyp83B1 (also called atr4 and superroot2) (see discussion below) f) atr1D and/or atr2D (see discussion below). 2) Dominant mutations in the Myb-encoding ATR1 (AtMyb34) gene
cause increased expression of CYP79B2 and CYP79B3 and originally were discovered for causing 5-methyltryptophan (5MT) resistance. In collaboration with Dr. Judith Bender (Johns Hopkins University) we have found that the atr1D mutant has elevated IAA and indole glucosinolate levels and that the atr1-2 loss of function allele has a reduced level of indole glucosinolates. These and related findings have been published (Plant Physiol. 137:253-262). Currently we are investigating interactions between CYP79B2, CYP79B3 and other Myb-encoding genes related to ATR1 as well as interactions with another transcription factor encoded by ATR2. 3) In collaboration with Karin Ljung and Goran Sandberg (Umea Plant Science Center, Sweden) we measured IAA synthesis rates in the apical 4 mm root tip sections from whole seedlings and excised roots of Arabidopsis wt and cyp79B2 cyp79B3 that had been incubated in deuterium oxide. There was a small but significantly lower rate of IAA synthesis in tips of
excised roots in the cyp79B2 cyp79B3 double mutant, which is consistent with the slight but significantly lower levels of IAA that are found in whole seedlings of this mutant. This data, along with the root localized expression patterns of CYP79B2 and CYP79B3, support a role for the IAOx pathway in roots. These findings have been published in Plant Cell. 4) We have created transgenic tobacco that express CYP79B2 from the CaMV35S promoter. These lines show increased 5-methyltryptophan resistance and show a dramatic increase in adventitious rooting. We have found a several fold increase in free IAA in these lines. 5) We are continuing temperature shift experiments in which the utilization of the Trp-dependent and Trp-independent IAA biosynthetic pathways will be compared between CYP79B2OEX, cyp79B2 cyp79B3, and WT Columbia grown at different temperatures. Root and hypocotyl length are being measured as well. 6) We have initiated several ongoing genetic screens to identify other
components of the IAOx pathway and components of other Trp metabolic pathways. Several candidates from each screen have been retested and are now undergoing genetic characterization.
Impacts
Our results to date have confirmed our hypothesis that CYP79B2 and CYP79B3 have roles in both indole-3-acetic acid (IAA) biosynthesis and indole glucosinolate (IG) biosynthesis. By combining the cyp79B2 cyp79B3 double mutant with other mutants defective in tryptophan metabolism we have being able to determine the relationship of these other genes to CYP79B2 and CYP79B3. Our results suggest that the CYP79B2/B3 pathway to indole glucosinolates is the predominate route of Trp catabolism in wild-type Arabidopsis and that there likely are regulatory circuits specific for different tryptophan-derived secondary products. Analysis of the temperature regulation of IAA biosynthesis in combination with our collaborative efforts with the Bender and Sandberg labs are beginning to unravel the regulatory pathways that govern the CYP79B2 and CYP79B3 pathway in response to environmental and metabolic signals. In addition, the cyp79B2 cyp79B3 double mutant has been used to help identify
a role for these genes in IAA synthesis in Arabidopsis roots. Finally, expression of CYP79B2 in transgenic tobacco will allow us to determine the role of the indole-3-acetaldoxime pathway in non-glucosinolate producing plants.
Publications
- Calio, J., Tam, Y.Y. and Normanly, J. 2006. Auxin Biology and Biosynthesis in Recent Advances in Phytochemistry: Integrative Plant Biochemistry, ed, J. Romeo, Elsevier, vol 40: 287-305.
- Celenza, J. L., Quiel, J.A., Smolen, G.A., Merrikh, H., Silvestro, A., Normanly, J. and Bender, J. 2005. The Arabidopsis ATR1 Myb transcription factor controls indolic glucosinolate homeostasis. Plant Physiol. 137:253-262.
- Ljung, K., Hull, A. K., Celenza, J., Yamada, M., Estelle, M., Normanly, J. and Sandberg, G. 2005. Sites and regulation of auxin biosynthesis in Arabidopsis roots. Plant Cell 17:1090-1104.
- Normanly, J., Slovin, J.P. and Cohen, J.D. 2005. Auxin Metabolism in Plant Hormones: Biosynthesis, Signal Transduction, Action! 3rd edition. P.J. Davies, ed. Kluwer Academic Publishers: Dordrecht, The Netherlands. pp 36-62.
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Progress 10/01/03 to 09/30/04
Outputs
1) We have constructed a series of mutant combinations between the cyp79B2 and cyp79B3 mutants and mutants with altered tryptophan (Trp), indole glucosinolate and/or IAA biosynthesis. Below are listed the combinations with the cyp79B2 cyp79B3 double mutant and summary of the results found: a) trp3 - cyp79B2 cyp79B3 partially suppresses the conditional tryptophan auxotroph phenotype b) alf3 - no genetic interactions were observed d) alf1, an allele of superroot1 - cyp79B2 cyp79B3 suppresses the superroot phenotype e) yucca these are constructed and are being analyzed now c) cyp83B1 (also called atr4 and superroot2) (see discussion below) f) atr1D and/or atr2D (see discussion below). 2) Dominant mutations in the myb-encoding ATR1 gene cause increased expression of CYP79B2 and CYP79B3. In collaboration with Dr. Judith Bender (Johns Hopkins University) we have found that the atr1D mutant has elevated IAA and indole glucosinolate levels and that the atr1-2 loss of function
allele has reduced level of indole glucosinolates. In addition, we have found that this increase in Trp secondary metabolism depends on the presence of a functional CYP79B2 or CYP79B3 gene; hence a cyp79B2 cyp79B3 atr1D triple mutant displays a phenotype similar to the cyp79B2 cyp79B3 double mutant. We also found that the atr1-2 mutation suppresses cyp83B1 superroot phenotype. These findings have been submitted to Plant Physiology. We are also investigating interactions between the atr2D mutant and CYP79B2 and CYP79B3. atr2D causes a phenotype similar to atr1D, but CYP79B2 or CYP79B3 only are required partially for the mutant phenotype. 3) In a collaboration with Karin Ljung and Goran Sandberg (Umea Plant Science Center, Sweden) we measured IAA synthesis rates in the apical 4 mm root tip sections from whole seedlings and excised roots of Arabidopsis wt and cyp79B2 cyp79B3 that had been incubated in deuterium oxide. There was a small but significantly lower rate of IAA synthesis in
tips of excised roots in the cyp79B2 cyp79B3 double mutant, which is consistent with the slight but significantly lower levels of IAA that are found in whole seedlings of this mutant. This data, along with the root localized expression patterns of CYP79B2 and CYP79B3, support a role for the IAOx pathway in roots. These findings have been submitted to Plant Cell. 4) We have created transgenic tobacco that express CYP79B2 from the CaMV35S promoter. These lines show increased 5-methyltryptophan resistance and show a dramatic increase in adventitious rooting t. We have found a several fold increase in free IAA in these lines. 5) We are continuing temperature shift experiments in which the utilization of the Trp-dependent and Trp-independent IAA biosynthetic pathways will be compared between CYP79B2Oex, cyp79B2 cyp79B3, and WT Columbia grown at different temperatures. Root and hypocotyl length are being measured as well. 6) In the previous report we described two genetic screens to
identify other components of the IAOx pathway and components of other Trp metabolic pathways. Several candidates from each screen are now undergoing genetic characterization.
Impacts
Our results to date support our hypothesis that CYP79B2 and CYP79B3 have roles in both indole-3-acetic acid (IAA) biosynthesis and indole glucosinolate (IG) biosynthesis. By combining the cyp79B2 cyp79B3 double mutant with other mutants defective in tryptophan metabolism we have being able to determine the relationship of these genes to CYP79B2 and CYP79B3. Analysis of the temperature regulation of IAA biosynthesis in combination with our collaborative efforts with Bender and Sandberg labs are beginning to unravel the regulatory pathways that govern the CYP79B2 and CYP79B3 pathway in response to environmental and metabolic signals. In addition, the cyp79B2 cyp79B3 double mutant has been used to help identify a role for these genes in IAA synthesis in Arabidopsis roots. Finally, expression of CYP79B2 in transgenic tobacco will allow us to determine the role of the indole-3-acetaldoxime pathway in non-glucosinolate producing plants.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
1) We are constructing a series of mutant combinations between CYP79B2 and CYP79B3 and genes which have roles in tryptophan (Trp), indole glucosinolate and/or IAA biosynthesis. Most of these combinations are completed and we are beginning phenotypic and metabolite analyses. These include combinations of cyp79B2 cyp79B3 with: a) trp3 b) alf3 c) cyp83B1 (also called atr4 and superroot2) d) alf1, an allele of superroot1 e) yucca f) atr1D and/or atr2D (see discussion below). 2) Dominant mutations in the myb-encoding ATR1 gene cause increased expression of CYP79B2 and CYP79B3. In collaboration with Dr. Judith Bender (Johns Hopkins University) we have found that the atr1D mutant has elevated IAA and indole glucosinolate levels. In addition, we have found that this increase in Trp secondary metabolism depends on the presence of a functional CYP79B2 or CYP79B3 gene; hence a cyp79B2 cyp79B3 atr1D triple mutant displays a phenotype similar to the cyp79B2 cyp79B3 double mutant.
3) We have created transgenic tobacco that express CYP79B2 from the CaMV35S promoter. These lines show increased 5-methyltryptophan (5MT) resistance and show a dramatic increase in adventitious rooting similar to that found in the Arabidopsis rooty mutant. Preliminary results indicate an increase in free IAA in these overexpressing lines. 4) We have initiated two genetic screens to identify other components of the IAOx pathway and components of other Trp metabolic pathways. First, we have screened mutagenized populations derived from CYP79B2OEX line 1J3 for suppression of the Trp-dependent adventitious rooting phenotype. In the second screen, suppressors of the 5MT sensitivity conferred by the cyp79B2 cyp79B3 double mutant were screened for. Several candidates from each screen have been retested and are now undergoing genetic characterization. 5) We have constructed a CYP79B2::GFP fusion that creates a translational fusion between CYP79B2 amino acid 1-55 and GFP. This fusion is
similar to one we created for GUS which showed plastid localization of GUS. We are currently analyzing several independent transgenic lines. 6) Conditions were determined for temperature shift experiments in which the utilization of the Trp-dependent and Trp-independent IAA biosynthetic pathways will be compared. Wild type Arabidopsis seedlings were sown on sterile agar medium, kept at 4C for 48 hours and then transferred to constant illumination at 22C. After 3 days, root and hypocotyl length were measured and the plates were either transferred to 15C, 29C or kept at 22 C. Root and hypocotyl length were measured at days 4, 5, 6, and 7 after transfer from 4C. We observed a significant increase in root and hypocotyl elongation in seedlings grown at 29C at all time points measured. Samples were collected for IAA analysis at each temperature at days 4, 5, 6 and 7 after transfer from 4C. 7) In 2002, co-PI Normanly spent a sabbatical in the laboratory of Dr. Goran Sandberg in order to
learn the microscale methodology for quantification of IAA in 1 mg tissue samples. This method has now been set up in co-PI Normanly's lab.
Impacts
Our results to date support our hypothesis that CYP79B2 and CYP79B3 have roles in both indole-3-acetic acid (IAA) biosynthesis and indole glucosinolate (IG) biosynthesis. By combining the cyp79B2 cyp79B3 double mutant with other mutants defective in tryptophan metabolism we anticipate being able to determine if CYP79B2 and CYP79B3 are required by these other mutants to display their phenotype. These results will help determine if CYP79B2 and CYP79B3 function in the same pathway as these other genes. Analysis of the temperature regulation of IAA biosynthesis in combination with our collaborative effort Dr. Bender is expected to lead to an understanding of the regulatory pathways that govern the CYP79B2 and CYP79B3 pathway in response to environmental and metabolic signals. Expression of CYP79B2 in transgenic tobacco will allow us to determine the role of the indole-3-acetaldoxime pathway in non-glucosinolate producing plants.
Publications
- Waxman, D. J. and Celenza , J. L. 2003. Sexual dimorphism of hepatic gene expression: Novel biological role of KRAB zinc finger repressors revealed. Genes Dev. 17:2607-2613.
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