Progress 09/01/01 to 08/31/04
Outputs
NON-TECHNICAL SUMMARY: Brassinosteroids are essential plant hormones regulating growth and development in all plants. The purpose of this project was to understand the molecular mechanisms by which brassinosteroids regulate growth in plants, using advanced biochemical techniques such as mass spectrometry. OBJECTIVES: Brassinosteroids (BRs) are growth-promoting natural products found at low levels in pollen, seeds and young vegetative tissues throughout the plant kingdom. Recent identification of BR-insensitive and BR-deficient mutants, has shown that these steroids are essential signals controlling plant growth and development. We identified a BR-insensitive mutant in Arabidopsis thaliana (bri1) that confers pleiotropic phenotypic effects including severely dwarfed stature. BRI1 encodes a Leu-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. We have
examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro which may be important predictors of in vivo function. Specific objectives of this rerearch were: (1) Identification of in vivo autophosphorylation sites of the BRI1 kinase domain of Arabidopsis thaliana, and (2) Continued biochemical characterization of the BRI1 kinase domain. APPROACH: The overall scheme followed in this experiment was immunoprecipitation of BRI1 from purified Arabidopsis plasma membranes followed by SDS-PAGE, gel extraction of the BRI1 band, digestion with trypsin, and injection of the digest into a Thermoquest/Finnigan LCQ Deca ESI-ion trap instrument equipped with a nanoelectrospray interface and capillary HPLC unit. KEYWORDS: brassinosteroids; arabidopsis thaliana; plant growth; plant development; receptors; kinases; mass spectrometry; phosphorylation; signal transduction; mechanism of action; plant biology; plant biochemistry; molecular biology; mutants; pleiotropic
effects; phenotypes; apical dominance; dwarfing; flowering; senescence; male sterility; gene analysis; plant genetics; pathways; plasma membranes; gel electrophoresis; polyclonal antibodies; enzyme inhibitors; purification; liquid chromatography; baseline studies; immunoprecipitation; plant evaluation; mutagenesis; recombinant proteins; peptides PROGRESS: 2001/09 TO 2004/08 During the funding period we identified eleven sites of in vivo autophosphorylation in BRI1 by several techniques employing mass spectrometry and BRI1 protein immunoprecipitated from purified plant membranes. We have also shown that treatment of plants with brassinolide, greatly increases the level of autophosphorylation of BRI. Functional analysis of each site was undertaken in vitro and in vivo by site-directed mutagenesis. Two sites in the activation loop of the kinase domain were essential for biological function and kinase activity. Mutation of phosphorylation sites in the juxtamembrane and carboxy terminus of
of BRI1 altered substrate phosphorylation but not autophosphorylation. Thus, BRI1 appears to have a mechanism of action similar to several animal receptor kinases.
Impacts
We have now accomplished the most detailed examination of in vivo authophosphorylation sites of a plant receptor kinase to date. Given that BRs are found throughout the plant kingdom, it is likely that greater understanding of the molecular mechanisms of BR action could have practical impact on generating crop plants of many species with altered growth properties.
Publications
- Clouse, S.D. 2003. Recent Advances in Brassinosteroid Research: From Molecular Mechanisms to Practical Applications. J Plant Growth Regul. 22:273-275.
- Hernandez, S.C., Hardin, S.C., Clouse, S.D., Keiber, J.J. and Huber, S.C. 2004. Identification of a new motif for CDPK phosphorylation in vitro that suggests ACC synthase may be a CDPK substrate. Arch. Biochem. Biophys. 428:81-91.
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Progress 10/01/02 to 09/30/03
Outputs
BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a Leu-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In previous work we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro which may be important predictors of in vivo function. We found that recombinant BRI1-KD autophosphorylated on Ser and Thr residues and also phosphorylated certain synthetic peptides in vitro. For optimum phosphorylation by BRI1-KD, positive residues at P-3, P-4, P-6 and P+5 (relative to the phosphorylated Ser at P=0), and a hydrophobic residue at P-5 were required. The putative BRI1-KD substrate recognition motif [KRH]-[LIVMFY]-[KR]-[KR]-X(2)-S-X(4)-[KRH], occurred in several Arabidopsis proteins with proposed roles in signal transduction, providing candidates for further molecular evaluation of possible BRI1-KD cytoplasmic binding partners involved
in downstream signaling. Ultimately, in vivo characterization of specific autophosphorylation sites is necessary for a complete molecular understanding of receptor kinase function. During the past year we have begun analysis of the in vivo autophosphorylation sites of BRI1 by several techniques employing mass spectrometry. We have now identified six sites of autophosphorylation in BRI1 using protein immunoprecipitated from purified plant membranes. Five out of the six sites match those previously identified in vitro. We have also shown that treatment of plants with brassinolide, greatly increases the level of autophosphorylation of BRI1, as would be expected based on the current model of BR signal transduction.
Impacts
We have now accomplished the most detailed examination of in vivo authophosphorylation sites of a plant receptor kinase to date. Given that BRs are found throughout the plant kingdom, it is likely that greater understanding of the molecular mechanisms of BR action could have practical impact on generating crop plants of many species with altered growth properties.
Publications
- No publications reported this period
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Progress 10/01/01 to 09/30/02
Outputs
The physiological, biochemical and genetic characterization of several brassinosteroid (BR) biosynthetic mutants has resulted in the widespread acceptance of BRs as a new class of plant hormone, as essential in controlling normal plant development as auxins and gibberellins. Like their animal steroid counterparts, BRs have been shown to regulate gene expression, stimulate cell division and differentiation, and modulate reproductive biology. BRs also mediate growth responses unique to plants, including promotion of cell elongation in the presence of a complex cell wall and coordinating multiple developmental responses to darkness and light. BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leu-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In previous work we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro
which may be important predictors of in vivo function. We found that recombinant BRI1-KD autophosphorylated on Ser and Thr residues and also phosphorylated certain synthetic peptides in vitro. For optimum phosphorylation by BRI1-KD, positive residues at P-3, P-4, P-6 and P+5 (relative to the phosphorylated Ser at P=0), and a hydrophobic residue at P-5 were required. The putative BRI1-KD substrate recognition motif [KRH]-[LIVMFY]-[KR]-[KR]-X(2)-S-X(4)-[KRH], occurred in several Arabidopsis proteins with proposed roles in signal transduction, providing candidates for further molecular evaluation of possible BRI1-KD cytoplasmic binding partners involved in downstream signaling. We have evaluated a number of new synthetic peptides and are refining the critical residues which allow optimum phosphorylation by BRI1. Ultimately, in vivo characterization of specific autophosphorylation sites is necessary for a complete molecular understanding of receptor kinase function, We are now determining
the in vivo sites of autophosphorylation in BRI1 using immunoprecipitation of BRI1 from purified plasma membranes followed by capillary liquid chromatography interfaced with nanoelectrospray tandem mass spectrometry. After identifying specific autophosphorylation sites, we will mutate each in turn and observe the effect on the ability of the mutated receptor kinase to rescue a BR-insensitive mutant. We will also characterize the dependence of in vivo autophosphorylation on BR and determine its effect on substrate phosphorylation in vitro.
Impacts
The proposed work will be the most detailed examination of in vivo authophosphorylation sites of a plant receptor kinase to date. Given that BRs are found throughout the plant kingdom, it is likely that greater understanding of the molecular mechanisms of BR action could have practical impact on generating crop plants of many species with altered growth properties.
Publications
- Clouse, S.D. 2002. Brassinosteroid Signal Transduction: Clarifying the Pathway from Ligand Perception to Gene Expression. Molecular Cell. 10:973-982.
- Clouse, S.D. 2002. Arabidopsis Mutants Reveal Multiple Roles for Sterols in Plant Development. Plant Cell 14: 1995-2000.
- Clouse, S.D. 2002. Brassinosteroid Signaling: Novel Downstream Components Emerge. Current Biology 12:R485-R487.
- Iliev, E., Xu, W., Polisensky, D.H., Oh, M.H., Torisky, R.S., Clouse, S.D. and Braam, J. 2002. Transcriptional and post-transcriptional regulation of Arabidopsis TCH4 expression by diverse stimuli: Roles of cis regions and brassinosteroids. Plant Physiol 130:770-783.
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Progress 10/01/00 to 09/30/01
Outputs
Since the project began on 09/01/2001 there is very little to report at this time, i.e. between 09/01/2001 and 09/30/2001. Therefore, a summary of the work underway is provided. Brassinosteroids (BRs) are growth-promoting natural products found at low levels in pollen, seeds and young vegetative tissues throughout the plant kingdom. Detailed studies of BR biosynthesis and metabolism, coupled with the recent identification of BR-insensitive and BR-deficient mutants, has shown that these steroids are essential signals controlling plant growth and development. We identified a BR-insensitive mutant in Arabidopsis thaliana (bri1) that confers pleiotropic phenotypic effects including severely dwarfed stature, reduced apical dominance, delayed flowering and senescence, male sterility and nearly complete insensitivity to BRs in a variety of assays. BRI1 has been shown by others to encode a membrane-bound leucine-rich repeat receptor kinase. A critical step in the biochemical
characterization of any receptor kinase is to determine the specific amino acid residues that are autophosphorylated. Ligand-dependent autophosphorylation of these residues leads to activation of the cytoplasmic domain, including competence to bind intracellular signal transduction partners and further phosphorylation of downstream components. To address this issue, we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro which may be important predictors of in vivo function. Recombinant BRI1-KD autophosphorylated on Ser and Thr residues with pSer predominating. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry identified a minimum of twelve sites of autophosphorylation in the cytoplasmic domain of BRI1, including five in the juxtamembrane region (N-terminal to the catalytic KD), five in the KD (one each in subdomain I and VIa and three in subdomain VIII), and two in the carboxy terminal region. To identify possible structural elements
required for substrate recognition by BRI1-KD, a series of synthetic peptides were evaluated, indicating that optimum phosphorylation of the peptide by BRI1-KD required R or K residues at P-3, P-4, P+5, and P+6 (relative to the phosphorylated Ser at P=0). Ultimately, in vivo characterization of specific autophosphorylation sites is necessary for a complete molecular understanding of receptor kinase function, We are now determining the in vivo sites of autophosphorylation in BRI1 using immunoprecipitation of BRI1 from purified plasma membranes followed by capillary liquid chromatography interfaced with nanoelectrospray tandem mass spectrometry. After identifying specific autophosphorylation sites, we will mutate each in turn and observe the effect on the ability of the mutated receptor kinase to rescue a BR-insensitive mutant. We will also characterize the dependence of in vivo autophosphorylation on BR and determine its effect on substrate phosphorylation in vitro.
Impacts
The proposed work would be the most detailed examination of in vivo authophosphorylation sites of a plant receptor kinase to date. Given that BRs are found throughout the plant kingdom, it is likely that greater understanding of the molecular mechanisms of BR action could have practical impact on generating crop plants of many species with altered growth properties.
Publications
- No publications reported this period
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