Source: WASHINGTON UNIV submitted to
BIOCHEMICAL AND PHYSIOLOGICAL ANALYSIS OF BRASSINOSTEROID-INACTIVATING CYTOCHROME P450S THAT MODULATE PLANT DEVELOPMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0204510
Grant No.
2005-35318-16214
Project No.
MOR-2005-02524
Proposal No.
2005-02524
Multistate No.
(N/A)
Program Code
54.3
Project Start Date
Aug 1, 2005
Project End Date
Aug 31, 2008
Grant Year
2005
Project Director
Thornton, L. E.
Recipient Organization
WASHINGTON UNIV
#1 NORTH BROOKINGS DRIVE, CAMPUS BOX 1137
ST LOUIS,MO 63130
Performing Department
(N/A)
Non Technical Summary
The biochemical processes required to control the hormones that regulate plant growth are not well understood. Brassinosteroids are a family of steroid hormones that promote the growth and development of virtually all tissues through out the life cycle of the plant. Cytochrome P450 monooxygenases (P450s) are a family of enzymes found in all organisms with a diverse array of biochemical roles. Previous work has identified two brassinosteroid-inactivating P450 enzymes in the model genetic plant, Arabidopsis thaliana, whose over abundance causes dwarf plants. When both of these P450s are genetically removed, the plants are larger and have larger seed pods. There are four very closely related proteins that may have a similar brassinosteroid-inactivating role in the important crop plant, rice. The goal of this project is to determine the biochemical function of the rice brassinosteroid-inactivating proteins. The research will determine specifically how this type of P450 enzyme binds and inactivates brassinosteroid targets. This study will establish the structural features unique to each P450 that confer subtle differences in activity. It is anticipated that this research will result in a better understanding of brassinosteroid regulation through inactivation in rice so that these proteins can be used as a means of optimizing crop yield.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2061530100020%
2061530104020%
2061530208010%
2062420100020%
2062420104020%
2062420208010%
Goals / Objectives
One method of enhancing economic opportunities for agricultural producers is to determine the conditions that produce the highest yield per acre of crop plants. This study addresses that need by exploring the mode of action of brassinosteroid-inactivating enzymes. Brassinosteroids are essential plant hormones that determine stature and seed size. Previous studies identified Arabidopsis thaliana cytochrome P450 enzymes, CYP734A1 and CYP72C1, important for the regulation of brassinosteroids through inactivation. CYP734A1 and CYP72C1 appear to work on the same substrates but produce different products. When these proteins are over-expressed, brassinosteroid levels are diminished, conferring a reduction in plant size. When both of these proteins are genetically knocked out, then brassinosteroid levels are increased and the plant is larger than normal. There are four proteins in rice that are overall more similar to CYP734A1, but they share some similarities with CYP72C1 in the area that is predicted to be important for substrate recognition. This project will determine the biochemical function of the rice cytochrome P450 enzymes in the CYP734A family and examine their potential for regulating endogenous brassinosteroid levels via inactivation. It will also determine the mechanism of enzyme function by identifying the amino acids required for substrate binding and hydroxylation. An understanding of the biochemical function of the rice proteins will allow us to explore the possibility of creating rice plants with decreased lodging and increased seed yield.
Project Methods
The CYP734A cDNAs from rice will be cloned and expressed in yeast for purification and biochemical analysis of the proteins. The proteins will be assayed for their ability to bind and hydroxylate brassinosteroids as compared to the activity of the similar proteins from Arabidopsis. Models of the tertiary structure will be created by comparing the proteins to P450s whose crystal structures have been solved. The models will be used to predict substrate orientation and potential products, as well as the amino acids involved in substrate binding. Site-directed mutagenesis and subsequent in vitro biochemical studies will establish the mechanistic importance of amino acids identified in the models. The proteins will also be expressed in Arabidopsis for seedling experiments that indicate the efficiency of activity in the plant.

Progress 08/01/05 to 08/31/08

Outputs
OUTPUTS: Brassinosteroids are growth-promoting hormones necessary for normal development in plants. This work examines the role that brassionsteroid-inactivating cytochrome P450 enzymes play in regulating levels of these hormones. Findings from these studies show which rice genes are the best candidates for manipulating brassinosteroid levels in crop plants, such as rice, leading to higher yield via increased biomass or decreased lodging. This work has been presented at scientific conferences; the American Society of Plant Biologists annual conference (2007, 2010) and the International Symposium on Cytochrome P450 Biodiversity (2008, 2010). It was also accepted for publication in the journal Plant Molecular Biology. It is also under review for publication in the journal Planta. PARTICIPANTS: PD: Thornton, LE Partner Organization: Washington University in St. Louis, MO Collaborators: Michael M. Neff, Mary A. Schuler, Sangeewa Rupasinghe, Hao Peng This project provided post doctoral training for the PD in the labs of Michael Neff and Mary Schuler. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
In further studying the mode of action of brassinosteroid (BR)-inactivating enzymes in plants, we have examined Arabidopsis proteins in vitro and expressed individual rice proteins in Arabidopsis. We also studied endogenous gene expression levels of the rice genes when seedlings were treated with external brassinolide. By studying both Arabidopsis and rice cytochrome P450 enzymes (P450s), we contributed new knowledge on the role of plant P450s in BR inactivation. Of the rice genes in the CYP734A family, CYP734A2, CYP734A5, and CYP734A6 expression is up-regulated in response to exogenous brassinolide, implicating them in BR inactivation. The Arabidopsis CYP734A1 protein and its functional homolog, CYP72C1, have been examined by three-dimensional structure modeling. The model was used to dock potential substrates in the active site to determine the atomically favorable position of the substrate. BR molecules consist of four rings, made of carbons 1 through 17, and a branch made of carbons 20 through 28. These are bulky molecules, and their position in the P450 binding site determines which carbon is closest to the reactive heme molecule for hydroxylation. BR positioning in the CYP734A1 active site of the model supports the biochemical finding for this protein being a carbon-26 hydroxylase, suggesting that the models can predict substrate/protein interactions. The model has allowed us to determine likely amino acid residues that are interacting with the substrate. Docking experiments have been done with the active BRs, brassinolide and castasterone, as well as some of the inactive precursors. Substrate positioning does not appear to be affected by the small differences in the various BRs. We have also expressed CYP734A1 in bacculovirus for in vitro substrate binding experiments. The purified protein gives a type I binding spectral shift for several BRs, but the active BRs, brassinolide and castasterone, show the best response. Clones of the rice CYP734A enzymes expressed in Arabidopsis indicate that all three have BR-inactivating activity. CYP734A5 contains an active site domain not typical to the CYP734A subfamily when sequences from many plants are compared, but the sequence still allows efficient inactivation of BRs. CYP734A2 is encoded by a gene that is not efficiently spliced in Arabidopsis, but the cDNA encodes an enzyme that efficiently inactivates BRs. The gene for CYP734A6 appears to have two splice variants; one encodes a BR inactivating enzyme and one does not. The USDA award provided the means to clone and begin characterizing the rice genes and for functional characterization of the Arabidopsis gene. As a result of this grant, the PI and collaborators are working to apply the information to other economically important plants. The work has also been published.

Publications

  • Thornton, LE, Rupasinghe, S, Schuler, MA, and Neff, MM. (2010) Arabidopsis CYP72C1 is an atypical cytochrome P450 that inactivates brassinosteroids. Plant Mol Bio. 74:167-181


Progress 08/01/06 to 07/31/07

Outputs
OUTPUTS: In further studying the mode of action of brassinosteroid-inactivating enzymes in plants, we have examined Arabidopsis proteins in vitro and expressed individual rice proteins in Arabidopsis. By studying both Arabidopsis and rice cytochrome P450 enzymes (P450s), we can determine the role of plant P450s in BR inactivation. Of the rice genes in the CYP734A family, only CYP734A2 and A5 appear to be expressed under normal growth conditions. CYP734A6 may be expressed under some conditions. The Arabidopsis CYP734A1 protein has been examined by three-dimensional structure modeling. The model was used to dock potential substrates in the active site to determine the atomically favorable position of the substrate. BR molecules consist of four rings, made of carbons 1 through 17, and a branch made of carbons 20 through 28. These are bulky molecules, and their position in the P450 binding site determines which carbon is closest to the reactive heme molecule for hydroxylation. BR positioning in the CYP734A1 active site of the model supports the biochemical finding for this protein being a carbon-26 hydroxylase, suggesting that the models can predict substrate/protein interactions. The model has allowed us to determine likely amino acid residues that are interacting with the substrate. Docking experiments have been done with the active BRs, brassinolide and castasterone, as well as some of the inactive precursors. Substrate positioning does not appear to be affected by the small differences in the various BRs. We have also expressed CYP734A1 in bacculovirus for in vitro substrate binding experiments. The purified protein gives a type I binding spectral shift for several BRs, but the active BRs, brassinolide and castasterone, show the best response. Expression data and sequence similarity suggest that CYP734A2 from rice is more likely than CYP734A5 to have the same biochemical function as the Arabidopsis CYP734A1. Semi-quantitative RT-PCR shows that gene expression for both CYP734A2 and CYP734A5 is elevated in response to exogenous brassinolide. These data suggest that both proteins are BR-regulating enzymes. We have expressed both rice proteins in Arabidopsis to determine if they are able to inactivate brassinosteroids in planta. Preliminary data from 20 transgenic plants indicates that CYP734A2 has no BR-inactivating activity and CYP734A5 has a high level of activity. These data are contrary to the expected results, so we are screening more plants and testing gene expression levels in multiple lines to verify the result. This work was presented at the annual meeting of the American Society of Plant Biologists in July 2007. It was presented in the form of a poster and discussed with other plant biologists interested in hormone control of plant develelopment. PARTICIPANTS: PD: Thornton, LE Partner Organization: Washington University in St. Louis, MO Collaborators: Michael M. Neff, Mary A. Schuler, Sangeewa Rupasinghe, Zhimou Wen PROJECT MODIFICATIONS: Aim 1 of the project required that the rice CYP734A genes be cloned and heterologously expressed. Due to the gene structure of the rice genes and other cloning challenges, this part of the project was significantly delayed. The genes were cloned in the last year and expressed in Arabidopsis to examine their individual effects on brassinosteroid accumulation in a different plant. Expression in yeast or insect cells is not proving possible under the time constraints of this project. Therefore, comparison of substrate specificity for Aim 2 will rely on computer modeling of the three dimensional structure for each protein.

Impacts
Brassinosteroids are growth-promoting hormones necessary for normal development in plants. This work examines the role that brassionsteroid-inactivating cytochrome P450 enzymes play in regulating levels of these hormones. Findings from these studies show which rice genes are the best candidates for manipulating brassinosteroid levels in crop plants, such as rice, leading to higher yield via increased biomass or decreased lodging.

Publications

  • No publications reported this period


Progress 08/01/05 to 07/31/06

Outputs
Brassinosteroids (BRs) are growth promoting plant hormones. This study examines the mode of action of BR-inactivating enzymes in plants. We are studying an Arabidopsis cytochrome P450 enzyme (P450), CYP734A1, to determine the role of P450s in BR inactivation. This information will help us characterize the closely related P450s from rice to determine how those proteins modulate BRs. Of the rice genes in this family, only CYP734A2 and A5 appear to be real genes. The other genes are pseudogenes or appear to not be expressed. The Arabidopsis CYP734A1 protein has been examined by three-dimensional structure modeling. The model was used to dock potential substrates in the active site to determine the atomically favorable position of the substrate. BR molecules consist of four rings, made of carbons 1 through 17, and a branch made of carbons 20 through 28. These are bulky molecules, and their position in the P450 binding site determines which carbon is closest to the reactive heme molecule for hydroxylation. BR positioning in the CYP734A1 active site of the model supports the biochemical finding for this protein being a carbon-26 hydroxylase, suggesting that the models can predict substrate/protein interactions. The model has allowed us to determine likely amino acid residues that are interacting with the substrate. Docking experiments have been done with the active BRs, brassinolide and castasterone, as well as some of the inactive precursors. Substrate positioning does not appear to be affected by the small differences in the various BRs. However, when 26-OH-brassinolide is docked in the CYP734A1 model, the molecular interactions create a 'bad fit', further supporting the finding that CYP734A1 is a 26-hydroxylase. We have three lines of evidence that CYP734A2 is a BR-inactivating protein in rice. First of all, CYP734A2 is expressed, whereas several of the other family members have no EST or MPSS data available. Furthermore, the CYP734A family from rice has about 50% overall identity to CYP734A1 from Arabidopsis. When looking at the residues potentially involved in substrate binding for CYP734A1, 74% are conserved in the rice CYP734A2, while only about 50% are conserved in CYP734A5. These data suggest that CYP734A5 is less likely to have the same function as CYP734A1, and that CYP734A2 has the residues that are sufficient for brassinolide hydroxylation at carbon 26. Expression of the rice CYP734A2 gene has been examined by semi-quantitative RT-PCR. The Arabidopsis BR-inactivating P450s have increased expression in response to exogenous application of the hormone. Rice seedlings were grown on media with and without added brassinolide to determine if the presence of this hormone modifies the expression pattern of the CYP734A2 gene in rice. RNA was extracted from 12-day old root and shoot tissue. CYP734A2 is expressed more abundantly in the roots than in the shoot, and its transcript levels increase in both tissues in response to external application of brassinolide. These results suggest that CYP734A2 is a BR-regulating enzyme in rice, and it should be examined further to determine its mode of action.

Impacts
Brassinosteroids are growth-promoting hormones necessary for normal development in plants. This work examines the role that brassionsteroid-inactivating cytochrome P450 enzymes play in regulating levels of these hormones. Findings from these studies will facilitate the regulation of brassinosteroid levels in crop plants, such as rice, leading to higher yield via increased biomass or decreased lodging.

Publications

  • No publications reported this period