GENETIC ANALYSES OF ROP GTPASE SIGNALING IN ARABIDOPSIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0187089
• Grant No.: 2001-35304-09894
• Proposal No.: 2000-01586
• Project Start Date: Nov 15, 2000
• Project End Date: Nov 14, 2003
• Grant Year: 2001
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521

Performing Department
BOTANY AND PLANT SCIENCES

Non Technical Summary
In plants, proper development and growth requires coordinate communications between cells, tissues, and organs. Such communications involve cell surface receptors and subsequent intracellular signaling cascades. The goal of this proposed research is to understand how Rop GTPases, which belong to a class of guanine nucleotide binding proteins that act as important intracellular signaling switches, relay extracellular signals from the putative cell surface receptor CLV1. CLV1 controls the maintenance of the shoot apical meristem (SAM) in Arabidopsis. Our preliminary studies have provided biochemical evidence that Rop may transmit signals CLV1. Two major objectives will be achieved in the proposed research. First, the specific Rop GTPase involved in the CLV1 signaling will be identified using combined functional genomics and genetic approaches. Second, we will use clv1 mutant plants that are defective in CLV1 kinase activity to investigate whether CLV1 passes signals onto Rop to through CLV-dependent phosphorylation of Rop, CLV1-dependent switching on Rop GTPase, and/or CLV1-dependent recruitment of Rop to the plasma membrane. The study described in this proposal will not only provide important basic understanding of signal transduction and molecular mechanisms for the control of meristem activity in plants, but also help to develop novel means to genetically manipulate plant signaling pathways for the improvement of crop production and food quality.

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
201	2499	1050	50%
206	2420	1030	50%

Knowledge Area
206 - Basic Plant Biology; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
2420 - Noncrop plant research; 2499 - Plant research, general;

Field Of Science
1030 - Cellular biology; 1050 - Developmental biology;

Keywords

signal transduction

guanosine triphosphates

binding proteins

receptors

kinases

meristems

phosphorylation

gene analysis

arabidopsis

plant genetics

plant biology

plant development

cell biology

genetic regulation

gene expression

spatial distribution

mutants

phenotypes

biochemical mechanisms

gene function

metabolic pathways

plant biochemistry

green fluorescent protein

plant enzymes

Goals / Objectives
1) To Identify Rop gene(s) involved in the CLV1 pathway--We will use three complementary approaches to identify RopSam gene(s) that participate in the CLV pathway in the control of SAM maintenance. First, we will determine which of the 11 Rop genes are expressed in SAM in a spatial pattern that is similar or identical to that of the CLV1 gene. Second, we will isolate T-DNA insertional mutants for the Rop genes with the said expression pattern and characterize SAM phenotypes in these mutants. This work will be carried out in coordination with the project that is currently funded by Department of Energy, which is aimed at isolating knockout mutants for the whole Rop gene family. Finally, we will analyze the phenotype of ropSa/clv1 double mutants. This genetic analysis will confirm that Ropsam indeed acts in the CLV pathway and will set the stage for understanding the mechanism by which CLV1 regulates Ropsam in Objective II. A more detailed genetic study will be carried out under the DOE project to elucidate functional relationship between Ropsam and other components of the CLV pathway. 2) To elucidate the mechanism by which the CLV1 kinase regulates Ropsam--We will investigate how CLV1 regulates Ropsam at the biochemical and subcellular levels. First, we will determine whether CLV1 phosphorylates Ropsam using both in vitro and in vivo assays. Second, we will determine whether CLV1 regulates the signaling activity of Ropsam by analyzing the amount of GTP-bound form of Ropsam in the clv1 mutant background. Finally, we will GFP-tagged Ropsam to test the hypothesis that CLV1 functions to recruit Ropsam to the plasma membrane.

Project Methods
A combination of reverse genetic and biochemical approaches will be used to investigate whether Rop GTPase participates in the control of shoot meristem maintanence.

Progress 11/15/00 to 11/14/03

Outputs
The goal of this project was to understand the function of the ROP GTPase gene family using genetic approaches. To achieve this goal, transgenic plants expressing dominant mutants of ROPs and genetic knockout mutants were isolated. Using these genetic stocks the function of several ROPs, including ROP2 and ROP9, have been investigated. ROP2 has been shown to regulate several developmental processes as well as plant responses to environmental stresses. ROP9 has been shown to specifically participate in the negative regulation of Arabidopsis responses to ABA. These studies have demonstrated a versatile role for ROP GTPases in plant signal transduction and have built a framework for future studies aimed at dissecting specific ROP-mediated signaling pathways.

Impacts
The results have led to a better understanding of how plant growth, development and responses to the environment is controlled by G protein signaling proteins. This knowledge may be useful for genetically engineering plants (especially crop plants) that have improved productivity and tolerance to environmental stresses. In particular, transgenic expression of dominant negative ROP10 mutants may be used to enhance crops capacity to ABA, a stress hormone produced under various stress conditions. Such plants may have improved tolerance to stresses such as drought stress. Such the work will most likely have an impact in agriculture productivity.

Publications

• Baxter-Burrel, A., Z. Yang, P. Springer, and J. Bailey-Serres. 2002. RopGAP4-dependent Rop GTPase rheostat controls Arabidopsis oxygen deprivation tolerance. Science. 296:2026-2028.
• Yang, Z. 2002. Small GTPases: Versatile Signaling Switches in Plants. Plant Cell 14: S375-S388.
• Li, H., J. Shen, Z. Zheng, Y. Lin, and Z. Yang. 2001. The Rop GTPase switch controls multiple distinct developmental processes in Arabidopsis. Plant Physiol. 126:670-684.
• Zheng, Z.-L., M. Nafisi, H. Li, A. Tam, D.N. Crowell, S.N. Chary, J. Shen, J. I. Schroeder, and Z.Yang. 2002. The Arabidopsis small GTPase ROP10 is a specific negative regulator of phytohormone abscisic acid ABA responses. Plant Cell. 14: 2787-2797

Progress 01/01/01 to 12/31/01

Outputs
The initial goal of this project was to determine whether Rop GTPases are involved in relaying signals from the CLV1 receptor. We have not made significant progress toward this goal, because no rop mutants showing clv1 mutant-like phenotypes are available yet. However, we have focused on a related aspect of Rop signaling. We have shown that Rop apparently regulates the responses to or biosynthesis of brassinolide hormones. Furthermore, we have shown that brassinolides cross-talk with auxin in their control of plant development including seed germination, lateral formation, shoot development and leaf expansion.

Impacts
The results obtained from this period helps understand the regulation of hormone functions and inter-relationship between different plant hormones.

Publications

• Yang, Z. 2002. Small GTPases: Versatile Signaling Switches in Plants. Plant Cell. In press.
• Fu, Y. and Z. Yang. 2001. The Rop GTPase: A master switch of cell polarity development in plants. Trends in Plant Science 6:545-547