SMALL-MOLECULE TRANSPORT PROTEINS AND THEIR ROLE IN LONG-DISTANCE SIGNALING AND DEVELOPMENT

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 0221601
• Project Start Date: Mar 1, 2010
• Project End Date: Feb 28, 2015
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
Biochemistry & Molecular Biology

Non Technical Summary
The role of the plant phloem has changed from that of simple assimilate transport to a trafficking system for pathogen response and developmental regulators. We have obtained enriched phloem exudates from Arabidopsis thaliana and characterized proteins, metabolites and mRNAs. We discovered several fatty acids, lipids and lipid-binding proteins in the phloem. These phloem lipids are not artifacts from membranes but intrinsic to phloem exudates. As the phloem is an aqueous environment, the lipids may be bound to proteins to increase solubility. This is not without precedence in biological systems: human blood contains a variety of lipids. They are bound to proteins and transported to other tissues for use, storage, modification, or degradation (e.g. vitamins, cholesterol), or serve as messengers and have transcription factor activity. It raises the possibility that lipids and the respective lipid-binding proteins in the phloem serve similar functions in plants.We are investigating the role of lipid-binding phloem proteins in the regulation of plant growth and development and in the response to biotic and abiotic stress.

Animal Health Component

15%

Research Effort Categories

Basic

75%

Applied

15%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	2499	1000	34%
203	2499	1020	33%
204	1510	1040	33%

Knowledge Area
206 - Basic Plant Biology; 204 - Plant Product Quality and Utility (Preharvest); 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1510 - Corn; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology; 1000 - Biochemistry and biophysics; 1020 - Physiology;

Keywords

plant phloem

long-distance signaling

lipid-binding proteins

lipids

biotic and abiotic stress

growth and development

Goals / Objectives
Plants have developed two transport systems: the xylem for water and mineral distribution, and the phloem, which was thought to be responsible only for the transport of assimilates. The view of the plant phloem has changed drastically in the past decade: rather than a simple assimilate transport system it is now considered a major trafficking path for proteins, viruses, RNA, hormones and metabolites. It has been called an "information superhighway" with an important role in plant development and in the response to biotic and abiotic stress. We have obtained enriched phloem exudates from both, Perilla crispa and Arabidopsis thaliana, and subsequently characterized proteins, metabolites and mRNAs in those samples. We discovered several fatty acids, lipids and lipid-binding proteins in the phloem. Lipids and fatty acids are not typically expected in the aqueous environment present in sieve elements. Their presence and that of proteins associated with lipids/lipid metabolism had not been anticipated. One exception in the past has been the movement of methyl jasmonate (JA), a wound-response signal, in the phloem. Finding hydrophobic compounds in an aqueous system is not without precedence in biological systems: The human blood contains a variety of lipids, many of which play a significant role in human health. These lipids are transported bound to proteins. Some lipid-protein complexes are transported to other tissues for storage, modification, or degradation (e.g.cholesterol), others transport essential vitamins. In several cases these lipid-protein complexes serve as messengers and have transcription factor activity. It raises the possibility that lipids and the respective lipid-binding proteins in the phloem serve similar functions in plants and may play an important role in stress related and developmental signaling beyond what is already known about JA. Our goals are to identify and characterize some of the phloem-associated lipids and lipid-binding proteins and identify their role in plant development and pathogen defense response. The first manuscript describing the protein identification is currently in preparation. We anticipate the completion of the mutant and lipid analysis as well lipid overlays for the LAFP within the first year. At that point we will submit a grant proposal to the National Science Foundation (NSF/BIO - IOS), which will be geared towards the functional characterization of the LAFP protein and its role in plant development and stress-response. By the end of year two we hope to have succeeded in cloning the HSA-like protein, which will constitute a separate NSF grant proposal.

Project Methods
I) Identification of lipids present in the phloem exudates Arabidopsis and Perilla phloem exudate will be harvested and lipids purified and separated on TLC. The bands will be analyzed using MALDI-TOF-MS and LC-ESI-MS/MS. We will use a mass spectrometry technique called MALDI imaging, which allows for the detection of individual molecules within the intact tissue. This approach will confirm the localization of the phloem lipids. Lipid-overlay or liposome-binding assays will be used to investigate if our proteins of interest bind to the newly-identified phloem lipids, or if not, which proteins from phloem exudates do bind. II) Characterization of lipid-binding proteins from Arabidopsis phloem exudate: (a)Recombinant protein We will over express the protein with a His-tag in E. coli. The purified protein will then be used to study the lipid binding as described under I. The protein will also be used to prepare antibodies. (b)Localization studies YFP constructs will be used to test the localization in transgenic tobacco leaves. Preliminary studies suggest that LAFP is indeed expressed in the secretory pathway/ along the perimeter of the cell rather than the chloroplast (Fig.1). Antibodies will be used to confirm/ complement the localization of the protein using TEM -immunolocalization. (c)Characterization of T-DNA knock-out mutants We obtained Arabidopsis T-DNA knock-out mutants for LAFP and GRP7. We are planning to compare the leaf, petiole and stem ultrastructure and the lipid profile of the mutants to wild-type plants as well as investigate the plant growth and gene expression (RT-PCR) throughout the plant and under various stress conditions. We will express a HA-tagged cDNA behind a Suc2 (phloem-specific) or 35S promoter (constitutive) in the knock-out mutant to investigate if it can complement the mutant and use anti-HA antibodies to follow its localization and transport throughout the plant. The promoter region will be identified and cloned in front of truncated LAFP-YFP to visualize the uptake into and transport through the phloem. Using the Suc2 promoter in front of LAFP-YFP will serve as a positive control. This will tell us if these proteins will be taken up into the SE from the companion cell and identify the endogenous promoter/protein sequences which control expression in the phloem and targeting to the SE.

Progress 03/01/10 to 02/28/15

Outputs
OUTPUTS: The work was performed with the help of an LBC-BMB undergraduate student (Urs Benning)and a technician (Banita Tamot). The student presented his data in poster format at the Annual Meeting of the Midwest Section of the American Society of Plant Biologists (MWASPB), where he won an "outstanding poster 3rd place" award, at the International Symposium on Plant Lipids, and at the MSU-UURAF; I gave oral presentations of the work at the Plant Vascular Biology Meeting and the International Symposium on Plant Lipids and presented a poster at the Annual Meeting of the American Society of Plant Biologists. Abstracts of the presentations are available online, on CD, and/or in abstract books which are available to meeting participants. Parts of the project were also presented to incoming graduate students, BMB seniors during the BMB 495 senior seminar and to BMB freshmen during the BMB 100 class. For the two classes the slides had been posted on ANGEL and were available to all class participants. PARTICIPANTS: Susanne Hoffmann-Benning, principal investigator Banita Tamot, Technician Urs Benning, undergraduate research aide Collaborators/ Contacts, internal: Bev Chamberlin/Dan Jones MSU-Mass Spectrometry facility Alicia Pastor/Melinda Frame MSU-Center for Advanced Microscopy Collaborators/ Contacts, external: Jocelyn Malamy (University of Chicago) Teun Munnik (University of Amsterdam) Ivo Feussner (University of Goettingen) Training Urs Benning - undergraduate student TARGET AUDIENCES: Individual lectures in BMB 100 and BMB 495 Outreach to area highschools to recruit interns for summer research Plants provide food, shelter, fuel, building materials, and medicine. Without plants we could not exist. This project tries to better understand the long-distance signalling in plants and how it relates to plant development and stress response. Results have a potential impact on how we grow and protect plants and as such affect everyone. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We were able to identify 62 proteins present in the Arabidopsis phloem exudate. Twelve of these proteins play a role in lipid/fatty acid metabolism, storage binding, and signaling. Along with the putative lipid-binding proteins we were able to identify several lipids and fatty acids.One of the phloem lipid-binding proteins, the lipid-associated family protein (PLAFP) contains a PLAT/LH2 domain. This domain is proposed to mediate interaction with lipids or membrane-bound proteins. PLAFP-YFP labeling shows its localization is consistent with proteins traveling through plasmodesmata, a prerequisite for proteins entering the phloem stream. PLAFP knock-out mutants have a low germination rate, delayed development of young leaves, and reduced seed set leading to a lower survival rate compared to wild-type plants. Root and root-hair development also appear delayed. The analysis of phloem lipids suggests a shift in the lipid profile. Electron microscopy has shown that chloroplast development is impaired in young leaves. However, in older leaves, chloroplasts adjacent to the sieve elements appear normal. We will present the effect of PLAFP on development, ultrastructure, gas exchange, phloem lipid- and metabolite profiles as well as its localization and lipid-binding properties. We suggest that PLAFP is a protein, which after binding to lipids, regulates proper development of chloroplasts and possibly other plant organs. These data have been used as the basis for a grant proposal which was submitted to NSF and a manuscript which should be ready for submission before the end of the year.

Publications

• Abstracts for Conferences: Benning, U.F. and Hoffmann-Benning, S. 2010 (poster; * won 3rd place outstanding undergraduate poster award).Identification of Lipid Binding Proteins in the Phloem and their Role in Plant Development and Stress Response. Annual Meeting of the Midwestern Section of the American Society of Plant Biologists in Purdue, Indiana
• Benning, U.F., Tamot, B., Guelette, B.S.,and Hoffmann-Benning, S. 2010(Poster).Identification of Lipid binding proteins in the phloem and their role in plant development and stress response. Program and abstract book Annual meeting of the American Society of Plant Biologists in Montreal, Canada, p.49, abstract on CD or online at aspb.org
• Benning, U.F., Tamot, B., Guelette, B.S.,and Hoffmann-Benning, S. 2010(Oral and Poster).Identification of Lipid binding proteins in the phloem and their role in plant development and stress response. Program and abstract book 19th International Symposium on Plant Lipids in Cairns, Australia, p.8, p.30
• Benning, U.F., Tamot, B., Guelette, B.S.,and Hoffmann-Benning, S. 2010(Oral and Poster).Identification of Lipid binding proteins in the phloem and their role in plant development and stress response. Program and abstract book International Conference on Plant Vascular Biology 2010 in Columbus, OH, p.5, p.47