FRIMP1 and FRIMP2: novel membrane proteins required for light-regulated development of Arabidopsis

Lead Research Organisation: University of Southampton
Department Name: School of Biological Sciences

Abstract

The ability of a plant to respond to its light environment is critical to its survival. Light controls many aspects of plant growth and development and is important throughout the plant life cycle. Examples of responses under the regulation of light include germination, the development of green, photosynthesizing seedlings, regulation of the architecture of the plant and control of flowering time. All of these processes are crucial to agricultural productivity and an understanding of how they are regulated has great long-term importance. Plants have a range of photoreceptors that perceive light including the phytochromes that absorb red (R) and far-red (FR) light and the cryptochromes and phototropins that respond to blue/UVA. Much of what we know about how plants respond to light has come from studies on the model plant Arabidopsis thaliana that has five phytochromes. How these phytochromes pass on their light signal to regulate plant development has been an area of great interest in recent years. We now know that in light all phytochromes relocate from the cytoplasm of the cell to the nucleus, the organelle that contains the majority of the cell's genetic information. Once in the nucleus they interact with a number of signalling proteins to change the expression of many genes that lead to changes in plant growth and development. However, this is not the full story, and there is also some biochemical and physiological evidence that phytochrome signals to proteins in the cytoplasm and in cellular membranes. To date though there is little direct genetic evidence to back up a role for membrane proteins in phytochrome regulation of plant development. We have attempted to address this anomaly by trying to identify and characterize membrane proteins with a role in light regulation of plant development. To do this we have first identified predicted membrane protein genes that are light regulated by examining data sets of all light-regulated genes in Arabidopsis. Using this approach we have identified a number of membrane transporter genes that appear to have a role in seedling development. We have also identified some membrane proteins of unknown function and one of these is a FR light-induced membrane protein we have called FRIMP1. FRIMP1 and its close counterpart, FRIMP2, appear to be important for both seedling development and leaf development in Arabidopsis. We have identified frimp1 and frimp2 mutants that lack the FRIMP1 and FRIMP2 proteins and these mutants show a long hypocotyl under FR light and large cotyledons under R light, indicating that FRIMP1 and FRIMP2 are required for normal development of these plant tissues in the light. Interestingly, the FRIMP proteins are members of a completely new membrane protein family with close relatives in all multicellular eukaryotes including humans. As they have not been investigated in any of these organisms, what we learn about them in plants may be of much broader significance. The main aim of this project is to understand the function of FRIMP1 and FRIMP2 in response to light by determining all of the physiological processes they regulate and where they are located. We will do this by examining in detail the physiological responses of the frimp1 and frimp2 mutants, a frimp1frimp2 double mutant we have produced, and plants in which the levels of FRIMP1 and FRIMP2 proteins have been artificially increased. This will tell us the full range of responses in which FRIMP1 and FRIMP2 are involved. We will also determine what genes show altered expression in frimp1 and frimp2 mutants and use this information to find out how FRIMP1 and FRIMP2 interact with different signalling pathways within the plant. Finally, we will use two types of reporter proteins to show where FRIMP1 and FRIMP2 are located in the plant and also where they are within the cell. We will then be in a position to develop testable hypotheses about how FRIMP1 and FRIMP2 function at the molecular level.

Technical Summary

To identify key membrane proteins involved in mediating the critical process of seedling de-etiolation we have used microarray analysis to screen for membrane protein genes that are regulated by light. Using this approach, we identified a FR light-induced membrane protein, FRIMP1, that is part of a new and highly conserved membrane protein family in multicellular eukaryotes. KO mutants of FRIMP1 and its close Arabidopsis homologue FRIMP2 have a long hypocotyl under far-red light and large cotyledons under red light, indicating these proteins are required for phytochrome signalling. The frimp mutants also exhibit a range of leaf abnormalities in mature plants with frimp1 showing a strong epinastic response and frimp2 an unusual leaf shape. Both mutants have more leaves than WT plants. The aim of this project is to understand the physiological function of FRIMP1 and FRIMP2 by determining what processes and genes they regulate and where they are located. Firstly, we will make a detailed phenotypic characterization of the frimp1 and frimp2 mutants, a frimp1frimp2 double mutant, and plants in which we have overexpressed FRIMP1 and FRIMP2 proteins. Secondly, we will determine whether FRIMP1 and FRIMP2 act directly in light signalling or whether they are light-responsive components of an alternative signalling pathway. We will do this initially by using a transcriptomics approach to determine the effect of FRIMP1 and FRIMP2 on R and FR light-regulated genes and comparing these data sets with profiles of genes regulated by other signalling pathways. Significant overlap of FRIMP-regulated genes with those regulated by other pathways may indicate a role for FRIMP proteins in this pathway and we will test this experimentally. Finally, to gain insight into the mechanism of FRIMP signalling, we will use enzyme and fluorescent reporter proteins to determine where FRIMP1 and FRIMP2 are localized at the tissue and cellular level.

Publications

10 25 50
 
Description We identified and thoroughly characterised single and double mutants in both Col and Ws Arabidopsis backgrounds for the novel membrane proteins AtGTG1 and AtGTG2 (originally designated FRIMPs) using both physiological and molecular approaches, including microarray analysis. gtg1gtg2 double mutants, but not single mutants, showed defects in fertility, seedling growth and responses to light, sugars and ions with clear disruption of cellular shape resulting in irregular, distended cells, particularly evident in the epidermal layer. Stable expression of reporter genes in Arabidopsis showed that GTG1 and GTG2 are expressed throughout the plant and that GTG1 is localized primarily to Golgi bodies and to the endoplasmic reticulum. This intracellular localization pattern was supported by transient expression of fluorescently-labelled GTG1 in tobacco.
Exploitation Route GTG proteins are important for both plant growth and fertility. Understanding fundamental mechanisms for these processes will be important in the long term as we look to increase plant growth for improved food and energy crops. As the World population increases and fossil fuel energy sources are depleted we will increasingly rely on improved plants to provide our food and energy. GTG/FRIMP proteins are also important for cell function in other eucaryotes and could have potential as pharmaceutical targets.
Sectors Agriculture, Food and Drink,Energy,Pharmaceuticals and Medical Biotechnology

 
Description This grant provided major new information of the GTG family of membrane proteins that was published in Jaffe et al (Plant Cell, 2012, 24, 3649-3668) and presented at international academic conferences (see engagement activities). It has also been used to secure further funding from the BBSRC and Gerald Kerkut Charitable Trust.
First Year Of Impact 2012
Sector Agriculture, Food and Drink,Education
 
Description Are GTGs a new class of plant anion channels regulating pH in the endomembrane system? Co-I with Dr L Williams, Dr I O'Kelly
Amount £45,193 (GBP)
Organisation The Gerald Kerkut Charitable Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 09/2018
 
Description Research grant (responsive mode)
Amount £429,776 (GBP)
Funding ID BB/L010313/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2014 
End 09/2017
 
Description The physiological function of the GTG/GPHRs, a highly conserved family of eukaryotic membrane proteins Co-I with Dr LE Williams and Dr N Smyth
Amount £85,000 (GBP)
Organisation The Gerald Kerkut Charitable Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2010 
End 07/2014
 
Description The physiological role of G protein coupled receptor type-G proteins (GTGs) in plants in response to stress Co-I with Dr LE Williams
Amount £28,101 (GBP)
Organisation The Gerald Kerkut Charitable Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 09/2019
 
Title Mutant and overexpressor lines for Arabidopsis GTG proteins 
Description We have made new gtg1-2gtg2-2 and gtg1-3gtg2-3 double mutants of Arabidopsis and constructed the following transgenic lines: 35Spro:GTG1 and 35Spro:GTG2 GTG1pro:GUS (for ß-glucuronidase) and GTG1pro:GUS GTG1pro:GTG-GUS and GTG2pro:GTG-GUS 35Spro:GTG1-GFP (for green fluorescent protein) and 35Spro:GTG2-GFP These lines are published in Jaffé et al (2012, Plant Cell 24, 3649-3668) and are available to the research community on request. 
Type Of Material Cell line 
Year Produced 2012 
Provided To Others? Yes  
Impact Development of these research tools enabled the publication of a major research paper and the other outcomes documented in this submission. 
 
Title Microarray analysis of gene expression in gtg1gtg2 mutants of Arabidopsis 
Description Microarray data set showing gene expression changes in the gtg1gtg2 double mutant compared to wild-type Arabidopsis. 
Type Of Material Database/Collection of data 
Year Produced 2012 
Provided To Others? Yes  
Impact This dataset has been published in Jaffé et al. (2012, Plant Cell 24, 3649-3668) and is available to the academic community. 
 
Description Localisation of GTG proteins in Arabidopsis and tobacco 
Organisation Oxford Brookes University
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration with Oxford Brookes University on localisation of GTG (FRIMP) proteins in Arabidopsis and tobacco.
Collaborator Contribution Provided expertise on studying the localization of GTG (FRIMP) protein in the plant Golgi.
Impact Jaffé FW, Freschet G-EC, Valdes BM, Runions J, Terry MJ and Williams LE (2012) G protein-coupled receptor type G proteins (GTGs) are required for light-dependent seedling growth and fertility in Arabidopsis. Plant Cell, 24, 3649-3668.
Start Year 2008
 
Description Searching for membrane proteins important during seedling de-etiolation: towards a systems biology approach (Peking U) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Seminar at College of Life Sciences, Peking University, China on April 23rd, 2007. Fostering of international collaboration.

Informal links still maintained.
Year(s) Of Engagement Activity 2007
 
Description Thinking of Studying Biological Sciences? (Woking College) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk to A-level students at Woking College about a career in biological sciences including some recent research results on localization of GTG proteins.

No documentable impacts realised to date.
Year(s) Of Engagement Activity 2010