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Genetic dissection of PIN polarity and endocytic cycling in Arabidopsis

Elena Feraru UGent (2011)
abstract
Auxin is the plant hormone that coordinates plant development and more, by a directional flow through the plant tissues, it mediates the polarity of tissues and organs. The directional auxin flow (polar auxin transport) is achieved by the subcellular localization of auxin influx and efflux carriers. The PINFORMED (PIN) proteins have been identified and characterized as key regulators of a multitude of auxin-mediated developmental processes. Five of them are plasma membrane proteins that act as auxin efflux carriers and have mainly a polar localization that correlates with, and is required for the direction of auxin flow. The polar targeting of PIN proteins has a clear developmental output, because the polarity of PIN localization at the single cell level determines the direction of intercellular auxin transport and the directional signalling to neighbouring cells. Hence, the importance of PIN proteins for plant development and the necessity of studying the PINs on different aspects in order to better understand their functionality. Research performed over the past years has shown that PIN activity is controlled at different levels, such as transcription, constitutive cycling, phosphorylation and degradation. However, little is still known about the regulatory mechanisms, therefore, the purpose of this PhD study was to contribute to the elucidation of two features of PIN proteins: polarity and trafficking. Questions such as, how PIN polarity is established and maintained and how is PIN trafficking regulated, have been addressed and we tried to answer them by using a combination of genetic and pharmacological approaches. In the review entitled “PIN polar targeting” (Chapter 1) we discuss about the PIN proteins as polarity markers that play essential role in auxin-dependent regulation of plant growth and development and make an update on the role of phosphorylation, sterol composition of plasma membrane, secretion, recycling and transcytosis in the regulation of PIN polar targeting. Two prominent mechanisms were shown so far to be crucial for PIN polar targeting: ARF GEF GNOM-dependent constitutive recycling and PINOID/PP2A-dependent phosphorylation /dephosphorylation. Thus, to identify novel molecular components of PIN targeting, and especially of the mechanisms that regulate the establishment and maintenance of PIN polarity we performed a forward genetic screen for the identification of PIN polarity regulators in Arabidopsis. We used PIN2::PIN1-HA in the pin2 mutant background, a transgenic line that has PIN1 mis-localized predominantly at the basal side of the epidermal cells and as a result of the defective PIN1 localization 95 it shows agravitropic growth. We screened for mutants that restored the gravitropic growth to PIN2::PIN1-HA;pin2, considering that the rescue will be possible only if PIN1 will (re)localize in PIN2-like manner and functionally replace PIN2 in its basipetal auxin transport activity. In the research article entitled “PIN polarity maintenance by the cell wall in Arabidopsis” (Chapter 2) we show the identification of a cell wall component (CESA3) as a regulator of PIN polarity and present insights into the cell wall-dependent maintenance of PIN polarity in plant cells. Since plant cells lack tight junctions that define polar domains in animal cells, and the mechanism that keeps the PINs at the correct plasma membrane domains is largely unknown, the role of cell wall in polarity maintenance opens new directions of research regarding the polarity of PIN proteins. In another complementary project, we aimed to investigate the subcellular trafficking of PIN1 protein and to identify novel regulators at different endosomal compartments along the endocytic pathway. By performing a PIN1-GFP fluorescence imaging-based forward genetic screen we looked for abnormal intracellular accumulation of PIN1-GFP in the root cells and identified pat (protein affected trafficking) mutants. The research article entitled “The AP-3 β adaptin mediates the biogenesis and function of lytic vacuoles in Arabidopsis” (Chapter 3) presents the identification and characterization of pat2, a mutant defective in the putative β subunit of the AP-3 complex. pat2 shows abnormal protein accumulation in mis-shaped lytic vacuoles, aberrant vacuolation of prevacuolar complexes (PVCs), abnormal morphology of protein storage vacuoles (PSVs) and impaired transition from storage to lytic vacuoles. It also shows arrested growth on medium lacking sucrose, indicating defects in trafficking from PVC to lytic and/or storage vacuoles. Hence, we identified PAT2/AP-3 β as a novel regulator of lytic vacuole biogenesis and function in Arabidopsis. PIN1 constitutive cycling between plasma membrane and endosomes is crucial for polar auxin transport. PIN1 is internalized from the plasma membrane by clathrin-dependent endocytosis and recycled back to the plasma membrane by the fungal brefeldin A (BFA)-sensitive ARF GEF GNOMdependent exocytosis. In order to identify new components of PIN1 constitutive cycling machinery we performed a PIN1-GFP fluorescence imaging-based forward genetic screen and looked for mutants that showed abnormal BFA-induced PIN1-GFP intracellular accumulation after long treatment. In the manuscript entitled “Genetic dissection of PIN constitutive cycling in Arabidopsis identifies BEX1/RabA1b regulator of protein recycling” (Chapter 4) we show the identification and partial characterization of bex1 (BFA-visualized exocytic trafficking defective 1) mutant. bex1-1 shows intracellular ectopic accumulation of constitutively endocytosed plasma membrane proteins into abnormal BFA compartments, indicating defective exocytosis and transcytosis. BEX1 encodes for the 96 small GTP binding protein, RabA1b that localizes at domains of TGN and seems to play role in trafficking of proteins to the plasma membrane. All together, this PhD research brings novel insights into the mechanisms of PIN polarity maintenance and vesicle trafficking. We identified and characterized several mutants that will definitely contribute to the elucidation of PIN polarity and trafficking pathways in Arabidopsis. Although we wished for the identification of specific regulators of PIN trafficking and this was not the case, we found so far unknown regulators of vacuole biogenesis and function, and protein recycling. Furthermore, finding the cell wall as an essential component required for the maintenance of the PIN polarity opens indeed novel directions in studying cell polarity in plants.
Please use this url to cite or link to this publication:
author
promoter
UGent
organization
year
type
dissertation (monograph)
subject
pages
103 pages
publisher
Ghent University. Faculty of Sciences
place of publication
Ghent, Belgium
defense location
Zwijnaarde : Technologiepark (FSVM building)
defense date
2011-03-04 16:00
language
English
UGent publication?
yes
classification
D1
additional info
dissertation consists of copyrighted material
copyright statement
I have transferred the copyright for this publication to the publisher
id
3006323
handle
http://hdl.handle.net/1854/LU-3006323
date created
2012-10-04 12:36:02
date last changed
2012-10-08 09:05:30
@phdthesis{3006323,
  abstract     = {Auxin is the plant hormone that coordinates plant development and more, by a directional flow through the plant tissues, it mediates the polarity of tissues and organs. The directional auxin flow (polar auxin transport) is achieved by the subcellular localization of auxin influx and efflux carriers. The PINFORMED (PIN) proteins have been identified and characterized as key regulators of a multitude of auxin-mediated developmental processes. Five of them are plasma membrane proteins that act as auxin efflux carriers and have mainly a polar localization that correlates with, and is required for the direction of auxin flow. The polar targeting of PIN proteins has a clear developmental output, because the polarity of PIN localization at the single cell level determines the direction of intercellular auxin transport and the directional signalling to neighbouring cells. Hence, the importance of PIN proteins for plant development and the necessity of studying the PINs on different aspects in order to better understand their functionality. Research performed over the past years has shown that PIN activity is controlled at different levels, such as transcription, constitutive cycling, phosphorylation and degradation. However, little is still known about the regulatory mechanisms, therefore, the purpose of this PhD study was to contribute to the elucidation of two features of PIN proteins: polarity and trafficking. Questions such as, how PIN polarity is established and maintained and how is PIN trafficking regulated, have been addressed and we tried to answer them by using a combination of genetic and pharmacological approaches. In the review entitled {\textquotedblleft}PIN polar targeting{\textquotedblright} (Chapter 1) we discuss about the PIN proteins as polarity markers that play essential role in auxin-dependent regulation of plant growth and development and make an update on the role of phosphorylation, sterol composition of plasma membrane, secretion, recycling and transcytosis in the regulation of PIN polar targeting. Two prominent mechanisms were shown so far to be crucial for PIN polar targeting: ARF GEF GNOM-dependent constitutive recycling and PINOID/PP2A-dependent phosphorylation /dephosphorylation. Thus, to identify novel molecular components of PIN targeting, and especially of the mechanisms that regulate the establishment and maintenance of PIN polarity we performed a forward genetic screen for the identification of PIN polarity regulators in Arabidopsis. We used PIN2::PIN1-HA in the pin2 mutant background, a transgenic line that has PIN1 mis-localized predominantly at the basal side of the epidermal cells and as a result of the defective PIN1 localization 95 it shows agravitropic growth. We screened for mutants that restored the gravitropic growth to PIN2::PIN1-HA;pin2, considering that the rescue will be possible only if PIN1 will (re)localize in PIN2-like manner and functionally replace PIN2 in its basipetal auxin transport activity. In the research article entitled {\textquotedblleft}PIN polarity maintenance by the cell wall in Arabidopsis{\textquotedblright} (Chapter 2) we show the identification of a cell wall component (CESA3) as a regulator of PIN polarity and present insights into the cell wall-dependent maintenance of PIN polarity in plant cells. Since plant cells lack tight junctions that define polar domains in animal cells, and the mechanism that keeps the PINs at the correct plasma membrane domains is largely unknown, the role of cell wall in polarity maintenance opens new directions of research regarding the polarity of PIN proteins. In another complementary project, we aimed to investigate the subcellular trafficking of PIN1 protein and to identify novel regulators at different endosomal compartments along the endocytic pathway. By performing a PIN1-GFP fluorescence imaging-based forward genetic screen we looked for abnormal intracellular accumulation of PIN1-GFP in the root cells and identified pat (protein affected trafficking) mutants. The research article entitled {\textquotedblleft}The AP-3 \ensuremath{\beta} adaptin mediates the biogenesis and function of lytic vacuoles in Arabidopsis{\textquotedblright} (Chapter 3) presents the identification and characterization of pat2, a mutant defective in the putative \ensuremath{\beta} subunit of the AP-3 complex. pat2 shows abnormal protein accumulation in mis-shaped lytic vacuoles, aberrant vacuolation of prevacuolar complexes (PVCs), abnormal morphology of protein storage vacuoles (PSVs) and impaired transition from storage to lytic vacuoles. It also shows arrested growth on medium lacking sucrose, indicating defects in trafficking from PVC to lytic and/or storage vacuoles. Hence, we identified PAT2/AP-3 \ensuremath{\beta} as a novel regulator of lytic vacuole biogenesis and function in Arabidopsis. PIN1 constitutive cycling between plasma membrane and endosomes is crucial for polar auxin transport. PIN1 is internalized from the plasma membrane by clathrin-dependent endocytosis and recycled back to the plasma membrane by the fungal brefeldin A (BFA)-sensitive ARF GEF GNOMdependent exocytosis. In order to identify new components of PIN1 constitutive cycling machinery we performed a PIN1-GFP fluorescence imaging-based forward genetic screen and looked for mutants that showed abnormal BFA-induced PIN1-GFP intracellular accumulation after long treatment. In the manuscript entitled {\textquotedblleft}Genetic dissection of PIN constitutive cycling in Arabidopsis identifies BEX1/RabA1b regulator of protein recycling{\textquotedblright} (Chapter 4) we show the identification and partial characterization of bex1 (BFA-visualized exocytic trafficking defective 1) mutant. bex1-1 shows intracellular ectopic accumulation of constitutively endocytosed plasma membrane proteins into abnormal BFA compartments, indicating defective exocytosis and transcytosis. BEX1 encodes for the 96 small GTP binding protein, RabA1b that localizes at domains of TGN and seems to play role in trafficking of proteins to the plasma membrane. All together, this PhD research brings novel insights into the mechanisms of PIN polarity maintenance and vesicle trafficking. We identified and characterized several mutants that will definitely contribute to the elucidation of PIN polarity and trafficking pathways in Arabidopsis. Although we wished for the identification of specific regulators of PIN trafficking and this was not the case, we found so far unknown regulators of vacuole biogenesis and function, and protein recycling. Furthermore, finding the cell wall as an essential component required for the maintenance of the PIN polarity opens indeed novel directions in studying cell polarity in plants.},
  author       = {Feraru, Elena},
  language     = {eng},
  pages        = {103},
  publisher    = {Ghent University. Faculty of Sciences},
  school       = {Ghent University},
  title        = {Genetic dissection of PIN polarity and endocytic cycling in Arabidopsis},
  year         = {2011},
}

Chicago
Feraru, Elena. 2011. “Genetic Dissection of PIN Polarity and Endocytic Cycling in Arabidopsis”. Ghent, Belgium: Ghent University. Faculty of Sciences.
APA
Feraru, E. (2011). Genetic dissection of PIN polarity and endocytic cycling in Arabidopsis. Ghent University. Faculty of Sciences, Ghent, Belgium.
Vancouver
1.
Feraru E. Genetic dissection of PIN polarity and endocytic cycling in Arabidopsis. [Ghent, Belgium]: Ghent University. Faculty of Sciences; 2011.
MLA
Feraru, Elena. “Genetic Dissection of PIN Polarity and Endocytic Cycling in Arabidopsis.” 2011 : n. pag. Print.