The Role of Proteases in Plant Development Maribel García-Lorenzo Department of Chemistry, Umeå University Umeå 2007 i Department of Chemistry Umeå University SE - 901 87 Umeå, Sweden Copyright © 2007 by Maribel García-Lorenzo ISBN: 978-91-7264-422-9 Printed in Sweden by VMC-KBC Umeå University, Umeå 2007 ii Organization Document name UMEÅ UNIVERSITY DOCTORAL DISSERTATION Department of Chemistry SE - 901 87 Umeå, Sweden Date of issue October 2007 Author Maribel García-Lorenzo Title The Role of Proteases in Plant Development. Abstract Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution. Systematic comparative analysis of the available sequenced genomes of two model organisms led to the identification of an increasing number of protease genes, giving insights about protein sequences that are conserved in the different species, and thus are likely to have common functions in them and the acquisition of new genes, elucidate issues concerning non-functionalization, neofunctionalization and subfunctionalization. The involvement of proteases in senescence and PCD was investigated. While PCD in woody tissues shows the importance of vacuole proteases in the process, the senescence in leaves demonstrate to be a slower and more ordered mechanism starting in the chloroplast where the proteases there localized become important. The light-harvesting complex of Photosystem II is very susceptible to protease attack during leaf senescence. We were able to show that a metallo-protease belonging to the FtsH family is involved on the process in vitro. Arabidopsis knockout mutants confirmed the function of FtsH6 in vivo. Key words: Comparative genomics, protease, PCD, leaf senescence, FtsH, Arabidopsis, Populus Language: English ISBN: 978-91-7264-422-9 Number of pages: 45 + 5 papers iii En este altar antiguo que levanté a lo alto de mis horas quiero subir, como polen nuevo me quiero esparcir, en un total abandono. Candiles de aceite habrá que encender, Sin llaves y a las puertas del instante estoy. “Sin llaves” Manolo García & Quimi Portet On this ancient altar that I have built at the summit of my days I yearn to climb, wanting to fling myself like new pollen, scattered in total abandon. Oil lamps shall be lit, Without keys, at the doors of instant I stand. “Without keys” Manolo García & Quimi Portet iv Table of Contents 1. List of Papers.................................................................................2 2. Abbreviations.................................................................................3 3. Introduction...................................................................................4 4. Model organisms and comparative studies ..................................6 4.1 Cyanobacteria, model organisms for chloroplast studies.................. 7 4.2 Comparative Genomics, a method to understand evolution............ 8 4.3 Genome duplication, the force that drives evolution ........................ 8 4.4 Phylogenetic trees: picturing evolution..............................................10 5. Plant Proteases .............................................................................11 5.1 Cytosolic proteases................................................................................11 5.1.1 Proteasome (ThrPro) ....................................................................11 5.1.2 Metacaspases (C14) .......................................................................13 5.2 Endoplasmic reticulum (ER) and ER-derived compartments .......14 5.3 Vacuolar proteases ................................................................................15 5.4 Mitochondria..........................................................................................17 5.4.1 ATP-independent proteases.........................................................18 5.4.2 ATP-dependent proteases............................................................20 5.5 Chloroplast.............................................................................................22 5.5.1 ATP-independent proteases.........................................................22 5.5.2 ATP-dependent proteases............................................................23 6. Proteases involved in senescence and PCD............................... 26 6.1 Proteases during plant development (Paper IV)...............................26 6.2 Proteases involved in wood formation (Paper I)..............................29 6.3 FtsH proteases involved in the degradation of the photosynthetic light-harvesting antenna (Papers II and III) ............................................30 7. Conclusions and future perspectives .......................................... 32 8. Acknowledgements ..................................................................... 34 9. Reference list ............................................................................... 36 1 . List of Papers The thesis is based on the following publications listed below and will be referred to in the text by their corresponding Roman numerals I. C. Moreau, N. Aksenov, M García-Lorenzo, B. Segerman, C. Funk, P. Nilsson, S. Jansson, H. Tuominen A genomic approach to investigate developmental cell death in woody tissues of Populus trees. Genome Biol. 2005; 6(4):R34 II. A. Zelisko, M. García-Lorenzo, G. Jackowski, S. Jansson, C. Funk AtFtsH6 is involved in the degradation of the light-harvesting complex II during high-light acclimation and senescence. Proc Natl Acad Sci U S A. 2005; 102(38):13699-704 III. M. García-Lorenzo, A. Zelisko, G. Jackowski, C. Funk Degradation of the main Photosystem II light-harvesting complex. Photochem Photobiol Sci. 2005; 4(12):1065-71 IV. M. García-Lorenzo, A. Sjödin, S. Jansson, C. Funk Protease gene families in Populus and Arabidopsis. BMC Plant Biol. 2006; 20,6:30 V. M. García-Lorenzo, A. Pruzinska, C. Funk ATP-dependent proteases in the chloroplast. Eva Kutejova (ed.) ATP-dependent proteases, Research Signpost, Kerala, India, accepted for publication 2 2. Abbreviations AAA ATPase associated with various activites AspPro Aspartic protease CP Proteasome core particle CtpA C-terminal processing peptidase CysPro Cystein protease EGY Ethylene-dependent gravitrospism-deficient and yellow green ER Endoplasmic reticulum EST Expressed sequence tag HL High light HR Hypersensitive response Htr High temperature requirement I-CLiP Intra-membrane cleaving proteases IM Inner membrane IMP Inner membrane peptidase LHC Light Harvesting Complex LV Lytic vacuoles MetalloPro Metalloprotease MIP Mitochondrial intermediate protease MPP Mitochondrial processing peptidase OM Outer membrane Oma Overlapping with m-AAA protease PCD Programmed cell death PreP Presequence protease PSII Photosystem II PSV Protein storage vacuoles RIP Regulated intramembrane proteolysis ROS Reactive oxygen species RP Proteasome regulatory particle SAG Senescence associated gene SerPro Serine protease SPP Stromal processing peptidase TE Tracheary element ThrPro Threonine protease TIM Translocase of the inner membrane TOM Translocase of the outer membrane TPP Thylakoid processing peptidase Ub Ubiquitin VPE Vacuolar processing enzyme 3 3. Introduction Proteolysis is the mechanism by which the degradation of a protein occurs. This process is ruled by hydrolytic enzymes called proteases. Proteases are found in every cellular compartment. Their working mechanism varies very much among all families and groups of proteases. Some of them work on their own, some of them in cooperation with other proteases (e.g. cascades) and some of them form complexes and constitute an active proteolytic machine. The cleavage of peptide bonds is not limited to the degradation of mature proteins to free amino acids, it also is relevant for modification and maturation of proteins. Therefore the proteolysis performed by proteases can be divided in two categories (Figure 1): 1. limited proteolysis: in which just some amino acids or a minor part of the protein are detached 2. unlimited proteolysis: in which a protein is totally degraded. Limited proteolysis usually modifies proteins post-translationally at highly specific sites. This process is ruled by the so called processing peptidases and the purpose of the limited proteolysis is the activation and maturation of proteins or removal of signal or transit peptides (e.g. under subcellular targeting). Unlimited proteolysis results in the total degradation of damaged, misfolded and potentially harmful proteins, providing free amino acids that will be recycled for the synthesis of new proteins. The proteases performing this kind of proteolysis are considered housekeeping proteases; they “clean” the compartment of malfunctional proteins, maintain stoichiometric amounts of protein subunits or control metabolic pathways. Limited and unlimited proteolysis usually is performed by different proteases using different regulatory mechanisms [1]. Proteases are involved in key aspects of plant growth, development and defense, as well as in senescence and plant cell death, the ultimate fate of cells, organs or the whole organism. Changes in light quality and intensity, temperature, nutrient supply, drought stress and pathogen or herbivore attack generally lead to a reorganization of cell morphology, metabolism and even membrane structure.