. i z a .11.. 14.2: 5.7131“... (a l ll:ll.l .- 43 .ciifiCu. .n :5... . z s .23 ...... :3. 1.91.2. 3.513.: ..ux9..o...iil!::\.. . .. 31.3.}. I: 1.. . i... 1. ..: 3.54... ,. 2 niézkr‘b, , 1" 1: . .Q a, 531.2. 73.151? :1 3.34;}: “91.... .2 3.23213 24.....- 2 ’/ LIE-"T “ «Y 7 m 0 Michigan State University This is to certify that the dissertation entitled ANALYSIS OF THE SUBCHLOROPLASTIC DISTRIBUTION OF GENOMES UNCOUPLED 4 AND MAGNESIUM CHELATASE presented by NEIL D. ADHIKARI has been accepted towards fulfillment of the requirements for the PhD. degree in Genetics wit/Z» Major Wssor’s‘Signature 6/30/2010 Date MSU is an Affinnative Action/Equal Opportunity Employer PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:lProjIAoc&Pres/ClRC/DateDue.indd AX.- ANALYSIS OF THE SUBCHLOROPLASTIC DISTRIBUTION OF GENOMES UNCOUPLED 4 AND MAGNESIUM CHELATASE By Neil D. Adhikari A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics 2010 'I AM I A ‘ C hloropl and man; contribut light reg ofthcir p Species (1 0X5gen.i referred 1 binding ll PTOdUCI. 1 magnesit Chloroph: Channel I On Chlore “'3 Predit Chloropla panlcipal magnesiu pomhil’ir ABSTRACT ANALYSIS OF THE SUBCHLOROPLASTIC DISTRIBUTION OF GENOMES UNCOUPLED 4 AND MAGNESIUM CHELATASE By Neil D. Adhikari Chlorophyll is the primary light harvesting pigment for photosynthesis in higher plants and many other organisms that perform oxygenic photosynthesis. Twenty three enzymes contribute to chlorophyll metabolism at different stages of plant growth and development. Tight regulation of chlorophyll biosynthesis is important because chlorophylls and some of their precursors are strong photosensitizers that can produce toxic reactive oxygen species (ROS) if porphyrins that are exposed to bright light collide with molecular oxygen. The GENOMES UNCOUPLED 4 protein from Arabidopsis thaliana (hereafter referred to as GUN4) stimulates magnesium chelatase by a mechanism that involves binding the Cth subunit of magnesium chelatase and its porphyrin substrate and product, the photosensitizing chlorophyll intermediates protoporphyrin IX and magnesium protoporphyrin IX, respectively. We hypothesized that GUN4 stimulates chlorophyll biosynthesis not only by activating magnesium chelatase but also by helping channel protoporphyrin IX into complexes of enzymes that drive chlorophyll biosynthesis on chloroplast membranes—the site of chlorophyll biosynthesis. From this hypothesis, we predicted that the porphyrin-bound form of GUN4 would more stably associate with chloroplast membranes by interacting with chloroplast membrane lipids or enzymes that participate in chlorophyll biosynthesis. Also, by binding protoporphyrin IX and magnesium protoporphyrin IX, GUN4 was previously hypothesized to shield these porphyrins from collisions with molecular oxygen thereby contributing to photooxidative stress ‘ conser Show stimula acid sul GENJ: that 3110 protopor \‘CISIOI‘IS prcx‘iousl in Vito, I chloropla; Addition: inhibit 1h: [Tansl‘om] GI..\~I ex] 3&9th 0n . blOSymhe: Mg‘chelar Stress [Ole] SignIfiCam stress tolerance. To test these hypotheses, I used site-directed mutagenesis to change conserved amino acid residues of GUN4. These amino acid substitutions were previously shown to cause deficiencies in the porphyrin-binding activity and the Mg-chelatase- stimulatory activity of a Synechocystis relative of GUN4. I found that some of the amino acid substitutions that cause porphyrin-binding defects in the Synechocystis relative of GUN4 also cause porphyrin-binding defects in GUN4. I also developed a binding assay that allowed me to show for the first time that GUN4 binds its natural ligands—— protoporphyrin IX and Mg-protoporphyrin IX. I used these porphyrin-binding deficient versions of GUN4 to test whether the porphyrin-binding activity of GUN4 that was previously demonstrated for cyanobacterial relatives of GUN4 in vitro is also significant in vivo. I found that porphyrins promote the association of GUN4 and Cth with chloroplast membranes and induce Mg-chelatase activity on chloroplast membranes. Additionally, I found that defects in porphyrin binding and defects in Cth function inhibit the association of GUN4 with chloroplast membranes. Finally, I found that stably transformed Arabidopsis plants that express porphyrin-binding-deficient versions of GUN4 exhibit higher expression levels of ROS-inducible genes compared to wild type. Based on these results, I conclude that GUN4 helps channel porphyrins into chlorophyll biosynthesis by binding porphyrins and Cth on chloroplast membranes and stimulating Mg—chelatase activity. I further conclude that these activities contribute to photooxidative stress tolerance. These findings indicate that the porphyrin-binding activity of GUN4 significantly contributes to chlorophyll biosynthesis and photooxidative stress tolerance in vivo. Ber-one. would c‘ project highh 1 Erp. Hi gradual SIIUHQ j ACKNOWLEDGEMENTS I would like to thank all my committee members, Drs. Gregg Howe, J ianping Hu. Beronda Montgomery, John Ohlrogge and Robert Larkin for their support and guidance. I would especially like to thank Dr. Larkin for letting me work on this very interesting project and providing excellent guidance. I would like to thank Dr. John F roehlich and Robert Orler for an excellent and highly productive collaboration during this entire project. I would like to thank all my friends from Michigan State, especially Harrie van Erp, Hiroshi Maeda, Mike Ruckle and Hong Luo for their friendship and making graduate school life a fun and enjoyable experience. Lastly, I would like to thank Kaori Ando and my family for their continued strong support along this journey, which made all the difference. iv [ET IEI LET CHAI CHAP lhch CHAP IIIIErac TABLE OF CONTENTS LIST OF TABLES .............................................................................................................. vi LIST OF FIGURES ........................................................................................................... vii LIST OF ABBREVIATIONS ............................................................................................. x CHAPTER 1: Introduction Tetrapyrrole biosynthesis in higher plants ............................................................... 1 . Regulation of tetrapyrrole biosynthesis ................................................................. 18 Impact of chlorophyll metabolism on other cellular processes ............................. 27 GENOMES UNCOUPLED 4 ................................................................................. 30 CHAPTER 2: Porphyrins promote the association of GENOMES UNCOUPLED 4 and a Mg-chelatase subunit with chloroplast membranes. Abstract .................................................................................................................. 39 Introduction ............................................................................................................ 40 Materials and methods ........................................................................................... 42 Results .................................................................................................................... 52 Discussion .............................................................................................................. 94 CHAPTER 3: The porphyrin-binding activity of GUN4 promotes GUN4-Cth interactions and photooxidative stress tolerance in Arabidopsis thaliana. Abstract ................................................................................................................ 106 Introduction .......................................................................................................... 1 07 Materials and methods ......................................................................................... 111 Results .................................................................................................................. 115 Discussion ............................................................................................................ l 39 CHAPTER 4: Conclusions and future perspectives ........................................................ 150 REFERENCES ................................................................................................................ 156 Table Z-f Table 2-1 substituti Table 2-3 Table 2-4 indicated Table 2-5 chloropla: Table 3-1 Table 3-2 computed Table 3.3. following LIST OF TABLES Table 2-1. Oligonucleotides used for site-directed mutagenesis ....................................... 44 Table 2-2. Solubilities of GST-GUN4A1-69 containing the indicated amino acid substitutions when expressed in E. coli ............................................................................. 68 Table 2-3. Quantitation of porphyrin binding by GUN4 .................................................. 70 Table 2-4. Quantitation of DPIX and Mg-DPIX binding by GUN4 containing the indicated amino acid substitutions ..................................................................................... 76 Table 2-5. Quantitation of Mg-chelatase activity in supematants prepared from lysed chloroplasts ........................................................................................................................ 92 Table 3-1. SSLP and CAPS markers used for mapping gun5-101 ................................. 1 13 Table 3—2. Percent decrease in the membrane association of Cth in untreated samples compated to WT ............................................................................................................... 130 Table 3-3. Percent increase in GUN4 protein in the membrane-containing pellet fraction following PPIX feeding ................................................................................................... 134 vi LIST OF FIGURES Figure 1-1. Tetrapyrrole biosynthesis pathway in higher plants ......................................... 3 Figure 2-1. Distribution of GUN4, Tic40, and SS in fractionated chloroplasts ............... 54 Figure 2-2. Subchloroplastic distribution of PPIX, Mg-PPIX, and GUN4 after ALA feeding ................................................................................................................................ 56 Figure 2-3. Subchloroplastic distribution of Tic40, SS, and total protein after ALA feeding ............................................................................................................................... 58 Figure 2-4. Analysis of GUN4—chloroplast membrane interactions in chloroplasts that were fed or not fed ALA .................................................................................................... 60 Figure 2-5. Subchloroplastic distribution of GUN4 following post-import ALA feeding ................................................................................................................................ 62 Figure 2-6. Distribution of GUN4 in the chloroplast envelope, thylakoid, and stroma fractions after ALA feeding ............................................................................................... 64 Figure 2-7. Subchloroplastic distribution of pea GUN4 and porphyrin levels after ALA feeding ................................................................................................................................ 65 Figure 2-8. Quantitative analysis of GUN4-binding DPIX and Mg-DPIX ...................... 71 Figure 2-9. Quantitative analysis of GUN4 binding PPIX, Mg-PPIX, and Mg-PPIX ME ...................................................................................................................................... 73 Figure 2-10. Quantitative analysis of GUN4 binding uroporphyrin III, coproporphyrin III, hemin, and pheophorbide a .......................................................................................... 74 Figure 2-11. Quantitative analysis of V123A, F191A, and R211A binding DPIX and Mg—DPIX ........................................................................................................................... 77 Figure 2-12. Subchloroplastic distribution of porphyrin-binding-deficient GUN4 afier ALA feeding ...................................................................................................................... 80 Figure 2-13. Mg—chelatase activity associated with chloroplast membranes after ALA feeding ................................................................................................................................ 81 Figure 2-14. Characterization of affinity-purified anti-Cth A1-823 antibodies ............. 83 Figure 2-15. Subchloroplastic distribution of pea Cth after ALA feeding ................... 85 vii Figure 2-16. Characterization of affinity-purified anti-ChlI A1-60 antibodies. A, Wild type and cs mutants ............................................................................................................ 86 Figure 2-17. Subchloroplastic distribution of ChlI and Cth after ALA feeding. A, Subchloroplastic distribution of ChlI after ALA feeding .................................................. 88 Figure 2-18. Characterization of affinity-purified anti-Cth A1-516 antibodies. A, Wild- type and cth mutants ....................................................................................................... 89 Figure 2-19. Solubility of pea GUN4 and pea Cth in chloroplast-membrane-depleted Mg-chelatase assays ........................................................................................................... 93 Figure 2-20. Subchloroplastic distribution of GUN4 after feeding with ALA or various porphyrins .......................................................................................................................... 95 Figure 2-21. Analysis of leaf senescence in gun4-1 ....................................................... 102 Figure 3-1. Analysis of stably transformed Arabidopsis plants containing GUN4-related transgenes ......................................................................................................................... 117 Figure 3-2. Analysis of chlorophyll levels in gun4 and cth/gun5 mutants grown under different fluence rates ...................................................................................................... 119 Figure 3-3. Images of seedlings grown in 100 umol m"2 3" white for 7d ....................... 120 Figure 3-4. Images of seedlings grown in l00 umol m'2 3" white light for 4d and then shifted to 850 umol m'2 5'1 white light for 3d .................................................................. 121 Figure 3-5. Positional cloning and sequence analysis of gun5 -1 01 ................................ 123 Figure 3-6. Analysis of GUN4 and Mg-chelatase subunit levels in 100 umol m'2 s'1 and 850 umol m'2 s" white light ............................................................................................. 124 Figure 3-7. Distribution of GUN4 in lysed and fractionated chloroplasts that were purified from gun4 and cth/gun5 mutants and were either fed or not fed with PPIX... 126 Figure 3-8. Statistical analysis of GUN4 in membrane-containing pellet fractions derived from purified and fractionated chloroplasts ..................................................................... 127 Figure 3-9. Distribution of GUN4 in soluble and membrane-containing pellet fractions derived from wild type, gun5, and cs chloroplasts .......................................................... 129 Figure 3-10. Distribution of Cth/GUN5 in lysed and fractionated chloroplasts that were purified from gun4 and chlI-l/gunS mutants and were either fed or not fed with PPIX...133 viii TE"; Figure 3-ll. umol m" s" 1 Figure 3-12. shift .............. Figure 3-13. light ............... Figure 3-11. Analysis of chlorophyll levels in gun4 and chlI-I/gunS mutants grown in 10 umol m'2 3’1 white light .................................................................................................... 136 Figure 3-12. Induction of WRKY40 and ZATlO expression during a fluence-rate shift .................................................................................................................................. 138 Figure 3-13. Analysis of WRKY40 and ZATI 0 expression in diurnal and continuous light .................................................................................................................................. 140 ix GUN4 ROS ALA PPIX big-PPIX Mg-PPIX-T POR ace PAO CAO 1(b H303 OH' 0;- Lheb GUN4 ROS ALA PPIX Mg-PPIX Mg-PPIX-ME POR RCC PAO CAO I ()2 H202 OH' 02.. Lhcb LIST OF ABBREVIATIONS Genomes Uncoupled 4 Reactive Oxygen Species 5-aminolevulinic acid Protoporphyrin IX Magnesium protoporphyrin IX Magnesium protoporphyrin IX monomethyl ester Protochlorophylide oxidoreductase Red chlorophyll catabolite Pheophorbide a oxygenase Chlorophyllide a oxygenase Singlet oxygen Hydrogen peroxide Hydroxyl radical Superoxide Light harvesting chlorophyll a/b binding protein joined r compou detelopr biosyntli chlomph bacteria. Which are Contribute NADPH c COfaCIOr it Of anrgan IOr Pbmos) OfPhOIorec Simhesizinl Simullaneot Si'ntheSiZe f. Chlorophillg All le field (ALA). f CHAPTER 1 INTRODUCTION TETRAPYRROLE BIOSYNTHESIS IN HIGHER PLANTS Chlorophylls are tetrapyrroles; tetrapyrroles are compounds consisting of four joined pyrrole rings which can either be linear or cyclic. Tetrapyrroles are essential compounds for all organisms and perform many functions during growth and development. They form a large and diverse family of molecules that are biosynthetically related. Examples of tetrapyrroles found in nature include heme, chlorophyll, bilins, phycobilins, siroheme, vitamin B12, and factor F 430 of methanogenic bacteria. These molecules serve as the prosthetic groups in many proteins, most of which are essential. For example, heme is the prosthetic group in proteins that contribute to respiration (cytochrome), oxygen metabolism (catalase, peroxidase and NADPH oxidase) and oxygen binding (leghemoglobin). Siroheme is an important cofactor in enzymes such as nitrite and sulfite reductases, which function in assimilation of inorganic nitrogen and sulphur. Chlorophylls are the major light harvesting pigments for photosynthesis. Phytochromobilin is the chromophore for phytochromes—a family of photoreceptors that perceive red and far-red light. No organism is capable of synthesizing all tetrapyrroles, but many synthesize two or more major products either simultaneously or at different stages in development (Beale, 1999). Plants can synthesize four classes of tetrapyrroles: siroheme, hemes, phytochromobilin, and chlorophylls. All tetrapyrroles are synthesized from a common precursor, 5-aminolevulinic acid (ALA). ALA can be synthesized by two different pathways. ALA synthase catalyzes eukaryote (Gibson et alpha-pron utilizes glu Castelfrane glutamate. g IRVA (Kanr by glutamjl semialdeb} d; glutamate l-s I978: Hoobei Three enz; m0l€eule of u: 1999). rim. t moliCUléS of}. porpllObllangL catalyzes ALA biosynthesis in organisms belonging to the alpha-proteobacteria and all eukaryotes that lack chloroplasts by condensing glycine and succinyl-coenzyme A (Gibson et al., 1958; Kikuchi et al., 1958). Plants, algae and all bacteria besides the alpha-proteobacterial group, utilize an entirely distinct ALA biosynthetic pathway that utilizes glutamate rather than glycine and succinyl-coenzyme A (Beale and Castelfranco, 1974; Beale et al., 1975; Meller et al., 1975). To synthesize ALA from glutamate, glutamyl tRN A synthetase activates glutamate by ligating it to glutamyl tRNA (Kannangara et al., 1984). The glutamyl tRN A is committed to ALA biosynthesis by glutamyl tRNA reductase, which converts the glutamyl tRNA to glutamate l- semialdehyde (GSA) (Pontoppidan and Kannangara, 1994). GSA is transaminated by glutamate l-semialdehyde aminotransferase yielding ALA (Kannangara and Gough, 1978; Hoober et al., 1988) (Figure 1-1). Stgs conserved in biosynthesis of all tetrapyrroles (the ‘common pathway’) Three enzymes perform the sequential conversion of eight ALA molecules into one molecule of uroporphyrinogen III—the first closed macrocyclic tetrapyrrole (Beale, 1999). First, ALA dehydratase performs an asymmetric condensation that converts two molecules of ALA into porphobilinogen (Jordan and Seehra, 1980). Next, porphobilinogen deaminase polymerizes four molecules of porphobilinogen yielding H000 HO- tRNA( New W Glutamate Glutamyl tRNA2 H Glutamate 1- N (35emialdehyde (4) COOH HOOC Porphobilinogen 5 5— Aminolevuliciiic acid () COOH COOH HOOC \ / \ NH HN / COOH HO / NH HN \ HOOC / \ COOH Hydroxymethylbilane HOOC (fill COOH Figure 1-1. Tetrapyrrole biosynthesis pathway in higher plants. Numbers indicate enzymes that catalyze each reaction as follows: (1) Glutamyl t-RNA synthetase, (2) Glutamyl t-RNA reductase, (3) Glutamate l-semialdehyde aminotransferase, (4) ALA dehydratase, (5) Porphobilinogen deaminase, (6) Uroporphorinogen III synthase, (7a) Uroporphorinogen III methylase, (7b) Oxidase, (7c) Siroheme ferrochelatase, (7d) Uroporphorinogen III decarboxylase, (8) Coproporphyrinogen III decarboxylase, (9) Protoporphyrinogen IX oxidase, (10a) Mg- chelatase, (10b) F errochelatase, (11) Mg-protoporphyrin IX methyltransferase, (12) Mg-proroporphyrin IX monomethylester oxidative cyclase, (13) Protochlorophyllide oxidoreductase, (l4) Divinyl Chlorophyllide reductase, (15a) Chlorophyll synthase, (15b) Chlorophyllase, (16) Hydroxymethyl Chlorophyllide a, (17) Chlorophyll b reductase, (l8) 7-Hydroxymethyl chlorophyll a reductase, (19) Mg-dechelating substance, (20) Pheophytinase, (21) Pheophorbide a oxygenase, (22) Red chlorophyll catabolite reductase. Hal n. E COOH COOH HOOC COOH (7a) HOOC COOH COOH COOH Uroporphyrinogen Ill l (N) COOH CH3 ego COOH H3C CH3 COOH COOH Coproporphyrinogen III Dihydrosirohydrochlorin 1 (7b) Sirohydrochlorin (7c) Figure 1-1 Tetrapyrrole biosynthesis pathway in higher plants (continued). ihC (7C) H3O COOH COOH COOH tr Protoporphyrinogen IX Q) COOH Siroheme COOH COOH Figure 1-1 Tetrapyrrole biosynthesis pathway in higher plants (continued). CH2 H3C H3C COOH COOH Mg-protoporphyrin IX COOH COOH Protoporphyrin IX laws) (11) 0-12 CH3 H3C CH2 H3C CH3 COOH COOH Fe-protoporphyrin IX (heme) Figure 1-1 Tetrapyrrole biosynthesis pathway in higher plants (continued). COOH COOH COOCH3 Mg-protoporphyrin lX monomethyl ester Divinyl Chlorophyllide a Figure 1-1 Tetrapyrrole biosynthesis pathway in higher plants (continued). Thu 7m (15a) Chlorophyllide b ———> nail Hydroxymethyl chlorophvllide a O\coocongg Chlorophyll b it”) Hydroxymethyl chlorophyll a Monovinyl Chlorophyllide a /CH2 H3C““"" CH3 O\COOCZOH39 Chlorophyll a Figure 1-1 Tetrapyrrole biosynthesis pathway in higher plants (continued). H3C H3C\\\“"' HOOC o H3C 0 \ \cooconsg \ NH N\ CH3 Pheophytin a N\ HN H3C\\\w" // CH3 H3COOC$ o HOOC Red Chlorophyll Catabolite l<22> Primary Fluorescent Chlorophyll Catabolite (pFCC) Nonfluorescent Chlorophyll Catabolite (NCC) Figure l-l Tetrapyrrole biosynthesis pathway in higher plants (continued). hydroxymethylbilane, the first linear tetrapyrrole. Free hydroxymethylbilane rapidly undergoes a spontaneous and irreversible cyclization that yields uroporphyrinogen I, a biologically non-relevant product (Battersby et al., 1979). Uroporphyrinogen III synthase, directs the conversion of hydroxymethylbilane to the correct isomer— uroporphyrinogen III—either during or immediately after the formation of hydromethylbilane (Hart and Battersby, 1985; Tsai et al., 1987). Uroporphyrinogen III is a branch-point substrate that can serve as a precursor for either Siroheme or protoporphyrin IX. For the biosynthesis of siroheme, uroporphyrinogen III methylase converts uroporhyrinogen III to dihydrosirohydrochlorin (Leustek et al., 1997), which is then converted to Sirohydrochlorin by an oxidase; the gene that encodes this oxidase has not yet been identified (Tanaka and Tanaka, 2007). Siroheme ferrochelatase then inserts Fe2+ into the porphyrin ring of Sirohydrochlorin yielding Siroheme (Raux-Deery et al., 2005). For the biosynthesis of protoporphyrin IX, uroporphyrinogen III decarboxylase catalyzes the stepwise decarboxylation of uroporphyrinogen Ill yielding coproporphyrinogen III (Luo and Lim, 1993). Coproporphyrinogen III oxidative decarboxylase catalyzes the oxidative decarboxylation of two of the four propionate residues on rings A and B of coproporphyrinogen III, yielding protoporphyrinogen IX (Cavaleiro et al., 1974), which is then converted to protoporphyrin IX by protoporphyrinogen IX oxidase (Klemm and Barton, 1987). Uroporphyrinogen III is hydrophilic, exhibits a low affinity for metals, and is photochemically unreactive. By contrast, protoporphyrin IX is hydrophobic, exhibits a much higher affinity for metals than uroporphryin III, and is photochemically reactive. 10 Tfibfl ’lfll /-. In fact, diphenyl ether herbicides cause lethal photooxidative stress by causing the buildup of protoporphyrin IX (Witkowski and Halling, 1988, 1989). Additionally, Arabidopsis mutants that exhibit misregulated porphyrin metabolism suffer from potentially lethal photooxidative damage (Kim et al., 2008). Thus, porphyrins are thought to be synthesized from porphyrinogens at the last possible moment to guard against photooxidative stress (Beale, 1999). The photosensitizing effects of porphyrins are not limited to plants. In humans, porphyric diseases cause severe blistering of the skin on exposure to light (Kauppinen, 2005; Puy et al., 2010). Chlorophle biosynthesis In plants, protoporphyrin IX is a branch-point substrate that can be utilized for the biosynthesis of either hemes or chlorophylls (Figure 1-1). F errochelatase inserts Fe2+ into protoporphyrin IX yielding heme; Mg-chelatase commits protoporphyrin IX to chlorophyll biosynthesis by inserting Mg2+ into protoporphyrin IX, yielding Mg- protoporphyrin IX. Mg-chelatase is an ATP-dependent enzyme that contains three subunits, Cth, ChlI and Cth (Willows, 2003). In contrast, ferrochelatase consists of a single subunit and does not require ATP (Loeb, 1995). The difference in ATP requirements between ferrochelatase and Mg-chelatase are thought to be related to the distinct energy requirements for inserting Mg2+ and F e2+ into protopoprhryin IX. Inserting Mg2+ into protoporphyrin IX requires more energy than inserting Fe2+ because removal of water molecules coordinated to Mg2+ is an energy-intensive process (Fleischer et al., 1964; Hambright, 1975). ll THEE 7m 1 Mg-protoporphyrin IX methyltransferase methylates the carboxyl group of the ring C propionate side chain of Mg-protoporphyrin IX, yielding the next intermediate, Mg- protoporphyrin IX monomethyl ester (Yee et al., 1989; Block et al., 2002). Next, the Magnesium protoporphyrin IX monomethyl ester oxidative cyclase catalyzes an oxygen-dependent reaction that yields divinyl protochlorophyllide by synthesizing a fifth ring to magnesium protoporphyrin IX monomethyl ester. This oxidative cyclase is a multisubunit enzyme that contains a non-heme iron (Bollivar and Beale, 1996). Protochlorophyllide oxidoreductase (POR) reduces ring D of divinyl protochlorophyllide, yielding divinyl Chlorophyllide. POR is a light-dependent enzyme in angiosperrns. In contrast, cyanobacteria and algae encode a light-independent POR that shares no sequence similarity with POR. Gymnospenns have both light-dependent as well as light-independent PORs (Masuda et al., 2003; Bollivar, 2006). The vinyl group on ring B of divinyl Chlorophyllide is reduced by divinyl Chlorophyllide reductase yielding monovinyl Chlorophyllide a (N agata et al., 2005; Nakanishi et al., 2005). In the final step of chlorophyll a biosynthesis, chlorophyll synthase esterifies monovinyl Chlorophyllide a with phytol-pyrophosphate, yielding chlorophyll a. The chlorophyll cycle A portion of chlorophyll a pool is converted to chlorophyll b by the action of Chlorophyllide a oxygenase. The phytyl group from chlorophyll a is assumed to be removed by Chlorophyllase, which then provides monovinyl Chlorophyllide a as a substrate for Chlorophyllide a oxygenase (Figure 1-1). Chlorophyll a oxygenase then 12 converts monovinyl chorophyllide a to Chlorophyllide b in a two-step reaction (Oster et al., 2000). Chlorophyllide b is then phytylated by chlorophyll synthase yielding chlorophyll b (Oster et al., 2000; Eggink et al., 2004). Thus, plants can increase the chlorophyll b pool without de novo synthesis of Chlorophyllide a—for example, in the dark. Chlorophyll b is reversibly inter-converted to chlorophyll a via the intermediate 7- hydroxymethyl chlorophyll a by the enzymes chlorophyll b reductase (Horie et al., 2009; Sato et al., 2009) and 7-hydroxymethyl chlorophyll a reductase, respectively (Scheumann et al., 1996; Nagane et al., 2010). Plants use the chlorophyll cycle to adjust the stoichiometry of chlorophyll a and b for optimal light harvesting in various light environments. In addition, conversion of chlorophyll b to a is an important first step in the chlorophyll degradation pathway (Tanaka and Tanaka, 2007). Biosvnthesis of hemes Protoporphyrin IX is converted to heme by ferrochelatase. There are several biologically important hemes that are distinguished by modifications of protoheme (also referred to as heme or heme b). Following heme b, heme a and heme c are the most commonly observed forms of heme (Severance and Harnza, 2009). Heme a is synthesized by the substitution of a vinyl side chain with a l7-carbon isoprenoid side chain and the oxidation of a methyl side chain to a formyl group. Heme 0 belongs to the C-type hemoproteins such as cytochrome c in which the two vinyl side chains of heme b are covalently attached to the protein (Severance and Hamza, 2009). Heme oxygenase catalyzes the oxidation and ring opening of heme, resulting in the formation of the linear tetrapyrrole biliverdin IXa. Phytochromobilin synthase then converts this product 13 to the 3(Z) isomer of phytochromobilin (Terry et al., 1995; Kohchi et al., 2001). An isomerase, the molecular identity of which is still unclear, is implicated in the conversion of 3(Z) phytochromobilin to 3(E)-phytochromobilin, which assembles with the apophytochrome in the cytoplasm to form phytochrome (Terry et al., 1993). Localization of tetrapyrrole biosynthesis In metazoans, ALA is synthesized in the mitochondria and is then transported to the cytosol. Coproporphyrinogen III is synthesized from ALA in the cytosol. Coproporphyrinogen III is transported into the mitochondria by an unknown mechanism, and converted to heme (Severance and Harnza, 2009). In plants, tetrapyrrole biosynthesis is thought to take place exclusively in the plastids, although there have been conflicting reports about the localization of the heme branch. Some studies indicate that heme biosynthesis occurs solely in the plastids (Masuda et al., 2003) while others report partial heme biosynthesis takes place in the mitochondria (Jacobs and Jacobs, 1987; Comah et al., 2002). Recent analyses of the mitochondrial proteome failed to detect either of the two isoforrns of ferrochelatase in Arabidopsis and rice (Heazlewood et al., 2004; Huang et al., 2009), thus favoring the model of exclusive chloroplast—localization of tetrapyrrole biosynthesis in higher plants. Phytochromobilin synthesis takes place entirely in the plastids as well (Kohchi et al., 2001), but the phytochrome apoproteins bind the phytochromobilin in the cytosol, yielding the functional photoreceptor (Terry et al., 2002). Within the plastids, biosynthesis of the hydrophilic precursors from glutamate to protoporphyrinogen IX takes place in the stroma. Biosynthesis of the hydrophobic l4 Trtbi 9m tetrapyrroles protoporphyrin IX (Watanabe et al., 2001), heme (Suzuki et al., 2002), Mg-protoporphyrin IX (Fuesler et al., 1984; Nakayama et al., 1998), Mg- protoporphyrin IX monomethyl ester (Block et al., 2002), protochlorophyllide (Tottey et al., 2003), Chlorophyllide (Barthelemy et al., 2000; Masuda et al., 2003) and chlorophyll (Eggink et al., 2004) is localized to the thylakoid and envelope membranes. Nonetheless, chlorophyll only accumulates in the thylakoid and not in the envelope membranes. The rationale for chlorophyll biosynthesis occurring in the both the thylakoid and envelope membranes is a matter of speculation. One hypothesis is that chlorophyll biosynthesis in the envelope facilitates the formation of pigment protein complexes as chlorophyll precursor- and chlorophyll-binding proteins are imported into the chloroplast (Reinbothe et al., 1995; Tottey et al., 2003; Kim et al., 2005). Another possibility is that the chlorophyll metabolism in the envelope could act as plastid- derived signals (Roderrnel and Park, 2003; Nott et al., 2006; Ankele et al., 2007; Koussevitzky et al., 2007). Mdation of chlorophyll The long standing model for chlorophyll degradation is that the first step in the degradation of chlorophyll is the conversion of chlorophyll b to chlorophyll a (Eckhardt et al., 2004; Hortensteiner, 2006; Tanaka and Tanaka, 2007). The next step is removal of the phytol chain from chlorophyll a by chlorophyllase to yield Chlorophyllide a (Jacob-Wilk et al., 1999; Tsuchiya et al., 1999; Hortensteiner, 2006), followed by the release of the Mg2+ atom that is catalyzed by a low molecular weight magnesium dechelating substance (MCS). The molecular identity of MCS remains unknown, and 15 THE 7 o the exact mechanism of demetallation remains unclear (Suzuki et al., 2002; Pruzinska et al., 2003; Suzuki et al., 2005; Suzuki et al., 2005). Demetallation yields pheophorbide a from Chlorophyllide a. The recent discovery of a novel, plastid-localized pheophytinase encoded by the PPH gene indicates that the pathway of chlorophyll degradation to pheophorbide a is complex (Schelbert et al., 2009). Chlorophyll a can lose its Mg2+ yielding pheophytin a. In vitro, pheophytinase specifically dephytylates pheophytin a yielding pheophorbide a. pph-I , a knockout mutant, exhibits a stay-green phenotype and accumulates pheophytin a when senescence is induced by prolonged incubations of leaves in the dark— indicating a block in chlorophyll degradation. Based on these findings, PPH has been proposed to be an important component of the chlorophyll degradation pathway. Based on the observation that chlorophyll degradation can proceed at similar rates in wild type Arabidopsis and an Arabidopsis double knockout mutant that lacks both isoforms of chlorophyllase, chlorophyllase is no longer considered to be essential for the degradation of chlorophyll (Schenk et al., 2007). In the most recent model for the degradation of chlorophyll, removal of the Mg2+ atom from chlorophyll a by MCS yielding pheophytin a is most likely the first step. Next, pheophytinase dephytylates pheophytin a yielding pheophorbide a. The fact that Chlorophyllide is most likely not an intermediate of chlorophyll breakdown suggests that chlorophyll synthesis and breakdown are metabolically separated during leaf senescence, contrary to what was previously believed (Schelbert et al., 2009). Pheophorbide a oxygenase (PAO) catalyzes another key step in chlorophyll degradation by opening the porphyrin macrocycle of pheophorbide a yielding the red 16 chlorophyll catabolite (RCC) (Hortensteiner, 1998; Pruzinska, 2003). RCC is subsequently reduced to primary fluorescent chlorophyll catabolite (pFCC) by red chlorophyll catabolite reductase (RCCR) (Rodoni et al., 1997; Hortensteiner et al., 2000). pFCCs then undergo further modifications in the vacuole, presumably by the acidic environment of the vacuole, to form nonfluorescent chlorophyll catabolites (NCC) (Krautler, 2008). Subcellular localization of chlorophyll degradation Chlorophyllase localization is unclear; some reports suggest its localization in the chloroplast inner envelope (Brandis et al., 1996; Matile et al., 1997; Tsuchiya et al., 1999) while others indicate an entirely non-plastidic localization (Hortensteiner, 2006; Schenk et al., 2007). Currently, the identity and localization of the magnesium dechelating substance is also unknown (Eckhardt et al., 2004; Hortensteiner, 2006). Pheophorbide a oxygenase (PAO) has been suggested to be localized in gerontoplast envelopes based on activity measured in purified barley gerontoplast envelope membranes (Matile and Schellenberg, 1996). The upregulation of PAO gene expression during the conversion of chloroplasts to gerontoplasts is an important mechanism that regulates the degradation of chlorophyll (Hortensteiner, 2006). Red chlorophyll catabolite reductase (RCCR) contains a predicted chloroplast transit peptide, can be imported into chloroplasts in vitro (Wuthrich et al., 2000), and is found in the stroma. In contrast to PAO, RCCR is constitutively expressed in chloroplasts as well as gerontoplasts (Rodoni et al., 1997; Matile et al., 1999; Yao and Greenberg, 2006). Localization of RCCR in the cell seems to depend on the developmental stage of the 17 plant. In young seedlings, it is localized in both plastids and mitochondria whereas in mature leaves it is present primarily in the chloroplasts (Mach et al., 2001). Upon induction of cell death by bacterial infection or protoporphyrin IX treatment, RCCR localization was observed to change from mostly localization in the chloroplast to localization in the chloroplast, mitochondria and cytosol (Yao and Greenberg, 2006). One explanation for this variation in subcellular localization of RCCR could be that during the induction of stress-inducing events such as bacterial infection or porphyrin accumulation, RCCR may bind and/or reduce porphyrins or porphyrin-related molecules in mitochondria and possibly chloroplasts, which could generate light- dependent reactive oxygen species. This could cause an alteration in organelle behavior and activate a cascade leading to programmed cell death (Yao and Greenberg, 2006). REGULATION OF TETRAPYRROLE BIOSYNTHESIS Developmental stage Chloroplast biogenesis and function plays a major role in the regulation of tetrapyrrole biosynthesis. During chloroplast biogenesis, there is a massive demand for chlorophyll and a massive induction of tetrapyrrole biosynthesis. At this stage, inducing the expression of genes that encode proteins contributing to tetrapyrrole metabolism is a major mechanism driving this robust biosynthesis of tetrapyrroles. After chloroplast biogenesis is completed, the level of tetrapyrroles must be finely regulated for optimal chloroplast function. Regulated gene expression continues to be an important mechanism that coarsely regulates tetrapyrrole biosynthesis in mature chloroplasts. As 18 described in more detail below, feedback regulation at the posttranslational level is critical for the fine regulation of tetrapyrrole biosynthesis (Masuda and Fujita, 2008). Light plays a major role in the development of photosynthetically active chloroplasts in angiosperms. When seedlings are grown in dark, chloroplast biogenesis is blocked. These dark grown seedlings contain etioplasts rather than chloroplasts. Etioplasts are prechloroplasts that form in the dark because many of the light-regulated genes that are required for proper chloroplast biogenesis are expressed at levels that are inadequate to support chloroplast biogenesis and because chlorophyll biosynthesis does not occur in the dark due to a lack of POR activity; POR is a light-dependent enzyme. In the dark, protochlorophyllide accumulates bound to POR in the prolamellar body of the etioplast. The accumulation of protochlorophyllide in dark indicates that all the enzymes participating in tetrapyrrole biosynthesis are synthesized and are active even in the dark in the early developmental stages of the seedling (Masuda and F ujita, 2008). Protochlorophyllide accumulates to a threshold level in the dark, and then protochlorophyllide biosynthesis is inhibited. This down regulation of protochlorophyllide biosynthesis is critical to the survival of plants; accumulation of excess amounts of protochlorophyllide that are not bound by POR can yield potentially lethal levels of reactive oxygen species when dark-grown seedlings are exposed to light (Meskauskiene et al., 2001). As described in more detail below, this buildup of protochlorophyllide inhibits the ALA synthesis by mechanisms that include feedback inhibition of glutamyl tRN A reductase and inhibition of HEMAI expression—the gene 19 that encodes glutamyl tRN A reductase. For further growth, the developing seedling has to synthesize increasing amounts of chlorophyll upon exposure to light. To do so, flux down the entire tetrapyrrole biosynthetic pathway has to be increased, especially so down the chlorophyll branch. At the same time, there has to be a tight coordination between chlorophylls and the chlorophyll binding proteins for assembly of the photosynthetic complexes. Transcmional regulation of chlorophyll synthesis during chloroplast biogenesis The expression of several genes that encode enzymes required for chlorophyll biosynthesis are induced to very high levels during chloroplast biogenesis. Glutamyl tRNA reductase is a major target for the transcriptional regulation of chlorophyll biosynthesis. Glutamyl tRNA reductase is encoded by two genes in Arabidopsis, HEMAI and HEMAZ. HEMAI is predominantly expressed in photosynthetic tissues, rapidly induced by light, and is thought to mostly contribute to chlorophyll biosynthesis. HEMAZ isoform is ubiquitously and constitutively expressed and is thought to predominantly drive tetrapyrrole biosynthesis in nonphotosynthetic tissues (Ilag et al., 1994; Bougri and Grimm, 1996; Tanaka et al., 1996; Goslings et al., 2004; Matsumoto et al., 2004). HEMAI, CHLH (encoding the porphyrin-binding and Mg2+ binding subunit of magnesium chelatase), CHL27/CRDI (encoding a subunit of magnesium protoporphyrin IX monomethylester cyclase), CAO (chlorophyll a oxygenase) belong to a cluster of genes that get rapidly upregulated by light within 1 hour of illumination during the onset of greening of etiolated seedlings and reaching a plateau after 3 hours, suggesting that these genes are key for regulating the biosynthesis and flux through the 20 chlorophyll branch (Matsumoto et al., 2004). GENOMES UNCOUPLED 4, a novel regulator of magnesium chelatase, also follows a similar expression pattern (Stephenson and Terry, 2008). Genes encoding the rest of the enzymes in the common tetrapyrrole pathway and most of the chlorophyll branch belong to a separate cluster based on expression pattern. They are slowly upregulated after seedlings are exposed to light. Their expression reaches a maximum, approximately 9 to 12 hours after de-etiolation begins (Matsumoto et al., 2004). PORA and PORB genes, which encode protochlorophyllide oxidoreductase, are regulated very differently by light under the above conditions compared to rest of the genes in the chlorophyll branch. The levels of the mRNAs that encode PORA and PORB accumulate in the dark to relatively high levels, but are reduced to much lower levels within 3 to 6 hours after dark-grown seedlings are exposed to light (Armstrong et al., 1995; Matsumoto et al., 2004). This may be a mechanism by which flow through the chlorophyll branch is regulated so as to prevent an abnormally high accumulation of Chlorophyllide or chlorophyll, which could be phototoxic to the seedlings. The expression pattern of HEMA I , CHLH, GUN4, CRDI and CAO matches that of Lhcb] , suggesting that these genes are important for coordinating chlorophyll biosynthesis to a functional photosynthetic apparatus during de-etiolation (Matsumoto et al., 2004; Stephenson and Terry, 2008). Feedback regulation of glutamyl tRNA reductase Negative feedback regulation of enzymes is another important mechanism that regulates chlorophyll biosynthesis. Glutamyl tRNA reductase is a major target of feedback regulation. Heme regulates ALA biosynthesis by feedback inhibition of 21 glutamyl tRNA reductase, the enzyme that converts glutamyl tRN A to ALA (Pontoppidan and Kannangara, 1994; Vothknecht et al., 1996). Additionally, the FLU protein inhibits glutamyl tRNA reductase if excess protochlorophyllide accumulates. FLU was first identified from an Arabidopsis conditional fluorescence (flu) mutant and has been localized to the chloroplast membranes. Protochlorophyllide accumulates to higher than wild type levels in flu when grown in dark, and flu seedlings are photobleached on exposure to light. Repression of glutamyl tRNA reductase by FLU has been shown to occur independently of the feedback inhibition by heme (Meskauskiene et al., 2001; Goslings et al., 2004). Thus, FLU prevents excess protochlorophyllide from accumulating in the dark. Upon illumination, protochlorophyllide levels decrease as it gets converted to chlorophyll, and so do levels of heme thereby relieving the feedback inhibition of glutamyl tRNA reductase and stimulating ALA synthesis. FLU has been shown to interact directly and specifically with glutamyl tRNA reductase encoded by HEMA], which is expressed in photosynthetically active tissues, using a yeast two hybrid system. qu3, a suppressor of the flu mutation was identified. This suppressor reduces ALA synthesis and protochlorophyllide accumulation, and is allelic to hyI , the gene encoding heme oxygenase. This data supports a model in which heme antagonizes the effect of the flu mutation by independently inhibiting glutamyl tRNA reductase (Goslings et al., 2004). Regulation of chlorophyll in mature chloroplasts In mature chloroplasts, synthesis of new chlorophyll is needed mainly to replace the chlorophyll that becomes degraded during photosynthesis, as compared to a massive 22 was; ’/ 701T. burst in chlorophyll biosynthesis that is required during greening. Maintenance of the photosynthetic apparatus is very important for sustaining the photoautotrophic grth of higher plants. Light and circadian clock are major regulators of chlorophyll biosynthesis and the accumulation of chlorophyll-binding proteins (Beator and Kloppstech, 1993). In addition, the demand for chlorophyll in the cell varies depending on the photosynthetic activity. Similar to regulation of chlorophyll biosynthesis during chloroplast biogenesis, regulating ALA biosynthesis is one major mechanism for regulating chlorophyll synthesis in mature chloroplasts. Both diurnal cycle and circadian clock were found to influence HEMA] expression (Kruse et al., 1997; Matsumoto et al., 2004) and activity (Kruse et al., 1997). Similar to HEMA] upregulation, CHLH, CRDI, CAO and Lhcb] genes, which were found to be rapidly upregulated during chloroplast biogenesis, also get rapidly upregulated in mature green leaves following dark to light shift. In contrast to their expression pattern during chloroplast biogenesis, PORA and PORB genes also get upregulated following a dark to light shift, although their expression levels peak slower than HEMA I, CHLH, CR0] and CA 0. In addition to regulation by the diurnal cycle, all of these genes are also regulated by the circadian clock (Matsumoto et al., 2004). The remaining genes encoding enzymes that contribute to the common part of the tetrapyrrole pathway and the chlorophyll branch are under a diurnal regulation but not the circadian clock (Matsumoto et al., 2004). These data suggest that regulating the expression of HEMA] , CHLH, CRDI and CAO are important for coordinating chlorophyll biosynthesis with assembly of a functional photosynthetic apparatus in mature green leaves, while PORA and PORB may fianction in maintaining chlorophyll biosynthesis (Matsumoto et al., 23 THE! 7-“! 2004). These data also indicate that light and circadian rhythm regulates the synthesis of chlorophyll and chlorophyll-binding proteins, as has been proposed before (Beator and Kloppstech, 1993). Other regulators of chlorophyll-biosynthesis-related gene expression. Phytochrome interacting factors: Phytochrome interacting factors (PIFs) belong to the basic helix-loop-helix (bHLH) family of transcription factors that are capable of activating or repressing gene expression (Castillon et al 2007). PIFl and PIF 3 play roles as critical modulators by which plants coordinate chlorophyll biosynthesis in response to light conditions and developmental stage (Huq et al., 2004). PIFl is a negative regulator of chlorophyll biosynthesis in the dark because pif] mutant seedlings accumulate protochlorophyllide when grown in the dark and are photobleached when transferred to the light (Huq et al., 2004). Negative regulation by PIFI is in turn at least partially attenuated by light, which would allow plants to perform higher rates of chlorophyll biosynthesis in light. In contrast to PIFl , PIF3 positively regulates chlorophyll biosynthesis in the initial hours of de-etiolation (Kim et al., 2003; Monte et al., 2004). However, recent reports indicate that pifI, pif3 and piprif3 double mutants accumulate protochlorophyllide when grown in the dark. These mutants also accumulate more chlorophyll than wild type in the initial 2 hours following de- etiolation , suggesting a model in which both PIFl and PIF3 act similarly and are negative regulators of chlorophyll biosynthesis in the dark (Stephenson et al., 2009). 24 GoldenZ-like: GoldenZ-Iike (GLK) genes belong to the GARP family of transcription factors (Riechmann et al., 2000) that were originally identified in maize and have been shown to promote chloroplast biogenesis in Arabidopsis, maize and Physcomitrella patens (Rossini et al., 2001; Fitter et al., 2002; Yasumura et al., 2005). Arabidopsis has two GLK genes, GLKI and GLKZ. glkI glk2 double mutants have reduced transcript and protein levels derived from genes participating in chlorophyll biosynthesis and light harvesting. These mutants are significantly pale green and have poorly developed chloroplasts (Fitter et al., 2002). Transcriptome analysis of transgenic plants containing transgenes that inducibly express GLKI and GLK2 indicates that a major function of GLKs is to induce the expression of genes that encode enzymes in chlorophyll biosynthesis (e.g., HEMA], CHLH, CR0] and CA 0) and other processes related to the light reactions of photosynthesis (Waters et al., 2009). Overexpression of GLKI and GLKZ in stably transformed plants causes an increase in chlorophyll levels (Waters et al., 2008; Waters et al., 2009). Based on these findings the GLKs have been proposed to help co-regulate gene expression that is related to the light reactions of photosynthesis. Regulation of Mg-chelatase Mg-chelatase consists of three subunits that are conserved in all organisms that perform photosynthesis. These subunits are often named BchI, BchD, and BchH in organisms that synthesize bacteriochlorophyll, and ChlI, Cth, and Cth in organisms that synthesize chlorophyll. In Arabidopsis, their masses are approximately 40 kDa, 80 kDa and 140 kDa, respectively (Willows et al., 2003). Cth is the porphyrin-binding and Mg2+-binding subunit of this enzyme whereas ChlI and Cth belong to the AAA 25 THH / 7m (ATPases Associated with a variety of Activities) class of proteins (Fodje et al., 2001; Willows et al., 2004). Chll and Cth provide the energy that drives the metalation reaction. Circadian regulation: Like many other genes involved in tetrapyrrole biosynthesis, CHLH transcript levels oscillate significantly during the diurnal cycle and are regulated by the circadian clock (Gibson et al., 1996; Nakayama et al., 1998; Matsumoto et al., 2004). Mg-chelatase activity mirrors the expression of the Cth subunit (Papenbrock et al., 1999). liming Qf CAB expression 1 (TOCI), a key component of circadian regulation, binds the promoter of the CHLH gene and controls its circadian expression (Legnaioli et al., 2009). Magnesium and ATP: Mg2+ and ATP are two substrates for Mg-chelatase. The concentration of Mg2+ in the chloroplast stroma and the ATP/ADP ratio in the . chloroplast fluctuates during the diurnal cycle. Mg2+ binding promotes the cooperative binding of both ATP and porphyrins (Reid and Hunter, 2004). Mg2+ was previously reported to affect the subchloroplastic distribution of Mg-chelatase. When chloroplasts are lysed and fractionated in the presence of 5 mM Mg”, Cth localizes predominantly to the chloroplast envelopes. When chloroplasts are lysed and fractionated in lmM Mg2+, Cth localizes to the soluble fraction. Possible interpretations of this data are that a Mg2+-induced conformational change in Cth promotes the association of Cth with chloroplast envelope membranes (Gibson et al., 1996; Nakayama et al., 1998) or that Mg2+ causes the enzyme to pellet. ATP has also been reported to affect the competition between Mg-chelatase and ferrochelatase because ATP is essential for Mg-chelatase activity (Jensen et al., 1999; Reid and Hunter, 2004), but ATP inhibits the activity of 26 THE ferrochelatase (Comah et al., 2002). Thus, changes in the ATP/ADP ratios in the day/night cycle have been proposed to affect the channeling of protoporphyrin IX into the heme or the chlorophyll branch (Comah et al., 2003). Re_d_g)_<: Activities of a number of enzymes involved in photosynthesis are coupled to the photosynthetic electron transport via the thioredoxin system (Buchanan and Balmer, 2005). Similarly, the Chll subunit of Mg-chelatase is a target for thioredoxin (Balmer et al., 2003; Ikegami et al., 2007). The ATPase activity of Chll from Arabidopsis is fully inactivated by oxidation but readily reactivated by a thioredoxin-assisted reduction (Ikegami et al., 2007). Also, the ATPase activity of Chll was inhibited by N-ethylmaleimide, a thiol-modifying reagent (Ikegami et al., 2007), which was shown to bind Chll specifically, (Jensen et al., 2000). Based on these data, Chll is proposed to be regulated by thioredoxin (Ikegami et al., 2007; Masuda and Fujita, 2008). IMPACT OF CHLOROPHYLL ON OTHER CELLULAR PROCESSES In addition to its role in the light harvesting reactions of photosynthesis, recent reports have indicated the involvement of chlorophyll metabolism in the regulation of particular physiological processes in plants such as (1) regulating the size of photosystem antenna, (2) chloroplast senescence, and (3) plastid-to-nucleus signaling (Tanaka and Tanaka, 2006). In green plants, antenna size is determined by the amount of chlorophyll that is associated with the photosystems. For example, in response to high light intensity, plants decrease the size of the antenna complexes that surround the photosystems and 27 vice versa. However, chlorophyll a oxygenase (CA 0)-overexpressing plants are unable to decrease the size of their antenna complexes in response to high light intensity, in contrast to wild type. Additionally, the chlorophyll a/b ratio increased in wild type plants acclimated from low light to high light, but in transgenic plants overexpressing CA 0, chlorophyll a/b ratios remained low under high light conditions, and the antenna proteins Lhcbl , Lhcb3 and Lhcb6 proteins accumulated compared to wild type under these same conditions. On the other hand, a defect in chlorophyll b biosynthesis in the ch] -1 mutant is correlated with a decrease in the accumulation of Lhcb l, 3 and 6. Based on these data, chlorophyll b biosynthesis was suggested to play a role in regulating antenna size of photosystem II (Tanaka and Tanaka, 2005). Evidence that chlorophyll metabolism can affect senescence comes from an analysis of the maize pao mutant, which has defects in chlorophyll catabolism. Slower rates of degradation were reported for the chlorophyll-binding light harvesting complex protein 11 (Lhcp 11) relative to other proteins in pao relative to wild type (Pruzinska et al., 2003). Similar reports have shown that pheophorbide a oxygenase (PA 0) knockout mutants of Arabidopsis show a stay-green phenotype after dark-induced senescence compared to wild type in both maize and Arabidopsis (Pruzinska et al., 2003; Pruzinska etal., 2005). The chlorophyll precursors Mg-protoporphyrin IX (Mg-PPIX) and Mg- Protoporphyrin IX monomethyl ester (Mg-PPIX ME) have been suggested to affect plastid-to-nucleus signaling. Feeding Mg-PPIX to Arabidopsis protoplasts in dim light causes Lhcb levels to decrease. In this same report, treating chlorophyll precursors with norflurazon caused Mg-PPIX levels to increase and repress the expression of a large 28 number of nuclear genes, most of which are involved in photosynthesis (Strand et al., 2003). Consistent with this model, Arabidopsis mutants with defects in Mg-PPIX and Mg-PPIX ME biosynthesis have defects in the plastid regulation of nuclear gene expression (Mochizuki et al., 2001; Strand et al., 2003). Buildup of Mg-PPIX is controversial; using sensitive methods such as liquid chromatography-mass spectrometry, recent reports find no buildup of Mg-PPIX, Mg-PPIX MB or any other chlorophyll biosynthetic intermediate under conditions in which nuclear gene expression is repressed (Mochizuki et al., 2008; Moulin et al., 2008). The mechanism by which the metabolism of Mg-PPIX and Mg-PPIX ME might affect nuclear gene expression remains an open question (Voigt et al., 2009). In green algae, it was observed that direct feeding of either Mg—protoporphyrin TX or Mg—protoporphyrin IX monomethyl ester, but not protoporphyrin IX or protochlorophyllide or Chlorophyllide, induced expression of Heat Shock Protein 70 (HSP 70) genes whose product is involved in protecting photosystem II from high light induced damage (Kropat et al 1997). Mg—PPIX has been shown to activate nuclear DNA replication via the A-type cyclin dependent kinase (CDKA), which is a regulatory component of the eukaryotic cell cycle in the red alga Cyanidioschyzon merolae and in tobacco cell cultures (Kobayashi et al., 2009). For such a system to work, it seems logical that a cytosolic component has to be present to perceive a tetrapyrrole signal. These findings suggest a link between tetrapyrrole metabolism in the plastid and the regulation of the cell cycle. 29 THEE 7m GENOMES UNCOUPLED 4 (GUN4) In Arabidopsis, the expression of many nuclear genes that encode photosynthesis-related proteins requires the biogenesis of functional chloroplasts (N ott et al., 2006; Larkin and Ruckle, 2008; Woodson and Chory, 2008). To identify these “plastid signals” and plastid signaling mechanisms, Joanne Chory and her colleagues developed a screen for Arabidopsis mutants that have defects in the plastid regulation of photosynthesis-related nuclear genes. These mutants were named genomes uncoupled (gun) because the coordinated expression of the plastid and nuclear genomes is disrupted in these mutants (Susek and Chory, 1992; Susek et al., 1993). The gun4-1 mutant was isolated from the first gun mutant screen (Susek et al., 1993; Mochizuki et. al., 2001). The gun4-1 allele was cloned by map-based cloning. As summarized below, GUN4 encodes a novel positive regulator of chlorophyll biosynthesis (Larkin et al., 2003). In Arabidopsis, leaky gun4 mutants are partially chlorophyll deficient and knockout mutants are albino under normal growth conditions. However, knockout mutants can accumulate readily observable quantities of chlorophyll in dim light (Larkin et al., 2003). Based on these data Larkin et a1. (2003) concluded that GUN4 encodes a positive regulator of chlorophyll biosynthesis that is not absolutely required for chlorophyll accumulation in vivo. GUN4 knockout mutants in Synechocystis sp. PCC 6803 were subsequently reported to not accumulate chlorophyll (Sobotka et al., 2008). Based on these data, the major biological fimction of GUN4 appears to be conserved between cyanobacteria and plants. 30 G UN4 is a nuclear gene that encodes a chloroplast-localized protein in plants. After import into the chloroplast, GUN4 protein is 22 kDa. GUN4 is found mainly in the stroma of chloroplasts that are purified from rosette leaves of ca. one-month-old Arabidopsis plants. Lesser but readily detectable quantities of GUN4 are found in envelope and thylakoid membranes of these same leaves. GUN4 is monomeric in the chloroplast stroma, but GUN4 is part of large multisubunit complexes in solubilized thylakoid and envelope membranes. These complexes range in size from 500 kDa to greater than 1 MDa. A GUN4 complex was purified from solubilized chloroplast membranes and found to contain Cth (aka, the 140 kDa subunit of Mg-chelatase). Based on these data and the chlorophyll-deficient phenotype of gun4 mutants, GUN4 was hypothesized to stimulate chlorophyll biosynthesis by activating Mg-chelatase. Indeed, in quantitative enzyme assays, Synechocystis sp. PCC 6803 GUN4 (hereafter referred to as SynGUN4) was found to stimulate Mg-chelatase activity threefold. Porphyrin binding assays and qualitative enzyme assays provided evidence that SynGUN4 stimulates Mg-chelatase by a mechanism in which GUN4 binds both the porphyrin substrate and product of Mg-chelatase (Larkin et al., 2003). The Cth subunit of Mg-chelatase was subsequently shown to associate with SynGUN4 (Sobotka etaL,2008) Crystal structures are available for GUN4 relatives from Synechocystis sp. PCC 6803 (V erdecia et al., 2005) and Thermosynechococcus elongatus (Davison et al., 2005). The porphyrin-binding domain of GUN4 from SynGUN4 was identified using NMR and extensive-site-directed mutagenesis. This porphyrin-binding domain of SynGUN4 is conserved among all GUN4 relatives and is hereafter referred to as the 31 ”1&8 791‘ GUN4 core domain (Verdecia et al., 2005). Although GUN4 is encoded by single copy genes in plants and many cyanobacteria, particular cyanobacteria may contain multiple genes that encode the GUN4 core domain. Cyanobacteria may contain distinct amino- terminal domains fused to the GUN4 core domain. These amino-terminal domains may contain no sequence similarity to known proteins or may contain similarity to kinase, protease, or protein interaction domains. These findings are consistent with GUN4- related proteins performing diverse functions in particular cyanobacteria. In plants, chloroplast transit peptides are fused to the amino terminus of the GUN4 core domain and ca. 35 residues are fused to the carboxy-terminus of the GUN4 core domain (Larkin et al., 2003; Davison et al., 2005; Verdecia et al., 2005). Based on genetic and biochemical data, GUN4 from cyanobacteria and plants is expected to bind the substrate and product of Mg-chelatase, PPIX and Mg-PPIX (Larkin et al., 2003; Wilde et al., 2004; Davison et al., 2005; Verdecia et al., 2005; Sobotka et al., 2008). There is no published data that provides compelling evidence that the GUN4 protein binds any porphyrin besides PPIX and Mg-PPIX in vivo. Because of the low water solubility of PPIX and Mg-PPIX, the porphyrin-binding activity of GUN4 has been studied using deuteroporphyrin IX (DPIX) and Mg-deuteroporphyrin IX (Mg- DPIX). DPIX and Mg-DPIX lack two vinyl groups found in PPIX and Mg-PPIX and are significantly more water-soluble than PPIX and Mg-PPIX (Larkin et al., 2003; Davison et al., 2005; Verdecia et al., 2005). The KdMg'DP'X and the depix were found to be 0.26 :t 0.029 uM and 2.2 i 0.3 uM, respectively (Larkin et al., 2003). Using different binding assays, essentially these same KdMg'PP'X and KdPP'X values were 32 THEE yo! obtained for SynGUN4 and T hermosynechococcus elongatus GUN4 (Davison et al., 2005; Verdecia et al., 2005). Metalated porphyrins like heme and Mg-PPIX are rigidly planar whereas porphyrins that lack metals such as PPIX are more malleable and assume a more ruffled or puckered conformation. Structural studies with Bacillus subtilis ferrochelatase suggest that the conversion of the more malleable puckered PPIX to the rigid and planar heme drives the dissociation of heme from the product-binding site (Lecerof et al., 2000; Sigfiidsson and Ryde, 2003). GUN4 and Mg-chelatase are different from ferrochelatase in that they bind Mg-DPIX and in the case of GUN4, bind Mg-DPIX with a higher affinity than DPIX (Karger et al., 2001; Larkin et al., 2003; Davison et al., 2005; Verdecia et al., 2005). Based on the much lower KdMg'DP'X than KdDP'x, SynGUN4, was proposed to facilitate the dissociation of Mg-PPIX from the product- binding site of Mg-chelatase (Larkin et al., 2003). This model is supported by the observation that amino acid substitutions that specifically lower the affinity of SynGUN4 for Mg—DPIX but not DPIX also significantly reduce the Mg-chelatase stimulatory activity of SynGUN4 (Verdecia et al., 2005). The porphyrin-binding specificity of SynGUN4 was investigated using 6 artificial and natural porphyrins. The conclusion from this work is that SynGUN4 has a higher affinity for planar porphyrins than porphyrins with nonplanar conformations and that SynGUN4 has a higher affinity for porphyrins containing particular metals (Verdecia et al., 2005). Nonetheless, because of technical limitations, prior to the work published from this thesis, there was no report that compared the binding affinity of GUN4 from any species for the natural ligands PPIX and Mg-PPIX to other porphyrins. Such experiments are important 33 because results from these experiments would indicate whether GUN4 binds PPIX and Mg-PPIX and might suggest whether the interpretations of data obtained with DPIX and Mg-DPIX should be modified. Results from such experiments might help indicate whether GUN4 binds porphyrins other than PPIX and Mg-PPIX in vivo. A proteinaceous cofactor that stimulates an enzyme by a mechanism that involves binding the enzyme as well as one of its substrates and products is novel enzymology. Mg-chelatase may require such a novel cofactor to protect itself from the ROS that are generated when 02 collides with porphyrins that have been exposed to the light. 02 in its ground state is a relatively stable molecule but can be converted to highly reactive and damaging forms such as singlet oxygen ('02), hydrogen peroxide (H202), superoxide or hydroxyl radicals (OH) upon energy transfer or by electron transfer reactions (Halliwell, 1999; Huq et al., 2004; Krieger-Liszkay, 2005). Absorbing a quanta of light excites chlorophyll. If long lived electronically excited states of chlorophyll known as triplet chlorophyll are not quenched by carotenoids, they may transfer energy to 02 after colliding with O2 yielding 102, 102 can be converted into other ROS such as superoxide anion (02") and hydrogen peroxide (H202), the highly toxic hydroxyl radical (OH') or perhydroxyl radicals (02H°) (Gomes et al., 2005; Flors et al., 2006; Gadjev et al., 2006). Such ROS can lead to apoptotic and necrotic cell death (Kim et al., 2008). GUN4 may be necessary to prevent ROS stress by binding porphyrins and helping channel PPIX into chlorophyll biosynthesis. Additionally, by channeling PPIX into chlorophyll biosynthesis, GUN4 might also help Mg-chelatase compete with ferrochelatase for PPIX. 34 THEE Nu In addition to channeling PPIX into chlorophyll biosynthesis, GUN4 has been proposed to bind readily detectable pools of PPIX and Mg-PPIX. Plants perform a burst of PPIX and Mg-PPIX biosynthesis at dawn and readily detectable levels of these porphyrins accumulate throughout the day (Popperl et al., 1998; Papenbrock et al., 1999; Mochizuki et al., 2008; Moulin et al., 2008). GUN4 or a GUN4-Cth complex has been hypothesized to bind the PPIX and Mg-PPIX that accumulates during the diurnal cycle and shield these porphyrins from collisions with 02 thereby protecting plants from ROS-triggered apoptotic and necrotic cell death (Larkin et al., 2003). Indeed, a high-resolution crystal structure and extensive site-directed mutagenesis support a model in which GUN4 envelopes porphyrins thereby shielding them from collisions with O2 (Verdecia et al., 2005). The photosensitizing properties of PPIX were previously shown to inactivate Mg-chelatase in Rhodobacter capsulatus by driving the formation of covalent adducts between the R. capsulatus relative of Cth and the PPIX it binds. This inactivation of Mg-chelatase appears to facilitate the rapid down regulation of bacteriochlorophyll biosynthesis and photosynthesis in R. capsulatus, which performs only anoxygenic photosynthesis (Willows et al., 2003). Rhodobacter species were found to lack a relative of GUN4 (Larkin et al., 2003). Thus, GUN4 was proposed to protect Mg- chelatase from forming covalent adducts with bound PPIX in the presence of bright light and 02in organisms that perform oxygenic photosynthesis (Verdecia et al., 2005). The observation that GUN4 is required for the accumulation of chlorophyll only in bright light but not in dim light (Larkin et al., 2003), and the observation that gun4 mutants exhibit greater sensitivity to light when grown in diurnal cycles that cause 35 l ‘ fl PPIX and Mg-PPIX to accumulate at higher levels relative to continuous light (Peter and Grimm, 2009) are consistent with a role for GUN4 in photoprotection. In this thesis, I report several conceptual advances for GUN4. (l) I report on a technical advance to porphyrin-binding assays that allows the first demonstration that GUN4 can bind PPIX and Mg-PPIX. Using this new binding assay, I compare the binding affinity of GUN4 for PPIX and Mg-PPIX to other natural and unnatural porphyrins. Based on these data and a review of published genetic and biochemical data, I conclude that the major and possibly only role of GUN4 in plants is stimulating Mg-chelatase and binding PPIX and Mg-PPIX in vivo. (2) I show that the porphyrin binding promotes the association of GUN4 with chloroplast membranes—the site of chlorophyll biosynthesis. Similar results are reported for Cth, although they are not explored to the same depth as GUN4. These findings are consistent with GUN4 stimulating chlorophyll biosynthesis not only by activating Mg-chelatase but also by promoting interactions between Cth and chloroplast membranes. (3) I developed stably transformed Arabidopsis plants that express new gun4 alleles that were engineered using site-directed mutagenesis and encode single amino acid substitutions. These single amino acid substitutions lower the affinity of GUN4 for PPIX and Mg- PPIX. I use these site-directed mutants to show that the porphyrin-binding activity of GUN4 that was previously only demonstrated in vitro is also important in vivo. Specifically, I show that this porphyrin-binding activity of GUN4 contributes to the binding of GUN4 to chloroplast membranes and photooxidative stress tolerance. Using cth mutants I show that Cth activity also helps GUN4 to associate with chloroplast membranes and to attenuate the production of ROS. My data indicate that GUN4 helps 36 protect plants from photooxidative stress that can be generated when plants are exposed to high fluence rates of light and that GUN4 attenuates photooxidative stress by binding porphyrins and Cth on chloroplast membranes. 37 CHAPTER 2 PORPHYRINS PROMOTE THE ASSOCIATION OF GENOMES UNCOUPLED 4 AND A MG-CHELATASE SUBUNIT WITH CHLOROPLAST MEMBRANES This research was originally published in the Journal of Biological Chemistry. Adbikari N, Orler R, Chory J, Froehlich J, Larkin R. Porphyrins Promote the Association of GENOMES UNCOUPLED 4 and a Mg-chelatase Subunit with Chloroplast Membranes. Journal of Biological Chemistry. 2009; Vol. 284: pp. 24783-24796. © The American Society for Biochemistry and Molecular Biology. 38 THEE 7m PORPHYRINS PROMOTE THE ASSOCIATION OF GENOMES UNCOUPLED 4 AND A MG-CHELATASE SUBUNIT WITH CHLOROPLAST MEMBRANES Abstract In plants, chlorophylls and other tetrapyrroles are synthesized from a branched pathway that is located within chloroplasts. GENOMES UNCOUPLED 4 (GUN4) stimulates chlorophyll biosynthesis by activating Mg-chelatase, the enzyme that commits porphyrins to the chlorophyll branch. GUN4 stimulates Mg-chelatase by a mechanism that involves binding the Cth subunit of Mg-chelatase, as well as a substrate (protoporphyrin IX) and product (Mg-protoporphyrin IX) of Mg-chelatase. We chose to test whether GUN4 might also affect interactions between Mg-chelatase and chloroplast membranes—the site of chlorophyll biosynthesis. To test this idea, we induced chlorophyll precursor levels in purified pea chloroplasts by feeding these chloroplasts with 5-aminolevulinic acid, determined the relative levels of GUN4 and Mg-chelatase subunits in soluble and membrane-containing fractions derived from these chloroplasts, and quantitated Mg-chelatase activity in membranes isolated from these chloroplasts. We also monitored GUN4 levels in the soluble and membrane-containing fractions derived from chloroplasts fed with various porphyrins. Our results indicate that 5- arninolevulinic acid feeding stimulates Mg-chelatase activity in chloroplast membranes and that the porphyrin-bound forms of GUN4 and possibly Cth associate most stably with chloroplast membranes. These findings are consistent with GUN4 stimulating chlorophyll biosynthesis not only by activating Mg-chelatase but also by promoting interactions between Cth and chloroplast membranes. 39 INTRODUCTION Chlorophylls are produced from a branched pathway located within plastids that also produces heme, siroheme, and phytochromobilin. In photosynthetic organisms, the universal tetrapyrrole precursor 5-aminolevulinic acid (ALA) is derived from glutamyl- tRNA and subsequently converted into protoporphyrinogen IX in the chloroplast stroma. Protoporphyrinogen TX is converted to protoporphyrin IX (PPIX), then ultimately to chlorophylls on plastid membranes. Almost all the genes encoding chlorophyll biosynthetic enzymes have been identified. Transcriptional control provides coarse regulation of this pathway and the regulation of enzyme activities provides fine regulation (Tanaka and Tanaka, 2007; Stephenson and Terry, 2008). Arabidopsis GUN4 (hereafter referred to as GUN4) was identified from a screen for plastid-to-nucleus signaling mutants (Susek et al., 1993; Mochizuki et al., 2001; Larkin et al., 2003). GUN4 is a major positive regulator of chlorophyll biosynthesis, but is not absolutely required for the accumulation of chlorophyll in Arabidopsis (Larkin et al., 2003). In Synechocystis, one of the GUN4 relatives, 3110558 (hereafter referred to as SynGUN4), was subsequently shown also to be required for the accumulation of chlorophyll (Wilde et al., 2004; Sobotka et al., 2008). The 140-kDa subunit of Mg-chelatase copurifies with the 22-kDa GUN4 from solubilized Arabidopsis thylakoid membranes (Larkin et al., 2003); similar results were subsequently reported using Synechocystis (Sobotka et al., 2008). Mg-chelatase catalyzes the insertion of Mg2+ into PPIX, yielding Mg-protoporphyrin IX (Mg-PPIX). This reaction diverts PPIX from heme biosynthesis and commits this porphyrin to chlorophyll biosynthesis. Mg-chelatase requires three subunits in vitro and in vivo. 40 THE W These three subunits are conserved from prokaryotes to plants and are commonly referred to as BchH or Cth, BchD or Cth, and Bchl or Chll. In Arabidopsis, these subunits are 140, 79, and 40 kDa, respectively. Cth is the porphyrin-binding subunit and is likely the Mg2+-binding subunit of Mg-chelatase. ChlI and Cth are related to AAA-type ATPases and form two associating hexameric rings that interact with Cth and drive the ATP-dependent metalation of PPIX (Elmlund et al., 2008; Masuda, 2008). SynGUN4 stimulates Synechocystis Mg-chelatase (Larkin et al., 2003; Davison et al., 2005; Verdecia et al., 2005). Cyanobacteria] relatives of GUN4 bind deuteroporphyrin IX (DPIX) and Mg—deuteroporphyrin IX (Mg-DPIX) (Larkin etal., 2003; Davison et al., 2005; Verdecia et al., 2005), which are more water-soluble derivatives of PPIX and Mg-PPIX. Crystal structures of SynGUN4 and Thermosynechococcus elongatus GUN4 indicate a novel fold that resembles a "cupped hand" that binds DPIX and Mg-DPIX (Davison et al., 2005; Verdecia et al., 2005). Preincubation experiments indicate that a SynGUN4-DPIX complex stimulates Mg-chelatase more potently than SynGUN4 (Larkin et al., 2003). SynGUN4 was found to lower the KmDPIX of Synechocystis Mg- chelatase (Verdecia et al., 2005) and to cause a striking increase in the apparent first- order rate constant for DPIX-Mg-chelatase interactions, an effect that is particularly striking at low Mg2+ concentrations (Davison et al., 2005). The Mg-DPIX-binding activity of SynGUN4 was also found to be essential for stimulating Mg-chelatase (Verdecia et al., 2005). GUN4 and Mg-chelatase subunits have been found in both soluble and membrane-containing fractions of purified chloroplasts (Gibson et al., 1996; Guo et al., 1998; Nakayama et al., 1998; Luo et al., 1999; Larkin et al., 2003). In contrast, 41 ~ .ll ‘3 protoporphyrinogen IX oxidase (PO) and Mg-PPIX methyl transferase (Mg-PPIX MT), which function immediately upstream and downstream of Mg-chelatase in the chlorophyll biosynthetic pathway, are found only in the membrane-containing fractions and not in stromal fractions when purified chloroplasts are lysed and fractionated (Lennontova et al., 1997; Che et al., 2000; Watanabe et al., 2001; Block et al., 2002; van Lis et al., 2005). PPIX and Mg-PPIX accumulate in chloroplast membranes rather than soluble fractions, which provides more evidence that these chlorophyll precursors are synthesized on chloroplast membranes (Mohapatra and Tripathy, 2007). If GUN4 promotes chlorophyll biosynthesis by not only stimulating Mg-chelatase activity but also promoting the formation of enzyme complexes that channel porphyrins into chlorophyll biosynthesis, GUN4 would be expected to more stably associate with chloroplast membranes by interacting with chloroplast membrane lipids or chlorophyll biosynthetic enzymes after binding porphyrins. In the following, we provide experimental evidence supporting this model. MATERIALS AND METHODS Construction of plasmids and strains- For in vitro transcription/translation experiments, the entire GUN4 open reading frame (ORF) was amplified from bacterial artificial chromosome clone T1G3 (Arabidopsis Biological Resource Center, Ohio State University, Columbus OH) using CGGGATCCTATCTTCCCCTGACGTGAC, AACTGCAGAAAGACATCAGAAGCTGTAATTTG, and PfuTurbo® DNA polymerase (Stratagene, La J olla CA). The resulting PCR product was ligated into pCMX-PLI (Umesono et al., 1991) between BamH I and Pst I. In vitro transcription 42 Pita ‘lll if. and translation of the control protein translocon at the inner envelope 40 (Tic40), the small subunit of Rubisco (SS), and a light-harvesting chlorophyll a/b-binding protein (LHCP) were as previously described (Olsen and Keegstra, 1992; Tripp et al., 2007). A glutathione S-transferase (GST)-tagged GUN4 deletion mutant that lacks the predicted 69-residue transit peptide (GST-GUN4 A1-69) was used for the expression and purification of GUN4 from E. coli, as previously described (Larkin et al., 2003). Site- directed mutagenesis was performed on each of these plasmids using the QuickChange® XL Site-Directed Mutagenesis Kit (Stratagene) and Oligonucleotides that were designed according to the manufacturer’s recommendations (Table 2-1). All mutations were confirmed by sequencing at the Research Technology Support Facility (RTSF) (Michigan State University, East Lansing MI). Isolation of pea chloroplasts- Intact chloroplasts were isolated from 6- to 8-day- old pea seedlings and purified over a Percoll gradient as previously described (Bruce et al., 1994). Intact pea chloroplasts were reisolated and resuspended in import buffer (330 mM sorbitol, 50 mM HEPES-KOH, pH 8.0) at a chlorophyll concentration of 1 mg/ml. Protein import was performed as previously described (Bruce et al., 1994). In vitro translation of precursor protein- All precursor proteins used in this study were either radiolabeled with [3SS]-methionine or [3H]-leucine and translated with the TNT® Coupled Reticulocyte Lysate System (Promega, Madison WI) according to the manufacturer’s recommendations. Import Assays- Large-scale import assays contained 50 mM HEPES-KOH, pH 8.0, 330 mM Sorbitol, 4 mM Mg-ATP, 100 pl chloroplasts with a chlorophyll concentration of 1 mg/ml, and labeled precursor protein at a final volume of 300 pl. 43 TN“ to: Table 2-1. Oligonucleotides used for site-directed mutagenesis Substitution Primers used for site-directed mutagenesis L88F L103A F120W V123A L131A I134A F191A E194A R211A Q214A Q214E L216G TCGACGTTCTGGAGAACCATI I IGTCAATCAAAACTTCAGACAAG/ CTTGTCTGAAGI I I IGATTGACAAAATGGTTCTCCAGAACGTCGA AGCCGACGAGGAGACACGGAGATTAGCTATTCAGATATCCGGAGAA GCCG/ CGGCTTCTCCGGATATCTGAATAGCTAATCTCCGTGTCTCCTCGTCGG CT AAACGTGGCTACGI I l ICTGGTCCGAGGTGAAAACAATCTCCCC/ GGGGAGATTGI I I ICACCTCGGACCAGAAAACGTAGCCACGT’IT TGGCTACGI I I ICTTCTCCGAGGCTAAAACAATCTCCCCCGAAGATC/ GATCTTCGGGGGAGATTGI I l IAGCCTCGGAGAAGAAAACGTAGCCA AAAACAATCTCCCCCGAAGATGCTCAAGCTATCGACAATCTATGG/ CCATAGATTGTCGATAGCTTGAGCATCTTCGGGGGAGATTGI I I I TCCCCCGAAGATCTTCAAGCTGCTGACAATCTATGGATTAAACAC/ GTG I I lAATCCATAGATTGTCAGCAGCTI‘GAAGATCTTCGGGGGA TACAGAGCGTTTCCTGACGAAGCTAAGTGGGAGCTTAACGATG/ CATCGTTAAGCTCCCACTTAGCTTCGTCAGGAAACGCTCTGTA TTTCCTGACGAAT’TCAAGTGGGCTCTTAACGATGAAACGCCTTTAG/ CTAAAGGCGTTTCATCGTTAAGAGCCCACTTGAATTCGTCAGGAAA TTACCGCTCACAAACGCCTTGGCTGGAACGCAGCTTCTGAAATGC/ GCAI I ICAGAAGCTGCGTTCCAGCCAAGGCGI I IGTGAGCGGTAA ACAAACGCCTTGAGAGGAACGGCTCTTCTGAAATGCGl I I IAAGC/ GCTTAAAACGCAI I ICAGAAGAGCCGTTCCTCTCAAGGCGI I IGT ACAAACGCCTTGAGAGGAACGGAACTTCTGAAATGCGI I I IAAGC/ GCTTAAAACGCATTTCAGAAGTTCCGTTCCTCTCAAGGCGI I IGT ACGCCTTGAGAGGAACGCAGCTTGGAAAATGCGTI I IAAGCCATCCT GC/ GCAGGATGGCTTAAAACGCAI I I ICCAAGCTGCGTTCCTCTCAAGGCG T 44 After a 30-min incubation at room temperature under white light provided by broad- spectrum fluorescent tube lamps at 75 umol m'2 5", the import assay was divided into two 150-111 aliquots. One aliquot was not further treated. For this aliquot, intact chloroplasts were directly recovered by centrifugation through a 40% Percoll cushion. The remaining aliquot was incubated with trypsin for 30 min on ice as previously described (Jackson et al., 1998). After stopping the protease treatment with trypsin inhibitor as previously described (Jackson et al., 1998), we again recovered chloroplasts by centrifugation through a 40% Percoll cushion. Recovered intact chloroplasts were resuspended in 200 pl of lysis buffer (25 mM HEPES-KOH, pH 8.0, 4 mM MgCl2), incubated on ice for 20 min, and then fractionated into a soluble and membrane- containing pellet fraction by centrifugation at 16,000 X g for 5 min. The pellet fraction contains the outer envelope, inner envelope, and thylakoid membranes. All fractions were analyzed using SDS-PAGE and subjected to fluorography. Fluorograms were exposed to X-ray film (Eastman Kodak, Rochester NY) for 1 to 7 days. Import assays were quantitated by scanning developed films with the VersaDoc 4000 MP Imaging System and Quantity One software, as recommended by the manufacturer (Bio-Rad, Hercules CA). ALA and porphyrin feeding- Prior to import, intact chloroplasts were incubated in import buffer (330 mM sorbitol, 50 mM HEPES-KOH, pH 8.0) that contained or lacked 10 mM ALA (Sigma-Aldrich, St. Louis M0) for 15 min at 26°C in the dark, unless indicated otherwise. PPIX, Mg-PPIX, uroporphyrin III, coproporphyrin III, hemin, and pheophorbide a were all purchased from Frontier Scientific (Logan UT). These porphyrins were first dissolved in DMSO and their concentrations were 45 determined spectrophotometrically as previously described (Rimington, 1960; Brown and Lantzke, 1969, 1969; Eichwurzel et al., 2000; Rebeiz, 2002). These stock solutions were diluted with import buffer, giving final porphyrin concentrations of 20 uM and final DMSO concentrations of l-2%. Intact chloroplasts were incubated in these solutions exactly as described for ALA. Fractionation of chloroplasts into stroma, thylakoid, and envelope fiactions- Fractionation of chloroplasts was performed as previously described (Keegstra and Yousif, 1986), with modifications. First, large-scale import assays were performed with (+) or without (-) an ALA pretreatment as described above. After import, intact chloroplasts were recovered by centrifugation through a 40% Percoll cushion. The intact chloroplasts were then resuspended in 0.6 M sucrose containing 25 mM HEPES- KOH, pH 8.0, 2 mM MgCl2, 8 mM EDTA. The suspension was placed on ice for 20 min and then placed at -20°C overnight. Subsequently, the suspension was thawed at room temperature, gently mixed, and then diluted with 2 volumes of dilution buffer (25 mM HEPES-KOH, pH 8.0, 2 mM MgCl2, 8 mM EDTA). This suspension was then centrifuged at 1,500 X g for 5 min. The resulting pellet predominantly contained the thylakoid fraction and was diluted twofold with 2X SDS-PAGE loading buffer (Sambrook and Russell, 2001). The remaining supernatant was then centrifuged at 100,000 X g for 1 hr. The resulting pellet fraction predominantly contained envelopes and was diluted twofold in 2X SDS-PAGE loading buffer. Cold acetone was added to the supernatant fraction to a final concentration of 80% and incubated on ice for 30 min, then centrifuged at 15,000 x g for 5 min. The precipitated soluble protein fraction was 46 resuspended in 2X SDS-PAGE loading buffer. All fractions were analyzed by SDS- PAGE. Analysis of porphyrins in purified chloroplasts- PPIX and Mg-PPIX levels in purified chloroplasts were quantitated following ALA feeding and mock protein import. 0.1-ml aliquots of chloroplasts were collected by centrifugation at 1,900 X g for 5 min at 4°C through a 40% Percoll cushion. Recovered chloroplasts were lysed by resuspension in 700 pl of acetone: 0.1 M NH40H (9:1, vol/vol). These lysates were clarified by centrifugation at 16,000 X g for 10 min at 4°C. Chlorophyll was removed from the resulting supematants by hexane extraction as previously described (Rebeiz, 2002). We quantitated the amount of PPIX and Mg-PPIX in these hexane-extracted supematants using fluorescence spectroscopy with a QuantaMasterTM spectrofluorometer (Photon Technology International, Inc., London Ontario) as previously described (Rebeiz, 2002). PPIX and Mg-PPIX, purchased from Frontier Scientific, were used to construct standard curves. Quantitative analysis of porphyrin binding— GUN4 131-69 and versions of GUN4 [31-69 that contain amino acid substitutions were expressed and purified from E. coli as previously described (Larkin et al., 2003). Binding constants were measured by quantitating the quenching of tryptophan fluorescence in GUN4 Al -69 by bound porphyrins essentially as described for the cyanobacterial relatives of GUN4 (Larkin et al., 2003; Davison et al., 2005; Verdecia et al., 2005). Binding reactions were in 20 mM MOPS-KOH, pH 7.9, 1 mM DTT, 300 mM glycerol and contained 200 nM GUN4 A1-69 or GUN4 A1-69 with the indicated single amino acid changes and variable concentrations of DPIX and Mg-DPIX (Frontier Scientific). We determined binding 47 constants for PPIX, Mg-PPIX, and Mg-PPIX ME, uroporphyrin III, coproporphyrin III, hemin, and pheophorbide a using the same conditions except that binding reactions also contained 1% DMSO. Stock solutions of DPIX and Mg-DPIX were prepared as previously described (Karger et al., 2001). Stock solutions of all other porphyrins were prepared as described in ALA and porphyrin feeding. We calculated binding constants using DYNAF IT (Kuzrnic, 1996) as previously described (Karger et al., 2001). The data fit best with a model that predicts a single binding site. Mg-chelatase assays- Chloroplasts were purified from pea and subjected to hypotonic lysis as described above, except that lysis buffer also contained 1 mM DTT and 2 mM Pefabloc (Roche, Indianapolis IN). Supematants were flash frozen in liquid nitrogen and stored at -80°C. We assayed pellet fractions for Mg-chelatase activity immediately by resuspending them in a Mg-chelatase assay buffer (50 mM Tricine- KOH, pH 7.8, 1 mM EDTA, 9 mM MgCl2, 4 mM MgATP, 1 mM DTT, 0.25% BSA, 5% glycerol, 60 mM phosphocreatine, 4 U/ml creatine phosphokinase) and incubating the resuspended pellets for 30 min at 30°C as previously recommended (Walker and Weinstein, 1991; Guo et al., 1998). PPIX dissolved in DMSO was added to particular reactions as indicated in the text. The final concentrations of PPIX and DMSO were 1.5 pM and 2%, respectively, as previously recommended (Walker and Weinstein, 1991). Aliquots of 8 pl were removed at 5-min intervals during a 30-min incubation and diluted into 200 pl of 90% acetone: 0.1 M NH4OH (9:1) and vortexed to terminate the reaction. The terminated reactions were centrifuged for 10 min at 16,000 X g at 4°C. Mg—PPIX in the resulting supematants was quantitated as described in Quantitative analysis of porphyrin binding, above. We subtracted the amount of Mg-PPIX in the 48 membranes before the reactions were initiated from the amount of Mg-PPIX at the end of each time point. Three replicates were analyzed for each time point. Mg-PPIX accumulated linearly for the entire 30-min assay. To assay supematants for Mg- chelatase activity, supematants were rapidly thawed, clarified at 16,000 X g at 4°C for 10 min, and then concentrated nearly 5-fold using an Amicon Ultra Centrifugal Filter Device with a nominal molecular weight limit of 10,000 (Millipore, Billerica MA). The concentrated supematants were diluted into a concentrated Mg-chelatase assay buffer yielding the same assay conditions described for pellets. Reactions were initiated by adding 1.5 uM PPIX. Polyclonal anti-Cth, anti-Chll, and anti-Cth antibody development- Poly(A)+ mRNA was isolated from Arabidopsis thaliana (Columbia-0 ecotype) using the Absolutely mRN A Kit (Stratagene). We prepared first-strand cDNA using Superscript II (Invitrogen, Carlsbad CA). A cDNA encoding a 62-kDa fragment of Cth that lacks the first 823 amino acid residues (ChIH A1-823) was amplified from this first-strand cDNA as described for GUN4 A1-69, except that CCGGAATTCGCTGTGGCCACACTGGTCAAC and TCGCGTCGACTTATCGATCGATCCCTTCGATCTTGTC were used. To express Cth A1-823 as a His-tagged protein in E. coli, this PCR product was ligated into pHIS8-3 (Jez et al., 2000) between EcoR I and Sal l. The resulting plasmid was sequenced at the RTSF to confirm that no mutations were introduced during PCR. The His-tagged Cth A1-823 protein was expressed from the resulting plasmid in the E. coli strain BL21-CodonPlus® (DE3)-RIL (Stratagene) at 18°C in terrific broth (Sambrook and Russell, 2001). We induced expression by adding 1 mM isopropyl B-D-l- 49 thiogalactopyranoside (Sigma-Aldrich) when the ODéoo was 0.8. All subsequent steps were performed at 4°C, unless indicated otherwise. Cells were harvested by centrifugation at 6,000 X g for 10 min and resuspended in 20 ml buffer A (50 mM Tris- acetate, pH 7.9, 500 mM potassium acetate, 20 mM imidazole, 20 mM [3- mercaptoethanol, 20% glycerol, 1% Triton X-100) per gram of bacterial pellet. Cells were lysed by sonication. The resulting lysate was clarified by centrifugation at 10,000 X g for 20 min. The supernatant was batch bound to Ni-NTA agarose (Qiagen, Valencia CA) equilibrated in buffer A. Bound proteins were batch washed twice with buffer A and twice with buffer A lacking Triton X-100. Ni-NTA agarose was poured into an Econo-Pac column (Bio-Rad, Hercules CA) and proteins were step eluted using buffer B (20 mM Tris-acetate, pH 7.9, 500 mM potassium acetate, 250 mM imidazole, 20 mM B-mercaptoethanol, 20% glycerol). Eluted proteins were dialyzed against buffer C (20 mM Tris-acetate, pH 7.9, 150 mM potassium acetate, 2.5 mM CaCl2, 20 mM [3- mercaptoethanol, 20% glycerol), digested with thrombin (Sigma-Aldrich) at room temperature and applied to the aforementioned Ni-NTA agarose column equilibrated in buffer A. Proteins in the flow-through fraction were dialyzed against buffer D (20 mM Tris-acetate, pH 7.9, 100 mM potassium acetate, 1 mM EDTA, 1 mM DTT, 20 % glycerol), applied to a HiPrepTM 16/10 Q FF column (GE Healthcare, Piscataway NJ) that was equilibrated in buffer D at a flow rate of 1.0 ml/min, and eluted with a 200-ml linear gradient to buffer B (20 mM Tris acetate, pH 7.9, 1000 mM potassium acetate, 1 mM EDTA, 1 mM DTT, 20% glycerol) also at a flow rate of 1.0 ml/min. Fractions of 2.5 ml containing Cth A1-823 were pooled, concentrated using an Amicon Ultra-15 centrifugal filter unit with a nominal molecular weight limit of 30,000 (Millipore), 50 dialyzed against storage buffer (50 mM Tricine-KOH, pH 7.9, 1 mM DTT, 50% glycerol), flash frozen with liquid N2, and stored in small aliquots at -—80°C. For polyclonal antibody development, purified Cth A1-823 was dialyzed extensively against phosphate-buffered saline, pH 7.4 (Sambrook and Russell, 2001) and used to develop anti-Cth A1-823 polyclonal antisera in New Zealand white rabbits at Strategic Diagnostics, Inc. (Newark DE). IgGs were purified from these antisera using Affi-Gel protein A (Bio-Rad) as recommended by Harlow and Lane (Harlow and Lane, 1999). Anti-Cth A1-823 antibodies were affinity-purified from total IgGs on Cth A1-823 columns that were constructed by linking purified Cth Al-823 to Affi-Gel 15 (Bio- Rad) at approximately 15 mg/ml. Antibodies were eluted from the Cth Al-823 columns in buffer F (100 mM glycine-HCI, pH 2.5, 50% ethylene glycol) and immediately mixed with 1/10 volume 1M Tris-HCI, pH 8.0, as recommended by Harlow and Lane (Harlow and Lane, 1999). Protein-containing fractions were pooled, dialyzed against PBS, concentrated using Amicon Ultra-15 centrifiigal filter units as described above, flash frozen with liquid N2, and stored at -80°C in small aliquots. For anti-Chll antibody development, Chll was expressed and purified as described for Cth 131-823, except that a cDNA encoding a Chll ORF that lacks the predicted transit peptide (Chll [31—60) was amplified using CCGGAATTCGCTGTGGCCACACTGGTCAAC and TCGCGTCGACTTATCGATCGATCCCTTCGATCTTGTC. Chll A1-60 antibody development and affinity purification were as described for Cth A1-823, except that Chll A1-60, rather than Cth A1-823, was linked to Affi-Gel 15. 51 THE For anti-Cth antibody development, Cth was expressed and purified as described for Cth A1-823, except that a cDNA encoding a Cth ORF that lacks the first 516 residues (Cth A1-516) was amplified using GCGGGATCCACCCTTAGAGCAGCTGCACCATAC and TCGCGTCGACTCAAGAATTCTTCAGATCAGATAGTGCATCC and ligated into pHIS8-3 using BamHI and SalI. Another difference was that after elution from Ni- NTA agarose and thrombin digestion, Cth A1-516 was further purified by fractionating on a HiLoadTM 26/60 SuperdexTM 200 prep-grade column equilibrated in buffer G (Tris-HCI, pH 7.9, 500 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol) at 2 ml/min and at 4°C. Anti-Cth A1-516 antibodies were developed and purified as for anti-Cth A1-823 and anti-Chll A1-60 except that affinity purification was performed using Cth A1-516 linked to Affi-Gel 10 rather than Affi-Gel 15. All immunoblotting was done as previously described (Larkin et al., 2003) using SuperSignal® West Dura Extended Duration Substrate (Pierce, Rockford IL). We quantified immunoreactive bands with the VersaDoc 4000 MP and Quantity One software (Bio-Rad). RESULTS In vitro import of GUN4 into pea chloroplasts. To test whether the porphyrin- binding activity of GUN4 affects interactions between GUN4 and chloroplast membranes, we imported GUN4 into purified pea chloroplasts in vitro. Because chlorophyll biosynthesis is well conserved among plant species (Eckhardt et al., 2004; Tanaka and Tanaka, 2007), we expected that GUN4 would interact similarly with 52 F1 /nl/. proteins associated with chloroplast membranes such as Cth from pea and Arabidopsis. The full-length GUN4 precursor containing the transit peptide was produced by in vitro translation. During SDS-PAGE, this translation product migrated like a 30-kDa protein (Figure 2-1A), which was expected, based on the mass calculated from the derived amino acid sequence of GUN4 containing the predicted transit peptide (Larkin et al., 2003). We tested whether GUN4 could be imported into pea chloroplasts as judged by (i) a mobility shift that is consistent with the removal of the predicted 69-residue transit peptide (Larkin et al., 2003), and (ii) resistance to a trypsin protease treatment, which cannot digest proteins that are transported across the inner envelope of pea chloroplasts (Jackson et al., 1998). After import into the pea chloroplast, GUN4 migrates as a doublet in SDS gels, with the predominant band migrating like a 22-kDa protein, consistent with the removal of the predicted transit peptide (Figure 2-1A). A similar doublet was previously observed when whole cell and various chloroplast extracts from Arabidopsis were analyzed by immunoblotting with affinity-purified anti-GUN4 antibodies (Larkin et al., 2003). After GUN4 was imported, chloroplasts were digested with trypsin. Intact chloroplasts were then recovered using Percoll gradients, subjected to hypotonic lysis, and the soluble and membrane-containing fractions of the chloroplast were separated by centrifugation. GUN4 was observed in both the soluble and the membrane-containing pellet fractions (Figure 2-1), which indicates that GUN4 was imported into these chloroplasts and not digested by trypsin. Two control proteins, Tic40 and SS, accumulated in membrane and soluble fractions, respectively (Figure 2-lB and C), as has been previously demonstrated (Tripp et al., 2007). 53 - + Trypsin P .5 .. A 4- prGUN4 W a... 4— mGUN4 B 4- prTio40 h v ' - 37 _ . u <- mTic40 C a.» ‘ <- prss 15 - ' an: , W...” 4-mSS Figure 2-1. Distribution of GUN4, Tic40, and SS in fractionated chloroplasts. A. Distribution of GUN4 in fractionated pea chloroplasts. [3H]-labeled GUN4 leucine was imported into intact pea chloroplasts. Following the import reaction, chloroplasts were treated with (+) or without (-) trypsin. Intact chloroplasts were recovered by centrifugation through a 40% Percoll cushion, lysed, and fractionated into soluble (S) and membrane-containing pellet (P) fractions. All fractions were then analyzed by SDS-PAGE and fluorography. TP represents approximately 10% of the precursor added to an import assay. The position of the GUN4 precursor containing the transit peptide (prGUN4) and the major form of mature GUN4 generated by proteolytic removal of the transit peptide during import into the chloroplast (mGUN4) and a minor form of mature GUN4 (*) are indicated. B. Distribution of Tic 40 in fractionated pea chloroplasts. Chloroplast import and analysis were as described in A except that [35S]-labeled Tic40 was used rather than [3H]-labeled GUN4. C. Distribution of SS in fractionated pea chloroplasts. Chloroplast import and analysis were as described in A except that [”8]- labeled SS was used rather than [3H]-labeled GUN4. Masses of protein standards are indicated at the left in kDa. 54 Subchloroplastic distribution of GUN4 in ALA-fed chloroplasts. To test whether porphyrin binding might affect the interactions between GUN4 and chloroplast membranes, we imported GUN4 into pea chloroplasts that were fed with ALA prior to initiating protein import. ALA feeding was previously reported to induce the levels of PPIX and Mg—PPIX in whole plants (Granick, 1961; Gough, 1972) and to increase the levels of heme efflux from purified chloroplasts (Thomas and Weinstein, 1990). Consistent with these previous reports, we found that PPIX and Mg-PPIX levels increased 20- to 30-fold when purified chloroplasts were fed ALA under these conditions (Figure 2-2A) and that these porphyrins accumulated in the membrane- containing pellet fraction (Figure 2—2B). We found that half of GUN4 associated with the membrane fraction in unfed chloroplasts, and that the amount of GUN4 in the membrane fraction increased by 50% in ALA-fed chloroplasts (Figure 2-2C and D). In contrast, the distribution of Tic40 and SS did not change after ALA feeding (Figure 2- 1A and B). Additionally, ALA feeding did not appear to change the total protein profile of the soluble and pellet fractions (Figure 2-3 C). ALA feeding did not affect the nature of GUN4-chloroplast membrane interactions as judged by extracting chloroplast membranes with either Na2CO3, pH 11, or NP40 (Figure 2-4A and B). These data are consistent with (i) elevated porphyrin levels causing GUN4 to accumulate in the membrane-containing pellet fraction after GUN4 has been imported into the chloroplast, but also with (ii) the distinct targeting of GUN4 to the stroma and to the chloroplast membranes and ALA feeding inhibiting stromal targeting. To distinguish between these possibilities, we fed ALA to chloroplasts following the import of GUN4. We found that ALA feeding subsequent to the import of GUN4 leads to increased levels 55 '5. fizoo DMg-PPIX '8 15.0. EPPIX T $310.0. l j E j , g 5. o~ T . 1+. g E 00......“ 0 1 10 [ALA] mM B -1200- 3. c1 Mg-PPIX S1ooo~ E3 PPIX =3 800‘ 3 8 600‘ E E 400- E- 8” 200‘ -ALA (P) -ALA (S) +AI.A (P) +ALA (S) Figure 2-2. Subchloroplastic distribution of PPIX, Mg-PPIX, and GUN4 after ALA feeding. A. Quantitation of PPIX and Mg-PPIX levels in ALA-fed chloroplasts. PPIX and Mg-PPIX levels were quantitated in intact chloroplasts that had been treated with 0 mM, 1.0 mM, or 10 mM ALA. N=3. Error bars represent standard error. B. Distribution of PPIX and Mg-PPIX in fractionated ALA-fed chloroplasts. Chloroplasts were fed with 10 mM ALA as in A. After ALA feeding, chloroplasts were lysed and separated in soluble (S) and membrane-containing pellet (P) fractions. PPIX and Mg- PPIX levels were determined in each fraction. N=3. C. Distribution of GUN4 in fractionated chloroplasts after ALA feeding. Chloroplasts were fed with ALA as described in A. Import and post-import analysis of GUN4 was performed as described in Figure 2-1A. Masses of protein standards are indicated at the left in kDa. D. Quantitation of GUN4 in soluble and membrane fractions after ALA feeding. The amounts of radiolabeled GUN4 in soluble (S) and membrane- containing pellet (P) fractions were quantitated 1n independent experiments that were performed as in C, using different preparations of chloroplasts. The amount of [3 H]- GUN4 found either 1n the soluble (S) or pellet (P) fraction IS presented as a percentage of total imported [3H]- GUN4. Column numbers correspond to lane numbers in B. N: 3. Error bars are as in A. 56 - - + 10mMALA - + - 1mMALA 123456 0 Percent GUN4 Figure 2-2 (continued). Subchloroplastic distribution of PPIX, Mg-PPIX, and GUN4 afier ALA feeding. 57 A -ALA Import NaZCO3 NP40 TPPSP’S’P’S’ «prGUN4 25 * d-mGUN4 20 B +ALA Import NaZCO3 NP40 TPPSP'S’P‘S' ‘- prGUN4 25 * 4- mGUN4 20 Figure 2-3. Subchloroplastic distribution of Tic40, SS, and total protein after ALA feeding. A. Subchloroplastic distribution of Tic40 after ALA feeding. ALA feeding was as described in Figure 2-2A. Import, chloroplast fractionation, and analysis of Tic40 were as described in Figure 2-1B. B. Subchloroplastic distribution of SS after ALA feeding. ALA feeding was as described in Figure 2-2A. Import and analysis of SS was as described in Figure 2-lB. C. Subchloroplastic distribution of total chloroplast protein after ALA feeding. ALA feeding was as described in Figure 2-2A. After ALA feeding, a mock import was performed as described in Figure 2-1A, without radiolabeled precursor proteins. All fractions were analyzed by SDS-PAGE and Coomassie blue staining. Masses of protein standards are indicated at the left in kDa 58 THE 11 {I n - + 10mMALA Figure 2-3 (continued). Subchloroplastic distribution of Tic40, SS, and total protein afier ALA feeding. 59 A -ALA Import NaZCO3 NP40 TPPSP’S'P’S’ «prGUN4 25 * mGUN4 20 B +ALA Import N32003 NP40 TPPS P'S’P'S' prGUN4 25 * 4- mGUN4 20 Figure 2-4. Analysis of GUN4-chloroplast membrane interactions in chloroplasts that were fed or not fed ALA. A. Analysis of interactions between GUN4 and chloroplast membranes in chloroplasts that were not fed ALA. [3H]-GUN4 was imported into pea chloroplasts. Afier import and chloroplast fractionation, a portion of the pellet fraction (P) was extracted with either Na2C03 or NP40 for 30 min on ice and centrifuged. A pellet fraction (P’) containing integral membrane proteins and a soluble fraction (8’) containing extracted proteins were obtained. All fractions were then analyzed by SDS- PAGE and fluorography. TP represents approximately 10% of precursor added to an import assay. B. Analysis of GUN4-chloroplast membrane interactions in ALA-fed chloroplasts. [3H]-GUN4 was imported into chloroplasts that had been pretreated with ALA. Analysis of the ALA-fed chloroplasts was as described in A. Masses of protein standards are indicated at the left in kDa. of PPIX and Mg-PPIX and causes GUN4 to redistribute to the membrane-containing pellet fraction (Figure 2-5A, B, and C). Based on these data, we conclude that inducing a rise in PPIX and Mg-PPIX levels by ALA feeding causes GUN4 to accumulate in the pellet fraction rather than blocking the import of GUN4 into the stroma. GUN4 was previously detected in stroma, envelope, and thylakoid fractions derived from purified Arabidopsis chloroplasts (Larkin et al., 2003). To test whether ALA feeding preferentially causes the accumulation of GUN4 in the chloroplast envelope or thylakoid membranes, we imported GUN4 into chloroplasts that were either fed or not fed ALA and then compared the levels of GUN4 in the stromal, envelope, and thylakoid fractions. We found that ALA feeding caused GUN4 levels to increase in the envelope and thylakoid membranes (Figure 2-6A and B). In this experiment, Tic40, LHCP, and SS accumulated in the envelope (Figure 2-6C), thylakoids (Figure 2-6D), and stroma (Figure 2-6E), respectively, as previously reported (Tripp et al., 2007). The levels and distributions of Tic40, LHCP, and SS were not different from those previously reported after ALA feeding (Figure 2-6C, D, and E). To test whether the tendency of ALA to cause accumulation of GUN4 in the membrane-containing fractions was unique to the newly imported radiolabeled GUN4 or whether a similar affect might be observed with the endogenous pea GUN4, we monitored the distribution of pea GUN4 in the soluble and membrane fractions of purified pea chloroplasts afier ALA feeding. We detected an immunoreactive band with essentially the same mobility as GUN4 during SDS- PAGE and found a 22% increase in the membrane-containing fraction and the same fold decrease in the soluble fraction after ALA feeding that caused PPIX and Mg-PPIX levels to increase (Figure 2-7A, B, C). 61 Chase -ALA Chase +ALA A - + - + Chase - - - + ALA TP P s P s P s P s prGUN4 3<-mGUN4 1 2 3 4 5 6 7 8 B 80"; 70. g 60: 8 so E 40+ 8 . 5 30. ‘L 20~j 104 oi 1 2 3 4 5 6 7 8 Chase -ALA Chase +ALA Figure 2-5. Subchloroplastic distribution of GUN4 following post-import ALA feeding. A. Representative fluorogram showing the subchloroplastic distribution of GUN4 following post-import ALA feeding. [ H]-GUN4 was imported for 15 min into pea chloroplasts. Intact chloroplasts were recovered by centrifugation through a 40% Percoll cushion. A portion of the recovered chloroplasts was directly lysed and fractionated into either total soluble (S) or total membrane (P) fractions (Lanes 1-2 and 5-6). The remaining portion of chloroplasts was resuspended in import buffer containing ALA (Lanes 7-8) or lacking ALA (Lanes 3-4) and incubated for an additional 15 min at 26°C (Chase). The ‘Chase’ reactions were terminated by centrifuging the chloroplasts through a 40% Percoll cushion. Chloroplasts were then lysed and fractionated. Membrane-containing pellet fractions (P) or soluble (S) fractions were analyzed by SDS-PAGE and fluorography. TP represents approximately 10% of precursor added to an import assay. B. Quantitation of the subchloroplastic distribution of GUN4 following post-import ALA feeding. GUN4 found in the soluble (S) or pellet (P) fraction is presented as a percentage of the total amount of imported protein. Column numbers 1-8 correspond to lane numbers 1-8 in A. Error bars represent standard error. N=3. C. Quantitation of PPIX and Mg-PPIX in intact chloroplasts represented in A. Column numbers 1-8 correspond to lane numbers 1-8 in A. Error bars are as in B. 62 DMg-PPIX 5 ElPPIX 1 ‘r-{jlr'fl'r-E' I 12 34 56 78 pmol porphyrin/pg chlorophyll co Chase -ALA Chase -ALA Figure 2-5 (continued). Subchloroplastic distribution of GUN4 following post-import ALA feeding. 63 A TP P S Env Str Thy prGUN4 mGUN4 B prGUN4 mGUN4 C D prLHCP mLHCP E 4- prSS mSS Figure 2-6. Distribution of GUN4 in the chloroplast envelope, thylakoid, and stroma fractions afier ALA feeding. A, Chloroplasts were either pretreated without, or B, with ALA prior to the import of [3H]GUN4. Control import assays with ALA-fed chloroplasts were likewise performed with C, [3SS]-prTic4O; D, [3H]prLHCP; and E, [3SS]-prSS. After import, chloroplasts were lysed and fractionated into soluble (S) and membrane-containing pellet fractions (P), or chloroplasts were separated into envelope (Env), stromal (Str) and thylakoid (Thy) fractions. All fractions were analyzed by SDS- PAGE as an equal load, using fluorography. 64 100 . * up —I— 305 'S V a T 2 :3 , .L 0 60 *5 1 40* r . s—I— 20~ i ' . 0 .. ~ -ALA +ALA Figure 2-7. Subchloroplastic distribution of pea GUN4 and porphyrin levels after ALA feeding. A. Subchloroplastic distribution of pea GUN4 following ALA feeding. Intact pea chloroplasts were either fed (+ALA) or not fed (-ALA) ALA and then subjected to a mock import assay that lacked radiolabeled proteins. These chloroplasts were subsequently lysed and fractionated. Samples of 7 pg of protein from soluble (S) and membrane-containing pellet (P) fractions were analyzed by immunoblotting with affinity-purified anti-GUN4 antibodies. B. Quantitation of the subchloroplastic distribution of pea GUN4 after ALA feeding. N=5. Error bars represent standard error. The statistical significance of GUN4 redistributing to the pellet during ALA feeding was tested using a paired t-test. * indicates a very significant difference (P=0.008). C. Quantitation of PPIX and Mg-PPIX levels in ALA-fed chloroplasts. Porphyrin levels were quantitated as described in Figure 2-2A after ALA feeding as described in Figure 2-6A and B. N=5. Error bars are as in B. 65 O = 2000 ' E D Mg-PPIX ,_ 9 E PPIX 2 1500 4 .c O E’ g 1000 - 5‘ 8- 500 " .1. '5 T E f n. o 4 m -ALA +ALA Figure 2-7 (continued). Subchloroplastic distribution of pea GUN4 and porphyrin levels after ALA feeding. 66 [fi- / Q . Quantitation of porphyrin binding by GUN4. The above findings are consistent with either porphyrin binding causing GUN4 to accumulate in the membrane-containing pellet fraction or ALA feeding somehow promoting interactions between GUN4 and chloroplast membranes by some other mechanism. To distinguish between these possibilities, we took advantage of a previous structure-function analysis of SynGUN4 (Verdecia et al., 2005). Based on this previous work, we made 11 single amino acid changes in GUN4 using site-directed mutagenesis (Table 2-2). Homologous amino acid substitutions in SynGUN4 cause general defects in porphyrin binding or specific defects in binding either DPIX or Mg-DPIX (Verdecia et al., 2005). We also introduced the L88F substitution from the gun4-1 missense allele. This amino acid substitution causes the GUN4 protein to accumulate at much lower levels in vivo compared to the wild type. An F substitution at the homologous L residue in T hermosynechococcus elongatus GUN4 and SynGUN4 causes a 6- to lS-fold increase in the affinities for DPIX and Mg-DPIX without affecting folding in the case of T. elongatus GUN4 (Davison et al., 2005). We expressed these site-directed mutants as GST-fusion proteins without the predicted 69-residue transit peptide, as previously described (Larkin et al., 2003). Six of these amino acid substitutions, including the L88F, caused GST-GUN4 [31-69 to accumulate in the insoluble fraction (Table 2-2) and were not analyzed When The remaining seven amino acid substitutions did not affect the solubility of GST- GUN4 A1-69 in E. coli (Table 2-2) and were purified. We determined the KdDP'x and KdMg'DP [X for GUN4 (Table 2-3; Figure 2-8). During the course of these studies, we observed that including 1% DMSO in binding DPIX Mg-DPIX assays does not significantly affect the Kd or the Kd and that including 1% 67 Table 2-2. Solubilities of GST-GUN4A1 -69 containing the indicated amino acid substitutions when expressed in E. coli. Amino acid substitutions Homologous amino acid Solubilities of GST-GUN4 in SynGUN4 that affect substitutions in GUN4 A1-69 with the indicated porphyrin binding amino acid substitutions Wild type Wild type soluble L1 00F L88F insoluble L1 16A L103A insoluble F132W F12OW soluble V135A V123A soluble L143A L131A insoluble 1146A 1134A insoluble F 196A F191A soluble D1 99A E1 94A insoluble R214A R21 1 A soluble R21 7A Q214A soluble R217E Q214E soluble A21 9G L216G insoluble All amino acid substitutions in SynGUN4 that affect either or both KdDPIX and KdMg'DPIX were reported by (Verdecia et al., 2005). L88F is the amino acid substitution caused by the gun4-1 missense allele (Larkin et al., 2003). GST-GUN4 A1-69 accumulated in either the soluble or the insoluble fraction when expressed in E. coli. 68 DMSO in binding assays solubilizes PPIX and Mg-PPIX sufficiently for us to perform binding assays with these natural ligands, which has not been reported for GUN4 from PM was almost twofold higher than KdDPlX and any species. We determined that the Kd that the KdMg'PP'x was 1.5-fold lower than KdMg'DP'X (Table 2-3; Figure 2-9). Based on previously published biochemical and genetic data, we suggest that the major function of GUN4 in vivo is to stimulate Mg-PPIX biosynthesis (Larkin et al., 2003; Davison et al., 2005; Verdecia et al., 2005). Nonetheless, we cannot rule out that GUN4 might participate in other reactions. To begin exploring this possibility, we tested whether GUN4 might bind other porphyrins. We performed binding assays with Mg-PPIX ME, which is the next chlorophyll precursor downstream of Mg-PPIX in the chlorophyll biosynthetic pathway. In this preparation of Mg-PPIX ME, either carboxyl group is methylated in a roughly 1:] ratio. In contrast, only the carboxyl group associated with ring C is methylated in nature (Tanaka and Tanaka, 2007). We found that the KdMg'PP'x ME was more than twofold higher than KdMg'PP'x (Table 2-3; Figure 2- 9). We found that GUN4 also binds uroporphyrin III, coproporphyrin III, hemin, and pheophorbide a (Table 2-3; Figure 2-10) and that the affinities of GUN4 for these porphyrins is intermediate between Mg-PPIX ME and PPIX. The amino acid substitutions F 120W, E194A, and Q214E in GUN4 did not affect KdDP'x and KdMg'DPlX (N.D.A., unpublished data). The homologous substitutions (i.e., F132W, D199A, and R217E) significantly reduced the affinity of SynGUN4 for porphryins (Verdecia et al., 2005). Amino acid substitutions V123A, F191A, and R211A caused both KdDP'X or KdMg'DP'x to increase in GUN4 (Table 2-4; Figure 2-1 1A, B, and C), although the degrees of the porphyrin-binding defects were not exactly as 69 Table 2-3. Quantitation of porphyrin binding by GUN4 Porphyrin 1% Kd (11M) DMSO DPIX — 6.4 :1: 0.21 DPIX + 6.0 :t 0.39 Mg-DPIX — 2.7 i 0.29 Mg-DPIX + 2.2 :1: 0.30 PPIX + 11 d: 0.50 Mg—PPIX + 1.6 i 0.17 Mg-PPIX ME + 4.0 i 0.20 Hemin + 8.1 i 0.75 Uroporphyrin 111 + 10 d: 0.57 Coproporphyrin 111 + 15 d: 0.82 Pheophorbide a + 3.8 i 0.33 Binding reactions were performed with (+) or without (—) 1% DMSO. 70 > a. 8. N 0 Protein fluorescence x 105 cut .8 O 0'! 0| o w r r v 1 290 300 320 340 360 380 400 Wavelength (nm) N 25' 201 15‘ 10“ Protein fluorescence x 105 - 0 . l 290 300 320 340 360 380 400 Wavelength (nm) Figure 2-8. Quantitative analysis of GUN4-binding DPIX and Mg-DPIX. A. Emission spectra of GUN4 with various amounts of Mg-DPIX. Emission spectra were recorded at 25°C using an excitation wavelength of 280 nm. A series of spectra that show the quenching of GUN4 protein fluorescence by increasing concentrations of Mg-DPIX is presented. B. Emission spectra of GUN4 with various amounts of DPIX. Emission spectra were recorded and are presented as in A, except that DPIX, rather than Mg- DPIX, was used to quench GUN4 protein fluorescence. C. GUN4 fluorescence quenching by Mg—DPIX. GUN4 protein fluorescence was measured in the presence of increasing concentrations of Mg-DPIX. The inset shows 20 pg of purified GUN4 analyzed by SDS-PAGE and Coomassie staining. D. GUN4 fluorescence quenching by DPIX. GUN4 protein fluorescence was measured as in C, except that protein fluorescence was quenched by increasing concentrations of DPIX. 71 '/ 7 N O N O o 1 A (’1 C) O 1 Fluorescence x 105 8 8 0 50 100 150 200 [Mg—Deuteroporphyrin IX] 11M U N o .3 01 Fluorescence x 105 O 0 50 100 150 200 [Deuteroporphyrin IX] pM Figure 2-8 (continued). Quantitative analysis of GUN4-binding DPIX and Mg-DPIX. A. Emission spectra of GUN4 with various amounts of Mg—DPIX. 72 3 il “3320 “525- ‘— T- 4’ X X 020" §15 2 8 8 (I) 111151 gm g g “1.101 c .5 173 5‘ 9 5 - 9 2 o. o. 0 . . . . 0 . . . . . . o 50 100 150 200 0 1o 20 30 4o 50 50 [PPIX] 11M [Mg-PPIX IX] 11M '2, 20 ,_ X c 15 m o (I) 9 10 o 3 c C '6 51 4...: o h 0. 0 o 50 100 150 200 250 300 [Mg-PPIX ME] 11M Figure 2-9. Quantitative analysis of GUN4 binding PPIX, Mg-PPIX, and Mg-PPIX ME. A. GUN4 fluorescence quenching by PPIX. GUN4 protein fluorescence was measured in the presence of increasing concentrations of PPIX. B. GUN4 fluorescence quenching by Mg-PPIX. GUN4 protein fluorescence was measured in the presence increasing concentrations of Mg-PPIX. C. GUN4 fluorescence quenching by Mg-PPIX ME. GUN4 protein fluorescence was measured in the presence of increasing concentrations of Mg-PPIX ME. 73 > [D . LO 1 "(’3 2511 o 25 v- ‘- 1’ x 20 j’ x it 8 g 20 1‘1 . 8 , 3 15 a) 15 (D d) h h 8 1oJ 3 1 ~ 11—— 11—— 0 .E .E 93 5+ «“3 5 8 9 ll ' ' O. 0 1 1 1 1 1 1 1 0 1 f 1 1 1 f 1 0 50 100 150 200 250 300 350 O 50 100 150 200 250 300 350 [Coproporphyrin Ill] 11M [Uroporphyrin III] (M C D m 'I o 20 0 $2 25 '- 0 x x 11 a) 20 0 8 15‘ g u 5 a1 ‘1’ o . 0 a) 15 (D q) 1 g 10 5 3 g 10 ‘ i .g '5 5 ‘ (D . 1.1 "" 5 o 2 L- n_ A A n J 0. n 1 1 1 1 1 1 0 T 1 1 W 1 1 1 O O 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 [Hemin] 11M [Pheophorbide 3] 11M Figure 2-10. Quantitative analysis of GUN4 binding uroporphyrin III, coproporphyrin III, hemin, and pheophorbide a. A. GUN4 fluorescence quenching by uroporphyrin III. GUN4 protein fluorescence was measured in the presence of increasing concentrations of uroporphyrin III. B. GUN4 fluorescence quenching by coproporphryin III. GUN4 protein fluorescence was measured in the presence of increasing concentrations of coproporphryin III. C. GUN4 fluorescence quenching by hemin. GUN4 protein fluorescence was measured in the presence of increasing concentrations of hemin. D. GUN4 fluorescence quenching by pheophorbide a. GUN4 protein fluorescence was measured in the presence of increasing concentrations of pheophorbide a. 74 observed for the homologous residues (i.e., V135A, F 196A, and R214A) in SynGUN4 (Verdecia et al., 2005). The solubilities of PPIX and Mg-PPIX were not sufficient for us to quantitate the affinities of F191A, V123A, and R211A for these natural ligands (N.D.A., unpublished data). Subchloroplastic distribution of G UN4 proteins with porphyrin-binding defects. To test whether porphyrin-binding defects might affect interactions between GUN4 and chloroplast membranes, we imported F191A, V123A, and R211A into ALA-fed chloroplasts and fractionated these chloroplasts into soluble and membrane-containing pellet fractions. A smaller percentage of V123A associated with the membrane- containing pellet fraction compared to the wild-type GUN4, and barely detectable levels of F 191A and R211A were found in the pellet fraction (Figure 2-12). Additionally, and in contrast to wild-type GUN4, ALA feeding did not affect the subchloroplastic distribution ofFl9lA, V123A, or R211A (Figure 2-12). Mg-chelatase activity in chloroplast membranes of ALA -fed chloroplasts. Because GUN4 binds Cth and stimulates Mg-chelatase (Larkin et al., 2003; Davison etal., 2005; Verdecia et al., 2005), the finding that boosting PPIX and Mg-PPIX levels in purified chloroplasts by ALA feeding causes GUN4 to accumulate in the membrane- containing pellet fraction suggests that ALA feeding might affect the Mg-chelatase activity that was previously reported to associate with pea chloroplast membranes (Walker and Weinstein, 1991). 75 \Q". i Table 2-4. Quantitation of DPIX and Mg-DPIX . binding by GUN4 containing the indicated amino acid substitutions Substitution KdDP'X(pM) KdMg'DP'X (11M) V123A 9.6 i 0.44 8.0 i 0.44 F191A 12:I: 0.77 7.7i0.37 R211A 14i0.83 14i0.64 76 \:'\'. 200 160 - 110- 80- 60- 50- 40' 30- 20‘ 15- 10% 3.5‘ _x 01 15‘ A v _L O 103’. Fluorescence x 105 01 O 0o 1'00 260 500 460 0 50 100150 260 250 [Mg-Deuteroporphyrin IX] 11M [Deuteroporphyrin IX] 11M Figure 2-11. Quantitative analysis of V123A, F 191A, and R211A binding DPIX and Mg—DPIX. A. Quantitative analysis of V123A binding Mg-DPIX and DPIX. Purified GUN4 (20 pg) containing the V123A substitution was analyzed by SDS-PAGE and Coomassie staining (left). Fluorescence of GUN4 containing the V123A substitution was measured in the presence of increasing concentrations of Mg-DPIX (middle) or DPIX (right). B. Quantitative analysis of F191A binding Mg-DPIX and DPIX. Purified GUN4 (20 pg) containing the F 191A substitution was analyzed by SDS-PAGE and Coomassie staining (left). Fluorescence of GUN4 containing the F191A substitution was measured in the presence of increasing concentrations of Mg-DPIX (middle) or DPIX (right). C. Quantitative analysis of R21 1A binding Mg-DPIX and DPIX. Purified GUN4 (20 pg) containing the R211A substitution was analyzed by SDS-PAGE and Coomassie staining (left). Fluorescence of GUN4 containing the R211A substitution was measured in the presence of increasing concentrations of Mg-DPIX (middle) or DPIX (right). 77 TH' raj 200 ' 160 - 1 10 - 80 - 60 - 50 30- 205 15- 10- 3.5 - N 9 A v 1511 A c; 01 Fluorescence x 105 0o 51) 160 1502110 250 [Mg-Deuteroporphyrin IX] 11M 20- 10 0 o 50 too 150 200 2750 [Deuteroporphyrin IX] 1.1M Figure 2-11 (continued). Quantitative analysis of V123A, F191A, and R211A binding DPIX and Mg-DPIX. 78 Tre. / l 2004 160- 110— 80- 50.. 50- 4o- 3.5- % 15‘ “E: 20‘ \- ‘- X 1’ x 15‘ 810‘ 8 1’ 5 5 o 0 10 w 5, m 9 9 5. O O 2 2 LL . . 1 a . 1 LL 0 . . . . . 0 0 50 100 150 200 250 0 50 100 150 200 250 [Deuteroporphyrin IX] 11M [Deuteroporphyrin IX] 11M Figure 2-11 (continued). Quantitative analysis of V123A, F191A, and R211A binding DPIX and Mg-DPIX. 79 .. + .. + ALA TPPSPS TPPSPS 4- prGUN4 4- mGUN4 V123A F191A Wild type R211A Figure 2-12. Subchloroplastic distribution of porphyrin-binding—deficient GUN4 alter ALA feeding. GUN4 mutants with DPIX- and Mg—DPIX-binding defects were imported into chloroplasts that had been pretreated with or without ALA. After import, chloroplasts were lysed, fractionated, and analyzed as in Figure 2-1A. The position of the GUN4 precursor containing the transit peptide (prGUN4), the major form of mature GUN4 generated by proteolytic removal of the transit peptide during import into the chloroplast (mGUN4), and a minor form of mature GUN4 (*) are indicated. Representative fluorograms from three independent experiments are shown for GUN4 containing the amino acid sequence found in wild type and for GUN4 containing the amino acid substitutionsV123A, F191A and R211A. 80 0'! O 1——-—4 I——— -1 Col-O GUN4-14 F191 F191A-14 R211A-2.2 R211A-2.5 SGS3 RP+LP sgs3 RP+LBa1 GUN4 RP+LP gun4-2 RP+LBa1 a! GUN4-I4 Vgun4-I . F191A-1 3.,F191A-14 R211A-2.2 C? T: U 1 .1 R211A-2.5 Figure 3-1. Analysis of stably transformed Arabidopsis plants containing GUN4-related transgenes. (A) Screening of stably transformed Arabidopsis plants containing GUN4- related transgenes for sgs3 and gun4-2 T-DNA insertion alleles. Genomic DNA was extracted from the indicated mutant or the indicated stably transformed line. Transgenic plants were from the T5 generation or a subsequent generation. Plants were screened for wild type and T-DNA insertion alleles by PCR-based genotyping with Oligonucleotides that can amplify PCR products from SGS3 (SGS3 RP + LP), GUN4 (GUN4 RP +LP) or particular T-DNA insertion alleles (sgs3 RP + LBaI or gun4-2 RP + LBal). PCR products were identified by electrophoresis in agarose gels followed by staining with ethidium bromide. (B) Analysis of GUN4 protein levels in stably transformed Arabidopsis plants containing GUN4-related transgenes. Protein was extracted from the indicated mutant or the indicated stably transformed line grown in 100 pmol m'2 s'l broad spectrum white light. Aliquots of these whole seedling extracts that contained 10 pg of protein were analyzed by immunoblotting using anti-GUN4 antibodies (upper panel). After immunoblotting each membrane was stained with Coomassie blue (lower panel). Mass standards are indicated at the left in kDa. 117 Based on previous analyses of GUN4 and SynGUN4 (Verdecia et al., 2005; Adhikari et al., 2009), we predicted that lines expressing F191A and R211A would contain reduced Mg-chelatase activity and therefore would exhibit chlorophyll deficiencies relative to wild type. Indeed, we found that, like the chlorophyll-deficient gun4-1 (Vinti et al., 2000; Mochizuki et al., 2001), the F191A- and R211A-expressing lines accumulate 1.3- to 2.0-fold less chlorophyll per mg fresh weight than wild type when these seedlings were grown for 7 d in 100 pmol m'2 3'1 white light (Figure 3-2; Figure 3-3A and B). Under these conditions, gun4-1 accumulated 2.9-fold less chlorophyll per mg fresh weight than wild type (Figure 3-2; Figure 3-3), which is consistent with previous analyses of gun4-I (V inti et al., 2000; Mochizuki et al., 2001). Mutants that are impaired in their ability to synthesize chlorophyll were previously reported to exhibit more severe chlorophyll deficiencies after fluence rates were increased (F albel et al., 1996). To test whether these F 191A- and R211A-expressing lines exhibit light-sensitive phenotypes similar to other chlorophyll-deficient mutants, we transferred them to a higher fluence rate. We found that high-intensity light enhances chlorophyll deficiencies in gun4-1 and the F191A- and R211A-expressing lines. When 3-d-old seedlings were transferred from 100 pmol m'2 s'1 to 850 pmol In2 S'1 and grown for an additional 4 d, the transgenic lines contained two- to 4-fold less chlorophyll than wild type and gun4-1 contained 40-fold less chlorophyll than wild type (Figure 3-2; Figure 3-4A and B). Next, we tested whether available Arabidopsis cth mutants exhibited light-sensitive phenotypes similar to gun4-1, R211A, and F191A. For these experiments, we tested 118 €1.4- 9 £1.21 1: 31.0 D 50.8- %] 1 gas §O.4~ i 0.2- 2 [L 00.04 VFFVN‘D “ #:3703538? E3555< “:5 D [21.2 a (D ug§ D Figure 3-2. Analysis of chlorophyll levels in gun4 and cth/gun5 mutants grown under different fluence rates. Seedlings were grown in continuous 100 pmol rn'2 3'1 white light for 7d (white bars) or seedlings were grown for 3d in continuouleO pmol In2 5'1 white light then 4d in continuous 850 pmol m'2 3‘1 white light (gray bars). Chlorophyll was extracted from at least three biological replicates for each mutant or line in each condition. Error bars indicate 95% confidence intervals. 119 gun5—101 Col-O GUN4-14 R211A-2.2 F191A-14 vol“? 3 ‘T'T‘t‘P.’ S s'r‘.‘<<<< ‘T 92v553: torn BDg—mefigg UOmmutrtIQUtOt Figure 3-3. Images of seedlings grown in 100 pmol m'2 s" white for 7d. (A) Representative plate containing seedlings grown in 100 pmol rn'2 s'l white light for 7d. Each sector contains >20 seedlings. Wild type (Col-0), mutants, and transgenic lines are indicated. (B) Representative individual seedlings grown in 100 pmol m'2 s'l white light for 7d. Wild type (Col-0), mutants, and transgenic lines are indicated. 120 gun5—101 COI-O I 1 gun4-1 cch F191A-1 R211A-2.2 F191A-14 Figure 34. Images of seedlings grown in 100 pmol m'2 s'I white light for 4d and then shifted to 850 pmol m'2 s'l white light for 3d. (A) Representative plate containing seedlings grown in 100 pmol rn'2 s'l white light for 4d then shifted to 850 pmol m"2 3'1 white light for 3d. Each sector contains >20 seedlings. Wild type (Col-0), mutants, and transgenic lines are indicated. (B) Representative individual seedlings grown in 100 pmol m'2 s'l white light for 4d then shifted to 850 pmol m'2 5‘1 white light for 3d. Wild type (Col-0), mutants, and transgenic lines are indicated. 121 gun5 and cch mutants, which are missense alleles of the Cth gene in Arabidopsis (Mochizuki et al., 2001). We hereafter refer to this gene as Cth/GUN5 to avoid the confusion that can come from different nomenclature. We also tested the Arabidopsis gun5-101 mutant, which we isolated from a new gun mutant screen (Ruckle et al., 2007). gun5-101 was mapped (Figure 3-5A) and found to be a novel missense allele that causes a P450L substitution in the derived amino acid sequence of Cth/GUNS (Figure 3-5B). We also tested the cs mutant, which is strikingly deficient in chlorophyll because of a defect in the ChlI subunit of Mg-chelatase (Koncz et al., 1990; Mochizuki et al., 2001; Adhikari et al., 2009). gun5, cch, and gun5-101 accumulate 2.0- to 6.7-fold less chlorophyll than wild type when seedlings are grown in 100 pmol rn’2 s'l for 7 d (Figure 3-2; Figure 3-3A and B). gun5 and gun5-101 accumulate 10- and 120-fold less chlorophyll, respectively, than wild type when seedlings are grown in 100 pmol m"2 s'1 for 3 d and then transferred to 850 pmol rn'2 s'I for 4 (I (Figure 3-2; Figure 3-4A and B). We could not extract detectable levels of chlorophyll when cch was grown under these conditions (Figure 3-2; Figure 3-4A and B). The cs mutant contains 2-fold less chlorophyll than wild type when seedlings are grown in 100 pmol m'2 5" for 7 d and 8- fold less chlorophyll than wild type when 3-d-old seedlings are transferred from 100 pmol rn'2 s'l to 850 pmol m'2 s'1 and grown for an additional 4 (1. Thus, all of the transgenic lines exhibit chlorophyll deficiencies compared to wild type, as previously observed for chlorophyll-deficient mutants. To explore the mechanism underlying these light-sensitive phenotypes, we next monitored the levels of GUN4 and Mg-chelatase subunit levels by immunoblotting. We observed a striking reduction in GUN4 protein levels in R211A, F191A, gun4-1, cch, and 122 gun5-101 T24H18 T3185 T22N19 T6|14 MXE10 MAC12 MUA22 < l l l l l l i» (——200 kb -—--——-> Recombinants 9 2 0 0 0 0 0 Recombinants 0 0 0 0 0 1 5 B gun5-101 P450L cch gun5 CCT->CTT P642L A990V 11 1 Figure 3-5. Positional cloning and sequence analysis of gun5-101 . (A) Positional cloning of gun5-1 01 . The chlorophyll-deficient phenotype was mapped based on an analysis of 21 10 chromosomes from F2 progeny that resulted from a gun5-101 >< Landsberg erecta cross and exhibited a chlorophyll-deficient phenotype similar to gun5- 101. Chromosomes were analyzed using SSLP and CAPS markers (12). The relative positions and names of bacterial artificial chromosome clones from which each marker was derived are indicated. The recombinants that are centromere distal (top) and centromere proximal (bottom) relative to gun5-101 are indicated. The location of gun5- 101 within the 200 kb interval that is defined by thee recombinants is indicated. (B) Nucleotide and derived amino acid sequences of gun5-1 01 . Lines and boxes indicate introns and exons, respectively. Light gray boxes indicate untranslated exons. The altered codon and the substitution in the derived amino acid sequence found in gun5- 101 are indicated. The positions of the single nucleotide substitutions and the substitutions in the derived amino acid sequence found in gun5 and cch are also indicated. 123 GUN4-14 gun4-1 R211A 2 2 R211A-2.5 F191A-1 F191A-14 gun5 gun5-101 Si 8 § 8 LL HL LL HL LL HL LL HL LL HL LL HL LL HL LL HL LL HL LL HL LL HL _ ' +14 ' GUN4 .- .111... u on Cth "' Chll can . ~ Cth Figure 3-6. Analysis of GUN4 and Mg—chelatase subunit levels in 100 pmol m'2 s‘1 and 850 pmol m'2 5’1 white light. Seedlings were grown in 100 pmol m'2 s'] for 7d (LL) and 100 pmol m’2 s'] for 3d and 850 pmol m'2 5'1 white light for 4d (HL) as indicated in Figure 3-2. Protein was extracted from the indicated mutant or the indicated stably transformed line. Aliquots of these whole seedling extracts that contained 10 pg of protein were analyzed by immunoblotting using anti-GUN4 antibodies, anti-Cth antibodies, anti-ChlI antibodies, or anti-Cth antibodies. Exposures were adjusted so that the faint bands in HL extracts are observable. 124 gun5-101 relative to wild type after seedlings are transferred from 100 pmol m'2 s'1 to 850 pmol m'2 s'1 (Figure 3-6). In contrast, ChlI—I/GUNS, ChlI, and Cth levels were either unaffected or only slightly reduced following these fluence-rate shifts (Figure 3- 6). Transferring cs and gun5 to higher fluence rates had no effect on the accumulation of the GUN4 protein (Figure 3-6). These data indicate that the striking reduction of GUN4 protein levels that occurs in bright light is dependent on particular amino acid substitutions in GUN4 and Cth; weak loss-of-fimction alleles of Cth, such as gun5, and strong loss-of—function alleles of ChlI, such as cs, have no effect (Figure 3-6). Analysis of interactions between GUN4, Cth/GUNS, and chloroplast membranes Previous findings indicate that porphyrin binding promotes the association of GUN4 with chloroplast membranes (Adhikari et al., 2009). Based on these findings, we expected that F191A and R211A would not associate with chloroplast membranes as stably as GUN4. We also analyzed gun4-I (Larkin et al., 2003). A similar or greater percentage of GUN4 is expected in the membrane-containing pellet fractions derived from gun4-1 and wild type because amino acid substitutions homologous to L88F increase the porphyrin-binding affinities of Thermosynechococcus elongatus GUN4 and Synechocystis GUN4 (Davison et al., 2005). Indeed, we found that when purified chloroplasts were lysed and fractionated, significantly less GUN4 was associated with the membrane-containing pellet fractions derived from R211A and F191 A than with those derived from wild type and that the distribution of GUN4 in soluble and membrane-containing pellet fractions was similar in gun4-1 and wild type (Figure 3-7A and B; Figure 3-8). 125 N 3, at “ < 11 5 .- 5 93 " 0 or u. E - + - + - + - + CD -I A 8 '8 ‘33 Percent GUN4 a 8 concern 1\ ”Nun's. gun E18311!” I h ‘e‘ 3 D Figure 3-7. Distribution of GUN4 in lysed and fractionated chloroplasts that were purified from gun4 and cth/gun5 mutants and were either fed or not fed with PPIX. (A) Representative immunoblots showing the distribution of GUN4 in lysed and fractionated chloroplasts that were purified from gun4 and cth/gun5 mutants and fed or not fed with PPIX. Purified intact chloroplasts (200 pg) were either fed (+) or not fed (-) with 20 pM PPIX. These chloroplasts were then fractionated into soluble and membrane-containing pellet fractions of equal volume. Equal volumes were analyzed by SDS-PAGE and immunoblotting with anti-GUN4 antibodies. (B) Quantitative analysis of GUN4 immunoblots showing the distribution of GUN4 in fractionated chloroplasts that were purified from gun4 and cth/gun5 mutants and fed or not fed with PPIX. The percent of GUN4 in the pellet (white bars) and supernatant (light-gray bars) fractions derived from chloroplasts that were not fed PPIX and the percent GUN4 in the pellet (medium-gray bars) and supernatant (dark-gray bars) fractions derived from chloroplasts that were fed PPIX are indicated for wild type (Col-O) and each mutant and transgenic line. Results from at least 2 independent experiments are shown. Error bars represent 95% confidence intervals. 126 gun5 Col-O gun4-1 F191A-14 El-t o R211A-2.2 30 b 100* _E_ C q. z a 3 801 o {— c E 60* 3 40- O. 201 0 4:_ . 8 § 16 § 61 Figure 3-8. Statistical analysis of GUN4 in membrane-containing pellet fractions derived from purified and fractionated chloroplasts. Chloroplasts were purified from wild type (Col-0) or the indicated mutants or transgenic lines. These chloroplasts were fractionated and GUN4 protein levels determined in membrane-containing pellet fractions as described in Figure 4. Significance differences among genetic backgrounds were tested among replicates and independent experiments using the unpaired t-test. Data from at least three and as many as 11 independent experiments are shown. a, GUN4 protein levels in the membrane-containing pellet fractions derived from Col-0 chloroplasts were found to be very significantly different from those derived from gun4— ], F191A-14, R211A-2.2, cch, gun5-101 (P<0.005) but not gun5 (P=0.2). b, GUN4 protein levels in the membrane-containing pellet fractions derived from gun4-1 were found to be significantly different from those derived from gun5 (P=0.05) or very significantly different from those derived from gun4-1, F191A-14, R211A-2.2, cch, gun5-101 (P<0.009). c, GUN4 protein levels in the membrane-containing pellet fractions derived from gun4-1, F 191A-14, R211A-2.2, cch, gun5, and gun5-101 were not significantly different from each other (P201). 127 Although previous findings indicate that GUN4 associates with Cth and that both proteins interact with chloroplast membranes, whether interaction between these two proteins is required for their association with chloroplast membranes is not clear (Larkin et al., 2003; Adhikari et al., 2009). To test whether the interactions between GUN4 and chloroplast membranes depend on Cth/GUNS, we purified chloroplasts from gun5, cch, and gun5-101, then lysed and fractionated them into soluble and membrane- containing pellet fractions. We found that significantly less GUN4 associated with the .V" membrane-containing pellet fractions derived from cch and gun5-101 than with those derived from wild type (Figure 3-7A and B; Figure 3-8). The weakness of the gun5 allele relative to gun5-101 and cch (Vinti et al., 2000; Mochizuki et al., 2001) likely explains the lack of a significant effect in gun5 relative to wild type (Figure 3-7; Figure 3-8). This effect on the membrane association of GUN4 appears specific to strong alleles of gun4 and gun5 because we did not find significantly less GUN4 protein in the membrane-containing pellet fractions derived from either gun5 or cs relative to wild type (Figure 3-9). In these same experiments, more Cth/GUN5 accumulated in the supernatant and less in the membrane-containing pellet fraction among these transgenic lines, mutants, and wild type (Figure 3-10; Table 3-2). The reduction in the association between Cth/GUNS and chloroplast membranes in these transgenic lines, mutants, and wild type is somewhat muted compared to our findings with GUN4. The distribution of pea GUN4 and in vitro translated and imported GUN4 are similar in fractionated pea chloroplasts to those reported here for fractionated Arabidopsis 128 11.5 o o I l 1 Percent GUN4 s a e N O 1 O Col-0 Figure 3-9. Distribution of GUN4 in soluble and membrane-containing pellet fractions derived from wild type, gun5, and cs chloroplasts. Intact chloroplasts were purified from wild type (Col-0), gun5, and cs. Purified chloroplasts were fed (+) or not fed (-) with PPIX, fractionated, and analyzed by immunoblotting with anti-GUN4 antibodies as described in Figure 3-7. The membrane-containing pellet (P or white bars) and supernatant fractions (S or gray bars) in the resulting immunoblot (above) were quantified by chemiluminescence from the immunoreactive bands (below). 129 Table 3-2. Percent decrease in the membrane association of Cth in untreated samples compared to WT. Error is represented by standard error of 2 independent experiments. Decrease in membrane Line association of Cth in untreated samples gun4-1 13 (i6) % F191A14 9(16)% R211A2-2 14(:I:1)% cch 20 (i1) % gun5 17 (i5) % gun5-101 20 (i2) % 130 chloroplasts. Additionally, the interactions between pea chloroplast membranes and both pea GUN4 and the in vitro translated and imported GUN4 were previously reported to be enhanced by inducing an increase in the porphyrin levels of purified chloroplasts. In contrast, in vitro translated and imported R211A or F191A do not associate with pea chloroplast membranes, regardless of whether porphyrin levels are increased (Adhikari et al., 2009). The inability of elevated porphyrin levels to promote interactions between pea chloroplast membranes and either R211A or F 191A is consistent with (1) R211A and F191A not only affecting porphyrin binding but also disrupting interactions between GUN4 and chloroplast membranes by inhibiting some other function of GUN4 besides porphyrin binding or (2) a technical limitation of the pea system such as the vast molar excess of pea GUN4 competing more effectively with in vitro translated and imported R211A and F191A than with in vitro translated and imported wild-type GUN4. To distinguish between these possibilities, we induced an increase in the porphyrin levels of chloroplasts that were purified from gun4 and gun5/cth mutants by feeding these chloroplasts with PPIX, as previously described by Adhikari et al. (2009). PPIX feeding did not have a major impact on the membrane association of GUN4 protein in wild type, gun4-1, or cs (Figure 3-7A and B; Figure 3- 9). Further promotion by PPIX of interactions between GUN4 and chloroplast membranes may not be possible in wild type, gun4-1, and cs because the bulk of GUN4 already stably associates with the membrane-containing pellet fractions of unfed chloroplasts purified from wild type, gun4-I, and cs when chloroplasts are lysed and fractionated under these conditions (Figure 3-7A and B; Figure 3-9). In contrast, feeding PPIX to chloroplasts purified from the F19lA- and R211A-expressing lines 131 caused a 20% and 45% increase in the amount of F 191A and R211A, respectively, retained in the membrane-containing pellet fraction (Figure 3-7A and B; Table 3-3). Based on these findings, we conclude that R211A and F 191A can disrupt porphyrin binding in vivo. We also conclude that in vitro translated and imported R211A or F 191A probably do not associate with pea chloroplast membranes regardless of whether porphyrin levels are increased, because the vast molar excess of pea GUN4 competes more effectively with R211A and F 191A than with wild type GUN4, as previously g suggested by Adhikari et al. (2009). We also observed that PPIX feeding of chloroplasts purified from gun5 and gun5-101—but not cch—caused at least a 20% increase in L, GUN4 protein levels in the membrane-containing pellet fractions (Figure 3-7; Table 3- 3). Based on these findings, we conclude that interactions between GUN4 and chloroplast membranes are promoted by GUN4 binding both PPIX and ChlI-I/GUNS. ' By analyzing these same fractions by immunoblotting with anti-Cth/GUNS antibodies, we found that the interactions between GUNS/Cth and chloroplast membranes are disrupted in all of these mutants relative to wild type. However, in contrast to GUN4, PPIX feeding did not affect the interactions between GUNS/Cth and chloroplast membranes in any of these mutants (Figure 3-10). Analysis of ROS-inducible gene expression Because porphyrins are photosensitizers, misregulated chlorophyll biosynthesis driving elevated ROS-induced cellular damage was previously suggested to in part explain the light sensitivity of chlorophyll-biosynthesis mutants (Falbel et al., 1996). However, ROS is produced not only from collisions between chlorophyll precursors and Oz in 132 Col-0 F191A-14 R211A-2.2 Percent ChIH E o Col-0 gun4-1 ,1 g 4 . .- 91A—14 —_ , _ . mg i.) l I it : ii i! 1 -1 .t j N. In “ N g e < c: 115 ‘- t "‘ 3 or E E Figure 3-10. Distribution of Cth/GUNS in lysed and fractionated chloroplasts that were purified from gun4 and cth/gun5 mutants and were either fed or not fed with PPIX. (A) Representative immunoblots showing the distribution of Cth/GUNS in lysed and fractionated chloroplasts that were purified from gun4 and cth/gun5 mutants and fed (+) or not fed (-) with PPIX. The same fractions described in Figure 4 were analyzed by immunoblotting with anti-Cth/GUNS antibodies. As described for Figure 4, 200 pg of purified intact chloroplasts were either fed (+) or not fed (-) with 20 pM PPIX. These chloroplasts were then fractionated into soluble and membrane-containing pellet fractions of equal volume. Equal volumes were analyzed by SDS-PAGE and immunoblotting. (B) Quantitative analysis of Cth/GUNS immunoblots showing the distribution of GUN4 in chloroplasts that were purified from gun4 and cth/gun5 mutants and fed or not fed with PPIX. The percent Cth/GUNS in the pellet (white bars) and supernatant (light-gray bars) fractions derived from chloroplasts that were not fed PPIX and the percent Cth/GUN5 in the pellet (medium-gray bars) and supernatant (dark-gray bars) fractions derived from chloroplasts that were fed PPIX are indicated for wild type (Col-0) and each mutant and transgenic line. Results from at least two independent experiments are shown. Error bars represent 95% confidence intervals. 133 Table 3-3. Percent increase in GUN4 protein in the membrane -containing pellet fraction following PPIX feeding. Line Percent increase in GUN4 protein in the membrane- containing pellet fraction following PPIX feeding Col-O l3iS% gun4-I 1i2% F191A 21:1:2% R211A 45:1:O% cch 4i2% gun5 20i16% F gun5-101 23i10% Error is represented by 95% confidence intervals. N22 134 bright light but can also be produced from photosystems and the photosynthetic electron transfer (N iyogi, 1999; Li et al., 2009). To distinguish between these possibilities, we grew the gun4 and cth/gun5 mutants under 10 pmol m'2 s'l white light. The levels of chlorophyll, levels and compositions of the photosystems, and thylakoid ultrastructures were previously reported to be more similar in comparisons between particular chlorophyll-deficient mutants and wild type when plants are grown under low fluence rates (Allen et al., 1988; Falbel et al., 1996). We noted that in 10 pmol m'2 5'1 white light, the chlorophyll levels of gun4-1 , both F191A lines, both R211A lines, gun5, and gun5-101 are essentially the same as in wild type (Figure 3-11A, B, and C). Chlorophyll levels in cch were slightly less than wild type (Figure 3-11A, B, and C). Therefore, in this 10 pmol rn'2 s'l light and shortly after seedlings are transferred to 850 pmol m‘2 3'1 white light, we expect that differences in ROS levels among these mutants, transgenic lines, and wild type will largely result from misregulated chlorophyll metabolism and that different electron transport activities will be responsible for a smaller proportion of the ROS. To test whether elevated ROS might contribute to the light sensitivities of these mutants and transgenic lines, we monitored the expression levels of ROS-inducible genes following fluence rate increases. We chose to monitor the expression of WRK 1’40 and ZA T10 because the expression of these genes is induced 10- to 250-fold in response to diverse ROS (Gadjev et al., 2006) and the expression levels of these genes range from the 60th to 88th percentile based on publicly available microarray data (N DA, unpublished observations). Therefore, monitoring the expression of these genes provides a sensitive 135 A gun5«101 Col-0 cch R211A-2.5 / F191A-1 R211A-2.2 F191A-14 3 ‘T'T‘t‘ct‘ 0 s'r“<<<< ‘T 9273553: "8‘2 33:1- N :1: OOmuEmgéuu .1 O 0.6 ' Chlorophyll (nmol/ mg fresh weight) e$$$§$ :55; Col-O E gun4-1 3 F191A-1 3 F191A-14 :E—l R211A-2.2 E R211A-2.5 2—1 cch :1 gun5 E gun5-101 ‘E Figure 3-11. Analysis of chlorophyll levels in gun4 and cth/gun5 mutants grown in 10 pmol m'2 s'I white light. (A) Representative plate containing seedlings grown in 10 pmol rn'2 s'I white light. Seedlings were grown in 10 pmol m"2 5'1 white light for 7d. (B) Representative cotyledons from seedlings described in A. (C) Quantitative analysis of chlorophyll levels in seedlings described in A. Seedlings were grown as described in A. Chlorophyll was extracted from at least three biological replicates for wild type (Col-0) and for each of the indicated mutants and transgenic lines. Error bars indicate 95% confidence intervals. 136 assay for the production of ROS. We grew the seedlings in 10 pmol m‘2 3'1 white light for 7 d and then transferred them to 850 pmol m'2 s'1 white light. We extracted RNA from seedlings that were collected immediately before this fluence-rate shift (0 h) and at 0.5 h, 1 h, and 3 h after this shift. We then quantified the levels of mRN A transcribed from WRKY40 and ZAT10. Consistent with this previous work (Gadjev et al., 2006), we found that WRK Y 4 0 and ZA T1 O-derived transcripts accumulate in wild type after the fluence-rate shift (Figure 3-12). Additionally, we found that the mRNAs transcribed from WRKY40 and ZAT10 were present at elevated levels in R211A and F191A relative to wild type when seedlings were grown in 10 pmol in2 3'1 white light and at levels similar to wild type after this fluence-rate shift (Figure 3-12). To further test whether the induced expression of WRKY40 and ZA T10 that was observed in 10 pmol m'2 s'l is triggered by porphyrin-derived ROS, we monitored their expression in continuous and diurnal light. There .is a burst of PPIX and Mg-PPIX biosynthesis at dawn that causes PPIX and Mg-PPIX to accumulate to readily detectable levels (Popperl et al., 1998; Papenbrock et al., 1999); this buildup of porphyrins in diurnal relative to continuous light causes photooxidative stress in mutants with defects in porphyrin metabolism (Meskauskiene et al., 2001; Peter and Grim, 2009). (1) To test whether GUN4 and/or Cth/GUNS perform porphyrin-binding functions that are distinct from their chlorophyll biosynthetic functions and (2) to further test whether these elevated levels of WRK 1’40 and ZAT10 expression are triggered by porphyrin- derived ROS, we quantified their expression levels in a diurnal cycle that contained 12 h of 2 pmol m'2 3" white light followed by 12 h of darkness. If GUN4 or a GUN4- 137 30 1 WRKY40 (A11 980840) 25‘ l 20‘ Relative expression :1 R21A-2.2 35 - ZAT10 (A11927730) 25- 20- 15- Relative expression 10 '0 33' t . it . .-.~. ‘1‘- $5 . 1?! 1“ 161. "'1 Col-0 F191A-14 R211A-2.2 Figure 3-12. Induction of WRK 1’40 and ZATI 0 expression during a fluence-rate shift. Wild type (Col-0), F191A-14, and R211A-2.2 were grown for 7d in 10 pmol In2 S'1 white light and then transferred to 850 pmol In2 S". Transcripts from WRKY40 (At1g80840; upper panel) and ZATIO (At1g27730; lower panel) were quantified by means of qRT-PCR at 0h (white bars), 0.5h (light-gray bars), 1h (medium-gray bars), and 3h (dark gray bars) after the fluence rate shift. Expression is reported relative to Col-0 at 0h, which was assigned a value of 1. Four biological replicates were analyzed at each time point. Error bars represent standard error. 138 Cth/GUN5 complex binds these pools of porphyrins that accumulate during the diurnal cycle to shield them from collisions with 02 and if these GUN4-porphyrin and GUN4-Cth/GUN5-porphyrin complexes are a distinct pool relative to the GUN4- porphyrin and the GUN4-Cth/GUN5-porphyrin complexes that are associated with the chlorophyll biosynthetic pathway, R211A and cch should accumulate more ROS than es and the mRNAs transcribed from WRK Y 40 and ZAT10 should accumulate to higher levels in R211A and cch relative to cs in both continuous and diurnal light. Any ROS derived from only impaired Mg-chelatase activity is expected to be similar in R211A- 2.2 and cs because these mutants have similar chlorophyll-deficient phenotypes (Figure 3-2). The expression of both WRKY40 and ZATI 0 was significantly induced 6- to 17- fold in R211A-2.2, cs, and cch relative to wild type under these diurnal conditions (Figure 3-13). Neither R211A nor cch accumulated significantly more ROS than cs. Based on the gene expression assay used here, we conclude that transgenic lines that express only porphyrin-binding-deficient versions of GUN4 and mutants with defects in either Cth/GUNS or ChlI exhibit similar ROS phenotypes. DISCUSSION Mg-chelatase is an unusual enzyme in that in most species, it requires a regulatory protein that binds one of its subunits, one of its substrates, and one of its products for robust activity. This proteinaceous cofactor, GUN4, was proposed to have evolved in part to attenuate the production of ROS by shielding PPIX and Mg-PPIX from collisions with 02 (Larkin et al., 2003; Verdecia et al., 2005) and to channel PPIX and Mg-PPIX into chlorophyll biosynthesis by binding PPIX, Mg-PPIX, and Cth/GUNS I39 251 WRKY40 (At1980840) : 20- .Q U) 8 ~ ~— 15 15- X Q) m % 1% 10- E * Q) 0: 5. 1 1. c.> N. s s 6 “1‘ 0 0 .<. K CE 20- ZAT10(At1927730) .5 151 '1 (D 0) 2 ~- 0. * * 1’13 10 ll- 01 .2 l. 311' o [I 5~ 0 1 i O N U) C _1 - 0 0 o “1‘ 0 0 55 K (I Figure 3-13. Analysis of WRK Y 40 and ZATI 0 expression in diurnal and continuous light. Seedlings were grown for 7d in 2 pmol m'2 3" white light that was a diurnal cycle containingl2h of light and12 h of dark (white bars) or was continuous light (gray bars). All seedlings were harvested 1h after dawn. Transcripts from WRK Y 40 (At1g80840; upper panel) and ZAT10(At1g27730; lower panel) were quantified by means of qRT- PCR. Expression is reported relative to Col-0 in continuous light, which was assigned a value of 1. Four biological replicates were analyzed at each time point. Error bars represent standard error. * , indicates a significant difference (P<0.05) relative to wild type according to the unpaired t-test. 140 on chloroplast membranes (Adhikari et al., 2009). These ideas cannot be tested with the previously available gun4 mutants because these mutants contain either null alleles or severe loss-of—function alleles that accumulate barely detectable levels of GUN4 protein. These mutants exhibit severe and pleiotropic phenotypes that are characterized by both severe chlorophyll deficiencies or albinism and abnormal photosynthetic membranes (Vinti et al., 2000; Mochizuki et al., 2001; Larkin et al., 2003; Wilde et al., 2004; Sobotka et al., 2008). Therefore, the analysis of the first set of gun4 alleles in E Arabidopsis and Synechocystis did not indicate whether the loss of the porphyrin- binding activity or the loss of some other function of GUN4 might be responsible for these extreme phenotypes. Additionally, data collected from a heterologous pea system and reported by Adhikari et al. (2009) did not clearly indicate whether amino acid substitutions that disrupt the porphyrin-binding activity of GUN4 in vitro also disrupt the porphyrin-binding activity of GUN4 in vivo. In this report, we tested whether the porphyrin-binding activity that was previously determined to be important for GUN4 activity in vitro (Adhikari etal., 2009) contributes to the porphyrin-binding activity of GUN4 in vivo and whether this activity is significant in vivo. We expressed gun4 ' alleles encoding versions of GUN4 that contain single amino acid substitutions that were previously shown to significantly inhibit the porphyrin-binding activity of GUN4 in vitro (Adhikari et al., 2009) and expressed these porphyrin-binding—deficient versions of GUN4 in stably transformed gun4 knockout mutant gun4-2. We found that not only do these amino acid substitutions inhibit the porphyrin binding activity of GUN4 in vivo, but that transgenic plants expressing only the porphyrin-binding-deficient versions of GUN4 contain lower levels of chlorophyll than wild type, lower levels of GUN4 141 associated with chloroplast membranes than wild type, and elevated levels of ROS relative to wild type. GUN4 helps channel porphyrins into chlorophyll biosynthesis by binding Cth/GUNS on chloroplast membranes When purified chloroplasts were lysed and fractionated, we observed that F 191A and R211A—the porphyrin-binding-deficient versions of GUN4—accumulated to higher levels in soluble fractions and lower levels in membrane-containing pellet fractions than wild-type GUN4. Additionally, we observed that wild-type GUN4 accumulated to elevated levels in soluble fractions and lower levels in membrane-containing pellet fractions derived from chloroplasts that were purified from strong loss-of-function cth/gun5 mutants. Based on these data, we conclude that Cth/GUNS activity and the porphyrin-binding activity of GUN4 promote interactions between GUN4 and chloroplast membranes. The most parsimonious interpretation of these data is that a significant fraction of GUN4-porphyrin complexes is tethered to chloroplast membranes by binding active Cth/GUNS. To test whether the loss of the porphyrin-binding activity or the loss of some other function of F191A and R211A impairs interactions between GUN4 and both Cth/GUNS and chloroplast membranes, we fed PPIX to purified chloroplasts. The result was that F191A, R211A, and wild-type GUN4 in the gun5 and gun5-101 backgrounds accumulated in the membrane-containing pellet fractions of these chloroplasts. Based on these findings, we conclude that GUN4-porphyrin complexes 142 bind Cth/GUN5 and have a higher affinity for ChlI-I/GUNS on chloroplast membranes than does free GUN4. This effect of PPIX feeding on the affinity of GUN4 for chloroplast membranes does not occur in cch. The P642L substitution encoded by the cch allele may cause pleiotropic dysfimction in Cth/GUNS that includes an inability to distinguish between free and porphyrin-bound GUN4. Alternatively, cch contains significantly less active Cth/GUNS than wild type that may already be saturated with GUN4-porphyrin complexes prior to PPIX feeding. Another possible interpretation is that indirect effects that attenuate the activities of GUN4 and Cth/GUNS and are promoted by chlorophyll deficiencies may be more robust in cch relative to both gun5 and gun5-101 because cch is more severely chlorophyll deficient than both gun5 and gun5-101. Porphyrins were previously reported to promote the association of in vitro translated and imported GUN4, pea GUN4, and pea Cth/GUNS with pea chloroplast membranes. However, this effect was more robust with in vitro translated GUN4 and pea GUN4 than with pea Cth/GUNS. One possible interpretation of these data is that Cth/GUNS associates with chloroplast membranes by a mechanism that does not entirely depend on GUN4, and that GUN4-porphyrin complexes can nonetheless promote interactions between Cth/GUNS and chloroplast membranes (Adhikari et al., 2009). Here, we tested this idea using Arabidopsis mutants and transgenic plants. We expect that the interactions between chloroplast membranes and Cth/GUNS are conserved between Arabidopsis and pea. We found that ca. 70% of Cth/GUNS was in the membrane-containing pellet fraction when chloroplasts purified from wild-type 143 Arabidopsis chloroplasts were fractionated. In contrast, pea ChlI-I/GUNS was only ca. 50% associated with the membrane-containing pellet fraction when pea chloroplasts were fractionated; a rise in porphyrin levels caused ca. 70% of pea ChlI-I/GUNS to associate with the membrane-containing pellet fraction (Adhikari et al., 2009). Thus, we expect that Cth/GUNS already maximally associates with chloroplast membranes in the wild-type Arabidopsis system used here, thereby preventing PPIX feeding from further promoting interactions between chloroplast membranes and ChlI-I/GUNS in wild type. we found 40 to 50% of Cth/GUN5 in supernatant fractions and 50 to 60% in the membrane—containing pellet fractions when chloroplasts purified from the transgenic lines, gun4-1, and all cth/gun5 mutants tested were fractionated. This reduction in the membrane association of Cth/GUNS was somewhat muted compared to GUN4. Also, in contrast to GUN4, PPIX feeding had no effect on the interactions between Cth/GUNS and chloroplast membranes. These findings indicate that although GUN4 can promote interactions between Cth/GUNS and chloroplast membranes, Cth/GUNS likely associates with chloroplast membranes using a mechanism that depends relatively less on GUN4 than the mechanism used by GUN4 to associate with chloroplast membranes, which depends relatively more on Cth/GUNS. Nonetheless, GUN4 can promote interactions between ChlI-I/GUNS and chloroplast membranes. The inability of PPIX feeding to promote interactions between Cth/GUNS and chloroplast membranes in any of the transgenic lines, gun4-1, gun5, cch, and gun5-101 provides evidence that porphyrin binding is less important for promoting interactions between chloroplast membranes and Cth/GUNS than for promoting interactions between chloroplast membranes and GUN4. Even so, the previous work of Adhikari et al. (2009) 144 indicates that porphyrins can promote interactions between Cth/GUNS and chloroplast membranes, albeit significantly less than for GUN4, consistent with the work reported here. A complex mechanism likely explains the light sensitivities of chlorophyll-deficient mutants Similar to other mutants with defects in chlorophyll biosynthesis (Falbel et al., 1996), the transgenic lines expressing F191A and R211A, gun4-1, and all of the cth/gun5 mutants used in this study exhibited significant chlorophyll deficiencies relative to wild type when they were transferred from 100 to 850 pmol rn'2 s'I white light. GUN4 protein levels decreased to very low or undetectable levels in gun4-1, F 191A, R211A, gun5-101, and cch but not in gun5 and cs mutants following these fluence rate shifts. These lower levels of GUN4 protein could be caused by an enhanced turnover of GUN4 protein and/or reduced expression of the G UN4 gene. This striking reduction of GUN4 protein levels was specific to GUN4; similar reductions were not observed for the Cth/GUNS, ChlI, or Cth subunits of Mg—chelatase. Consistent with these findings, Peter and Grim (2009) did not observe a striking reduction in Mg-chelatase subunit levels in gun4 mutants. Based on previous kinetic analysis of SynGUN4 and Synechocystis Mg-chelatase (Larkin et al., 2003; Davison et al., 2005; Verdecia et al., 2005), we expect that this reduction in GUN4 protein levels will significantly attenuate Mg-chelatase activity. It will be interesting to test whether this reduction in GUN4 protein levels is a rapid response that contributes significantly to the down-regulation of chlorophyll biosynthesis when tetrapyrrole metabolism is misregulated. 145 We also monitored the expression levels of Z4T10 and WRK Y 4 0, two ROS-inducible genes, and found evidence that F 191A, R211A, and wild type accumulate similar levels of ROS following a fluence rate shift. We found that ROS production increases when wild type and all the mutants tested were transferred from 10 to 850 pmol rn'2 s'l white light. Our findings, however, do not support a model in which the enhanced I chlorophyll-deficient phenotypes of the F191A- and R211A-expressing lines in bright light are explained by more ROS accumulating in these transgenic lines relative to wild type following fluence-rate shifts fi'om dim to bright light. On the contrary, although we found that the F 191A- and R211A-expressing lines, cch, and cs mutants accumulate more ROS than wild type in 2 to 10 pmol m'2 s'l continuous white light and in 2 pmol m'2 s'1 diurnal-dim light, our data indicate that these F191A- and R211A-expressing lines and wild type accumulate similar levels of ROS after a fluence-rate shift from 10 pmol 111'2 s'1 to 850 pmol rn'2 3". Therefore, we propose that the enhanced chlorophyll deficiencies of mutants with these defects in chlorophyll biosynthesis may result from a complex mechanism that depends not only on light-induced production of ROS but also other light-regulated processes. (1) Following a fluence-rate shift, the lower rates of chlorophyll biosynthesis that occur in these chlorophyll biosynthesis mutants relative to wild type may be insufficient to compensate for the chlorophyll a that is turned over; the rate of chlorophyll a turnover can increase in high-intensity light (Beisel et al., 2010). The striking reductions in the levels of GUN4 protein observed in gun4-1, F191A, R211A, gun5-101, and cch reported here is expected to further attenuate rates of chlorophyll biosynthesis, thereby further promoting chlorophyll deficiencies. (2) If 146 some effect besides lower rates of chlorophyll biosynthesis is responsible for the light sensitivities of these mutants, this effect would not appear to be elevated ROS because WRKY40 and ZATI 0 expression is not elevated in R211A and F191A following a fluence-rate shift. The data reported here are consistent with these Mg-chelatase- deficient mutants exhibiting an uncoupling of ROS production and some other light- intensity-dependent process such as photoreceptor-based light signaling. The light sensitivities of these chlorophyll-deficient mutants may be explained by effects on photoreceptor-based light signaling if the elevated ROS observed in 2 and 10 pmol m'2 s'1 white light reprograrns light signaling to promote chlorophyll deficiencies when the mutants are transferred to 850 pmol rn'2 s'l light. Consistent with this interpretation, elevated chloroplastic ROS that is derived from the over-accumulation of chlorophyll precursors induces albinism by a mechanism that depends on the blue-light receptor cryptochrome 1 (Danon et al., 2006). Also, plastid dysfunction triggered by inhibitors of chloroplast biogenesis have been reported to convert cryptochrome l and other photoreceptors from positive to negative regulators of photosynthesis-related genes (Ruckle et al., 2007). These effects of plastid dysfunction on light signaling were proposed to protect chloroplasts from stress and dysfunction by helping to balance processes that promote and processes that attenuate photooxidative stress within the chloroplast (Ruckle et al., 2007 ; Larkin and Ruckle, 2008) or in extreme cases promote cell death (Danon et al., 2006; Kim et al., 2008). The findings that GUN4-porphyrin complexes predominantly associate with Cth/GUN5 on chloroplast membranes in vivo and that transgenic Arabidopsis plants 147 that express only porphyrin-binding-deficient versions of GUN4 exhibit elevated ROS- inducible gene expression have two major implications. First, these findings support a role for the porphyrin-binding activity of GUN4 in the channeling of PPIX into chlorophyll biosynthesis. Based on previous kinetic analysis of SynGUN4 and Synechocystis Mg-chelatase, GUN4-PPIX-Cth/GUN5 complexes that accumulate on chloroplast membranes are expected to be converted to GUN4-Cth/GUN5-Mg-PPIX complexes after interacting with the ChlI and Cth subunits of Mg-chelatase (Larkin et al., 2003; Davison et al., 2005; Verdecia et al., 2005). Second, these findings provide insight into the mechanism by which plants protect themselves from the photooxidative stress derived from the PPIX and Mg-PPIX that accumulate to readily detectable levels during the light-phase of diurnal cycles (Pbpperl et al., 1998; Papenbrock et al., 1999; Mochizuki et al., 2008; Moulin et al., 2008). We tested whether GUN4 or a GUN4- Cth/GUNS complex might perform distinct roles (1) in binding pools of PPIX and Mg-PPIX associated with chlorophyll biosynthesis and (2) in binding separate pools of diurnally accumulating PPIX and Mg-PPIX with the purpose of protecting plants from ROS by shielding these porphyrins from collisions with 02. The only source of porphyrin-derived ROS in the es mutant is from porphyrins immediately associated with chlorophyll biosynthesis and not from a separate pool of porphyrins that is not associated with the chlorophyll biosynthetic pathway because cs mutants have defects in ChlI, which does not bind porphyrins. In contrast, in R211A and cch, porphyrin- derived ROS could be derived from either the PPIX and Mg-PPIX associated with the chlorophyll biosynthetic pathway or a separate pool of diurnally accumulating PPIX and Mg-PPIX, because R211A and cch have defects in GUN4 and Chth/GUNS—two 148 PPIX- and Mg-PPIX-binding proteins. Therefore, if a significant fraction of the diurnally accumulating PPIX and Mg-PPIX forms a pool that is distinct from the PPIX and Mg-PPIX that associates with the chlorophyll biosynthetic pathway, R211A or both R211A and cch would accumulate higher levels of ROS than cs, and higher levels of mRNAs from WRKY40 and ZAT10 than cs. Further, R211A would accumulate higher levels of mRNAs transcribed from WRK Y 40 and ZA T10 than cch if GUN4 contributed to photooxidative stress tolerance independently of Cth. The finding that R221A, cch, and cs express similar amounts of mRNA from WRKY40 and ZATI 0 is most consistent with the model in which the PPIX and Mg-PPIX that accumulates during the diurnal cycle does not associate with GUN4 or a ChlI-I/GUNS-GUN4 complex in pools that are distinct from the pools of PPIX and Mg-PPIX that are associated with the chlorophyll biosynthetic pathway. Rather, our findings are most consistent with a model in which the PPIX and Mg-PPIX that builds up during the day associate with the GUN4 and ChlI-I/GUNS associated with the chlorophyll biosynthetic pathway. In this model, the PPIX and Mg-PPIX that accumulates to readily detectable levels during the day remains bound to the substrate and product binding sites of chlorophyll biosynthetic enzymes; this association with enzymes of the chlorophyll biosynthetic pathway shields these chlorophyll precursors from collisions with 02, thereby protecting plants from the production of ROS that can cause photooxidative damage. We propose that some event such as the accumulation of pigment-binding apoproteins somehow triggers (l) the conversion of these chlorophyll precursors into chlorophyll and (2) the rapid sequestration of chlorophyll into pigment-protein complexes. I49 CHAPTER 4 CONCLUSIONS AND FUTURE PERSPECTIVES In this thesis, I tested several hypotheses and report several conceptual advances for GUN4. First, I report on a technical advance to porphyrin-binding assays that led to the first demonstration that GUN4 can bind PPIX and Mg-PPIX. Previous quantitative analysis of porphyrin binding utilized cyanobacterial relatives of GUN4 (Larkin et al., 2003; Davison et al., 2005; Verdecia et al., 2005); GUN4 was previously only shown to bind porphyrins in qualitative assays (Larkin et al., 2003). Also, previous quantitative binding assays utilized the unnatural ligands deuteroporphryin IX (DPIX) and Mg- deuteroporphyin IX (Mg-DPIX). DPIX and Mg-DPIX lack two vinyl groups found in PPIX and Mg-PPIX and are therefore more water soluble than PPIX and Mg-PPIX. Thus, no data was previously published showing that GUN4 or any GUN4 relative can actually bind PPIX and Mg-PPIX. I modified the previously used porphyrin—binding assay (Karger et al., 2001; Larkin et al., 2003; Davison et al., 2005; Verdecia et al., 2005) by adding 1% dimethyl sulfoxide (DMSO). When 1% DMSO was added to these binding assays, the solubilities of PPIX and Mg-PPIX are increased enough to quantify their binding to GUN4. Additionally, based on results from binding assays that utilized DPD( and Mg-DPIX :I: 1% DMSO, we concluded that 1% DMSO may not affect the affinity of GUN4 for its natural porphyrin ligands. Using this new porphyrin binding assay, the binding constants of GUN4 for protoporphyrin IX, Mg-protoporphyrin IX and a variety of other natural porphyrins were reported for the first time. GUN4 was found to bind Mg-PPIX with a 2-9 fold higher affinity than other natural porphyrins. These findings are consistent with previous findings showing that cyanobacterial 150 relatives of GUN4 bind Mg-DPIX with higher affinities than other porphyrins. This technical advance may be useful for quantifying the porphyrin binding activity of other proteins or quantifiying the binding activity of proteins to other hydrophobic molecules besides porphryins. Second, I provide evidence that GUN4 regulates chlorophyll biosynthesis by a novel I regulatory mechanism. Previously, cyanobacterial relatives of GUN4 were shown to stimulate Mg-chelatase activity in vitro. Based on sequence similarity, the copurification of Cth with GUN4, and the chlorophyll-deficient phenotype of gun4 mutants, GUN4 was also proposed to stimulate Mg-chelatase activity in plants. In this thesis, I tested whether poprhryin binding might promote interactions between GUN4 and Mg-chelatase subunits with chloroplast membranes. The first set of experiments followed subchloroplastic distribution of in vitro translated and imported GUN4, pea GUN4, and pea Mg-chelatase subunits after inducing a rise in porphyrin levels in purified pea chloroplasts—the site of chlorophyll biosynthesis. I show that porphyrin binding promotes the association of in vitro translated and imported GUN4, pea GUN4, pea Cth with pea chloroplast membranes. These findings are consistent with GUN4 promoting chlorophyll biosynthesis not only by stimulating Mg-chelatase activity but also by channeling porphyrins into chlorophyll biosynthesis. These findings are also consistent with GUN4 stimulating chlorophyll biosynthesis not only by activating Mg- chelatase but also by promoting interactions between Cth and chloroplast membranes. 151 Third, I developed a genetic approach to test whether the porphyrin-dependent binding of GUN4 depends on Cth and vice versa. This genetic approach was also useful for testing whether the porphyrin- and chloroplast-membrane-binding activity of GUN4 helps protect plants from photooxidative stress. Additionally, this genetic approach was useful for resolving an issue from my work with pea chloroplasts. Based on the work with pea, we could not conclude whether residues in GUN4 that were shown to be important for porphyrin-binding activity measured in vitro were also important for porphyrin binding in vivo or whether my inability to demonstrate that these residues are important for binding porphyrins in pea chloroplasts is due to a technical barrier of this system (Chapter 2). I developed transgenic Arabidopsis lines that stably express gun4 alleles encoding single amino acid substitutions that lower the affinity of GUN4 for porphyrins. I used these lines to show that residues in GUN4 that contribute to porphyrin binding in vitro also contribute to porphyrin binding in vivo. Further I used cth/gun5 mutants to show that the interactions between GUN4 and chloroplast membranes depend on Cth and that these interactions are strengthened by the porphyrin binding activity of GUN4. Also, I found that interactions between Cth and chloroplast membranes either do not depend on GUN4 or do not depend on GUN4 to the same degree as interactions between GUN4 and chloroplast membranes depend on Cth. Based on the data reported in Chapter 3, I further conclude that the residues that are important for porphyrin binding in vitro do not appear important for porphyrin binding in the heterologous pea system because of technical limitations of this system. Using the homologous Arabidopsis system, I showed residues that contribute to porphyrin binding in vitro also contribute to porphyrin binding in vivo. Further, based 152 on the chlorophyll-deficient phenotypes of these transgenic lines, I conclude that the porphyrin-binding activity of GUN4 that was quantified in vitro contributes to its activity in vivo. Thus, one major conclusion from the work presented in chapters 2 and 3 is that porphyrin binding promotes interactions between GUN4 and Cth on chloroplast membranes. In chapter 2, I report that increases in these complexes on chloroplast membranes are correlated with increases in Mg-chelatase activity on chloroplast membranes. Therefore, these data strongly support a role for GUN4 in channeling porphyrins into chlorophyll biosynthesis by forming GUN4-PPIX-Cth complexes on chloroplast membranes. I used this genetic approach to test whether the porphyrin-binding activity of GUN4 might help protect plants against photooxidative stress as was previously proposed (Larkin et al., 2003; Verdecia et al., 2005). Chloroplastic photooxidative stress can cause necrosis and apoptosis (Kim et al., 2008) and inactivation of Cth (Willows et al., 2003). Indeed, we found that residues of GUN4 that are important for porphyrin binding are also important for photooxidative stress tolerance in Arabidopsis. For these experiments I used the same transgenic Arabidopsis lines that stably express gun4 alleles encoding single amino acid substitutions that lower the affinity of GUN4 for porphyrins that I used in my subchloroplastic distribution experiments. I found that these lines and Mg-chelatase subunit gene mutants exhibit enhanced chlorophyll deficiencies when they are transferred to high-intensity light. I also found that these 153 transgenic lines and mutants exhibit elevated levels of ROS-inducible genes relative to wild type in dim light but not in high intensity light. Based on these data, I conclude that these transgenic lines and mutants contain elevated levels of ROS relative to wild type. After absorbing light, particular electronically excited states of porphyrins can transfer energy to Oz yielding singlet oxygen that subsequently yields other ROS. Yields of porphyrin-derived ROS increase as fluence rates increase. Therefore, one surprising result was that according to our ROS-inducible gene expression assay, these transgenic lines and mutants exhibit elevated levels of ROS relative to wild type in dim white light (e. g., 2 to 10 pmol m"2 s’l white light) but similar levels of ROS relative to u: wild type after plants are transferred to high intensity light (e. g., 850 pmol m'2 5'1 white light). Mutants with defects in chlorophyll biosynthesis have long been known to be sensitive to bright light. This light sensitivity has long been proposed to result from (1) enhanced chlorophyll turnover and inefficient chlorophyll biosynthesis in bright light and (2) ROS derived from free porphyrins in bright light. Based on the findings reported here, we conclude that elevated levels of ROS in bright light do not fully explain the light sensitivities of these chlorophyll-deficient mutants because ROS-inducible genes are not elevated in these chlorophyll biosynthesis-deficient transgenic lines and mutants when plants are shifted into intense light. An alternative explanation is that a light signaling that controls chloroplast function is reprogrammed by chloroplastic in dim light and this light signaling network promotes chlorophyll deficiencies when the transgenic lines and mutants are shifted into bright light. Consistent with this proposal, 154 elevated chloroplastic ROS triggered by misregulated chlorophyll metabolism induces albinism by a mechanism that depends on the blue-light receptor cryptochrome l (Danon et al., 2006). Also, plastid dysfunction triggered by inhibitors of chloroplast biogenesis have been reported to convert cryptochrome 1 and phytochrome B from positive to negative regulators of photosynthesis-related genes (Ruckle et al., 2007). These effects of plastid dysfunction on light signaling were proposed to protect chloroplasts from stress and dysfunction by balancing processes that promote and attenuate photooxidative stress within the chloroplast (Larkin and Ruckle, 2008). The approach that I developed for expressing engineered gun4 alleles in gun4-2 sgs3 double mutants resolves a previous major technical barrier; GUN4 expressing transgenes exhibit robust cosuppression (Larkin et al., 2003). This technical advance that will allow researchers to test other hypotheses related to the function of GUN4 in vivo in the future. 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