A Study of Chloroplast Biogenesis Within Variegated Mutants of Arabidopsis Andrew Alexander Foudree Iowa State University

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2012 A study of chloroplast biogenesis within variegated mutants of Arabidopsis Andrew Alexander Foudree Iowa State University

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A study of chloroplast biogenesis within variegated mutants of Arabidopsis

Andrew Alexander Foudree

A thesis submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Major: Plant Biology

Program of Study Committee: Steven R. Rodermel, Major Professor Martin Spalding David Oliver

Iowa State University

Ames, Iowa

2012

DEDICATION

I would like to dedicate this thesis to all of my teachers who without their support I would not have been able to complete this work.

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TABLE OF CONTENTS

LIST OF FIGURES ...... vii

ACKNOWLEDGEMENTS ...... ix

ABSTRACT ...... x

CHAPTER 1. Introduction: overview of immutans...... 1

1.1 Review of immutans...... 2

1.2 Generation of ROS in immutans and the process of photobleaching...... 4

1.3 Literature cited ...... 7

CHAPTER 2. Two approaches to the study of chloroplast development...... 11

2.1 Electron microscopy...... 11

2.2 Transcriptome profiling...... 12

2.3 Literature cited...... 14

CHAPTER 3. A brassinosteroid transcriptional network revealed by genome-wide identification of BES1 target genes in Arabidopsis thaliana...... 15

3.1 Research overview...... 15

3.2 Literature cited...... 18

3.3 Figures...... 19

CHAPTER 4. Chloroplast photooxidation-induced transcriptome reprogramming in Arabidopsis immutans white leaf sectors...... 21

4.1 Research overview...... 21

4.2 Literature cited...... 27

4.3 Figures...... 29

CHAPTER 5. Suppressor of variegation4, a new var2 suppressor locus, encodes a pioneer protein that is required for chloroplast biogenesis...... 33

5.1 Research overview...... 33

5.2 Literature cited...... 36

5.3 Figures...... 37

CHAPTER 6. PDS activity acts as a rheostat of retrograde signaling during early chloroplast biogenesis...... 38

6.1 Abstract...... 38

6.2 Bidirectional signaling between the chloroplast and the nucleus...... 39

6.3 Phytoene desaturase and photoprotection...... 40

6.4 Comparison of three Arabidopsis photooxidized tissues impaired in PDS function. . . . .42

6.5 A hypothesis: the redox state of the PQ pool acts as a rheostat of retrograde signaling...... 44

6.6 Acknowledgements...... 46

6.7 Literature cited...... 47

6.8 Figures...... 50

CHAPTER 7. Background: mechanism of variegation in immutans ...... 52

7.1 Introduction...... 52

7.2 Proplastid-to-chloroplast conversion: embryogenesis...... 52

7.3 Proplastid-to-chloroplast conversion: meristem, leaf primordia and early leaf development...... 55

7.4 Etioplast-to-chloroplast conversion: greening by de-etiolation...... 57

7.5 Literature cited...... 60

7.6 Figures...... 63 v

CHAPTER 8. The mechanism of variegation in immutans provides insight into chloroplast biogenesis...... 70

8.1 Abstract...... 70

8.2 Introduction...... 71

8.3 immutans...... 72

8.4 PTOX function...... 73

8.5 Mechanism of variegation: the threshold hypothesis...... 79

8.5.1 Studies using mature leaves...... 79

8.5.2 Studies using greening as a model system...... 81

8.6 Compensating factors...... 83

8.7 How do the green and white sectors of im differ?...... 84

8.8 Second-site suppressors of im variegation...... 85

8.9 AOX2 and AOX1a suppressors...... 87

8.10 pgr5 and crr2-2 suppressors...... 89

8.11 Acknowledgments...... 91

8.12 Literature cited...... 92

8.13 Figures...... 106

CHAPTER 9. Dark-grown immutans reveals IMMUTANS is essential for normal chloroplast development...... 109

9.1 Abstract...... 109

9.2 Introduction...... 110

9.3 Results...... 115

9.4 Discussion...... 120

9.5 Acknowledgements...... 124 vi

9.6 Literature cited...... 125

9.7 Figures...... 132

CHAPTER 10. Conclusions ...... 145

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LIST OF FIGURES

Figure 3.1 Total chlorophyll levels of wild-type and bes1-D mutant...... 19

Figure 3.2 Transmission electron micrographs of wild-type and bes1-D...... 20

Figure 4.1 Functional classes of genes repressed and induced in imW sectors...... 29

Figure 4.2 MapMan display of transcript profiling data...... 30

Figure 4.3 Schematic representation of the metabolic adaptations corresponding to major transcript changes in imW leaf sectors ...... 31

Figure 4.4 Variegated phenotype of wild-type Arabidopsis plants treated with NF...... 32

Figure 5.1 Chloroplast ultrastructure of wild-type and svr4-2...... 37

Figure 6.1 Model depicting the effect of PDS on plastoquinone redox status in immutans, NF-treated tissues and the pds3 mutant...... 50

Figure 6.2 A comparison of genes differentially expressed in white tissues...... 51

Figure 7.1 Heart stage embryo development in immutans and wild-type ...... 63

Figure 7.2 immutans and wild-type torpedo stage embryos ...... 64

Figure 7.3 im and im x arc6 protoplasts isolated from the leaves of 3 month old plants ...... 65

Figure 7.4 arc6 x im plastid ...... 66

Figure 7.5 Tissue section of arc6 x im variegated tissue...... 67

Figure 7.6 Chlorophyll and carotenoid delay during de-etiolation...... 68

Figure 8.1 Confocal microscopy of leaf primordia ...... 106

Figure 8.2 Model of PTOX as a cofactor of phytoene desaturase (PDS) ...... 107

Figure 8.3 Ultrastructural analysis of immutans ...... 108

Figure 9.1 Phenotypes of wild-type and immutans grown for three weeks on MS plates...... 132

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Figure 9.2 De-etiolated phenotype of wild-type and immutans during greening...... 133

Figure 9.3 Carotenoid levels for wild-type and immutans that were grown in the dark and exposed to light for 48 hours...... 134

Figure 9.4 Chlorophyll levels for wild-type and immutans that were grown in the dark and exposed to light for 48 hours...... 135

Figure 9.5 Confocal auto-fluorescence of immutans and wild-type during greening...... 136

Figure 9.6 Confocal autofluorescence of cotyledons taken at 5 hours of light exposure...... 137

Figure 9.7 Western blot of common chloroplast membrane proteins that are found in thylakoids ...... 138

Figure 9.8 TEM images of etiolated and 24 hour light exposed plastids and plastoglobule analysis...... 139

Figure 9.9 Transmission electron micrographs of immutans and wild-type plastids that were grown in the dark for 7 days...... 140

Figure 9.10 Western blot analysis of dark grown etiolated wild-type and immutans seedlings for plastoglobulin (PGL-35)...... 141

Figure 9.11 HPLC analysis of phytoene in immutans and wild-type seedlings grown in the dark as well as light exposed for 24 hours...... 142

Figure 9.12 Etiolated wild-type seedlings are grown on MS and MS with NF...... 143

Figure 9.13 ROS staining of wild-type and immutans seedlings that were light exposed for three hours after being grown in the dark ...... 144

ACKNOWLEDGEMENTS

I would like to take this opportunity to express my thanks to those who helped me with various aspects of conducting research and the writing of this thesis. First and foremost,

Dr. Steve Rodermel for his guidance throughout my research and the writing of this thesis.

His insights and writing expertise have contributed significantly to the assembly of this thesis. I would also like to thank my committee members for their efforts and contributions to this work: Dr. Steve Rodermel, Dr. David Oliver, and Dr. Martin Spalding. In addition I would like to thank Dr. Steve Whitham who served on my committee for a majority of my graduate school education. I would additionally like to thank Dr. Gustavo MacIntosh for his guidance throughout the initial stages of my graduate career as well as Dr. Harry Horner and

Tracey Pepper for their electron microscopy expertise and positive inspiration. I would also like to thank my friends and family for their positive reinforcement and financial assistance during the writing of this work. As well, I would like to thank the undergraduates that have worked on these projects: Jason Pierce, Burak Demirci, Michael Rand and Jenna Fussell.

Finally, I would like to thank the current and past colleagues in my lab for their support.

ABSTRACT

immutans (im) is a recessive Arabidopsis mutant that is involved in chloroplast biogenesis. In the absence of IMMUTANS (IM) green and white leaf sectors form. This thesis is concerned with the development of these green and white sectors and the methods used to investigate the mechanism of their formation. IM resides in the chloroplast thylakoid membrane and has been implicated in the adjustment of electron flux through the photosynthetic electron transport chain. Without IM white sector formation occurs due to the inability of im to produce colored carotenoids. It is still a mystery as to how green sectors are able to form in the absence of IM. This thesis presents im as a developmental chloroplast defect and discusses the need for IM very early in plastid development. The requirement for IM most likely starts within progenitor plastids known as proplastids.

CHAPTER 1. Introduction: overview of immutans

Variegation mutants have been used as a tool to observe how organelles interact with nuclear programmed cells to genetically control cellular development. Variegations can occur through mutations in the nuclear, mitochondrial, or chloroplast genomes and result in cells that are able to form normal chloroplasts while other chloroplasts become defective. It is important for these three organellar genomes to communicate in order for development to proceed normally. For this reason, variegation mutations are an excellent example for the study of organelle signaling and interaction. Causes of variegations occasionally occur by way of transposons, but the most well studied variegation mutants can be divided into two different categories: those that are caused by nuclear mutations and those that are caused by plastid mutations. Examples of variegation mutants that are plastid defects include IOJAP in maize and Albostrians in barley that result from a lack of chloroplast ribosomes (Han et al.,

1992; Han and Martienssen, 1995; Hess et al., 1994). An example of a nuclear mutation that results in variegation is that of the maize nonchromosomal stripe mutant, which is caused by a mitochondrial defect (Rousell et al., 1991; Gu et al., 1993). Several different nuclear variegation mutants have been discovered in the model system Arabidopsis and include var1, var2, chloroplast mutator, and immutans (Yu et al., 2007). However, relative to the known number of mutations in Arabidopsis, this is a small number. This thesis is primarily concerned with the variegated Arabidopsis mutant immutans that was originally discovered and studied by Rédei and Röbbelen in the early 1960s (Rédei, 1963; Rédei, 1967; Röbbelen,

1968).

1.1 Review of immutans

immutans is a nuclear recessive mutation that results in white and green sectors that contain defective and normal chloroplasts respectively (Wetzel et al., 1994; Wu et al., 1999).

As a rare event, in white cell sectors, green plastids have been found (Wetzel et al., 1994).

This indicates that the immutans defect can be plastid autonomous. While green sectors expand and develop a normal cellular morphology, white sectors fail to expand to normal size and exhibit irregular looking cells (Aluru et al., 2001). immutans is highly light sensitive during early development and must therefore be germinated under low light in order to survive. If immutans is germinated under high light, the plastids within the cotyledons become photobleached and result in albino plants that eventually die. immutans has also been found to be influenced by temperature during growth, with plants becoming more variegated when grown in cold temperatures due to lower activity of enzymes in downstream processes (e.g. Calvin cycle) (Rosso et al., 2006).

IMMUTANS was originally cloned by our lab and was found to be located on the fourth chromosome in Arabidopsis (Wu et al., 1999). An EMS generated allele that was used to do the chromosomal walk, and is predominantly called “Spotty”, contains a G to A mutation in the fourth exon of IMMUTANS. IMMUTANS encodes a nuclear gene that shows similarities to the mitochondrial alternative oxidase protein (AOX) (Carol and Kuntz.,

2001; Wu et al., 1999). Mitochondrial AOX consists of a family of genes that are found in many plant, algae, fungal, and protist species (McDonald, 2008; Moore and Albury, 2008).

In mitochondria, AOX is an oxidase that is involved in the control of the electron transport chain at the step of ubiquinone, and is known as the cyanide resistance pathway 3

(Vanlerberghe and Mcintosh, 1997; Siedow and Umbach, 2000). Therefore, due to its

similarity with AOX, immutans was labeled as a Plastid Terminal Oxidase or PTOX (Josse et al., 2000; Joet et al., 2002). IMMUTANS has two transmembrane domains and is found as a peripheral, stromal facing, thylakoid membrane protein. Similar to AOX, IMMUTANS is a diiron protein in which the iron atoms receive electrons from the photosynthetic electron transport chain (Fu et al., 2005). However, IMMUTANS lacks a dimerization domain and contains an exon 8 sequence that is not found in AOX (Fu et al., 2005). Experiments involving IMMUTANS using EPR and site directed mutagenesis have enforced the structural similarities that exist between IMMUTANS and mitochondrial AOX (Siedow et al., 1995; Fu et al., 2009).

Mitochondrial AOX is involved in the transfer of electrons from the ubiquinone pool to molecular oxygen to form water, thus preventing the ubiquinone pool from becoming over-reduced when electron flow is high or downstream electron carriers become blocked or limiting. On this basis, it was hypothesized that the chloroplast protein IMMUTANS also plays a similar role, and is hypothesized to act as the terminal oxidase in chlororespiration

(Wu et al., 1999). In the case of the chloroplast, it is thought that IMMUTANS is able to relieve electron “excitation” pressure that occurs during high light scenarios (Rosso et al.,

2006). However, this process most likely occurs during early development and not when leaves have become fully developed (Rosso et al., 2006; Aluru et al., 2006). While conducting metabolite analysis of the white sectors versus the green sectors, it was observed that the white sectors of immutans accumulated the colorless carotenoid phytoene (Wetzel et al., 1994). This observation pointed to a blockage in the carotenoid pathway at the site of the enzyme phytoene desaturase (PDS), however IMMUTANS does not encode the gene for 4

PDS. Interestingly, PDS needs oxidized plastoquinone (PQ) in order to catalyze this step in

carotenogenesis (Norris et al., 1995). The link between plastoquinone and PDS was

previously provided by albino mutants that were thought to be defects of PDS gene, but were

actually mutations in the plastoquinone biosynthesis pathway (Norris et al., 1995). It was

therefore hypothesized that without IMMUTANS, the plastoquinone pool would become

over-reduced during high light exposure, and indirectly block the function of PDS (Wu et al.,

1999; Aluru et al., 2006). This would cause the activity of PDS to be leaky because some

small amount of oxidized PQ would still be present depending on the PQ turnover rate at

photosystem I (PSI). This would result in low PDS activity that would only be able to

produce small levels of carotenoids. The protective nature of these low levels of colored

carotenoids appears to be most critical during the early development of immutans. With a

much lower amount of carotenoids, the photosynthetic apparatus fails to develop and

immutans plants photobleach and die without protection during germination (Rosso et al.,

2009). However, if immutans plants are germinated and grown under low light for a

relatively long amount of time after germination (e.g. 3-4 weeks), normal looking green

plants will develop completely green leaves with no white sectors and will stay green

irrespective of the light conditions (Rosso et al., 2006).

1.2 Generation of ROS in immutans and the process of photobleaching

Photooxidative damage of cell structures has long been investigated and has been

shown to be a product of our current oxygenic environment. Radical Oxygen Species (ROS)

are formed in the chloroplast by way of triplet chlorophyll. A large majority of ROS in leaves 5 are therefore generated at PSI and PSII within the thylakoid membrane. It has become

necessary for plants to evolve a system to respond to oxidative damage, and it has been

proposed that several preventative measures have resulted from this evolutionary process. It

is evident that compounds that protect against ROS, such as carotenoids, are crucial to the

generation of green sectors in immutans. Synthesis, membrane integration, and storage of carotenoids are intimately linked to the variegation process within immutans. It has also been

proposed that ROS play a role not only in photobleaching, but also in the process of

retrograde signaling between the chloroplast and the nucleus (either directly or indirectly)

(Meskauskiene et al., 2001). Formation of chloroplasts depends on the translation of multiple

nuclear genes that require coordination so that they can be made at the proper time with the

proper stoichiometry to balance chloroplast metabolism and assembly. In order for this to

occur, signals relating the overall status of the chloroplast must be conferred to the nucleus

from the chloroplast to balance nuclear transcription with chloroplast translation. This

process defines retrograde signaling. Research has revealed that retrograde signaling is

complex and most likely involves multiple pathways (Beck, 2005). The first direct evidence

of retrograde signaling was founded upon the discovery that the nuclear-encoded light

harvesting complex (LHC) protein is down-regulated in photobleached tissues. This finding

led to initial studies that pointed to chlorophyll precursors as possible signaling molecules

(Strand et al., 2003). However, now it is thought that the chlorophyll precursors may not

serve directly as signaling molecules and that perhaps the signals may be indirectly related to

these compounds (Moulin et al., 2008, Mochizuki et al., 2008). Interestingly, unbound

chlorophyll precursors are able to generate ROS and could possibly be the indirect

component of chlorophyll precursor retrograde signaling. One type of ROS signal that has 6 been shown to be involved during chloroplast development is singlet oxygen (Meskauskiene

et al., 2001). Discovery of the flu mutant is seminal in the research and elucidation of this

process. Since FLU is unable to perform feedback inhibition of the production of

protochlorophyllide (chlorophyll precursor that is a photosensitizer) when grown in the dark,

it photobleaches and turns white on exposure to light. It is therefore similar to immutans

with respect to the nature of photobleaching, and that they are both developmental mutants.

Since immutans develops both white and green sectors, the green sectors must somehow be

able to form even though they lack IMMUTANS. It is possible that retrograde signaling

differs between the green and white tissue types and therefore compensating factors are

activated to a greater extent within the green sectors. It is also possible that green sectors can

form because they are able to generate plastids with a higher ability to withstand ROS

species (e.g. more colored carotenoids). It is known that after sectors are developed, either

green or white, high light and low light respectively cannot change this sectoring. Whatever

mechanism plastids use to protect from ROS in immutans, it must occur early on during

plastid development to determine if a plastid will become white or green. This thesis sets the

groundwork for the role that carotenoid levels and ROS formation within immutans play in

the development of white and green sectors during early plastid development.

1.3 Literature cited

Aluru, M., Bae, H., Wu, D., and Rodermel, S. (2001). The Arabidopsis immutans Mutation

Affects Plastid Differentiation and the Morophogenesis of White and Green Sectors in

Variegated Plants. Plant Physiol. 127, 67-77.

Aluru, M., Yu, F., Fu, A., and Rodermel, S. (2006). Arabidopsis variegation mutants: new

insights into chloroplast biogenesis. J Exp Bot. 57:9, 1871-1881.

Beck, C.F. (2005). Signaling pathways from the chloroplast to the nucleus. Planta. 222, 743-

756.

Carol, P., and Kuntz, M. (2001). A plastid terminal oxidase comes to light: implications for

carotenoid biosynthesis and chlororespiration. Trends in Plant Science. 6, 31-36.

Fu, A., Aluru, M., and Rodermel, S. (2009). Conserved Active Site Sequences in Arabidopsis

Plastid Terminal Oxidase (PTOX). J Biol Chem. 284, 22625-22632.

Fu, A., Park, S., and Rodermel, S. (2005). Sequences Required for the Activity of PTOX

(IMMUTANS), a Plastid Terminal Oxidase. J Biol Chem. 280, 42489-42496.

Gu, J., Miles, D., and Kewton, K.J. (1993). Analysis of leaf sectors in the NCS6

mitochondrial mutant of maize. Plant Cell. 5, 963-971.

Han, C.D., Coe, E.H., and Martienssen, R.A. (1992). Molecular cloning and characterization

of iojap (ij), a pattern striping gene of maize. EMBO J. 11, 4037-4046.

Han, C.D., and Martienssen, R.A. (1995). The iojap protein (IJ) is associated with 50S

chloroplast ribosome subunits. Maize Coop Newsl. 69, 32.

Hess, W.R., Muller, A., and Borner, T. (1994). Ribosome-deficient plastids affect

transcription of light-induced nuclear genes: Genetic evidence for a plastid-derived 8

signal. Mol Gen Genet. 242, 305-312.

Joet, T., Genty, B., Josse, E.M., Kuntz, M., Cournac, L., and Peltier, G. (2002). Involvement

of a Plastid Terminal Oxidase in Plastoquione Oxidation as Evidenced by Expression

of Arabidopsis thaliana Enzyme in Tobacco. J Biol Chem. 277, 31623-31630.

Josse, E., Simkin, A.J., Gaffe, J., Laboure, A-M., Kuntz, M., and Carol, P. (2000). A Plastid

Terminal Oxidase Associated with Carotenoid Desaturation during Chromoplast

Differentiation. Plant Physiol. 123:4, 1427-1436.

McDonald, A. E. (2008). Alternative oxidase: an inter-kingdom perspective on the function

and regulation of this broadly distributed “cyanideresistant” terminal oxidase. Funct

Plant Biol. 35, 535–552.

Meskauskiene, R., Nater, M., Goslings, D., Kessler, F., op den Camp, R., and Apel, K.

(2001). FLU: A negative regulator of chlorophyll biosynthesis in Arabidopsis

thaliana. Proc Natl Acad Sci. 98:22, 12826-12831.

Mochizuki, N., Tanaka, R., Tanaka, A., Masuda, T., and Nagatani, A. (2008). The steady-state

level of Mg-protoporphyrin IX is not a determinant of plastid-to-nucleus signaling in

Arabidopsis. Proc Natl Acad Sci. 105:39, 15184-15189.

Moore, A.L., and Albury, M.S. (2008). Further insights into the structure of the alternative

oxidase: from plants to parasites. Biochem Soc Trans. 36, 1022-1026.

Moulin, M., McCormac, A., Terry, M., and Smith, A. (2008). Tetrapyrrole profiling in

Arabidopsis seedlings reveals that retrograde plastid nuclear signaling is not due to

Mg-protoporphyrin IX accumulation. Proc Natl Acad Sci. 105:39, 15178-15183.

Norris, S.R., Barrette, T.R., and DellaPenna, D. (1995). Genetic Dissection of Carotenoid

Synthesis in Arabidopsis Defines Plastoquinone as an Essential Component of 9

Phytoene Desaturation. The Plant Cell. 7:12, 2139-2149.

Rédei, G.P. (1963). Somatic instability caused by a cysteine-sensitive gene locus in

Arabidopsis. Mutat Res. 18, 149-162.

Rédei, G.P. (1967). Biochemical aspects of a genetically determined variegation in

Arabidopsis. Genetics. 56, 431-443.

Röbbelen, G. (1968). Genbedingte Rotlicht-Empfindlichkeit der

Chloroplastendifferenzierung be Arabidopsis. Planta. 80, 237-254.

Rosso, D., Bode, R., Li. W., Krol, M., Saccon, D., Wang, S., Schillaci, L.A., Rodermel, S.,

Maxwell, D., and Huner, N. (2009). The Plant Cell. Photosynthetic Redox Imbalance

Governs Leaf Sectoring in the Arabidopsis thaliana Variegation Mutants immutans,

spotty, var1, and var2. The Plant Cell. 21, 3473-3492.

Rosso, D., Ivanov, A.G., Fu, A., Geisler-Lee, J., Hendrickson, L., Geisler, G.S., Krol, M.,

Hurry, V., Rodermel, S., Maxwell, D.P., and Huner, N. (2006). IMMUTANS Does

Not Act as a Stress-Induced Valve in the Protection of the Photosynthetic Apparatus

of Arabidopsis during Steady-State Photosynthesis. Plant Physiol. 142, 574-584.

Rousell, D.L., Thompson, D.L., Pallardy, S.G., Miles, D., and Newton, K.J. (1991).

Chloroplast structure and function is altered in the NCS2 maize mitochondrial

mutant. Plant Physiol. 96, 232-238.

Siedow, J.N., and Umbach A.L. (2000). The mitochondrial cyanide-resistant oxidase:

structural conservation amid regulatory diversity. Biochim Biophys Acta. 1459, 432-

439.

Siedow, J.N., Umbach, A.L., and Moore, A.L. (1995). The active site of the cyanide-resistant

oxidase from plant mitochondria contains a binuclear iron center. FEBS Lett. 362, 10- 10

14.

Strand, A., Asami, T., Alonso, J., Ecker, J.R., and Chory, J. (2003). Chloroplast to nucleus

communication triggered by accumulation of Mg-protoporphorin IX. Nature. 421, 79-

83.

Vanlerberghe, G.C., and Mcintosh, L. (1997). Alternative oxidase: from gene to function.

Annu Rev Plant Physiol Plant Mol Biol. 48, 703-734.

Wetzel, C.M., Jiang, C., Meehan, L., Voytas, D. and Rodermel, S. (1994). Nuclear-organelle

interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous

and impaired in carotenoid biosynthesis. The Plant J. 6:2, 161-175.

Wu, D., Wright, D.A., Wetzel, C., Voytas, D.F. and Rodermel, S. (1999). The IMMUTANS

Variegation Locus of Arabidopsis Defines a Mitochondrial Alternative Oxidase

Homolog That Functions during Early Chloroplast Biogenesis. The Plant Cell. 11,

43-55.

Yu, F., Fu, A., Aluru, M., Park, S., Xu, Y., Liu, H., Liu, X., Foudree, A., Nambogga, M., and

Rodermel, S. (2007). Variegation mutants and mechanisms of chloroplast biogenesis.

Plant Cell Environ. 30:3, 350-365.

CHAPTER 2. Two approaches to the study of

chloroplast development

2.1 Electron microscopy

Over the course of my time as a graduate student I have been involved in a number of projects. These projects have led me to become proficient in two techniques. My facility with electron microscopy has allowed me to contribute to two publications. My contributions will be described in the context of these publications (Chapters 3 and 5). I also learned how to analyze microarrays, and these analyses formed the basis of a first author paper in Plant

Signaling & Behavior (Chapter 6) and authorship on another in Plant Physiology (Chapter 4).

Electron microscopy gives one the ability to look directly into cellular processes. To use transmission electron microscopy (TEM) on plant tissues the material must be fixed and embedded in resin or cryogenically frozen and sectioned. This thesis will use the former technique, which has been used for many years and was originally perfected in the early

1960's (Sabatini et al., 1963). This technique involves fixing the tissue in buffered 2% gluteraldehyde and 2% paraformaldehyde. It is important to fix the samples and embed them quickly to reduce the generation of artifacts. Samples are then stained and fixed with osmium tetraoxide in cacodylate buffer followed by an en bloc stain, uranyl acetate. Once this is accomplished, samples are dehydrated by adding a gradient of alcohol concentrations and acetone. Finally, the acetone is replaced in each of the samples with resin. Spurr's resin is more amenable for plant tissues since plants are harder to infiltrate due to the presence of cuticular waxes, cellulose, and hemicellulose. Once the plant material is embedded and the 12 resin has hardened, samples can be sectioned in 70-80 nm sections and applied to copper grids for viewing.

Once the section coated grids are placed in the TEM, they can be viewed by way of an electron beam. The electron beam, created by a lanthanum hexaboride (LaB6) crystal at the top of the microscope column, is focused onto the sample using magnetic lenses. Out of focus electrons are eliminated by the use of several apertures along the electron path. Once the electron beam hits the sample it proceeds down the microscope column and is projected onto a phosphor screen for direct visualization or a film media/camera for image capture.

When viewing a sample through an electron microscope it is important to remember that one is only able to visualize electron dense materials. Therefore, only cellular components that are able to bind the osmium or that contain heavy atoms can be seen in these images; osmium and uranyl acetate bind to proteins and lipids. This is why membranes (i.e. thylakoids) and lipid bodies appear dark in TEM images while cytosol, vacuoles, DNA, and starch granules do not.

2.2 Transcriptome profiling

Using the technique of electron microscopy to study plastids gives a snapshot of what

is occurring at the time the sample is embedded. Another way to obtain an idea of what is

occurring in these tissues at a specific time point is by studying mRNA expression profiles.

To examine the transcriptome, the RNA must be extracted and applied to a microarray chip

for hybridization. Once RNA is hybridized, it can be read and normalized by controls that are

on the chip. Normalization consists of a background adjustment, normalization, and 13 summarization (Lim et al., 2007). It is standard practice to have three replicates of each

treatment. Statistical analysis of the data uses a cutoff of at least two-fold or can be analyzed

using methods that are similar to a t-test pairwise comparison. Once the data are acquired,

there are several programs that can be used to analyze the gene expression patterns (e.g.

Genespring GX by Agilent).

After genes have been determined to be differentially-regulated, they can be assigned

functional categories and gene networks can be constructed. There are many ways of

visualizing the data that come from expression profiling, “Mapman” being one of the most

common (Usadel et al., 2009). Visualization of this kind of data can lead to many insights

into what processes are significant during plastid development. More commonly, critical

analysis of gene expression profiles leads to the search for commonalities within promoter

regions that play a role in the mutant or treatment being analyzed.

2.3 Literature cited

Lim, W.K., Wang, K., Lefebvre, C., and Califano, A. (2007). Comparative analysis of

microarray normalization procedures: effects on reverse engineering gene networks.

Bioinformatics. 23:13, i282-i288.

Sabatini, D.D., Bensch, K., and Barrnett, R.J. (1963). Cytochemistry and electron

microscopy. The preservation of cellular ultrastructure and enzymatic activity by

aldehyde fixation. Tissue and Cell. 17, 19-58.

Usadel, B., Poree, F., Nagel, A., Lohse, M., Czedik-Eysenberg, A., and Stitt, M. (2009). A

guide to using MapMan to visualize and compare Omics data in plants: a case study

in the crop species, Maize. Plant Cell Environ. 32, 1211-1229.

CHAPTER 3. A brassinosteroid transcriptional network revealed by genome-wide

identification of BES1 target genes in Arabidopsis thaliana

3.1 Research overview

Brassinosteroids are plant signaling molecules derived from cholesterol that have many different roles within plants including processes like development, stress response, and

cell differentiation (Clouse and Sasse, 1998). Brassinosteroids act by way of a signaling

pathway that controls transcription factors of the BES1 and BZR1 family (Li and Deng,

2005). The questions posed in this research were what are the gene targets for the BES1

transcription factor in Arabidopsis and what are its binding sites? In order to address these

questions, Chip-chip and gene expression experiments were conducted using the gain of

function bes1-D mutant (Yin et al., 2002). bes1-D constitutively expresses BES1 and is therefore very likely to be found bound to most of the gene promoter regions that are regulated by this transcription factor, thus making this mutant ideal for a Chip-chip study of promotor binding sites.

BES1 antibodies were used to precipitate crosslinked DNA, and the precipitate was then purified by affinity column chromatography. Probing using an Affymetrix gene tiling array yielded 1609 positive BES1 target genes. It was found that 404 of these BES1 target genes could be differentially expressed, either up- or down-regulated, in response to brassinolide treatment. Therefore, this indicates that BES1 can serve as an inducer or repressor of target genes. Characterization of BES1 binding domains using gel shift mobility assays showed that it binds CACGTG and CACTTG regions (brassinosteroid response 16 element and E-box sequences, respectively). BES1 target genes included genes important in

hormone signaling of IAA, ABA, and GA pathways, showing an overlap between well-

known signaling hormones with brassinosteroids.

To dissect these gene interactions a network was formed using ARACNE (Algorithm for the Reconstruction of Accurate Cellular Networks) (Margolin et al., 2006). Many genes involved in chloroplast biogenesis appeared to be related to BES1 in this network including

GLK1, GLK2 and PIL6. This suggested that BES1 is involved in this process. To test this hypothesis, bes1-D mutants and wild-type plants were grown side by side in order to perform chlorophyll measurements. Plants were grown for 2 weeks at 22 degrees centigrade under continuous light of 50 µmol photons m-2s-1. I found that chloroplasts within the mutant accumulate less chlorophyll (Figure 3.1), suggesting that the mutant is perturbed in its ability to accumulate chlorophyll-protein complexes. To identify the cause of the chloroplast defect,

I performed TEM of leaves of the mutant and wild-type. Figure 3.2 shows that plastids in the bes1-D leaves are generally smaller than normal, but are able to form normal thylakoid stacks as well as starch granules. However, they also accumulated large plastoglobules.

Plastoglobules have been shown to be involved in regulating the redox status of the thylakoid membrane and contain photoprotective compounds such as carotenoids, tocopherols, and quinones (Bréhélin and Kessler, 2008). They also contain enzymes for the synthesis of these compounds (Lundquist et al., 2012). The increase in plastoglobules in bes1-D might be a sign of stress, or might simply reflect lipophilic molecules that have not yet been integrated into chloroplast membranes. Therefore genes involved in the coordinated assembly and formation of new chloroplasts are regulated in part by way of brassinosteroid-related transcription factors. This finding as well as a down-regulation of chloroplast proteins, including the 17 chloroplast Lhcb gene, shows that chloroplast biogenesis is indeed impaired in the gain of

function bes1-D mutant. My experiments have set the stage for further studies to define how

BES1-D controls this process.

3.2 Literature cited

Bréhélin, C., and Kessler, F. (2008). The Plastoglobule: A Bag Full of Lipid Biochemistry

Tricks. Photochem Photobiol. 84, 1388-1394.

Clouse, S.D., and Sasse, J.M. (1998). Brassinosteroids: Essential Regulators of Plant Growth

and Development. Plant Biology. 49, 427-451.

Li, L., and Deng, W. (2005). It runs in the family: regulation of brassinosteroid signaling by

the BZR1–BES1 class of transcription factors. Trends in Plant Science. 10:6, 266-

268.

Lichtenthaler, H. (1987). Chlorophylls and carotenoids: Pigments of photosynthetic

biomembranes. Meth Enz. 148, 350-382.

Lundquist, P.K., Poliakov, A., Bhuiyan, N.H., Zybailov, B., Sun, Q., and van Wijk, K.J.

(2012). The Functional Network of the Arabidopsis Plastoglobule Proteome Based on

Quantitative Proteomics and Genome-Wide Coexpression Analysis. Plant Physiol.

158, 1172-1192.

Margolin, A.A., Nemenman, I., Basso, K., Wiggins, C., Stolovitzky, G., Favera, R.D., and

Califano, A. (2006). ARACNE: An Algorithm for the Reconstruction of Gene

Regulatory Networks in a Mammalian Cellular Context. BMC Bioinformatics. 7,

Suppl 1:S7.

Yin, Y., Wang, ZY., Mora-Garcia, S., Li, J., Yoshida, S., Asami, T., and Chory, J. (2002).

Bes1 Accumulates in the Nucleus in Response to Brassinosteroids to Regulate Gene

Expression and Promote Stem Elongation. Cell. 109:2, 181-191.

3.3 Figures

2500

2000

1500

1000

Total chlorophyll chlorophyll (ug/g) Total 500

0 WT bes1-D

Figure 3.1 Total chlorophyll levels of wild-type and bes1-D mutant. Chlorophyll contents were determined as described by Lichtenthaler using wild-type and mutant seedlings. Plants were grown for 2 weeks at 22 degrees centigrade under continuous light of

50 µmol photons m-2s-1. Chlorophyll was extracted from seedlings based on fresh weight using 95% ethanol. Total chlorophyll was based on absorbancies of 664nm and 649nm

(Lichtenthaler, 1987).

Figure 3.2 Transmission electron micrographs of wild-type (C) and bes1-D (D).

Plastoglobules are indicated by arrows and stain dark black in osmium. Plants were grown

for 2 weeks at 22 degrees centigrade under continuous light of 50 µmol photons m-2s-1. For

TEM analysis, mutant and wild-type cotyledon tissue was fixed in a solution consisting of

2% glutaraldehyde/2% paraformaldehyde in a 0.1 mM cacodylate buffer (pH 7.2). Samples were post-fixed in 1% buffered osmium tetraoxide. The tissue was en bloc stained with 3% uranyl acetate. Samples were dehydrated in an ethanol series followed by a 100% acetone dehydration step. Directly after dehydration, samples were infiltrated with a ratio of acetone to Spurr’s resin mixture. Samples were polymerized in Spurr’s resin at 60°C. Resin was sectioned on a Reichert Ultracut S ultramicrotome with a glass knife to a thickness of 70 nm and placed on formvar coated copper grids. Images were captured on a JEOL 2100 STEM.

Scale bars represent 0.5 µm.

CHAPTER 4. Chloroplast photooxidation-induced transcriptome reprogramming in

Arabidopsis immutans white leaf sectors

4.1 Research overview

Arabidopsis immutans is a variegated leaf mutant that forms white and green tissue sectors (Figure 6.1C). This mutant is light sensitive and forms sectors during early leaf and cotyledon development (Aluru et al., 2001). Green sectors contain chloroplasts with a normal morphology while white sector chloroplasts lack thylakoid stacks and appear vacuolated (Aluru et al., 2001). In order to dissect the differences between white and green sectors, mRNA was extracted from each and probed with Affymetrix 22K ATH1 microarrays.

In addition wild-type Arabidopsis tissue treated with the carotenoid inhibitor norflurazon

(NF) was used due to its similarity in nature to the proposed mechanism for white sector formation in immutans (Bartels and Watson, 1978). Both immutans and norflurazon (NF) block carotenoid biosynthesis at the phytoene desaturase (PDS) step in carotenogenesis (Wu et al., 1999). However, differences do exist: immutans is a genetic lesion and acts prior to the stage of germination, while NF is a biochemical inhibitor and must be applied after some developmental progress. Also, immutans can be considered leaky due to the fact that the PDS step is only partially blocked in this mutant, whereas NF causes an almost complete blockage of the carotenoid pathway.

Here I will discuss the following questions: how do green sectors form and what are the relationships between the green and white sectors of immutans? To address these questions, expression profiles were performed using immutans white, immutans green, and 22

NF-treated wild-type tissues. Wild-type tissue was used for comparison, and experiments

were done in triplicate. Analysis of the data included use of the MAS 5.0 algorithm for

normalization, and genes were selected based on a 2-fold cutoff as well as p values of less

than or equal to 0.05 (Hubbell et al., 2002). Once significant genes from the replicates were

identified, they were characterized by MIPS classification into functional biological classes

(Figure 4.1). Mapman was used to view differentially-expressed genes across metabolic

pathways within the cell (Figure 4.2).

Genes determined to be differentially expressed when immutans white sectors were

compared to green sectors numbered 1,434 and of these, 747 were induced and 687 were

repressed. Several of these genes were selected and regulation was confirmed by RT-PCR.

RT-PCR expression data concurred with selected genes from that of the microarray. Of the

total gene data set, genes fell into many different categories including: photosynthesis,

carbohydrate metabolism, energy production, nitrogen metabolism, sulfur metabolism, fatty

acid metabolism, and plant defense. This shows that affected plastids of immutans have far

reaching and multi-factorial cellular consequences (Figure 4.3).

Because immutans is a light sensitive chloroplast mutant, it makes sense with respect

to this thesis to focus on genes that involve photosynthesis or are responsive to oxidative

stress. A large number of genes that are involved in photosynthesis were down-regulated in

immutans white sectors. Also, genes involved in pigment biosynthesis, such as chlorophyll

and carotenoids, were down-regulated. These results were expected due to the lack of

thylakoids and photosynthesis in the white tissue of immutans. In this context it is important

to note that a down-regulation of certain genes involved in photosynthesis, such as Lhcb, are

used as indicators of retrograde signaling (Fernandez and Strand, 2008). Therefore, the white 23 sectors of immutans are a good system for the study of retrograde signaling in vivo.

We also found that starch synthesis enzymes are down regulated in immutans white

sectors, while sucrose degradation genes are strongly induced. Several genes involved in

sugar transport were induced in immutans white and green sectors including BMY7,

BFRUCT1, and STP4, lending credence to the idea of a source-sink relationship in which

green sectors feed the white within the immutans leaf tissue. Genes for enzymes of glycolysis in white sectors of immutans were down-regulated, while genes for fermentation were up- regulated, perhaps making these tissues energetically less efficient. The opposite was found in green sectors of immutans, where genes important in aerobic respiration were up- regulated. Mitochondrial metabolite transporter genes as well as AOX were induced in immutans white sectors, indicating a possible compensatory role played by mitochondria in this tissue type (reducing equivalents can be transported to the mitochondria by way of triose phosphate transporters).

One main category of gene expression that this research focused on was that of plant defense and stress response due to an overlap between these responses and those seen in photosynthesis mutants. A lack of carotenoids in white sectors of immutans is thought to lead to a higher level of ROS and photobleached tissues. Consistent with this idea several superoxide dismutase genes were induced in the white sectors along with HSP70, while interestingly, catalase 3 was repressed. Genes involved in the glutathione cycle were up- regulated, while ascorbate peroxidase genes were up-regulated except for thylakoid ascorbate peroxidase. This differentially-regulated set of ROS sensitive genes indicates that fine tuning of these pathways is complex and therefore likely requires elaborate communication within the cells of immutans. 24

To gain a better understanding of how white sectors form, immutans expression

profiles of white tissues were compared with those of NF-treated tissue-types. Both types of

tissue would be expected to be similar due to their impairment in the same enzyme in the

carotenoid biosynthesis pathway. Due to a blockage of phytoene desaturase in both

immutans and NF-treated tissue, there is a lack of carotenoids and therefore both tissue-types

might have similar signaling and response events. It is interesting to note that the block at the

PDS step results in white tissue formation, and not light green (similar to bes1-D) or yellow

(similar to svr4-1). This indicates that colored carotenoids are not present in the plastids of

white sectors to any appreciable extent (Farre et al., 2010). The inherent nature of immutans

leads us to believe that this mutation occurs very early in plastid and plant development (leaf

patterning includes the oldest sections of leaf tissue) (Wu et al., 2011). Yet, one main

difference that exists between NF and immutans tissue-types is that the white sectors of immutans form due to a genetic lesion that causes an indirect effect on PDS activity and the white sectors of norflurazon-treated tissues form due to a chemical inhibitor. Therefore, there is a difference in the timing and extent of the lack of PDS activity. This occurs throughout development in the case of immutans, while it depends on the time of application of NF; NF inhibition is complete, while inhibition by immutans is conditional and depends on factors such as light. This raises the possibility that NF-treated and immutans white tissue-types have

similar but not identical expression profiles. We found this to be the case and our expression

profiles showed that 759 genes were repressed and 205 genes were induced in both. In

comparisons of the two tissue-types using significantly up- and down-regulated genes, no

genes were induced in one tissue type while being repressed in the other, indicating that the

two tissues have high similarity. 25

To investigate in greater detail the similarity between immutans white and NF white

tissues, wild-type NF-treated plants were plated on MS media to see if it was possible to

mimic the variegation phenotype of immutans. At 0.025 µmol concentration, wild-type plants grown on NF supplemented MS media showed a variegated phenotype (Figure 4.4). In addition, NF-treated wild-type plants were smaller than untreated wild-type, similar to the phenotype that we observe in immutans. NF-treated wild-type differed from the immutans in that the white sectoring did not involve the formation of large uniform sectors and was found in smaller patches. This small difference could be due to effects that occur in immutans prior to germination (NF takes effect only after germination because it is present in the MS media).

Further experimentation will be needed to confirm this hypothesis.

In immutans, white sectors develop from photooxidized plastids that occur early during development. These plastids continue to grow and divide throughout leaf growth and expansion. This, in addition to the fact that the cells within white sectors are still transcriptionally active, indicate that both the cells and plastids within them are generally still alive and do not undergo programmed cell death (Aluru et al., 2009). It is therefore essential for the green sectors to transfer photosynthate to the white sectors as well as increase mitochondrial activity in order to supply ATP to white sectors during high metabolic growth periods. White sectors of immutans have a higher level of gene expression related to fermentation, possibly due to a lack of intracellular spaces in white tissue, and are therefore likely to be undergoing anaerobic generation of energy.

Comparison of white tissue sectors of NF-treated wild-type and immutans white sectors show that oxidative stress genes are induced in both. Both tissue types show an increase in photooxidative response genes, yet immutans white sectors also have an induction 26 of developmental genes that may play a role in green sector formation. While immutans and

NF show a high similarity in their gene expression patterning, some differences do exist and are most likely due to differential changes in early development. These early developmental genes are crucial to understanding what makes immutans different than a chemical blockage of PDS, and will be important in future studies.

In summary, in this expression profile study it was found that Lhcb was down- regulated to a higher extent in NF-treated tissues versus immutans. This indicates that a

retrograde signaling event may be occurring and that it may be different between the two

tissue types. It is also important that future studies look at early stages and not fully-

expanded leaves. immutans causes a plastid developmental defect that occurs very early in

chloroplast development from the initial proplastid stage. Early growth stages may yield a

better and more informative view of how these sectors interact as well as any plastid-to-

nuclear signaling that may exist.

4.2 Literature cited

Aluru, M., Bae, H., Wu, D., and Rodermel, S. (2001). The Arabidopsis immutans Mutation

Affects Plastid Differentiation and the Morophogenesis of White and Green Sectors in

Variegated Plants. Plant Physiol. 127, 67-77.

Aluru, M., Zola, J., Foudree, A., and Rodermel, S. (2009). Chloroplast Photooxidation-

Induced Transcriptome Reprogramming in Arabidopsis immutans White Leaf Sectors.

Plant Physiol. 150, 904-923.

Bartels, P.G., and Watson, C.W. (1978). Inhibition of Carotenoids Synthesis by Fluridone and

Norflurazon. Weed Science. 26:2, 198-203.

Farre, G., Sanahuja, G., Naqvi, S., Bai, C., Capell, T., Zhu, C., and Christou, P. (2010). Travel

advice on the road to carotenoids in plants. Plant Science. 179, 28-48.

Fernandez, A.P., and Strand, A. (2008). Retrograde signaling and plant stress: plastid signals

initiate cellular stress responses. Curr Opinion in Plant Biol. 11:5, 509-513.

Hubbell, E., Liu, WM., and Mei, R. (2002). Robust estimators for expression analysis.

Bioinformatics. 18:12, 1585-1592.

Thimm, O., Blasing, O., Gibon, Y., Nagel, A., Meyer, S., Kruger, P., Selbig, J., Muller, L.A.,

Rhee, S.Y., and Stitt, M. (2004). MAPMAN: a user-driven tool to display genomics

data sets onto diagrams of metabolic pathways and other biological processes. Plant J.

37:6, 914-939.

Wu, D., Wright, D.A., Wetzel, C., Voytas, D.F. and Rodermel, S. (1999). The IMMUTANS

Variegation Locus of Arabidopsis Defines a Mitochondrial Alternative Oxidase

Homolog That Functions during Early Chloroplast Biogenesis. The Plant Cell. 11, 28

43-55.

Wu, N.S., Gordon, M.J., Graham, S.R., Rossel, J.B., Badger, M.R., and Pogson, B.J. (2011).

A mutant in the puring biosynthetic enzyme LACTASE2 impacts high light signaling

and acclimation responses in green and chlorotic sectors of Arabidopsis leaves. Funct

Plant Biol. 38, 401-419.

4.3 Figures

Figure 4.1 Functional classes of genes. The 1,434 immutans-responsive genes were assigned to functional classes based on the Arabidopsis MIPS classification scheme

(http://mips.gsf.de/proj/thal/db/) and gene ontology searches.

Figure 4.2 MapMan display of transcript profiling data. MapMan software (Thimm et al., 2004) was used to visualize changes in transcript abundance of the differentially regulated genes associated with major metabolic pathways from imW tissues. Induced genes are indicated in blue and repressed genes are shown in red. White squares represent genes whose expression is unaltered versus the wild type. CHO, Carbohydrate.

Figure 4.3 Schematic representation of the metabolic adaptations corresponding to major transcript changes in imW leaf sectors. Red arrows indicate up-regulation, while blue arrows indicate down-regulation of metabolic pathways and/or steps in the metabolic pathway. Dashed lines represent hypothetical changes occurring in the imW tissues. Circles represent transporters: 1, sugar transporter; 2, metabolite transporter; 3, ATP/ADP translocator; 4, amino acid or oligopeptide transporter. ETC, Electron transport chain; OPPP, oxidative pentose-P pathway 32

Figure 4.4 Variegated phenotype of wild-type Arabidopsis plants treated with NF.

Seeds from wild-type Arabidopsis plants were germinated and grown on Murashige and

Skoog plates with or without NF for 3 weeks at 200 µmol m−2 s−1 continuous illumination.

Image A, wild-type. Image B, wild-type with 0.025 µm NF. Image C, Individual leaf from A.

Image D, Individual leaf from B. Bars = 2 mm.

CHAPTER 5. Suppressor of variegation4, a new var2 suppressor locus, encodes a

pioneer protein that is required for chloroplast biogenesis

5.1 Research overview

One way to study the mechanism of plastid biogenesis is to characterize the

variegated mutant var2 (Yu et al., 2007). To discover new genes important in the process of

chloroplast formation, suppressor analysis of var2 can be a powerful tool. var2 is a thylakoid

membrane FtsH-type metalloprotease protein that was initially found to be important in D1

turnover and repair in the cyanobacteria Synechocystis (Silva et al., 2003). To understand the

mechanism of var2 variegation, this question was asked: what is the role of a suppressor of

variegation, designated svr4 (suppressor of variegation 4), in allowing proper chloroplast development within var2? The svr4 mutant is virescent meaning that it develops yellow leaves that slowly green during elongation and expansion. svr4/var2 double mutants are virescent but not variegated meaning that svr4 is partially able to compensate for a lack of var2. The svr4 suppressor was mapped to the gene locus of At4g28590, and it was found to have a G to A mutation in exon 3.

Target P, a program that uses an algorithm that locates chloroplast target sequences, was used on SVR4 and was able to find an N-terminal chloroplast targeting sequence. To determine that this protein was indeed targeted to the chloroplast, SVR4 was tagged with green fluorescent protein (GFP) and co-localized to the chloroplast within protoplasts by way of confocal microscopy. Western blot analysis of this mutant revealed that the main photosynthetic membrane proteins in svr4 accumulated to a level similar to wild-type. 34

However, svr4 was found to be slightly impaired in photosynthetic electron transport while at the same time to have a higher level of non-photochemical quenching (NPQ), as indicated by fluorescent measurements. A tDNA line of SVR4 was ordered that had an insertion in exon3.

This mutant was designated svr4-2 since it is the second allele to be discovered. Observed phenotypes of this segregating tDNA line showed approximately 25 percent albino progeny.

Complementation analysis supported the linkage of this phenotype with the SVR4 gene mutation. The observation that svr4-1 is virescent and svr4-2 is albino is supportive of svr4-1

being a leaky allele while svr4-2 is a null allele. When these two alleles were crossed, the

svr4-2/svr4-1 phenotype was more virescent than that of svr4-1 individually. svr4-2 was

analyzed further by Western blot analysis of photosynthetic proteins, and all of these proteins

were found to be present in much lower quantities with the exception of PsbS and Lhcb2.

My contribution to this research involved conducting transmission electron

microscopy of svr4-2 cotyledons to observe plastid morphology (Figure 5.1). These plastids

were vacuolated and contained several clusters of plastoglobules. This phenotype has similar

plastids as immutans in that they are vacuolated and show clusters of plastoglobules. These

images show that thylakoid membranes are unable to form in a majority of plastids within

svr4-2 which points to the necessity of this gene in the process of chloroplast biogenesis.

Clusters of plastoglobules indicate that there is a buildup of lipophilic compounds such as

carotenoids, tocopherols, or quinones in these plastids. Also of interest, in Figure 5.1B, is the

residual build up of phytoferritin which can be seen as small dark circular structures that

cluster near the nucleoid. The accumulation of phytoferritin indicates that svr4-2 plastids are

unable to integrate iron as well as wild-type plastids. This is interesting because many

proteins in thylakoid membranes require iron to function properly, including immutans. svr4- 35

2 has a reduced thylakoid protein accumulation and therefore may not be able to integrate iron into redox proteins.

The vacuolated structures of svr4-2 plastids indicate that membranes may be attempting to form but are being aborted, and therefore components needed to form normal thylakoids are unable to assemble properly. Interestingly, one plastid was found in svr4-2 that was able to partially form a thylakoid stack. This rare occurrence shows that there are proteins present that can promote the formation of a thylakoid membrane, yet they are most likely too low in abundance to do so in a majority of the plastids. This is a crucial finding for two reasons. First, it shows that the formation of defective chloroplasts versus fully-formed functional chloroplasts are not mutually exclusive processes, but there is a continuum, depending on availability of substrates. Second, it indicates that the amount of developmentally important proteins present in the plastid is a crucial determining factor in the development of white versus green plastids, which may be applicable to all variegated

Arabidopsis mutant lines.

The exact function of SVR4 has yet to be determined other than its ability to suppress variegation in var2. It has been proposed that SVR4 mediates some aspect of membrane

formation that allows for NPQ in developing seedlings. In the knockout svr4-2 mutant

vacuolated structures form similarly to those seen in immutans. It may be noted that this

similarity, as well as the need for SVR4 during early plastid biogenesis (as shown in

seedlings), makes this an interesting mutant to study possible interactions with immutans.

SVR4’s ability to enhance NPQ and its role in thylakoid membrane structure formation

shows that it plays an important role in avoidance of photodamage within developing plastids

5.2 Literature cited

Silva, P., Thompson, E., Bailey, S., Kruse, O., Mullineaux, C.W., Robinson, C., Mann, N.H.,

and Nixon, P.J. (2003). FtsH Is Involved in the Early Stages of Repair of Photosystem

II in Synechocystis sp PCC 6803. The Plant Cell. 15:9, 2152-2164.

Yu, F., Aigen, F., Aluru, M., Park, S., Xu, Y., Liu, H., Foudree, A., Nambogga, M., and

Rodermel, S.R. (2007). Variegation mutants and mechanisms of chloroplast

biogenesis. Plant Cell Environ. 30:3, 350-365.

5.3 Figures

Figures

Figure 5.1 Chloroplast ultrastructure in wild-type and svr4-2. Transmission electron

microscopy (TEM) was performed using cotyledons from 10-day-old wild-type (A) and svr4-

2 (B, C). Materials and methods are identical to those used in Figure 3.2 of this thesis. TEM sections were additionally post-stained with lead citrate for these images (Sato’s method).

CHAPTER 6. PDS activity acts as a rheostat of retrograde signaling during early

chloroplast biogenesis

A paper published in Plant Signaling & Behavior

Andrew Foudree, Maneesha Aluru, and Steve Rodermel

6.1 Abstract

Chloroplasts are crucial for the process of photosynthesis as well as for developmental and environmental sensing. One of the important mechanisms of sensing is

retrograde (plastid-to-nucleus) signaling, whereby the state of the chloroplast is signaled to

the nucleus, resulting in alterations in gene expression for chloroplast proteins, usually at the

transcriptional level. Retrograde signaling was early studied in carotenoid-deficient plants

that contain, upon exposure to high light, photooxidized plastids that arise because of an

inability to quench ROS produced during the light reactions of photosynthesis. Phytoene

desaturase (PDS) is required for one of the early steps of the carotenogenic pathway, and

impaired PDS activity during early chloroplast biogenesis results in a highly reduced

plastoquinone pool (high excitation pressure), accumulation of the colorless C40

intermediate, phytoene and white photooxidized plastids. Here we discuss results from global

transcript profiling of white leaf tissues of Arabidopsis that are blocked at the PDS step in

three different ways – two by mutation (immutans & pds3) and one by inhibitor treatment

(norflurazon). We show that the molecular phenotypes of the three tissues bear many 39 similarities, but there are also significant tissue-specific differences. We propose that PDS

acts as a rheostat of excitation pressure-mediated retrograde signaling during chloroplast

development and speculate that whether the rheostat is set high (as in pds3 and NF-treated

seedlings), intermediate (as in im) or low (as in WT) is a crucial determinant of the suite of

genes that is expressed during chloroplast biogenesis.

6.2 Bidirectional signaling between the chloroplast and the nucleus

Chloroplast proteins are the products of genes located in the nuclear genome (~3,000)

and the plastid genome (~150) (Kleine et al., 2009). Bidirectional signaling mechanisms have evolved to coordinate the expression of genes in these compartments. Anterograde (nucleus- to-chloroplast) signaling is elicited in response to exogenous and endogenous cues, and it regulates the expression of nuclear genes whose products are required for transcriptional or post-transcriptional events in the plastid (Kleine et al., 2009; Pesaresi et al., 2007).

Retrograde (plastid-to-nucleus) signals, on the other hand, are generated in the plastid in response to its developmental and/or metabolic state, and they are transduced to the nucleus, where they regulate the expression of nuclear genes for chloroplast proteins, usually at the level of transcription (Rodermel, 2001). A number of candidate plastid signals have been identified, including chlorophyll biosynthesis intermediates and reactive oxygen species

(ROS). Although the primary signals have not been identified, other pathways have been postulated that are sensitive to the redox state of the thylakoids, the sugar status of the plastid, and plastid gene expression (Kleine et al., 2009; Rodermel, 2001; Pogson et al.,

2008; Woodson and Chory, 2008; Jung and Chory, 2010). A potential complicating factor in 40 understanding mechanisms of retrograde signaling is convergence of the pathways. In addition, there appears to be considerable crosstalk between retrograde signaling and other signaling pathways that play a role in chloroplast biogenesis, such as light (Ruckle et al.,

2007).

6.3 Phytoene desaturase and photoprotection

One of the earliest pieces of evidence supporting the notion of a plastid signal came from studies of plants and algae treated with norflurazon (NF), a bleaching herbicide

(Rodermel, 2001). NF is a competitive inhibitor for the plastoquinone cofactor binding site of phytoene desaturase (PDS), which converts phytoene (a colorless C40 intermediate) to zeta- carotene in an early step of the carotenoid biosynthesis pathway (Breitenbach et al., 2001).

All the steps of carotenogenesis take place in plastids by nuclear-encoded enzymes (Tanaka et al., 2008), and the primary function of carotenoids is to protect the chloroplast against

ROS-induced photooxidation by quenching triplet chlorophyll and singlet oxygen generated as a result of excess light captured by the photosynthetic antennae (Li et al., 2009). The inhibition of PDS by NF results in an accumulation of phytoene at the expense of downstream colored (photoprotective) carotenoids (Figure 6.1B). This leads to photooxidation of the contents of the organelle. Early observations that NF-induced photooxidation is accompanied by down-regulation of transcription of a number of nuclear genes for chloroplast proteins provided compelling evidence that nuclear gene expression is feedback-regulated by the metabolic status of the plastid, arguing for the existence of plastid- to-nucleus signaling. 41

In addition to NF inhibition, there are two other ways that white, photooxidized

plastids have been generated in Arabidopsis by manipulating the PDS step of

carotenogenesis. One of these is by abolition of the enzyme, as in the pds3 knockout mutant

(Figure 6.1D) (Qin et al., 2007). Like NF-treated seedlings, pds3 accumulates phytoene and

has an albino phenotype due to chloroplast photooxidation. The other example is the

immutans (im) variegation mutant (Fig. 6.1C) (Wetzel et al., 1994; Carol et al., 1999; Wu et al., 1999). The green sectors of im contain cells with morphologically normal chloroplasts (as well as normal carotenoid levels), whereas plastids in cells of the white leaf sectors lack pigments and accumulate phytoene (Wetzel et al., 1994). IMMUTANS (IM) functions as a versatile plastoquinol terminal oxidase in plastid membranes, where it transfers electrons from the plastoquinone (PQ) pool to molecular oxygen. In recognition of its biochemical activity, IM has been called PTOX, (plastid terminal oxidase). One of IM's functions is to serve as a component of a redox pathway that desaturates phytoene. In this pathway, electrons are transferred from phytoene to PQ via PDS, and from PQ to oxygen via IM

(Figure 6.1A) (Rosso et al., 2009). As demonstrated by the im phenotype, this role of IM is crucial during early chloroplast biogenesis when the electron transport chain is not yet fully assembled and functional. At this time, a lack of IM causes over-reduction of the PQ pool and, hence, flux into this pool becomes restricted. This causes phytoene to accumulate and a lack of colored carotenoid production, resulting in plastid photooxidation and the generation of white cells and white sectors as leaf development proceeds. It may be noted that green sectors are thought to arise in im because of factors that are able to compensate for a lack of

IM in some developing plastids but not others (Rosso et al., 2009).

6.4 Comparison of three Arabidopsis photooxidized tissues impaired in PDS function

By a number of morphological, biochemical and molecular criteria, the white tissues of im, pds3 and NF-treated Arabidopsis are similar. For instance, the plastids in all three accumulate phytoene and lack lamellar structures; the palisade cells fail to expand; the mesophyll cells are smaller than normal; and the extent of white tissue formation is light- dependent (Aluru et al., 2001, 2009). The purpose of our original studies was to compare the molecular phenotypes of the white leaf tissues of im (imW) with those from NF-treated

Arabidopsis, with the possibility that these studies might lend insight into mechanisms of retrograde signaling. For these experiments we took advantage of Arabidopsis full genome chips (the Affymetrix ATH1 oligoarray containing ~22,500 genes) and of bioinformatics tools to categorize chloroplast metabolic pathways (Aluru et al., 2009). These studies identified a total of 1,434 differentially regulated genes in imW (compared to WT) and 1,044 differentially regulated genes in NF-treated tissues (compared to WT). Of these, 964 genes had similar expression trends in both white tissues (Figure 6.2). This represents 67% of the total genes in imW that are differentially regulated versus 92% of the total differentially- regulated genes in the NF-treated tissues. Genes with similar expression trends included genes involved in photosynthesis, photorespiration, sucrose and starch metabolism, amino acid biosynthesis, pigment biosynthesis and nitrate and sulfate assimilation, all of which were repressed in both tissues. Genes involved in amino acid catabolism and ammonia assimilation, on the other hand, were induced in both tissues.

Despite these similarities, there were distinct differences between the two tissue types. For example, a number of genes involved in mitochondrial respiration, plant 43 development and defense were induced only in imW, and more genes of some metabolic

pathways were repressed in NF-treated than in imW, particularly tetrapyrrole biosynthesis.

Taken together, the profiling experiments revealed that im and NF-treated white tissues have

very similar but not identical molecular phenotypes. This suggests that both tissues have

similar broad strategies of response to chloroplast photooxidation, but that embedded within

these are tissue-specific strategies. We hypothesize that the differences between the two

tissues reflect, in part, differences in retrograde signaling.

To further understand the impact of PDS inhibition on retrograde signaling, we

compared our microarray data (Aluru et al., 2009) to those of Qin et al. with the pds3 mutant

(Qin et al., 2007). They reported that 596 genes are differentially regulated two-fold or more

in pds3 compared to WT Arabidopsis. This is a smaller number of genes than found in our

studies, one reason being that ~66% fewer genes were surveyed in their experiments.

Surprisingly, there were very few similarities among the 596 differentially regulated genes in

pds3 compared to imW or to NF-treated tissues. Expression of only 76 genes was similar in

imW and pds3, while expression of only 49 was similar in NF-treated tissues and pds3

(Figure 6.2). Interestingly, the percentage of differentially regulated genes that are unique to

pds3 was 85% – a remarkably high number compared to the other two tissues (30% unique

genes in imW versus 6% in NF-treated seedlings). Most of the similarly-expressed genes are

involved in photosynthesis, pigment biosynthesis, carbohydrate and amino acid metabolism,

nitrogen assimilation and plant stress, whereas many of the differences are genes involved in

plant development, redox signaling, and defense. For instance, the state transition signaling

gene STN7 (Pesaresi et al., 2009) is down-regulated specifically in pds3, while FLU,

EXECUTER1 and EXECUTER3, genes that mediate singlet oxygen retrograde signaling (Lee 44 et al., 2007), are uniquely induced in imW. imW also has unique induction of several ROS

signaling genes, such as CAT1, AOX1A and AOX1D (Strodtkotter et al., 2009). Interestingly, only 37 genes were similarly regulated in all three tissues. Nearly half of these were genes for photosynthesis and defense, while the remainder were for a variety of other processes.

The pds3 studies showed that there is a pronounced down-regulation of genes for enzymes of the carotenogenesis pathway in this mutant, including the gibberelic acid (GA) synthesis branch of the IPP pathway. Qin et al. suggested that this reflects feedback regulation of the pathway (Qin et al., 2007). This is supported by the observation that pds3 mutants exhibit dwarfism that can be overcome by the addition of GA. Even though NF- treated plants and im grow slowly and are smaller than WT Arabidopsis, our global profiling experiments did not reveal expression changes for any of the genes in the GA pathway in these tissues.

6.5 A hypothesis: the redox state of the PQ pool acts as a rheostat of retrograde

signaling

Our comparisons of global expression profiles of three white tissues that lack PDS

activity revealed some striking and consistent similarities in gene expression in response to

chloroplast photooxidation but also significant tissue-specific differences. Rosso et al. have

shown that the extent of white sector formation in im is controlled by the redox state of the

PQ pool (excitation pressure) during chloroplast biogenesis – a time when retrograde

signaling is crucial to coordinate gene expression between the nucleus and the chloroplast

(Rosso et al., 2009; Pogson et al., 2008). In fact, it has been demonstrated that the redox state 45 of the PQ pool is a potent initiator of retrograde signaling, and although the nature of the

signaling pathway(s) is unclear (Pfannschmidt et al., 2009), an attractive hypothesis is that

PDS activity exerts considerable control on excitation pressure, especially during chloroplast

biogenesis when the photosynthetic electron transport chain is not yet fully functional and

electrons from the desaturation reactions of carotenogenesis cannot be transferred efficiently

to acceptors downstream of the PQ pool.

In the present studies PDS activity was manipulated in three different ways, resulting

in plants with all-white leaves (pds3), green and white leaves (the application of NF turns

developing leaves white, but developed leaves remain green) or all-variegated leaves (im).

This range of phenotypes is consistent with the idea that developing thylakoids in the three

plants have different excitation pressures, with a full inhibition of PDS activity resulting in

the highest excitation pressures (pds3 and NF), a leaky PDS activity resulting in moderate

excitation pressures (im), and normal PDS activities resulting in the lowest excitation

pressures (WT). This correlation between PDS activity and phenotype is supported by the

observation that PDS antisense and RNAi mutants, both of which have partial down-

regulation of PDS, have variegated phenotypes (Busch et al., 2002; Wang et al., 2005), and

by our demonstration that variegated Arabidopsis can be produced by treatment of WT with

low concentrations of NF, again partially inhibiting PDS activity (Aluru et al., 2009). If our assumption is correct that plastid phenotype can be determined via an impact of PDS activity on excitation pressure, then it can be hypothesized that retrograde signaling varies in a qualitative or quantitative manner depending on the overall excitation pressure that is present in the developing plastids in a cell. We therefore propose that the redox state of the PQ pool acts as a rheostat of retrograde signaling, and speculate that whether the rheostat is set high 46

(as in pds3), intermediate (as in im) or low (as in WT) is a crucial determinant of the suite of

genes that is expressed during chloroplast biogenesis.

One drawback of the present experiments is that the three white tissue samples were

from fully-expanded leaves. Thus they represent the “adapted” state of the tissue to

chloroplast photooxidation, which occurred very early in leaf development, i.e., during

chloroplast biogenesis in the leaf primordium. It is therefore likely that some of the

differences in transcript profiles among the three tissues are due to an effect of

developmental and physiological factors on nuclear gene expression. For example, we have

found via transcript profiling and biochemistry that the green and white sectors of im have

source-sink interactions (Aluru et al., 2007). These complications argue for the use of an

inducible system and kinetic studies to study mechanism(s) of retrograde signaling.

6.6 Acknowledgements

This work was supported by the Office of Basic Energy Sciences, Chemical Sciences

Division, US Department of Energy (contract DE-FG02-94ER20147).

6.7 Literature cited

Aluru, M.R., Bae, H., Wu, D., and Rodermel, S.R. (2001). The Arabidopsis immutans

mutation affects plastid differentiation and the morphogenesis of white and green

sectors in variegated plants. Plant Physiol. 127, 67-77.

Aluru, M.R., Stessman, D., Spalding, M.H., and Rodermel, S.R. (2007). Alterations in

photosynthesis in Arabidopsis lacking IMMUTANS, a chloroplast terminal oxidase.

Photosyn Res. 91, 11-23.

Aluru, M.R., Zola, J., Foudree, A., and Rodermel, S.R. (2009). Chloroplast photooxidation-

induced transcriptome reprogramming in Arabidopsis immutans white leaf sectors.

Plant Physiol. 150, 904-923.

Breitenbach, J., Zhu, C., and Sandmann, G. (2001). Bleaching herbicide norflurazon inhibits

phytoene desaturase by competition with the cofactors. J Agric Food Chem. 49, 5270-

5272.

Busch, M., Seuter, A., and Hain, R. (2002). Functional analyses of the early steps of

carotenoid biosynthesis in tobacco. Plant Physiol. 128, 439-453.

Carol, P., Stevenson, D., Bisanz, C., Breitenbach, J., Sandmann, G., Mache, R., et al. (1999).

Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by

inactivating a chloroplast terminal oxidase associated with phytoene desaturation.

Plant Cell. 11, 57-68.

Jung, H.S., and Chory, J. (2010). Signaling between chloroplasts and the nucleus: can a

systems biology approach bring clarity to a complex and highly regulated pathway? 48

Plant Physiol. 152, 453-459.

Kleine, T., Maier, U.G., and Leister, D. (2009). DNA transfer from organelles to the nucleus:

the idiosyncratic genetics of endosymbiosis. Annu Rev Plant Biol. 60, 115-138.

Lee, K.P., Kim, C., Landgraf, F., and Apel, K. (2007). EXECUTER1- and EXECUTER2-

dependent transfer of stress-related signals from the plastid to the nucleus of

Arabidopsis thaliana. Proc Natl Acad Sci USA. 104, 10270-10275.

Li, Z., Wakao, S., Fischer, B.B., and Niyogi, K.K. (2009). Sensing and responding to excess

light. Annu Rev Plant Biol. 60, 239-260.

Pesaresi, P., Schneider, A., Kleine, T., and Leister, D. (2007). Interorganellar communication.

Curr Opin Plant Biol. 10, 600-606.

Pesaresi, P., Hertle, A., Pribil, M., Kleine, T., Wagner, R., Strissel, H., et al. (2009).

Arabidopsis STN7 kinase provides a link between short- and long-term

photosynthetic acclimation. Plant Cell. 21, 2402-2423.

Pfannschmidt, T., Brautigam, K., Wagner, R., Dietzel, L., Schroter, Y., Steiner, S., et al.

(2009). Potential regulation of gene expression in photosynthetic cells by redox and

energy state: approaches towards better understanding. Ann Bot. 103, 599-607.

Pogson, B.J., Woo, N.S., Forster, B., and Small, I.D. (2008). Plastid signaling to the nucleus

and beyond. Trends Plant Sci. 13, 602-609.

Qin, G., Gu, H., Ma, L., Peng, Y., Deng, X.W., Chen, Z., et al. (2007). Disruption of phytoene

desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing

chlorophyll, carotenoid and gibberellin biosynthesis. Cell Research. 17, 471-482.

Rodermel, S. (2001). Pathways of plastid-to-nucleus signaling. Trends Plant Sci. 6, 471-478.

Rosso, D., Bode, R., Li, W., Krol, M., Saccon, D., Wang, S., et al. (2009). Photosynthetic 49

redox imbalance governs leaf sectoring in the Arabidopsis thaliana variegation

mutants immutans, spotty, var1 and var2. Plant Cell. 21, 3473-3492.

Ruckle, M.E., DeMarco, S.M., and Larkin, R.M. (2007). Plastid signals remodel light

signaling networks and are essential for efficient chloroplast biogenesis in

Arabidopsis. Plant Cell. 19, 3944-3960.

Strodtkotter, I., Padmasree, K., Dinakar, C., Speth, B., Niazi, P.S., Wojtera, J., et al. (2009).

Induction of the AOX1D isoform of alternative oxidase in A. thaliana T-DNA

insertion lines lacking isoform AOX1A is insufficient to optimize photosynthesis

when treated with antimycin A. Mol Plant. 2, 284-297.

Tanaka, Y., Sasaki, N., and Ohmiya, A. (2008). Biosynthesis of plant pigments: anthocyanins,

betalains and carotenoids. Plant J. 54, 733-749.

Wang, T., Iyer, L.M., Pancholy, R., Shi, X., and Hall, T.C. (2005). Assessment of penetrance

and expressivity of RNAi-mediated silencing of the Arabidopsis phytoene desaturase

gene. New Phytol. 167, 751-760.

Wetzel, C., Jiang, C., Meehan, L.J., Voytas, D.F., and Rodermel, S.R. (1994). Nuclear-

organelle interactions: the immutans variegation mutant of Arabidopsis is plastid

autonomous and impaired in carotenoid biosynthesis. Plant J. 6, 161-175.

Woodson, J.D., and Chory, J. (2008). Coordination of gene expression between organellar

and nuclear genomes. Nature Rev Genetics. 9, 383-395.

Wu, D., Wright, D.A., Wetzel, C., Voytas, D.F., and Rodermel, S.R. (1999). The immutans

variegation locus of Arabidopsis defines a mitochondrial alternative oxidase homolog

that functions during early chloroplast biogenesis. Plant Cell. 11, 43-55.

6.8 Figures

Figure 6.1 (A) Model depicting the effect of PDS on plastoquinone redox status in immutans, NF-treated tissues, and the pds3 mutant. NF inhibits phytoene desaturase activity by competing for the PQ cofactor biding site, while the pds3 mutant is null and completely lacks the PDS enzyme. IMMUTANS causes an indirect inhibition of PDS activity by causing an over-reduced PQ pool. (B) Phenotypes of wild-type Arabidopsis (a), Norflurazon-treated

Arabidopsis (b), immutans (c), and the pds3 mutant (d).

Figure 6.2 A comparison of genes differentially expressed two-fold or more in immutans

white tissues (imW), Norflurazon-treated tissues (NF), and pds3 mutant tissues. Comparisons we made between data obtained from Aluru et al. (2009) and Qin et al. (2007).

CHAPTER 7. Background: mechanism of variegation in immutans

7.1 Introduction

This chapter serves as a foundation for the discussion of immutans in a developmental context. Multiple stages of development can be used to study immutans due to the fact that during the life cycle of Arabidopsis, there are three different developmental contexts during which chloroplasts can form: embryogenesis, development of the shoot apical meristem, and de-etiolation. A review by our laboratory that has been accepted recently for publication is included in Chapter 8 due to its inclusion of data I obtained that pertains to these developmental stages, as well as the mechanism of immutans variegation. A majority of this review was written by Dr. Steve Rodermel. It grew out of observations I made on immutans embryogenesis, shoot apical meristem development, and de-etiolation. Therefore I am first author of this submitted publication. This chapter serves as supplemental information to that which is provided within the review in Chapter 8.

7.2 Proplastid-to-chloroplast conversion: embryogenesis

During gametogenesis proplastids are present when the functional megaspore divides to form the seven cell ovule. The egg cell contains proplastids with irregular grana and occasional starch granules. They can be found predominantly surrounding the nucleus.

Plastids in the egg cell are much less defined and are found at a much lower quantity than those of the central cell, which contains well-differentiated plastids and large starch granules 53

(Mansfield and Briarty, 1991). Proplastids are maternally inherited in Arabidopsis, and hence proplastids within the egg become the progenitors to all of the plastids in the developing embryo. Upon fertilization of the egg, an apical and a basal cell form after the first cellular division. A suspensor then forms from the basal cell, and apical, central, and basal regions develop from the apical cell, generating the globular embryo. Often proplastids can be found surrounding the nucleus during early development in Arabidopsis as well as other plant species (Mansfield and Briarty, 1991; Robertson et al., 1995). This close proximity may aid in the process of protein targeting as well as transmission of retrograde signals.

Proplastids start to develop into chloroplasts in the late globular stage of embryogenesis (Mansfield and Briarty, 1991). This is followed by a triangular shaped embryo in which proplastids are still relatively unchanged. During the next stage, the heart stage, chlorophyll starts to accumulate and thylakoid grana stacks become more complex such that at the end of the heart stage, chloroplasts are fully-developed (Figure 7.1).

Chloroplasts remain in this state during the torpedo stage, as the cells of the embryo continue to expand and divide until a majority of the ovule is filled by the embryo. As the embryo reaches its full size, chloroplasts de-differentiate into proplastids, and desiccation occurs within the developing seed. Proplastids can again be seen about 12 hours after imbibition and will start to green after cotyledons emerge from the seed, about 42 hours after imbibition starts (Mansfield and Briarty, 1996).

Whether PTOX plays a role during embryogenesis has not been examined. im siliques develop normally and can be either green, white or variegated. However, white siliques are smaller and contain fewer seeds than green siliques (Aluru et al., 2001). Within 54 each silique ovules can vary in their amount of pigmentation and can be green, all-white, or

shades of green. This is interesting because all of the ovules within the silique seem to be

evenly illuminated. Embryo morphology appears normal in im, even though all plastids do

not develop normally (Figure 7.2). Development of a seed coat and seeds that can germinate

also seem to proceed normally in all siliques, independent of whether or not the silique is

green, white or variegated. This suggests that the process of seed development occurs

normally in im, and that white im plastids can be converted into proplastids during

desiccation. im is expressed at a high level in the funiculus of the young seed and to a lesser

extent within the seed interior which suggests that PTOX is no longer needed after plastid de-

differentiation and desiccation has occurred in mature seeds (Aluru et al., 2001).

In summary, my observations suggest that im is not essential for embryo and seed

development. It has been previously shown that seeds of im germinate normally under all

light conditions (Aluru et al., 2001). It has also been shown that embryo development does

not require photosynthesis if grown in the presence of sucrose (Ruppel and Hangarter, 2007).

However, im may have less carbon sequestration within developing seeds and hence the de-

differentiation of chloroplasts to proplastids could be impaired, as in the variegated mutant

sco1 (Ruppel and Hangarter, 2007). Even though embryogenesis and seed formation involve

dynamic transitions to and from the proplastid stage and may offer insight into plastid

development, it has some pitfalls when used to study im that are discussed within the next

section.

7.3 Proplastid-to-chloroplast conversion: meristem, leaf primordia and early leaf

development

Nearly all molecular and biochemical studies on immutans have been conducted on fully-expanded rosette leaves in which the green and white sectors have been separated by dissection or via fluorescence-activated cell sorting (FACS) (e.g., Wetzel et al., 1994;

Meehan et al., 1996; Aluru et al., 2009). Early fluorescence and electron microscopic studies revealed that the white sectors of im are heteroplastidic and contain rare chloroplasts in addition to vacuolated plastids that lack organized lamellar structures (Wetzel et al., 1994).

This is in contrast to the green sectors, which contain normal-appearing chloroplasts. These observations gave rise to the concept that im behaves in a plastid autonomous manner inasmuch as plastid phenotypes within a cell appear to be determined independently in the im background.

Some of the first studies I conducted as a graduate student were to examine when variegation first appears during leaf development from the shoot apical meristem (SAM). In

Arabidopsis, a SAM first develops during the torpedo stage of embryogenesis (Van

Lijsebettens and Clarke, 1998). It is composed of approximately 50 to 70 cells, and can be divided into three layers (L1, L2, and L3) (Furner and Pumfrey, 1992; Medford, 1992). As mentioned above, chloroplasts develop from proplastids, and upon completion of embryo development, chloroplasts in the embryonic cotyledons de-differentiate back into proplastids during desiccation. It has been suggested that forming chloroplasts temporarily during embryogenesis occurs because it is a way of supplementing maternally-derived carbon sources with internally generated carbon during seed formation (Mansfield et al., 1991; 56

Mansfield and Briarty, 1991). Just prior to germination the seed contains an embryo with two cotyledons (with proplastids and a SAM). Upon germination and light exposure each cell within the SAM contains about 4 proplastids that divide approximately three to four times during primordia development and leaf expansion (Robertson et al., 1995; Possingham,

1983). Leaf primordia develop from the L2 layer, and phylotaxis of developing leaves occurs in a clockwise or counterclockwise manner. As the cotyledons emerge, approximately

42 hours after imbibition, proplastids begin to convert into chloroplasts within the forming leaf primordia (formed at 96 hours post-imbibition) (Mansfield and Briarty, 1996; Pien et al.,

2001; Irish and Sussex, 1992).

The main difference between cotyledon development and leaf development from the

SAM is that cells within cotyledons do not divide (Mansfield and Briarty, 1996). Comparison of variegated cotyledons and leaves can therefore be used to investigate the connection between cell division and variegation. Towards this end, I have crossed im with a chloroplast division mutant (arc6). Within this double mutant chloroplasts cannot divide and develop one large green or non-colored plastid per cell. arc6 is a developmental mutant that affects both the proplastid stage as well as the chloroplast stage of plastid development (Robertson et al.,

1995). ARC6 is involved in the positioning of the ftsZ ring during plastid division (Glynn et al., 2008). The im x arc6 double mutant has a phenotype that is identical to that of im. The optimal way of observing chloroplasts in im x arc6 is by using protoplasts (Figure 7.3).

Isolated protoplasts indicate that non-colored plastids of the im x arc6 double mutant are alive and have the ability to expand in size. TEM images also support this finding in im x arc6 (Figure 7.4). This double mutant can be used to investigate the response of im to high light due to the inability of large arc6 chloroplasts to move throughout the cell to avoid high 57 light. Preliminary observations of im x arc6 tissue show that within these mutants hybrid chloroplasts may exist (half normal and half vacuolated/defective) (Figure 7.5). Even though this finding has yet to be proven, large hybrid chloroplasts have implications for the mechanism of organellar signaling and partitioning of chloroplast proteins within im. Here I show that chloroplast division does not affect variegation, but cell division and sorting of plastids may be the determining factor in the variegation of im.

As discussed in the following review, variegation can be seen within the leaf

primordia as it develops from the L2 layer of the shoot apical meristem (Figure 8.1). One of

the main drawbacks to the use of the proplastid-to-chloroplast conversion within the shoot

apical meristem for studies of im variegation is that plastid development cannot be

synchronized and thus large numbers of plastids at the same developmental stage cannot be

obtained for study. There is also a drawback to the use of mature leaves for studies of PTOX

function. It is difficult to discern primary effects of the absence of PTOX in the leaf

primordium from those that occur later in leaf development due to acclimation of the leaf to

its absence, events that probably involve complex physiological and developmental

interactions.

7.4 Etioplast-to-chloroplast conversion: greening by de-etiolation

Although functional analysis showed that PTOX does not act as a safety valve in

mature Arabidopsis leaves (Rosso et al., 2006), we hypothesize that PTOX function is

important early in chloroplast biogenesis. The development of chloroplasts in the leaf

primordium from proplastids in the leaf meristem is technically challenging to study in dicots 58 because it is impossible to obtain a homogeneous population of developing plastids all at the

same stage of development. To overcome this difficulty we turned to Arabidopsis greening

(de-etiolation) as an experimental system. In this system light can be used to synchronize

chloroplast development.

Etioplasts in dark-grown angiosperm seedlings have large prolamellar bodies (PLB)

that accumulate Pchlide (Biswal and Raval, 2003; Masuda et al., 2009; Denev et al. 2005).

Upon exposure to light, Pchlide is converted into Chlide and the membranes of the PLB

disperse to form thylakoids and organized grana (Philippar et al., 2007). The greening

process is characterized by a tight coordination between the de novo synthesis of pigments,

lipids, and protein components of the photosynthetic apparatus, as well as coordinated

expression of chloroplast and nuclear genes for chloroplast proteins (Sakamoto et al., 2008).

Cotyledons that are observed in the de-etiolated system are previously formed in the

developing seeds. After germination, cotyledons enlarge and expand but do not divide

(Mansfield and Briarty, 1996). This removes cell division from the developmental equation

which is an added benefit when attempting to discover the underpinning of a tissue specific

process, such as leaf variegation. The conversion of etioplasts to chloroplasts is fast and is

usually completed within 24 hours. Therefore experiments can be repeated quickly without

having to complete a full plant life cycle, which can take up to 16 weeks in Arabidopsis.

Etioplasts are less complex than chloroplasts since they are devoid of grana as well as PSI

and PSII, but they still contain a majority of the proteins that are found in fully-developed

chloroplasts.

Using this system, I have found that the immutans mutant of Arabidopsis develops

chlorophyll and carotenoids at a slower rate than that of wild-type (Figure 7.6). This indicates 59 that immutans is defective in its ability to develop chloroplasts from etioplasts. This topic

will be discussed in detail in Chapter 9, but the delay could be due to multiple factors that

include lack of a proper complement of carotenoids, a retrograde signal that delays cotyledon

expansion, or an impaired redox status of prothylakoid membranes. It is apparent that

etioplast development is impaired in immutans since I have observed plastoglobule clustering

in etioplasts. Plastoglobules are lipophilic and contain carotenoids, tocopherols, and

quinones, among other compounds. It has been suggested that storage of carotenoids in

plastoglobules is a way to maintain their stability (Howitt and Pogson, 2006), and consistent

with this idea, plastoglobules might be crucial in immutans for storage of carotenoids before

light exposure, so that they may be used to stabilize newly-forming membranes. This

hypothesis is backed by the observation that most cotyledons of dark-grown immutans appear

visually green after light exposure, even under high light conditions. Therefore, the leaky

nature of im may allow for the buildup of carotenoids prior to light exposure. Our hypothesis

of im variegation is that some of these plastids may perish under high light, but those that

have enough compensating factors built up during dark growth may live. This suggests that

there may be some level of optimization of dark-grown immutans between carotenoid

production and compensating factors. It is therefore evident that the conversion from

etioplasts to chloroplasts through the process of de-etiolation will shed more light on the

nature of variegation formation and chloroplast biogenesis in immutans.

7.5 Literature cited

Aluru, M.R., Bae, H., Wu, D., and Rodermel, S.R. (2001). The Arabidopsis immutans

mutation affects plastid differentiation and the morphogenesis of white and green

sectors in variegated plants. Plant Physiol. 127, 67-77.

Aluru, M.R., Zola, J., Foudree, A., and Rodermel, S.R. (2009). Chloroplast photooxidation-

induced transcriptome reprogramming in Arabidopsis immutans white leaf sectors.

Plant Physiol. 150, 904-923.

Biswal, U. C., and Raval, M. K. (2003). Chloroplast biogenesis: from proplastid to

gerontoplast. Springer.

Denev, I., Tahubyan, G., Minkov, I., and Sundqvist, C. (2005). Organization of

protochlorophyllide oxidoreductase in prolamellar bodies isolated from etiolated

carotenoid-deficient wheat leaves as revealed by fluorescence probes. Bio et Biophys

Acta. 1716, 97-103.

Furner, J. and Pumpfrey, J.E. (1992). Cell fate in the shoot apical meristem of Arabidopsis

thaliana. Development. 115:3, 755-764.

Glynn, J., Froehlich, J., and Osteryoung, K. (2008). Arabidopsis arc6 Coordinates the

Division Machineries of the Inner and Outer Chloroplast Membranes through

Interaction with PDV2 in the Intermembrane Space. The Plant Cell. 20:9, 2460-2470.

Howitt, C., and Pogson, B. (2006). Carotenoid accumulation and function in seeds and non-

green tissues. Plant Cell Environ. 29:3, 435-445.

Irish, V.F. and Sussex, I.M. (1992). A fate map of the Arabidopsis embryonic shoot apical

meristem. Development. 115:3, 745-753. 61

Mansfield, S.G., Briarty, L.G., Erni, S. (1991). Early embryogenesis in Arabidopsis thaliana.

I. The mature embryo sac. Canadian Jornal of Bot. 69:3, 447-460.

Mansfield, S.G., and Briarty, L.G. (1991). Early embryogenesis in Arabidopsis thaliana. II.

The developing embryo. Can Jour of Bot. 69:3, 461-476.

Mansfield, S.G., and Briarty, L.G. (1996). The dynamics of seedling and cotyledon cell

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7.6 Figures

Chlorophyll auto-fluorescence Wild-type

Figure 7.1 Heart stage embryo development in immutans and wild-type Arabidopsis.

Images in the left column show chlorophyll auto-fluorescence. Images in the right column are bright field images. Variegated siliques were dissected and ovules were removed using a dissecting microscope. Ovules were then punctured under a dissecting microscope and embryos were removed from the ovules. Auto-fluorescence images were taken with a Zeiss

Axioplan II fluorescence microscope using a TRITC filter.

A B

Figure 7.2 immutans and wild-type torpedo stage embryos. (A) immutans and (B) wild-type auto-fluorescence confocal images overlayed on transmitted light (non-confocal) images. Similar late torpedo stage embryo development showing the variegated phenotype of immutans. Variegated siliques were dissected and ovules were removed using a dissecting microscope. Ovules were then punctured under a dissecting microscope and embryos were removed from the ovules.

(A) (B)

Figure 7.3 im and arc6 x im protoplasts isolated from the leaves of 3 month old plants. (A) im protoplasts with green and (B) non-colored plastids have smaller and more numerous plastids than does (C) arc6 x im green and (D) non-colored plastids which contain one large plastid per cell. Protoplasts were isolated by digestion of leaf tissue in buffered cellulose/macerozyme solution. Protoplasts were then filtered through 100 µm nylon mesh.

Figure 7.4 arc6 x im plastid. Variegated arc6 x im tissue was isolated and embedded in Spurr’s resin for visualization by transmission electron microscopy. Several starch granules are present (light colored ovals) as well as plastoglobules (small black spheres). Scale bar represents 5 µm. Materials and methods are identical to those used in Figure 3.2 of this thesis.

Figure 7.5 Tissue section of arc6 x im variegated tissue. Image shows one large chloroplast per cell. Some plastids appear vacuolated and some appear normal and contain dark purple starch granules. A black box highlights a plastid that appears to have starch granules as well as vacuolation (hybrid plastid). 1 µm sections were taken from samples embedded in Spurr’s resin and were stained with toluidine blue with heat for 5 seconds. Sample was fixed to a frosted slide with permount and xylene.

Figure 7.6 Chlorophyll and carotenoid delay during de-etiolation. (A) Carotenoid accumulation of wild-type (WT) and immutans (im) exposed to high light (H) and low light

(L) for 48 hours. (B) Chlorophyll accumulation of wild-type (WT) and immutans (im)

Figure 7.6 continued. exposed to high light (H) and low light (L) for 48 hours. Light exposure conditions were continuous low light (15 µmol photons m-2s-1) or high light with respect to im (50 µmol photons m-2s-1) for each time point.

CHAPTER 8. The mechanism of variegation in immutans provides insight into

chloroplast biogenesis

A paper published in Frontiers in Plant Physiology Journal

Andrew Foudree, Aarthi Putarjunan, Sekhar Kambakam, Trevor Nolan, Jenna Fussell,

Gennady Pogorelko, and Steve Rodermel

8.1 Abstract

The immutans (im) variegation mutant of Arabidopsis has green and white-sectored leaves due to the absence of fully functional PTOX (plastid terminal oxidase), a plastoquinol oxidase in thylakoid membranes. PTOX appears to be at the nexus of a growing number of biochemical pathways in the plastid, including carotenoid biosynthesis, PSI cyclic electron flow, and chlororespiration. During the early steps of chloroplast biogenesis, PTOX serves as an alternate electron sink and is a prime determinant of the redox poise of the developing photosynthetic apparatus. Whereas a lack of PTOX causes the formation of photooxidized plastids in the white sectors of im, compensating mechanisms allow the green sectors to escape the effects of the mutation. This manuscript provides an update on im variegation, new data about the mechanism of im variegation, and findings about im compensatory mechanisms.

8.2 Introduction

Variegation mutants provide an excellent system to explore the mechanisms of chloroplast biogenesis and the nature of communication between the nucleus-cytoplasm, chloroplast and mitochondrion (Rodermel, 2001; Yu et al., 2007). The leaves of these mutants contain green and chlorotic (white, yellow) sectors that arise as a consequence of mutations in nuclear or organelle genes (Tilney-Bassett, 1975). Whereas cells in the green sectors have morphologically normal chloroplasts, cells in the chlorotic sectors contain pigment-deficient plastids with abnormal membrane structures (Aluru et al., 2001). One common mechanism of variegation involves the induction of defective chloroplasts (or mitochondria) by mutations in nuclear genes that code for organelle proteins. In some cases the green and white cells have different genotypes. For example, transposable element activity can inactivate a gene required for normal chloroplast function in some cells (chlorotic cells) while excision can reconstitute gene function (green cells). In other cases of variegation, the two types of cells have the same (mutant) genotype, indicating that the gene product is required in some, but not all cells of the mutant. This raises the question of how the green cells escape this requirement. Is there a compensating activity? Our operating hypothesis is that answers to this question will provide insight into mechanisms of variegation, as well as into pathways and regulatory networks of chloroplast biogenesis

The past decade has witnessed a growing interest in the use of variegation mutants as systems to address fundamental questions in photosynthesis and chloroplast biogenesis (Yu et al., 2007; Liu et al., 2010). Yet, mechanisms of variegation are still poorly understood. Here we focus on the mechanism of variegation in immutans, which defines the gene for PTOX 72

(plastid terminal oxidase), a versatile quinol oxidase in plastid membranes. Early studies led

to the notion that this mechanism involves the attainment of a “threshold” by developing

plastids (the threshold hypothesis). In this manuscript, we review the underlying

assumptions and experiments in support of this hypothesis and conclude that immutans offers

a paradigm to understand plant variegation.

8.3 immutans

The immutans (im) variegation mutant is one of the oldest Arabidopsis mutants,

reported independently by Rédei and Röbbelen in the 1960s (Rédei, 1963; Röbbelen, 1968).

Cells in the green sectors of im have morphologically normal chloroplasts, whereas cells in

the white sectors are heteroplastidic and contain abnormal plastids that lack pigments and

organized lamellae, as well as rare, normal-appearing chloroplasts (Wetzel et al., 1994). The

extent of white sector formation in im is promoted by high light and low temperature (Rédei,

1963, 1967; Röbbelen, 1968; Wetzel et al., 1994; Rosso et al., 2009). HPLC analyses

showed that the white sectors accumulate the colorless C40 carotenoid intermediate, phytoene, indicating that im is impaired in the activity of phytoene desaturase (PDS), the enzyme which converts phytoene to zeta-carotene (Wetzel et al., 1994). All the steps of carotenogenesis take place in the plastid and are mediated by nuclear-encoded enzymes that are imported into the organelle post-translationally, and PDS mediates an early step of the pathway (DellaPenna and Pogson, 2006). An inhibition of PDS activity would thus result in lack of accumulation of downstream, colored (photoprotective) carotenoids, and under high light conditions, would be anticipated to give rise to white, photooxidized plastids. A defect 73 of this sort is consistent with the light-sensitivity of im; as in other carotenoid mutants, the

higher the light intensity, the greater the extent of photooxidation (white sector formation)

(Mayfield and Taylor, 2005; Oelmüller, 1989). The presence of normal-appearing

chloroplasts in im, on the other hand, suggests that some plastids are able to bypass the

requirement for the IMMUTANS gene product, likely because of compensating activities that

make them less susceptible to photooxidation early in their development.

8.4 PTOX function

IMMUTANS (IM) was cloned in Arabidopsis by both map-based methods (Wu et al.,

1999) and T-DNA tagging (Carol et al., 1999). The gene product (IM) was discovered to be a

plastid membrane protein that is distantly-related (37% amino acid similarity) to alternative

oxidase (AOX), a mitochondrial inner membrane protein which functions in the alternative

(cyanide-resistant) pathway of respiration, where it transfers electrons from ubiquinol to

molecular oxygen (Wu et al., 1999). Central among its physiological functions, AOX is an

important sensor of cellular redox balance (Giraud et al., 2008; McDonald, 2008). Similar to

AOX, IM has quinol oxidase activity in vivo and in vitro and, consequently, it has been

designated PTOX (plastid terminal oxidase) (Joët et al., 2002; Josse et al., 2003). PTOX is found in some cyanophages, which might serve as vectors for transfer of PTOX among cyanobacteria, but otherwise appears to be limited to oxygenic photosynthetic prokaryotes and eukaryotes, where it is found in all lineages (McDonald et al., 2011). IM is generally present as a single copy gene, although two copies are found in some cyanobacteria, red algae and green algae (Wang et al., 2009). In chloroplasts, PTOX is bound to the stromal 74 lamellae of thylakoids and is modeled as an interfacial membrane protein whose active site

faces the stroma (Berthold et al., 2000; Joët et al., 2002; Lennon et al., 2003). It does not

appear to be present in chloroplast envelope membranes.

Several functions have been ascribed to PTOX. The phytoene accumulation

phenotype of im led to the suggestion that PTOX serves as the terminal oxidase of an

oxygen-dependent redox pathway that desaturates phytoene (Beyer et al., 1989; Mayer et al.,

1990, 1992; Hugueney et al., 1992; Schulz et al., 1993; Nievelstein et al., 1995; Norris et al.,

1995; Al-Babili et al., 1996). This pathway is thought to involve transfer of electrons from

phytoene to plastoquinone (PQ) via PDS, forming zeta-carotene and plastoquinol (PQH2), then from PQH2 to molecular oxygen via PTOX, forming water and PQ (Carol et al., 1999;

Wu et al., 1999; Rosso et al., 2009). Thylakoids of developing im plastids have over-reduced

PQ pools (Rosso et al., 2009), and according to this pathway, the accumulation of phytoene in im can be explained by a decreased supply of PQ available to PDS, as suggested by

Okegawa et al. (2010), and/or because electron transfer from phytoene into an over-reduced

PQ pool is not energetically favorable (Rochaix, 2011). In addition to the phytoene- accumulation phenotype of mutants that lack PTOX (im in Arabidopsis and the orthologous ghost mutant in tomato) (Wetzel et al., 1994; Josse et al., 2000; Barr et al., 2004; Shahbazi et al., 2007), an involvement of PTOX in carotenogenesis is suggested by the close coordination between PTOX expression and carotenoid production in a number of systems, perhaps most strikingly in chromoplasts during the ripening of tomato, citrus and pepper fruit

(Josse et al., 2000; Barr et al., 2004). Another noteworthy example is the up-regulation of

PTOX expression in etiolated seedlings of Arabidopsis treated with paclobutrazol (PAC), an inhibitor of gibberellin biosynthesis; PAC causes an increase in carotenoid production 75

(Rodríguez-Villalon et al., 2009).

It might be noted that all the enzymes of carotenogenesis are present in chloroplast

envelopes, with the exception of PDS which is also found in thylakoids (Joyard et al., 2009).

The involvement of PTOX in carotenogenesis is therefore puzzling given its apparent

exclusive location in thylakoids (Lennon et al., 2003). This suggests that carotenoid

intermediates are trafficked between the envelope and thylakoids, perhaps via vesicles or by

connections between the envelope and thylakoids (Adam et al., 2011). Alternatively, there

might be PTOX-independent mechanisms of plastoquinol oxidation in envelopes, such as

PQH2 auto-oxidation (Khorobrykh and Ivanov, 2002), the activity of another plastoquinol

oxidase (Buchel and Garab, 1995, Lajko et al., 1997, Joët et al., 2002), or perhaps a plastoquinol peroxidase (Casano et al., 2000; Rumeau et al., 2007).

IM mRNAs are ubiquitously expressed in Arabidopsis, and the anatomies of various

plastid types are affected in im (Aluru et al., 2001), as well as in tomato ghost (Josse et al.,

2000; Barr et al., 2004). However, a positive correlation between IM mRNA levels and

carotenoid accumulation does not always hold, and IM is expressed highly in some tissues

with low carotenoid accumulation (Aluru et al., 2001). Considered together, these

observations argue that the role of PTOX in plastid metabolism is not limited to

carotenogenesis. In fact, since its discovery over 13 years ago, it has become apparent that

PTOX resides at the nexus of a growing number of redox pathways in the plastid. For

instance, in vitro and in vivo studies have demonstrated that PTOX is the terminal oxidase of

chlororespiration (nonphotochemical reduction of the PQ pool) (Peltier and Cournac, 2002;

Rumeau et al., 2007). Chlororespiration involves the transfer of electrons from NAD(P)H

(and/or Fd) to PQ via a thylakoid NAD(P)H dehydrogenase (NDH) complex, and from PQH2 76 to molecular oxygen via PTOX (Peng et al., 2012). One purpose of chlororespiration is to

help poise the redox state of the PQ pool during cyclic electron flow (CEF) around PSI

(Okegawa et al., 2010). In addition to chlororespiration, PTOX mediates chromorespiration,

the analogous process in chromoplasts (Josse et al., 2000; Barr et al., 2004; Rodrigo et al.,

2004; Shahbazi et al., 2007).

Safety valves are photoprotective mechanisms that dissipate excess photons and

electrons (Niyogi, 2000). They include nonphotochemical quenching mechanisms and

alternative electron acceptors that prevent over-reduction of photosynthetic electron carriers,

thereby protecting PSI and PSII from photodamage (Aro et al., 1993; Allahverdiyeva et al.,

2005). Soon after its discovery, Niyogi (2000) proposed that PTOX-mediated electron flow

from PQH2 to O2 might act as a safety valve. Early support for this hypothesis came from

studies showing that the green leaf sectors of im have morphological, biochemical and molecular adaptations similar to plants acclimated to growth in high light, even when grown in permissive (low light) conditions (Aluru et al., 2001). Early studies also showed that IM mRNAs and proteins are significantly up-regulated in high light conditions in antisense mutants of tobacco that lack both ascorbate peroxidase and catalase (Rizhsky et al., 2002). A number of experiments have since supported the safety valve hypothesis in systems as diverse as the alpine species Ranunculus glacialis acclimated to low temperature (Streb et al.,

2005); oat exposed to heat and high light (Quiles, 2006); mature green leaves and green fruit of the tomato ghost mutant subjected to high light stress (Shahbazi et al., 2007); wild species of Brassica fruticulosa versus the agricultural species Brassica oleracea adapted to both heat and high light (Díaz et al., 2007); the salt-stressed halophyte Thellungiella (Stepien and

Johnson, 2009); and cold-acclimated Lodgepole pine (Savitch et al., 2010). In addition, 77

PTOX is up-regulated in various photosynthetic mutants, including the gun4 mutant of

Chlamydomonas and the tobacco rbcL and psbA deletion mutants. The gun4 mutants lack

GUN4, a regulatory subunit of Mg-chelatase, and it was suggested that enhanced PTOX

activities in these mutants play a physiological role in decreasing PSII excitation pressures

(Formighieri et al., 2012). The rbcL mutants lack Rubisco and consequently two major

electron sinks, photosynthesis and photorespiration. Elevated PTOX levels in these mutants

might reduce oxidative pressure on both PSI and PSII (Allahverdiyeva et al., 2005). In the

psbA mutants, which lack the D1 protein of PSII, it was suggested that enhanced PTOX

levels primarily support increased carotenoid production that is needed to quench singlet

oxygen generated by free LHCII accumulation (Baena-González et al., 2003).

The demonstration that PTOX serves as a safety valve in a particular system is not

straightforward and must rely on more than gene expression data, since there are many ways

of preventing over-reduction of the electron transport chain, such as upregulation of

downstream electron sinks, as found in some cold-tolerant plants, or upregulation of the

Mehler reaction (e.g., Gray et al., 1997; Savitch et al., 2000). Hence, care must be taken to

show that induction of PTOX expression is not simply a correlation rather than a cause-and-

effect. It is also clear that there are compelling cases where PTOX does not act as a safety

valve. For example, overexpression of Arabidopsis PTOX does not impart increased

resistance to photoinhibition in mature tobacco leaves (Joët et al., 2002), nor does it regulate

the redox state of the PQ pool during steady state photosynthesis in Arabidopsis during stress

and acclimation (Rosso et al., 2006). These findings are consistent with the observation that

the steady state flux of electrons through PTOX is relatively minor - estimated to be <2% of

the flux through the photosynthetic electron transport chain (PETC) (Ort and Baker, 2002). 78

On the other hand, these same studies showed that PTOX is important in preventing over-

reduction of PSII acceptors during dark to light transients (Joët et al., 2002), and that it

protects against PSI photoinhibition (Rosso et al., 2006). The latter has been confirmed in

recent experiments showing that PTOX acts as a safety valve in Arabidopsis under stress

conditions of low temperature and high light, where it protects against both PSII and PSI

photoinhibition (Ivanov at al., 2012).

In this context it is important to note that in contrast to PSII, where there are a number

of photochemical and non-photochemical mechanisms to avoid over-reduction of PSII

acceptors, the repertoire is rather more limited for PSI and includes, most prominently, the

water-water cycle (reviewed in Rumeau et al., 2007). PTOX might thus turn out to be an

important mechanism to reduce electron pressure on PSI, as during stress when NADPH/ATP

ratios are high and CEF around PSI is activated; in concert with NDH, PTOX might serve to

poise the redox state of intersystem carriers by recycling electrons to PQ. This might also be

the case during chloroplast biogenesis when it has been demonstrated that PTOX is a central

regulator of thylakoid redox and PSII excitation pressure (Rosso et al., 2009)

Although not within the scope of this review, it might be mentioned that the functions

described for PTOX in cyanobacteria and eukaryotic algae are similar to those in higher

plants (reviewed by McDonald et al., 2011). These include carotenogenesis and response to

diverse environmental stresses, such as high light, temperature, salt, iron, and phosphate

deprivation (Moseley et al., 2006; Bailey et al., 2008; Wang et al., 2009). One difference is

that cyanobacterial respiratory and photosynthetic electron transport chains share some

components, which offer the possibility that PTOX controls both activities.

In summary, there is growing evidence that PTOX is a versatile terminal oxidase in 79 chloroplast metabolism and that its primary physiological role varies from taxon to taxon.

This might not be surprising, since PTOX is only one element of a large network of factors

involved in stress tolerance, pigment biosynthesis and photosynthetic control (regulation of

ATP/NADPH ratios) (Kramer and Evens, 2011; Foyer et al., 2012). In such a network, it

might be anticipated that the relative importance of a given element varies according to

developmental and/or physiological context. For instance, PTOX might turn out to play a

more significant regulatory role in PSI cyclic electron transport in bundle sheath cells of C4

plants than in mesophyll cells of C3 plants; in contrast to C3 plants, cyclic electron transport

is a key regulator of ATP synthesis during C4 photosynthesis (Rumeau et al., 2007; Foyer et

al., 2012). Given the ubiquity of PTOX expression, it might also be predicted that novel

functions will be found for PTOX, especially in non-green plastids. A schematic diagram of

PTOX in thylakoids is shown in Figure 8.2.

8.5 Mechanism of variegation: the threshold hypothesis

8.5.1 Studies using mature leaves

Nearly all molecular and biochemical studies on immutans have been conducted using fully-expanded rosette leaves; for comparative studies, green versus white sectors have been obtained by dissection or via fluorescence-activated cell sorting (FACS) (Wetzel et al.,

1994; Meehan et al., 1996; Aluru et al., 2009). Alternatively, all-green im leaves have been produced by altering light conditions during early seedling development (Rosso et al., 2009).

The notion that im variegation is due to a threshold came from two sets of 80 observations. The first was that the white sectors of im are heteroplastidic, indicating that im

behaves in a plastid autonomous manner, i.e., plastid phenotypes within a cell are determined

independently in the im background. The second was that intermediate plastid phenotypes

are not seen; plastids in the green sectors look similar to one another, as do abnormal plastids

in the white sectors (Figure 8.3). Taken together with the finding that the white (but not

green) sectors accumulate phytoene, the first iteration of the threshold hypothesis held that

plastid phenotype in im is determined by the attainment of a threshold of PDS activity during

chloroplast biogenesis: developing plastids with above-threshold activities are able to

produce enough downstream, photoprotective carotenoids to form normal-appearing

chloroplasts (green sectors), whereas plastids with below-threshold PDS activities are

deficient in colored carotenoid production and susceptible to photooxidation (white plastids

and sectors) (Wetzel et al., 1994; Wetzel and Rodermel, 1998). This hypothesis was later

refined after IM was cloned and its function was defined (described in greater detail later).

Chloroplast biogenesis involves the differentiation of a small number of proplastids in

the shoot apical meristem into chloroplasts early in leaf primordial development (Mullet,

1988). This process is accompanied by several rounds of plastid division (Robertson et al.,

1995; Pyke and López-Juez, 1999). In C3 plants like Arabidopsis, cell expansion along the

proximal-distal axis results from divisions at the base of the leaf, while expansion in the

lateral direction is caused by cell divisions originating at the center of the leaf and extending

outward (Pyke et al., 1991; Van Lijsebettens and Clarke 1998). Hence, the oldest

chloroplasts are present in cells at the tips and margins of the leaf, whereas the youngest ones

are found in cells at the base of the leaf and at the midveins. Although plastid number

depends on cell size (Possingham and Lawrence, 1983), a typical Arabidopsis meristem cell 81 contains a dozen or so proplastids whereas a mesophyll cell contains from 100-120 mature

chloroplasts (Mullet, 1988; Pyke and Lopez-Juez, 1999). The numbers of plastids in im

green and white sectors have not yet been reported.

Wetzel et al. (1994) performed light-shift experiments to determine when PTOX

activity is first required following germination. These studies showed that cotyledon

pigmentation (all-green, all-white, or variegated) is influenced by the intensity of light (low

or high) perceived between 0 and 24 hours after seed coat breakage; the intensity perceived

before or after this interval does not matter. During this period, proplastids differentiate into

chloroplasts within the developing cotyledon (Mansfield and Briarty, 1996). The light shift

experiments thus indicate that PTOX plays a crucial role in early chloroplast biogenesis.

We have recently exploited confocal microscopy to visualize im variegation during

leaf primordia development. Figure 8.1 shows the red autofluoresence from chloroplasts in

cells of wild type and im leaf primordia; each primordium is located between the two

cotyledons. The figure dramatically illustrates that im cotyledons and primordia have fewer

chloroplasts than wild-type, and that im variegation develops very early in leaf development.

8.5.2 Studies using greening as a model system

Etioplasts in dark-grown angiosperm seedlings have prolamellar bodies (PLB) that

contain abundant ternary complexes of protochlorophyllide reductase (POR), NADPH and

Pchlide (Solymosi and Schoefs, 2010). Upon exposure to light, Pchlide is converted to

Chlide and the membranes of the PLB disperse to form thylakoids and organized grana

(Adam, 2011). Most of the protein components of the photosynthetic apparatus are present 82 in etioplasts, and hence de-etiolation (greening) primarily involves the coordinated synthesis

and assembly of pigments and pigment-binding proteins to form functional electron transport

chains (Blomquist et al., 2008; Kanervo et al., 2008). Photosynthetic competence is attained

in Arabidopsis ~5 hours after light induction at 22oC and 100 µmol photons m-2s-1 (Rosso et al., 2009).

Using Arabidopsis de-etiolation as a model system, Hüner’s group found that white sector formation in im is positively correlated with elevated excitation pressures during early greening, irrespective of whether excitation pressures are modulated by light and/or temperature (Rosso et al., 2009). Excitation pressure is a relative measure of the reduction state of QA, the first stable electron acceptor of PSII (Dietz et al., 1985; Hüner et al., 1998), and is measured by chlorophyll a fluorometry (Krause and Weis, 1991). The elevated excitation pressures in im were accompanied by delayed biogenesis of the thylakoid membrane, as monitored by the abundance of protein markers of chloroplast development

(Rosso et al., 2009). Consistent with these findings, we have found that the first visual observation of variegation in im is coincident with the ability to measure excitation pressures

(5-6 hours after illumination), indicative of functional PSII units and the acquisition of electron transport capacity (A. Foudree and S. Rodermel, unpublished data). This indicates that variegation is established prior to this time.

Given these considerations, our current working model of im variegation assumes that above-threshold excitation pressures predispose developing plastids to photooxidation, whereas im plastids with below-threshold excitation pressures have the capacity to develop into normal chloroplasts. This model is based on the twin notions of plastid autonomy and plastid heterogeneity. We surmise that excitation pressures vary from plastid-to-plastid in the 83 developing leaf primordium because of intrinsic differences in plastid biochemistry caused,

for example, by gradients in the leaf, sometimes steep, of light and of determinants of light

capture and use (Smith et al., 1997), including cell-specific circadian rhythms that dictate

ROS scavenging capacities (Velez-Ramirez et al., 2011). Such heterogeneity appears to be

widespread and has recently been reported in species as diverse as trees (Solymosi et al.,

2012). One of the advantages of this model is that it can account for the phytoene-

accumulation phenotype of im, as well as for the ultrastructural defects of im white plastids.

For instance, one of the primary targets of over-reduction might be membrane biogenesis

since a prominent effect of ROS formation is lipid peroxidation of polyunsaturated fatty acids

(Moller et al., 2007). A lack of plastid galactotlipids coupled with a lack of stabilizing

carotenoids might impede membrane formation, giving rise to the large vesicles seen in im

white plastids (Figure 8.3).

In summary, our current model of im variegation proposes that the pattern of

green/white sectoring is a reflection of the pattern of leaf development in C3 plants, coupled

with a heterogeneity in plastid-to-plastid excitation pressure during early cell and plastid

development, i.e., the chaotic sectoring in the mature leaf is established in the leaf

primordium. Recent support for the threshold hypothesis has come from the alx13

variegation mutant of Arabidopsis (Woo et al., 2011).

8.6 Compensating factors

Because the green sectors of immutans contain morphologically normal chloroplasts,

we have proposed that they arise from plastids that escape the effects of the im/im mutation 84 early in chloroplast biogenesis, presumably by the action of compensating factors that affect

excitation pressure thresholds, directly or indirectly. Two approaches have been used to gain

insight into these factors: 1) molecular characterization of green versus white im sectors; and

2) identification and characterization of second-site suppressors of im variegation. These

approaches will be summarized briefly.

8.7 How do the green and white sectors of im differ?

The green leaf sectors of im have higher photosynthetic rates than wild type leaves,

14 monitored either by rates of O2 evolution or CO2 uptake (Aluru et al., 2001, 2007). They also have elevated chlorophyll a/b ratios and enhanced chlorophyll amounts; FACS analyses have revealed these increases are due to elevated chloroplasts numbers (per unit volume) rather than to enhanced amounts of chlorophyll per chloroplast (Meehan et al., 1996).

Accompanying these changes, the anatomies of the green leaf sectors are reminiscent of leaves adapted to growth in high-light conditions, e.g., they are thicker than normal due to enlarged air spaces, mesophyll cells and epidermal cells (Aluru et al., 2001). On the other hand, the white leaf sectors have a normal thickness, but their palisade cells fail to expand.

This perturbation is consistent with an impairment in retrograde signaling that regulates leaf developmental programming (Rodermel, 2001; López-Juez, 2009; Ruckle and Larkin, 2009).

We have made a similar proposal to explain reduced fruit size and disruptions in pericarp tissue morphogenesis in the tomato ghost mutant (Barr et al., 2004).

Consistent with elevated rates of photosynthesis, the green leaf sectors have increased

Rubisco and sucrose phosphate synthase activities, enhanced starch and sucrose pool sizes, 85 and an altered pattern of carbohydrate partitioning that favors sucrose over starch (Aluru et

al., 2007). By contrast, the white sectors accumulate low levels of sucrose and have

increased acid invertase activities. These observations suggest that sucrose moves along a

gradient from the green to the white cells, where it is hydrolyzed and used for growth. It is

therefore possible that photosynthetic rates in the green sectors are controlled, in part, by sink

demand, and that the elevation of photosynthetic rates in the green sectors is part of a growth

strategy to compensate for reductions in total source tissue (Aluru et al., 2007).

Global transcriptomics experiments revealed that some of the differences between the

green and white im sectors are likely due to alterations in transcript abundance (Aluru et al.,

2009). In particular, genes for photosynthesis and photosynthesis-related processes are

repressed in im white tissues, while genes for sucrose catabolism, transport, mitochondrial

electron transport, and fermentation are induced. These results suggest that energy is derived

via aerobic and anaerobic metabolism of imported sugar in the im white cells; these data are

in accord with findings from the biochemical studies, discussed above. The profiling studies

also showed that oxidative stress response genes are generally induced in both the green and

white im tissues, but that some stress genes are significantly more upregulated in the green

than white sectors. These genes are targets for investigation as potential compensating

factors.

8.8 Second-site suppressors of im variegation

The above studies have been conducted with mature leaves, and though they have provided valuable information about im, they are limited in their usefulness regarding 86 information about the primary lesion in im because they describe developmental, physiological, and biochemical states that represent long term adaptations to the lack of

PTOX in the leaf. They do not provide information about the primary alterations that occur when the mutation first becomes active during chloroplast biogenesis and when compensating mechanisms might first come into play.

Given that all alleles of im isolated to date are null (Carol et al., 1999; Wu et al.,

1999), an attractive strategy to identify compensating factors is to characterize second-site suppressors of im variegation; the rationale being that since these factors define elements and/or processes that are able to substitute for or bypass the requirement for PTOX activity early during chloroplast biogenesis, these factors might not easily be accessed by any other means. We assume that later in chloroplast biogenesis these factors include the acquisition of photosynthetic competence, when fully-functional photosynthetic electron transport chains are able to oxidize the PQ pool, hence obviating the need for PTOX. This would be consistent with the observation that PTOX is required during chloroplast biogenesis but not during steady state photosynthesis (Rosso et al., 2006, 2009). Yet, since variegation is present early in development of the leaf primordium (Figure 8.1) and by the time chlorophyll fluorescence assays can first be taken after transfer of etiolated seedling to the light (~5 hours) (A. Foudree and S. Rodermel, unpublished data), we assume that much of the variegation pattern is established prior to the acquisition of photosynthetic competence.

Hence, we propose that during this time compensating factors are present that permit the desaturation reactions of carotenogenesis to occur in a PTOX-independent manner. For example, there might be PTOX-independent pathways of PQH2 re-oxidation coupled to PDS; other mechanisms of PQH2 re-oxidation coupled to the photosynthetic membrane (Casano et 87 al., 2000); or chemical re-oxidation of plastoquinol (Khorobrykh and Ivanov, 2002).

Shahbazi et al. (2007) provided early support for this idea on the basis of experiments

showing that phytoene did not accumulate following exposure of mature green leaf or fruit

tissues of the ghost mutant to high light: carotenoid synthesis occurred normally in the absence of PTOX. In this context, it might be noted that early studies showing an O2 requirement for PDS activity were based on in vitro systems from non-photosynthetic chromoplasts (Beyer et al., 1989; Mayer et al., 1990); we suggest that this requirement reflects a need for PTOX activity in these systems where, interestingly, an “oxidoreductase fraction” was proposed to act as an intermediate between PDS and O2 (Beyer et al., 1989).

8.9 AOX2 and AOX1a suppressors

To identify im suppressors, we have conducted EMS and activation-tagging mutagenesis of im and screened for mutant plants with a non-variegated phenotype.

Molecular characterization of one suppressor line (designated ATG791) has been reported

(Fu et al., 2012). This line is all-green and, surprisingly, carries an activation-tagged

(overexpressed) version of mitochondrial AOX2. Arabidopsis contains five members of the

AOX gene family, all of which are thought to be exclusively mitochondrial in location

(AOX1a-d and AOX2); AOX2 is a low abundance, seed-specific member of this family

(Saisho et al., 1997; Saisho et al., 2001; Clifton et al., 2006; Winter et al., 2007; Polidoros et al., 2009).

We found that AOX2 is targeted to chloroplast thylakoids of ATG791, where its activity replaces that of PTOX in the desaturation steps of carotenogenesis, i.e., it rescues the 88 phytoene-accumulation defect of im, restoring carotenoid biosynthesis to normal. This

restoration is accompanied by a normalization of excitation pressures, suggesting that the two

processes are linked (Fu et al., 2012). It is therefore likely that elevated carotenoids in the

suppressor lines are directly responsible for rescue of the variegation phenotype, and hence

for recovery of the capacity to undergo normal chloroplast biogenesis. Results similar to

ATG791 were obtained when AOX2 was overexpressed using the CaMV 35S promoter, or

when AOX1a, the most highly and ubiquitously expressed member of the Arabidopsis AOX

gene family, was re-engineered to target the plastid; overexpressed AOX1a is found

exclusively in mitochondria (Clifton et al., 2006; Winter et al., 2007; Polidoros et al., 2009).

In both cases, the im-induced defects in phytoene accumulation and chloroplast biogenesis

were reversed.

Further experiments with the AOX2 and AOX1a overexpression lines showed that

chloroplast-localized AOX2, but not AOX1a, formed monomers and dimers in the thylakoid

membrane, reminiscent of AOX regulation in mitochondrial inner membranes. In addition,

both proteins accumulated as higher molecular weight complexes in thylakoids, though these

complexes differed in size and number between the two lines. Interestingly, photosynthetic

activities, as monitored by chlorophyll fluorescence, were not generally perturbed in the

overexpression lines, nor was growth altered. This suggests that the presence of AOX1a and

AOX2 complexes in thylakoids does not significantly perturb steady state photosynthesis, at

least under non-stress conditions. Our operating assumption is that the suppressor lines

define novel electron transport paths in the chloroplast.

AOX2 has previously been localized to mitochondria (Saisho et al., 2001), and

because it was imported into chloroplasts using its own transitpeptide in the AOX2 89 overexpression lines, as well as in transient expression assays, our current working

hypothesis is that AOX2 is a dual-targeted protein that functions in plastids to supplement

PTOX activity during the early events of chloroplast biogenesis. Although the idea that

AOX2 is normally an im-compensating activity needs further confirmation, the ability of

AOX1a to substitute for PTOX in the correct physiological and developmental contexts is a

dramatic example of the capacity of a mitochondrial protein to replace the function of a

chloroplast protein, and illustrates the plasticity of the photosynthetic apparatus. This is all

the more remarkable given the striking differences in regulation, substrate-specificity

(ubiquinol in mitochondria versus plastoquinol in plastids) and phylogenetic distance

between AOX and PTOX (McDonald et al., 2011; Fu et al., 2012). It will therefore be of

great interest to explore further the function of chloroplast AOX1a and AOX2 in

photosynthesis.

8.10 pgr5 and crr2-2 suppressors

Shikanai’s group has long been studying mechanisms of PSI cyclic electron transport, a process that contributes to ∆pH formation across the thylakoid, but not to NAD(P)H accumulation, thus providing a way of manipulating ATP/NAD(P)H ratios (Shikanai, 2007).

Okegawa et al. (2010) reported that two mutants which are perturbed in this process - crr2-2

(chlororespiratory reduction) (Hashimoto et al., 2003) and pgr5 (proton gradient regulation5) (Munekage et al., 2002) are able to suppress im variegation. The pgr5 mutant is defective in electron transport, has a reduced ∆pH, an over-reduced stroma (a decreased

ATP/NADPH), an oxidized P700+, and a loss of qE (Munekage et al., 2002; DalCorso et al., 90

2008; Okegawa et al., 2008). The crr2-2 mutant, on the other hand, is defective in NDH

activity due to aberrant expression of the plastid ndhB gene (Hashimoto et al., 2003); it has

photosynthetic characteristics similar to pgr5 (Okegawa et al., 2010). Neither of these mutants has a chloroplast development phenotype (Munekage et al., 2004).

Okegawa et al. (2010) assumed that crr2-2 and pgr5 define separate pathways of PSI

cyclic electron transport, and given this assumption, the goal of their experiments was to take

a genetic approach to examine interactions between these pathways and PTOX, all of which

share the PQ pool. Although it was found that loss of either one of these pathways (in crr2-2

or pgr5) or both (in crr2-2/pgr5) was able to suppress im variegation, the data did not permit

a clear explanation of the suppression mechanism: the authors proposed that it might involve

remodeling of the thylakoid membrane, or perhaps elevation of the ATP/NADPH ratio

(Okegawa et al., 2010).

One factor complicating an understanding of the mechanism of im suppression in

these studies is that considerable controversy surrounds the function of PGR5 and whether it

is even involved in PSI cyclic electron transport (reviewed in Kramer and Evans, 2011). The

only agreement seems to be that PGR5 plays a role in fluctuating light conditions (Tikkanen

et al., 2010). However, if the threshold model of im variegation is correct, it is likely that

crr2-2 and pgr5 exert their effects by lowering excitation pressures during early chloroplast

development, perhaps by reducing the flow of electrons into the PQ pool. Although further

studies will be needed to define the interactions between PTOX, PGR5 and CRR2, it is clear

from the present data that interactions occur between these proteins very early in chloroplast

development. All three are also present in etioplasts (Aluru et al., 2001; Long et al., 2008;

Peng et al., 2008), perhaps to help poise the PQ pool for photosynthesis. 91

In summary, suppressor analysis holds great promise as a tool to understand the function of PTOX and mechanisms of compensation that occur when the protein is lost. The data in this paper highlight the significance of im suppressor analysis for understanding pathways and interactions that mediate early chloroplast development, and they include the identification of novel proteins (AOX2) and pathways (PSI cyclic electron transport).

8.11 Acknowledgements

We extend our thanks to Margaret Carter of the Iowa State Image Analysis Facility.

Additionally, we thank Dr. Harry Horner and Tracey Pepper of the Iowa State Microscopy and Nanoimaging Facility. This work was supported by funding to S.R. from the Chemical

Sciences, Geosciences, and Biosciences Division, U.S. Department of Energy (DE-FG02-

94ER20147).

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8.13 Figures

Figure 8.1 Confocal microscopy of leaf primordia. The images show first leaf primordia

(LP) and flanking cotyledons (Cot) from wild-type (A) and immutans (B). Images were captured by taking a confocal image of autofluorescence (red) and overlaying it with a non- confocal transmitted light image (gray). Plants were grown at 22oC under continuous illumination for 3 days at 15 µmol photons m-2s-1 followed by 2 days at light intensity 100

µmol photons m-2s-1. White scale bars represent 25 µm.

107

Figure 8.2 Model of PTOX as a cofactor of phytoene desaturase (PDS). PTOX is a plastoquinol terminal oxidase that regulates the redox state of the plastoquinone pool (PQ) during early chloroplast biogenesis. Electrons from the linear electron flow, cyclic electron flow mediated by either NDH or the Fd dependent PGR5 pathway, and the desaturation of phytoene feed into the PQ pool. PTOX plays a pivotal role in transferring the electrons from the PQ pool to molecular oxygen thus keeping the pool oxidized.

108

Figure 8.3 Ultrastructural analysis of immutans (A) A representative immutans plant grown at 22oC under continuous illumination for 7 days at 5 µmol photons m-2s-1 followed by 3 weeks at 100 µmol photons m-2s-1. (B) Representative plastids from an im white sector. The plastids lack organized lamellar structures and contain vacuoles and numerous plastoglobules. (C) Representative chloroplast from an im green sector. TEM Materials and methods are identical to those used in Figure 3.2 of this thesis.

109

CHAPTER 9. Dark-grown immutans reveals IMMUTANS is essential for normal

chloroplast development

A paper to be published

Andrew Foudree, Sekhar Kambakam, Steve Rodermel

9.1 Abstract

Chloroplasts develop by the coordination of nuclear gene expression with that of the chloroplast genome. This coordination is required to form proper protein complexes within the chloroplast and photosynthetic apparatus. Using the chloroplast variegation mutant immutans, I show that variegation is influenced at a very early stage in development by a lack of IMMUTANS. Early in development the complex process of assembling the thylakoid membranes of immutans results in abnormal or normal chloroplast development. At an early stage, using an etiolated system, dark grown plastids (etioplasts) of immutans are impaired in their ability to form a proper amount of colored carotenoids, and they develop improperly upon movement into the light (de-etiolation). During the de-etiolation process, within the chloroplast thylakoids, proteins are inserted into the membrane while radical oxygen species are generated by the inability of electrons to be quenched non-photochemically or photochemically. Experiments involving these processes point to the requirement of

IMMUTANS in the dark to set the stage for proper membrane assembly in the light.

110

9.2 Introduction

immutans is a recessive nuclear mutation which causes variegated leaf formation in

Arabidopsis (green and white tissue sectoring) (Figure 9.1). Variegation mutants, such as immutans, have been studied for 50 years and have helped contribute much to the understanding of chloroplast biogenesis (Rédei, 1963; Rédei, 1967; Rédei, 1975; Röbbelen,

1968). immutans is important due to its unique green and white sectoring that can be used to further our basic understanding of chloroplast development as well as that of the bioenergetic process of photosynthesis. The immutans mutant is light sensitive and must be germinated under low light conditions in order to form functional chloroplasts and avoid photobleaching

(Wetzel, et al., 1994; Aluru, et al., 2001). IMMUTANS was found to have homology with the mitochondrial alternative oxidase (AOX), and because of this similarity it is also referred to as the plastid terminal oxidase (PTOX) (Siedow, et al., 1995; Fu, et al., 2005, 2009).

IMMUTANS has been shown to be critical in carotenoid biosynthesis and is linked tightly to the oxidative state of the plastoquinone pool (Wu, et al. 1999). The link between carotenoid biosynthesis and the plastoquinone pool comes at the phytoene desaturase (PDS) step of carotenogenesis (Norris, et al., 1995). PDS requires an oxidized plastoquinone pool to donate electrons to in order to function properly. It has previously been shown that in the white sectors of immutans, phytoene accumulates, thus implying a block in the PDS step and an over-reduced plastoquinone pool (Wetzel, et al., 1994). Mechanistically, IMMUTANS plays a role in controlling the redox state of the plastoquinone pool by taking excess electrons from plastohydroquinone (PQH2), the reduced form of PQ, and transferring them to molecular oxygen under high excitation pressure conditions to form water. This mechanism therefore 111 protects the photosynthetic electron transport chain from over-reduction and oxygen radical

formation while at the same time keeping the plastoquinone pool oxidized so that it can be

used as an electron sink by biosynthetic enzymes such as PDS. Because PTOX does not

appear to be a “safety valve” during steady state photosynthesis (Rosso, et al., 2006), I

hypothesize that it plays this role much earlier in development when nascent thylakoid

membranes are being formed (Aluru, et al. 2006). Previous work involving mutants of PQ

biosynthesis have shown a similar phenotype to that of immutans during early development,

indicating that the role of PQ is central in photoprotection during early plastid biogenesis

(Norris, et al., 1995, 1998; Motohashi, et al., 2003).

One way of looking at early development of chloroplasts is to observe their

development during the transition of shoot apical meristems into true leaves of Arabidopsis.

Meristematic proplastids that exist in these early leaves undergo division and maturation into

fully functional chloroplasts as cells divide and expand. In Arabidopsis meristems cells

contain between 10 and 15 proplastids that divide approximately three times such that mature

cells have approximately 80 to 120 chloroplasts. Within the shoot apical meristem of

developing leaves chloroplasts at differing stages of development are present. This makes

these leaves heterogeneous with respect to their stage of chloroplast development.

A second way of looking at early chloroplast development is to use an etiolated

system. Etioplasts are a natural stage of chloroplast development and occur in seedlings that

germinate underground and have yet to be exposed to light (Gunning, 1965). Etioplasts occur

in dark grown seedlings and characteristically form a paracrystalline body known as the

prolemellar body (PLB). The PLB consists primarily of protochlorophyllide oxidoreductase

(POR) that is complexed with protochlorophyllide (Pchlide) and NADPH. Also present in the 112 etioplasts are several photosynthetic proteins, chlorophyll biosynthetic enzymes, carotenoid

biosynthesis enzymes as well as carotenoids (Welsch, et al., 2000; Bréhélin and Kessler;

2008, Park, et al., 2002). Pigment compositions of the envelope membranes of non-green

plastids compared to green chloroplasts are in most cases very similar (Douce and Joyard

1979, 1984). Recent proteomics data suggest that within chloroplasts carotenoid biosynthesis

enzymes reside on the inner envelope membrane (Joyard, et al., 2009). Plastoquinone is also

synthesized on the chloroplast inner membrane, which implies that plastoquinone is in close

proximity to PDS and can be used during the generation of carotenoids within developing

etioplasts (Motohashi, et al., 2003; Jeffery, et al., 1974; Douce and Joyard, 1990). Within

etioplasts carotenoids are synthesized on the inner envelope membrane and stored within the

PLB (Park, et al., 2002; Liljenberg, 1980; Joyard, et al., 2009).

Inner envelope membranes, prothylakoids, and PLB serve as a continuous unit, even

though differential partitioning of polypeptides between them may occur (Gunning, 1965;

Sundqvist and Dahlin, 1997; Block, et al., 1983; Cline, et al., 1981; Murata, et al., 1981;

Carde, et al., 1982). This is important because it implies that interplay between carotenoid

synthesis, plastoquinone synthesis, and IMMUTANS resides within a spatially linked inner

envelope, prothylakoid and PLB membrane. Upon illumination, the PLB body starts to

degrade and the light dependent enzyme POR converts protochlorophyllide to chlorophyllide

resulting in the formation of thylakoid stacks called grana. This process, known as

photomorphogenesis, occurs very rapidly and the transition into a chloroplast is usually

complete within twenty-four hours of light exposure. Etioplasts are considered to be an

intermediate step between proplastids and chloroplasts and are therefore conducive to the

study of chloroplast development. This etiolated system provides a homogeneous population 113 of plastids within the cotyledon that are synchronized in their developmental stage. Previous

research has shown that etiolated plastids of Arabidopsis plants are subject to reducing

conditions and that plastoquinone may become over-reduced in the absence of light (Perez-

Ruiz, et al., 2006; Moro, et al., 2004, Wu, et al. 2011).

During the processes of both etioplast and meristematic development of chloroplasts,

the synthesis and insertion of photosynthetic protein complexes into the membranes of the

thylakoids must result from the concerted effort between the nuclear and chloroplast

genomes. Inhibition of chloroplast protein import such as that of the ppi2 mutant shows a

very similar phenotype to that of immutans in that they are both arrested at the cotyledon

stage under normal light conditions (Bauer, et al., 2000). At early stages in development,

newly forming chloroplast membranes are particularly susceptible to oxidative damage. This

is because they lack downstream components needed for photochemical quenching while at

the same time carotenoid biosynthesis and other metabolic pathways are adding electron

pressure to the system by using the plastoquinone pool as an electron sink. Also, the light

dependent xanthophyll cycle may not yet be efficient enough due to the requirement of

prolonged light exposure for its function (Demming-Adams and Adams, 1992; Horton, et al.,

1996). Research has shown that excitation pressure is the key to variegation in immutans

(Rosso, et al., 2009). Also, research on etiolated plastids of Arabidopsis plants have shown

that they are subject to reducing conditions and that plastoquinone may become over-reduced

even in the absence of light (Perez-Ruiz, et al., 2006; Moro, et al., 2004). If the newly

forming thylakoid membrane is not at the proper redox state, then insertions of protein

complexes occur at reduced rates (Zhang, et al., 2000; Zhang and Aro, 2002; Russell, et al.,

1995). This membrane susceptibility is only exacerbated by the fact that, upon light 114 exposure, unbound chlorophyll and/or Pchlide will act as photosensitizers and generate toxic

oxygen radicals (ROS). Carotenoids are crucial during this early stage of membrane

formation for the protection against generated ROS (Nyogi, 1999; Li, et al., 2000). During

the conversion of dark to light, when IMMUTANS is absent, thylakoid membranes will

undergo light stress conditions immediately upon movement into the light. Knowledge of

thylakoid photosynthetic gene expression as well as the dynamics of photosynthetic proteins

present during the conversion of etioplasts to chloroplasts are important to understanding the

effects of IMMUTANS during development and have yet to be fully elucidated.

Under stress conditions, such as high light, chloroplasts accumulate plastoglobules.

Plastoglobules are lipid-rich bodies that form at the curved edges of thylakoid membranes

and serve as dynamic reservoirs for many compounds such as carotenoids, alpha-tocopherol,

and plastohydroquinone (Austin, et al., 2006; Szymanska and Kruk, 2010). They are

important to the study of immutans due to their storage of compounds that have been directly

linked to immutans function. Under most stress conditions, a concomitant reduction of the

thylakoid membrane occurs alongside plastoglobule formation (Bréhélin and Kessler, 2008).

This phenomenon of thylakoid membrane reduction is similar to that which occurs in

immutans under high light, when thylakoids have high excitation pressure (Rosso et al.,

2009). During events that involve thylakoid membrane degradation such as those that occur

during stress, senescence, and chloroplast-to-chromoplast transitions, plastoglobules have

been shown to increase in number and size (Vidi, et al., 2006; Bréhélin and Kessler, 2008).

Therefore, plastoglobules can serve as an indicator of membrane degradation under stress

conditions (e.g. an over-reduced membrane).

White chloroplasts of fully-developed immutans leaves are vacuolated and unable to 115 form normal thylakoid membrane structures, and they resemble chloroplasts that have been

under high light stress or are undergoing senescence (Aluru, et al., 2001). Several groups

have studied the proteomics of plastoglobules and have shown that they may act directly in

several metabolic pathways (Kanervo et al., 2008; Yitterberg, et al., 2006; Vidi, et al., 2006).

Not only do plastoglobules accumulate carotenoids and plastoquinone, but they also contain

the carotenoid metabolism enzyme atCCD4 (Yitterberg, et al., 2006). The role of carotenoid

cleavage dioxygenases such as CCD4 is still under investigation in several plant species

(Huang, et al., 2009). Because plastids of immutans are faced with high excitation pressure,

determining the incidence and morphology of plastoglobules within this mutant are important

to understanding the underlying mechanism of its variegation. In addition, ROS play a role in

chloroplast sectoring prior to chlorophyll insertion and membrane competencey.

9.3 Results

Due to the light sensitivity of immutans cotyledons as well as previous knowledge

about the development of immutans variegation, I hypothesized that the variegation lesion

was formed at an early stage of chloroplast development. I therefore looked at chlorophyll

auto-fluorescence during the development of the first true leaves in immutans plants versus

wild-type plants (Figure 8.1). Images of the first true leaves that initiate between the

cotyledons were then taken by a confocal microscope. For these experiments seedlings were

grown under low light for three days and then transferred into normal light for two days. By

looking at the pattern and intensity of auto-fluorescence in these leaves, it is apparent that the

chloroplasts of immutans at this stage do not develop properly and that they appear to be less 116 fluorescent and less homogeneous than those of wild-type. This result indicates that

IMMUTANS is essential for normal growth at very early stages during chloroplast

differentiation, confirming earlier observations (Wetzel et al., 1994).

Expanding first true leaves of Arabidopsis contain a heterogeneous population of

developing chloroplasts. Therefore I used a de-etiolation system in which the chloroplasts

were at the same stage of development (etioplast stage) prior to illumination, which acts to

synchronize their development. For these experiments dark grown plants were exposed to the

light for differing amounts of time: 0 hour, 3 hours, 6 hours, 12 hours, and 24 hours. The

changes in phenotype over this 24 hour period were captured using a dissecting microscope

(Figure 9.2). These photographs show that upon light exposure, the cotyledons greened at a

slower rate in immutans than in wild-type. In wild-type chlorophyll starts to accumulate

between the 3 hour and 6 hour time points, whereas in immutans green cotyledons did not

appear until 12 hours in the light. At 24 hours both wild-type and immutans cotyledons were

mostly green, but after about two weeks, immutans cotyledons became variegated.

Cotyledons during this stage expanded normally in both wild-type and the mutant. On

occasion variegated sectoring was observed in immutans at 24 hours of light exposure. It is

important to note that wild-type and immutans cotyledons at time 0 hours were both orange

and were therefore able to synthesize colored carotenoids in the dark (even though immutans

appeared to be less orange than wild-type).

These observations were confirmed and expanded by analysis of chlorophyll and

carotenoid pigment concentrations (Figure 9.3 and 9.4). Samples of seedlings were pooled

and pigments were measured using a spectrophotometer using the maximum absorbance for

chlorophyll and carotenoids. The data show that during de-etiolation cotyledons of immutans 117 have a lower amount of chlorophyll and carotenoids than wild-type, and that they accumulate

at a slower rate than wild-type during greening. Confocal images at each of these given time

points were also taken. In wild-type, chlorophyll accumulates at 3 hours while in immutans it

accumulates at 6 hours (Figure 9.5).

In order to see if variegation occurs at the cellular level throughout the de-etiolated

stages, I used confocal microscopy to look at the cotyledons of immutans and wild-type. I

used a white light laser to excite chloroplasts in both wild-type and immutans and observed

auto-fluorescence of developing chloroplasts. Images were taken at intervals between 3 and 6

hours of light exposure to assure that enough auto-fluorescent signal would be present to

discern a difference between chloroplasts. Corresponding bright field images were also taken

to illustrate the section of tissue being used. These images clearly showed that variegation

occurred at the 5 hour time point post light exposure of etiolated cotyledons (Figure 9.6).

Optical slices through the cotyledon tissue confirm that those segments that lacked auto-

fluorescence were indeed devoid of chlorophyll-containing chloroplasts.

To understand the overall state of the thylakoid membrane and its components during

de-etiolation, I observed the protein levels of various photosynthetic components. Time

points of 0 hour, 3 hours, and 6 hours post light exposure were chosen to observe the

dynamics of this system. During these developmental time points, I observed a decrease in

overall protein for Var2 and ATPase between immutans and wild-type (Figure 9.7). POR is

shown to decrease and then increase over time, which is concurrent with the shifting of

PORA to PORC. Equal protein was loaded in each Western blot and corresponding

coomassie gels were run in parallel. Coomassie gels show that overall protein accumulation

occurs rapidly over this time interval. 118

This delay in chlorophyll and protein integration during de-etiolation indicated the impairment at a very early time point of chloroplast membrane development and led us to hypothesize that IMMUTANS may be playing a role even earlier in development. In order to observe what was happening prior to the de-etiolation of immutans, I chose to look at etioplasts within dark grown plants. To observe if a defect was present, I used transmission electron microscope images of etioplasts in both immutans and wild-type cotyledons that were grown in the dark for 3 days. Large black oil bodies were prevalent in the cytoplasm of all of these etioplast images and serve as an energy store while these cells grow under strictly heterotrophic conditions. Both immutans and wild-type etioplasts were able to form prolamellar bodies with a normal crystalline pattern; however, they differed in the amount and size of their plastoglobules. immutans had more plastoglobules than wild-type, and they tended to occur in a clustered formation (Figure 9.8). While clusters were more commonly observed, they were not always present. These clusters contained plastoglobules that were less osmiophilic and larger than those of wild-type, and they were usually associated with the etioplast PLB. These images show a defect in immutans etioplasts when compared to wild- type. To see if etioplast development is affected by the amount of time seedlings are grown in the dark, I grew them for 7 days in the dark and embedded these samples for TEM analysis.

A similar phenotype was seen at seven days in the dark compared to that at 4 days in the dark

(Figure 9.9).

To verify that plastoglobules were indeed more prevalent in immutans versus wild- type, a Western blot containing protein of equal amounts was run using the antibody for fibrillin. Fibrillin or PGL-35 is a protein that is specific to plastoglobules and is thought to be involved in the prevention of plastoglobule coalescence within plastids (Venkatasalam, 2012; 119

Vidi, et al., 2006). This Western blot shows that PGL-35 is more abundant in immutans than in wild-type dark grown cotyledons (Figure 9.10).

To determine if the dark grown etioplasts of immutans were similar to defective white sector plastids in fully expanded leaves, Dr. Sekhar Kambakam used HPLC to detect if phytoene accumulates in the dark. He found that when compared to wild-type, etiolated immutans seedlings accumulate phytoene to a much larger extent (Figure 9.11). This implies that the PDS step in carotenogenesis is blocked in immutans in the dark, and suggests that one reason plastoglobules occur at a higher frequency in immutans is because they serve as carotenoid storage reservoirs and may be storing excess quantities of phytoene.

Plastoglobules were often found associated with the PLB, which has previously been determined to be the location in the etioplast where carotenoids are stored (Steinmüller and

Tevini, 1985).

Since the level of colored carotenoids was impaired during de-etiolation of immutans

I wanted to grow wild-type seedlings on 0.1 µmol Norflurazon to see whether NF-treatment would have a similar effect on phenotype as it did on immutans. NF-treated seedlings were completely white and died upon movement into the light, indicating that they were devoid of photoprotective carotenoids and became photooxidized (Figure 9.12). TEM images of plastids in NF-treated dark-grown cotyledons showed that they had a similar phenotype to that of immutans. This indicates that a block in the PDS step of carotenogenesis in the dark can lead to an accumulation of plastoglobules. Interestingly, etioplasts from the NF-treated seedlings had a greater prevalence of plastoglobules which were lighter and had less osmiophilic staining than those from wild-type. These plastoglobules were similar to those in immutans. My results are similar to previously published data that show plastids accumulate 120 plastoglobules when there is a block in the PDS step (Moro, et al., 2004).

I hypothesize that due to impaired carotenoid biosynthesis in dark-grown immutans,

movement of immutans into the light generates ROS that contribute to the variegation

phenotype of the mutant. To determine the role that ROS plays in this process, etiolated wild-

type and immutans seedlings were grown in the dark for 3 days and then transferred to light.

Several time series were conducted using ROS sensitive dyes. ROS were detected by NBT,

DAB, and SOSG dyes which detect super oxide, hydrogen peroxide and singlet oxygen

respectively. NBT, DAB, and SOSG all showed a maximum amount of ROS accumulation at

3 hours of light exposure post dark-growth (Figure 9.13). Also, immutans showed a higher

incidence of ROS generation at this time point than did wild-type.

9.4 Discussion

In order to study the mechanism of variegation in immutans I focused on early

development. Since IMMUTANS does not function in fully-expanded leaves to relieve

electron excitation pressure, our hypothesis is that it plays a role in early development

between the proplastid and the chloroplast developmental stages. To investigate this process I

looked at the development of the first true leaves of immutans and wild-type to see if

variegation was visible as proplastids divided in the meristem and developing leaf primordia.

Confocal images of these leaves show that, when compared to wild-type, variegation is

occurring in the early developing leaf primordia of immutans. This supports our hypothesis

that as the proplastids transition into chloroplasts, signals such as singlet oxygen may be

occurring at different levels in each of the cells, which in turn lead to the development of 121 white or green chloroplasts.

I then turned to dark grown seedlings and a de-etiolated system to study the etioplast

to chloroplast transition. By using a de-etiolated system I was able to take advantage of

cotyledons that contained a homogeneous population of etioplasts at a uniform stage of

development. This allowed me to use a standardized population of plastids that were

synchronized in their development. De-etiolation of immutans and wild-type showed that

immutans developed chlorophyll at a slower rate and was not as green when exposed to light.

During the transition from dark to light, POR-Pchlide-NADPH complexes of the PLB

disassemble, and the PLB membranes begin to transform into flattened stacks that will give

rise to thylakoids. The presence of POR during this process protects the membranes from

ROS production originating from Pchlide (which is a photosensitizer). At the same time that

proteins are being integrated into the developing thylakoid membrane and photosystems are

being assembled, fully-functional PSI and downstream components are not yet present to

serve as photochemical quenchers of the system. Therefore I speculate that variegation starts

to form even before chloroplasts build up enough chlorophyll to autofluoresce.

The thylakoid membrane allows for the generation of an electrochemical gradient that

results in the synthesis of ATP and NADPH. Control of the electrical component (as opposed

to the chemical component) of this gradient is important in order to prevent over-reduction

and subsequent production of ROS during light conditions. Control of the electrical

component of this pathway can occur by photochemical and non-photochemical quenching

with respect to photosynthesis and by way of thioredoxin reductase with respect to the Calvin

cycle. However, the chemical component of this system is also important. A fine balance of

pH exists between the lumen and the stroma. This pH difference (6.8 and 8 respectively 122 under normal conditions) regulates the activities of the cytochrome b6f complex/non-

photochemical quenching and Calvin cycle enzymes. Proper pH is important for protein

import and insertion into newly forming thylakoids. During early development when

proplastids or etioplasts are developing into chloroplasts, fully-formed thylakoids do not yet

exist and ∆pH has yet to be generated. Hence at this time carotenoids that have built up in the dark serve to protect newly forming thylakoid membranes. However, in immutans, colored carotenoids are not present at the levels needed to defend against ROS that are produced during the first 6 hours of development. Higher ROS were detected in immutans versus wild- type within the first three hours of development. These ROS most likely cause irreparable damage to the membrane-formation ability of plastids that are to become white sectors within immutans. Once this damage has occurred, the chloroplasts remain white and are unable to form thylakoid stacks. Some plastids are able to make it through this stage, turn green, and form normal chloroplasts. This is most likely due to their development of a slightly larger carotenoid content in the dark than their neighboring etioplasts.

During the transition between proplastids and chloroplasts, thylakoid membrane

development is crucial. If thylakoid membranes are not formed properly during this

developmental stage, membrane deficient white plastids form and are permanently unable to

turn into normal chloroplasts. Here our experiments show that in the absence of IMMUTANS

some chloroplasts are unable to form properly between initial light exposure and three hours

of development. Chlorophyll and carotenoid concentrations within the chloroplast are

reduced during the greening process and variegation occurs at 5 hours of development post

initial light exposure. This follows the data indicating that chlorophyll does not accumulate to

appreciable levels until 6 hours of light exposure after dark growth. Therefore, during the 123 first 6 hours of growth, unbound chlorophyll precursors (such as Pchlide) are present and can

serve to generate species similar to triplet chlorophyll and reactive oxygen species. I also

show that important membrane-bound regulatory photosynthetic proteins are reduced during

this greening process. Therefore, I found that IMMUTANS is a vital component during early

chloroplast biogenesis when thylakoids are initially forming and that IMMUTANS is crucial

for the formation of thylakoid membranes during dark to light transitions.

I hypothesized that IMMUTANS is important during the initial stages of chloroplast

development when thylakoids are being formed, and it may also be working in the dark to

prepare proplastids for their transition into chloroplasts. Here I show that etioplasts of

immutans differ from wild-type in their phenotype as well as composition. Even though they

can occasionally form normal looking prolamellar bodies, immutans etioplasts contain more

plastoglobules than wild-type. The presence of plastoglobules in these plastids suggests that

they are accumulating compounds that are lipophilic, perhaps to deal with oxidative stress.

These lipophilic compounds may be forming due to an over-reduction of photosynthetic

components in the dark from electron sources like NADPH or carotenoid biosynthesis. It has

previously been shown that reducing conditions occur in the dark (Perez-Ruiz, et al., 2006).

Also, the accumulation of plastoglobules in dark-grown etioplasts has been supported by

previous experiments that show that Norflurazon-treated etioplasts accumulate plastoglobules

(Moro, et al., 2004). These studies have also shown that etioplast membrane stability is

compromised due to an altered association of Pchlide and POR, yet they are still able to form

normal PLBs (Moro, et al. 2004; Park, et al., 2002).

While it has previously been stated that carotenoid defects can cause a delay in greening (Park, et al., 2002), the accumulation of phytoene in dark-grown immutans may be a 124 secondary effect of the mutation and the absence of IMMUTANS may be the primary cause

of the delay. This is because IMMUTANS is directly involved with control of plastid

membrane redox status, and there is an impairment of protein insertion during the greening

process. Plastoglobule clustering and phytoene accumulation in the dark support my

hypothesis that IMMUTANS is functioning during the development of dark-grown etioplasts.

This conclusion implies that the stage for variegation has already been set before the onset of

light exposure. This fits with a model whereby IMMUTANS plays a role in the dark to keep

the plastoquinone pool oxidized which will prevent ROS formation when the lights are

turned on and chloroplast biogenesis occurs. This predisposition sets the stage for the light

exposure of etioplasts and the race that ensues between photo-protective pathways and photo-

oxidation to determine if they will develop into normal or defective chloroplasts.

9.5 Acknowledgements

This work was supported by the Office of Basic Energy Sciences, Chemical Sciences

Division, US Department of Energy (contract DE-FG02-94ER20147).

125

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