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Redefining cyclic electron flow around photosystem I (cef1): The induction, pathway, and role of cef1 in c3 plants

Dissertation
Author: Aaron Kyle Livingston
Abstract:
While traditional photosynthetic research has focused on the "linear" electron transfer pathway, alternative "cyclic" pathways have been proposed as a means to balance energy needs in plants. After decades of work, spanning a diverse field of techniques and ideas, controversy remains as to the pathway, role and regulation of cyclic electron flow around photosystem I (CEF1). CEF1 must be elucidated to understand how plants respond to and survive changing environmental stresses, such as drought, cold, heat and salt. We have isolated a new mutant phenotype where CEF1 is greatly increased with respect to normal photosynthesis or linear electron transfer. These high CEF1, or hcef, mutants provide a unique opportunity for answering key questions about the regulation, role, and pathway of CEF1. Through the utilization of map-based cloning, new spectroscopic techniques, and crossing with other known CEF1 mutants, we have determined that CEF1 is a highly dynamic, regulated, and large capacity pathway in plants. CEF1 in C 3 plants appears to run through the thylakoid NAD(P)H dehydrogenase (NDH) complex and not the once favored antimycin A-sensitive ferredoxin-plastoquinone oxidoreductase (FQR), or PGR5 (proton gradient regulator 5) dependent pathway. Furthermore, hydrogen peroxide has been found to be both an inducer of the formation of the NDH complex and activator of NDH mediated CEF1.

TABLE OF CONTENTS ABSTRACT ................................................................................................................................... iii PREFACE ....................................................................................................................................... 1 References ......................................................................................................................... 10 CHAPTER 1: An Arabidopsis mutant with high cyclic electron flow around photosystem I (hcef) involving the NADPH dehydrogenase complex ........................................................................... 19 Abstract. ............................................................................................................................ 19 Introduction ....................................................................................................................... 20 Results ............................................................................................................................... 23 Discussion ......................................................................................................................... 31 Methods ............................................................................................................................. 38 Figures ............................................................................................................................... 47 References ......................................................................................................................... 54 Supplemental Figures ........................................................................................................ 64 Supplemental References .................................................................................................. 71 CHAPTER 2: Regulation of Cyclic Electron Flow in C 3 Plants: Differential effects of limiting p hotosynthesis at Rubisco and Glyceraldehyde-3-phosphate Dehydrogenase ............................. 72 Abstract. ............................................................................................................................ 72 Introduction ....................................................................................................................... 73 Material and Methods........................................................................................................ 76 Results ............................................................................................................................... 78 Discussion ......................................................................................................................... 83

vi Table .................................................................................................................................. 89 Figures ............................................................................................................................... 90 References ......................................................................................................................... 94 CHAPTER 3: A mutation in glyceraldehyde-3-phosphate dehydrogenase subunit B induces cyclic electron flow around photosystem I ............................................................................................ 102 Abstract ........................................................................................................................... 102 Introduction ..................................................................................................................... 103 Material and Methods...................................................................................................... 105 Results ............................................................................................................................. 108 Discussion ....................................................................................................................... 114 Figures ............................................................................................................................. 118 References ....................................................................................................................... 123 CHAPTER 4: Regulation of Cyclic Electron Flow around Photosystem I in vivo by hydrogen peroxide. ...................................................................................................................................... 130 Abstract ........................................................................................................................... 130 Introduction ..................................................................................................................... 131 Material and Methods...................................................................................................... 134 Results ............................................................................................................................. 137 Discussion ....................................................................................................................... 141 Conclusion ....................................................................................................................... 144 Figures ............................................................................................................................. 146 References ....................................................................................................................... 153

1 PREFACE

Through the process of photosynthesis, photonic energy from the sun is converted into stored energy, as both reducing equivalents (NADPH) (Ort & Yocum, 1996) and an electrochemical gradient or proton motive force (pmf) (Cruz, Sacksteder, Kanazawa & Kramer, 2001, Kramer, Cruz & Kanazawa, 2003) through a process known as linear electron flow (LEF). The pmf has two separate functions. First, pmf can be used to make a stable form of energy in the conversion of ADP to ATP (Allen, 2002). Secondly, the pmf regulates the photosynthetic process by slowing the electron flow through the cytochrome b 6 f c omplex (Hope, Valente & Matthews, 1994, Takizawa, Cruz, Kanazawa & Kramer, 2007) and up-regulating a process to dissipate excess energy, q E (energy dependent quenching) (Horton, Ruban & Walters, 1996). T he pmf is built up by the translocation of protons into the thylakoid lumen during LEF. LEF uses light energy to transfer electrons through both photosystem II (PSII) and photosystem I (PSI) to ultimately produce NADPH. Since the electron and proton transfers are coupled together, LEF produces a fixed ratio of ATP to NADPH (1.3 ATP per NADPH) (Sacksteder, Kanazawa, Jacoby & Kramer, 2000, Seelert, Poetsch, Dencher, Engel, Stahlberg & Müller, 2000, Kramer, Avenson & Edwards, 2004). However, the rate at which a plant is predicted to consume ATP to NADPH is expected to be significantly higher (1.45 ATP per NADPH) than that produced (Noctor & Foyer, 1998, Kramer et al., 2004, Avenson, Kanazawa, Cruz, Takizawa, Ettinger & Kramer, 2005b). This short fall in the production of ATP must be corrected otherwise an over-abundance of reduced PSI electron acceptors would be created (Kramer et al., 2004). An over-abundance of reduced PSI acceptors can lead to superoxide production (Scandalios, 1993) and singlet oxygen species

2 (Macpherson, Telfer, Barber & Truscott, 1993, Hideg, Spetea & Vass, 1994) which can cause damage or death to the plant.

Methods of balancing the ATP/NADPH ratio

A shortfall in ATP production can be augmented by one of three mechanisms: the water- water cycle (WWC), the malate shunt, and cyclic electron flow around photosystem I (CEF1). In the WWC, instead of electrons from LEF being used to make NADPH, the electrons are pushed onto oxygen to make superoxide. The superoxide is scavenged into hydrogen peroxide (Macheroux, Kleweg, Massey, Söderlind, Stenberg & Lindqvist, 1993) and, by consuming NADPH, is, ultimately, converted back into water (Asada, 2000). The WWC increases the amount of pmf, while consuming NADPH, helping to increase the ATP to NADPH ratio. Unfortunately, the actual contribution of the WWC in energy balancing is still largely unknown (Miyake & Yokota, 2000, Heber, 2002). In the malate shunt, NADPH created by LEF is used inside the chloroplast to make malate. The malate is then transferred from the chloroplast to the mitochondria, where it is consumed to produce ATP (Scheibe, 2004). Essentially, the malate shunt functions as an exchange of NADPH for ATP. However, the shunt has a limited capacity (~1% of LEF) and probably has little effect on the actual ATP/NADPH ratio (Fridlyand, Backhausen & Scheibe, 1998). During CEF1 the electrons, instead of being used to make NADPH, are transferred back into LEF between PSII and PSI (Heber & Walker, 1992). The electrons can then circle around PSI, while translocating additional protons into the thylakoid lumen. The translocation of additional protons increases the pmf without any net increase in NADPH (Kramer et al., 2004,

3 Eberhard, Finazzi & Wollman, 2008). The increased pmf helps balance the ATP/NADPH ratio as well as regulating photosynthesis by enhancing photoprotection (q E ) and slowing LEF through t he cytochrome b 6 f c omplex (Heber & Walker, 1992, Kramer et al., 2004, Livingston, Cruz, Kohzuma, Dhingra & Kramer, 2010a, Chapter 1).

Controversy around CEF1

While some research groups have found significant increases in CEF1 under environmental stress, e.g. drought (Jia, Oguchi, Hope, Barber & Chow, 2008, Kohzuma, Cruz, Akashi, Munekage, Yokota & Kramer, 2008), high light (Baker & Ort, 1992), or during the induction of photosynthesis from prolonged dark (Joët, Cournac, Peltier & Havaux, 2002, Joliot & Joliot, 2002), other groups found that CEF1 is only activated in very small amounts, particularly under steady-state conditions (Genty, Briantais & Baker, 1989, Harbinson, Genty & Baker, 1989, Avenson, Cruz, Kanazawa & Kramer, 2005a). In C 4 plants (Kubicki, Funk, Westhoff & Steinmüller, 1996), cyanobacteria (Carpent ier, Larue & Leblanc, 1984) and green algae (Finazzi, Rappaport, Furia, Fleischmann, Rochaix, Zito & Forti, 2002), the amount of CEF1 is constantly high due to the increased ATP demand. The extra ATP is necessary to drive the organism’s CO 2 concentrating mechanisms. Since C 3 plants do not concentrate CO 2 there is no increased ATP demand, so under nonstressed conditions the p lant probably only requires a small contribution by CEF1 (Kramer et al., 2004, Avenson et al., 2005a). Until recently, the only way to study CEF1 was to use mutants that are CEF1 knockouts, either pgr5 (Munekage, Hojo, Meurer, Endo, Tasaka & Shikanai, 2002) a mutant deficient in the antimycin A (AA)-sensitive ferredoxin-plastoquinone oxidoreductase (FQR) pathway (Bendall

4 & Manasse, 1995), or crr2-2, which is impared in the NAD(P)H dehydrogenase (NDH) complex (Endo, Shikanai, Sato & Asada, 1998, Shikanai, Endo, Hashimoto, Yamada, Asada & Yokota, 1998, Nixon, 2000). Currently there is a push towards finding and using mutants with consistently high amounts of CEF1, such as a chloroplast fructose-1,6-bisphosphatase mutant (high cyclic electron flow mutant 1 or hcef1) (Livingston et al., 2010a, Chapter 1), a fructose-6- phosphate aldolase mutant (Gotoh, Matsumoto, Ogawa, Kobayashi & Tsyama, 2009) and mutants in glyceraldehydes-3-phosphate dehydrogenase (GAPDH), both an anti-sense tobacco mutant, gapR (Livingston, Kanazawa, Cruz & Kramer, 2010b) and a GAPDH subunit B Arabidopsis mutant (hcef2) (Chapter 3).

Activation of CEF1 by H 2 O 2

The following theories have been proposed about what induces CEF1: (1) the ATP /ADP ratio (Joliot & Joliot, 2002); (2) the redox status of PSI electron acceptors (NAD(P)H, ferredoxin) (Breyton, Nandha, Johnson, Joliot & Finazzi, 2006); (3) Calvin-Benson cycle intermediates; (Fan, Nie, Hope, Hillier, Pogson & Chow); (Fan et al.) and (4) the reactive oxygen species, hydrogen peroxide (H 2 O 2 ) (Lascano, Casano, Martin & Sabater, 2003, G ambarova, 2008). We compared these possibilities by constructing a large-scale metabolic analysis of mutants with both high levels of CEF1 and no CEF1 (see Chapter 3). In the high CEF1 mutants, hcef1 (Livingston et al., 2010a, Chapter 1) and gapR (Livingston et al., 2010b, Chapter 2), we found that the ATP/ADP levels were not significantly altered, however there was an increase in ATP/ADP level in the non-CEF1 mutant, an anti-sense Rubisco small subunit mutant (ssuR), suggesting that the ATP/ADP levels do not trigger CEF1.

5 Additionally, the metabolic comparison showed that only one Calvin-Benson Cycle intermediate, Rubisco 1,5-bisphosphate (RuBP), could possibly be the trigger of CEF1 (Livingston et al., 2010b, Chapter 2). RuBP increased in ssuR, which showed no CEF1, and decreased in the high CEF1 mutants. This suggests that RuBP could be an inhibitor of CEF1. However, when plants were subject to drought conditions, where RuBP is expected to accumulate, there was an increase in CEF1 (Jia et al., 2008, Kohzuma et al., 2008). Overall this suggests that CEF1 is not directly induced by any Calvin-Benson Cycle intermediate. Furthermore, a study on gapR showed no change in the NADP + /NADPH ratio compared t o wild-type (Ruuska, Andrews, Badger, Price & von Caemmerer, 2000), suggesting that NADPH is not the regulator of CEF1. Overall, this suggests that CEF1 is activated by some other intracellular messenger, such as H 2 O 2 . Studies have found that varying H 2 O 2 concentrations in C 3 plants can cause an array of ove rall physiological responses (reviewed in (Veal, Day & Morgan, 2007)), changes in the levels of protein transcription (Quinn, Findlay, Dawson, Jones, Morgan & Toone, 2002), and changes in the levels of enzyme activation (Casano, Martin & Sabater, 2001). Importantly, the high CEF1 mutant, hcef1, has very high levels of intercellular hydrogen peroxide (Chapter 4). To test the role of hydrogen peroxide on CEF1, wild-type plants where soaked in H 2 O 2 for two hour s, and an increase in CEF1 was observed (Chapter 4). Since hydrogen peroxide can cause many changes throughout the plant, mutants that have increased levels of hydrogen peroxide in the chloroplast only were also tested. These glycolate oxidase (GO) or GO mutants produce varying levels of additional H 2 O 2 in the chloroplast. The GO mutants also showed varying levels of CEF1 activation which correlates to the concentration of hydrogen peroxide produced

6 (Chapter 4). Taken together these data suggest that hydrogen peroxide in the chloroplast plays a role in the induction of CEF1. Previous work, suggests that the formation of a protein or protein complex is necessary to induce CEF1 (Chapter 1, 3 and 4). Following a time course, we found that if wild-type plants where subjected to H 2 O 2 it took at least 60 minutes to start and 105 minutes to achieve full i nduction of CEF1. Furthermore, if a plant is subjected to H 2 O 2 and lincomycin, which inhibits p rotein formation, CEF1 is never induced (Chapter 4). This suggests that H 2 O 2 induces the f ormation of the CEF1 protein complex. In fact under all conditions where elevated CEF1 is seen, cold (Apostol, Szalai, Sujbert, Popova & Janda, 2006), heat (Jin, Li, Hu & Wang, 2009), salt (Lu, Yang, He & Jiang, 2007), and drought (Kohzuma et al., 2008), we also find elevated levels of H 2 O 2 [cold (Dia, Huang, Zhou & Z hang, 2009), heat (Volkov, Panchuk, Mullineaux & Schöffl, 2006), salt (Xiong, Schumaker & Zhu, 2002, Mandhania, Madan & Sawhney, 2005), and drought (Xiong et al., 2002)]. Pre- treatment of plants to H 2 O 2 causes increased survival in all CEF1 inducing conditions: cold, heat, dr ought, and salt stress (Gong, Chen, Li & Guo, 2001).

Pathway of CEF1

Currently, there are two major pathways that CEF1 is proposed to run through: 1) the antimycin A (AA)-sensitive ferredoxin-PQ oxidoreductase (FQR) (Bendall & Manasse, 1995) which is inhibited in the pgr5 mutant; and 2) through the NADPH dehydrogenase (NDH) complex (Endo et al., 1998). Recent studies suggest that the NDH and PGR5 pathways are slow under non-stressed conditions, or that the two pathways can compensate for each other (Munekage et al., 2002, Avenson et al., 2005a).

7 Recent work with the high CEF1 mutants, hcef1 (Livingston et al., 2010a, Chapter 1) and hcef2 (Chapter 3), show no change in CEF1 rates when crossed with a FQR knockout (pgr5). This suggests that the AA-sensitive FQR pathway of CEF1 is not active under these circumstances. Additionally, when the FQR knockout mutant, pgr5, was subjected to H 2 O 2 CEF1 w as induced to approximately the same level as at that induced in wild-type + H 2 O 2 (Chapter 4). W hen protein content was looked at for the high CEF1 mutant, hcef1 (Livingston et al., 2010a, Chapter 1) the amount of PGR5 goes down, further suggesting that PGR5 does not have a role in CEF1. When the high CEF1 mutants hcef1 (Livingston et al., 2010a, Chapter 1), hcef2 (Chapter 3), and FBPaldolase (Gotoh et al., 2009) where crossed with a knockout of NDH, crr2-2, there was an almost complete loss of CEF1 function. Additionally, the protein levels of hcef1 (Livingston et al., 2010a, Chapter 1), showed a dramatic (10X) increase in NDH compared to wild-type. When the NDH knockout mutant, crr2-2, was subjected to H 2 O 2 there was no i nduction of CEF1 (Chapter 4). Furthermore when wild-type was subjected to the conditions that induce CEF1, i.e. cold (Lee, Henderson & Zhu, 2005, Hannah, Wiese, Freund, Fiehn, Heyer & Hincha, 2006), heat (Balasubramanian, Sureshkumar, Lempe & Weigel, 2006), drought (Abdeen, Schnell & Miki, 2010), and H 2 O 2 (Vandenbroucke, Robbens, Vandepoele, Inzé, Van de Peer & Van Breusegem, 2008), microarray data shows an increase in the expression levels of NDH subunits in Arabidopsis. This suggest that NDH is a necessary component of CEF1. A further correlation has been seen between the amount of CEF1 in C 4 plants and the expression l evel of NDH, but not the expression level of PGR5 (Sazanov, Burrows & Nixon, 1996, Quiles, 2005).

8 Roles of CEF1: Importance of energy balancing

Cyclic electron flow is proposed to have two purposes: (1) CEF1 is proposed to increase the output ratio of ATP to NADPH (ATP/NADPH) (Allen, 2003) balancing the energy deficit. (2) In photoprotection, CEF1 increases pmf to increase excess energy dissipation (Heber & Walker, 1992) and decrease the flow of electrons through LEF (Miyake, Shinzaki, Miyata & Tomizawa, 2004). We know that the amount of CEF1 is highly regulated, as seen in the anti- sense GAPDH tobacco mutant (Chapter 2) and that a high level of flexibility is necessary to fix the energy balance without causing damage to the plant (Kramer et al., 2004). Like under changing CO 2 conditions (Kanazawa & Kramer, 2002), plants rapidly i ncrease pmf, and thereby photoprotection, by simply altering the conductivity through the ATP synthase (g H + ). This means that a plant can rapidly engage and disengage photoprotecti on by changing g H + , whereas CEF1 takes hours to induce. Since CEF1 requires the formation of an a dditional protein complex that takes hours to fully induce (Chapter 4), CEF1 cannot rapidly respond to changing environmental conditions like altering g H + can. However, if the CEF1 c omplex is already induced it can be rapidly activated, as seen when high CEF1 mutants were subject to changing levels of oxygen (Chapter 4). This means that if CEF1 does play a role in photoprotection it would likely be for long term photoprotection in response to repeated or constant stress conditions. Additionally, in all the conditions we find elevated CEF1, i.e. drought, salt stress, heat stress and cold, we find increased ATP demand. Under drought conditions the amount of ATP decreases (Rezara, Mitchell, Driscoll & Lawlor, 1999, Flexas & Medrano, 2002) due to the loss of functioning ATP synthases (Lawlor & Tezara, 2009). Under cold stress, that ATP/ADP ratio decreases due to increased sucrose production (Savitch, Harney & Huner, 2003). Salt stress

9 induces the activation of a vacuolar ATPase proton/salt pumping mechanism which is necessary for the plant to survive the salt stress but costs additional ATP to run (Marlaux, Fischer-Schliebs, Lüttge & Ratajczak, 1997). High temperatures leads to leak of protons through the thylakoid membrane (Svintitskikh, Andrianov & Bulychev, 1985) or slip of protons through the ATP synthase (Groth & Junge, 1993). Leak and slip decreases in the amount of available pmf without effecting electron transfer, which causes a decrease in the amount of ATP produced per NADPH (Groth & Junge, 1993). Overall this suggests that CEF1 is likely induced to increase ATP production to balance the energy budget in C 3 plants, although CEF1 could be used for long term p hotoprotection under continuing stress conditions.

Conclusion

Overall this work suggests that CEF1 in C 3 plants is a finely tuned mechanism for b alancing the energy budget, which is induced by H 2 O 2 and runs through the NDH complex.

10 References

Abdeen A., Schnell J. & Miki B. (2010) Transcriptome analysis reveals absence of unintended effects in drought-tolerant transgenic plants overexpressing the transcription factor ABF3. BMC Genomics, 11, 1-21. Allen J.F. (2002) Photosynthesis of ATP- electrons, proton pumps, rotors, and poise. Cell, 110, 273-276. Allen J.F. (2003) Cyclic, pseudocyclic and noncyclic photophosphorylation: new links in the chain. Trends in Plant Science, 8, 15-19. Apostol S., Szalai G., Sujbert L., Popova L.P. & Janda T. (2006) Non-invasive monitoring of the light-induced cyclic photosynthetic electron flow during cold hardening in wheat leaves. Z Naturforsch, 61c, 734-740. Asada K. (2000) The water-water cycle as alternative photon and electron sinks. Philosophical Transactions of the Royal Society B: Biological Sciences, 355, 1419-1431. Avenson T.J., Cruz J.A., Kanazawa A. & Kramer D.M. (2005a) Regulating the proton budget of higher plant photosynthesis. Proceedings of the National Academy of Sciences, 102, 9709–9713. Avenson T.J., Kanazawa A., Cruz J.A., Takizawa K., Ettinger W.E. & Kramer D.M. (2005b) Integrating the proton circuit into photosynthesis: progress and challenges. Plant, Cell and Environment, 28, 97-109. Baker N.R. & Ort D.R. (1992) Light and crop photosynthetic performance. In Crop Photosynthesis: Spatial and Temporal Determinants (eds N.R. Baker & H. Thomas), Elsevier Science Publishers, Amsterdam, the Netherlands, 289-312.

11 Balasubramanian S., Sureshkumar S., Lempe J. & Weigel D. (2006) Potent induction of Arabidopsis thaliana flowering by elevated growth temperature. PLoS Genetics, 2, e106. Bendall D.S. & Manasse R.S. (1995) Cyclic photophosphorylation and electron transport. Biochemia Biophysica Acta, 1229, 23-38. Breyton C., Nandha B., Johnson G., Joliot P. & Finazzi G. (2006) Redox modulation of cyclic electron flow around Photosystem I in C3 plants. Biochemistry, 45, 13465-13475. Carpentier R., Larue B. & Leblanc R.M. (1984) Photoacoustic spectroscopy of Anacystis nidulans : III. Detection of photosynthetic activities. Archives of Biochemistry and Biophysics, 228, 534-543. Casano L.M., Martin M. & Sabater B. (2001) Hydrogen peroxide mediates the induction of chloroplastic Ndh complex under photooxidative stress in barley. Plant Physiology, 125, 1450-1458. Cruz J.A., Sacksteder C.A., Kanazawa A. & Kramer D.M. (2001) Contribution of electric field (∆ψ) to steady-state transthylakoid proton motive force (pmf) in vitro and in vivo. Control of pmf parsing into ∆ψ and ∆pH by ionic strength. Biochemistry, 40, 1226-1237. Dia F., Huang Y., Zhou M. & Zhang G. (2009) The influence of cold acclimation on antioxidative enzymes and antioxidants in sensitive and tolerant barley cultivars. Biologia Plantarum, 53, 257-262. Eberhard S., Finazzi G. & Wollman F.-A. (2008) The Dynamics of Photosynthesis. Annual Review of Genetics, 42, 463-515. Endo T., Shikanai T., Sato F. & Asada K. (1998) NAD(P)H dehydrogenase dependet, antimycin A-sensitive electron donation to plastoquinone in tobacco chloroplasts. Plant Cell Physiology, 39, 1226-1231.

12 Fan D.-Y., Nie Q., Hope A.B., Hillier W., Pogson B.J. & Chow W.S. (2007) Quantification of cyclic electron flow around Photosystem I in spinach leaves during photosynthetic induction. Photosynthesis Research, 94, 347-357. Finazzi G., Rappaport F., Furia A., Fleischmann M., Rochaix J.D., Zito F. & Forti G. (2002) Involvement of state transitions in the switch between linear and cyclic electron flow in Chlamydomonas reinhardtii. European Molecular Biology Organization, 3, 280–285. Flexas J. & Medrano H. (2002) Drought-inhibition of photosynthesis in C 3 plants: Stomatal and non -stomatal limitations revisted. Annals of Botany, 89, 183-189. Fridlyand L.E., Backhausen J.E. & Scheibe R. (1998) Flux control of the Malate Valve in leaf cells. Archives of Biochemistry and Biophysics, 349, 290-298. Gambarova N.G. (2008) Activity of photochemical reactions and accumulation of hydrogen peroxide in chloroplasts under stress conditions. Russian Agricultural Sciences, 34, 149- 151. Genty B., Briantais J.M. & Baker N.R. (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta, 990, 87-92. Gong M., Chen B.O., Li Z.G. & Guo L.H. (2001) Heat-shock-induced cross adaption to heat, chilling, drought and salt stress in maize seedlings and involvement of H 2 O 2 . J ournal of Plant Physiology, 158, 1125-1130. Gotoh E., Matsumoto M., Ogawa K., Kobayashi Y. & Tsyama M. (2009) A qualitative analysis of the regulation of cyclic electron flow around photosystem I form the post-illumination chlorophyll flyorescence transient in Arabidopsis: a new platform for th in vivo invesigation of the chloroplast redox state. Photosynthesis Research, 103, 111-123.

13 Groth G. & Junge W. (1993) Proton slip of the chloroplast ATPase: Its nucleotide dependence, energetic threshold, and relation to an alternating site mechanism of catalysis. Biochemistry, 32, 8103-8111. Hannah M.A., Wiese D., Freund S., Fiehn O., Heyer A.G. & Hincha D. (2006) Natural Genetic Variation of Freezing Tolerance in Arabidopsis. Plant Physiology, 142, 98-112. Harbinson J., Genty B. & Baker N.R. (1989) Relationship between the Quantum Efficiencies of Photosystems I and II in Pea Leaves. Plant Physiology, 90, 1029-1034. Heber U. (2002) Irrungen, Wirrungen? The Mehler reaction in relation to cyclic electron transport in C3 plants. Photosynthesis Research, 73, 23-231. Heber U. & Walker D. (1992) Concerning a dual function of coupled cyclic electron transport in leaves. Plant Physiology, 100, 1621-1626. Hideg É., Spetea C. & Vass I. (1994) Singlet oxygen production in thylakoid membranes during photoinhibition as detected by EPR spectroscopy. Photosynthesis Research, 39, 191-199. Hope A.B., Valente P. & Matthews D.B. (1994) Effects of pH on the kinetics of redox reactions in and around the cytochrome bf complex in an isolated system. Photosynthesis Research, 42, 111-120. Horton P., Ruban A. & Walters R. (1996) Regulation of light harvesting in green plants. Annual Review of Plant Physiology and Plant Molecular Biology, 47, 655-684. Jia H., Oguchi R., Hope A.B., Barber J. & Chow W.S. (2008) Differential effects of severe water stress on linear and cyclic electron fluxes through Photosystem I in spinach leaf discs in CO 2 -enriched air. P lanta, 228, 803-812.

14 Jin S.H., Li X.Q., Hu J.Y. & Wang J.G. (2009) Cyclic electron flow around photosystem I is required for adaption to high temperature in a subtropical forest tree, Ficus concinna. Journal of Zhejiang University Science, 10, 784-790. Joët T., Cournac L., Peltier G. & Havaux M. (2002) Cyclic electron flow around photosystem I in C3 plants. In vivo control by the redox state of chloroplasts and involvement of the NADH-dehydrogenase complex. Plant Physiology, 128, 760–769. Joliot P. & Joliot A. (2002) Cyclic electron transfer in plant leaf. Proceedings of the National Academy of Sciences, 99, 10209–10214. Kanazawa A. & Kramer D.M. (2002) In vivo modulation of nonphotochemical exciton quenching (NPQ) by regulation of the chloroplast ATP synthase. Proceedings of the National Academy of Sciences, 99, 12789-12794. Kohzuma K., Cruz J.A., Akashi K., Munekage Y., Yokota A. & Kramer D.M. (2008) The long- term responses of the photosynthetic proton circuit to drought. Plant, Cell & Environment, 32, 209-219. Kramer D.M., Avenson T.J. & Edwards G.E. (2004) Dynamic flexibility in the light reactions of photosynthesis governed by both electron and proton transfer reactions. Trends in Plant Science, 9, 349-357. Kramer D.M., Cruz J.A. & Kanazawa A. (2003) Balancing the central roles of the thylakoid proton gradient. Trends in Plant Science, 8, 27-32. Kubicki A., Funk E., Westhoff P. & Steinmüller K. (1996) Differential expression of plastome- encoded ndh genes in mesophyll and bundle-sheath chloroplasts of the C4 plant Sorghum bicolor indicates that the complex I-homologous NAD(P)H-plastoquinone oxidoreductase is involved in cyclic electron transport. Planta, 199, 276-281.

15 Lascano H.R., Casano L.M., Martin M. & Sabater B. (2003) The activity of the chloroplastic Ndh complex is regulated by phosphorylation of the NDH-F subunit. Plant Physiology, 132, 256-262. Lawlor D.W. & Tezara W. (2009) Causes of decreased photosynthetic rate and metabolic capacity in water-deficient leaf cells: a critical evaluation of mechanisms and integration of processes. Annals of Botany, 103, 561-579. Lee B., Henderson D.A. & Zhu J.K. (2005) The Arabidopsis cold-responsive transcriptome and its regulation by ICE. Plant Cell, 17, 3155-3157. Livingston A.K., Cruz J.A., Kohzuma K., Dhingra A. & Kramer D.M. (2010a) An Arabidopsis mutant with high cyclic electron flow around photosystem I (hcef) involving the NDH complex. Plant Cell, 22, 1-13. Livingston A.K., Kanazawa A., Cruz J.A. & Kramer D.M. (2010b) Regulation of Cyclic Electron Flow in C3 Plants: Differential effects of limiting photosynthesis at Rubisco and Glyceraldehyde-3-phosphate Dehydrogenase. Plant Cell and the Environment. Lu K.X., Yang Y., He Y. & Jiang D.A. (2007) Induction of cyclic electron flow around photosystem 1 and state transitions are correlated with salt tolerance in soybean. Photosynthetica, 46, 10-16. Macheroux P., Kleweg V., Massey V., Söderlind E., Stenberg K. & Lindqvist Y. (1993) Role of tyrosine 129 in the active site of spinach glycolate oxidase. European Journal of Biochemistry, 213, 1047-1054. Macpherson A.N., Telfer A., Barber J. & Truscott T.G. (1993) Direct detection of singlet oxygen from isolated Photosystem II reaction centres. Biochimica et Biophysica Acta, 1143, 301- 309.

16 Mandhania S., Madan S. & Sawhney V. (2005) Antioxidant defense mechanism under salt stress in wheat seedlings. Biologia Plantarum, 50, 227-231. Marlaux J., Fischer-Schliebs E., Lüttge U. & Ratajczak R. (1997) Dynamics of activity and structure of the tonoplast vacuolar-type H + -ATPase in plants with differing CAM e xpression and in a C 3 plant under salt stress. P rotoplasma, 196, 181-189. Miyake C., Shinzaki Y., Miyata M. & Tomizawa K.-i. (2004) Enhancement of Cyclic Electron Flow Around PSI at High Light and its Contribution to the Induction of Non- Photochemical Quenching of Chl Fluorescence in Intact Leaves of Tobacco Plants. Plant and Cell Physiology, 45, 1426-1433. Miyake C. & Yokota A. (2000) Determination of the Rate of Photoreduction of O2 in the Water-Water Cycle in Watermelon Leaves and Enhancement of the Rate by Limitation of Photosynthesis. Plant Cell Physiol., 41, 335-343. Munekage Y., Hojo M., Meurer J., Endo T., Tasaka M. & Shikanai T. (2002) PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis. Cell, 110, 361-371. Nixon P.J. (2000) Chlororespiration. Philosophical Transactions of the Royal Society B: Biological Sciences, 355, 1541-1547. Noctor G. & Foyer C. (1998) A re-evaluation of the ATP:NADPH budger during C3 photosynthesis: a contribution form nitrate assimilation and its associated respiratory activity. Journal of Experimental Botany, 49, 1895-1908. Ort D.R. & Yocum C.F. (1996) Light reactions of oxygenic photosynthesis. In: Oxygenic Photosynthesis: The Light Reactions (ed C.F. Yocum), pp. 1-9. Kluwer Academic Publishers, The Netherlands.

17 Quiles M.J. (2005) Regulation of the expression of chloroplast ndh genes by light intensity applied during oat plant growth. Plant Science, 168, 1561-1569. Quinn J., Findlay V.J., Dawson K., Jones N., Morgan B.A. & Toone W.M. (2002) Distinct regulatory proteins control the adaptive and acute response to H 2 O 2 in Sc hizosaccharomyces pombe. Molecular Biology of the Cell, 13, 805-816. Rezara W., Mitchell V.J., Driscoll S.D. & Lawlor D.W. (1999) Water stress inhibits plant photsynthesis by decreasing coupling factor and ATP. Nature, 401, 914-917. Ruuska S.A., Andrews T.J., Badger M.R., Price G.D. & von Caemmerer S. (2000) The role of chloroplast electron transport and metabolites in modulating rubisco activity in tobacco. Insights from transgenic plants with reduced amounts of cytochrome b/f complex or glyceraldehyde 3-phosphate dehydrogenase. Plant Physiology, 122, 491–504. Sacksteder C., Kanazawa A., Jacoby M.E. & Kramer D.M. (2000) The proton to electron stoichiometry of steady state photosynthesis in living plants: a proton-pumping Q-cycle is continuously engaged. Proceedings of the National Academy of Sciences, 97, 14283- 14288. Savitch L.V., Harney T. & Huner N.P.A. (2003) Sucrose metabolism in spring and winter wheat in response to high irradiance, cold stress and cold acclimation. Physiologia Plantarum, 108, 270-278. Sazanov L.A., Burrows P.A. & Nixon P.J. (1996) Detection and characterization of a complex I- like NADH-specific dehydrogenase from pea thylakoids. Biochemical Society Transactions, 24, 739-743. Scandalios J.G. (1993) Oxygen Stress and Superoxide Dismutases. Plant Physiology, 101, 7-12.

Full document contains 166 pages
Abstract: While traditional photosynthetic research has focused on the "linear" electron transfer pathway, alternative "cyclic" pathways have been proposed as a means to balance energy needs in plants. After decades of work, spanning a diverse field of techniques and ideas, controversy remains as to the pathway, role and regulation of cyclic electron flow around photosystem I (CEF1). CEF1 must be elucidated to understand how plants respond to and survive changing environmental stresses, such as drought, cold, heat and salt. We have isolated a new mutant phenotype where CEF1 is greatly increased with respect to normal photosynthesis or linear electron transfer. These high CEF1, or hcef, mutants provide a unique opportunity for answering key questions about the regulation, role, and pathway of CEF1. Through the utilization of map-based cloning, new spectroscopic techniques, and crossing with other known CEF1 mutants, we have determined that CEF1 is a highly dynamic, regulated, and large capacity pathway in plants. CEF1 in C 3 plants appears to run through the thylakoid NAD(P)H dehydrogenase (NDH) complex and not the once favored antimycin A-sensitive ferredoxin-plastoquinone oxidoreductase (FQR), or PGR5 (proton gradient regulator 5) dependent pathway. Furthermore, hydrogen peroxide has been found to be both an inducer of the formation of the NDH complex and activator of NDH mediated CEF1.