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Forschungsarbeit

An emerging new concept for the binding of ferredoxin-NADP(H) oxidoreductase to the thylakoids of higher plants

Von Johan Philipp Benz (28.01.2011)

Our life on this planet is absolutely dependent on the availability of oxygen (O2) in the air we breathe. This precious element is mainly produced by plants and algae as a by-product in a process called photosynthesis which is taking place in specialized organelles called chloroplasts. It is commonly accepted that chloroplasts represent a kind of “domesticated” photosynthetic bacterium that was engulfed by an ancestor of plants and algae but considered too useful to simply digest it. Instead, over time, the host cell transferred most of the bacterial genes to its own nucleus, thereby taking over control and transforming the “guest” to a cell organelle. However, this process necessitates that most of the chloroplastic proteins, that are now produced in the cell cytosol, have to be post-translationally imported into the organelle, a process that is mediated for most of these proteins by two translocons called TOC and TIC (translocon at the outer or inner envelope membrane of chloroplasts, respectively) sitting in the two membranes surrounding the chloroplast [1]. Intriguingly, there is a tight connection between the protein import machinery and photosynthesis, since one subunit of the TIC complex, called Tic62, and the final electron acceptor protein of the photosynthetic electron transfer chain, called ferredoxin: NADP(H) oxidoreductase (FNR), were found to directly interact at the inner envelope [2]. This was surprising, since photosynthesis is taking place at the thylakoids, which is a specialized membrane system, and not at the inner envelope membrane.

Figure  1: Schematic illustration of the redox-dependent Tic62 shuttling between inner envelope and stroma (For further information see PDF below).[Bildunterschrift / Subline]: Figure 1: Schematic illustration of the redox-dependent Tic62 shuttling between inner envelope and stroma (For further information see PDF below).

During my PhD studies, we analyzed the localization, structure, function, and regulation of the Tic62-FNR protein couple in detail. We found that Tic62 is an active NADP(H) dehydrogenase and is dynamically shuttling between the inner envelope and the chloroplast stroma depending on the chloroplast redox state, represented by the NADP+:NADPH ratio (Figure 1) [3]. Moreover, a third pool of Tic62 was identified at the thylakoid membranes (Figure 2), establishing that both Tic62 and FNR are present at multiple locations in the chloroplast, which seem to be connected by a dynamic shuttling behavior [4]. At the thylakoids, both proteins were found to form high-molecular-weight complexes (Figure 3) and structural analyses revealed that Tic62 binds to FNR in a novel binding mode for flavoproteins (Figure 4) [5]. The Tic62-FNR complexes, that represent about 50% of the thylakoid-bound FNR content, form and disassemble in response to light- and redox- signals, but are clearly not involved in photosynthetic electron transfer reactions. Moreover, our data indicate that also the other 50% of thylakoid-localized FNR is not bound to photosynthetic complexes, as commonly believed. Finally, in absence of Tic62 (i.e. in tic62 knock-out plants), membrane binding and stability of FNR was found to be drastically reduced.

Figure 2: Localization of Tic62, FNR and Tic110 in Arabidopsis protoplasts by confocal microscopy (For further information see PDF below).[Bildunterschrift / Subline]: Figure 2: Localization of Tic62, FNR and Tic110 in Arabidopsis protoplasts by confocal microscopy (For further information see PDF below).

We concluded that Tic62 represents a major FNR interaction partner not only at the envelope and in the stroma, but also at the thylakoid membranes of higher plants. Association with Tic62 stabilizes FNR during periods of photosynthetic inactivity (such as at night) and is involved in its dynamic and light-dependent membrane binding and relocation into the stroma.

Figure 3: Tic62 and FNR form high-molecular-weight complexes at the thylakoid membrane that are regulated by light (For further information see PDF below).[Bildunterschrift / Subline]: Figure 3: Tic62 and FNR form high-molecular-weight complexes at the thylakoid membrane that are regulated by light (For further information see PDF below).

Our data, in combination with observations made by other groups, prompted us to propose a model describing how the Tic62-dependent allocation of FNR between stroma and membranes affects the partitioning system of photosynthetic electrons in the chloroplast (Figure 5) [6]. Even though FNR is the connecting element between the photosynthetic electron chain (accepting electrons from photosystem I via ferredoxin (Fd)) and the metabolism (by production of NADPH), we believe that the major distributor of photosynthetic electrons is actually Fd. Several pathways are competing for reduced Fd, which donates electrons to various assimilatory pathways (FNR being one), the redox-regulatory system (thioredoxins), back into the photosynthetic electron chain, or to molecular O2. FNR clearly represents a major sink for Fd-derived electrons during the day, since a lot of NADPH is needed for the carbon fixation processes in the Calvin cycle. In our model, the soluble FNR in the stroma is the photosynthetically active pool, and therefore reversible assembly of FNR into (inactive) thylakoid complexes seems to provide an elegant way to store surplus molecules not needed at a given time (such as at night or during low-light conditions), thus allowing Fd to distribute electrons to other pathways. It is tempting to speculate that the evolution of this process might have helped to optimize the distribution of electrons to pathways that are active simultaneously, thus alleviating oxido-reductive stress. Transient storage would be a highly advantageous solution to this problem since it is much faster, easier to regulate, and more energy efficient than for example proteolytic degradation followed by new production. Current research in the laboratory is investigating what aspects of this thylakoid-to-stroma system might be transferable to the inner envelope membrane and the regulation of protein transport. Clearly, protein import in general is responsive to redox signals [7], but many exciting questions, in particular concerning the TIC complex, remain to be answered. 

Figure 4: Tic62/FNR structure as revealed by X-ray crystallography (For further information see PDF below).[Bildunterschrift / Subline]: Figure 4: Tic62/FNR structure as revealed by X-ray crystallography (For further information see PDF below).

In conclusion, our results provide further insight into how plants adapt to changing light conditions, and therefore also provide another piece in the puzzle to a better understanding of the complex processes of photosynthesis itself.

Figure 5: Working model illustrating the effect of Tic62-dependent FNR relocation on the electron distribution of Fd in chloroplasts (For further information see PDF below).[Bildunterschrift / Subline]: Figure 5: Working model illustrating the effect of Tic62-dependent FNR relocation on the electron distribution of Fd in chloroplasts (For further information see PDF below).

References:

[1] Benz JP, Soll J, Bölter B (2009) Protein transport in organelles: The composition, function and regulation of the Tic complex in chloroplast protein import. Febs Journal 276: 1166-1176.

[2] Küchler M, Decker S, Hörmann F, Soll J, Heins L (2002) Protein import into chloroplasts involves redox-regulated proteins. Embo Journal 21: 6136-6145.

[3] Stengel A, Benz P, Balsera M, Soll J, Bölter B (2008) TIC62 redox-regulated translocon composition and dynamics. J Biol Chem 283: 6656-6667.

[4] Benz JP, Stengel A, Lintala M, Lee YH, Weber A, Philippar K, Gügel IL, Kaieda S, Ikegami T, Mulo P, Soll J, Bölter B. (2009): Arabidopsis Tic62 and ferredoxin-NADP(H) oxidoreductase form light-regulated complexes that are integrated into the chloroplast redox poise. Plant Cell, 21 (12): 3965-83.

[5] Alte F, Stengel A, Benz JP, Petersen E, Soll J, Groll M, Bölter B. (2010): Ferredoxin:NADP(H) Oxidoreductase is recruited to thylakoids by binding to a poly-proline type II helix in a pH-dependent manner. PNAS, accepted for publication.

[6] Benz JP, Lintala M, Soll J, Mulo P, Bölter B. (2010): A new concept for ferredoxin-NADP(H) oxidoreductase binding to plant thylakoids. Trends Plant Sci. [Epub ahead of print].

[7] Stengel A, Benz JP, Buchanan B, Soll J, Bölter B. (2009): Preprotein import into chloroplasts via the Toc and Tic complexes is regulated by redox signals in Pisum sativum. Molecular Plant, 2 (6): 1181-97.


Stationen
  • 04.2010 - dato
  • Postdoc in the group of Prof. Dr. Chris Somerville at the Energy Biosciences Institute, University of California at Berkeley, CA, USA
  • 07.2009 – 03.2010
  • Postdoc in the group of Prof. Dr. Jürgen Soll, Plant Biochemistry and Physiology, Ludwig-Maximilians-University Munich, GER
  • 10.2004 – 06.2009
  • PhD graduate student in the group of Prof. Dr. Jürgen Soll, Plant Biochemistry and Physiology, Ludwig-Maximilians-University Munich, GER
  • 11.2003 – 08.2004
  • Scientific Lab Assistant in the lab of Prof. Dr. Nigel M. Crawford, Section of Cell and Developmental Biology, University of California at San Diego, CA, USA
  • 10.1998 – 08.2003
  • Diploma student in Biology at the TU Braunschweig, GER; Preparation of Diploma thesis in the lab of Prof. Dr. Nigel M. Crawford, University of California at San Diego, CA, USA; Supervisor: Prof. Dr. R.R. Mendel (TU Braunschweig, GER)

Stipendien und Auslandsaufenthalte
  • 2010
  • Award of a Feodor Lynen research fellowship by the ´Alexander von Humboldt Stiftung` for a long-term postdoctoral research project in the group of Prof. Dr. Chris Somerville at the Energy Biosciences Institute, UC Berkeley, CA, USA
  • 2009
  • Benz et al. (2009) Plant Cell was evaluated as one of the most interesting papers recently published in the biological sciences based on its scientific merit and selected for the Faculty of 1000 Biology.
  • 2008
  • Best student poster award at the XVI Congress of the Federation of European Societies of Plant Biology (FESPB); 17.08.-22.08.2008; Tampere, FIN
  • 2001-2003
  • Alumnus of the ´Studienstiftung des deutschen Volkes` (German National Academic Foundation). Admission on the basis of the prediploma grade average including a graduate student scholarship
  • 2003
  • Award of a short-term grant for research abroad by the ´Deutscher Akademischer Austausch Dienst e.V.` (DAAD; German Academic Exchange Service)

Veröffentlichungen
  • *Alte F, Stengel A, Benz JP, Petersen E, Soll J, Groll M, Bölter B. (2010) Ferredoxin:NADP(H) Oxidoreductase is recruited to thylakoids by binding to a poly-proline type II helix in a pH-dependent manner. PNAS, Oct. 25 [Epub ahead of print].
  • *Benz JP, Lintala M, Soll J, Mulo P, Bölter B. (2010): A new concept for ferredoxin-NADP(H) oxidoreductase binding to plant thylakoids. Trends in Plant Sciences, Sep.17 [Epub ahead of print].
  • *Stengel A, Benz JP, Buchanan B, Soll J, Bölter B. (2010): Redox-regulation of protein import into chloroplasts and mitochondria: similarities and differences. Plant Signaling & Behavior, 5 (2): 15-20.
  • *Benz JP, Stengel A, Lintala M, Lee YH, Weber A, Philippar K, Gügel IL, Kaieda S, Ikegami T, Mulo P, Soll J, Bölter B. (2009): Arabidopsis Tic62 and ferredoxin-NADP(H) oxidoreductase form light-regulated complexes that are integrated
  • into the chloroplast redox poise. Plant Cell, 21 (12): 3965-83.
  • *Benz JP, Soll J, Bölter B. (2009): Protein transport in organelles: The composition, function and regulation of the Tic complex in chloroplast protein import. FEBS J., 276 (5): 1166-76.
  • *Stengel A, Benz JP, Buchanan B, Soll J, Bölter B. (2009): Preprotein import into chloroplasts via the Toc and Tic complexes is regulated by redox signals in Pisum sativum. Molecular Plant, 2 (6): 1181-97.
  • *Stengel A*, Benz P*, Balsera M, Soll J, Bölter B. (2008): Tic62 - redox-regulated translocon composition and dynamics. J Biol Chem., 283 (11): 6656-67*(contributed equally).
  • *Benz P, Soll J, Bölter B. (2007): The Role of the Tic Machinery in Chloroplast Protein Import. In The Enzymes, Vol. 25; Molecular Machines Involved in Protein Transport across Cellular Membranes
  • (Dalbey R, Koehler C & Kamanoi F, eds.), Academic Press/Elsevier: 439-62.