Guy Hanke


“I know of nothing sublime that is not some modification of power”   Edmund Burke

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Research Philosophy

The aim of my research is to identify the molecular and genetic mechanisms that control energy distribution in crop plants and cyanobacteria, and to manipulate them for the improvement of agronomic traits. We depend upon agricultural plants to supply our food and fossil plants to support most of our energy needs. This is possible because their autotrophic activity provides the energy to assimilate inorganic molecules and synthesize an incredible variety of useful products. Since the inception of agriculture, plant breeding has selected for advantageous traits. Current systems and ‘omics approaches provide the opportunity to precisely identify the genetics of the regulatory and metabolic pathways that produce specific desirable compounds. A fundamental part of improving traditional agricultural crops and generating new microbial bioenergy crops will be to optimize energy distribution into metabolically engineered pathways.

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Photosynthetic electron transport proteins and complexes.

electrons are generated by water splitting at photosystem II (green) and transfered by plastoquinone to the cytochrome b6f complex (orange) before passing via plastocyanin (blue) to photosystem I (purple). They are accepted from photosystem I by ferredoxin (brown), which can then donate them to many enzymes, including the ferredoxin:NADP(H) oxidoreductase responsible for NADPH production.

Background

In oxygenic photosynthesis, the flow of electrons through membrane complexes in the thylakoids of chloroplasts and cyanobacteria generates a proton gradient, which drives ATP synthesis, and a supply of electrons. The reducing power of these electrons is harnessed primarily in fixation of C, but also in bioassimilation of S and N, biosynthesis of amino and fatty acids, co-factors and pigments, and in redox signaling and stress remediation, among many other processes. Electrons generated at the membrane are connected to soluble enzymes by an electron transfer protein: ferredoxin (Fd). Fd can distribute these electrons to many different enzymes, including the Fd:NADP(H) oxidoreductase (FNR) that supplies the NADPH used in C fixation. There is great diversity in Fds and FNRs, sometimes conserved across great evolutionary distance, and sometimes restricted to specific tissues, such as the bundle sheath of C4 plants. My research uses a combination of genetic, ‘omics, structural, cell biology and biophysical techniques to understand the specific functions behind this diversity.

Project 1. Diversity of ferredoxin genes and functions


The purpose of this project is:


1.       To identify the functions of novel, conserved Fd proteins

2.       To understand regulation of expression of different Fd genes

3.       To investigate the molecular mechanisms by which different Fd proteins partition electrons into different pathways

4.       To understand the impact of this differential electron partitioning on gene expression


Members and collaborators:

  • Dr. Ana Esteves (PDRA, Global Challenges Research Fund, Queen Mary University of London)

  • Dr.Maxie Roessler (PI in SBCS, Queen Mary University of London)

  • Michael Schorsch (PhD student, Queen Mary University of London)

  • Dr. Tatjana Goss (former PhD student, University of Osnabrueck, DFG grant HA 5921/1-1)

  • Dr. Nico Blanco (previous short stay visiting Scientist and collaborator)



Research Background


             Fd is represented in higher plant, algeal and cyanobacterial genomes by genes encoding multiple iso-proteins (see Figure 1 below). Among sequenced genomes, 6, 8, 5, 6 and 5 different plant type Fds can be identified in Arabidopsis, maize, rice, Chlamydomonas and Synechocystis, respectively.  The diversity of enzymes with which Fd interacts, combined with the diversity in Fd iso-proteins in all photosynthetic organisms for which we have significant sequence information, has led to speculation that different Fds may partition electrons into different areas of metabolism.

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Figure 1. Physiological functions of Fd.

Fd connects PET to soluble metabolism. Photosynthetically generated electrons are donated by PSI to Fd, which then transfers them to a wide range of soluble enzymes, including, but not limited to those listed here. Abbreviations: Fd:thiredoxin reductase (FTR), nitrate reductase (NaR), nitrite reductase (NiR) sulfite reductase (SiR), glutamine oxoglutarate amino transferase (GOGAT), acy-acyl carrier protein desaturase (Acyl-ACP desaturase),  heme oxygenase (HO).


       We are using RNAi techniques to knock down Fd genes in Arabidopsis, and analyzing their phenotypes using physiological, transcriptomic and proteomic techniques. We are identifying new Fd-interaction partners by Y2H and binding assays for weak, electrostatic interactions. We are recombinantly expressing Fds and Fd-dependent enzymes cloned from a range of organisms to examine their interaction and redox transfer properties. Results of investigations into specific photosynthetic Fds are shown in Figure 2.

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Figure 2: Variable function and physiological role of Arabidopsis Fds. 

A, Relative protein abundance of different classical Fd isoforms in Arabidopsis leaves. B, electron transport ability of Arabidopsis Fd1 (blue circles) Fd2 (green circles) and Fd3 (brown squares) in photosynthetic electron transport (top panel) and heterotrophic (root type) electron transport (bottom panel). C, Basic phenotypes of two independent lines each of Arabidopsis plants with RNAi knockdown of the  Fd1, Fd2, and Fd3 genes. D, Photosynthetic electron transport rates in intact leaves, measured by chlorophyll fluorescence. Filled triangles show wt plants and open symbols show two independent lines each of fd1:RNAi (panel), fd2:RNAi (middle panel) and fd3:RNAi (bottom panel). Adapted from Hanke & Hase (2008) and Hanke et al. (2004).

Recent Publications from the Project

  1. Kim J.Y., Kinoshita M., Kume S., Hanke, G.T., Sugiki T., Ladbury J.E., Kojima C., Ikegami T., Kurisu G., Goto Y., Hase T., Lee Y.H. (2016) Noncovalent forces tune the electron transfer complex between ferredoxin and sulfite reductase to optimize enzymatic activity. Biochem J. pii: BCJ20160658. [Epub ahead of print]
  2. Hachiya T., Ueda N., Kitagawa M., Hanke G., Suzuki A., Hase T., Sakakibara H. (2016) Arabidopsis Root-Type Ferredoxin:NADP(H) Oxidoreductase 2 Is Involved in Detoxification of Nitrite in Roots. Plant Cell Physiol. pii: pcw158. [Epub ahead of print]
  3. Hanke, G.T., Mulo, P. (2013) Plant type ferredoxins and ferredoxin-dependent metabolism. Plant Cell Environ. 36 (6): 1071-1084.
  4. Blanco, N.E., Ceccoli, R.D., Dalla Vía, M.V., Voss,I., Segretin, M.E., Bravo-Almonacid, F.F., Melzer, M., Hajirezaei, M., Scheibe, R., Hanke G.T. (2013) Expression of the minor isoform pea ferredoxin in tobacco alters photosynthetic electron partitioning and enhances cyclic electron flow. Plant Phys. 161 (2):866-879. 

Project 2. Control of redox metabolism by FNR location


The purpose of this project is:


1.       To identify the molecular mechanisms that controls FNR association with different membrane complexes.

2.       To Analyze how association of FNR with different membrane complexes impacts on electron flow and free radical evolution

3.       To understand how changes in electron transport impact on gene expression


Members and collaborators


  • Manuela Kramer (PhD student, formerly DFG grant SFB 944. P2, now Bayer Stipendium, Queen Mary University of London)

  • Manuel Twachtmann (Former PhD student, Osnabrueck, DFG grant SFB 944. P2)

  • Dr Marina Kozuleva (previous short stay visiting Scientist, DFG grant HA 5921/2-1, and collaborator)

Research Background

 

         It has been reported that FNR associates with membrane complexes at many sites on the thylakoid membrane (See Figure 1), and that this association and dissociation can be regulated by several factors, including stromal pH. We have found that in maize the bundle sheath and mesophyll cells (which conduct predominantly cyclic and linear electron flow respectively) contain FNR proteins with variable ability to bind to different membrane complexes.

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Figure 1, Localisation of FNR on the thylakoid membrane.

In linear PET (solid arrows): electrons flow from photosystem II (PSII), through plastoquinone (PQ) and the cytochrome b6f (Cytb6f) to plastocyanin (PC) and then to PSI, where they are donated to Fd and used by FNR to reduce NADP+. FNR that is soluble, associated with PSI, or tethered by the FNR binding proteins Tic62 and TROL could mediate this. Electrons may also be returned to the thylakoid in a cyclic flow (hashed arrows), either by Fd directly donating electrons to the Cyt b6f, or an as yet unidentified Fd:quinine reductase (FQR?). Alternatively, cyclic PET may occur over NADPH, via the NAD(P)H dehydrogenase complex (NDH). FNR has been localized to both the Cyt b6f complex and the NDH complex.


 

          We are probing the structure:function relationship in FNRs by transforming Arabidopsis with constructs for chimeric and truncated versions of different maize FNR genes (See Figure 2). Protein:protein interactions are being investigated in vitro using recombinant proteins and in vivo using fluorescence microscopy. FNRs which localize to specific locations on the thylakoid are being isolated in Arabidopsis null background to examine the impact on photosynthetic electron flow and ROS production.


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Figure 2. N-terminal structure controls FNR recruitment to the thylakoid membrane. Adapted from Twachtmann et al. (2012).

Cartoons of the crystal structures of maize FNR1 (A) and maize FNR3 (B) indicating the main structural features. Protein is shown in red (α-helices) and yellow (β-sheets), with unstructured regions in green, and the FAD co-factor in blue ball and stick. The positions of the N-termini are highlighted with pink circles. C, Membrane association of maize FNR iso-proteins following transgenic introduction to Arabidopsis. Genes encoding maize FNR1 (ZmFNR1), maize FNR3 (ZmFNR3) and a chimeric construct encoding ZmFNR3 with its N-terminal exchanged for that of ZmFNR1 (ZmFNR1-3) were expressed in Arabidopsis. Protein extracts from transgenic plants were separated into soluble and membrane fractions, before western blotting to detect the introduced proteins. Densitometry was performed on the western blots to calculate the ratio of the expressed protein that was bound to the membrane. Calculated from data published in Twachtmann et al (2012).



 

 

Recent publications from the project


  1. Kozuleva M., Goss T., Twachtmann M., Rudi K., Trapka J., Selinski J., Ivanov B., Garapathi P., Steinhoff H.J., Hase T., Scheibe R., Klare J.P., Hanke G.* (2016) Ferredoxin:NADP(H) oxidoreductase abundance and location influences redox poise and stress tolerance. Plant Physiol. pii: pp.01084.2016. [Epub ahead of print]
  2. Busch, K.B., Deckers-Hebestreit, G., Hanke, G.T.*, Mulkidjanian, A,Y. Dynamics of bioenergetic membranes (2013) Biol Chem. 161 (2):866-879.
  3. Altmann, B., Twachtmann, M., Muraki, N., Okutani, S., Voss, I., Kurisu, G., Hase, T., and Hanke, G.T*.(2012) N-terminal structure of ferredoxin:NADP+ reductase determines recruitment into different thylakoid membrane complexes. Plant Cell.24(7), 2979-2991.