My research efforts focus on the photosynthetic electron transfer reactions in the thylakoid membranes of cyanobacteria and plants. This important biological process provides the primary biological currency for life on earth by converting solar energy into biochemical energy and molecular oxygen. The enzymes responsible for these reactions are Photosystem II (PSII), cytochrome b6f complex, Photosystem I (PSI), and ATP synthase. The photosystems utilize light-harvesting antenna complexes to maximize the capture of solar energy. These are Light-harvesting antenna complexes I and II (LHCI and LHCII), which are integral membrane protein complexes within the thylakoid membranes of plants, and the Phycobilisome, a giant soluble complex on the cytoplasmic side of the thylakoid membrane, in cyanobacteria. In addition, the thylakoid membranes also contain complexes for alternative electron transfer pathways: NADPH dehydrogenase complexes (NDH), Fd-PQ oxidoreductase (FQR), and a plastid terminal oxidase (PTOX). All of these molecular machines collectively maintain optimal electron flow for biochemical energy conversion. The alternative electron transfer pathways help to fine tune the functioning of this biological engine. Extensive regulatory networks govern these components allowing them to respond to constantly changing environmental conditions and developmental needs of photosynthetic organisms.
I have been specifically involved in studies of the Photosystem II (PSII) enzyme, which is the component that uses light energy to split water apart into protons and the oxygen we breathe. The PSII enzyme consists of more than 26 protein subunits and associated cofactors. A high resolution crystal structure of PSII is available and has provided a wealth of information, but it doesn’t give away all of PSII’s secrets. It’s true the Devil is in the details.
Some of the more bedeviling details to me include…
PSII’s elaborate structure requires an intricate assembly process. Moreover, PSII must constantly undergo a cycle of assembly and disassembly due to damage inherent to electron transfer reactions it performs. The static structural image of the fully-assembled and functional complex doesn’t tell us about the steps involved in assembling the components together and disassembling them for damage repair. Also, the structure represents a cyanobacterial PSII complex. While many of the main components are universal among photosynthetic organisms, there are some key differences, which limit the usefulness of the structure for research in other organisms like plants. One particular difference relevant to my work involves the proteins associated with the lumenal side of PSII (where the water-splitting reaction occurs).
A number of extrinsic protein subunits are associated with the lumenal side of the PSII complex. These subunits contribute to the stability of the water oxidation complex and optimize electron transfer within the enzyme. These proteins have also been shown to optimize the accumulation of fully assembled PSII complexes. Collectively, these proteins are essential for PSII function under physiological conditions. In contrast to the high degree of conservation of the PSII transmembrane subunits across oxygenic photosynthetic organisms, the lumenal side of PSII is structurally more diverse. The complement of lumenal proteins associated with PSII complexes differs between cyanobacteria and plants.
The PsbO protein is common among all oxygenic photosynthetic organisms, but the number of copies associated with each PSII complex differs between plants and cyanobacteria. The PsbR protein is unique to plants. Both plants and cyanobacteria contain the PsbP and PsbQ proteins, but their roles and modes of association with PSII complexes differ significantly between the two systems. Finally, cyanobacterial PSII complexes also contain the PsbU and PsbV proteins. There is also another small extrinsic protein called Psb27. (PSII has so many subunits, that researches ran out of letters of the alphabet to name them. After PsbA-PsbZ names had been taken, we decided to start numbering them- hence Psb27.) While this protein is also common among many photosynthetic organisms, it is not found as part of the fully assembled complex, nor is it present in the current structural model.
Again, because of the subunit composition differences and the dynamic nature of PSII, there are still many unknowns about PSII- especially when it comes to the lumenal side of the complex. So, I have spent more than a decade fascinated with the molecular details of this part of this enzyme. Think I should get out more (scientifically, that is)? Well, don’t worry. Keep reading because scientific projects can take you to interesting places you don’t anticipate at the outset of investigations.
My graduate work in Himadri Pakrasi’s lab at Washington University in St. Louis focused on the assembly of cyanobacterial PSII. Using a combination of approaches including mutational analysis, biochemical preparations and biophysical measurements, I investigated a number of steps on the pathway to PSII assembly in cyanobacteria. All of this was done in the organism Synechocystis sp. PCC 6803, which is a real workhorse when it comes to lab work in photosynthesis research- great for molecular biology work (making any kind of directed mutation), genetics, and biochemistry. Also, fun fact, this cyanobacterium was the first photosynthetic organism to have its genome completely sequenced in the distant past at 1996. Here are the highlights of my work (with elaboration to follow in future posts):
- D1 processing is an early and essential step in the assembly of the lumenal side of PSII. (Roose and Pakrasi, 2004)
- PsbP and PsbQ proteins are present in cyanobacterial PSII, but their roles may be different than those of their plant counterparts. (Thornton et al, 2004; Kashino et al, 2006; Roose et al 2007)
- The Psb27 protein is an assembly factor that facilitates the assembly of PSII lumenal components (Roose et al, 2008; Liu et al 2011)
As a postdoc in Terry Bricker‘s lab at Louisiana State University, I’ve focused on PSII in plants, but still on the lumenal side of the thylakoid membrane. Spinach (yes, the bags of Fresh Express or, even better, the dark curly-leaves in bunches) is an excellent system for biochemical studies of PSII. Thylakoid membranes containing lots of PSII can be isolated in just a couple of hours. This preparation is a great starting point for picking apart the roles of the extrinsic proteins because these proteins can be easily removed and added back. Using molecular biology, all varieties of mutant versions of these proteins can be made in the bacterium E. coli and purified for use with our PSII membranes in biochemical studies. The research I conducted using the spinach PSII membrane system was done in collaboration with Hana Popelkova and Charles Yocum at the University of Michigan. Here are the highlights (with elaboration to follow in future posts).
- Removing the extrinsic proteins from one side of PSII can have effects on electron transfer on the other side of the PSII enzyme. (Roose et al, 2010)
- Investigation of the Aspartate 157 residue of the PsbO protein: Complex effects on the water-splitting function of PSII (Roose et al, 2010)
- How the binding of one or two copies of PsbO to PSII affects function (Roose et al 2011)
One ongoing investigation involves the PsbP family of lumenal proteins in plants. The extrinsic protein PsbP has been extensively characterized as a component of PSII- meaning we know how important it is and it is part of PSII. However, PsbP is also one member of an extensive family of PsbP-related proteins, which includes two PsbP-like (PPL) and seven PsbP-domain (PPD) proteins. These are distinct proteins with corresponding homologs in all plant genomes analyzed to date. Many of the PPL and PPD proteins have also been identified as expressed proteins in the thylakoid lumen of different plant species. In other words, we’re not just talking about similar sequences found among tons of new genomic data, but real detectable proteins found in the same compartment as PsbP.
What are all these other proteins doing? Well, they aren’t able to replace PsbP function because mutants of PsbP are very sick plants. (If any of the other proteins could replace PsbP, the plants would be no different from the wild-type plants.) The functions of the PPL proteins have been described previously. The PPL1 protein has been shown to be involved in PSII assembly and the repair cycle, but we don’t know anything more specific. The PPL2 protein is required for NDH complex accumulation. These examples indicate that the proteins of the PsbP family can have a wide range of possible functions, including those unrelated to PSII.
Little is known about the functions of the seven PPD proteins. My research has focused on investigating how the PPD proteins contribute to optimal photosynthetic performance using the model plant Arabidopsis. This plant offers a lot of genetic tools that spinach doesn’t, making it the organism of choice for tackling the functions of an entire protein family. Seeds of insertion mutants in the PPL and PPD genes are just an internet order away at the ABRC. In the event that those mutants aren’t useful or available, another technique called RNAi can be used to generate the necessary mutants. Arabidopsis plants are also OK for biochemical work, although material can be limiting at times. The goal of this research was to characterize Arabidopsis mutants in each of the PPDs with respect to photosynthetic function in order to gain some insights as to what these PPD proteins were doing in the plants. This project was supported by a fellowship from the United States Department of Agriculture. My findings suggest that the PPD proteins function in capacities other than PSII function. Nevertheless, these proteins appear to impact photosynthesis via effects on other thylakoid protein complexes and the redox state of the plastoquinone pool. Here’s how that project is going, stay tuned for more details:
Loss of the PPD5 protein affects plant development through a plant hormone synthesized in plastids. (Roose et al, 2011).
My work in photosynthesis has employed both cyanobacteria and plant model systems and uses diverse techniques to answer the relevant biological questions. PSII has been the focus of my research, and investigating the contributions of its extrinsically-associated proteins to optimal function and assembly of PSII remain active areas of interest for my research program. However, my future studies will certainly extend beyond this enzyme to the other aspects of photosynthesis and plant development. Beyond these initial studies, the extent to which lumenal proteins optimize electron transfer within the thylakoid membranes and affect whole plant physiology will be a longer term area of research interest.
Photosynthetic Electron Transfer
Photosystem II Structure and Function
Photosystem II Assembly and Repair Cycle Review
Photosystem II Extrinsic Proteins
The PsbP Family of Proteins
- Roose, J.L., Frankel, L.K., Bricker, T.M. The PsbP-Domain Protein 1 Functions in the Assembly of Lumenal Domains in Photosystem I, Journal of Biological Chemistry, submitted
- Bricker, T. M., Roose, J. L., Zhang, P., and Frankel, L. K. (2013) The PsbP family of proteins. Photosynth Res doi 10.1007/s11120-013-9820-7
- Bricker, T.M., Roose, J.L., Fagerlund, R.D., Frankel, L.K., and Eaton-Rye, J.J. (2012) The extrinsic proteins of Photosystem II. Biochimica et Biophysica Acta. 1817: 121-142.
- Roose, J.L., Frankel, L.K., and Bricker, T.M. (2011) Developmental defects in mutants of the PsbP domain protein 5 in Arabidopsis thaliana. PloS One 6:e28624.
- Liu, H., Roose, J.L., Cameron, J.C., and Pakrasi, H.B. (2011) A genetically tagged Psb27 protein allows purification of two consecutive Photosystem II (PSII) assembly intermediates in Synechocystis 6803, a cyanobacterium. Journal of Biological Chemistry. 286: 24865 – 24871.
- Roose, J.L., Yocum, C.F., and Popelkova, H. (2011) Binding stoichiometry and affinity of the Manganese-Stabilizing Protein affects redox reactions on the oxidizing side of Photosystem II. Biochemistry 50: 5988 – 5998.
- Roose, J.L., Yocum, C.F., Popelkova, H. (2010) Function of PsbO, the Photosystem II Manganese-Stabilizing Protein: Probing the role of aspartic acid 157. Biochemistry 49: 6042 – 6051.
- Roose, J.L., Frankel, L.K., Bricker, T.M. (2010) Documentation of significant electron transport defects on the reducing side of Photosystem II upon removal of the PsbP and PsbQ extrinsic proteins. Biochemistry 49: 36 – 41.
- Roose, J.L., and Pakrasi, H.B. (2008) The Psb27 protein facilitates manganese cluster assembly in Photosystem II. Journal of Biological Chemistry. 283: 4044 – 4050.
- Roose, J.L., Wegener, K.M. and Pakrasi, H.B. (2007) The extrinsic proteins of Photosystem II. Photosynthesis Research. 92(3):369 – 387.
- Roose, J.L., Kashino, Y. and Pakrasi, H.B. (2007) The PsbQ protein defines cyanobacterial Photosystem II complexes with highest activity and stability. Proceedings of the National Academy of Sciences USA. 104: 2548 – 2553.
- Kashino, Y., Inoue-Kashino, N., Roose, J.L., and Pakrasi, H.B. (2006). Absence of the PsbQ protein results in destabilization of the PsbV protein and decreased oxygen evolution activity in cyanobacterial Photosystem II. Journal of Biological Chemistry. 281: 20834 – 20841.
- Thornton, L.E., Roose, J.L., Ikeuchi, M. and Pakrasi, H.B. (2005) The Low Molecular Components: New Insights from Proteomics, Genomics, and Structural Studies. In Photosystem II: The Water/Plastoquinone Oxido-Reductase In Photosynthesis pp. 121-137 (Wydrzynski, T. and Satoh, K., eds.), Springer, The Netherlands.
- Roose, J.L. and Pakrasi, H.B. (2004) Evidence that D1 processing is required for manganese binding and extrinsic protein assembly into Photosystem II. Journal of Biological Chemistry. 279: 45417 – 45422.
- Thornton, L.E., Ohkawa, H., Roose, J.L., Kashino, Y., Keren, N. and Pakrasi, H.B. (2004) Homologs of plant PsbP and PsbQ proteins are necessary for regulation of Photosystem II activity in the cyanobacterium, Synechocystis 6803. Plant Cell 16:2164 – 2175.
- Bricker, T.M., Prevost, M., Vu, V., LaBorde, S., Womack, J., Frankel, L.K. (2000) Isolation of lumenal proteins from spinach thylakoid membranes by Triton X-114 phase partitioning. Biochemica et Biophysica Acta. 1503:350 – 356.