Our last journal club was a throwback article of mine on D1 processing and PSII assembly in cyanobacteria. Just this week, a new report in PNAS describes what happens in plants when the D1 protein isn’t processed. Che and co-authors still found that D1 processing is essential for PSII assembly, but there are some new details that reveal differences between the cyanobacterial and plant systems. Check out my previous post for more background info.
C-terminal processing of reaction center protein D1 is essential for the function and assembly of photosystem II in Arabidopsis* Che et al PNAS 110: 16247-16252
Here’s how the science breaks down:
Observations: Processing of the precursor D1 (pD1) protein in cyanobacteria is critical for PSII assembly and photosynthetic growth in cyanobacteria. This processing step does not occur in mutants of the C-terminal processing protease (CtpA), leaving only the pD1 protein in the thylakoid membranes. Cyanobacterial PSII complexes containing only the pD1 protein have specific problems with the assembly of the lumenal side of the complex. There are some differences in PSII between cyanobacteria and plants, so what are the consequences of the absence of pD1 processing in plants?
Hypothesis: Based on the genome sequence of Arabidopsis, there are a few genes that could encode C-terminal processing proteases. The Arabidopsis CtpA (AtCtpA) protein (responsible for pD1 processing) will be found in the thylakoid lumen and mutants (atctpa) lacking this protein will accumulate the pD1 protein and not be able to grow photosynthetically. The extent to which PSII complexes are assembled in the atctpa mutants may be the same or different relative to the previously described cyanobacterial mutants.
Experiment: Identify the AtCtpA gene based on sequence signatures that predict lumenal targeting. Confirm that the AtCtpA candidate is present in the thylakoid lumen and characterize mutants for PSII assembly and function.
Results: Che and co-workers were able to identify AtCtpA and show it was found in the thylakoid lumen. There were two mutants atctpa-1 and atctpa-2 available for the gene they identified. The atctpa-1 mutant did not accumulate and AtCtpA protein, but the atctpa-2 mutant accumulated ~2% of the amount of AtCtpA found in wild-type plants. The atctpa-1 plants could only be grown in the presence of sucrose and had defects in chloroplast development. The atctpa-2 mutant could grow without sucrose, but was sensitive to high light. The PSII complexes in these mutants were analyzed by native gel electrophoresis, which separates the protein complexes in a mixture. Using this technique the authors found that the mutants had fewer PSII dimers and PSII-LHCII complexes, which are the forms of the fully assembled and functional complexes in plants. Using this technique, they did not see significant differences in the assembly state of the lumenal extrinsic proteins.
Conclusions: Processing of the pD1 protein is an essential step in PSII assembly in plants. In the absence of AtCtpA (as in the atctpa-1 mutant), plants cannot assemble PSII complexes sufficient to support photosynthetic growth. Only a small amount of AtCtpA is necessary to support some photosynthetic growth (as in the atctpa-2 mutant), but small amounts are insufficient to keep up with the pD1 processing demands plants face under high light conditions where replacement of the D1 protein must occur more frequently. The main difference in the PSII assembly state of pD1-containing complexes vs. normal was that the pD1-containing complexes did not assemble into the larger dimer forms with the LHCII antenna attached. This means that pD1 processing is required for attachment of the antenna, which is something we didn’t know before and didn’t really expect. However, this makes sense because it would be problematic for plant cells to attach a light-harvesting antenna to a partially assembled complex that would otherwise be sensitive to light.
Think Ahead: Future studies will focus on exactly how the presence of the pD1 protein prevents attachment of the LHCII light-harvesting antenna complex. The authors did not have any data on the presence of the Psb27 protein in their pD1-containing PSII complexes. They reported on just the PSII components known to be part of the assembled complex. I would hypothesize that this extrinsic protein accumulates on pD1-containing PSII in plants as well and should be detectable using a specific antibody.
This study emphasizes the need to use different model organisms to provide information about a seemingly universal process in photosynthetic organisms. My work in cyanobacteria gave certain kinds of information and the work in Arabidopsis present in this report gave slightly different information. The sources of these differences come from a couple of possible places. The first obvious reason is that there could be inherent differences in the way the process works in cyanobacteria vs. plants. I’ve outlined in the My Research section that the lumenal portions of the PSII enzyme have different protein compositions in cyanobacteria vs. plants. The C-terminal extension of the pD1 protein is also different in cyanobacteria (16 amino acid extension) vs. plants (9 amino acid extension). Plants and cyanobacteria also have very different light-harvesting antenna systems. Plants have the LHCII antenna complex, which is comprise of integral membrane proteins that bind chlorophyll pigments, while cyanobacteria have phycobilisome complexes, which are comprised of soluble, extrinsically-associated bilin pigment-containing proteins attached to the cytoplasmic face of the complex. Thus, any effects (however unexpected) on the antenna system relationship with PSII would be expected to be quite different between these two types of photosynthetic organisms.
The second reason is that the experimental techniques available in these two model organisms are different and come with their own technical limitations. Cyanobacteria allow for precise genetic manipulation and tagging of certain proteins, allowing me to selectively purify PSII complexes containing a histidine-tagged CP47 protein. These researchers didn’t have that option with Arabidopsis and had to rely on native gel electrophoresis to separate the PSII complexes from the other protein complexes in the thylakoids of their mutants. This different experimental method can provide different information with respect to protein composition of the complexes. For example, the extrinsic lumenal proteins largely migrated as free proteins in both wild type and their atctpa-1 mutant. This may be because the electrostatic interactions that hold these proteins to the complex do not survive in an electric field, resulting in their release during electrophoresis. This makes it difficult to say how strongly the PsbO, PsbP and PsbQ proteins are attached to their pD1-containing complexes. However, they are able to detect a small amount of PsbO associated with these complexes, which is more than I was able to find in the cyanobacterial equivalent.
Finally, I just have to say ‘Bravo!’ to the researchers for taking on this kind of biochemical work in Arabidopsis mutants. If you read the footnotes on my post, I complained about how difficult it was to grow my cyanobacterial mutant and isolate such small amounts of its PSII. I cannot communicate to you how much more difficult the biochemistry must have been in this study. Arabidopsis is a great model plant for genetic reasons, but even wild-type plants are on the small side for providing adequate material for biochemical work. The atctpa-1 mutants were much smaller than wild-type and had to be maintained on sucrose-containing medium. I shudder to think of how many of those small plants went into the one lane of the native gel figure in the paper (and consequently how many lab-hours that required).
Find out more information on some of the techniques used in this paper on the Experiments in Photosynthesis Research page.
* It’s open access!