Stroma and thylakoid membrane degradomes of DEG2

As shown in Figure 5.4 and reported previously [81, 235], DEG2 is distributed between stroma and thylakoid fractions. Therefore, we expect that DEG2 substrates should be located in these two com-partments. To search for physiological substrates of DEG2 we performed comparative proteomics on stroma fractions isolated from wild-type anddeg2knock-out plants. Proteins of purified stroma fractions were separated by isoelectric focusing (IEF) in the first dimension in combination with denaturing SDS-PAGE in the second dimension. We expected that in the absence of the DEG2

DEG2

Figure 5.4:Distribution of DEG2 in a total protein ex-tract separated into soluble and membrane fractions by ultracentrifugation. Immunoblots of a subunit of the ATP synthase (CF1α) and the large subunit of Rubisco (RbcL) are shown to demonstrate the quality of the separation.

protease, specific substrates will accumulate in the deg2 knock-out mutants. Our colleagues successfully applied this strategy in the past to identify Clp protease substrates [223]. The re-sulting two dimensional (2D)-PAGE gels were scanned and spot intensities were analysed us-ing Applied-Maths Bionumericsv5.1. Observed differences were tested for significance using statistic software Rv.2.13. 8 spots with highly significant different intensities were extracted from the gel, digested by trypsin and analysed by mass spectrometry. We were able to identify 4 subunits of the photosynthetic apparatus, such as photosystem II subunit P1 (PsbP1), photosystem I subunit E (PsaE) and subunit N (PsaN) and plastocyanin (PC), whereby PsaN, PC and PsbP1 are up-regulated and PsaE down-regulated indeg2 mutants as compared to wild-type plants (Figure 5.5). The PsaE protein, located at the reducing side of photosystem I and involved in docking the soluble electron acceptor ferredoxin [105], is the only potential direct interaction partner of DEG2. It was reported that the ferredoxin-docking site, includ-ing PsaE is especially sensitive to ROS. The strongly reduced amount of PsaE indeg2knock-out

WT

Figure 5.5:Comparative proteomics of stroma fractions from wild-type anddeg2knock-out plants.AStroma fractions were isolated as described in Material & Methods and proteins separated by isoelectric focusing (IEF) in the 1st dimension and denaturating SDS-PAGE in the 2nd dimension. Gels were scanned and spots were analysed using Applied-Maths Bionumericsv5.1. Protein identities in selected spots were analysed by mass spectrometry. Replicates N = 4,1PsbP1,2PsaE,3PsaN,4PCBStatistical comparison of selected spot volumes; * significance level: p-value: * =p≤0.05, ** =p≤0.005, *** =p≤0.0005CPurity of stromal preparations. 1, 5 and 10 µg purified stroma of wild-typeA. thalianaplants was tested with representative antibodies of the different chloroplastic compartments. Lhcb2 represents an insoluble, thylakoid associated protein and was only detected after long exposures (>30 min) in very weak quantities (not shown). Detection of PsbP1 protein indicated a contamination of soluble, originally luminal located proteins. Rubisco large subunit RbcL served as stroma control.

5.3 Results and Discussion

mutants might be due to a required chaperone function of DEG2 for a proper PsaE assembly. PsbP1, PC and PsaN are located in the thylakoid lumen or attached to the luminal side of the thylakoid membrane, thus they rather represent contaminations caused by ruptured thylakoids during the stroma purification procedure than DEG2 substratesin vivo. It is plausible that they are usually recognised by DEG1, which level is strongly down-regulated indeg2knock-out mutants [143] (Fig 5.6), due to a common DEG protease recognition motif. It was shown earlier that down-regulation of DEG1 led to the reduced amount of DEG2 [37]. Since DEG2 is peripherally associated with the stromal side of the thylakoid membrane [81], while DEG1 is localised in the thylakoid lumen [98], it is unlikely for these two proteases to physically interact but rather being regulated at a superordinate level. Redox regulation (see Chapter 6) across the thylakoid membrane, as reported previously by Motohashi and Hisabori [161], represents one possible explanation for such an unusual interaction.

The authors describe the transfer of reducing equivalents across the thylakoid membrane through the thioreduxin-like, membrane-anchored protein HCF164. Interestingly, since one of the identified targets of HCF164 was PsaN [161], and DEG1, DEG2 and PsbP1 were identified as beeing redox regulated previously [232] (Chapter 6), we could speculate that our findings catched a tiny glimpse of a thioreduxin mediated redox regulation of DEG proteases and photosystem subunits within the chloroplast. Anyhow, our data, in accordance with previous publications [37, 143] (Figure 5.6), emphasises that DEG1 and DEG2 need each other for stability.

WT deg2

Figure 5.6: DEG1 level is strongly reduced in deg2 knock-out plants (sample 4-6), as indicated by immunoblotting. 5 µg total protein was loaded in each lane. An unspecific signal of the DEG1 an-tisera (confirmed by Agrisera through massspec-trometry as neither DEG nor protease-specific) was used as loading control.

Our work presents an important step forward in understanding the complex relationship of DEG proteases within the chloroplast. We were able to demonstrate that DEG2 transcript and protein level remain unaffected by light stress, therefore questioning an involvement in plant stress response. Nevertheless, an effect on root growth as well as on protein level of several subunits of the photosynthetic apparatus could be observed. Our ability to complement the dramatic root phenotype ofdeg2ko mutants with sucrose or NAA brought up speculations about DEG2 being involved in carbon metabolism or hormone homeostasis, even though preliminary results indicated

that DEG2 has no effect on starch accumulation. Disappearance of the photosystem I subunit PsaE in the chloroplast stroma indeg2ko mutants strongly supports the hypothesis of a chaperone function of DEG2, while up-regulation of the lumenal proteins PsaN, PC and PsbP1 could be explained by recognition and degradation through DEG1. A positive correlation between DEG2 and luminal located DEG1 was already described [112, 143]. This indicates that DEG2 is part of a complex machinery within the chloroplast, affecting not only stromal proteins directly, but rather influencing a multitude of pathways. In this context, the observed root phenotype further supports the importance of DEG2in planta. We were able to shed a little bit of light onto the complex correlations of chloroplast proteins, nevertheless the precise function of DEG2 in the plant chloroplast remains to be elucidated.