difficulty be aligned to the sequences of PsbO from Thermo- synechococcus, for which experimental structures are available (PDB IDs: 1FE1, 1LX, and 1IZL). The sequence identity between these proteins is 35% or higher. This high degree of similarity of the sequences of cyanobacterial PsbO and spinach PsbO indicates that the folds of these proteins are most likely very similar and that spinach PsbO is a useful model for the proteolytic cleavage of cyanobacterial PsbO by Deg proteases. For purification of PsbO, spinach has the advantage that it has a simpler arrangement of PSII that allows isolation of a homoge- neous PsbO fraction. Multi-gene families coding for PsbO isoforms have been reported in Arabidopsis [11,12,58], pea [59], tomato [60], and tobacco [61]. Genomic sequencing of other plant species, such as rice and wheat, has also revealed multiple PSBO genes, coding for highly similar PsbO proteins. However, spinach has only one known PsbO gene product, and there is no evidence for the presence of other PsbO proteins in this plant [11]. The extrinsic PsbO protein binds to the lumen side of PSII, and it is also present as soluble unassembled protein in the thylakoid lumen [44]. It is well known that the intrinsic proteolytic activity in the thylakoid lumen is low under non-reducing conditions, and most proteins of this compartment are not significantly degraded in vitro [62]. The recently reported observation that both PsbO subunits are degraded in Arabidopsis lumen samples in the presence of reduced thioredoxin [20] points to a potential participation of lumenal proteases in this process. To investigate if redox-dependent degradation of PsbO could also be observed in the thylakoid lumen of spinach, lumenal proteins were isolated from both spinach and Arabidopsis, and incubated in the presence of the complete thioredoxin system (Figure 3A). Control assays were performed using the thioredoxin system without the electron donor b-NADPH or vice versa. Even when some basal level of proteolysis was detected in the controls, PsbO degradation was clearly enhanced in the presence of reduced thioredoxin. However, redox-dependent degradation of PsbO was slower in the fraction of lumenal proteins from spinach than in that from Arabidopsis. Densitometric analysis of the corresponding bands revealed that about 61% of the initial PsbO amount remained in the spinach sample after 3 h of incubation in the presence of the complete thioredoxin system while for PsbO1 and PsbO2 from Arabidopsis 46 and 41% remained, respectively. As previously reported, redox-dependent (TL17) and -independent (Cyp38) protein degradation has been observed in the lumen of Arabidopsis [20] and redox-dependent degradation of TL17 is also clearly detectable in the lumenal fraction from spinach. The ability of reduced thioredoxin to reduce the disulfide of PsbO was confirmed using monobromobimane (mBBr), which allows specific fluorescence labeling of sulfhydryl groups (Figure 3B). rHhoA is able to degrade spinach PsbO in a redox- dependent manner In order to test the susceptibility of spinach PsbO to Synechocystis Deg proteases, PsbO isolated from spinach leaves was incubated for 5 h at 37uC in the presence of the recombinant proteases rHhoA, rHhoB, and rHtrA (Figure 4A). No degradation was observed after 5 h in non-reducing conditions (panel 1, lanes 1 and 2); the weak bands visible in these lanes were not immunostained by the antibody directed against PsbO nor did mass spectrometric analysis identify any PsbO fragments (not shown). In the presence of the thioredoxin system (PsbOred) some residual degradation was observed that might be caused by low amounts of cross-contamination by spinach proteases (panel 1, lane 3). Longer incubation times were tested up to 10 h, and the time course degradation of PsbO was quantified from the CBB Redox-Dependent Degradation by HhoA stained gels using the Image J software (Figure S3). Results showed that the fastest PsbO degradation was achieved in the presence of reduced thioredoxin and rHhoA in a ratio of 1:10 enzyme to substrate (56). In this case, about 38% of the initial PsbO remained after 5 h of incubation and less than 7% after 10 h. Concomitant with the decrease of the PsbO band at 33 kDa, an additional band with a molecular mass of 29 kDa was observed in the CBB-stained gel. Addition of recombinant HhoA to reduced PsbO using an enzyme to substrate ratio of 1:50 resulted in further, strong degradation of the PsbO protein (panel 2, lane 2). Four degradation products were observed with apparent molec- ular masses of around 32 kDa (F1), 29 kDa (F2), 27 kDa (F3), and 24 kDa (F4). At higher rHhoA concentration (56) the 24-kDa band accumulated, while the 29-kDa band disappeared (panel 2, lane 3). It is important to note that addition of recombinant HhoB or HtrA did not lead to any redox-dependent degradation of PsbO, apart from the residual degradation that was observed as background activity when no protease was added (panels 1, 3, and 4). This background activity was always around 20% of degradation (80% of initial PsbO remaining) even in the presence of recombinant HhoB and HtrA both at standard or high protease concentrations (56) and after 10 h of incubation (Figure S3). Also addition of CaCl2 to the buffer to stimulate the activity of these proteases did not lead to higher degradation rates (not shown) [50]. Of the four fragments detected, F1 and F4 were specific bands, which only appeared in the presence of the thioredoxin system and rHhoA. F3 already was visible in unreduced PsbO preparations, but accumulated over time in the presence of the thioredoxin system and rHhoA (Figure 4A, panel 2, lane 3). In contrast, F2 was produced in the presence of the thioredoxin system without addition of recombinant proteases, and most likely it was the product of background protease activity. Interestingly fragment F3 was further degraded by rHhoA; its intensity in the CBB-stained gel was reduced over time and totally disappeared at higher rHhoA concentrations (Figure 4A, panel 2, lane 3). The pro- teolytic fragments of the redox-dependent PsbO degradation by recombinant HhoA were N-terminally sequenced using the Edman degradation method (Figure 4B). The cleavage sites in spinach PsbO were identified as Val19–Lys20 (F1), Lys48–Lys49 (F2), Lys60–Ala61 (F3), and Met78–Thr79 (F4) (Figure 4B). The rHhoA-specific cleavage sites in fragments F1 (Tyr-Leu-Glu- ValQLys-Gly-Thr-Gly) and F4 (Thr-Lys-Leu-MetQThr-Arg- Leu-Thr) are not in the Merops peptidase database, indicating that HhoA belongs to a subfamily of the chymotrypsin family of peptidases that has its own unique substrate specificity. The identified cleavage sites are located near to the N-terminus of the protein (Figure 4B) and large fragments (32 to 24 kDa) remain after rHhoA action. Currently, we cannot exclude the need for additional factors/proteases to enhance or continue with the redox-dependent degradation of PsbO. However, addition of different combinations of recombinant Deg proteases (rHtrA or rHhoB with rHhoA) to reduced PsbO did not lead to a faster or further degradation of the protein in spinach (Figure S4). To test the substrate specificity of the redox-dependent degradation performed by rHhoA, another extrinsic protein of the manganese-stabilizing complex of PSII, the PsbQ protein was isolated from PSII membrane fragments and incubated for 5 h at 37uC in the presence or absence of the thioredoxin system (denoted PsbQred and PsbQ, respectively). As shown in Figure 5, no degradation was observed, either on the CBB-stained gel (upper panels) or in the immunoblot using antibodies directed against PsbQ (lower panels); the additional band with molecular mass of 12 kDa in the PsbQred fractions corresponded to PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45713