Modulation of PSBS and LHCSR contents in Physcomitrella patens by generation of KO and OE mosses
Reverse genetic studies of PSBS, LHCSR1 and LHCSR2 in Physcomitrella patens had previously shown that these gene products are all active in NPQ (Alboresi et al., 2010). Here, different KO mutations were combined to generate plants retaining only one of these subunits. As the triple psbs lhcsr1 lhcsr2 KO mutant lacks NPQ, the double KO mutants psbs lhcsr1 KO, psbs lhcsr2 KO and lhcsr1 lhcsr2 KO rely for their NPQ exclusively on LHCSR2, LHCSR1 and PSBS, respectively. These double mutants were obtained from single KO, as reported in Alboresi et al. (2010), exploiting the capacity of P. patens for homologous recombination. Transformations led to the isolation of several independent stably resistant colonies which, upon PCR analysis, were confirmed to carry an insertion at the expected target site. Individuals with single copy insertions were retained as described in Alboresi et al. (2010) and in the Supporting Information (Fig. S1). All PCR fragments were sequenced as further controls and at least two independent single insertion lines for each transformation were analyzed in detail for their phenotype. However, all lines showed indistinguishable phenotypes, so only results for one line for each mutation are reported here. In the selected lines, the accumulation of PSBS/LHCSR proteins was first determined by western blotting (Fig. 1a). As expected, only PSBS was detectable in lhcsr1 lhcsr2 KO plants, and only LHCSR was present in psbs lhcsr2 KO. In the case of psbs lhcsr1 KO, LHCSR2 accumulated at low levels, matching previous results (Alboresi et al., 2010).
Figure 1. PSBS (Photosystem II subunit S) and LHCSR (Light harvesting complex stress-related) content in knock-out (KO) and over-expressing (OE) Physcomitrella patens lines. Western blotting with antibodies against PSBS and LHCSR polypeptides in thylakoid extracts of selected lines was carried out. Four micrograms of Chl was loaded for each sample for PSBS detection, except for lhcsr1 lhcsr2 KO-PSBS OE line 1 (3 μg) and line 2 (0.65 μg) and psbs KO-PSBS OE line 1 (3 μg) and line 2 (2 μg). One microgram of Chl was loaded in all cases for LHCSR. (a) Wild type (WT) and double KO mutants (lhcsr1 lhcsr2 KO, psbs lhcsr1 KO and psbs lhcsr2 KO). (b) Two PSBS over-expressing lines compared with WT and background lhcsr1 lhcsr2 KO. (c) Two PSBS over-expressing lines compared with WT and background psbs KO. Dashed line: other lanes present in same blot. (d) Two LHCSR1 over-expressing lines compared with WT and background psbs KO. Where western blotting is not shown, protein was verified to be absent.
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In order to assess the dependence of NPQ amplitude on the relative amounts of PSBS and LHCSR, these KO plants were used as starting material for the generation of over-expressers for PSBS or LHCSR1. PSBS was over-expressed in lhcsr1 lhcsr2 KO and psbs KO backgrounds, to obtain lines with different PSBS contents in the absence or presence of LHCSR. LHCSR1 was also over-expressed in psbs KO to produce mosses over-accumulating LHCSR. All over-expression lines were generated by transformation with a construct driving the insertion of DNA in a region of the P. patens genome (called BS213) not encoding proteins, thus avoiding phenotypes caused by inactivation of endogenous genes (Schaefer & Zryd, 1997). In the plasmid employed (pMAK1), heterologous gene expression is under the control of a constitutive promoter derived from 35S (called 7113; Mitsuhara et al., 1996). At least five independent stable resistant lines were isolated for each transformation and retained for detailed analysis. The insertion of exogenous DNA was first verified by PCR (Fig. S2) and, in all lines showing positive PCR results, PSBS and LHCSR accumulation was evaluated with specific antibodies (Fig. 1b–d). Fig. 1(b) shows data from two independent lines over-expressing PSBS in the lhcsr1 lhcsr2 KO background (lhcsr1 lhcsr2 KO-PSBS OE), demonstrating that both these clones had increased PSBS accumulation with respect to WT and to the lhcsr1 lhcsr2 KO background which, instead, had a similar PSBS level. Over-accumulation is particularly strong in the case of line 2, also as a result of the lower amount of sample loaded on the gel. As transformation occurs by homologous recombination (Fig. S2), this strong over-expression is attributable to multiple insertions of the transgene during recombination, which are known to occur often in Physcomitrella patens (Kamisugi et al., 2006).
In order to assess possible differences in PSBS over-expression in the presence of LHCSR, psbs KO plants were also transformed with the same construct (psbs KO-PSBS OE). Fig. 1(c) shows two lines with different levels of PSBS over-expression. Despite their large differences in PSBS accumulation, the WT, KO and PSBS over-expressing lines showed no significant alterations in LHCSR accumulation, indicating that the latter is not affected by modulation of PSBS expression.
LHCSR1 was also over-expressed in a psbs KO line (psbs KO-LHCSR1 OE), generating lines where NPQ was totally dependent on LHCSR. Analysis of protein accumulation by western blotting showed increased LHCSR content with respect to the psbs KO background (Fig. 1d). In this case, however, no line with strong protein over-expression was identified, despite the analysis of a similar number of resistant lines. In P. patens, multiple insertions during homologous recombination are estimated to occur in c. 50% of cases (Kamisugi et al., 2005, 2006) and this number is consistent with our observations of PSBS over-expressing lines. It is thus likely that, out of the six independent lines over-expressing LHCSR, at least one carries a multiple insertion, as also indicated by PCR analysis (Fig. S2). Thus, failure to find lines with strong LHCSR accumulation is probably the result of an unidentified mechanism of post-translational regulation active for this protein.
Nonphotochemical quenching in mutants with altered PSBS/LHCSR contents
No mosses having variable accumulations of PSBS and/or LHCSR generated as described above, when grown in control conditions, showed a visible phenotype. Photochemical efficiency, estimated from Fv/Fm values, was indistinguishable from that of WT, as also observed previously for the triple mutant psbs lhcsr1 lhcsr2 KO (Table 1; Alboresi et al., 2010). No major differences were detectable in Chl a : b and Chl : Car ratios either (Table 1), which suggests that no substantial alterations in thylakoid composition or, at least, in the antenna/reaction center ratio took place.
Table 1. Photosystem II (PSII) quantum yield and pigment content of Physcomitrella patens wild type (WT), double knock-out (KO) and over-expresser (OE) lines grown in control conditions
| ||Fv/Fm||Chl a : b||Chl : Car|
|psbs lhcsr1 KO||0.80||2.3||3.7|
|psbs lhcsr2 KO||0.82||2.5||3.8|
|lhcsr1 lhcsr2 KO||0.82||2.5||4.0|
|lhcsr1 lhcsr2 KO-PSBS OE line 1||0.82||2.5||4.1|
|lhcsr1 lhcsr2 KO-PSBS OE line 2||0.81||2.5||4.2|
|psbs KO-LHCSR1 OE line 1||0.81||2.5||4.2|
|psbs KO-LHCSR1 OE line 2||0.81||2.5||4.1|
|psbs KO-PSBS OE line 1||0.81||2.5||4.2|
|psbs KO-PSBS OE line 2||0.81||2.5||4.2|
Instead, major alterations were observed in NPQ kinetics. All double KO mutants, which express only PSBS, LHCSR1 or LHCSR2, showed reduced NPQ with respect to WT and to the single KO mutant used as background, psbs lhcsr2 KO showing the most intense residual NPQ (Figs 2, S3). In all cases, residual NPQ rapidly relaxed in the dark and was thus identifiable as the qE component, indicating that PSBS and both LHCSR isoforms were all active, although to different extents, independently of the presence of the other proteins (Fig. 2).
Figure 2. Nonphotochemical quenching (NPQ) phenotype double knock-out (KO) mutants of Physcomitrella patens. NPQ kinetics of double KO mutants lhcsr1lhcsr2 KO (blue diamonds), psbs lhcsr1 KO (red circles), and psbs lhcsr2 KO (green triangles) compared with wild-type (WT) plants (black squares). Actinic light was switched off after 8 min. Data are presented as means ± SD (n ≥ 3).
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It was observed that lhcsr1 lhcsr2 KO showed very low NPQ activity with respect to the other lines, raising the question as to whether P. patens PSBS protein is less active in triggering NPQ than vascular plant isoforms and/or whether the photosynthetic apparatus of P. patens is not able to fully undergo the structural rearrangements required for PSBS-dependent NPQ activation (Betterle et al., 2009; Johnson et al., 2011). Analysis of the lines over-expressing PSBS in the lhcsr1 lhcsr2 KO background (lhcsr1 lhcsr2 KO-PSBS OE) clarified this point. In both lhcsr1 lhcsr2 KO-PSBS OE lines a strong increase in NPQ capacity was observed with respect to the KO background of the transformation (Fig. 3a). Interestingly, NPQ amplitude depended on the level of PSBS accumulation and was stronger in over-expressing line 2, where PSBS was more abundant. In all over-expressing lines, most NPQ was rapidly relaxed in the dark and may thus qualify as the fast qE component, similar to the case of WT plants. These results clearly suggest that the low PSBS-dependent NPQ activity of lhcsr1 lhcsr2 KO is attributable to the low PSBS accumulation in Physcomitrella patens, while the protein is fully capable of activating strong NPQ even in the absence of LHCSR. It is also worth noting that this PSBS-only-dependent NPQ has a time-course very similar to that of Arabidopsis thaliana plants (Fig. S4), confirming that, once enough PSBS is present, quenching in mosses occurs as well as in vascular plants.
Figure 3. Nonphotochemical quenching (NPQ) kinetics of Physcomitrella patens lines over-expressing PSBS (Photosystem II subunit S) or LHCSR1 (Light harvesting complex stress-related 1). (a) NPQ kinetics of wild type (WT; black squares), lhcsr1 lhcsr2 KO (background of over-expressing (OE) lines; red circles), lhcsr1 lhcsr2 KO-PSBS OE line 1 (blue triangles) and 2 (green triangles). (b) NPQ kinetics of WT (black squares), psbs KO (background of over-expressing lines; red triangles), psbs KO-PSBS OE line 1 (green diamonds) and 2 (blue circles). (c) NPQ kinetics of WT (black squares), psbs KO (background of over-expressing lines; red triangles), psbs KO-LHCSR1 OE line 1 (blue diamonds) and 2 (green circles). In all cases, actinic light was switched off after 8 min. Data are presented as means ± SD (n ≥ 3).
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PSBS over-expression in psbs KO plants, which retain WT levels of LHCSR, yielded similar results. psbs KO-PSBS OE line 1, with a lower PSBS accumulation, also showed the smallest increase in NPQ induction (Fig. 3b). Instead, line 2, in which western blotting showed higher PSBS accumulation, was able to induce an NPQ response stronger than that of WT (Fig. 3b). As LHCSR protein content showed no significant differences in any of the lines tested (Fig. 1c), these results – the increased NPQ capacity of these clones – are consistent with the activity being attributable only to the larger accumulation of PSBS.
Both LHCSR isoforms alone were fully capable of inducing NPQ, as shown by the KO mutants' phenotype. The over-expression of LHCSR1 in psbs KO, where NPQ is only LHCSR-dependent, led to lines with increased protein accumulation which also showed increased NPQ capacity with respect to the background (Fig. 3c). The NPQ induced in the LHCSR over-expression lines could be identified as qE, owing to its fast relaxation in the dark. As observed previously, NPQ values were higher in genotypes with the highest LHCSR accumulation, indicating that the level of LHCSR influences NPQ in P. patens, as observed above in PSBS over-expression. In this case, however, the increase in protein accumulation observed with western blotting was lower, and also the increase in NPQ was not as strong as in mosses over-expressing PSBS.
Zeaxanthin accumulation is well known to enhance NPQ intensity (Niyogi et al., 1997, 1998). In order to verify if the differences observed in mutants above were at least partially attributable to alterations in zeaxanthin synthesis, carotenoid composition was monitored in mutant and OE lines during NPQ activation. As shown in Table 2 and Fig. S5, all lines, independently of their PSBS/LHCSR accumulation levels, showed a similar accumulation of zeaxanthin under illumination, showing that differences in NPQ levels are not attributable to alteration in xanthophyll cycle activation.
Table 2. Light-induced zeaxanthin formation in Physcomitrella patens
| ||Dark-adapted||Light-treated||Light-treated + DTT|
|WT||0.15 ± 0.04||0.41 ± 0.05||0.13 ± 0.02|
|psbs KO-PSBS OE line2||0.11 ± 0.02||0.52 ± 0.06||0.16 ± 0.05|
|lhcsr1 lhcsr2 KO-PSBS OE line2||0.11 ± 0.02||0.47 ± 0.09||0.12 ± 0.03|
|psbs KO-LHCSR1 OE line1||0.13 ± 0.04||0.48 ± 0.15||0.11 ± 0.02|
|psbs lhcsr1 lhcsr2 KO ||0.14 ± 0.02||0.49 ± 0.07||0.19 ± 0.05|
In order to verify if this increased NPQ was effective in protecting P. patens from excess illumination, KO and OE lines were exposed to strong illumination, and PSII radiation damage was monitored using the Fv/Fm parameter, reductions in which are indicative of light-induced damage. psbs KO mutants showed no significant differences from the WT (Fig. 4a), suggesting that residual NPQ was sufficient to ensure resistance in the experimental conditions tested, as also observed in Alboresi et al. (2010). In contrast, lhcsr1 lhcsr2 KO, which showed a stronger reduction in NPQ, also showed increased light sensitivity (Fig. 4a). When PSBS was over-expressed in these lines (lhcsr1 lhcsr2 KO-PSBS OE), not only was NPQ intensity recovered but their capacity to withstand strong illumination treatments was also restored (Fig. 4b). Instead, psbs KO-PSBS OE and psbs KO-LHCSR1 OE showed a response to the light treatment similar to that of WT mosses and their backgrounds, as expected (Fig. S6).
Figure 4. Light sensitivity of Physcomitrella patens lines. The light sensitivity of Physcomitrella patens plants was tested by treating plants with 800 μmol m−2 s−1 light and monitoring Fv/Fm as a signal of light-induced damage. (a) Wild type (WT), psbs knock-out (KO), lhcsr1 lhcsr2 KO and psbs lhcsr1 lhcsr2 KO are shown, respectively, as black squares, red circles, green diamonds and blue triangles. (b) WT, lhcsr1 lhcsr2 KO, and lhcsr1 lhcsr2 KO- PSBS OE lines 1 and 2 are shown as black squares, green diamonds, blue triangles and red circles, respectively. Data are presented as means ± SD (n ≥ 6).
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Correlation of LHCSR and PSBS accumulation with NPQ values
Combining the various KO and over-expressing lines presented here and in Alboresi et al. (2010), a set of 20 moss lines with varying PSBS and/or LHCSR accumulation levels and altered NPQ values is now available. For a more general picture of the dependence of NPQ on accumulation of these proteins, the latter was more precisely quantified, by using specific antibodies (Fig. 5a). The same procedure was also performed for mutants acclimated to varying light conditions, in which PSBS and LHCSR were differentially accumulated (Gerotto et al., 2011). Only low-light-acclimated samples (grown at 10 μmol m−2 s−1) were considered in order to avoid the NPQ-enhancing effect of zeaxanthin, which is also accumulated in high light conditions (Niyogi et al., 1998).
Figure 5. Correlation between PSBS (Photosystem II subunit S) and LHCSR (Light harvesting complex stress-related) accumulation and nonphotochemical quenching (NPQ) values. LHCSR and PSBS contents were quantified in all available LHCSR1/2 and PSBS knock-out (KO)/overexpressing (OE) lines. Contents in wild-type (WT) Physcomitrella patens plants grown in control conditions were set at 1. Different amounts of thylakoids were loaded to achieve the same antibody signal as in WT and thus avoid signal saturation. (a) Example of western blotting. Numbers over each lane indicate the amount of Chl (in micrograms) loaded. (b) Correlation of PSBS amount and NPQ. NPQ values were obtained from the NPQ kinetics of each clone (values reached at the end of light induction). A total of 27 lines were analyzed (all shown in Supporting Information Fig. S7); 16 of these, with LHCSR content 0 (red circles), one-tenth that of WT (green triangles) or equal to WT (black squares), are included in the graph. R2 of the linear correlations shown in the figure are 0.996, 0.982 and 0.885, respectively. (c) Correlation of LHCSR amount and NPQ. NPQ values were obtained from the NPQ kinetics of each clone (values reached at the end of light induction). Sixteen of the 27 lines analyzed (shown in Fig. S7), with PSBS content 0 (red circles) or equal to WT (black squares), are included in the graph. R2 of the linear correlations shown in the figure are 0.873 and 0.983, respectively. Data are presented as means ± SD (n ≥ 3).
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Altogether, a total of 27 different samples with variable PSBS/LHCSR contents were analyzed, allowing us to explore the quantitative relationship between the accumulation of PSBS and LHCSR vs NPQ intensity. All data are reported in Fig. S7, whereas Fig. 5(b,c) shows details of the dependence of NPQ values on PSBS and LHCSR contents, respectively. In the case of PSBS (Fig. 5b), many samples with large differences in PSBS expression levels were available, ranging from 0 to c. 27-fold the WT level. The data distribution identifies three groups, differing in LHCSR accumulation: one group includes all lines without LHCSR, another those with WT LHCSR levels (1 ± 0.1), and a third, smaller group includes lhcsr1 KO lines, which have only residual LHCSR2 expression (0.08 ± 0.05 of WT level).
In all cases, the NPQ values showed a positive correlation with the amount of PSBS. In the range from 0 up to five times WT content, data could be fitted linearly, and the slope of the curve was clearly higher when LHCSR was present (Fig. 5b). In cases with the strongest PSBS accumulation, NPQ deviated from linearity, reaching saturation.
Similar results were obtained in the case of NPQ dependence on LHCSR content (Fig. 5c): two distinct groups of samples, depleted in or having WT levels of PSBS, were analyzed. As we found above for PSBS, LHCSR accumulation level also positively correlated with the NPQ capacity of the samples, both in genotypes where PSBS is absent and in those exhibiting a WT level of PSBS accumulation. As in the previous case, the slope of the linear fit was higher in samples accumulating both PSBS and LHCSR.
In view of the complex nature of NPQ, some caution should be employed to avoid over-interpreting this correlation between PSBS/LHCSR and NPQ. The data presented here, however, clearly indicate that PSBS and LHCSR accumulation levels are limiting for NPQ, also suggesting that a similar increase in PSBS/LHCSR is more effective when both subunits are present.