Light levels affected the degree o f variegation o f Var2-2 strains. Whereas Col-0 plants would normally be grown at 130 pmol/m^/s in our growth chambers, when optimising conditions for growth o f the Var2-2 plants it became clear that they required light levels o f approx. 30 pmol/m^/s for best growth. Takechi et al. (2000) later reported that they found it necessary to grow four strains o f Var2 (Var2-1, Var 2-6, Var2-7 and Var 2-8) at
70 |Imol/m^/s. Interestingly, it has also been noted that high temperatures and low light conditions retard the growth o f vait2T mutants and result in plants that are nearly all-green [Chen et a l, 2000] so there is clearly some light-related effect o f the mutation o f this FtsH.
To investigate the ability o f the Var2-2 A . thaliana to respond to an altered light environment, ColO and Var2-2 plants were subjected to moderate and extreme light intensity and their response monitored using 77K fluorescence emission spectroscopy. Work in parallel by S. Bailey measured the ratio o f maximum to variable fluorescence
(F^/F^; a measure o f PSII efficiency) to monitor photoinhibition [for review o f method, see Hall & Rao, 1999], also using Western blots to follow the repair o f the PSII D1 protein.
8.4.1 Fluorescence spectra in higb-intensity ligh t
Plants typically contain equimolar amounts o f active photosystems, but those grown under high light do increase the amount o f PSII (along with the chlorophyll cr.h ratio) while the amount o f LHCII drops. This adaptation in the form o f a changing photosystem
stoichiometry is more often seen in young than old plants [for review, see Chitnis, 2001], so young plants were used for assays o f response to high-intensity light here. For high light treatment, 25-day-old seedlings grown in half-strength Murashige and Skoog agar were subjected to approx. 1600 (imol/m^/s hght in a water-cooled apparatus for approx. 2 h. 77K fluorescence emission spectra were then recorded as described above, using thylakoid membranes prepared from ColO and Var2-2 leaves.
Ü 0.5
L D
^ 0.4
ColOLL-HL-LL
yTTZm Var2LL-HL-LL
Low light High light Low hght
Figure 8.10. Mean PSILPSI fluorescence ratio o f plate-grown Arahidopsis thaliana ColO and Var2-2 mutant during low hght (IX), high hght (2 h at 1600 gmol/m^/s) and LL recovery for approx. 4 h. Fluorescence emission measured from 77K fluorescence emission spectra with 435 nm excitation hght. Differences between mean fluorescence ratio in these plants not significant by t test.
In both strains, the mean fluorescence ratio from PSILPSI was reduced after high-hght treatment for approx. 2 h, although the mean ratio was shghtly lower in Var2-2 seedhngs than ColO seedlings [Figure 8.10]. The difference was not significant, however [Table 8.7]. Neither strain increased the activity o f PSII relative to PSI during the following 4 or 5 h recovery under standard (low-intensity hght) growth conditions, the mean PSILPSI fluorescence ratio again remaining rather lower in Var2-2 than in ColO.
Table 8.7. Fluorescence emission from ColO and N7lt2-2 Arahidopsis thaliana after 2 h high-intensity
Strain
Mean PSILPSI fluorescence ratio (± SEM)*
LL HL LL recovery
ColO 0.52±0.04 0.43±0.04 0.4210.03
Var2-2 0.50±0.04 0.40±0.02 0.4010.03
P=0.7129 P=0.5655 P=0.6128
*None significantly different (12 degrees of freedom in t test).
PS, Photosystem, SEM, standard error of the mean; LL, low hght (30 gmol/m^/s); ILL, high hght (approx. 1600 pmol/m^/s).
The peaks at 685 and 695 nm in 77K fluorescence emission spectra correspond to emission from CP43 and CP47, which funnel excitation energy from the light-harvesting antenna to PSII. The reduction in fluorescence in both ColO and Var2-2 from PSII within 2 h o f very high light intensity may correspond with a drop in LHCII content, reducing the energy directed to CP43 and CP47, thus reducing the PSILPSI fluorescence ratio. PSII damage would also occur under this high-intensity hght (1600 [xmol/m^/s), contributing to the decrease in PSII activity.
The 77K fluorescence emission spectra in high hght suggested that the Var2-2 mutant could sense high light conditions and regulate light-harvesting and photosynthetic function in the absence o f this FtsH. The fluorescence kinetics (F^/F^ o f Var2-2 and ColO plants were also comparable after growth under standard conditions (data not shown). When detached leaves were subjected to high hght intensities, however, F^/F^ measurements in the FtsH mutant revealed a severe photoinhibition phenotype. Photoinhibition was monitored using a fluorimeter (work by S. Bailey) to measure room-temperature
fluorescence and calculate F^/F^. These data showed that Var2-2 leaves suffered a greater magnitude o f photoinhibition at moderate (300 pmol/m^/s) and at very high (1800 [imol/m^/s) light, and recovery from photoinhibition was slower in Var2-2 than in ColO [Figure 8.11] (S. Bailey, personal communication, 2001). In ColO the drop in F^/F^ levelled out after approx. 2 h, whereas in Var2-2 there was a drop in F^/F^ but no recovery. Using a protein synthesis inhibitor, lincomycin, to prevent new D1 protein being made, and thus repair PSII damaged by high hght, the F^/F^ response o f ColO and Var2-2 became
comparable, showing a drop without any subsequent recovery. This suggests that the D1 repair cycle is always faulty in Var2-2: even without lincomycin, the mutant seemed unable to repair damaged PSII. Western blots by S. Bailey [Figure 8.11] using plants from this laboratory confirmed that the D1 protein was correctly degraded in ColO in high light In Var2, in contrast, the D1 protein was normally present but after photoinhibitory treatment it was not removed. Interestingly, there was some effect on the D 2 protein also. It has been su^ ested that the degradation o f D 2 and D1 are indeed coupled [Jansen et al.^ 1999].
-ûOmins Omins 30mms 120mms 240niins 360mins 1200mins
— A - Col0 300
----Var2 300
— A - ColO 1800
- O - Var2 1800
Duration o f light/dark treatm ent (min) 0- A - ColO —# - Var2 —A - Cok) lincomycin — • - Var2 hncomycin
120 180 240 D unoon o f photomhibilDiy inadiance (min)
w u n tre a te d ColO + L inc + L in c/U L Var2 u n tre a te d + L in c + L in c/H L D l m D2 m m * PsbS — - w u n tre a te d +HjO + H jO /H L D1
Figure 8.11. {a, b) Maximum photochemical efficiency o f photosystem II (PSII) in ColO and Var2-2 Arahidopsis thaliana (mean±standard error). [Work by S. Bailey, University o f Warwick.] {d) ColO F^/F„ is stable at moderate light (300 pmol/m^/s; A ), but decreases in high Hght (HL; 1800 pmol/m^/s; A ) before recovering. Var2-2 is more susceptible than ColO to photoinhibition o f PSII at both moderate light (O) and HL ( • ) . -60 min, overnight dark adaptation; 0 min, end o f 1-h Hght treatment, {fy) F^/F^ o f PSII in ColO (A ) and Var2-2 (O) leaves after HL with or without Hncomycin. {p) Western blot. With Hncomycin, ColO leaves have decreased D1 protein relative to untreated leaves. After HL and Hncomycin, D1 is almost absent. With no Hncomycin, D1 is replaced in ColO even in HL (see lower panel). In Var2-2, levels o f D1 remain stable. Even in HL with Hncomycin, D1 is not lost. Turnover o f the D2 protein mirrors that o f D l, but the minor subunit PsbS remained constant showing that the reaction centre as a whole was not faulty.