Regulation of Elip and Sep2 Expression by the Redox State of the PQ/PQH2 Pool

It has been shown that the redox status of the PQ/PQH2 pool in the chloroplast regulates gene expression (for recent reviews see Baier and Dietz 2005; Beck 2005; Ogawa 2005; Leister 2005;

Pfannschmidt and Liere 2005; Nott et al. 2006). To test whether and on which level redox poise is involved in the regulation of Elip1, Elip2 and Sep2 expressions, we carried out studies with inhibitors of photosynthetic electron transport. Electron flow from PSII to PQ was inhibited by the addition of DCMU, which prevented the reduction of PQ and mimicked low light conditions (Figure 20A). The inhibitor DBMIB blocked electron flow from PQ to cytochrome (cyt) b6/f, preventing the oxidation of the

Figure 19: Differential accumulation of Elips and Sep2 in response to increasing light intensities.

Mature Arabidopsis leaves were exposed to increasing light intensities for 3 h prior to isolation of total membrane proteins and immunoblotting using polyclonal antibodies raised against Elip1, Elip2 and Sep2.

PQ pool and mirrored the situation that occurred under light stress conditions. To proof whether the external addition of DCMU and DBMIB indeed inhibited the photosynthetic electron flow in the chloroplast we assayed changes in Chl fluorescence in leaves exposed to low or high light intensities.

Images of leaves with various Chl fluorescence parameters, such as maximal quantum yield of PSII (Fv/Fm) and effective quantum yield of PSII (PSII yield) are depicted in false colors coding from 0.0 (black) to 1.0 (purple) and are shown in Figure 20B. The mean values for Fv/Fm and PSII yield were calculated for selected leaf areas and are shown in Table 3. A significant inhibition of photosynthetic activity (64% or 0% of control values) was observed in DCMU-treated leaves exposed to low light as calculated for Fv/Fm or PSII yield, respectively (Table 3).

A less pronounced effect was observed for DBMIB-treated leaves, where this inhibitor at low light did not significantly influence Fv/Fm values.

However, PSII yield was reduced to 86% in the presence of DBMIB as compared to control leaves incubated in its absence. Under high light conditions, both fluorescence parameters decreased to 0%, regardless of the presence or the absence of DCMU or DBMIB. Two barley mutants, vir-zb63 (Simpson and Wettstein 1980; Hiller et al. 1980; Knoetzel et al.

1992; Skovgaard Nilsen et al. 1996) and vir-115 (Simpson et al. 1989; Gamble and Mullet 1989) with impaired assembly of PSI or PSII, respectively, were used in our studies. Mutant vir-zb63 has the PQ/PQH2 pool and cyt b6/f complex in reduced form while mutant vir-115 was characterized by oxidized electron transport chain components.

Dot blot analysis revealed (Figure 21A) that only small amounts of Elip9 (a member of low molecular mass Elips in barley) and Elip5 (a member of high molecular mass Elips in barley) transcripts were detected in WT or vir-115 mutant under low light conditions and the level of these transcripts accumulated in response to light stress. In contrast, strongly enhanced amounts of Elip9 and Elip5 transcripts were detected in vir-zb63 mutant under low light conditions and the amounts of these transcripts did not changed (for Elip9) or was induced eight-fold during exposure to high intensity light. No differences in the expression of Cab-2 and RbcS transcripts were detected between both mutants and WT. Accumulation of Elips in the thylakoid membrane was investigated by immunoblotting. Unfortunately, antibodies raised against the low molecular mass Elip6 (Pötter and Kloppstech 1993) could not distinguished between individual Elip species due to their high degree of identity at the amino acid level and identical molecular masses of 13.5 kDa (Grimm and Kloppstech 1987; Grimm et al. 1989; Pötter and Kloppstech 1993).

The data showed that low molecular mass Elips were not detected in thylakoid membranes under low light conditions but they accumulated in response to light stress (Figure 21B). However, approximately ten-fold higher amounts of these proteins (Figure 21C) were detected in vir-zb63 mutant as compared with vir-115 mutant or WT. Interestingly, the enhanced level of low molecular mass Elips

TABLE 3

Chl fluorescence parameters measured in leaves treated with DCMU and DBMIB and exposed to low or light light intensities.

Petioles of detached Arabidopsis leaves were immersed in water in the absence (-) or in the presence (+) of 10 µM DCMU or DBMIB and preincubated for 2 h at low light (10 µmol of photons m^-2s^-1) to allow the uptake of inhibitors.

The solutions were changed and leaves incubated further at low light (10 µmol of photons m^-2s^-1) or moved to high light (1.500 µmol of photons m^-2s^-1) for 3 h prior to measurements of Chl fluorescence parameters as described in Materials and Methods. Values shown for the maximal quantum yield (Fv/Fm) or effective quantum yield of PSII (PSII yield) are means ± S.D.

in vir-zb63 mutant was not observed, when leaves were exposed to a higher light intensity of 3.500 µmol of photons m^-2s^-1 (not shown). Comparable amounts of Cab-2, CP24 and PsbS were detected in mutants and WT under low and high light conditions (Figure 21B).

Figure 20: Regulation of Elip and Sep2 expression by the redox state of the PQ/PQH2 pool.

Detached Arabidopsis leaves were preincubated at low light (10 µmolof photons m^-2s^-1) for 2 h in the absence (-) or in the presence (+) of inhibitors of photosynthetic electron flow, DCMU or DBMIB (added at the final concentration of 10 µM), to allow the uptake of solutions. Then leaves were exposed either to low (Control, 10 µmolof photonsm^-2s^-1) or moved to high (light stress, 1.500 µmol of photons m^-2s^-1) light conditions for 3 h.

A: DCMU binds to the QB site on the D1 protein and inhibits electron flow from PSII to plastoquinone (PQ) keeping the PQ/PQH2 pool in the oxidized state. DBMIB inhibits electron flow from plastoquinol (PQH2) to cytochrome b6/f (cyt b6/f) keeping the PQ/PQH2 pool in the reduced state.

B: Images of chlorophyll fluorescence parameters, Fv/Fm (maximal quantum yield of PSII) and PSII yield (effective quantum yield of PSII), assayed for leaves treated with DCMU or DBMIB and exposed to low light or high light conditions.

C: The expression of Elip1, Elip2 and Sep2 mRNAs analyzed by dot blot hybridization. As a reference, the rRNA pattern in the gel, visualized by staining with ethidium bromide, is shown.

Signals obtained with 2.5 µg RNAs are shown in this panel.

D: The amount of Elips and Sep2 in thylakoid membranes assayed by immunoblotting. As a reference, the α and ß subunits of the CF1-ATP-synthase complex (CF1-α/ß), are shown.

Figure 21: Expression of Elips in viridis mutants of barley exposed to light stress.

Deatched leaves of vir-zb63 mutant deficient in PSII reaction center and with constantly oxidized PQ/PQH2 pool and vir-115 mutant deficient in PSI reaction center and with constantly reduced PQ/PQH2 pool were exposed to low (C, 100 µmol of photons m^-2s^-1) or high (HL, 2.000 µmol of photons m^-2s^-1) light conditions for 6 h. Green leaves of wild type (WT) seedlings were used as a control.

A: The expression of transcripts for low molecular mass Elip9, high molecular mass Elip5, the Chl a/b-binding protein (Cab-2) of PSII and the small subunit of ribulose-1,5-bisphosphate carboxylase (RbcS) assayed by dot blot hybridization.

B: The amount of low molecular mass Elips, Cab-2, Chl-binding protein of 24 kDa (CP24) and PsbS protein from PSII in thylakoid membranes assayed by immunoblotting.

C: Following dilutions of HL-treated samples were loaded for WT and vir-zb63 mutant and Elip amounts were assayed by immunoblotting: 10 µg (line 1), 5 µg (line 2) and 1 µg (line 3) protein.