©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Post-translational Modification of the Photosystem II Subunit CP29 Protects Maize from Cold Stress (*)

Elisabetta Bergantino (1), Paola Dainese (1), Zoran Cerovic (2), Salvatore Sechi (3), Roberto Bassi (4)(§)

From the (1) Dipartimento di Biologia, Universit di Padova, via Trieste 75, 35121 Padova, Italy, the (2) Laboratoire pour l'Utilization du Rayonnement Electromagnétique, Centre Universitaire Paris-Sud, 91405 Orsay Cedex, France, the (3) Laboratory of Experimental Carcinogenesis, NCI, National Institutes of Health, Bethesda, Maryland 20892, and the (4) Facolt di Scienze Matematiche, Fisiche e Naturali, Universit di Verona, Strada Le Grazie, 37134 Verona, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The resistance of maize plants to cold stress has been associated with the appearance of a new chlorophyll a/b binding protein in the thylakoid membrane following chilling treatment in the light. The cold-induced protein has been isolated, characterized by amino acid sequencing, and pulse labeled with radioactive precursors, showing that it is the product of post-translational modification by phosphorylation of the minor chlorophyll a/b protein CP29 rather than the product of a cold-regulated gene or an unprocessed CP29 precursor. We show here that the CP29 kinase activity displays unique characteristics differing from previously described thylakoid kinases and is regulated by the redox state of a quinonic site. Finally, we show that maize plants unable to perform phosphorylation have enhanced sensitivity to cold-induced photoinhibition.


INTRODUCTION

In the photosynthetic apparatus of higher plants, the efficiency of the electron transport and light-harvesting functions is regulated in response to environmental and metabolic conditions. The earliest described mechanism is the state I-state II transition and consists of the phosphorylation-induced redistribution of light-harvesting complex II (LHCII)()(1) and cytochrome b /f (2) complexes between grana- and stroma-exposed membranes, thus yielding a modulation of cyclic versus linear photophosphorylation (2) . A second mechanism has been described for the thermal dissipation of the excitation energy excess. This process is dependent on the deepoxidation of the xanthophyll violaxanthin to zeaxanthin (3) , which is mainly located in the minor photosystem II (PSII) subunits CP26 and CP24 (4, 5) .

Recently, a new regulatory process has been reported that is elicited by strongly photoinhibitory conditions such as those produced by illumination of maize plants at low temperature. Under these conditions, a new thylakoid protein appears in cold-resistant cultivars and is absent from cold-sensitive plants. While the protein has been isolated and characterized as a chlorophyll a/b protein located close to the PSII reaction center (RC), its origin is still unclear.() In particular, it is not known whether the cold-induced SDS-PAGE band is the product of a previously silent lhcb gene or a post-translational modification of an existing chlorophyll protein such as CP29, with which it shares epitopes (6, 7) and pigment composition.In this study, we have characterized the cold-induced protein by amino acid sequencing and pulse-chase with radioactive precursors, showing that it is the result of CP29 phosphorylation. Moreover, we report that this phosphorylation induces a change in the CP29 conformation and that the kinase activity is controlled by a quinonic site closely associated to photosystem II RC. Finally, we show that maize plants unable to perform CP29 phosphorylation are more sensitive to photoinhibition.

This is the first report of CP29 phosphorylation and suggests that the reversible modification of membrane-intrinsic, chlorophyll a/b binding proteins can be a mechanism for the modulation of light harvesting and excitation energy transfer to the reaction center.


EXPERIMENTAL PROCEDURES

Preparation of Thylakoid Membranes

Zea mays seedlings (cvs. Adon, DK300, Oh7N, and H93, supplied by Dekalb Co., IL) were grown for 2 weeks in a growth chamber at 28/21 °C day/night at a light intensity of 200 µE msand 80% humidity. Leaves from 2-week-old plants were harvested at the end of a 6-h illumination period at 25 or 4 °C, and thylakoids from mesophyll chloroplasts were prepared as previously described (8) . PSII membranes (BBY particles) were obtained according to the method of Berthold et al. (9) with the modification described in Ref. 10. Aliquots were suspended in 50 m M Hepes/KOH, pH 7.5, 5 m M MgCl, 50% glycerol and frozen at -80 °C until required. Membrane yield was determined by measuring chlorophyll content in 80% acetone, using the equations of Porra et al. (11) .

PAGE and Immunoblotting

For the purification of CP29 and CP34 apoproteins, two consecutive preparative SDS-PAGE were used to avoid minor contaminations. The first elecrophoresis was run on a 12-18% polyacrylamide gradient gel containing 6 M urea with the Tris/sulfate buffer system, as previously described (8) . Bands were excised and rerun on a second gel of uniform polyacrylamide concentration (10%) without urea in the Tris/tricine buffer (12) . Two-dimensional electrophoresis was performed using the non-denaturing Deriphat-PAGE (on a 5-12% polyacrylamide gradient gel) (13) for the first dimension and the SDS-urea-PAGE (9-16% gradient gel) according to Laemmli (14) , with double buffer concentration for the second dimension. Individual spots in the two-dimensional gels were identified by immunoblotting with specific antibodies as previously described (13) . One-dimensional electrophoresis was always performed by the Tris/sulfate, 6 M urea system mentioned above. For immunoblot assays, samples were separated by PAGE and electro-transferred to nitrocellulose filters (Hoefer). Filters were then probed with -CP29 antibodies, and antibody binding was detected by alkaline phosphatase-conjugated -rabbit IgG (Boehringer Mannheim). Antibodies were raised in rabbits, using poly(A)poly(U) as adjuvant, and characterized as previously described (15) .

Purification of Proteolytic Peptides and Amino Acid Sequencing

The samples were purified by SDS-PAGE as described above and electrotransferred to Immobilon-P membranes (Millipore) (17) . The proteins were then visualized by staining with 0.2% Ponceau S in 2% acetic acid. The bands of interest were excised and digested 24 h with trypsin in 0.1 M ammonium bicarbonate containing 5 m M calcium chloride and 8% acetonitrile. The resulting tryptic fragments were first purified by utilizing an ABI 130 HPLC and an aquapore RP-300 column equilibrated with 0.1 M potassium phosphate, pH 7.0, followed by a 17-min linear gradient from 0 to 40% acetonitrile. Individual peaks were then rechromatographed using the same column equilibrated with 0.1% (v/v) trifluoroacetic acid and a linear gradient from 0 to 40% acetonitrile in 0.07% (v/v) trifluoroacetic acid. Samples were sequenced with an Applied Biosystems model 475A sequencer equipped with an ``on line'' model 120A PTH amino acid analyzer.

In Vivo Labeling

Maize seedlings of 2-week-old plants were cut under water at the base of their epicotyl and quickly immersed into 0.5 ml of 40 m M Hepes/KOH (pH 7.5), in small glass vials (height, 40 mm; diameter, 5 mm). 300 µCi of [S]methionine or 200 µCi of [P]orthophosphoric acid were added to each vial, and seedlings were allowed to incorporate radioactivity for 3 h at 25 °C in the light, with the evaporative demand increased by cool air from a hair drier. When solutions became low during this period, 40 m M Hepes/KOH was added to each vial. Following incorporation, seedlings were kept in the light for 6 h at either 4 or 25 °C. Leaves, apart from the first one, were then cut, and thylakoids were immediately prepared as described (8) . Densitometry of the relative Coomassie-stained gels (treated with ENHANCEautoradiography enhancer, DuPont NEN) and autoradiographs (Hyperfilm-Amersham films) were performed with a Bio-Rad GS670 imaging densitometer according to the company's protocol.

Limited Proteolysis of Thylakoid Membranes

Thylakoid membranes (25 µg of chlorophyll) were washed once in 5 m M EDTA, 10 m M Hepes/KOH, pH 7.5, and resuspended in 0.1% DM, 10 m M Hepes/KOH, pH 7.5, at a chlorophyll concentration of 0.5 mg/ml. Proteolytic digestion was carried out at 37 or 25 °C for 30 min by addition of chymotrypsin to a final concentration of 100 µg/ml. The reaction was stopped by adding 1 volume of solubilization buffer (containing 6 M urea, 5% SDS, 1% 2-mercaptoethanol, and 20% glycerol), and the fragments were loaded on a 12% acrylamide gel using the Tris/tricine system.

In Vitro Dephosphorylation

Membranes, in aliquots of 5 µg of chlorophyll, were suspended in 50 m M Tris/HCl (pH 7.0 or 8.5) and incubated with 0.5 units of alkaline phosphatase (type VII-SA, Sigma) at 30 °C. Where indicated, membranes were previously washed twice in 50 m M Tricine, pH 7.8, 5 m M EDTA, and/or MgClwas added to the sample to a final concentration of 5 m M. After incubation, the reactions were stopped by addition of the solubilization buffer; samples were boiled for 2 min and loaded onto the gel.

Fluorescence Analysis

The cultivars Oh7N and H93 were grown exactly in the same conditions described for the preparation of thylakoid membranes, in a Haereus growth cabinet at a light intensity of 300 µE ms. Changes in chlorophyll fluorescence yield were measured using a commercial pulsed fluorimeter (PAM 2000, Walz, Effeltrich, Germany) with the light guide that delivers the measuring and saturating light held at 5 mm from the upper side of the third maize leaf. The leaf was fixed on a copper support while still in the pot in a cold room. For the photoinhibitory treatment at 4 °C, strong white light (1000 µE msat the surface of the leaf) was delivered by a cold light illuminator (KL1500 electronic, Schott, France). The temperature of the leaf (measured by a thermocouple) could be rapidly changed from 4 to 25 °C. The minimum ( F) and maximum ( F) fluorescence yield in the dark, and the ratio ( F- F)/ F= F/ F, which reflects the potential yield of the photochemical reaction of photosystem II (18) was recorded at 25 °C before each chilling period. The standard protocol consisted of four periods of 2 h of light at 4 °C, separated by 15 min of dark at 25 °C. During this dark period, that part of non-photochemical quenching not related to photoinhibition could relax (19) . After the last photoinhibitory treatment, the plants were allowed to recover at 25 °C for 15 h, and the fluorescence parameters were measured again (24 h after the first measurements).


RESULTS

Characterization of the CP34 Protein

When maize leaves are chilled in the light, a new SDS-PAGE band appears at a position corresponding to 34 kDa. The process is completely reversible upon recovery at room temperature for 1 h (Fig. 1). This protein is related to the chlorophyll a/b binding protein CP29, as recognized by immunological cross-reaction (6, 7) and spectroscopic analysis of the isolated native protein. This band was called CP34 on the basis of its apparent molecular weight.The relationship between the CP29 and CP34 proteins is unclear. To assess the origin of this cold-induced thylakoid protein, we first determined the primary sequence of both CP29 and CP34.

The 34-kDa polypeptide was purified from thylakoid membranes of cold-treated maize leaves (6 h at 4 °C in the presence of high light). Since several thylakoid polypeptides have molecular masses in the 28-35-kDa range, we isolated the protein by two consecutive preparative SDS-PAGE systems to avoid the presence of minor contaminations. The same procedure was applied to CP29.

The polypeptides were then blotted onto polyvinylidene difluoride filters and submitted to automated Edman degradation. As they were N-terminally blocked, peptide fragments were generated by limited proteolysis using trypsin. Following digestion, two of the resulting peptides were purified by HPLC and sequenced in the case of CP34, and three peptides were purified and sequenced in the case of CP29. The results of this analysis are shown in Fig. 2. Fragments I and II of both CP29 and CP34 gave sequences of 32 and 20 amino acids and were identical in the two apoproteins. They matched almost exactly with segments of the protein sequence deduced from the lhcb4 cDNA clone of barley encoding the CAB protein CP29 (20) . The two stretches of residues span, respectively, amino acids 102-131 and 212-231 of the barley apoprotein precursor. Only few differences could be observed for fragments I and II, as shown in Fig. 2. Fragment III, sequenced only for CP29, differed more from the corresponding segment of its barley counterpart, especially at its N-terminal side (see Fig. 2).


Figure 2: N-terminal sequences of tryptic fragments from digestions of CP29 and CP34 apoproteins. The alignment was obtained with the FASTA program. Vertical bars stress amino acid identities. Amino acid positions on the primary sequence of the barley protein (20) are indicated. X, undetermined amino acid.



These results show that the two SDS-PAGE bands recognized by the -CP29 antibodies in cold-treated maize thylakoids are the products of genes very similar if not identical to lhcb4. As far as the sequence analysis, CP29 and CP34 are identical. In Vivo Labeling of Thylakoid Proteins with [S]Methionine-In the light of the above results, we could then address questions on the biogenesis of CP34, i.e. whether it was ( a) the product of an alternative splicing of the lhcb4 gene transcript, ( b) the unprocessed precursor of CP29, or ( c) a post-translationally modified CP29. The first two hypotheses imply that the cold-induced protein is synthesized de novo following cold stress, while a post-translational modification would act on a protein already existing in the thylakoids.

To test these possibilities, we examined the patterns of thylakoid proteins synthesized at 25 and 4 °C in the presence of light. We labeled plastid proteins in vivo by feeding [S]methionine to seedlings. Maize seedlings, loaded with [S]methionine for 3 h at 25 °C, were then incubated for 6 h at either 25 or 4 °C under 200 µE msillumination. Following this treatment, thylakoids were isolated from mesophyll chloroplasts, and their polypeptide content was analyzed by two-dimensional gel electrophoresis and autoradiography. Membranes were first fractionated on non-denaturing Deriphat-PAGE (13, 21) and then by SDS-urea-PAGE in the second dimension. This combined electrophoretic system allowed a clear-cut separation of CP29 from the D1 apoprotein, a PSII core component comigrating with CP29, which is known to have a very fast turnover, and was therefore expected to be heavily labeled (22, 23) . As for their amino acid content, CP29 and D1 proteins, respectively, contain 3 and 12 methionines in their primary sequences in barley (20, 24, 25) .

Fig. 3, a and b, show the results of this analysis. In particular, it was clear that the chilling treatment did not cause de novo synthesis of the CP34 protein, since the labeling of this polypeptide was as low as that of CP29 on a protein quantity basis, as determined by densitometry of gels and relative autoradiographs. The low [S]methionine incorporation in CP34 following induction by cold shows that this polypeptide is neither the precursor of CP29 nor the product of an alternative splicing of the lhcb4 maize messenger, since both cases imply de novo synthesis and consequent incorporation of radioactivity.

The autoradiographic pattern was very similar in chilled and control samples. However, incorporation in all of the polypeptides was lower in the cold-exposed plants as a result of the effect of low temperature on protein synthesis (26, 27) . The D1 protein is an exception to this rule; its labeling was higher in the cold-treated membranes, consistent with the photoinhibitory conditions applied (28, 29) .

In Vivo Labeling of Thylakoid Proteins with H333PO

We then checked the remaining hypothesis, aiming at identifying the specific post-translational modification of CP29. It has been already reported that the cold-induced polypeptide is not glycosylated (7) . Moreover, the putative modification appeared to be rapidly inducible and reversible. This was shown by the rate of appearance of the 34-kDa band on exposing leaves to chilling and high light,as well as the rate of degradation of the polypeptide on returning leaves to 25 °C (7) (Fig. 1). These kinetics are consistent with a phosphorylation-dephosphorylation mechanism that could also explain the electrophoretic upshift, a behavior common to other phosphoproteins (30) . In vitro phosphorylation of the purified, non-denatured form of the CP29 protein by various kinases was not successful (not shown). We then proceeded to in vivo labeling of maize leaves with H333PO. The radioactive phosphate was fed to excised seedlings as described for [S]methionine. Seedlings were then exposed to chilling treatment in the light, and thylakoid membranes were analyzed by two-dimensional PAGE and autoradiography. The results are shown in Fig. 3 c; CP34, but not CP29, was heavily labeled. The two-dimensional analysis allowed a sharp distinction between CP34 and the phosphorylated form of the D2 apoprotein, which comigrate in one-dimensional PAGE.


Figure 1: Polypeptide profile and immunoblot of thylakoid membranes from control ( C), cold-treated ( T), and recovered at room temperature for 1 h ( RT) maize leaves. Coomassie-stained SDS-PAGE ( A) and immunoblot assayed with -CP29 polyclonal antibodies ( B) are shown; the chlorophyll a/b binding protein CP29 and the cold-induced 34-kDa band are indicated, together with some other major bands.



In the autoradiograph, all of the known major thylakoid phosphoproteins (31, 32) could be identified: the light-harvesting chlorophyll a/b proteins (three between 28 and 30 kDa) (8) and the four proteins of the PSII core CP43 (43 kDa), D2 (34 kDa), D1 (32 kDa), and the 9-kDa product of the psbH gene (not visible in the figure but evident at the lower edge of the gel in the original film). Apart from CP34, the autoradiographic pattern of control and cold-treated samples also differed with respect to the relative intensity of the labeling of the other subunits; LHCII apoproteins were more heavily phosphorylated in the cold-treated samples by a factor of 2.5, as determined by densitometry of the film. Moreover, labeling of the PSII core subunits CP43, D1, D2, and 9 kDa was barely detectable in the control sample, while it was very strong in the cold-treated one. It is worth noting that only the PSII core subunits deriving from the monomeric form of the complex are phosphorylated, while those associated to the dimeric form are not.

When we performed the above experiment with maize lines, which produce very little or any CP34 upon cold stress in the light, essentially no differences were observed in the phosphorylation pattern apart from the lower labeling of the CP34 band (not shown).

Limited Proteolysis of CP29 and CP34 in Destacked Thylakoids

Which is the effect of the phosphorylation on CP29? Protein phosphorylation has been shown to be one of the most general mechanisms in the regulation of protein function through changes in their conformational state (30) . To verify if this is also the case for CP29, we analyzed the two proteins by limited proteolysis at 37 and 25 °C. Control and cold-treated membranes were partially solubilized with 0.1% DM and digested with chymotrypsin. The results of the proteolytic treatment (identical at the two temperatures) were analyzed by SDS-PAGE and immunoblotting (Fig. 4). It was shown that the digestion produced different patterns in the two samples; the smaller fragment detected (about 16 kDa) was more abundant in the control membranes, while an additional 23-kDa band appeared in the cold-treated sample containing CP34. This may indicate that at least one proteolytic site was protected in the phosphorylated form of the protein due to a conformational change induced by the phosphorylation. Similar results, in fact, were obtained with other endoproteinases cutting at different sites (not shown), ruling out the possibility that the added phosphate group could sterically constrain proteolysis at a nearby target site.

In Vitro Dephosphorylation of CP34

As shown above, the phosphorylation of CP29 is accompanied by a change of its mobility in SDS-urea-PAGE possibly due to changes in the secondary structure of the polypeptide in the gel, owing to the addition of the highly charged phosphate group. It can be asked whether the change of electrophoretic mobility is exclusively due to phosphorylation or if other modifications such as aggregation with surrounding small peptides are involved, similar to the case of D1 and the cytochrome b-subunits (33) .

To clarify this point, we treated thylakoid membranes containing CP34 with phosphatase enzymes in vitro. Both acidic and alkaline phosphatases were used, and the former was less active than the latter at neutral pH (not shown). When treated with alkaline phosphatase at pH 7.00, the CP34 band, detected by immunoblotting, disappears, indicating that the change in mobility is only due to phosphorylation (Fig. 5 A). Surprisingly, the dephosphorylation was shown to be slower at higher pH, 8.5 versus 7.0, closer to the alkaline phosphatase optimum. Moreover, the addition of the mild detergent DM increases the dephosphorylation rate at both pH values, thus suggesting that external (stromal) pH changes may induce differences in the accessibility of the phosphorylation site. We designed an experiment to ascertain if the domain of the antenna complex involved in phosphorylation-dephosphorylation events was exposed on the stromal or lumenal site of the membrane. Thylakoids and PSII membranes (BBY particles), exposing, respectively, the stromal and the lumenal domains of the polypeptide to the solvent, were used. When thylakoids were treated with alkaline phosphatase, dephosphorylation was more effective if membranes were previously destacked by EDTA washing to increase the accessibility to the stromal surface of grana membranes. Dephosphorylation was more efficient when alkaline phosphatase was added before restacking with Mg. Accordingly, the treatment of BBY particles was ineffective. These results, shown in Fig. 5B, indicate that the phosphorylation site is most probably exposed on the stromal side of the membrane in the stacked domain.


Figure 5:In vitro dephosphorylation of CP34. Immunoblot analysis with -CP29 polyclonals of thylakoid membrane samples from cold-treated leaves, after in vitro dephosphorylation treatment is shown. A, membranes were suspended in Tris buffer at the indicated pH values and incubated with alkaline phosphatase at 30 °C for different times, in the absence or presence ( +DM) of 0.1% dodecyl maltoside. B, EDTA-washed thylakoids or PSII membranes were incubated for 20 min with Mg, alkaline phosphatase ( AP), or both (in these cases, the first component was added, the sample was kept for 5 min on ice, and the incubation was started as the second component was mixed). Control membranes, not treated and kept for 20 min at 30 °C, are indicated by C.



Identification of the Site Controlling CP29 Phosphorylation

Previous work on thylakoid kinases showed that the activity of the LHCII-specific enzyme is controlled by the redox state of the plastoquinone pool, thus acting in the regulation of the electron transport between PSII and PSI (1) . Activation of the kinase appears to be dependent on the formation of a complex with the cytochrome b /f (34, 35) . We tested the effect of blocking the photosynthetic electron transport chain at different sites. This was performed by using a mutated line and treatments with chemical inhibitors.

In a first approach, we blocked electron transport by depleting CO, the terminal electron acceptor of photosynthesis; seedlings were illuminated in Natmosphere for 10 min at room temperature. Thylakoids were then isolated and analyzed by SDS-PAGE and immunoblotted with -CP29 polyclonals, showing that the phosphorylated form of CP29 was induced in those conditions even without cold treatment (Fig. 6, panel A, lane c). This suggests that phosphorylation is activated by the reduction of a component of the electron transport chain. Further insights were obtained by comparing the wild type with hcf2 mutant seedlings lacking cytochrome b /f (36) . In this mutant, CP34 was present following illumination, even at room temperature ( lanes f and g). 20 µ M DCMU was used to block electron transport between PSII reaction center and plastoquinone. This treatment completely prevented CP29 phosphorylation in both wild type and hcf2 mutant whether at 4 °C or room temperature ( lanes d, h, and i). These results suggest that the CP29 phosphorylation is controlled by the redox state of plastoquinone.

Fluorescence Analysis

Which is the role of cold-induced CP29 phosphorylation? A possible function would be protection against photoinhibition. We compared the sensitivity to photoinhibition of maize lines differing for their ability to phosphorylate CP29 following cold stress in the light. Several available lines were screened by exposing seedlings to the photoinhibitory treatment, followed by SDS-PAGE and immunoblot analysis (not shown) or fluorescence analysis. For example, the two lines H93 and Oh7N differed substantially in the level of CP34 after the cold treatment. In H93, CP34 represented 9% of the sum (CP29 + CP34), while in Oh7N CP34 represented 33%. As shown in Fig. 7, under high light (1000 µE s) at 4 °C, the F/ Ffluorescence ratio decreased rapidly in the line H93 (from 0.75 to 0.48 in 6 h), mainly due to a rapid decrease of F. The decrease in F/ Fwas much smaller in Oh7N, from 0.79 to only 0.68, even after 8 h of treatment. This shows that the photochemistry of PSII in the line Oh7N was much better protected under stress conditions than in the line H93. The recovery of the F/ Fratio after 15 h at room temperature confirms that the PSII reaction centers were more irreversibly photoinhibited at 4 °C in H93 than Oh7N. Moreover, the resistant line Oh7N has a capacity to recover from the stress, which is almost absent in H93. This overall sensitivity of the photochemistry to cold stress was observed in several lines and was correlated with their ability to phosphorylate CP29, suggesting that this modification of the protein could be involved in a photoprotective mechanism. Although fluorescence results must be cautiously compared between different leaves, we are confident that the differences in fluorescence induction values of the two maize lines are significant. In fact, the same figure was obtained for 14-, 18-, and 21-day-old plants while, for each age, chlorophyll content and deepoxidation state of xanthophyll at the end of the treatment showed very small differences, if any (within 3 and 2%, respectively).


DISCUSSION

The aim of this work was to characterize the nature of the 34-kDa (CP34) SDS-PAGE band, which appears in maize thylakoid membranes following exposure to cold in the presence of light. This cold-induced polypeptide had been shown to be immunologically related to the chlorophyll a/b binding protein CP29, one of the minor antenna complexes of PSII (7) . Moreover, Mauro et al.isolated the two com-plexes in their native forms and showed that the spectral properties and the pigment composition were very similar. We further characterized CP29 and CP34 by amino acid sequencing, showing that the two polypeptides, homologous to the barley CP29, were identical over the 32- and 20-amino acid-long stretches determined. This strongly suggested that CP34 was not the product of a different gene but rather of an alternative splicing of the lhcb4 messenger, the pre-cursor of CP29, or a post-translational modification of it.

The S labeling experiment discriminated between these hypotheses since the synthesis of CP34 was not enhanced, as would be the case if it was either the product of alternative splicing or the precursor complex. We thus concluded that CP34 is a post-translationally modified form of CP29 and proceeded to the identification of the nature of the modification. This was made difficult by the fact that only in vivo treatment is effective in inducing CP34 accumulation while treatment of isolated thylakoids is not. As suggested by the rapid formation and degradation of the protein, we succeeded in labeling CP34 by feeding H333PO to the plant prior to treatment in the light. Not only was CP34, in contrast to CP29, heavily labeled, but the treatment of the membranes with alkaline phosphatase restored CP29 mobility, thus confirming that the post-translational modification of CP29 was a phosphorylation.

Alkaline phosphatase treatment of thylakoids and PSII membranes, which, respectively, expose stromal and lumenal faces, showed that the phosphorylation site is located on the stromal side of the membrane. This post-translational modification is effective in modifying the protein structure as judged by the limited proteolysis experiment (Fig. 3) and by the change in SDS-PAGE mobility, similar to that described for other phosphoproteins (30) . We concluded that the SDS-PAGE band induced by the light treatment in the cold is the phosphorylation product of the chlorophyll a/b protein CP29.

Regulation of function of thylakoid proteins by phosphorylation is a well known mechanism that drives lateral migration of LHCII antenna complexes in state I-state II transitions. LHCII kinase has been shown to be activated by plastoquinol and inactivated by plastoquinone; the role of plastoquinol has been documented in the phosphorylation of all other thylakoid proteins, but authors are far from an agreement on the question of how many kinases exist in thylakoids (for a review, see Refs. 31 and 32). The redox sensitivity of the LHCII kinase is provided in vivo (37, 38, 39) , as well as in vitro (34) , by its association with the cytochrome b /f complex.

We tested if the same kinase was also responsible for the phosphorylation of CP29, another member of the CAB family. In addition to a wild type line, we analyzed the maize mutant hcf2, which is known to lack the activity of the LHCII kinase as a consequence of the absence of the cytochrome b /f complex, while the phosphorylation of the four PSII core proteins is largely unaffected (37, 39) . We noticed that the phospho-CP29 (CP34) was constitutively present in the thylakoid membranes of this mutant. A pretreatment with DCMU, which prevents all known thylakoid protein phosphorylation by blocking the reduction of plastoquinone (40) , totally abolished phosphorylation of CP29 in both wild type and hcf2 lines even when chilling treatment was applied. The sensitivity of the CP29 kinase to inhibition by DCMU points to a redox control at the level of plastoquinone, with plastoquinol being the activator of the enzyme. However, in contrast to the LHCII kinase, referred to as the 64-kDa kinase, and likewise the putative RC kinase of PSII core proteins, the activation of the CP29 kinase does not require an active cytochrome b /f complex.

On the other hand, PSII core proteins are phosphorylated in isolated thylakoids while CP29 is not (41) , thus suggesting that the complexes are not substrates of the same kinase. Finally, preliminary results of experiments with DBMIB revealed that this halogenated quinone analogue clearly enhances phosphorylation of CP29 while it inhibits both LHCII and PSII polypeptide phosphorylation (31, 32, 37, 38, 42) . DBMIB induces the CP29 kinase activity in the concentration range from 1 to 100 µ M, even in dim light without cold treatment. Taken altogether, our results bring new evidences for the existence of at least three thylakoid protein kinases.

Where is the CP29 kinase located? It has been reported that the 64-kDa LHCII kinase is localized in grana domains, particularly at the edges of the grana stacks, where it is associated to the cytochrome b /f complex, which modulates its activity (34) . In the mutant lacking this complex, we noticed that the CP29 kinase is constitutively activated, but it does retain its redox sensitivity since DCMU, i.e. oxidation of plastoquinol to plastoquinone, turns it off.

The redox sensitivity, the inactivation by DCMU, and the activation by DBMIB indicate that the CP29 kinase must be associated with at least one quinonic site. On one hand, results obtained with DCMU both in wild type and hcf2 clearly show that the kinase senses the electronic state of the Qsite of PSII. On the other hand, the activation by DBMIB suggests a negative control played by the Qsite of the cytochrome b /f, to which DBMIB binds in wild type (43) , displacing the quinone on the cytochrome complex and thus mimicking the hcf2 condition. We propose as a working hypothesis that the CP29 protein kinase is located in grana partitions, in between PSII (where it is in contact with the Qsite) and the cytochrome b /f complex (in contact with the Qsite). This would also mean an intermediate position between the RC kinase and the LHCII kinase, respectively. If we consider the models so far proposed for the topological organization of photosystem II (21, 44) , our hypothesis is consistent with the position of CP29 between the RC and LHCII, which are substrates of phosphorylation by the different kinase (Fig. 8). An integrated scheme for the redox control of thylakoid protein phosphorylation cannot be drawn as yet and is beyond the aim of this work.

Concomitant with the phosphorylation of CP29, cold treatment induces phosphorylation of the RC subunits CP43, D1, and D2 and 9 kDa in the monomeric form of PSII. Since this corresponds to a dissociation of RC dimers into monomers, it can be hypothesized that this phosphorylation event might be the cause for dissociation of PSII dimers. Phosphorylation-induced dissociation of neighboring chlorophyll-proteins has been previously described (45) ; similarly, both PSII RC subunits and CP29 phosphorylation can be related to a regulatory mechanism based on the dissociation of the PSII supermolecular complex. These events could be part of at least two regulation phenomena, the first being the dissociation of antenna complexes from PSII and their migration into the stroma membranes (1, 45, 46) , with consequent reduction of PSII antenna size, and the second being PSII cycling whose primary step could well be the RC dimer dissociation (47) .

Free lateral migration of LHCII and PSII RC subpopulations from the grana stacks and their margins to stroma-exposed thylakoid membrane is an obvious requirement for such mechanisms. Thylakoid membrane fluidity is strongly decreased at temperatures lower than 10 °C (48) ; we can therefore argue that, under conditions chosen for our experiments (4 °C), the phosphorylated RC subunits of monomeric PSII accumulate since subsequent steps in their cycling are impaired.

Which is the role of CP29 phosphorylation? We approached this question by using two maize lines phosphorylating CP29 to a different extent and evaluating their resistance to photoinhibition following treatment in cold and light. The results showed that the line more active in CP29 phosphorylation is also more resistant to photoinhibition. In fact, although CP29 is not the only protein that is phosphorylated in the cold, PSII core complex subunits and LHCII polypeptides are phosphorylated both in resistant and sensitive lines. This is in agreement with previous data obtained by the analysis of two lines sharing the same genetic background but differing in the level of CP34 induction.In that study, it was also shown that the protective effect was located in the antenna system rather than in the RC complex and consisted in a decreased efficiency in the energy transfer from the antennas to the RC. CP29 phosphorylation isolates an important portion (30%) of PSII antennas, which are connected to the RC through CP29 (32, 45) , and may influence chlorophyll and/or carotenoid organization within the complex, thus leading to a decreased energy transfer to PSII RC. While the disconnection of PSII chlorophyll proteins seems to be a general consequence of photoinibitory conditions (47) irrespective of CP29 phosphorylation, spectral differences were actually detected between CP29 and CP34,with a decrease in the absorption of the Chla spectral forms, which are more effective in the energy transfer to PSII RC. This hypothesis is consistent with the observed increase in Fduring the treatment in the cold (18) .

We therefore propose that CP29 phosphorylation is a novel photoprotection mechanism, based on the reorganization of pigments in the chlorophyll-protein complex, in such a way that a lower amount of excitation energy is funneled to RCII. Further experiments are in progress to obtain a better characterization of the spectral and structural changes induced by CP29 phosphorylation.


FOOTNOTES

*
This research was supported by the Italian Ministry of Agriculture and Forestry Grant 4.7240.90. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 39-45-8098916; Fax: 39-45-8098929.

The abbreviations used are: LHCII, light-harvesting complex II; PSII, photosystem II; PSI, photosystem I; RC, reaction center; DM, dodecyl maltoside; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone; HPLC, highpressure liquid chromatography; µE, microeinstein; PAGE, polyacrylamide gel electrophoresis.

Mauro, S., Dainese, P., and Bassi, R., submitted for publication.


ACKNOWLEDGEMENTS

We are grateful to Dr. David Simpson (Carlsberg Laboratory, Copenhagen, Denmark) and to Dr. Francis-Andre Wollman (Institut de Biologie Physico-chimique, Paris, France) for critically reading the manuscript. We thank Dr. Maria Ruzzene for testing CP29 phosphorylation in vitro by exogenous kinases and Prof. Arianna Donella Deana for helpful suggestions on the use of phosphatase enzymes (Dipartimento di Chimica Biologica, Padova, Italy). We also thank Dr. D. Miles (Dept. of Biology, University of Missouri, Tucker, Columbia) for the kind gift of the hcf2 maize mutant.


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