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Role of Chloroplast Protein Kinase Stt7 in LHCII Phosphorylation and State Transition in Chlamydomonas

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Science  07 Mar 2003:
Vol. 299, Issue 5612, pp. 1572-1575
DOI: 10.1126/science.1081397

Abstract

Photosynthetic organisms adapt to changes in light quality by redistributing light excitation energy between two photosystems through state transition. This reorganization of antenna systems leads to an enhanced photosynthetic yield. Using a genetic approach inChlamydomonas reinhardtii to dissect the signal transduction pathway of state transition, we identified a chloroplast thylakoid–associated serine-threonine protein kinase, Stt7, that has homologs in land plants. Stt7 is required for the phosphorylation of the major light-harvesting protein (LHCII) and for state transition.

Oxygenic photosynthetic organisms have the ability to adapt to changes in light quality and quantity. They balance energy input and consumption in the short term through dissipation of excess energy and regulate energy flow between the two photosystems through state transition. This reversible redistribution leads to an overall increase in photosynthetic quantum yield. State transition has been correlated with the reversible phosphorylation of the polypeptides of the light-harvesting complex II (LHCII) antenna complex (1). We used mutants of Chlamydomonas reinhardtii that were deficient in state transition to identify components of the signal transduction chain involved in this process. We found that two of these mutants are affected in the same nuclear gene encoding a chloroplast Ser-Thr kinase that is implicated in LHCII phosphorylation.

The kinetics of state transition match the pattern of LHCII phosphorylation-dephosphorylation: LHCII is phosphorylated under state II and dephosphorylated under state I conditions (1, 2). Numerous thylakoid proteins undergo phosphorylation-dephosphorylation cycles during state transition, although their functions are not clear (1). The activation of the thylakoid-bound kinase responsible for phosphorylation of light-harvesting chlorophyll-binding protein (LHCP) under state II depends on the redox state of the plastoquinone pool (1), which is part of the photosynthetic electron transport chain and located between photosystem II (PSII) and photosystem I (PSI). Activation of the LHCII kinase is regulated specifically by binding of plastoquinol in the QO pocket of the cytochrome b6f complex (3).

In plants induced to undergo state transition, a portion of LHCII becomes associated with the stromal arrays that are enriched in PSI (1). During transition to state II, the mobile part of LHCII becomes connected to PSI in a cytochrome b6f-controlled process (4). This connection requires the PsaH subunit (5). The lateral displacement of LHCII from the PSII-rich grana to the PSI-rich lamellar thylakoid regions results in transfer to PSI of about 80% of the excitation energy absorbed by LHCII in C. reinhardtii(4), a considerably higher amount than in land plants, in which only 15 to 20% of LHCII is mobile (1). The transition from state I to state II induces a switch from linear to cyclic electron flow in C. reinhardtii and reveals a correlation between the redistribution of antenna complexes during state transition and the onset of cyclic electron flow (6,7). C. reinhardtii is advantageous for analyzing state transition because its transition from state I to state II is accompanied by a large fluorescence decrease (8). Screens using fluorescence videoimaging have identified several mutants of C. reinhardtii that are unable to perform state transition, in particular stt7-1 (9,10).

Another allelic mutant, stt7-2, was isolated during this study (11). The fluorescence levels in the stt7-1and stt7-2 mutants remained unchanged during state transition (Fig. 1A). To test whether phosphorylation of LHCP is affected during a state I–to–state II transition, cells from both mutants and wild-typeC. reinhardtii were labeled with 33P-phosphate under state I and state II conditions (Fig. 1B). Whereas a marked increase in phosphorylation in state II was observed for the major LHCII polypeptide P11 and for the minor chlorophyll protein complex CP26 in wild-type cells, such an increase did not occur or was much reduced in the mutant cells (Fig. 1B). The phosphorylation level as measured by immunoblotting with an antiserum to phosphothreonine was reduced in stt7-1, more so than in stt7-2 (Fig. 1D). The residual level of phosphorylation of P11 in state II in stt7-1 was comparable to that observed in cytochrome b6f–deficient mutants that are blocked in state I (4, 9).

Figure 1

State transition is restored in thestt7-1 and stt7-2 mutants rescued with theStt7 gene. (A) Cells from wild-type,stt7-1, stt7-2, and the rescued mutant strains (stt7-1R and stt7-2R) in state I were transferred to the dark in a closed vessel of a pulse-amplitude-modulation fluorimeter (Hansatech). Under these conditions, respiration in the absence of photosynthesis leads to anaerobiosis. The increase of F0 (the fluorescence yield when the electron acceptors of PSII are fully oxidized, lower trace) indicates the reduction of the plastoquinone pool. The decrease of maximal fluorescence (measured by 0.7-s saturating light pulses given every 2 min and shown as spikes) indicates the occurrence of state II in wild-type and rescued strains, but not in the mutants that are blocked in state I. a.u., arbitrary units (B) Phosphorylation during state transition in wild-type, stt7-1 and stt7-2 mutant, andStt7-1R rescued strains. Cells were labeled with33P-phosphate under state I or state II conditions as described (9, 11). Thylakoid polypeptides were fractionated on a 6 M urea SDS-polyacrylamide gel (12 to 18%) and autoradiographed. (C) Coomassie blue staining corresponding to (B). (D) Thylakoid membranes were isolated from cells in state I and state II and the proteins were separated as in (B), transferred to nitrocellulose filters, and reacted with an antiserum to phosphothreonine. (E) Immunoblot of (D) with antiserum to the D2 protein.

To isolate the Stt7 gene, we constructed an indexed cosmid library with the Arg7 gene included in the cosmid vector (12, 13). Pools of genomic cosmid clones from this library were used to transform thestt7-1/arg7 mutant by selection for arginine prototrophy. Transformants were then screened with a fluorescence-videoimaging system (9) for their ability to restore state transition. One pool yielded one transformant with a wild-type state transition phenotype. From this pool, one cosmid was isolated that complemented the stt7 mutant allele, as determined by fluorescence and phosphorylation measurements taken in state I and state II (Fig. 1). A subclone of 10 kb of this cosmid rescued the stt7 mutant and was used as a probe to isolate a cDNA from a library of C. reinhardtii. The cDNA, called Stt7 cDNA, was fused to the PsaD promoter and used to transform stt7 cells. State transition was partially restored in the transformed strains, presumably because the cDNA was not properly expressed (14). The predicted Stt7 protein consists of 754 amino acids with a catalytic domain typical of Ser-Thr kinases with all of its hallmarks (Fig. 2). The protein is predicted to be localized in the chloroplast by the ChloroP (15) and Predotar (16) programs. It contains a putative 41–amino acid transit peptide at its NH2-terminal end and one potential transmembrane domain upstream of the catalytic kinase domain.

Figure 2

Sequence comparison of the Stt7 kinase and its homologs in Chlamydomonas and Arabidopsis. The protein sequences were aligned with Clustal W (22) and Boxshade. Stt7 (AY220122) and Stl1 (AY220123) are from C. reinhardtii; Stt7-AT1 and Stt7-AT2 correspond to At1g68830 and At5g01920 from A. thaliana. The arrow indicates the predicted cleavage site of the chloroplast targeting domain. The hydrophobic region is marked by a striped box above the amino acid sequence. The kinase catalytic domain is indicated by an open box above the amino acid sequence. The asterisks indicate the Cys residues that are potential targets for thioredoxin. The insertion site instt7-2 is marked by a wedge.

To test whether Stt7 was still expressed instt7-1, we used reverse transcription–polymerase chain reaction (RT-PCR) with primers specific for Stt7. A fragment with the expected size of 480 base pairs (bp) could be amplified from wild-type RNA, but not from stt7-1 RNA (Fig. 3). As a positive control, RT-PCR was also performed with primers specific for a G protein. The same primers were used for PCR on total DNA. A fragment of 480 bp was amplified from wild-type but notstt7-1 DNA. Thus, a DNA rearrangement occurred at theStt7 locus in the stt7-1 mutant. This rearrangement was confirmed by DNA blot analysis (11) (fig. S1).

Figure 3

(Left) RT-PCR analysis of the transcripts ofStt7 from the wild type and the mutants stt7-1and stt7-2. Total RNA from the wild type and mutants was subjected to RT-PCR with primers specific for Stt7 and for a cytosolic G protein gene as a positive control (23). (Right) The same primers were used for PCR on wild-type andstt7-1 genomic DNA. The products were (A) fractionated by agarose gel electrophoresis, stained with ethidium bromide, and detected by fluorescence, then (B) blotted and hybridized with a probe specific for Stt7 to verify that the amplified fragment originated from Stt7 DNA. m1, m2, and w refer to the stt7-1, stt7-2, and wild type, respectively; L, size markers. The RT-PCR in lane m2 was performed in a separate experiment and gave rise to a higher background.

A cosmid was isolated that was able to rescue thestt7-2 mutant by transformation (Fig. 1) (11). Analysis of the Stt7-1 and Stt7-2 mutant loci revealed that the two mutants were allelic and that the insertion was in the 3′ part of the Stt7 gene close to the COOH-terminal end of the kinase catalytic domain in stt7-2 (Fig. 2). A transcript corresponding to the 5′ part of Stt7 that includes most of the kinase domain was detectable by RT-PCR in this mutant (Fig. 3). The relatively intact kinase domain ofstt7-2 may be the cause of the residual phosphorylation shown in Fig. 1D.

The presence of an NH2-terminal transit peptide suggested that the Stt7 kinase was localized within the chloroplast. To verify its localization, we inserted six copies of the hemagglutinin (HA) epitope in frame upstream of the termination codon of the genomicStt7 gene. This construct was introduced into a plasmid containing the Arg7 gene and was used for transformingstt7-1/arg7 cells and selecting for arginine prototrophy. Transformants capable of state transition were isolated. Whole-cell extracts were separated into total, soluble, and membrane fractions; thylakoids; and chloroplast isolated, soluble, and membrane fractions. Each fraction was examined by immunoblotting with an antiserum to HA (Fig. 4A). A protein of the expected size, ∼85 kD, was identified in total extracts and was associated with total membrane fractions. The HA-tagged protein was enriched in chloroplasts and was present in both the membrane and soluble fractions. The chloroplast fractions were not substantially contaminated with cytosol, as shown by the immunoblot with an antiserum raised against cytosolic thioredoxin h (Trx) (Fig. 4A). As expected, the Stt7-HA construct was also detected in isolated thylakoids. Moreover, a slightly faster migrating band was detected in the soluble fraction of total cell extracts and chloroplasts. This band could have arisen through dephosphorylation of the protein in the membrane fractions. Indeed, treatment of thylakoids with calf intestinal phosphatase (CIP) converted the slower form to the faster migrating form (Fig. 4B). To test how tightly the Stt7 kinase was associated with the thylakoid membranes, these were isolated from the strain containing the HA-tagged Stt7 protein and were incubated in the presence of one of the following: 2 M NaCl, 0.1 M Na2C03, 0.1% Triton X-100, 0.1 N NaOH, or 6.8 M urea. Most of the Stt7 protein was released from the membrane by treatment with NaOH, but not by any of the other reagents (Fig. 4C). Thus, the membrane-associated Stt7 protein was firmly bound to the thylakoids.

Figure 4

Localization of Stt7 on the thylakoid membrane. (A) The stt7-1 mutant was complemented with the Stt7-HA gene. Proteins from total cell extracts from this strain (T), from the total soluble (S) and membrane (M) fractions, from thylakoids (Tk), from isolated chloroplasts (Tc), and from the chloroplast soluble (Sc) and membrane (Mc) fractions were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE), blotted onto nitrocellulose filters, and reacted with HA monoclonal antibody and with antisera raised against the thylakoid D2 protein of PSII, Trx, and the soluble chloroplast DnaK protein. (B) Thylakoid membranes (50 μg of protein) were treated for 5 min with 40 U of CIP; the proteins were then fractionated by SDS-PAGE and immunoblotted with antiserum to HA. The lower band probably represents a degradation product of Stt7. (C) Thylakoid membranes were isolated from the Stt7-HA strain and incubated for 30 min in 2 M NaCl, 0.1 M Na2CO3, 0.1% Triton X-100, 0.1 N NaOH, or 6.8 M urea. The membranes were recovered by centrifugation at 100,000g for 30 min (or 1 hour after the NaOH treatment) and the pellet (M) and supernatants (S) were fractionated by PAGE, blotted, and reacted with antibodies to HA. For quantitation, a thylakoid dilution series was run alongside.

A BLAST search with the Stt7 sequence revealed an expressed sequence tag from C. reinhardtii with substantial sequence similarity. The complete cDNA was isolated and calledStl1 (11). Its sequence predicts a protein kinase of 499 amino acids with 29% sequence identity and 43% sequence similarity to Stt7 (Fig. 2). ChloroP predicts a chloroplast localization of this protein. Stt7 is related to twoArabidopsis protein kinases of unknown function. The first, Stt7-AT1 (At1g68830), is 45% identical and 60% similar to Stt7; the second, Stt7-AT2 (At5g01920), is 37% identical and 43% similar to Stt7 (Fig. 2). When these aligned sequences were analyzed with the TMAP transmembrane prediction program (17), one transmembrane domain was predicted upstream of the kinase domain in all four proteins. On the basis of the sequence of their NH2-terminal ends, both Arabidopsis proteins are predicted to be localized within the chloroplast. Sequence similarity of Stt7 with cyanobacterial proteins was substantially lower.

It is possible that the NH2-terminal region of Stt7 is localized in the thylakoid lumen, whereas the bulk of the protein with its kinase domain is on the stromal side of the thylakoid membrane, where it would have access to its target phosphorylation site on the NH2-terminal end of the LHCII proteins that are known to be exposed to the stroma (1). Another possibility is that the protein is peripherally bound on the stromal side of the thylakoid membrane. The COOH-terminal end of Stt7 is characterized by a nearly continuous stretch of 13 Ala residues and by a long alternating Gly-Asn motif. This region does not appear to be essential for Stt7's function, because one cosmid that lacks the 164 COOH-terminal residues of Stt7 is still able to complement the stt7 mutation (13).

The LHCII kinase of higher plants is inactivated by high light in a process mediated by the ferredoxin-thioredoxin system (18). Target sites of thioredoxin include Cys separated by four residues (19). Among the seven Cys residues of Stt7, two are separated by four residues and located between the transit peptide and the hydrophobic region. They are also conserved in Stt7-AT1 (Fig. 2).

TAKs are a family of thylakoid-associated kinases that phosphorylate the LHC proteins (20, 21). The activity of these kinases is increased by reductant and they are associated with the reaction center of PSII, with the cytochrome b6f complex, and with LHCP. Decrease of TAK1 content by antisense expression leads to lower LHCII phosphorylation and to diminished state transition. There is no major sequence similarity between Stt7 and the TAK kinases, although a hydrophobic region is present between the chloroplast targeting region and the kinase domain in both proteins. TAK kinases display sequence similarity to the transforming growth factor–β1 receptors of metazoans (20), a feature that is not shared by Stt7. Whereas inactivation of TAK1 inArabidopsis leads to growth retardation and bleaching, the inactivation of Stt7 in Chlamydomonas did not appreciably affect the growth rate and did not lead to enhanced photosensitivity. Taken together, these findings raise the possibility that a cascade of kinases that are associated with the thylakoid membrane is part of the signal transduction chain of state transition. Although the role of the Stt7 kinase in the phosphorylation of LHCII is clear, it remains to be seen whether this kinase phosphorylates LHCII directly or whether it acts further upstream in this signaling pathway.

Supporting Online Material

www.sciencemag.org/cgi/content/full/299/5612/1572/DC1

Materials and Methods

Fig. S1

References and Notes

  • * To whom correspondence should be addressed. E-mail: jean-david.rochaix{at}molbio.unige.ch

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