The energetic metabolism of photosynthetic organisms is profoundly influenced by state transitions and cyclic electron flow around photosystem I. The former involve a reversible redistribution of the light-harvesting antenna between photosystem I and photosystem II and optimize light energy utilization in photosynthesis whereas the latter process modulates the photosynthetic yield. We have used the wild-type and three mutant strains of the green alga Chlamydomonas reinhardtiilocked in state I (stt7), lacking the photosystem II outer antennae (bf4) or accumulating low amounts of cytochrome b 6 f complex (A-AUU)-and measured electron flow though the cytochrome b 6 f complex, oxygen evolution rates and fluorescence emission during state transitions. The results demonstrate that the transition from state 1 to state 2 induces a switch from linear to cyclic electron flow in this alga and reveal a strict cause-effect relationship between the redistribution of antenna complexes during state transitions and the onset of cyclic electron flow.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b 6 f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b 6 f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b 6 f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LH-CII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.State transition has been originally described as a process whereby organisms performing oxygenic photosynthesis respond to changes in the spectral quality of light by changing the relative sizes of their photosystem I (PSI) 1 and photosystem II (PSII) antennae (for review, see Ref. 1). This leads to a redistribution of excitation energy between these two photosystems, which optimizes the photosynthetic yield. The reorganization of the antennae involves the displacement of the mobile fraction of a peripheral antenna complex, the light-harvesting antenna of photosystem II (LHCII), within the thylakoid membrane. During transition from state I to state II induced by PSII light (650 nm), the mobile fraction of LHCII is displaced from the PSII-enriched grana region to the PSI-enriched stromal lamellae. In the presence of PSI light (700 nm), the reverse migration of LHCII from PSI to PSII occurs. The redistribution of excitation energy between the photosystems results in changes in room temperature fluorescence with high and low fluorescence levels in state I and II, respectively, and in drastic alterations of the fluorescence emission spectrum at low temperature. As a consequence of this redistribution of energy between the two photosystems, state transition regulates the...

Insulin stimulates the transcription of the sterol regulatory- element binding protein (SREBP) 1/ADD1 gene in liver. Hepatocytes in primary culture were used to delineate the insulin signalling pathway for induction of SREBP1 gene expression. The inhibitors of phosphoinositide 3-kinase (PI 3-kinase), wortmannin and LY 294002, abolished the insulin-dependent increase in SREBP1 mRNA, whereas the inhibitor of the mitogen- activated protein kinase cascade, PD 98059, was without effect. To investigate the role of protein kinase B (PKB)/cAkt downstream of PI 3-kinase, hepatocytes were transduced with an adenovirus encoding a PKB--oestrogen receptor fusion protein. The PKB activity of this recombinant protein was rapidly activated in hepatocytes challenged with 4-hydroxytamoxifen (OHT), as was endogenous PKB in hepatocytes challenged with insulin. The addition of OHT to transduced hepatocytes resulted in accumulation of SREBP1 mRNA, with a time-course and magnitude similar to the effect of insulin in non-transduced cells. The level of SREBP1 mRNA was not increased by OHT in hepatocytes expressing a mutant form of the recombinant protein whose PKB activity was not activated by OHT. Thus acute activation of PKB is sufficient to induce SREBP1 mRNA accumulation in primary hepatocytes, and might be the major signalling event by which insulin induces SREBP1 gene expression in the liver.

The psbD mRNA of Chlamydomonas reinhardtii is one of the most abundant chloroplast transcripts and encodes the photosystem II reaction center polypeptide D2. This RNA exists in two forms with 5 Ј untranslated regions of 74 and 47 nucleotides. The shorter form, which is associated with polysomes, is likely to result from processing of the larger RNA. Using site-directed mutagenesis and biolistic transformation, we have identified two major RNA stability determinants within the first 12 nucleotides at the 5 Ј end and near position Ϫ 30 relative to the AUG initiation codon of psbD. Insertion of a polyguanosine tract at position Ϫ 60 did not appreciably interfere with translation of psbD mRNA. The same poly(G) insertion in the nac2-26 mutant, which is known to be deficient in psbD mRNA accumulation, stabilized the psbD RNA. However, the shorter psbD RNA did not accumulate, and the other psbD RNAs were not translated. Two other elements were found to affect translation but not RNA stability. The first comprises a highly U-rich sequence (positions Ϫ 20 to Ϫ 15), and the second, called PRB1 (positions Ϫ 14 to Ϫ 11), is complementary to the 3 Ј end of the 16S rRNA. Changing the PRB1 sequence from GGAG to AAAG had no detectable effect on psbD mRNA translation. However, changing this sequence to CCUC led to a fourfold diminished rate of D2 synthesis and accumulation. When the psbD initiation codon was changed to AUA or AUU, D2 synthesis was no longer detected, and psbD RNA accumulated to wild-type levels. The singular organization of the psbD 5 Ј untranslated region could play an important role in the control of initiation of psbD mRNA translation. INTRODUCTIONChloroplast gene expression has been shown to be regulated at various levels, including transcription and several post-transcriptional steps, such as RNA stabilization, RNA processing, and RNA splicing and translation. Genetic analysis of the unicellular green alga Chlamydomonas reinhardtii and higher plants suggests that these processes are controlled in large part by nucleus-encoded factors. These factors are synthesized in the cytosol and subsequently imported into the chloroplast, where they interact with their cognate target sites on either chloroplast RNAs or proteins to fulfill their function (Rochaix, 1996;Sugita and Sugiura, 1996; Goldschmidt-Clermont, 1998).The availability of a biolistic chloroplast transformation system (Boynton et al., 1988) has allowed us to analyze sitedirected chloroplast mutations and to use heterologous reporter systems. This has led to the identification of essential cis -acting RNA elements on chloroplast mRNAs that are involved in the regulation of gene expression. As an example, the analysis of chloroplast transformants revealed the importance of 5 Ј untranslated regions (UTRs) for the regulation of translation of the psbC (Zerges and Rochaix, 1994;Zerges et al., 1997), psbA (Mayfield et al., 1994), petD (Sakamoto et al., 1994), and psaB (Stampacchia et al., 1997) mRNAs in Chlamydomonas. They encode subunits of photosynthetic c...

The psbD mRNA of Chlamydomonas reinhardtii is one of the most abundant chloroplast transcripts and encodes the photosystem II reaction center polypeptide D2. This RNA exists in two forms with 5 Ј untranslated regions of 74 and 47 nucleotides. The shorter form, which is associated with polysomes, is likely to result from processing of the larger RNA. Using site-directed mutagenesis and biolistic transformation, we have identified two major RNA stability determinants within the first 12 nucleotides at the 5 Ј end and near position Ϫ 30 relative to the AUG initiation codon of psbD. Insertion of a polyguanosine tract at position Ϫ 60 did not appreciably interfere with translation of psbD mRNA. The same poly(G) insertion in the nac2-26 mutant, which is known to be deficient in psbD mRNA accumulation, stabilized the psbD RNA. However, the shorter psbD RNA did not accumulate, and the other psbD RNAs were not translated. Two other elements were found to affect translation but not RNA stability. The first comprises a highly U-rich sequence (positions Ϫ 20 to Ϫ 15), and the second, called PRB1 (positions Ϫ 14 to Ϫ 11), is complementary to the 3 Ј end of the 16S rRNA. Changing the PRB1 sequence from GGAG to AAAG had no detectable effect on psbD mRNA translation. However, changing this sequence to CCUC led to a fourfold diminished rate of D2 synthesis and accumulation. When the psbD initiation codon was changed to AUA or AUU, D2 synthesis was no longer detected, and psbD RNA accumulated to wild-type levels. The singular organization of the psbD 5 Ј untranslated region could play an important role in the control of initiation of psbD mRNA translation. INTRODUCTIONChloroplast gene expression has been shown to be regulated at various levels, including transcription and several post-transcriptional steps, such as RNA stabilization, RNA processing, and RNA splicing and translation. Genetic analysis of the unicellular green alga Chlamydomonas reinhardtii and higher plants suggests that these processes are controlled in large part by nucleus-encoded factors. These factors are synthesized in the cytosol and subsequently imported into the chloroplast, where they interact with their cognate target sites on either chloroplast RNAs or proteins to fulfill their function (Rochaix, 1996;Sugita and Sugiura, 1996; Goldschmidt-Clermont, 1998).The availability of a biolistic chloroplast transformation system (Boynton et al., 1988) has allowed us to analyze sitedirected chloroplast mutations and to use heterologous reporter systems. This has led to the identification of essential cis -acting RNA elements on chloroplast mRNAs that are involved in the regulation of gene expression. As an example, the analysis of chloroplast transformants revealed the importance of 5 Ј untranslated regions (UTRs) for the regulation of translation of the psbC (Zerges and Rochaix, 1994;Zerges et al., 1997), psbA (Mayfield et al., 1994), petD (Sakamoto et al., 1994), and psaB (Stampacchia et al., 1997) mRNAs in Chlamydomonas. They encode subunits of photosynthetic c...

Insulin stimulates the transcription of the sterol regulatory- element binding protein (SREBP) 1/ADD1 gene in liver. Hepatocytes in primary culture were used to delineate the insulin signalling pathway for induction of SREBP1 gene expression. The inhibitors of phosphoinositide 3-kinase (PI 3-kinase), wortmannin and LY 294002, abolished the insulin-dependent increase in SREBP1 mRNA, whereas the inhibitor of the mitogen- activated protein kinase cascade, PD 98059, was without effect. To investigate the role of protein kinase B (PKB)/cAkt downstream of PI 3-kinase, hepatocytes were transduced with an adenovirus encoding a PKB-oestrogen receptor fusion protein. The PKB activity of this recombinant protein was rapidly activated in hepatocytes challenged with 4-hydroxytamoxifen (OHT), as was endogenous PKB in hepatocytes challenged with insulin. The addition of OHT to transduced hepatocytes resulted in accumulation of SREBP1 mRNA, with a time-course and magnitude similar to the effect of insulin in non-transduced cells. The level of SREBP1 mRNA was not increased by OHT in hepatocytes expressing a mutant form of the recombinant protein whose PKB activity was not activated by OHT. Thus acute activation of PKB is sufficient to induce SREBP1 mRNA accumulation in primary hepatocytes, and might be the major signalling event by which insulin induces SREBP1 gene expression in the liver.

Inhibitors of signalling pathways were used to dissect the mechanism of insulin action on expression of the gene encoding glucokinase in cultured rat hepatocytes. Wortmannin and LY 294002 completely prevented the insulin-induced increase in glucokinase mRNA seen in unhibited cells, indicating that the phosphoinositide 3-kinase module has a key role. A ligand inducible protein kinase B (PKB, also termed cAkt) fusion protein was expressed by using adenoviral transduction of hepatocytes in primary culture. The PKB activity of this protein was shown to be activated in transduced hepatocytes within 30min of the addition of 4-hydroxytamoxifen and to stay high for 8h, as a result of serine phosphorylation at position 473 of PKB. The increase in PKB activity was reflected in the hyperphosphorylation of phosphorylated, heat and acid stable regulated by insulin protein (PHAS-I; also termed 4E-BP1, for eukaryotic initiation factor 4E-binding protein 1), a protein involved in the regulation of translation initiation. These effects were comparable to the insulin-induced activation of endogenous PKB and phosphorylation of PHAS-I in non-transduced hepatocytes. The addition of tamoxifen to transduced hepatocytes resulted in an induction of glucokinase mRNA with kinetics and magnitude similar to those of insulin-induced mRNA accumulation. The effect of tamoxifen depended on stimulated PKB activity because it did not occur in hepatocytes that were transduced with a mutant PKB fusion protein that was refractory to activation with tamoxifen. These results establish that acute activation of PKB is sufficient to produce an insulin-like induction of glucokinase in isolated hepatocytes. Together with the inhibition by phosphoinositide 3-kinase inhibitors, they suggest that the activation of PKB might be critical in mediating the induction of glucokinase by insulin. In addition, experiments showed that PD98059 decreased by half the increase in glucokinase mRNA brought about by insulin, suggesting a contributory role of the mitogen-activated protein kinase cascade.

We have changed the potential phosphorylation site, a threonine residue at position 2 of the D2 polypeptide of the photosystem II complex of Chlamydomonas reinhardtii, to alanine, valine, aspartate, proline, glycine, or glutamate. Mutants with neutral amino acid changes did not display any phenotype with regard to photoautotrophic growth, light sensitivity, fluorescence transients, or photoinhibition. Pulse labeling of these mutants with 32 P indicated that a phosphorylated protein of the same size as D2 is absent in these mutants, suggesting that threonine-2 is indeed the unique phosphorylation site of D2. In contrast, mutants in which threonine-2 has been replaced with acidic residues are deficient in photosystem II. Use of chimeric genes containing the psbD 5-untranslated region revealed that the initiation of translation was not affected in these mutants, but the mutations interfered with a later step of D2 synthesis and accumulation.