A key reaction in the biosynthesis of chlorophylls (Chls) a and b from cyanobacteria through higher plants is the strictly light-dependent reduction of protochlorophyllide (Pchlide) a to chlorophyllide (Chlide) a. Angiosperms, unlike other photosynthetic organisms, rely exclusively upon this mechanism to reduce Pchlide and hence require light to green. In Arabidopsis, light-dependent Pchlide reduction is mediated by three structurally related but differentially regulated NADPH:Pchlide oxidoreductases, denoted as PORA, PORB, and PORC. The PORA and PORB genes, but not PORC, are strongly expressed early in seedling development. In contrast, expression of PORB and PORC, but not PORA, is observed in older seedlings and adult plants. We have tested the hypothesis that PORB and PORC govern light-dependent Chl biosynthesis throughout most of the plant development by identifying porB and porC mutants of Arabidopsis, the first higher plant por mutants characterized. The porB-1 and porC-1 mutants lack the respective POR transcripts and specific POR isoforms because of the interruption of the corresponding genes by a derivative of the maize Dissociation (Ds ) transposable element. Single por mutants, grown photoperiodically, display no obvious phenotypes at the whole plant or chloroplast ultrastructural levels, although the porB-1 mutant has less extensive etioplast inner membranes. However, a light-grown porB-1 porC-1 double mutant develops a seedling-lethal xantha phenotype at the cotyledon stage, contains only small amounts of Chl a, and possesses chloroplasts with mostly unstacked thylakoid membranes. PORB and PORC thus seem to play redundant roles in maintaining light-dependent Chl biosynthesis in green plants, and are together essential for growth and development.

In higher plants, chloroplast-destined precursor proteins are thought to be phosphorylated. Mediated by a specific 14-3-3 protein, these phosphorylated proteins bind to the chloroplast surface and are subsequently imported into the chloroplast. We demonstrate that also in the green alga Chlamydomonas reinhardtii the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase becomes phosphorylated by a plant protein kinase and that the phosphorylation site is located in the transit peptide. The phosphorylation status of the precursor protein regulates its import into chloroplasts especially at an early step during this process. The possible physiological function is discussed.

By studying the import of radioactively labelled small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) into chloroplasts of the green alga C. reinhardtii cw-15 protein delivery to chloroplasts was found to vary during the cell cycle. Chloroplasts were isolated from highly synchronous cultures at different time points during the cell cycle. When pSS was imported into 'young' chloroplasts isolated early in the light period about three times less pSS was processed to small subunit SS than in 'mature' chloroplasts from the middle of the light period. In 'young' chloroplasts also, less pSS was bound to the envelope surface. During the second half of the light period the import competence of isolated chloroplasts decreased again when based on chlorophyll content or cell volume, but did not change significantly when related to chloroplast number. Measurements of pSS binding to the surface of chloroplasts of different age indicated that the adaptation of protein import competence during the cell cycle is due to a variation of the number of binding sites per chloroplast surface area, rather than to modulation of the binding constant.

During the sequencing of the genome of Arabidopsis thaliana a gene has been identified that encodes a novel NADPH-protochlorophyllide oxidoreductase (POR)-like protein (accession number AC 002560). This protein has been named POR C. We have expressed the POR C protein in Escherichia coli and have determined its in vitro activity. POR C shows the characteristics of a light-dependent and NADPH-requiring POR similar to POR A and POR B. The expression of the POR C gene differs markedly from that of the POR A and POR B genes. In contrast to the POR A and POR B mRNAs, the POR C mRNA has been shown previously to accumulate only after the beginning of illumination. In light-adapted mature plants only POR B and POR C mRNAs were detectable. The amounts of both mRNAs show pronounced diurnal rhythmic fluctuations. While the oscillations of POR B mRNA are under the control of the circadian clock, those of POR C mRNA are not. Another difference between POR B and POR C was found in seedlings that were grown under continuous white light. The concentration of POR C mRNA rapidly declined and soon dropped beyond the limit of detection, after these seedlings were transferred to the dark. On the other hand. POR B mRNA was unaffected by this light/dark shift. When seedlings were exposed to different light intensities, the amounts of POR B mRNA remained the same, while POR A and POR C mRNAs were modulated in an inverse way by these light intensity changes. POR A mRNA was still detectable in seedlings grown under low light intensities but disappeared at higher light intensities, while the mRNA concentration of POR C rose with increasing light intensities. These different responses to light suggest that the functions of the three PORs of Arabidopsis are not completely redundant, but may allow the plant to adapt its needs for chlorophyll biosynthesis more selectively by using preferentially one of the three enzymes under a given light regime.

Proteins synthesized in the cytoplasm and destined for importation into the chloroplast across the double envelope membrane contain an N-terminal transit sequence which upon import is cleaved off by a stromal-processing peptidase. Since for stromal-residing proteins no intermediates have ever been found in vivo, it is assumed that precursor proteins are cleaved to the mature size by one proteolytic event which occurs immediately after translocation across both envelope membranes. During import of the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) into isolated chloroplasts of Chlamydomonas we identified an intermediate-sized product, called iSS. It might be identical to a previously described iSS obtained in vitro by a partially purified soluble chloroplast protease [Su and Boschetti (1993) Eur. J. Biochem. 217, 1039-1047]. The kinetics of the formation of iSS in chloroplasts suggest that pSS is processed to the mature small subunit (SS) not by one, but by two steps via this intermediate product. Since, after an induction period, the ratio of iSS/SS was constant under various experimental conditions of import, the formation of iSS was considered not to be a side-reaction. The location of iSS in the intermembrane space of the envelope, as suggested by protease treatment of chloroplasts, questions the one-step translocation mechanism of precursor import into chloroplasts.

Using different precursors of chloroplast proteins and stromal extracts from both Chlamydomonas reinhardii and pea chloroplasts, we analysed the specificity of stroma-localized processing peptidases. By gel filtration of a stromal extract from isolated Chlamydomonas chloroplasts, fractions could be separated containing enzymic activities for processing the precursors of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) and of the protein OEE1 from the photosynthetic water-splitting complex (pOEE1). The enzymes differed not only in molecular size, but also in their sensitivity to inhibitors and in their pH optima. Obviously, in the stroma of Chlamydomonas chloroplasts different peptidases exist for processing of pSS and pOEE1, the latter being converted into an intermediate-sized form, iOEE1, which was found to be further processed to mature OEE1 by a thylakoid-associated protease. To study the species-specificity of the stromal peptidases, stromal extracts from Chlamydomonas and pea chloroplasts were incubated with pSS from either of these organisms. In the heterologous combinations, the precursors were partly hydrolysed, but not to the correct size. In importation assays, pSS from pea (but also the precursor of the ribosomal protein L12 from spinach) could not enter into chloroplasts from Chlamydomonas. In contrast, the algal pSS was imported into chloroplasts from pea, although it was not processed to mature SS. Our results indicate that the importation machinery and the pSS-processing enzymes in higher plants and green algae have different specificities and that in Chlamydomonas several stromal peptidases for different precursor proteins exist.

Two stromal peptidases (SPP-1 and SPP-2) were partially purified from chloroplasts of Chlamydomonas reinhardii. They specifically processed in vitro the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSS), which had been synthesized by using the cloned rbcS-2 gene of Chlamydomonas. SPP-1 shortened pSS to an intermediate-sized form (iSS), while SPP-2 cut pSS and iSS to the mature small subunit SS. N-terminal amino acid sequencing demonstrated that the reaction product obtained with SPP-2 had an N-terminus identical to natural SS, and that iSS derived from pSS by hydrolysis at the amino side of the methionine located within the transit sequence. By gel filtration, apparent molecular masses of 340 kDa and 90 kDa were determined for SPP-1 and SPP-2, respectively. The comparison of these molecular masses with the protein patterns obtained by SDS/PAGE of the partially purified enzymes suggested that at least SPP-1 was a multimeric protein. The enzymes differed also in their pH optima of about 8 (SPP-1) and 9 (SPP-2) and in their sensitivity to different inhibitors. However, both enzymes seem to be serine proteases as they were completely blocked by N-alpha-tosyl-L-lysinechloromethane or tosylphenylalaninechloromethane, respectively. Competition experiments, using either mature SS or a synthetic hexadecapeptide with 15 amino acids similar to the C-terminal end of the transit sequence of pSS, indicated that SPP-2 had some affinities not only to the transit sequence of pSS, but especially to sequences in the mature protein part. We conclude that SPP-2 in Chlamydomonas is the enzyme involved in import of pSS into chloroplasts and responsible for its processing by a one-step mechanism.

The binding affinity of the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) to isolated, intact chloroplasts and to isolated chloroplast envelopes from the green alga Chlamydomonas reinhardii was studied under conditions where no import into chloroplasts occurred. pSS bound to both chloroplasts and envelopes with equally high affinity. The dissociation constants were 5.9 +/- 2.1 x 10(-9) M and 2.9 +/- 1.4 x 10(-9) M, respectively. The number of binding sites per chloroplast was determined to be 8.1 +/- 4.1 x 10(4). Binding of pSS to isolated envelopes or intact chloroplasts was specific with respect to the type of the membrane and the presence of the transit sequence.