Ribosome-binding sites on chloroplastrbcL andpsbA mRNAs and light-induced initiation of D1 translation

Chloroplast ribosome-binding sites were identified on the plastidrbcL andpsbA mRNAs using toeprint analysis. TherbcL translation initiation domain is highly conserved and contains a prokaryotic Shine-Dalgarno (SD) sequence (GGAGG) located 4 to 12 nucleotides upstream of the initiator AUG. Toeprint analysis ofrbcL mRNA associated with plastid polysomes revealed strong toeprint signals 15 nucleotides downstream from the AUG indicating ribosome binding at the translation initiation site.Escherichia coli 30S ribosomes generated similar toeprint signals when mixed withrbcL mRNA in the presence of initiator tRNA. These results indicate that plastid SD sequences are functional in chloroplast translation initiation. ThepsbA initiator region lacks a SD sequence within 12 nucleotides of the initiator AUG. However, toeprint analysis of soluble and membrane polysome-associatedpsbA mRNA revealed ribosomes bound to the initiator region.E. coli 30S ribosomes did not associate with thepsbA translation initiation region.E. coli and chloroplast ribosomes bind to an upstream region which contains a conserved SD-like sequence. Therefore, translation initiation onpsbA mRNA may involve the transient binding of chloroplast ribosomes to this upstream SD-like sequence followed by scanning to localize the initiator AUG. Illumination 8-day-old dark-grown barley seedlings caused an increase in polysome-associatedpsbA mRNA and the abundance of initiation complexes bound topsbA mRNA. These results demonstrate that light modulates D1 translation initiation in plastids of older dark-grown barley seedlings.     

Vir-115 gene product is required to stabilize D1 translation intermediates in chloroplasts

The nuclear gene mutant of barley, vir-115, shows a developmentally induced loss of D1 synthesis that results in inactivation of Photosystem II. Translation in plastids isolated from 1 h illuminated vir-115 seedlings is similar to wild type. In wild-type barley, illumination of plants for 16 to 72 h results in increased radiolabel incorporation into the D1 translation intermediates of 15–24 kDa. In contrast, these D1 translation intermediates were not observed in vir-115 plastids isolated from plants illuminated for 16–72 h. In addition, after 72 h of illumination, radiolabel incorporation into D1 was undetectable in vir-115 plastids. The level and distribution ofpsbA mRNA in membrane-associated polysomes was similar in wild-type and vir-115 mutant plastids isolated from plants illuminated for 16–72 h. Toeprint analysis showed similar levels of translation initiation complexes onpsbA mRNA in vir-115 and wild-type plastids. These results indicate that translation initiation and elongation of D1 is not significantly altered in the mutant plastids. Ribosome pausing onpsbA mRNA was observed in wild-type and vir-115 mutant plastids. Therefore, the absence of D1 translation intermediates in mutant plastids is not due to a lack of ribosome pausing onpsbA mRNA. Based on these results, it is proposed that vir-115 lacks or contains a modified nuclear-encoded gene product which normally stabilizes the D1 translation intermediates. In wild-type plastids, ribosome pausing and stabilization of D1 translation intermediates is proposed to facilitate assembly of cofactors such as chlorophyll will D1 allowing continued D1 synthesis and accumulation in mature chloroplasts.     

Two NADPH:Protochlorophyllide Oxidoreductases in Barley: Evidence for the Selective Disappearance of PORA during the Light-Induced Greening of Etiolated Seedlings

Chlorophyll synthesis in barley is controlled by two different light-dependent NADPH:protochlorophyllide oxidoreductases, termed PORA and PORB. PORA is present abundantly in etioplasts but selectively disappears soon after the beginning of illumination. This negative light effect is mediated simultaneously at three different levels. First, the concentration of porA mRNA declines drastically during illumination of dark-grown seedlings. Second, the plastids' ability to import the precursor of PORA (pPORA) is reduced during the transition from etioplasts to chloroplasts. This effect is due to a rapid decline in the plastidic level of protochlorophyllide (Pchlide), which is required for the translocation of the pPORA. Third, PORA becomes selectively destabilized in illuminated seedlings. When illuminated, PORA-Pchlide-NADPH complexes formed in the dark photoreduce their Pchlide to Chlide and become simultaneously susceptible to attack by plastid proteases. The PORA-degrading protease activity is not detectable in etioplasts but is induced during illumination. In contrast to PORA, the second Pchlide-reducing enzyme, PORB, remains operative in both illuminated and green plants. Its translocation into plastids does not depend on its substrate, Pchlide.     

Translation and stability of proteins encoded by the plastid psbA and psbB genes are regulated by a nuclear gene during light-induced chloroplast development in barley

We have characterized a nuclear mutant of barley, viridis-115, lacking photosystem II (PSII) activity and compared it to wild-type seedlings during light-induced chloroplast development. Chloroplasts isolated from wild-type and viridis-115 seedlings illuminated for 1 h synthesized similar polypeptides and had similar protein composition. After 16 h of illumination, however, mutant plastids exhibited reduced ability to radiolabel D1, CP47, and several low Mr membrane polypeptides, and by 72 h, synthesis of these proteins was undetectable. Immunoblot analysis showed that plastids of dark-grown wild-type barley lacked several PSII proteins (D1, D2, CP47, and CP43) and that 16 h of illumination resulted in the accumulation of these polypeptides. In contrast, these polypeptides did not accumulate in illuminated viridis-115 seedlings, although mutant plastids accumulated two PSII proteins that participate in oxygen evolution, oxygen-evolving enhancers 1 and 3. Northern analysis showed that the levels of psbA and psbB mRNA in mutant plastids were equal to or greater than levels in wild-type plastids throughout the developmental period examined here. These results indicate that the nuclear mutation present in viridis-115 affects the translation and stability of the chloroplast-encoded D1 and CP47 polypeptides and that its influence is expressed after the onset of light-induced chloroplast development.     

Influence of nitrogen sources on chloroplast development in wheat seedlings

The effect of different nitrogen sources (ammonium, nitrate or both ions together) on plastid development in dark-grown and illuminated seedlings of wheat ( Triticum vulgare L. cv. Yecora) has been investigated. Plastids of plants grown in ammonium showed even in the dark a larger internal membrane length, higher ribulose bisphos-phate carboxylase activity and greater content of soluble proteins than plastids of plants grown in nitrate. After the first hour of illumination rudimentary thylakoids showing some joining points were observed in the ammonium plastids. After 10 h no prolamellar bodies were seen in the ammonium plastids, and the internal plastid membrane length was greater than in the other treatments. There was no light-induced increase in protein synthesis after illumination for 1 h. After 10 h the increase observed in protein synthesis was not followed by a response in the enzyme activity in any of the treatments. After 20 h the lag in the induction of ribulose bisphosphate carboxylase ceased, the enzyme activity and soluble proteins being higher in the leaves of ammonium seedlings than in those from nitrate. From the correlation obtained between the ultrastructural electron microscope observations and the enzymatic studies, it appears that ammonium nutrition has a positive influence on the formation of the plastid membrane system and on the onset of photosynthesis and, consequently, on the development of chloroplasts.     

Subunit control of Rubisco biosynthesis – a relic of an endosymbiotic past?

Subunits of multiprotein complexes in the chloroplasts of eukaryotic cells are frequently the products of protein synthesis in the nucleus-cytoplasm and the organelle. The mechanisms that integrate gene expression in the two compartments are poorly understood. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a model of nuclear-chloroplast interactions because it is a relatively simple example of a multimeric complex, being composed of nuclear DNA-encoded (RbcS) small subunits (SS) and chloroplast DNA-encoded ( rbcL) large subunits (LS). One means by which RbcS and rbcL expression are coordinated is by the adjustment of subunit stoichiometries in response to the abundance of unassembled subunits. This type of integration occurs by two principal mechanisms. When SS accumulation is limiting (as in antisense mutants of tobacco), LS levels are primarily adjusted to those of the SS at the level of rbcL mRNA translation initiation. On the other hand, when LS accumulation is limiting (as in some rbcL nonsense and missense mutants), SS levels are adjusted to those of the LS at the level of protein degradation. These two mechanisms may be ubiquitous and serve as either fine-tune or course controls during normal growth and development. Autogenous control is a central theme of prokaryotic gene regulation, and intergenomic regulation of RbcS and rbcL expression by subunit concentrations may be a relic of an endosymbiotic past.     

Formation of the small subunit in the absence of the large subunit of ribulose 1,5-bisphosphate carboxylase in 70 S ribosome-deficient rye leaves.

Immunological tests with monospecific antisera to ribulosebisphosphate carboxylase (EC 4.1.1.39) and to its large and small subunits indicated the presence of a protein with antigenic properties of the small subunit in the absence of the large subunit in the leaves of young rye plants (Secale cereale L.) with a high-temperature-induced (32 °C) deficiency of 70 S plastid ribosomes. The small subunit-like protein was isolated from crude extracts of plastid ribosome-deficient 32 °C-grown leaf tissue by the use of columns with immobilized antibody. The main polypeptide retained by the immobilized antibodies had the same mobility after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels as the small subunit of ribulosebisphosphate carboxylase and was also immunologically identical to the small subunit. The small subunit-like protein was present in the supernatant as well as in the membrane fraction of isolated 70 S ribosome-deficient plastids. At very young stages of normal leaves grown at a permissive temperature (22 °C) an excess of small subunit was observed that was also not integrated into the complete ribulosebisphosphate carboxylase molecule. From the results, we conclude that the synthesis of the small subunit occurs on cytoplasmic ribosomes and is not strictly coordinated with the translation of the large subunit in the chloroplast. During early leaf development, the formation of the large subunit seems to be the ratelimiting step in the synthesis of ribulosebisphosphate carboxylase.