Characterization of the mRNA transcripts of the maize, ribulose-1,5-bisphosphate carboxylase, large subunit gene

Erion, Jack L.

doi:10.1007/bf02418764

Cited by 24 publications

(6 citation statements)

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“…Two rbcL transcripts were resolved of which the larger is the primary transcript, and the smaller a processed version (Erion, 1985), which is likely stabilized by the maize ortholog of the RNA binding protein MRL1 (Johnson et al, 2010). While RBCS transcripts appeared to be slightly decreased in raf1-1 mutants, rbcL transcripts accumulated to more than three times the level of the wild type, a result that was confirmed by quantitative RT-PCR ( Figure 3B).…”

Section: Rubisco Subunit Genes Are Normally Transcribed and Translatementioning

confidence: 65%

Ribulose-1,5-Bis-Phosphate Carboxylase/Oxygenase Accumulation Factor1 Is Required for Holoenzyme Assembly in Maize

et al. 2012

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Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). Although the structurally comparable cyanobacterial Rubisco is amenable to in vitro assembly, the higher plant enzyme has been refractory to such manipulation due to poor understanding of its assembly pathway. Here, we report the identification of a chloroplast protein required for Rubisco accumulation in maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks any characterized functional domains. Maize lines lacking RAF1 due to Mutator transposon insertions are Rubisco deficient and seedling lethal. Analysis of transcripts and proteins showed that Rubisco large subunit synthesis in raf1 plants is not compromised; however, newly synthesized Rubisco large subunit appears in a high molecular weight form whose accumulation requires a specific chaperonin 60 isoform. Gel filtration analysis and blue native gels showed that endogenous and recombinant RAF1 are trimeric; however, following in vivo cross-linking, RAF1 copurifies with Rubisco large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a Rubisco accumulation function. Our results support the hypothesis that RAF1 acts during Rubisco assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process. INTRODUCTIONRibulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) is the major enzyme by which green plants, algae, cyanobacteria, and other autotrophic organisms sequester CO 2 into organic compounds via the Calvin-Benson pathway (Andersson and Backlund, 2008). Rubisco catalyzes the photosynthetic carbon reduction and the photorespiratory carbon oxidation reactions of the substrate ribulose-1,5-bisphosphate with CO 2 and O 2 , respectively. The inefficiency of Rubisco in fixing CO 2 has a limiting impact on agricultural productivity and in compensation, Rubisco accounts for as much as 20 to 30% and 4 to 9% of total nitrogen compounds in C 3 and C 4 higher plants, respectively (Feller et al., 2008).Attempts to improve the catalytic properties of plant Rubisco (reviewed in Parry et al., 2003;Mueller-Cajar and Whitney, 2008) have met with only modest success, which can be traced in part to the lack of a comprehensive knowledge of its biogenesis and the absence of an in vitro reconstitution system. Form I Rubisco, found in higher plants, algae, and cyanobacteria, is a hexadecamer composed of eight large (50-kD) and eight small (13-15 kD) subunits, denoted here as LS and SS, respectively. The genes encoding LS (rbcL) and SS (RBCS) are located in the chloroplast and nuclear genomes, respectively. SS is expressed as a preprotein that is translocated into the chloroplast, where its signal peptide is cleaved prior to its assembly with LS (Nishimura et al., 2008). The two subunits accumulate stoichiometrically in the chloroplast, a phenomenon that is mediated by feedback inhibi...

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Section: Rubisco Subunit Genes Are Normally Transcribed and Translatementioning

confidence: 65%

Ribulose-1,5-Bis-Phosphate Carboxylase/Oxygenase Accumulation Factor1 Is Required for Holoenzyme Assembly in Maize

et al. 2012

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“…Recent experiments, with S1 nuclease protection probes which extend. further upstream from the coding region, uncovered a second maize rbcL transcript with its 5' terminus at position -300 (21,41). Both transcripts were detected (Fig.…”

Section: Resultsmentioning

confidence: 89%

“…Northern hybridization analyses of transcripts from a broad spectrum of angiosperms indicate that the rbcL gene is generally transcribed as a 1.6-kilobase mRNA, although larger transcripts are also observed (46). Si nuclease protection experiments have revealed the presence of two 5' termini for the rbcL RNAs of maize, barley, spinach, and peas (16,21,41,49,62.) Tobacco has a single rbcL RNA with its 5' end at position -180 relative to the coding region (53).…”

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confidence: 99%

In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

Hanley‐Bowdoin¹,

Orozco²,

Chua³

1985

Mol. Cell. Biol.

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The large subunit gene (rbcL) of ribulose 1,5-bisphosphate carboxylase was transcribed in vitro by using maize and pea chloroplast extracts and a cloned plastid DNA template containing 172 base pairs (bp) of the maize rbcL protein-coding region and 791 bp of upstream sequences. Three major in vitro RNA species were synthesized which correspond to in vivo maize rbcL RNAs with 5' termini positioned 300, 100 to 105, and 63 nucleotides upstream of the protein-coding region. A deletion of 109 bp, including the "-300" 5' end (the 5' end at position -300), depressed all rbcL transcription in vitro. A plasmid DNA containing this 109-bp fragment was sufficient to direct correct transcription initiation in vitro. A cloned template, containing 191 bp of plastid DNA which includes the -105 and -63 rbcL termini, did not support transcription in vitro. Exogenously added -300 RNA could be converted to the -63 transcript by maize chloroplast extract. These results established that the -300 RNA is the primary maize rbcL transcript, the -63 RNA is a processed form of the -300 transcript, and synthesis of the -105 RNA is dependent on the -300 region. The promoter for the maize rbcL gene is located within the 109 bp flanking the -300 site. Mutagenesis of the 109-bp chloroplast sequence 11 bp upstream of the -300 transcription initiation site reduced rbcL promoter activity in vitro.Ribulose 1,5-bisphosphate carboxylase (RUBISCO) catalyzes CO2 fixation, the first step of the Calvin cycle (40). In higher plants, the chloroplast enzyme is composed of eight identical, catalytic polypeptides of 50,000 to 60,000 kilodaltons and eight smaller polypeptides of 12,000 to 20,000 kilodaltons (2). The small subunit, whose function is unknown, is encoded in the nucleus by a small multigene family (7,14,19). The large subunit gene (rbcL) is present as a single copy on the multicopy chloroplast genome (5). Under most conditions, the synthesis of the two RUBISCO subunits is coordinated (55) and regulated by light (15, 50) and cytokinins (23). In maize and other C4 plants, RUBISCO expression is leaf cell-type specific (30). The maize mRNAs encoding the two subunits are present in bundle sheath cells but not in mesophyll cells, which assimilate CO2 via ribulose 1,5-bisphosphate and C4 dicarboxylic acids, respectively (11,35).In chloroplast genomes of higher plants, the genes for the large subunit of RUBISCO and the beta subunit of ATP synthetase (atpB) are adjacent and transcribed divergently (31, 60). The 5' ends of the two genes are separated by approximately 150 base pairs (bp) (52). Northern hybridization analyses of transcripts from a broad spectrum of angiosperms indicate that the rbcL gene is generally transcribed as a 1.6-kilobase mRNA, although larger transcripts are also observed (46). Si nuclease protection experiments have revealed the presence of two 5' termini for the rbcL RNAs of maize, barley, spinach, and peas (16,21,41,49,62.) Tobacco has a single rbcL RNA with its 5' end at position -180 relative to the coding region (53). In maize ...

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“…A and by 5' processing in higher-plant chloroplasts. For example, rbcL and psbB mRNAs each have two 5' ends, one of which is generated by processing (8,10,21,32). Spinach atpB mRNA has five 5' termini, four of which result from alternative initiation sites.…”

Section: Resultsmentioning

confidence: 99%

petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

Sakamoto¹,

Sturm²,

Kindle³

et al. 1994

Mol. Cell. Biol.

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Complex processing of primary transcripts occurs during the expression of higher-plant chloroplast genes. In Chlamydomonas reinhardtii, most chloroplast genes appear to possess their own promoters, rather than being transcribed as part of multicistronic operons. By generating specific deletion mutants, we show that petD, which encodes subunit IV of the cytochrome b6lf complex, has an RNA processing site that is required for accumulation of monocistronic petD mRNA in petD promoter deletion mutants; in such mutants, transcription ofpetD originates from the upstream petA promoter. The 5' ends of transcripts initiated at the petD promoter are probably also generated by processing, since the 5' end of monocistronic petD mRNA is the same in wild-type strains as it is in the petD promoter mutants. The location and function of the processing site were further examined by inserting petD-uidAl fusion genes into the chloroplast genome (uid4 is an Escherichia coli gene that encodes ,-glucuronidase). When a promoterless petD-uidA fusion gene was inserted downstream of petA, a monocistronic uid4 transcript accumulated, which was apparently initiated at the petA promoter and was processed at a site corresponding precisely to the petD mRNA 5' end. When a construct including only sequences downstream of +25 relative to the mature mRNA 5' end was inserted into the same site, a dicistronic petA-uidA4 transcript accumulated but no monocistronic uid4 transcript could be detected, suggesting that a processing site lies at least partially within the region from -1 to +25. ,1-Glucuronidase activity was not detected in transformants that accumulated only the dicistronic petA-uid4 transcript, suggesting that the first 25 bp of the 5' untranslated region are required for translation initiation. One explanation for this translational defect is that Chlamydomonas chloroplasts cannot translate the second coding region of some dicistronic messages.Higher-plant chloroplast genes are organized as operons that are transcribed into polycistronic primary transcripts. Splicing and other endo-and exonucleolytic cleavages subsequently occur to generate mature transcripts (1,9,12,32). One such operon in higher plants contains the petD gene, which encodes subunit IV of the cytochrome bdlf complex. Its genomic location is conserved in higher plants (13,30) and cyanobacteria (15); however, its mode of expression varies. In spinach chloroplasts, a primary transcript containing the psbB, psbH, petB, and petD coding regions is processed into 18 RNA species, including monocistronicpsbB andpsbH transcripts and a dicistronic petB-petD transcript (32). As no monocistronic petD mRNA has been detected in spinach chloroplasts, the coding region is presumed to be translated from the dicistronic message. The transcription ofpetD RNA in maize chloroplasts also occurs as part of the psbB operon, but the major translatable form of the petD message is monocistronic. Nonetheless, polycistronic transcripts can be immunoprecipitated from maize polysomes with subunit IV-spe...

show abstract

Characterization of the mRNA transcripts of the maize, ribulose-1,5-bisphosphate carboxylase, large subunit gene

Cited by 24 publications

References 30 publications

Ribulose-1,5-Bis-Phosphate Carboxylase/Oxygenase Accumulation Factor1 Is Required for Holoenzyme Assembly in Maize

Ribulose-1,5-Bis-Phosphate Carboxylase/Oxygenase Accumulation Factor1 Is Required for Holoenzyme Assembly in Maize

In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

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