Increased zinc content in transplastomic tobacco plants expressing a polyhistidine-tagged Rubisco large subunit

Rubisco is a hexadecameric enzyme composed of two subunits: a small subunit (SSU) encoded by a nuclear gene (rbcS), and a large subunit (LSU) encoded by a plastid gene (rbcL). Due to its high abundance, Rubisco represents an interesting target to express peptides or small proteins as fusion products at high levels. In an attempt to modify the plant metal content, a polyhistidine sequence was fused to Rubisco, the most abundant protein of plants. Plastid transformation was used to express a polyhistidine (6x) fused to the C-terminal extremity of the tobacco LSU. Transplastomic tobacco plants were generated by cotransformation of polyethylene glycol-treated protoplasts using two vectors: one containing the 16SrDNA marker gene, conferring spectinomycin resistance, and the other the polyhistidine-tagged rbcL gene. Homoplasmic plants containing L8-(His)6S8 as a single enzyme species were obtained. These plants contained normal Rubisco amounts and activity and displayed normal photosynthetic properties and growth. Interestingly, transplastomic plants accumulated higher zinc amounts than the wild-type when grown on zinc-enriched media. The highest zinc increase observed exceeded the estimated chelating ability of the polyhistidine sequence, indicating a perturbation in intracellular zinc homeostasis. We discuss the possibility of using Rubisco to express foreign peptides as fusion products and to confer new properties to higher plants.     

羊草光合作用相关基因的克隆与分析

在构建了羊草叶片cDNA文库的基础上,利用M13载体通用引物筛选其亚文库,挑选阳性克隆进行测序,将测序结果在NCBI基因库中进行比对,得到一个Rubisco大亚基基因全长序列和Rubisco小亚基基因部分序列,并对其核苷酸及其编码的氨基酸序列进行分析。结果显示,Rubisco大亚基基因长度为1 796 bp,与禾本科大麦、小麦、野雀麦、粗山羊草、旱麦草、异形花草、黑麦等的核苷酸序列同源性达98%以上;羊草的Rubisco小亚基基因部分序列含有一个开放阅读框,其长度为186 bp,编码61个氨基酸,与禾本科的小麦、大麦、燕麦、黑麦以及扁穗雀麦Rubisco小亚基基因氨基酸序列的同源性分别为93%、93%、91%、91%、92%。羊草Rubisco基因的克隆与分析有利于进一步研究其光合作用效率。     

Polypeptide substrate specificity of PsLSMT. A set domain protein methyltransferase

Rubisco large subunit methyltransferase (PsLSMT) is a SET domain protein responsible for the trimethylation of Lys-14 in the large subunit of Rubisco. The polypeptide substrate specificity determinants for pea Rubisco large subunit methyltransferase were investigated using a fusion protein construct between the first 23 amino acids from the large subunit of Rubisco and human carbonic anhydrase II. A total of 40 conservative and non-conservative amino acid substitutions flanking the target Lys-14 methylation site (positions P(-3) to P(+3)) were engineered in the fusion protein. The catalytic efficiency (k(cat)/K(m)) of PsLSMT was determined using each of the substitutions and a polypeptide consensus recognition sequence deduced from the results. The consensus sequence, represented by X-(Gly/Ser)-(Phe/Tyr)-Lys-(Ala/Lys/Arg)-(Gly/Ser)-pi, where X is any residue, Lys is the methylation site, and pi is any aromatic or hydrophobic residue, was used to predict potential alternative substrates for PsLSMT. Four chloroplast-localized proteins were identified including gamma-tocopherol methyltransferase (gamma-TMT). In vitro methylation assays using PsLSMT and a bacterially expressed form of gamma-TMT from Perilla frutescens confirmed recognition and methylation of gamma-TMT by PsLSMT in vitro. RNA interference-mediated knockdown of the PsLSMT homologue (NtLSMT) in transgenic tobacco plants resulted in a 2-fold decrease of alpha-tocopherol, the product of gamma-TMT. The results demonstrate the efficacy of consensus sequence-driven identification of alternative substrates for PsLSMT as well as identification of functional attributes of protein methylation catalyzed by LSMT.     

Linked Rubisco subunits can assemble into functional oligomers without impeding catalytic performance

Although transgenic manipulation in higher plants of the catalytic large subunit (L) of the photosynthetic CO2-fixing enzyme ribulose 1,5-bisphospahte carboxylase/oxygenase (Rubisco) is now possible, the manipulation of its cognate small subunit (S) is frustrated by the nuclear location of its multiple gene copies. To examine whether L and S can be engineered simultaneously by fusing them together, the subunits from Synechococcus PCC6301 Rubisco were tethered together by different linker sequences, producing variant fusion peptides. In Escherichia coli the variant PCC6301 LS fusions assembled into catalytically functional octameric ([LS]8) and hexadecameric ([[LS]8]2) quaternary structures that excluded the integration of co-expressed unfused S. Assembly of the LS fusions into Rubisco complexes was impaired 50-90% relative to the assembly of unlinked L and S into L8S8 enzyme. Assembly in E. coli was not emulated using tobacco SL fusions that accumulated entirely as insoluble protein. Catalytic measurements showed the CO2/O2 specificity, carboxylation rate, and Michaelis constants for CO2 and ribulose 1,5-bisphosphate for the cyanobacterial Rubisco complexes comprising fusions where the S was linked to the N terminus of L closely matched those of the wild-type L8S8 enzyme. In contrast, the substrate affinities and carboxylation rate of the Rubisco complexes comprising fusions where L was fused to the N terminus of S or a six-histidine tag was appended to the C terminus of L were compromised. Overall this work provides a framework for implementing an alternative strategy for exploring simultaneous engineering of modified, or foreign, Rubisco L and S subunits in higher plant plastids.     

The gene for the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit relocated to the plastid genome of tobacco directs the synthesis of small subunits that assemble into Rubisco

To assess the extent to which a nuclear gene for a chloroplast protein retained the ability to be expressed in its presumed preendosymbiotic location, we relocated the RbcS gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to the tobacco plastid genome. Plastid RbcS transgenes, both with and without the transit presequence, were equipped with 3' hepta-histidine-encoding sequences and psbA promoter and terminator elements. Both transgenes were transcribed abundantly, and their products were translated into small subunit polypeptides that folded correctly and assembled into the Rubisco hexadecamer. When present, either the transit presequence was not translated or the transit peptide was cleaved completely. After assembly into Rubisco, transplastomic small subunits were relatively stable. The hepta-histidine sequence fused to the C terminus of a single small subunit was sufficient for isolation of the whole Rubisco hexadecamer by Ni(2)+ chelation. Small subunits produced by the plastid transgenes were not abundant, never exceeding approximately 1% of the total small subunits, and they differed from cytoplasmically synthesized small subunits in their N-terminal modifications. The scarcity of transplastomic small subunits might be caused by inefficient translation or assembly.     

小球藻Rubisco在叶绿体中的免疫金标定位

应用免疫技术对Rubisco在中国小球藻(Chlorellaspp.640909)叶绿体中进行了分子定位及Native-PAGE电泳、SDS-PAGE电泳及其Westen印迹分析,并对小球藻淀粉核(Pyrenoid)超微结构进行了观察.结果显示Native-PAGE电泳图谱主要为一条主带,Westen印迹反应证明该条带即为Rubisco酶,SDS-PAGE电泳及其Western印迹图谱显示Rubisco大亚基分子量大约为55kD.中国小球藻淀粉核为椭圆形,被淀粉鞘所包围,中央有一条由2个类囊体组成的纵向通道,并在蛋白核内段处稍膨胀.淀粉核与叶绿体基质存在多处联系.免疫分子定位显示Rubisco大亚基和全酶分子主要分布于叶绿体的淀粉核上,且Rubisco在淀粉鞘部位也有少量分布,极少部分分布在叶绿体基质中,表明叶绿体淀粉核与光合作用关系密切.Rubisco聚集于淀粉核可能有利于藻类对CO2固定.     

Rubisco small and large subunit N-methyltransferases. Bi- and mono-functional methyltransferases that methylate the small and large subunits of Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)is methylated at the alpha-amino group of the N-terminal methionine of the processed form of the small subunit (SS), and at the epsilon-amino group of lysine-14 of the large subunit (LS) in some species. The Rubisco LS methyltransferase (LSMT) gene has been cloned and expressed from pea and specifically methylates lysine-14 of the LS of Rubisco. We determine here that both pea and tobacco Rubisco LSMT also exhibit (alpha)N-methyltransferase activity toward the SS of Rubisco, suggesting that a single gene product can produce a bifunctional protein methyltransferase capable of catalyzing both (alpha)N-methylation of the SS and (epsilon)N-methylation of the LS. A homologue of the Rubisco LSMT gene (rbcMT-S) has also been identified in spinach that is closely related to Rubisco LSMT sequences from pea and tobacco. Two mRNAs are produced from rbcMT-S, and both long and short forms of the spinach cDNAs were expressed in Escherichia coli cells and shown to catalyze methylation of the alpha-amino group of the N-terminal methionine of the SS of Rubisco. Thus, the absence of lysine-14 methylation in species like spinach is apparently a consequence of a monofunctional protein methyltransferase incapable of methylating Lys-14, with activity limited to methylation of the SS.     

Immunogold localization of ribulose-1,5-bisphosphate carborylsae/oxygenase in chloroplasts of Chlorella]

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a first key enzyme in the Calvin Circle of plant cell photosynthesis. This paper mainly studied gold immunolocalization of Rubisco of Chlorella spp. 640909, and the Native-PAGE and, SDS-PAGE and Western bloting analysis, as well as the observation to pyrenoid ultra structure. The Native-PAGE result showed a main band, evidenced as the Rubisco band by the Western blot with the antibody against the Rubisco from C. prototothecoides, The special immunoacton of Rubisco from Chlorella spp. 640909 and the antibody to large subunit of Rubisco from C. prothecoides showed the large subunit proteins of Rubisco in the two species of Chlorella shared the high homology. The SDS-PAGE and Western blotting maps showed the molecule weight of the large subunit of Rubisco of Chlorella spp. 640909 was about 55 KD. The shape of pyrenoid ultra structure of the electronic microscope was oblong, and was embedded in starch sheath, with 2 swelling thylakoids through out a center portrait channel of the pyrenoid. There were some connections between pyrenoid and the chloroplast stroma. The distribution of the large subunits and the whole Rubisco in the chloroplast of Chrolella spp. 640909 was studied by immunoelectron microscopy by embedded sections with antibody to large subunit and whole enzyme followed by second antibody, goad anti-rabbit immunoglobulin G conjugated to 10 nm gold particles(Sigma production). The result showed the antibodies against large subunit and whole enzyme heavily labeled the pyrenoid, as well as starch sheath region, whereas the thylakoid region of the plastid was lightly labeled. And the whole Rubisco antibody labeled the pyrenoid surface more heavily than the large subunit antibody did. It is demonstrated the pyrenoid and starch sheath have the photosynthesis function. Rubisco concentrating in pyrenoid and starch sheath is valuable to fix CO2 for photosynthesis in algae.     

Construction of a tobacco master line to improve Rubisco engineering in chloroplasts

The inability to assemble Rubisco from any photosynthetic eukaryote within Escherichia coli has hampered structure-function studies of higher plant Rubisco. Precise genetic manipulation of the tobacco chloroplast genome (plastome) by homologous recombination has facilitated the successful production of transplastomic lines that have either mutated the Rubisco large subunit (L) gene, rbcL, or replaced it with foreign variants. Here the capacity of a new tobacco transplastomic line, (cm)trL, to augment future Rubisco engineering studies is demonstrated. Initially the rbcL was replaced with the selectable marker gene, aadA, and an artificial codon-modified (cm)rbcM gene that codes for the structurally novel Rubisco dimer (L(2), approximately 100 kDa) from Rhodosprillum rubrum. To obtain (cm)trL, the aadA was excised by transiently introducing a T-DNA encoding CRE recombinase biolistically. Selection using aadA enabled transplantation of mutated and wild-type tobacco Rubisco genes into the (cm)trL plastome with an efficiency that was 3- to 10-fold higher than comparable transformations into wild-type tobacco. Transformants producing the re-introduced form I tobacco Rubisco variants (hexadecamers comprising eight L and eight small subunits, approximately 520 kDa) were identified by non-denaturing PAGE with fully segregated homoplasmic lines (where no L(2) Rubisco was produced) obtained within 6-9 weeks after transformation which enabled their Rubisco kinetics to be quickly examined. Here the usefulness of (cm)trL in more readily examining the production, folding, and assembly capabilities of both mutated tobacco and foreign form I Rubisco subunits in tobacco plastids is discussed, and the feasibility of quickly assessing the kinetic properties of those that functionally assemble is demonstrated.     

Chimaeras of the Rubisco Large Subunit from Wheat and Anacystis do not Assemble into Active Enzyme in E. coli

A new restriction site was engineered in the cloned gene codingfor the large subunit polypeptide of ribulose 1, 5-bisphosphatecarboxylase (Rubisco) of the cyanobacterium Anacystis nidulans.This change resulted in the mutation of a phenylalanine residueto an isoleucine residue in the encoded polypeptide but hadno effect on the assembly or biochemical properties of Rubiscocontaining the polypeptide. The mutation was in a loop regionlinking highly structured domains at the N and C termini ofthe complete large subunit. Using the new restriction site, and a corresponding EcoRl restrictionsite in the cloned gene for the native large subunit polypeptideof wheat Rubisco, chimaeric genes were made encoding the polypeptidewith either the 140 residues of the N-terminal part of the wheatlarge subunit fused to the 336 residues forming the C-terminalregion of the A. nidulans large subunit, or the alternativeof 136 residues comprising of the N-terminal chains of A. nidulanssubunit and the 338 residue chain at the C-terminus of the wheatlarge subunit polypeptide. The chimaeric proteins expressedin E. coli, together with the small subunit of the A. nidulansRubisco, formed an insoluble inactive aggregate mainly in inclusionbodies. The possible reasons for the failure to obtain activeholoenzyme are discussed. Key words: Rubisco, protein engineering, site-directed mutagenesis     

A protein with an inactive pterin‐4a‐carbinolamine dehydratase domain is required for Rubisco biogenesis in plants

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin–Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co‐factor of hepatocyte nuclear factor 1 (DCoH)/pterin‐4α‐carbinolamine dehydratases (PCD)‐like protein is the causative mutation in a seedling‐lethal, Rubisco‐deficient mutant named Rubisco accumulation factor 2 (raf2‐1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high‐molecular weight complex, the formation of which requires a specific chaperonin 60‐kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross‐linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co‐immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co‐immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins.     

A point mutation in the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase affects holoenzyme assembly in Nicotiana tabacum.

In photosynthetic eukaryotes, the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is composed of eight large and eight small subunits. Chloroplast-coded large subunits are found in association with chaperonins (binding proteins) of 60-61 kd to form a high mol. wt pre-assembly complex (B-complex). We have isolated a heterotrophic, maternally-inherited mutant from Nicotiana tabacum var. Xanthi which accumulates the B-complex but contains no Rubisco holoenzyme. The B-complex of the mutant dissociates in the presence of ATP, as does that of the wild-type. Processing of the nuclear-coded small subunit takes place in the mutant and neither large nor small subunits accumulate. The large subunit gene from mutant and wild-type plants was cloned and sequenced. A single nucleotide difference was found between them predicting an amino acid change of serine to phenylalanine at position 112 in the mutant. Based on the resolved structure of N.tabacum Rubisco, it is argued that the alteration at position 112 prevents holoenzyme assembly by interfering with large subunit assembly.     

油菜BnrbcS基因超表达提高拟南芥种子重量和含油量

为深入分析光合作用关键酶二磷酸核酮糖羧化酶（Rubisco）小亚基在油菜等＂绿色种子＂发育过程中的作用,采用RT-PCR技术从油菜种胚中克隆了一个含Rubisco小亚基全长编码区的cDNA序列,命名为BnrbcS（GenBank登录号DQ242646）。BnrbcS编码181个氨基酸残基,其推导的氨基酸序列中包含典型的Rubisco小亚基功能域并与其他高等植物Rubisco小亚基具有很高的同源性,暗示BnrbcS基因产物与拟南芥等植物的Rubisco小亚基在结构和功能上可能十分相似。除了在叶、子叶、角果皮等光合器官中有很高表达外,BnrbcS在贮藏物质高速积累时期的未成熟油菜种子中亦有中等水平的表达,且其＂钟型＂表达模式与一些脂肪酸合成酶系基因的表达模式相类似。将NAPIN启动子驱动的BnrbcS种子特异超表达结构转入拟南芥,转化株系的种子含油量和种子重量有一定程度提高,显示BnrbcS在调控种子油脂等贮藏物质积累过程中具有作用。     

The role of the N-terminus of the large subunit of ribulose-bisphosphate carboxylase investigated by construction and expression of chimaeric genes

The genes for the large and small subunits of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Anacystis nidulans have been expressed in Escherichia coli under the control of the lac promoter to produce active enzyme. The enzyme can be purified from the cells to yield up to 200 mg Rubisco/l cultured bacteria, and is indistinguishable from the enzyme extracted from A. nidulans. In order to investigate the role of the N-terminus of the large subunit in catalysis, chimaeric genes were constructed where the DNA coding for the 12 N-terminal amino acids in A. nidulans was replaced by DNA encoding the equivalent, but poorly conserved, region of either the wheat or maize large subunit. These genes, in constructs also containing the gene for the A. nidulans small subunit, were expressed in E. coli and produced enzymes with similar catalytic properties to the wild-type Rubisco of A. nidulans. In contrast, when the N-terminal region of the large subunit was replaced by unrelated amino acids encoded by the pUC8 polylinker, enzyme activity of the expressed protein was reduced by 90% under standard assay conditions, due to an approximately tenfold rise in the Km for ribulose 1,5-bisphosphate. This confirms that the N-terminus of the large subunit has a function in catalysis, either directly in substrate binding or in maintaining the integrity of the active site.