Chloroplast Isozyme Research Articles

Genetic Diversity of Four Species of the Genus Astragalus L. Section Cenantrum Bunge (Fabaceae) in the Asian Part of Russia according to the Data of trnL–trnF Chloroplast DNA Variability and Isozyme Markers

Genetic Diversity of Four Species of the Genus Astragalus L. Section Cenantrum Bunge (Fabaceae) in the Asian Part of Russia according to the Data of trnL–trnF Chloroplast DNA Variability and Isozyme Markers

Setaria viridis chlorotic and seedling-lethal mutants define critical functions for chloroplast gene expression.

Deep insights into chloroplast biogenesis have been obtained by mutant analysis; however, in C4 plants a relevant mutant collection has only been developed and exploited for maize. Here, we report the initial characterization of an ethyl methyl sulfonate-induced mutant population for the C4 model Setaria viridis. Approximately 1000 M2 families were screened for the segregation of pale-green seedlings in the M3 generation, and a subset of these was identified to be deficient in post-transcriptional steps of chloroplast gene expression. Causative mutations were identified for three lines using deep sequencing-based bulked segregant analysis, and in one case confirmed by transgenic complementation. Using chloroplast RNA-sequencing and other molecular assays, we describe phenotypes of mutants deficient in PSRP7, a plastid-specific ribosomal protein, OTP86, an RNA editing factor, and cpPNP, the chloroplast isozyme of polynucleotide phosphorylase. The psrp mutant is globally defective in chloroplast translation, and has varying deficiencies in the accumulation of chloroplast-encoded proteins. The otp86 mutant, like its Arabidopsis counterpart, is specifically defective in editing of the rps14 mRNA; however, the conditional pale-green mutant phenotype contrasts with the normal growth of the Arabidopsis mutant. The pnp mutant exhibited multiple defects in 3' end maturation as well as other qualitative changes in the chloroplast RNA population. Overall, our collection opens the door to global analysis of photosynthesis and early seedling development in an emerging C4 model.

Comparison of Plant-Type Phospho enol pyruvate Carboxylases from Rice: Identification of Two Plant-Specific Regulatory Regions of the Allosteric Enzyme

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of primary metabolism in bacteria, algae and vascular plants, and it undergoes allosteric regulation by various metabolic effectors. Rice (Oryza sativa) has five plant-type PEPCs, four cytosolic and one chloroplastic. We investigated their kinetic properties using recombinant proteins and found that, like most plant-type PEPCs, rice cytosolic isozymes were activated by glucose 6-phosphate and by alkaline pH. In contrast, no such activation was observed for the chloroplastic isozyme, Osppc4. In addition, Osppc4 showed low affinity for the substrate phosphoenolpyruvate (PEP) and very low sensitivities to allosteric inhibitors aspartate and glutamate. By comparing the isozyme amino acid sequences and three-dimensional structures simulated on the basis of the reported crystal structures, we identified two regions where Osppc4 has unique features that can be expected to affect its kinetic properties. One is the N-terminal extension; replacement of the extension of Osppc2a (cytosolic) with that from Osppc4 reduced the aspartate and glutamate sensitivities to about one-tenth of the wild-type values but left the PEP affinity unaffected. The other is the N-terminal loop, in which a conserved lysine at the N-terminal end is replaced with a glutamate-alanine pair in Osppc4. Replacement of the lysine of Osppc2a with glutamate-alanine lowered the PEP affinity to a quarter of the wild-type level (down to the Osppc4 level), without affecting inhibitor sensitivity. Both the N-terminal extension and the N-terminal loop are specific to plant-type PEPCs, suggesting that plant-type isozymes acquired these regions so that their activity could be regulated properly at the sites where they function.

Regulation of Nitrogen Assimilation in Green Microalgae

AbstractGreen microalgae have evolved a diversity of regulatory mechanisms for assimilating inorganic and organic nitrogen compounds. Ammonium, which is a preferred nitrogen source under most conditions, is usually assimilated via the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. However, some green microalgae assimilate ammonium via NADP-dependent glutamate dehydrogenase (GDH) under high ammonium conditions (e.g., Chlorella species), or under conditions of restricted nitrogen supply (Stichococcus bacillaris, which possesses a high-affinity NADP-GDH). NAD-dependent glutamate dehydrogenase appears to function mainly under heterotrophic conditions, and when amino acids serve as sources of nitrogen, thus suggesting a catabolic role for this enzyme. In S. bacillaris, when ammonium assimilation occurs chiefly via the GS/GOGAT cycle, GS1 (cytosolic isozyme) appears to be responsible for glutamine synthesis in the dark, and GS2 (chloroplastic isozyme) in the light. S. bacillaris synthesizes GS2 and N...

Effect of leaf position on levels of the chloroplast and cytosolic fructose bisphosphatase isozymes in the pea leaf nucleus.

Immunocytolocalization experiments indicate that nuclear levels of the pea leaf cytosolic fructose bisphosphatase are higher in leaves located near the base of the plant and lower in expanded leaves at the apex. It seems possible that the cytosolic isozyme in the nucleus has a role in tissue aging. In contrast, there is no indication that leaf position or tissue age affects levels of the chloroplastic enzyme in the nucleus. The density of the chloroplast fructose bisphosphatase is higher in the nucleolus than in the nucleoplasm. Conversely, the density of the cytosolic isozyme is slightly higher in the nucleoplasm. Analysis of double immunolabeling experiments indicates that both isozymes are distributed nonrandomly with respect to DNA, and therefore colocalized with DNA, in the nucleus, and that the chloroplast isozyme is also distributed nonrandomly with respect to DNA in the chloroplast.

Non-Random Distribution of Four Chloroplastic Isozymes and the Corresponding Cytosolic Isozymes with respect to DNA in the Pea Leaf Nucleus

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

Subcellular localization, purification, and some catalitic properties of aspartate aminotransferase from Spirodela polyrhiza

Intracellular distribution of aspartate aminotransferase (AAT) in Spirodela polyrhiza (Lemnaceae), strain SJ, has been studied by differential centrifugation. The bulk of the enzyme (73% of total cellular content) was localized in the cytoplasm and 24% activity was localized in chloroplasts. Purified cytoplasmic and chloroplastic isozymes differed by their affinity for substrates. The reaction balance was shifted towards direct and reverse transamination in the cytoplasm and chloroplast, respectively. Competitive inhibition of AAT by excessive substrates and enzyme affinity modulation by certain intermediates of the tricarboxylic acid cycle (isocitrate, succinate, and citrate) were observed. Possible involvement of AAT isozymes in the coordination of carbon and nitrogen metabolism through the regulation of 2-oxoglutarate synthesis and utilization in different cellular compartments is discussed.

Effect of Leaf Position on Levels of the Chloroplast and Cytosolic Phosphofructoaldolase Isozymes in the Pea Leaf Nucleus

Immunocytolocalization experiments indicate that nuclear levels of the pea leaf cytosolic aldolase (EC 4.1.2.13) isozyme are higher in leaves located near the base of the plant and lower in leaves at the apex. In contrast, nuclear levels of the chloroplastic isozyme are highest in the apical leaf. Analysis of double‐immunolabeling experiments indicates that both isozymes are distributed nonrandomly with respect to DNA in the nucleus, and therefore are colocalized with DNA, and that the isozyme in the chloroplast is distributed nonrandomly with respect to DNA, and therefore is colocalized with plastid DNA. It seems possible that the chloroplast and cytosolic aldolase isozymes in the nucleus might function as sensors that link sugar metabolism in the cytoplasm to gene expression.

Both chloroplastic and cytosolic phosphofructoaldolase isozymes are present in the pea leaf nucleus.

In higher plants, fructose bisphosphate aldolase (EC 4.1.2.13) occurs in chloroplast, cytosol, and nucleus. Immunocytolocalization experiments with isozyme-directed antibodies indicate that both chloroplastic and cytosolic aldolase isoforms are present in the pea (Pisum sativum L.) leaf nucleus.

Correlation of viral RNA biosynthesis with glucose-6-phosphate dehydrogenase activity and host resistance.

Changes in glucose-6-phosphate dehydrogenase (G6P DH; EC 1.1.1.49) activity caused by infection of tobacco ( Nicotiana tabacum L.) leaves with potato virus Y (PVY), cucumber mosaic virus, potato virus X, tobacco rattle virus and turnip mosaic virus, the subcellular localisation of G6P DH isozymes in mesophyll protoplasts derived from healthy and PVY-infected tobacco leaves, as well as G6P DH control and the relationship of its isozymes with the degree of tobacco resistance to PVY multiplication, were studied. The activities of G6P DH were markedly increased in locally and systemically infected leaves and the time courses of the activity linearly correlated with those of virus multiplication. In leaves infected with PVY, the activity time courses of the crude and the partially purified G6P DH were coincident. This probably indicates the involvement of coarse regulation of the enzyme. PVY content linearly correlated with enhanced G6P DH activity in leaf discs derived from susceptible, tolerant and resistant cultivars of tobacco. The increased activity of the enzyme in infected protoplasts and plant tissues was predominantly caused by the increased activity of chloroplastic isozymes. This was confirmed by the specific staining of isozymes after electrophoretic separation of chloroplastic proteins of tobacco leaves. These findings enable the degree of resistance to virus multiplication to be quantified for the use of gene manipulation and breeding.

Differential Expression of Genes Encoding Enzymes Involved in Sulfur Assimilation Pathways in Response to Wounding and Jasmonate in Arabidopsis thaliana

Summary Effects of mechanical wounding on levels of transcripts encoding enzymes involved in the sulfur assimilation pathway were examined. RNA blot hybridization showed the sulfate transporter, ATP sulfurylase, adenosine phosphosulfate (APS) kinase, APS reductase and cysteine synthase, all but the first being chloroplastic isozymes differentially responding to wounding, and also to jasmonate, a powerful inducer ofwound responsive gene expression. When plants were exposed to jasmonate, transcripts for APS kinase were increased within 1h after treatment, while those for APS reductase were increased after 3 h. We also found the glucosinolate content to be 2-fold elevated at the latter time point. APS is directed to the secondary metabolic pathway by APS kinase, so that glucosinolates are synthesized, while it is allotted through the action ofAPS reductase to the primary metabolic pathway by which cysteine is synthesized. Consequently, our results suggest that wounded plants primarily deliver available sulfur to synthesize defense-related substances by activating genes involved in the secondary metabolic pathway.

Isolation and characterisation of human genomic sequences encoding cytosolic serine hydroxymethyltransferase

Serine hydroxymethyltransferase (EC 2.1.2. I , SHMT) is a ubiquitous enzyme found in all prokaryotes and eukaryotes. Bacteria have one gene coding for SHMT (XlyA) but in most eukaryotic there are cytosolic and mitochondrial isozyme forms; encoded by different genes @I$ and glyM). In plants there is evidence for a separate chloroplast isozyme [ I ] . The precise role each isozyme plays in the cell is still relatively poorly understood but is thought to be related in part to the compartmentalisation of folate metabolism between the cytosol and the mitochondria [2] In mammals SHMT is found in many different tissue types and expression of the different isoforms is tissue specific The major physiological role of SHMT is the transfer of a one carbon unit to tetrahydrofolate to form N5,Nl O-methylene tetrahydrofolate with the concomitant production of glycine from serine Both glycine and one carbon units are required for de m v o purine biosynthesis and SHMT is also involved in the thymidylate synthase cycle which converts dUMP to dTMP. It has been demonstrated that when human peripheral blood lymphocytes are mitogenically stimulated SHMT activity rises 4 to 9 fold and radiolabel from [3-14C] serine is incorporated into DNA [3,4]. SHMT activity has also been shown to be elevated in certain cancers [ 51. cDNA’s encoding the human cytosolic isozyme [6-81 and mitochondrial isozyme [6] of SHMT have been isolated. An incomplete genomic sequence encoding the human mitochondrial isozyme has also been reported [9]. A pseudogene of human cSHMT has been characterised [lo]. To date no genomic sequences encoding human cSHMT have been described. Human cSHMT cDNA’s which have been isolated are suggestive of alternative splicing producing 3 subforms of the enzyme [ 8 ] . cDNA’s isolated in our laboratory [7] indicate further potential splice sites. In order to characterise hrther the potential for alternative splicing of human cSHMT cDNA’s and to determine any potential regulation of gene expression, genomic sequences encoding human cSHMT are being isolated and characterised. PCR was used to amplify DNA sequences from a human genomic library (Clontech Human Promoter Finder Kit) using the manufacturer’s protocol The library consists of digested human DNA with sequences ligated at each end which can be used to prime one end of the PCR reaction using supplied PCR primers. To prime amplification of the reverse strand, PCR primers were designed based on the known sequence of human cSHMT cDNA. Genomic sequences including introns have been isolated using the primers supplied with the kit and primers complementary to 3’ cSHMT cDNA sequences. The sequence of a 1.2kb amplified genomic fragment has been obtained. The DNA is identical to human cSHMT cDNA but includes two introns. The first intron identified within this sequence occurs between the first and second nucleotides of the codon for glycine 391. The second intron is 358bp long and falls between the I A B C I I ’ I t t t t t t t

Chloroplast DNA and isozyme evidence on the evolution ofSenecio vulgaris (Asteraceae)

Chloroplast DNA (cpDNA) and isozyme variation were analyzed over a range of populations of two infraspecific taxa of the tetraploidSenecio vulgaris. The isozyme data were supportive of the hypothesis that the weedy and cosmopolitanS. vulgaris var.vulgaris is an evolutionary derivative ofS. vulgaris subsp.denticulatus from the coasts of W Europe and montane altitudes in S Spain and Sicily. The two taxa exhibited a very high genetic identity with subsp.denticulatus containing slightly more isozyme diversity than was found in var.vulgaris. — Three cpDNA haplotypes (A, B, C) already known from other Mediterranean diploid species ofSenecio were resolved in var.vulgaris, and an additional fourth haplotype (E) was found in subsp.denticulatus. Two alternative hypotheses were chosen to account for the origin and maintenance of the observed cpDNA composition ofS. vulgaris. It either reflects (1) the retention of an ancestral polymorphism which stems from the recurrent and polytopic formation of ancestral tetraploid lineages; or (2)S. vulgaris originally was characterized by haplotype E, and haplotypes A, B and C were acquired through repeated introgressive hybridization with related diploid species. The finding that very low levels of nuclear (isozyme) diversity were present in both taxa ofS. vulgaris examined supports the second of these two hypotheses; however, more detailed analysis of nuclear genetic diversity is required before a firm conclusion can be reached on this matter.

Congruence of chloroplast DNA restriction site characters with morphological and isozyme data in Solanum sect. Lasiocarpa

Chloroplast DNA restriction site characters were analyzed cladistically in species of Solanum sect. Lasiocarpa (subg. Leptostemonum), and results were compared with previously published analyses of morphological and isozyme characters. Cladistic analysis of the chloroplast DNA restriction site characters resulted in a single tree in which two main clades were detected: one comprised three morphologically distinct lowland Amazonian species, and the other included the Andean and the two Asian species. Character congruence among the three data sets was assessed with a previously described index and with a permutation test derived from this index. Taxonomic congruence was evaluated with a measure modified from that of another researcher. The three assessments of congruence indicate that the chloroplast DNA and isozyme analyses are the most incongruent, but they differ as to their evaluation of relative (in)congruence for the other comparisons. Combining the three data sets resulted in two most parsimonious trees whose topologies incorporate elements from each of the separate analyses. The combined chloroplast DNA and morphological analyses support a previously proposed hypothesis that the two Asian species are sister taxa and of a single origin, rather than having originated separately from two distinct South American groups. Key words: Solanum, chloroplast DNA, congruence, combined data, cladistics.

Analysis of the primary structure of the chloroplast isozyme of triosephosphate isomerase from rye leaves by protein and cDNA sequencing indicates a eukaryotic origin of its gene

The primary structure of the chloroplast isozyme of triosephosphate isomerase from rye leaves was identified by protein and cDNA sequencing and compared to the deduced amino acid sequence of a cDNA for the cytosolic isozyme. The mature cytosolic and chloroplast isozyme proteins share 64% amino acid sequence identity. The cDNA for the chloroplast isozyme codes for a precursor protein consisting of an N-terminal transit peptide of M r 4351 and a mature subunit of M r 27282. Southern blot analysis indicates that the two rye isozymes are encoded by two independent single genes. Amino acid residues or sequence regions of basic functional relevance in known triosephosphate isomerases are strictly conserved in the chloroplast isozyme. The chloroplast isozyme contains 6 cysteine residues, instead of 4 in the cytosolic isozyme. A cysteine at position 143 of the chloroplast isozyme appears to be modified. Phylogenetic trees constructed on the basis of sequence comparisons for triosephosphate isomerases from different species of all major taxonomic groups indicate that the chloroplast isozyme is much more closely related to eukaryotic cytosolic enzymes than to eubacterial enzymes. The results indicate that the nuclear gene for the chloroplast isozyme originated with that for the cytosolic isozyme through duplication of an ancestral eukaryotic gene, rather than through gene transfer from a prokaryotic endosymbiont.

Characterization of cis-acting elements in light regulation of the nuclear gene encoding the A subunit of chloroplast isozymes of glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana.

We have characterized cis-acting elements involved in light regulation of the nuclear gene (GapA) encoding the A subunit of chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Arabidopsis thaliana. Our results show that a 1.1-kb promoter fragment of the GapA gene is sufficient to confer light inducibility and organ specificity in transgenic Nicotiana tabacum (tobacco) plants, using the beta-glucuronidase gene of Escherichia coli as the reporter gene. Deletion analysis indicates that the -359 to -110 bp region of the GapA gene is necessary for light responsiveness. Within this region there are three copies of a decamer repeat (termed the Gap box) having the consensus sequence 5'-CAAATGAA(A/G)A-3', which has not been characterized in the promoter regions of other light-regulated genes. A deletion (to -247) producing loss of one copy of these elements from the GapA promoter reduces light induction by two- to threefold compared with a promoter deletion (to -359) with all three Gap boxes present, while deletion of all three Gap boxes (to -110) abolishes light induction completely. Gel mobility shift experiments using tobacco nuclei as the source of nuclear proteins show that GapA promoter fragments that contain these repeats bind strongly to a factor in the nuclear extract and that binding can be abolished by synthetic competitors consisting only of a monomer or dimer of the Gap box. Furthermore, a trimer, dimer, and monomer of the Gap box show binding activity and, like the authentic GapA promoter-derived probes, show binding activities that are correlated with Gap box copy number. These results strongly suggest that these repeats play important roles in light regulation of the GapA gene of A. thaliana.

Separate Assays Specific for Ascorbate Peroxidase and Guaiacol Peroxidase and for the Chloroplastic and Cytosolic Isozymes of Ascorbate Peroxidase in Plants

Ascorbate (AsA) peroxidase can be inactivated both by p-chloromercuribenzoate and by the depletion of AsA but guaiacol peroxidases, such as horseradish peroxidase, cannot. The cytosolic isozymes of AsA peroxidase are less sensitive to depletion of AsA than the chloroplastic isozymes, which include stromal [Chen and Asada (1989) Plant Cell Physiol. 30: 987] and thyla-koid-bound [Miyake and Asada (1992) Plant Cell Physiol. 33: 541] enzymes. Exploring these properties, we established simple methods for separate assays of AsA peroxidase and guaiacol peroxidase and of the three isozymes of AsA peroxidase in plant extracts. These methods were used to characterize the guaiacol peroxidases and isozymes of AsA peroxidase in plants and algae.

Isolation and characterization of a soybean cDNA clone encoding the plastid form of aspartate aminotransferase

Five aspartate aminotransferase (EC 2.6.1.1; AAT) isozymes were identified in soybean seedling extracts and designated AAT1 to AAT5 based on their rate of migration on non-denaturing electrophoretic gels. AAT1 was detected only in extracts of cotyledons from dark-grown seedlings. AAT3 and AAT4 were detected in crude extracts of leaves and in cotyledons of seedlings grown in the light. AAT2 and AAT5 were detected in all tissues examined. A soybean leaf cDNA clone, pSAT17, was identified by hybridization to a carrot AAT cDNA clone at low stringency. pSAT17 had an open reading frame which could encode a 50,581 Da protein. Alignment of the deduced amino acid sequence from the pSAT17 open reading frame with mature AAT protein sequences from rat disclosed a 60 amino acid N-terminal extension in the pSAT17 protein. This extension had characteristics of a plastid transit peptide. A plasmid, pEXAT17, was constructed which encoded the mature protein lacking the putative chloroplast transit polypeptide. Transformed Escherichia coli expressed a functional soybean AAT isozyme, which comigrated with the soybean AAT5 isozyme during agarose gel electrophoresis. Differential sucrose gradient sedimentation of soybean extracts indicated that AAT5 specifically cofractionated with chloroplasts. Antibodies raised against the pEXAT17-encoded AAT protein specifically reacted with the AAT5 isozyme of soybean and not with any of the other isozymes, indicating that the soybean cDNA clone, pSAT17, encodes the chloroplast isozyme, AAT5.

Chloroplast and cytoplasmic enzymes: Isolation and sequencing of cDNAs coding for two distinct pea chloroplast aldolases

Two cDNAs which correspond to two very similar Class I aldolases have been isolated from a pea ( Pisum sativum L.) cDNA library. With the exception of one codon they match the experimentally determined N-terminal sequence of a pea chloroplast aldolase. The deduced C-terminal sequence of one of these clones is unique among Class I aldolases. The deduced C-terminus of the other is more like the C-terminus of other eucaryotic Class I aldolases. Comparisons of sequence homology suggest that the pea chloroplast isozymes are only marginally more closely related to the anaerobically induced plant aldolases than to aldolases from animals.

The tomato Cu,Zn superoxide dismutase genes are developmentally regulated and respond to light and stress

The expression of the two Cu,Zn superoxide dismutase (SOD) genes of tomato was followed in different organs and plant developmental stages at the transcript and enzymatic activity levels. The cDNA clones used as probes code for the chloroplast Cu,Zn SOD (clone T1) and the cytosolic Cu,Zn SOD (clone P31). The two genes were found to display distinct expression patterns. While the T1 transcript was rare or absent from roots, stems and ripening fruits, the P31 transcript was very abundant in these organs. Shoot tips, flower buds, seedlings and young leaves contained high levels of the two mRNAs. During leaf expansion, the levels of both transcripts diminish markedly. Despite the diminished presence of transcripts, SOD activity levels of the corresponding cytosolic and chloroplast isozymes accumulated and were sustained throughout leaf expansion. In non-photosynthetic organs, the SOD-3 (cytosolic) isozyme contained most of the activity, while in the expanded leaf the SOD-1 (chloroplast) isozyme was more abundant. Light-regulated accumulation of both the P31 transcript (1.7-fold) and the T1 transcript (3-fold) was observed upon light exposure of etiolated seedlings. However, only SOD-1 activity was observed to increase, after a lag of a few hours. The levels of both transcripts increased in response to paraquat and mechanical wounding. The level of the cytosolic transcript and the respective isozyme activity increased dramatically during prolonged drought stress while the chloroplast transcript remained unaffected. The expression of both genes was enhanced by spraying tomato plants with ethephon--a compound that releases ethylene. Our data show that the expression of Cu,Zn SOD genes in tomato is modulated in response to a variety of factors and suggest the importance of oxyradical toxicity as well as the role of SOD in the defence mechanism of plants exposed to stress.