Soybean Chloroplasts Research Articles

GmPGL2, Encoding a Pentatricopeptide Repeat Protein, Is Essential for Chloroplast RNA Editing and Biogenesis in Soybean.

Chloroplast biogenesis and development are highly complex processes requiring interactions between plastids and nuclear genomic products. Pentatricopeptide repeat (PPR) proteins play an essential role in the development of chloroplasts; however, it remains unclear how RNA editing factors influence soybean development. In this study, a Glycine max pale green leaf 2 mutant (Gmpgl2) was identified with decreased chlorophyll contents. Genetic mapping revealed that a single-nucleotide deletion at position 1949 bp in the Glyma.05g132700 gene in the Gmpgl2 mutant, resulting in a truncated GmPGL2 protein. The nuclear-encoded GmPGL2 is a PLS-type PPR protein that localizes to the chloroplasts. The C-to-U editing efficiencies of rps16, rps18, ndhB, ndhD, ndhE, and ndhF were reduced in the Gmpgl2 mutant. RNA electrophoresis mobility shift assay (REMSA) analysis further revealed that GmPGL2 binds to the immediate upstream sequences at RNA editing sites of rps16 and ndhB in vitro, respectively. In addition, GmPGL2 was found to interact with GmMORF8, GmMORF9, and GmORRM6. These results suggest that GmPGL2 participates in C-to-U RNA editing via the formation of a complex RNA editosome in soybean chloroplasts.

Salt stress affects mRNA editing in soybean chloroplasts.

Soybean, a crop known by its economic and nutritional importance, has been the subject of several studies that assess the impact and the effective plant responses to abiotic stresses. Salt stress is one of the main environmental stresses and negatively impacts crop growth and yield. In this work, the RNA editing process in the chloroplast of soybean plants was evaluated in response to a salt stress. Bioinformatics approach using sRNA and mRNA libraries were employed to detect specific sites showing differences in editing efficiency. RT-qPCR was used to measure editing efficiency at selected sites. We observed that transcripts of NDHA, NDHB, RPS14 and RPS16 genes presented differences in coverage and editing rates between control and salt-treated libraries. RT-qPCR assays demonstrated an increase in editing efficiency of selected genes. The salt stress enhanced the RNA editing process in transcripts, indicating responses to components of the electron transfer chain, photosystem and translation complexes. These increases can be a response to keep the homeostasis of chloroplast protein functions in response to salt stress.

Measurement of Nitric Oxide (NO) Generation Rate by Chloroplasts Employing Electron Spin Resonance (ESR).

Chloroplasts are among the more active organelles involved in free energy transduction in plants (photophosphorylation). Nitric oxide (NO) generation by soybean (Glycine max, var ADM 4800) chloroplasts was measured as an endogenous product assessed by electron paramagnetic resonance (ESR) spin-trapping technique. ESR spectroscopy is a methodology employed to detect species with unpaired electrons (paramagnetic). This technology has been successfully applied to different plant tissues and subcellular compartments to asses both, NO content and generation. The spin trap MGD-Fe(2+) is extensively employed to efficiently detect NO. Here, we describe a simple methodology to asses NO generation rate by isolated chloroplasts in the presence of either L-Arginine or nitrite (NO2 (-)) as substrates, since these compounds are required for enzymatic activities considered as the possible sources of NO generation in plants.

Reactive Nitrogen Species-Dependent Effects on Soybean Chloroplasts

Nitric oxide (NO) generation by soybean (Glycine max, var ADM 4800) chloroplasts was studied by electron paramagnetic resonance (EPR) spin-trapping technique1. Both nitrite and L-arginine (arg) are the required substrates for enzymatic activities considered as possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 ± 0.2 nmol min-1 mg-1 protein in the presence of 1 mM NaNO2. Chloroplasts incubated with 1 mM arg showed a NO production of 0.76 ± 0.04 nmol min-1 mg-1 protein. This production was inhibited when chloroplasts were incubated in presence of NOS-inhibitors L-NAME and L-NNA. In vitro exposure of chloroplasts to a NO-donor (GSNO) decreased both ascorbyl radical content and the activity of ascorbate peroxidase, without modification of the total ascorbate content. Exposure of the isolated chloroplasts to a NO-donor decreased lipid radical content in membranes, however, incubation in the presence of 25 μM peroxynitrite (ONOO-) led to an increase in lipid-derived radicals (34%). The effect of ONOO- on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to control. Taken as a whole, NO seems to be an endogenous metabolite in soybean chloroplasts and reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplasts, since both a decreased lipid radical content in membranes and a decrease in the activity of ascorbate peroxidase were observed after exposure to a NO donor.

Chloroplasts as a nitric oxide cellular source. Effect of reactive nitrogen species on chloroplastic lipids and proteins.

Nitric oxide (NO) generation by soybean (Glycine max var. ADM 4800) chloroplasts was studied as an endogenous product assessed by the electron paramagnetic resonance spin-trapping technique. Nitrite and l-arginine (Arg) are substrates for enzymatic activities considered to be the possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 +/- 0.2 nmol min(-1) mg(-1) protein in the presence of 1 mm NaNO(2). Inhibition of photosynthetic electron flow by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea resulted in a lower rate (1.21 +/- 0.04 nmol min(-1) mg(-1) protein) of NO generation. Chloroplasts incubated with 1 mm Arg showed NO production of 0.76 +/- 0.04 nmol min(-1) mg(-1) protein that was not affected either by omission of Ca(2+) or by supplementation with Ca(2+) and calmodulin to the incubation medium. This production was inhibited when chloroplasts were incubated in the presence of NO synthase inhibitors N(omega)-nitro-l-Arg methyl ester hydrochloride and N(omega)-nitro-l-Arg. In vitro exposure of chloroplasts to an NO donor (250 mum S-nitrosoglutathione) decreased lipid radical content in membranes by 29%; however, incubation in the presence of 25 mum peroxynitrite (ONOO(-)) led to an increase in lipid-derived radicals (34%). The effect of ONOO(-) on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to controls. Moreover, ONOO(-) treatment significantly affected both O(2) evolution and chlorophyll fluorescence in thylakoids. Data reported here suggest that NO is an endogenous metabolite in soybean chloroplasts and that reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplast macromolecules.

The DNA-compacting protein DCP68 from soybean chloroplasts is ferredoxin:sulfite reductase and co-localizes with the organellar nucleoid.

The multiple copies of the chloroplast genome (plastome) are condensed and organized into nucleoids by a set of proteins. One of these, the DNA-binding protein DCP68 from soybean, has previously been shown to compact DNA and to inhibit DNA synthesis in vitro. N-terminal amino acid analysis and the absorption spectrum of the purified protein suggest that DCP68 is the siroheme protein sulfite reductase, a ferredoxin-dependent enzyme that participates in sulfur assimilation for cysteine and methionine biosynthesis. The in vivo association of this protein with chloroplast nucleoids was confirmed by immuno-colocalization with antibodies against sulfite reductase from Arabidopsis thaliana. These results suggest that DCP68 is a bifunctional chloroplast protein that participates in reductive sulfur assimilation and plays a role in organellar nucleoid organization. The fact that dephosphorylation by alkaline phosphatase affects the binding of purified DCP68 to DNA in vitro might be indicative of the way the interaction of the protein with the nucleoid is regulated in vivo.

Effect of iron on activity of soybean multi-subunit acetyl-coenzyme A carboxylase.

Multi-subunit acetyl-coenzyme A carboxylase (MS-ACCase; EC 6.4.1.2) isolated from soybean chloroplasts is a labile enzyme that loses activity during purification. We found that incubating the chloroplast stromal fraction under anaerobic conditions or in the presence of 5 mM FeSO4 stimulated ACCase (acetyl-CoA-->malonyl-CoA) and carboxyltransferase (malonyl-CoA-->acetyl-CoA) activity. Fe-stimulation of activity was associated with 59Fe binding to a stromal protein fraction. ACCase and carboxyltransferase activities measured in the stromal protein fraction containing bound 59Fe were 2-fold and 6-fold greater, respectively, than the control (stromal fraction not pretreated with FeSO4). Superose 6 gel filtration chromatography indicated 59Fe comigrated with stromal protein of approximately 180 kDa that exhibited carboxyltransferase activity, but lacked ACCase activity. Anion exchange (Mono-Q) chromatography of the Superose 6 fraction yielded a protein peak that was enriched in carboxyltransferase activity and contained protein-bound 59Fe. Denaturing gels of the Mono-Q fraction indicated that the 180-kDa protein was composed of a 56-kDa subunit that was bound by an antibody raised against a synthetic beta-carboxyltransferase (beta-CTase) peptide. Incubation of the Mono-Q carboxyltransferase fraction with increasing concentrations of iron at a fixed substrate concentration resulted in increased initial velocities that fit well to a single rectangular three parameter hyperbola (v=vo+Vmax[FeSO4]/Km+[FeSO4]) consistent with iron functioning as a bound activator of catalysis. UV/Vis spectroscopy of the partially purified fraction before and after iron incubation yielded spectra consistent with a protein-bound metal cluster. These results suggest that the beta-CTase subunit of MS-ACCase in soybean chloroplasts is an iron-containing enzyme, which may in part explain its labile nature.

The 68 kDa DNA compacting nucleoid protein from soybean chloroplasts inhibits DNA synthesis in vitro.

Nucleoids were purified from chloroplasts of dividing soybean cells and their polypeptide composition analyzed by SDS-polyacrylamide gel electrophoresis. Of the 15-20 nucleoid-associated polypeptides, several demonstrated DNA binding activity. Upon disruption of the nucleoids with high concentrations of NaCl, a subset of these proteins and the majority of chloroplast DNA were recovered in the supernatant after centrifugation. Removal of the salt by dialysis resulted in formation of nucleoprotein complexes resembling genuine nucleoids. Purification of these structures revealed three major proteins of 68, 35 and 18 kDa. After purification of the 68 kDa protein to homogeneity, this protein was able to compact purified chloroplast DNA into a nucleoid-like structure in a protein concentration-dependent fashion. Addition of the 68 kDa protein to an in vitro chloroplast DNA replication system resulted in complete inhibition of nucleotide incorporation at concentrations above 300 ng of 68 kDa protein per microg of template DNA. These results led to in situ immunofluorescence studies of chloroplasts replicating DNA which suggested that newly synthesized DNA is not co-localized with nucleoids. Presumably, either the plastid replication machinery has means of removing nucleoid proteins prior to replication or the concentration of nucleoid proteins is tightly regulated and the proteins turned over in order to allow replication to proceed.

A light-dependent redox signal participates in the regulation of ammonia fixation in chloroplasts of higher plants — ferredoxin: Glutamate synthase is a thioredoxin-dependent enzyme

Ferredoxin: glutamate synthase from spinach and soybean chloroplasts has been recognized as a new member of thioredoxin-dependent enzymes in higher plants. For this purpose, a new ferredoxin: glutamate synthase assay procedure had to be established. The assay mixture was filtered through size exclusion filter units to separate the low molecular substrates and products from the proteins that stop the reaction in the filtrate. This avoids the artificial generation of glutamate by boiling, which was frequendy applied to interrupt the reaction by denaturing the enzyme. The glutamate formation was determined by capillary zone electrophoresis. Indications of a thioredoxin-dependency of nitrite reductase and glutamine synthetase have not been observed in combination with different thioredoxin species. Here, we describe for the first time that the fixation of ammonium ions in chloroplasts of higher plants is under thioredoxin control.

Repair mechanisms of UV-induced DNA damage in soybean chloroplasts.

In order to better understand the biochemical mechanisms of DNA metabolism in chloroplasts, repair of UV induced plastome damage in vivo was determined by exposure of soybean suspension cells to UV light and subsequent quantitation of the damage remaining in nuclear and chloroplast encoded genes with time by quantitative polymerase chain reaction (QPCR). The kinetics of damage repair in the nuclear rbcS gene suggest that photoreactivation and dark mechanisms are active, while for the plastome encoded psbA gene only a light-dependent repair process was detected which is considerably slower than would be expected for photolyase-mediated photoreactivation.

Cloning, Subcellular Localization and Expression of chl1, a Subunit of Magnesium Chelatase in Soybean

Mg-insertion is the first committed step in chlorophyll synthesis and is catalyzed by Mg-chelatase. In photosynthetic bacteria, bchI gene product was suggested to be a subunit of Mg-chelatase. We isolated a bchI homolog from a soybean cDNA library and designated it as chlI. CHLI consisted of 421 amino acid residues and the sequence exhibited a high similarity to other BchI homologs. CHLI contained an ATP-binding motif found in other BchI homologs. CHLI was localized in the soluble fraction in soybean chloroplasts, suggesting that it was stromal subunit of Mg-chelatase. chlI mRNA in cell culture (SB-P) of soybean was reversibly induced by light.

Licorice-derived compounds inhibit linoleic acid (C:18:2ω6) desaturation in soybean chloroplasts

Although glycyrrhizic acid, a major constituent of licorice root, has important pharmacological effects in humans, the biological activity of glycyrrhizic acid and its aglycone glycyrrhetinic acid in plants is unknown. Here we report that these licorice-derived compounds and the analog carbenoxolone inhibit desaturation of linoleic acid (C18:2ω6) in soybean chloroplasts using monogalactosyldiacylglycerol and phosphatidylcholine substrates in an in vitro assay for desaturase activity. At 10 nM glycyrrhetinic acid, there is significant inhibition of desaturation of linoleic acid suggesting that licorice-derived compounds could prove useful in investigating biochemical pathways of linoleic acid desaturation in plant chloroplasts and plant desaturase regulation, which has application in modification of plant response to environmental stress, as well as optimization of oil seed composition.

Accuracy of Deoxynucleotide Incorporation by Soybean Chloroplast DNA Polymerases Is Independent of the Presence of a 3[prime] to 5[prime] Exonuclease.

DNA polymerase was purified from soybean (Glycine max) chloroplasts that were actively replicating DNA. The main form (form I) of the enzyme was associated with a low level of 3[prime] to 5[prime] exonuclease activity throughout purification, although the ratio of exonuclease to polymerase activity decreased with each successive purification step. A second form (form II) of DNA polymerase, which elutes from DEAE-cellulose at a higher salt concentration than form I, was devoid of any exonuclease activity. To assess the potential function of the 3[prime] to 5[prime] exonuclease in proofreading, the fidelity of deoxynucleotide incorporation was measured for form I DNA polymerase throughout purification. Despite the steadily decreasing ratio of 3[prime] to 5[prime] exonuclease to polymerase activity, the extent of misincorporation by form I enzyme remained unchanged during the final purification steps, suggesting that the exonuclease did not contribute to the accuracy of DNA synthesis by this polymerase. Fidelity of form I DNA polymerase, when compared with that of form II, revealed a higher level of misincorporation for form I enzyme, a finding that is consistent with the exonuclease playing little or no role in exonucleolytic proofreading.

Chloroacetamide-Induced Reduction of Fatty Acid Desaturation

The fatty acid profile of the green alga Scenedesmus acutus was strongly influenced after a 40-hr incubation with 5 μ M alachlor or metazachlor. The relative amounts of polyunsaturated fatty acids (C18:4 to C18:2 and C16:4 to C16:2) were reduced while C18:1 and C16:0 accumulated. The two successive desaturation steps between C18:1 and C18:3 were investigated. Desaturation of [ 14C]oleoyl-CoA (activated C18:1) by pea microsomes was reduced in the presence of 25 μ M alachlor and 50% inhibition was achieved with 100 μ M. However, C18:2 desaturation by broken soybean chloroplasts was not affected by chloroacetamides. Since inhibition of C18:1 desaturation may be responsible for the changes observed in the fatty acid profile in vivo, the incorporation of [ 14C]oleic acid (C18:1) was investigated with S. acutus. After a 5.5-hr incubation, over 10% of the total radioactivity was incorporated into a precipitate obtained after saponification of the cells. Radioactivity in that fraction was dramatically reduced in the presence of metazachlor. A herbicide concentration between 10 −7 and 10 −8 M was sufficient for a 50% inhibition.

Primary structure and sequence organization of the 16S–23S spacer in the ribosomal operon of soybean (Glycine max L.) chloroplast DNA

The nucleotide sequence of a spacer region between 16S and 23S rRNA genes from soybean chloroplasts has been determined. The spacer region is over 3000 bp long and contains two tRNA genes, coding for rRNA(Ile) and tRNA(Ala) which contain intervening sequences of 953 and 811 base pairs respectively. There is a strong homology between the two introns suggesting that they have a common origin. These spacer tRNAs are synthesized as part of a kb precursor molecule containing 16S and 23S rRNA sequences.

Callus formation of somatic hybrid of rice and soybean and characteristics of the hybrid callus.

Induction of calli from rice (strain A-58) and soybean (cv. Harosoy) protoplast mixtures treated by polyethyleneglycol (PEG) was successful and hybrid calli were able to be identified and selected by the observation of a blackish purple pigment of fravonoids in the rice calli and a softness in the soybean calli. However, the blackish purple pigment of hybrid calli disappeared rapidly after one or two subcultures. The peroxidase and esterase isozyme band patterns of all hybrid calli after several subcultures were that of soybean callus. From these results, it can be supposed that rice chromosomes are rapidly eliminated, while soybean nuclear genome may be retained in the hybrid calli. During subcultures of the hybrid calli, yellow, green and white calli have been segregated. The soybean calli were usually yellow, but sometimes turned green. The rice calli were a blackish purple which was so unstable that whitish yellow calli were often obtained. When all these calli were transferred onto the medium containing 1-naphthaleneacetic acid (NAA) and kinetin, the whitish yellow calli of rice and the white calli segregated from the hybrids did not usually turn green. Nevertheless the segregated white calli occasionally produced green parts in a mosaic pattern during long term cultures. On the other hand, soybean calli, and yellow and green calli segregated from the hybrids became green or remained green. Analysis of Fraction I protein showed that the segregated green calli have soybean chloroplasts, while the white calli have both of rice and soybean chloroplasts. Therefore, it may be considered that the segregated green calli have mainly soybean nuclei and soybean chloroplasts, while the white calli have soybean nuclei, and rice and soybean chloroplasts in a mosaic pattern.

Localization of four genes for three tRNA Leu's on the physical map of the soybean ( Glycine max L.) chloroplast genome

Three tRNAs Leu from soybean chloroplasts were isolated and hybridized to restriction fragments of soybean chloroplast DNA. Based on the hybridization pattern, the locations of four genes coding for tRNA 1 Ley, tRNA 2 Leu (two genes tRNA 2a Ley and tRNA 2b Leu, are present in the inverted repeat region) and tRNA 3 Leu were determined on the physical map of the soybean chloroplast genome.

Inhibition of deoxyribonuclease activity associated with soybean chloroplasts

The effects of spermidine, pH, ethylene diamine tetracetic acid (EDTA), and adenosine triphosphate (ATP) on deoxyribonuclease (DNase) activity associated with the chloroplasts of soybean (Glycine max (L.) Merr.) were investigated. Chloroplast DNase activity was found to be partially inhibited by either 10 mM spermidine, 20 mM EDTA, or 20 mM ATP. DNase activity was also partially inhibited at non-neutral pH's. Nearly complete inhibition was achieved with use of 30 mM EDTA, pH 10, or a combination of 10 mM spermidine and 10 mM EDTA.

Chloroplast Alkaline Fructose 1,6-Bisphosphatase Exists in a Membrane-Bound Form

An alkaline fructose 1,6-bisphosphatase activity associated with soybean (Glycine max cv Beeson) chloroplasts appears to be membrane-bound. The pH optimum of the membrane-associated activity corresponds to that found for activity associated with the stroma. Illumination of washed thylakoids results in an increase in alkaline fructose 1,6-bisphosphatase activity in the absence of any added stromal factors. Exposure to pH 8.0 results in a partial release of enzyme activity from the membrane. The activation status of the enzyme does not appear to alter its association with the membrane.

Morphometry of T219 Soybean Chloroplasts During Initial Exposure to Light

Strain T219 soybeans exhibit three genetically determined levels of photosynthetic pigments yielding plants characterized as dark green (DG), light green (LG) and lethal yellow (LY). Rapid light-induced changes in plastid morphology have been demonstrated (e.g. 1). Our previous observations indicated that the amount of change in morphology of T219 plastids following illumination after 12 hr. darkness varied in direct relation to the amount of chlorophyll present. DG plastids changed more than LG while LY plastids showed virtually no change (2). Change was characterized by flattening, loss of volume and diminution of starch immediately following illumination with a gradual recovery after 10-20 min. of light. In the present study changes in surface area of plastid boundary membranes and thylakoids were analyzed for all three types of strain T219 plants during initial exposure to light.