Cytoplasmic incompatibility following somatic cell fusion inGriffithsia pacifica Kylin,a red alga

Summary Somatic cell fusion between two isolates ofG. pacifica is followed by a cytoplasmic incompatibility reaction (CIR) in the cytoplasm donated by only one of the isolates. This CIR is characterized by the aggregation, fusion and lysis of chloroplasts of the sensitive strain; the chloroplasts of the other strain are unaffected. In addition, the nuclei of both strains retain a normal distribution during the fusion and lysis events. Cell elongation and nuclear division stop in CIR-affected cells. The CIR begins in the hybrid cell and then appears sequentially in adjacent cells of the sensitive strain; this transfer occurs only between living cells which share a crosswall. There is a lag between hybrid cell formation and the initiation of the CIR. This lag is more than 3 times as long at 17 C than at 24 C; over this range, the rate of movement of the CIR along a filament is temperature-insensitive. Thus it appears that a temperature dependent process, perhaps the synthesis of CIR-inducing agents, is required for the initiation of the CIR; subsequent movement of such agents appears to occur by diffusion.Abbreviations CIR Cytoplasmic incompatibility reaction - HC hybrid cell - 1st SC first shoot cell - 2nd SC second shoot cell - 3rd SC third shoot cell - Pac-PP Puerto Penasco isolate - Pac-BC British Columbia isolate This research was supported in part by National Science Foundation Grant PCM 7823240 to SDW; NICHD Developmental Biology Training Grant ST 32 HDO 71835, Traineeship to DJK and a State of Washington Graduate Opportunities for Women and Minorities to DJK.     

Properties of chloroplasts isolated by phase partition

Summary Chloroplasts from spinach can be separated into at least three different populations by countercurrent distribution using polymer two-phase systems. The chloroplast particles of the three populations differ in protein/chlorophyll ratio, ultrastructure and metabolism. One population, peak I, consists of intact chloroplasts surrounded by the chloroplast envelope; the second population, peak II, consists of chloroplasts, which have lost their envelopes and much of their stromal material; the third population, peak III, consists of particles containing intact chloroplasts surrounded by a membrane-bound cytoplasmic layer including mitochondria and peroxisomes.Rapid batch procedures of peak I chloroplasts incorporated¹⁴C almost entirely into glycolate and intermediates of the Calvin cycle and starch synthesis. Only small amounts were found in sucrose and amino acids. On the other hand preparations of peak III chloroplasts gave a much broader spectrum of¹⁴C-labelled products. Sucrose, malate and some amino acids contained about 40% of the¹⁴C incorporated. It is concluded from these experiments that sucrose is formed not within the chloroplast but in the cytoplasm from intermediates exported by the chloroplast.The origin of peak III particles and their use for studying the cooperation between the chloroplast and the surrounding cytoplasm including mitochondria and peroxisomes is discussed.An invited article.     

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.     

Isolation of active polyribosomes from the cytoplasm, mitochondria and chloroplasts of Euglena gracilis

1. A procedure is described for the isolation of intact polyribosomes from the cytoplasm, chloroplasts and mitochondria of Euglena gracilis. 2. All three polyribosomal preparations incorporated labelled amino acids in a system in vitro. The cytoplasmic system was inhibited by chcloheximide but not by chloramphenicol. Both the chloroplast and the mitochondrial systems, however, were inhibited by chloramphenicol but not by cycloheximide. It is shown that mitochondrial polyribosomes, like the polyribosomes from cytoplasm and chloroplasts, can participate directly in protein synthesis without supplementary mRNA being added to the synthesizing system, as in previously reported instances. 3. Sedimentation coefficients were measured for the ribosomes, ribosomal subunits, and rRNA of the cytoplasm, chloroplasts and mitochondria. 4. The G+C content was 55% for cytoplasmic rRNA, 50% for chloroplast rRNA, and 29% for mitochondrial rRNA. 5. The cytoplasmic ribosomal subunits contained a ribonuclease activity that was inhibited by heparin.     

Role of cyclosis in asymmetric formation of alkaline zones at the boundaries of illuminated region in <Emphasis Type="Italic">Chara</Emphasis> cells

The role of cytoplasmic streaming in pattern formation at the plasma membrane and chloroplast layer was examined with Chara corallina Klein ex Willd. cells exposed to nonuniform illumination. Our hypothesis was that the exchange of ions and metabolites between the chloroplasts and the cytoplasm in the illuminated cell area alters the composition of the cytosol while the flow of modified cytoplasm induces asymmetrical changes in the plasmalemmal transport and fluorescence of chloroplasts in the adjacent shaded areas. The hypothesis was tested by measuring the H⁺-transporting activity of plasmalemma and non-photochemical quenching (NPQ) in shaded areas of Chara cells at distances of 1–5 mm on either side of the illuminated region (white light, 1000 μmol/(m² s), beam width 2 mm). When measured at equal distances on opposite sides from the illuminated region, both pH and NPQ changes differed considerably depending on the direction of cytoplasmic movement at the light-shade boundary. In the region where the cytoplasm flowed out of irradiated area, the formation of alkaline zone (the plasma membrane domain with a high H⁺-conductance) and NPQ in chloroplasts was observed. In the vicinity of light-shade boundary where the flow was directed from the shade to the illuminated area, neither alkaline zone nor NPQ were formed. The results demonstrate the significance of cyclosis in the transfer of physiologically active intermediate that affects the membrane transport, the functional activity of chloroplasts, and the pattern formation in the plant cell.     

Chloroplast ultrastructure and fluorescence response of oilseed rape containing male-sterile radish cytoplasm

Summary The production of hybrid seed is facilitated if one parent possesses a male-sterile cytoplasm. Introduction of the cytoplasm of male-sterile radish (Raphanus sativus L.) into rapeseed (Brassica napus L.) results not only in transfer of the desirable male-sterile trait but induces a chlorophyll defect in the backcrossed male-sterile plants. In this study we show that the defect manifests itself in two different ways in the alloplasmic plants: a) smaller and fewer chloroplasts with an impaired ultrastructure and b) an increase in chlorophyll fluorescence. Defective chloroplasts were characterized by a reduction in both the number and size of grana, the latter due to poor stacking of thylakoids and with frequent discontinuity in the intergranal thylakoid systems. The changed chloroplast morphology and the increase in chlorophyll fluorescence are probably the cause of the lowered photosynthetic efficiency associated with the alloplasmic plants. We propose that the deficiency is the result of incompatibility between the genomes of the radish chloroplast and the rapeseed nucleus. Supporting this hypothesis are studies of male-sterile rapeseed plants in which, by protoplast fusion, the radish chloroplasts were substituted by those of normal male-fertile rapeseed. Such plants showed complete restoration of their photosynthetic potential and displayed both normal chloroplast ultrastructure and normal levels of chlorophyll fluorescence.     

Studies on {partial}-amino levulinic acid deydratase in radish cotyledons during chloroplast development

The subcellular localization and biosynthetic site of 8-aminolevulinic acid dehydratase [EC 4.2.1.24 [EC] , ALAD] were investigatedin relation to chloroplast development in radish cotyledons. ALAD was mainly located in the chloroplasts and cytoplasm. Mostof the ALAD in the chloroplasts was readily released by hypotonicshock. The enzyme was also found in the proplastids of etiolatedcotyledons. The normal increase in the activity of ALAD in the chloroplastsas well as the cytoplasm was inhibited by cycloheximide butunaffected by D-threo chloramphenicol and kanamycin during thegreening of radish cotyledons. We concluded that the ALAD inboth the cytoplasm and chloroplasts was synthesized on the cytoplasmic80S-ribosomes. This suggests that the ALAD formed on the 80S-ribosomesmight be incorporated into chloroplasts during their development. When etiolated radish seedlings were illuminated, ALAD in boththe cytoplasm and chloroplasts increased up to the point ofthe full development of the chloroplasts, and thereafter itdecreased. (Received August 20, 1975; )     

Cyclosis-related asymmetry of chloroplast–plasma membrane interactions at the margins of illuminated area in <Emphasis Type="Italic">Chara corallina</Emphasis> cells

Cytoplasmic streaming in plant cells is an effective means of intracellular transport. The cycling of ions and metabolites between the cytosol and chloroplasts in illuminated cell regions may alter the cytoplasm composition, while directional flow of this modified cytoplasm may affect the plasma membrane and chloroplast activities in cell regions residing downstream of the illumination area. The impact of local illumination is predicted to be asymmetric because the cell regions located downstream and upstream in the cytoplasmic flow with respect to illumination area would be exposed to flowing cytoplasm whose solute composition was influenced by photosynthetic or dark metabolism. This hypothesis was checked by measuring H⁺-transporting activity of plasmalemma and chlorophyll fluorescence of chloroplasts in shaded regions of Chara corallina internodal cells near opposite borders of illuminated region (white light, beam width 2 mm). Both the apoplastic pH and chlorophyll fluorescence, recorded in shade regions at equal distances from illuminated area, exhibited asymmetric light-on responses depending on orientation of cytoplasmic streaming at the light–shade boundary. In the region where the cytoplasm flowed from illuminated area to the measurement area, the alkaline zone (a zone with high plasma membrane conductance) was formed within 4-min illumination, whereas no alkaline zone was observed in the area where cytoplasm approached the boundary from darkened regions. The results emphasize significance of cyclosis in lateral distribution of a functionally active intermediate capable of affecting the membrane transport across the plasmalemma, the functional activity of chloroplasts, and pattern formation in the plant cell.     

Salicylhydroxamic acid enhances the NADH-oxidase activity of peroxidase in pea mitochondrial and chloroplast suspensions

Salicylhydroxamic acid (SHAM), an alternative oxidase inhibitor of plant mitochondria, enhances the NADH-oxidase activity in mitochondrial and chloroplast suspensions obtained from pea roots or leaves, respectively. This reaction is inhibited by the washing of mitochondria or chloroplasts and is observed in supernatants after the removal of the organelles by centrifugation. The reaction is sensitive to CN^– and to antioxidant propyl gallate. The NADH oxidation is also enhanced by 2,4-dichlorophenol or phenol, but not salicylic acid. The acceleration of NADH oxidation by phenolic compounds is observed with presence of commercial horseradish peroxidase and is connected with the involvement of these compounds in NADH-dependent peroxidase reaction. SHAM and 2,4-dichlorophenol significantly enhance the destruction of nuclei in guard cells of pea leaf epidermis caused by the generation of reactive oxygen species during the oxidation of exogenous NADH by apoplastic peroxidase.     

A light sensitive recipient for the effective transfer of chloroplast and mitochondrial traits by protoplast fusion in Nicotiana

Summary A light sensitive mutant was used as a recipient in the transfer of chloroplasts from a wildtype donor. Gamma irradiated (lethal dose) mesophyll protoplasts of Nicotiana gossei were fused with mesophyll protoplasts of a N. plumbaginifolia line carrying light sensitive plastids from a N. tabacum mutant. After fusion, colonies containing wild-type plastids from the cytoplasm donor were selected by their green colour. Most of the regenerated plants had N. plumbaginifolia morphology, but were a normal green in colour. The presence of donor-type plastids was confirmed by the restriction pattern of chloroplast DNA in each plant analysed. These cybrids were fully male sterile with an altered flower morphology typical of certain types of alloplasmic male sterility in Nicotiana. The use of the cytoplasmic light sensitive recipient proved to be suitable for effective interspecific transfer of wild-type chloroplasts. The recombinant-type mitochondrial DNA restriction patterns and the male sterility of the cybrids indicated the co-transfer of chloroplast and mitochondrial traits. On leave from: Department of Genetics, Section of Biosciences, Martin Luther University, Domplatz 1, DDR-4020 Halle/ S., German Democratic Republic     

Structural and functional properties of the coleoptile chloroplast: Photosynthesis and photosensory transduction

Recent studies have shown that guard cell and coleoptile chloroplasts appear to be involved in blue light photoreception during blue light-dependent stomatal opening and phototropic bending. The guard cell chloroplast has been studied in detail but the coleoptile chloroplast is poorly understood. The present study was aimed at the characterization of the corn coleoptile chloroplast, and its comparison with mesophyll and guard cell chloroplasts. Coleoptile chloroplasts operated the xanthophyll cycle, and their zeaxanthin content tracked incident rates of solar radiation throughout the day. Zeaxanthin formation was very sensitive to low incident fluence rates, and saturated at around 800–1000 mol m^–2 s^–1. Zeaxanthin formation in corn mesophyll chloroplasts was insensitive to low fluence rates and saturated at around 1800 mol m^–2 s^–1. Quenching rates of chlorophyll a fluorescence transients from coleoptile chloroplasts induced by saturating fluence rates of actinic red light increased as a function of zeaxanthin content. This implies that zeaxanthin plays a photoprotective role in the coleoptile chloroplast. Addition of low fluence rates of blue light to saturating red light also increased quenching rates in a zeaxanthin-dependent fashion. This blue light response of the coleoptile chloroplast is analogous to that of the guard cell chloroplast, and implicates these organelles in the sensory transduction of blue light. On a chlorophyll basis, coleoptile chloroplasts had high rates of photosynthetic oxygen evolution and low rates of photosynthetic carbon fixation, as compared with mesophyll chloroplasts. In contrast with the uniform chloroplast distribution in the leaf, coleoptile chloroplasts were predominately found in the outer cell layers of the coleoptile cortex, and had large starch grains and a moderate amount of stacked grana and stroma lamellae. Several key properties of the coleoptile chloroplast were different from those of mesophyll chloroplasts and resembled those of guard cell chloroplasts. We propose that the common properties of guard cell and coleoptile chloroplasts define a functional pattern characteristic of chloroplasts specialized in photosensory transduction.Abbreviations Ant or A antheraxanthin - dv/dt fluorescence quenching rate - Fm maximum yield of fluorescence with all PS II reaction centers closed - Fo yield of instantaneous fluorescence with all PS II reaction centers open - Vio or V violaxanthin - Zea or Z zeaxanthin     

The nucleotide sequences of barley cytoplasmic glutamate transfer RNAs and structural features essential for formation of δ-aminolevulinic acid

In chloroplasts and a number of prokaryotes, -aminolevulinic acid (ALA), the universal precursor of porphyrins, is synthesized by a multistep enzymatic pathway with glutamyl-tRNA^Glu as an intermediate. The ALA synthesizing system from barley chloroplasts is highly specific in its tRNA requirement for chloroplast tRNA^Glu; a number of other Glu-tRNAs are inactive in ALA formation although they can be glutamylated by chloroplast aminoacyl-tRNA synthetases. In order to obtain more information about the structural features defining the ability of a tRNA to be recognized by the ALA synthesizing enzymes, we purified and sequenced two cytoplasmic tRNA^Glu species from barley embryos which are inactive in ALA synthesis. By using glutamylated tRNAs as a substrate for the overall reaction, we showed that Glu-tRNA reductase is the enzyme responsible for tRNA discrimination.     

Inhibition of leucyl-tRNA-synthetases by modified ATP analogs

The interaction between modifying ATP analogs containing alkylating or phosphorylating groups in the polyphosphate moiety of the ATP molecule and leucyl-tRNA synthetases from cytoplasm and chloroplasts of Euglena gracilis (strain Z) was studied. It was shown that most of the ATP analogs irreversibly inhibit the cytoplasmic enzyme, having no inhibiting effect on the chloroplast synthetase. The kinetic constants K1 and k2 for the interaction between the most effective irreversible inhibitors and the cytoplasmic enzyme were determined. The data on the protection of the enzyme activity by substrates against irreversible inhibition suggest, that the effect of the adenosine 5'-(beta-chloroethyl phosphate) is directed to the ATP-binding site of the cytoplasmic enzyme, whereas the mixed anhydride of AMP and mesithylene carbonic acid acts predominantly on the binding site of 3'-terminal adenosine of the tRNALeu molecule. ATP analogs may be effectively used for affinity labelling of the cytoplasmic leucyl-tRNA synthetase.     

Rapid separation of the chloroplast and cytoplasmic fractions from intact leaf protoplasts

Intact protoplasts are ruptured by rapid centrifugation through a narrow-aperture nylon mesh and the intact chloroplasts are then separated from the cytoplasm by sedimentation through a layer of silicone oil below the mesh. Within 6 to 8 s of starting the centrifuge, 90% of the chloroplasts are separated into the pellet fraction which contains only 10 to 15% contamination by mitochondria and peroxisomes and less than 5% contamination by soluble cytoplasm as judged by the distribution of marker enzymes. This technique should allow determination of the distribution of metabolites between the chloroplast and cytoplasmic compartments of intact protoplasts.     

Evidence for the nuclear location of the gene for chloroplast elongation factor G.

The chloroplast protein synthesis factor responsible for the translocation step of polypeptide synthesis on chloroplast ribosomes (chloroplast elongation factor G [EF-G]) has been detected in whole cell extracts and in isolated chloroplasts from Euglena gracilis. This factor can be detected by its ability to catalyze translocation on 70 S prokaryotic ribosomes such as those from E. coli. Chloroplast EF-G is present in low levels when Euglena is grown in the dark and can be induced more than 20-fold when the organism is grown in the light. The induction of this factor by light is inhibited by cycloheximide, a specific inhibitor of protein synthesis on cytoplasmic ribosomes. However, inhibitors of chloroplast protein synthesis such as streptomycin or spectinomycin have no effect on the induction of this factor by light. Furthermore, chloroplast EF-G can be partially induced by light in an aplastidic mutant (strain W₃BUL) which has neither significant plastid structure nor detectable chloroplast DNA. These data strongly suggest that the genetic information for chloroplast EF-G resides in the nuclear genome, and that this protein is synthesized on cytoplasmic ribosomes prior to compartmentalization within the chloroplasts.     

Mechanosensitive Ca2+ release from intracellular stores in Nitella flexilis

We found previously that the cytoplasmic drop isolated from internodal cells of Nitella flexilis releases Ca2+ in response to hypotonic treatment and named the phenomenon hydration-induced Ca2+ release (HICR). The HICR is assumed to be a result of activation of Ca2+ permeable channels in the membrane of Ca2+ stores in a stretch-activated manner. To prove this idea, mechanical stimulus was applied to the drop by means of shooting isotonic/hypnotic medium or silicon oil into the drop, or compressing the drop. All these mechanical stimuli induced a rapid increase in the Ca2+ concentration of the drop. The chloroplast fraction isolated from the cytoplasmic drop released Ca2+ on compression, while the chloroplast-free cytoplasm did not. In Chara corallina, the cytoplasmic drop, which shows a very weak HICR, also responded weakly to the mechanical stimulus, but the chloroplast fraction was inert. When chloroplasts from Chara were added to the chloroplast-free cytoplasm of N. flexilis, the cytoplasm recovered the mechanoresponse. Starch grains were as effective as chloroplasts. The data indicate that Ca2+ permeable channels in the membrane of Ca2+ stores in N. flexilis are really mechano-sensitive.     

Biosynthetic cause of in vivo acquired thermotolerance of photosynthetic light reactions and metabolic responses of chloroplasts to heat stress

Thermotolerance of photosynthetic light reactions in vivo is correlated with a decrease in the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol and an increased incorporation into thylakoid membranes of saturated digalactosyl diacylglycerol species. Although electron transport remains virtually intact in thermotolerant chloroplasts, thylakoid protein phosphorylation is strongly inhibited. The opposite is shown for thermosensitive chloroplasts in vivo. Heat stress causes reversible and irreversible inactivation of chloroplast protein synthesis in heat-adapted and nonadapted plants, respectively, but doe not greatly affect formation of rapidly turned-over 32 kilodalton proteins of photosystem II. The formation on cytoplasmic ribosomes and import by chloroplasts of thylakoid and stroma proteins remain preserved, although decreased in rate, at supraoptimal temperatures. Thermotolerant chloroplasts accumulate heat shock proteins in the stroma among which 22 kilodalton polypeptides predominate. We suggest that interactions of heat shock proteins with the outer chloroplast envelope membrane might enhance formation of digalactosyl diacylglycerol species. Furthermore, a heat-induced recompartmentalization of the chloroplast matrix that ensures effective transport of ATP from thylakoid membranes towards those sites inside the chloroplast and the cytoplasm where photosynthetically indispensable components and heat shock proteins are being formed is proposed as a metabolic strategy of plant cells to survive and recover from heat stress.     

Estimation of rRNA synthesis and degradation rates in senescing wheat leaves

Changes in cytoplasmic and chloroplast rRNA content and rates of rRNA synthesis and degradation of detached wheat leaves were determined. It was found that rRNA loss is proportionally higher in chloroplasts than in cytoplasm. Rates of synthesis were measured by incorporation of large amounts of [3H]orotic acid into rRNA. This approach overcame size differences between pyrimidine pools of cells under different physiological status. Furthermore, these pools reached nearly the same specific radioactivity as that of the administered solution. Rates of degradation were estimated either as the difference between synthesis and net variation of rRNA or by disappearance of radioactivity from 32P-labeled rRNA. Results indicated a decrease in the net rRNA synthesis capacity of leaves after 48 h of detachment. However, the fractional rates of rRNA synthesis were maintained in both cytoplasm and chloroplasts. Ribosomal RNA degradation rates were 2.5-fold higher in chloroplast than in cytoplasm. The observed chloroplast rRNA loss is due to an increased degradation rate which is 15-fold higher than the synthesis rate 48 h after detachment.     

Oxygen activation by isolated chloroplasts from Euglena gracilis. Ferredoxin-dependent function of a fluorescent compound and photosynthetic electron transport close to photosystem.

Oxygen reduction by isolated chloroplast lamellae from spinach, yielding the superoxide free radical in the light, is stimulated by a fluorescent factor (“compound No. 4”, isolated from Euglena gracilis strain Z) in a ferredoxin-dependent reaction. This reaction is not observed with Euglena chloroplasts, although there is a stimulation by compound No. 4 of ferredoxin-dependent oxygen reduction at the expense of NADPH + H⁺ as electron donor in the dark. Evidence is provided that in Euglena chloroplasts in the absence of NADP as electron acceptor a cyclic electron transport is predominating, including photosystem I, ferredoxin, NADP-ferredoxin reductase, and cytochrome₅₅₂. Isolated spinach chloroplast lamellae show a similar “cyclic” electron transport after treatment with digitonin, depending on the addition of the above cofactors. This result might indicate that Euglena chloroplast lamellae show this cyclic electron transport only as an artifact due to the isolation procedure. The results furthermore indicate that the pteridine-like, fluorescent compound No. 4 is not active as the primary electron acceptor of photosystem I; it may however be involved in oxygen activation by Euglena gracilis chloroplasts.     

Use of Silica Sol Step Gradients to Prepare Bundle Sheath and Mesophyll Chloroplasts from Panicum maximum

The first method for the direct separation of mesophyll and bundle sheath chloroplasts from whole tissue homogenates of a C₄ plant is described. Centrifugation of mixed chloroplast preparations from Panicum maximum through low viscosity silica sol gradients effectively separates large, starch-containing chloroplasts from smaller plastids. The large chloroplasts are judged to be bundle sheath chloroplasts on the basis of microscopic appearance, the presence of starch grains, the protein complement displayed on sodium dodecyl sulfate acrylamide gels, and the exclusive localization of ribulose bisphosphate carboxylase activity in these plastids. As a measure of intactness both the large (bundle sheath) and small (mesophyll) chloroplasts contain glyceralde-hyde-3-phosphate NADP-dependent dehydrogenase activity that is greatly enhanced by plastid lysis and both chloroplast preparations are impermeable to deoxyribonuclease. Chloroplast enzyme activities are inhibited by silica sol due to the Mg²⁺ chelating activity of this reagent. However, well washed chloroplasts separated on silica gradients had enzyme activities similar to reported values in which silica sol gradients were not used.