Differentiation of Photorespiratory Activity between Mesophyll and Bundle Sheath Cells of C4 Plants I. Glycine Oxidation by Mitochondria

Mitochondria were isolated from mesophyll protoplasts and bundlesheath protoplasts or strands which were obtained by enzymaticdigestion of six C₄ species: Zea mays, Sorghum bicolor, Panicummiliaceum, Panicum capillare, Panicum maximum and Chloris gayana,representative of three C₄ types. Photorespiratory glycine oxidationand related enzyme activities of mesophyll and bundle sheathmitochondria were compared. Mesophyll mitochondria showed good P/O ratios with malate andsuccinate as substrate but lacked the ability to oxidize glycine.On the other hand, mitochondria isolated from bundle sheathprotoplasts of P. miliaceum and bundle sheath strands of Z.mays possessed glycine oxidation activity similar to that ofmitochondria from C₃ plant leaves. The two enzymes involvedin glycine metabolism in mitochondria, serine hydroxymethyltransferaseand glycine decarboxylase, were also assayed in the mitochondriaof the two cell types. The activities of the two enzymes inbundle sheath mitochondria were in the range found in C₃ mitochondria.In contrast, the activities in mesophyll mitochondria were eithernot detectable or far lower than those in bundle sheath mitochondriaand ascribed to contaminating bundle sheath mitochondria. The present results indicate the deficiency of a complete glycineoxidation system in mesophyll mitochondria and also a differentiationbetween mesophyll and bundle sheath cells of C₄ plants withrespect to the photorespiratory activities of the mitochondria. (Received June 8, 1983; Accepted August 29, 1983)     

Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein

Drosophila slitis a secreted protein involved in midline patterning. Three vertebrateorthologs of the fly slit gene, Slit1, 2, and3, have been isolated. Each displays overlapping, butdistinct, patterns of expression in the developing vertebrate centralnervous system, implying conservation of function. However,vertebrate Slit genes are also expressed in nonneuronaltissues where their cellular locations and functions areunknown. In this study, we characterized the cellular distribution andprocessing of mammalian Slit3 gene product, theleast evolutionarily conserved of the vertebrate Slit genes,in kidney epithelial cells, using both cellular fractionation andimmunolabeling. Slit3, but not Slit2, was predominantly localizedwithin the mitochondria. This localization was confirmed usingimmunoelectron microscopy in cell lines and in mouse kidney proximaltubule cells. In confluent epithelial monolayers, Slit3 was alsotransported to the cell surface. However, we found no evidence of Slit3proteolytic processing similar to that seen for Slit2. We demonstratedthat Slit3 contains an NH₂-terminal mitochondriallocalization signal that can direct a reporter green fluorescentprotein to the mitochondria. The equivalent region from Slit1 cannotelicit mitochondrial targeting. We conclude that Slit3 protein istargeted to and localized at two distinct sites within epithelialcells: the mitochondria, and then, in more confluent cells, the cellsurface. Targeting to both locations is driven by specificNH₂-terminal sequences. This is the first examination ofSlit protein localization in nonneuronal cells, and this study impliesthat Slit3 has potentially unique functions not shared by other Slit proteins.

     

Preliminary Studies on the Photosynthetic Structures of Trifolium alpinum L. as Related to Productivity

Trifolium alpinum L. is a high-quality alpine forage plant growingspontaneously from 1900 to 2800 m above sea level and is widelydistributed in Piedmont and the Valle d'Aosta (Italy), whereit can reach population frequencies of 90 per cent. Yields weredetermined on forage harvested in the Valle dell'Orco (Piedmont)and were comparable to cultivated clovers from higher latitudes;yields decreased progressively as the elevation increased. Thechemical and nutritional characteristics of the forage, thoughcomparable to clovers cultivated in the Po valley (Italy), were,however, more constant. The structure of the leaf lamina asrelated to elevation was investigated using light microscopy,TEM and SEM. This is complemented by data on chlorophyll concentration,succulence, specific leaf weight and area. At all elevationsT. alpinum lacks, apart from bundle sheath cell chloroplastsin a centrifugal arrangement, the structural characteristicsof C₄ plants. The chlorophyll a:b ratio (less than four) istypical of a C₂ plant. Succulence indices (S and S_m) were verylow, making CAM pathway photosynthesis unlikely. Unusual anddifficult to interpret structures included: small functionalchloroplasts in both the epidermises, stomata present almostexclusively in the upper epidermis and mitochondria enveloped(or enclosed) by chloroplasts. It was observed that, as theelevation increases, populations are selected which are well-adaptedfor gas exchange (increase in specific leaf area, stomatal densityand intercellular spaces) and characterized by a decrease inthe grana thylacoid:integrana thylacoid ratio (consistent withthe increase in the chlorophyll a:b ratio), the per cent water,S_m and the specific leaf weight. Trifolium alpinum L., alpine trefoil, leaf structures, photosynthesis, yield, elevation, C₂, C₄     

Cyanide-Insensitive Respiration in Thermogenic Flowers of Victoria and Nelumbo

The thermogenic carpellary appendages of the flowers of Victoriacruziana d'Orb. and the thermogenic staminal appendages of Nelumbolutea (Willd.) Pers. possess the cyanide-insensitive, ‘alternative’respiratory pathway. The presence of this pathway was demonstratedin tissue slices as well as in mitochondria. The thermogenicactivity was accompanied by ultrastructural changes in the Victoriamitochondria. Before anthesis, mitochondria with well-developedcristae were present in appendage tissue in large numbers. Duringanthesis, lamelliform cristae appeared in a different orientation. Key words: Victoria, Nelumbo, flowers, thermogenicity, mitochondria     

Ultrastructure and Secretion of the Secretory Cells of Two Species ofFagoniaL. (Zygophyllaceae)

The ultrastructure and secretion of the secretory cells of theglandular trichomes ofFagonia mollisandF. glutinosawere studied.The most important finding of this study is that two speciesof the same genus produce the lipophilic component of the secretorymaterial in completely different ways and at different siteswithin the cell. In the early stages of development of secretorycells ofF. mollis,numerous mitochondria, containing myelin-likestructures, occur in the basal part of the cell. Above them,highly-elongate elements, which are suspected to develop frommitochondria with myelin-like structures, are present. Thesehave been termed ‘modified mitochondria’. It issuggested that the myelin-like structures are precursors ofthe lipophilic material ofF. mollis.InF. glutinosa,the lipophilicmaterial appears first in the plastids as plastoglobuli. Polysaccharidesappear to be produced by dictyosomes in both species. Secretionof the secretory substance to the outside of the protoplastappears to be granulocrine.Copyright 1998 Annals of Botany Company Fagonia mollis,Fagonia glutinosa,glandular trichomes, secretory cell, mitochondria, modified mitochondria, plastids, dictyosomes, lipophilic material, myelin-like structures, polysaccharides     

Internal Changes in Hyphae of Rhizopus sexualis (Smith) Callen and Mucor hiemalis Wehm. associated with Zygospore Formation

The distribution of nucleic acids, nuclei, mitochondria, andreserve foods in vegetative hyphae, zygophores, and developingzygospores of Rhizopus sexualis and Mucor hiemalis were examinedby differential staining. The extreme tips and growing zones of vegetative hyphae containeda high concentration of RNA and numerous mitochondria. Nucleiwere not present at the extreme tip but were numerous just behindit. In older parts of the hyphae the concentration of RNA waslow and both nuclei and mitochondria were fewer than in thezone of elongation. Glycogen and lipids were present in all parts of the livinghyphae except the extreme tips and were more highly concentratedin the older parts of the hyphae. Young zygophores showed a much lower RNA/DNA ratio than thatfound in the vegetative hyphal tips. Transfer of colonies from20? C to temperatures of less than 10? C, which is known toprevent zygospore initiation, caused some but not all recognizablezygophores of R. sexualis, but not those of M. hiemalis, torevert to the RNA/DNA ratio characteristic of vegetative hyphae.Some zygophores of Rhizopus and most of those of Mucor developedinto sporangiophores at low temperature, retaining the relativelylow RNA/DNA ratio throughout development. It is suggested thata reduction in the RNA/DNA ratio is an early step in the changefrom the vegetative state to the reproductive one. At firstthis step is reversible, but soon becomes irreversible by anadditional step, the nature of which is unknown. For some timeafter this the reproductive hyphae are capable of either producingasexual sporangia or of conjugating to produce zygospores. Onceconjugation has taken place development either ceases or continuesuntil the spore is fully mature, but it cannot under any circumstancesthen be reversed. The development and maturation of the zygospore involves a greatincrease in number of both nuclei and mitochondria and in theconcentration of glycogen and lipids.     

Oxidative Phosphorylation in Germinating Lettuce Seeds

The effect of thiourea and coumarin, in vivo and in vitro, onthe phosphorylating activity of lettuce mitochondria was investigated,as well as the effect of coumarin on the P/O ratio. It was shownthat both substances in vitro inhibit phosphorylation; whilein vivo coumarin inhibits but thiourea under certain circumstancesstimulates. Coumarin was also shown to decrease the P/O ratioand therefore may be considered as an uncoupler. The difficulties in considering these effects of the substancesas a primary mechanism controlling germination are pointed outand discussed.     

Malate Decarboxylation by Mitochondria of the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Mitochondria isolated from leaves of Mesembryanthemum crystallinumoxidized malate by both NAD malic enzyme and NAD malate dehydrogenase.Rates of malate oxidation were higher in mitochondria from plantsgrown at 400 mil NaCl in the rooting medium and performing Crassulaceanacid metabolism (CAM) than in mitochondria from plants grownat 20 mM NaCl and exhibiting C₃-photosynthetic CO₂ fixation.The mitochondria isolated from plants both in the CAM and C₃modes were tightly coupled and gave high respiratory control.At optimum pH for malate oxidation (pH 7.0), pyruvate was themajor product in mitochondria from CAM-M. crystallinum, whereasmitochondria from C₃-M. crystallinum produced predominantlyoxaloacetate. Both the extracted NAD malic enzyme in the presenceof CoA and the oxidation of malate to pyruvate by the mitochondriafrom plants in the CAM mode had a pH optimum around 7.0 withactivity declining markedly above this pH. The activity of NAD-malicenzyme, expressed on a cytochrome c oxidase activity basis,was much higher in mitochondria from the CAM mode than the C₃mode. The results indicate that mitochondria of this speciesare adapted to decarboxylate malate at high rates during CAM. ¹Current address: Lehrstuhl für Botanik II, UniversitätWurzburg, Mittlerer Dallenbergweg 64, 8700 Würzburg, WestGermany. ²Current address: KD 120, Chemical Research Division, OntarioHydro, 800 Kipling Avenue, Toronto, Ontario M8Z5S4, Canada. ³Current address: Department of Botany, Washington State University,Pullman, Washington 99164-4230, U.S.A. (Received March 13, 1986; Accepted September 18, 1986)     

Purification and properties of cytochrome c and two peroxidases from spinach leaves

1. A simultaneous purification procedure of cytochrome c, peroxidases,ferredoxin, ferredoxin-NADP reductase and sulfite reductasefrom spinach leaves is described. Cytochrome c, ferredoxin andferredoxin-NADP reductase were prepared in crystalline states.The two peroxidases were obtained in homogeneous states as evidencedby their electrophoretic patterns on acrylamide gel and sedimentationanalysis. 2. Crystalline cytochrome c showed a molecular weight of 13,800and an E₀' of 270 mv at pH 7.0. In addition to these properties,its spectral pattern also indicated that this cytochrome c wasderived from mitochondria. 3. Two peroxidases were isolated in high spin forms after treatmentwith HgCl₂. They had a-peaks at 556 mµ in their reducedforms. Although both peroxidases showed small differences inchromatographic behavior on a carboxymethyl cellulose column,' they had similar spectral properties, dissociation constantsof peroxidase-cyanide complex and rate constants for peroxidasereactions. (Received December 24, 1970; )     

Observations on the Internal Structure of the Spermatozoid of Dictyota

The spermatozoid of Dictyota is shown to be structurally morelike a vegetative zoospore than was the case in Fucus. The mitochondria,golgi, overall shape of the nucleus, nuclear membrane, fat bodies,and miscellaneous vesicles are as in a zoospore; there is, however,a vestigial chromatophore without an eyespot and the ciliaryapparatus is specialized. In spite of the single flagellum thereare two basal bodies inside the cell, one of which is apparentlyvestigial, ending blindy in the cytoplasm; both the a similarinternal structure with nine fibres in the wall. A fibrous ‘root’,possibly homologous with the ‘proboscis’ of Fucus,arises near the outer side of the functional basal body; itis a band of about eight fibres passing through the cell withouttouching the nucleus but closely pressed to the inner facesof at least two mitochondria before ending in an unknown mannerat the cell surface. The internal structure of the flagellumis normal but the fact that the median strand is marked by arowspines has been used to demonstrate is a conclusive way thefacts of bilateral symmetry in the whole organ; Dictyota agreesexactly with Fucus and a previous error based on incompleteinformation has been corrected. The process of unwinding ofthe flagellum from the surface of the body after liberationfrom the antheridium is described and used to illustrate someunexpected properties of the surface membrane.     

A Comparative Ultrastructural Study of Cyanidium caldarium and the Unicellular Red Alga Rhodosorus marinus

The presence of nucleus, chloroplasts, mitochondria, microbodiesand a vacuole are confirmed in Cyanidium caldarium strain CCAP1355/1. Chloroplasts usually contain parallel rows of unstackedthylakoids surrounded by a peripheral thylakoid, although theconcentric arrangement has also been observed. The chloroplastenvelope consists of two closely appressed membranes which canonly be resolved after gluteraldehyde—osmium fixation.The chloroplast of Rhodosorus marinus also contains parallel,unstacked thylakoids surrounded by a peripheral thylakoid but,in addition, a prominent, stalked pyrenoid projects into thecytoplasm and is bounded by both the peripheral thylakoid andthe chloroplast envelope. This structure is covered by a capof starch grains and contains membranous vesicles in the matrix.Phycobilisomes are absent from the chloroplasts of both algae.The recognition of C. caldarium as a rhodophyte is supportedby these observations. Cyanidium caldarium, Rhodosorus marinus, chloroplast ultrastructure     

An Inhibitor of Mitochondrial Oxidations in Extracts Prepared from Senescing Leaves

Aqueous extracts were prepared from leaves of Ricinus Communis,L. by centrifuging homogenates at 18000 g for 20 min. Extractsprepared from yellow or brown leaves inhibited the oxidationof succinate by mitochondria prepared from Ricinus endosperm.Extracts became less inhibitory when dialysed or treated withbovine serum albumen. The inhibitory properties of the extractsare attributed to their fatty acid content.     

Respiration and Mitochondrial Activity in Zea mays Roots As Affected by Osmotic Stress

Studies were made of metabolism in highly vacuolated and slightlyvacuolated Zea mays root tissue both during and after plasmolysis. Plasmolysis resulted in decreased respiration and carbon dioxideevolution from glucose and an increased sucrose synthesis. Inhibitionof respiration during plasmolysis in both the highly vacuolatedand slightly vacuolated tissue was not relieved by supply ofglucose, organic acids, or uncouplers of oxidative phosphorylation.Mitochondria isolated from plasmolysed tissue were tightly coupled,but activity in vitro was inhibited by exposure to a high negativeosmotic potential. It is suggested that low TCA cycle activityin vivo must be due either to inhibition of mitochondrial activityor to reduced flow of carbon through the glycolytic pathway. A low potential for TCA cycle activity after deplasmolysis issuggested, as addition of pyruvate stimulated carbon dioxideevolution but not oxygen uptake, which was severely decreased.This was presumably due to severe mitochondrial damage as shownby their activity in vitro. However, it is not clear whetherrespiration in vivo is rate limited by rapid leakage of metabolicintermediate (reported earlier) or by lysis of mitochondria. Deplasmolysis did not damage mitochondria from slightly vacuolatedtissue, a result which was consistent with respiratory measurementsmade in vivo. The data show that mitochondria in vacuolated tissue are damagedduring and after deplasmolysis and not before. It is suggestedthat lysis of mitochondria occurs in vivo as a result of a sharpincrease in the osmotic potential of the cell fluids.     

Electron microscope studies of the vegetative cellular life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture II. Association of mitochondria and the chloroplast at an early developmental stage

At an intermediate stage in the growth phase of the cell cycleof Chlamydomonas reinhardi, mitochondria and the chloroplastassociated with each other in a characteristic manner. Aftertemporary association with the chloroplast, mitochondria seemto fuse into giant forms as reported previously. ¹ This work was reported in part at the 27th Annual Meetingof the Japanese Society of Electron Microscopy, May 1971. (Received June 7, 1972; )     

Respiration of Malate and Glutamate in Symbiotic Frankia Prepared from Alnus incana

Frankia vesicle clusters were prepared from root nodules ofAlnus incana (L.) Moench inoculated either with a local sourceof Frankia or with Frankia Cpll. The capacity of vesicle clustersto respire was investigated by respirometric and enzymologicalstudies. Simultaneous addition of malate, glutamate, and NAD⁺supported respiration in both types of Frankia, though at asmaller rate compared to the substrates NADH or 6-phosphogluconate.The saturating concentrations of malate and glutamate were alsomuch higher than with the other substrates. No respiration wassupported by succinate. Activity of the enzymes malate dehydrogenase(EC 1.1.1.37 [EC] ) and glutamate oxaloacetate transaminase (EC 2.6.1.1 [EC] )was demonstrated in crude extracts from both types of symbioticFrankia. Their maximum rates were high enough to account forthe respiration of malate and glutamate. This respiration wasinhibited by mersalylic acid, an inhibitor of the dicarboxylateshuttle in mitochondria, but it was shown that inhibition ofrespiration could be due to a direct effect on the enzymes.We conclude that respiration of malate and glutamate is mostlikely mediated by malate dehydrogenase and glutamate oxaloacetatetransaminase, but no explicit evidence for or against the presenceof a dicarboxylate carrier was found. The utilization of respiratorysubstrates was largely similar in the two types of Frankia,except for some differences in maximum rates and cofactor dependency. Key words: Actinorhizal symbioses, Alnus, dicarboxylate shuttle, Frankia, reducing power, respiration     

Microsporogenesis and the Mechanism of Cytoplasmic Male Sterility in Maize

Examination of the structural changes occurring during microsporogenesisin cms-T, -C and -S Zea mays L. with different nuclear backgroundsrevealed considerable variations in the stages at which abortionof the sporocytes occurred within each cytoplasmic genotype.This variation was not associated with the nuclear background.Significantly, we found that the mitochondria in cms-T plantsdid not always follow the pattern of degeneration describedpreviously. We conclude that these observations are not consistentwith a proposed mechanism of cytoplasmic male sterility involvingthe direct, antagonistic action of an anther produced substanceon an altered mitochondrion or plastid, that is functionallynormal in the rest of the plant. The value of microscopic studiesin detecting variation in apparently uniform material is emphasised,though it is recognized that other methods will be requiredto elucidate the mechanism of male sterility. male sterility, mitochondria, cytoplasmic inhertance, microsporogenesis, Zeamays L., maize, corn     

Effects of Light and Dark on the Ultrastructure of Lichen Algae

Quantitative ultrastructural observations have been made onthe algal cells (Trebouxia) in two lichens, Parmelia sulcataand P. laevigata, stored for 48 h in the dark or under a light/darkregime. The response of the alga was found to differ in theselichens. In P. sulcata the dark treatment caused a decreasein starch grains, lipid-rich pyrenoglobuli and peripheral cytoplasmicstorage bodies and an increase in pyrenoid and chloroplast proteinbodies. The algae in P. laevigata contained little starch andno chloroplast protein bodies. However, after dark treatment,starch, cytoplasmic storage bodies and pyrenoid dimensions sometimesdeclined, while pyrenoglobuli numbers increased. Some of theseapparent changes depended upon the units used for calculatingthe cross-sectional areas of structures, e.g. absolute units,percentage of cell wall, protoplast or chloroplast cross-sectionalarea. Chloroplast area increased in the dark (as a % of cellwall area) in both species while mitochondria were larger inthe dark in P. sulcata but not in P. laevigata. Ultrastructuralchanges were not clearly correlated with changes in photosyntheticand respiratory rates. These results directly support the suggestionthat some intra-cellular structures are energy-generating reservesthe dimensions of which can rapidly change. Parmelia sulcata, Parmelia laevigata, lichen algae, light and dark storage, starvation, reserve substances, organdie dimensions     

Salt-Induced Ultrastructural Damage to Mitochondria in Root Tips of a Salt-Sensitive Ecotype of Agrostis Stolonifera

In root tips of a salt-sensitive ecotype of Agrostis stolonifera,treatment with 100 mmol I^–1 NaCl for two weeks resultedin conspicuous structural damage to mitochondria, which becamedeficient in cristae, swelled and vacuolated. A salt-tolerantecotype maintained normal root tip mitochondrial structure underthe same salt treatment, in spite of accumulating higher levelsof sodium and chloride in its roots. The subcellular distributionof chloride, as judged by electron microscopy after precipitationwith silver ions, was identical in both ecotypes.     

Diurnal Phototropism in Leaves of Lavatera cretica L. under Conditions of Simulated Solar-Tracking

Diurnal laminar reorientation was followed in solar-trackingleaves of Lavatera cretica L. under simulated conditions. Asimulated ‘sun’ was moved over the lamina in a 180?arc in the vertical plane of the mid-vein, at an angular velocityof 15? h^–1 in a regime of 12-h photoperiods. In one groupof plants the petioles of the experimental leaves were arrangedto face ‘sunrise’, while in the other they werearranged to face ‘sunset’. At ‘sunrise’,the laminae in both groups, which were inclined towards theanticipated direction of ‘sunrise’, changed theirelevation towards the rising ‘sun’, resulting inprogressive reduction in the angle of incidence (AI) of lighton the laminar surface (AI= differential between laminar and‘solar’ elevation). As a result, laminar and ‘solar’elevation converged, and laminar reorientation gradually ceased,until the ‘solar’ elevation had passed the normalto the laminar surface (AI=0?). laminar reorientation was thenresumed, but its direction was reversed to follow the directionof ‘solar’ reorientation. During most of the remaining‘day’, laminar elevation (LE) trailed that of the‘sun’ by an average of 11?-14?. Laminar reorientationthen anticipated ‘sunset’ by starting to slow down60 to 90min in advance. During the 12-h dark period, the laminareoriented towards the anticipated direction of the subsequent‘sunrise’. The time-course of nocturnal reorientationwas qualitatively different in the two groups of experimentalplants. The time-course of diurnal phototropism under naturaland simulated conditions is analysed and compared and differencesand similarities between them are discussed. Key words: Diurnal phototropism, solar-tracking, vectorial excitation