Studies on the mechanism of action of isopropyl N-phenyl carbamate

The binding of [¹⁴C]isopropyl N-phenyl carbamate (IPC) to microtubular protein isolated from chick brains, and the effect of isopropyl N-phenyl carbamate (IPC) on the in vitro reassembly of microtubules was investigated. While [¹⁴C]colchicine binds to microtubular protein, [¹⁴C]IPC does not. Concentrations from 1 × 10⁻⁴ M to 1 × 10⁻³ M IPC do not prevent in vitro repolymerization of microtubular protein. IPC (1 × 10⁻⁴ M) does not affect the rate of reassembly of microtubules. We conclude that IPC does not exert its effect through an interaction with microtubular protein; we suggest that IPC probably interacts with microtubule organizing centers.     

<Emphasis Type="Italic">Nicotiana sylvestris</Emphasis> L. mutants resistant to isopropyl <Emphasis Type="Italic">N</Emphasis>-phenyl carbamate possess microtubule organizing centers of heightened stability

N. sylvestris mutants resistant to isopropyl N-phenyl carbamate (IPC), a herbicide belonging to the phenyl carbamate series, are obtained by means of in vitro selection using gamma radiation. A concentration of 30 μM IPC was found to be the maximum concentration at which mutants of the N. sylvestris line capable of regeneration and rooting under conditions of selection pressure could be selected. IPC resistance in the mutants obtained was confirmed by a number of tests, in particular, tests that measure the capacity of leaf explants of the mutant lines to regenerate plants and the ability of their callus cells to survive in media with a selective IPC concentration, as well as by means of genetic, morphometric, cytological, and immunofluorescent analyses. The results of these studies attest to increased resistance of the mutant plants to this antimitotic substance by comparison with a control. It is shown that resistance to IPC is based on the heightened resistance of the microtubule organizing centers of the cells of these lines. It is established that the acquired resistance trait inherited in the F₁ and F₂ generations of the mutants is a dominant nuclear trait.     

Cell shape in the algaPediastrum (Hydrodictyaceae; Chlorophyta)

Summary Species ofPediastrum, a genus in which the colonies assemble from aggregating zoospores, differ in the number and form of prongs on peripheral cells and the amount of space between cells of the colony; cell shape appears to be genetically based. Peripheral cells of theP. boryanum colony, for example, have two prongs per cell;P. simplex has one prong per cell. Prong extension is suppressed in the interior cells ofP. boryanum, but prong sites have been reported in scanning electron micrographs of the cell walls. A mutant unicellular strain in which cells of the colony separate after attaining typical form reveals several prong sites (6 or more) in each cell. Multiple suppressed prong sites are evident inP. simplex cells as well. Polyeders, 4- and 5-pronged unicells, occur in the life cycle ofP. simplex. Based on these observations and a recent report byMarchant (1979) of a microtubule organizing center associated with the prongs, it is suggested that several microtubule organizing centers are to be found in zoospores ofPediastrum species and may be related to species differences in cell shape.Research supported in part by Argonne Center for Educational Affairs, U.S. Department of Energy, under contract No. W-31-109-ENG-38.     

Atrazine-resistant cauliflower obtained by somatic hybridization between Brassica oleracea and ATR-B. napus

Summary Somatic hybridization between Brassica oleracea ssp. botrytis (cauliflower, 2n=18), carrying the Ogura (R1) male-sterile cytoplasm and B. napus (2n= 38), carrying a male-fertile, atrazine-resistant (ATR) cytoplasm, yielded three hybrids (2n=56) and six cauliflower cybrids (2n=18), which were selected for resistance to the herbicide in vitro. The hybrids and cybrids were male fertile and self-compatible. They contained both chloroplasts and mitochondria from the ATR cytoplasm. We found no evidence for mtDNA recombination in any of the regenerated plants. Selfed progeny of the B. oleracea atrazine-resistant cybrids were evaluated for tolerance to the herbicide in the field. Resistant plants exposed to 0.56–4.48 kg/ha (0.5–4.0 pounds/acre) atrazine in the soil showed no damage at any herbicide level, whereas plants of a susceptible alloplasmic line were severely damaged at the lowest level of herbicide application and killed at all higher levels. These atrazine-resistant cauliflower may have potential horticultural use, especially in fields where atrazine carry over is a serious problem.     

Ordering microtubules

How do cells order their cytoplasm? While microtubule organizing centers have long been considered essential to conferring order by virtue of their microtubule nucleating activity, attention has currently refocused on the role that microtubule motors play in organizing microtubules. An intriguing set of recent findings⁽¹⁾ reveals that cell fragments, lacking microtubule organizing centers, rapidly organize microtubules into a radial array during organelle transport driven by the microtubule motor, cytoplasmic dynein. Further, interaction of radial microtubules with the cell surface centers the array, revealing that centering information resides not with centrosomes but with organized microtubules.     

Involvement of chloroplasts in the programmed death of plant cells

The effect of cyanide, an apoptosis inducer, on pea leaf epidermal peels was investigated. Illumination stimulated the CN^–-induced destruction of guard cells (containing chloroplasts and mitochondria) but not of epidermal cells (containing mitochondria only). The process was prevented by antioxidants (-tocopherol, 2,5-di-tret-butyl-4-hydroxytoluene, and mannitol), by anaerobiosis, by the protein kinase C inhibitor staurosporine, and by cysteine and serine protease inhibitors. Electron acceptors (menadione, p-benzoquinone, diaminodurene, TMPD, DCPIP, and methyl viologen) suppressed CN^–-induced apoptosis of guard cells, but not epidermal cells. Methyl viologen had no influence on the removal of CN^–-induced nucleus destruction in guard cells under anaerobic conditions. The light activation of CN^–-induced apoptosis of guard cells was suppressed by DCMU (an inhibitor of the electron transfer in Photosystem II) and by DNP-INT (an antagonist of plastoquinol at the Q_o site of the chloroplast cytochrome b ₆ f complex). It is concluded that apoptosis initiation in guard cells depends on the simultaneous availability of two factors, ROS and reduced quinones of the electron transfer chain. The conditions for manifestation of programmed cell death in guard and epidermal cells of the pea leaf were significantly different.     

Role of the MT-MTOC complex in determination of the cellular locomotory unit inDictyostelium

Summary The microtubule inhibitors, ethyl-N-phenylcarbamate (EPC) and thiabendazole (TB), which disrupt cytoplasmic microtubules and induce giant cells inDictyostelium (Kitanishi et al. 1984), were found to induce the occurrence of multiple microtubule organizing centers (MTOCs) in these giant cells. Probing was done by indirect immunofluorescence using monoclonal anti--tubulin. The nuclear DNA content of the giant cells increased in parallel with an increase in the number of MTOCs, as shown by microspectrophotometory of cells stained with the fluorescent DNA stain DAPI (4,6-diamidino-2-phenylindole).Shortly after the inhibitors were removed, the MTOCs of the giant cell formed multiple mitotic spindles or synchronously reconstituted numerous cytoplasmic MT-networks. These events apparently reflected the cell-cycle dependent activities of the MTOCs at the time the inhibitors were removed. When multiple spindles were formed, numerous cytoplasmic MT-networks became organized subsequent to the breakdown of the spindles. In either case, reconstitution of the cytoplasmic MT-networks was followed by apparently normal cytokinesis resulting in the production of many daughter cells each containing a single MT-MTOC complex. The evidence suggested the possible mechanism of the induction of multiple MTOCs, and implied that the MT-MTOC complex is significant in the cytokinesis ofDictyostelium by determining the cell locomotory unit.     

Conversion and Distribution of Cobalamin in Euglena gracilis z, with Special Reference to Its Location and Probable Function within Chloroplasts

Cobalamin is essentially required for growth by Euglena gracilis and shown to be converted to coenzyme forms promptly after feeding cyanocobalamin. Concentrations of coenzymes, methylcobalamin, and 5′-deoxyadenosylcobalamin, reached about 1 femtomole/10⁶ cells 2 hours after feeding cyanocobalamin to cobalamin-limited cells. Cobalamins all were bound to proteins in Euglena cells and located in subcellular fractions of chloroplasts, mitochondria, microsomes, and cytosol. Incorporated cobalamin into chloroplasts was localized in thylakoids. Methylcobalamin existed in chloroplasts, mitochondria, and cytosol, while 5′-deoxyadenosylcobalamin was in mitochondria and the cytosol, 2 h after feeding cyanocobalamin to Euglena cells. Quantitative alterations of methylcobalamin and 5′-deoxyadenosylcobalamin in chloroplasts suggest their important functions as coenzymes in this organelle. The occurrence of functional cobalamins in chloroplasts has not been reported in other photosynthetic eukaryotes.     

Resistance to glyphosate in the cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> as result of pre-selective mutations

Adaptation of Microcystis aeruginosa (Cyanobacteria) to resist the herbicide glyphosate was analysed by using an experimental model. Growth of wild-type, glyphosate-sensitive (G^s) cells was inhibited when they were cultured with 120 ppm glyphosate, but after further incubation for several weeks, occasionally the growth of rare cells resistant (G^r) to the herbicide was found. A fluctuation analysis was carried out to distinguish between resistant cells arising from rare spontaneous mutations and resistant cells arising from other mechanisms of adaptation. Resistant cells arose by rare spontaneous mutations prior to the addition of glyphosate, with a rate ranging from 3.1 × 10⁻⁷ to 3.6 × 10⁻⁷ mutants per cell per generation in two strains of M. aeruginosa; the frequency of the G^r allele ranged from 6.14 × 10⁻⁴ to 6.54 × 10⁻⁴. The G^r mutants are slightly elliptical in outline, whereas the G^s cells are spherical. Since G^r mutants have a diminished growth rate, they may be maintained in uncontaminated waters as the result of a balance between new resistants arising from spontaneous mutation and resistants eliminated by natural selection. Thus, rare spontaneous pre-selective mutations may allow the survival of M. aeruginosa in glyphosate-polluted waters via G^r clone selection.     

Enzymes of nitrogen assimilation in maize roots

The intracellular distribution of enzymes involved in the Crassulacean acid metabolism (CAM) has been studied in Bryophyllum calycinum Salisb. and Crassula lycopodioides Lam. After separation of cell organelles by isopycnic centrifugation, enzymes of the Crassulacean acid metabolism were found in the following cell fractions: Phosphoenolpyruvate carboxylase in the chloroplasts; NAD-dependent malate dehydrogenase in the mitochondria and in the supernatant; NADP-dependent malate dehydrogenase and phosphoenolpyruvate carboxykinase in the chloroplasts; NADP-dependent malic enzyme in the supernatant and to a minor extent in the chloroplasts; NAD-dependent malic enzyme in the supernatant and to some degree in the mitochondria; and pyruvate; orthophosphate dikinase in the chloroplasts. The activity of the NAD-dependent malate dehydrogenase was due to three isoenzymes separated by (NH₄)₂SO₄ gradient solubilization. These isoenzymes represented 17, 78, and 5% of the activity recovered, respectively, in the order of elution. The isoenzyme eluting first was associated with the mitochondria and the second isoenzyme was of cytosolic origin, while the intracellular location of the third isoenzyme was probably the peroxisome. Based on these findings, the metabolic path of Crassulacean acid metabolism within cells of CAM plants is discussed. New address: Institut für Pflanzenphysiologie und Zellbiologie, Freie Universität Berlin, Königin-Luise-Straße 12-16a. D-1000 Berlin 33     

C4-Dicarboxylic acid metabolism in bundle-sheath chloroplasts,mitochondria and strands of Eriochloa borumensis Hack., a phosphoenolpyruvate-carboxykinase type C4 species

C₄-acid metabolism by isolated bundlesheath chloroplasts, mitochondria and strands of Eriochloa borumensis Hack., a phosphoennolpyruvate-carboxykinase (PEP-CK) species, was investigated. Aspartate, oxaloacetate (OAA) and malate were decarboxylated by strands with several-fold stimulation upon illumination. There was strictly light-dependent decarboxylation of OAA and malate by the chloroplasts, but the chloroplasts did not decarboxylate aspartate in light or dark. PEP was a primary product of OAA or malate decarboxylation by the chloroplasts and its formation was inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea or NH₄Cl. There was very little conversion of PEP to pyruvate by bundle-sheath chloroplasts, mitochondria or strands. Decarboxylation of the three C₄-acids by mitochondria was light-independent. Pyruvate was the only product of mitochondrial metabolism of C₄-acids, and was apparently transaminated in the cytoplasm since PEP and alanine were primarily exported out of the bundle-sheath strands. Light-dependent C₄-acid decarboxylation by the chloroplasts is suggested to be through the PEP-CK, while the mitochondrial C₄-acid decarboxylation may proceed through the NAD-malic enzyme (NAD-ME) system. In vivo both aspartate and malate are considered as transport metobolites from mesophyll to bundle-sheath cells in PEP-CK species. Aspartate would be metabolized by the mitochondria to OAA. Part of the OAA may be converted to malate and decarboxylated through NAD-ME, and part may be transported to the chloroplasts for decarboxylation through PEP-CK localized in the chloroplasts. Malate transported from mesophyll cells may serve as carboxyl donor to chloroplasts through the chloroplastic NAD-malate dehydrogenase and PEP-CK. Bundle-sheath strands and chloroplasts fixed ¹⁴CO₂ at high rates and exhibited C₄-acid-dependent O₂ evolution in the light. Studies with 3-mercaptopicolinic acid, a specific inhibitor of PEP-CK, have indicated that most (about 70%) of the OAA formed from aspartate is decarboxylated through the chloroplastic PEP-CK and the remaining (about 30%) OAA through the mitochondrial NAD-ME. Pyruvate stimulation of aspartate decarboxylation is discussed; a pyruvate-alanine shuttle and an aspartate-alanine shuttle are proposed between the mesophyll and bundle-sheath cells during aspartate decarboxylation through the PEP-CK and NAD-ME system respectively.Abbreviations CK carboxykinase - -Kg -ketoglutarate - ME malic enzyme - 3-MPA 3-mercaptopicolinic acid - OAA oxaloacetate - PEP phosphoenolpyruvate - R5P ribose-5-phosphate     

Ultrastructural aspects of kranz anatomy inDigitaria sanguinalis andSetaria viridis (poaceae)

Structural differentiation of Kranz anatomy has been investigated in leaf cross sections of two C-4 Poaceae:Digitaria sanguinalis andSetaria viridis. The study mainly focused on cellular and interfacial features of bundle sheath (BS) and mesophyll (MS) cells of the C-4 structure. Prominent BS, spaced by only two MS cells apart, were surrounded concentrically by a layer of MS cells. BS cells ofS. viridis had centrifugally arranged relatively large chloroplasts containing much starch, but the chloroplasts had agrana to rudimentary grana. Structural and size dimorphisms, when starch was present, were detected between BS and MS chloroplasts. Loosely arranged MS cells had peripherally displaced smaller chloroplasts containing little to none starch. BS chloroplasts ofD. sanguinalis were similar to those ofS. viridis, but had very little starch and well-developed long agranal stroma lamella. Features of MS cells were similar in both species, but well-defined peripheral reticulum (PR) was easily recognized in MS chloroplasts ofS. viridis. Virtually no PR was developed in BS chloroplasts examined. BS cells contained more mitochondria and microbodies, but no structural dimorphism was noticed. The electron-dense suberized lamella were often observed between BS and MS cells, especially in the primary wall of BS cells. It was most frequently found at the BS and MS cell interfaces and terminated in radial walls of the adjacent BS cells. Prominent pits with plasmodesmata (pd) were seen in the walls of both cells. There also were numerous pd in outer tangential walls of the BS cells. The number of pd ranged from 20 to 60. The pd trasversed a segment of cell wall much thinner than the adjacent wall. The current cellular data have been compared to the ultrastructural features known in leaves of other C-4 plants, especially NADP-ME species.     

Degradation of chloroplast DNA in second leaves of rice (Oryza sativa) before leaf yellowing

Summary The second leaf ofOryza sativa develops, grows and ages within the 10 days that follow imbibition under our controlled continuous-light conditions. Proplastids in the leaf cells develop, mature to become chloroplasts and then age and disintegrate. In an examination of this life process, we studied first the behavior and the number of copies of plastid DNA and levels of chlorophyll by epifluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI), and by fluorimetry with a video-intensified microscope photon-counting system (VIMPCS). The results indicated that the number of copies of the plastid DNA per plastid increased and reached to plateau value of approximately 100 at the time when the elongation of the mesophyll cells and the enlargement of chloroplasts ceased 96 h after imbibition. However, 24 h later, the number of copies of plastid DNA per chloroplast began to decrease and fell rapidly to approximately 30 copies within 168 h after imbibition. Our examination of the number of chloroplasts per mesophyll cell indicated that no division of chloroplasts occurred more than 72 h after imbibition. The results suggest that the decrease in number of copies of plastid DNA per chloroplast was not due to an increase in the number of chloroplasts, but that this decrease was caused by degradation by unidentified enzymes. Since visible senescence of leaves, which was characterized by development of a yellowish color, began 168 h after imbibition, the degradation of plastid DNA seemed to occur 48 h before the visible leaf senescence. When we tested the nucleolytic activities in the second leaves after imbibition by digestion of plasmids in vitro and DNA-SDS polyacrylamide gel electrophoresis, five Ca²⁺–, four Zn²⁺–, and four Mn²⁺–dependent nucleases were detected in the leaf blades, and one of the Ca²⁺–, two of the Zn²⁺–, and two of the Mn²⁺–dependent nucleases were also identified in a purified preparation of intact chloroplasts. When the activity of the Zn²⁺–dependent nucleases (51 kDa and 13 kDa) increased markedly, degradation of the plastid DNA occurred. These results suggest that the destruction of chloroplast DNA, which occurs approximately 48 h before leaf yellowing, could be due to the activation of some metallo-nucleases and, furthermore, this enzymatic degradation propels the leaf towards senescence.     

Effects of reactive oxygen species on α-tocopherol production in mitochondria and chloroplasts of Euglena gracilis

The effects of reactive oxygen species (ROS) on α-tocopherol production in mitochondria and chloroplasts of Euglena gracilis were investigated. Addition of an organic carbon source to the medium resulted in increased mitochondrial activity, intracellular O₂ ^- concentration and α-tocopherol productivity in E. gracilis W₁₄ZUL (a chloroplast deficient mutant). α-Tocopherol productivity of the wild-type strain (with both mitochondria and chloroplast) was higher than that of the W₁₄ZUL strain. In the case of the wild strain, the O₂ ⁻ generated in chloroplasts was efficiently scavenged by the α-tocopherol synthesized inside the chloroplast. In photoheterotrophic culture (with an organic carbon source), there was a positive correlation between α-tocopherol production and O₂ ⁻ generation. Addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of photosynthesis) resulted in increased O₂ ⁻ generation and α-tocopherol productivity. These results indicate that the ROS generated in mitochondria and chloroplasts play important roles in α-tocopherol production by E. gracilis. The presence of chloroplasts and generation of intracellular ROS are important for efficient production of α-tocopherol.     

Preparation of intact chloroplasts by chemically induced lysis of the green alga Dunaliella marina

A method for the isolation in high yield of intact chloroplasts from the unicellular green alga Dunaliella marina (Volvocales) is described. This procedure uses chemically induced lysis of cells with the polycationic macromolecules, DEAE-dextran (M=500,000) or poly-D,l-lysine (M=30,000-70,000). Reaction conditions were optimized with respect to obtaining a high yield of intact chloroplasts, after isopycnic centrifugation in a linear sucrose density gradient, by varying the concentration of polycation and the temperature and pH of incubation. Broken chloroplasts devoid of the stromal marker enzymes fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, but containing mitochondrial (fumarase) and microbody (catalase) contamination, were banded at a bouyant density of 1.18 g cm^-3. Intact chloroplasts, as indicated by their retention of alkaline fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, were found in 30% yield (chlorophyll in intact cells, 100%) at an equilibrium density of 1.24 g cm^-3. Contamination by cytoplasmic material (pyruvate kinase), mitochondria, and microbodies was less than 8% each.Abbreviations Chl chlorophyll - DEAE-dextran diethylaminoethyl-dextran - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - FBPase fructose-1,6-bisphosphate phosphatase, EC 3.1.3.11 - G6P-DH glucose 6-phosphate dehydrogenase, EC 1.1.1.49 - HEPES N-2-hydroxyethylpiperazine-N-ethanesulphonic acid - MES 2-(N-morpholino)ethanesulphonic acid - RuBP carboxylase D-ribulose-1,5-bisphosphate carboxylase or 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39     

NaCl胁迫对宁夏枸杞叶和幼根显微及超微结构的影响

以宁夏枸杞为材料,采用超薄切片技术制备样品,应用光学显微镜和透射电镜分析了不同浓度NaCl胁迫条件下宁夏枸杞叶和幼根显微及超微结构的变化。结果表明：随着NaCl胁迫的加重,(1)叶片上表皮细胞增厚,栅栏组织细胞出现缩短现象,排列疏松且紊乱;幼根的初生结构无明显变化。(2)叶片栅栏组织中叶绿体不再紧靠在细胞膜上,叶绿体双层膜破坏,基粒片层松散排列,杂乱无章,出现膨胀和空泡现象,淀粉粒和嗜锇颗粒增多,叶肉细胞中线粒体发生轻微变化;幼根中皮层薄壁细胞线粒体形状发生改变,结构破坏,内膜和外膜模糊甚至破裂,大多数嵴模糊,出现空泡现象;细胞核解体,基质外溢。研究表明, 不同浓度的NaCl胁迫对宁夏枸杞叶片和幼根细胞的显微及超微结构影响不同,NaCl浓度大于200 mmol/L时,宁夏枸杞叶片和幼根细胞的显微及超微结构发生了明显变化,且叶肉细胞中线粒体的变化没有叶绿体的变化显著,推测叶肉细胞中线粒体的耐盐性比叶绿体强。     

Effects of the herbicide isopropyl-N-phenyl carbamate on microtubules and MTOCs in lines of Nicotiana sylvestris resistant and sensitive to its action

To clarify the mechanism of isopropyl-N-phenyl carbamate (IPC) action on higher plant cells the sensitivity of microtubules (cortical network and mitotic arrays) and microtubule organizing centers to IPC treatment (30 microM) in IPC-resistant and sensitive Nicotiana sylvestris lines was studied. It was clearly demonstrated that IPC does not depolymerize plant MTs but causes the MTOC damage in cells, which results in MTOC fragmentation, splitting of the spindle poles and in abnormal division spindle formation. It was also found that IPC-resistance of mutant N. sylvestris line correlates not with tubulin resistance to IPC action but possibly with resistance of one of the proteins involved in MTOC composition.     

Influence of the herbicide oryzalin on cytoskeleton and growth ofFunaria hygrometrica protonemata

Summary Protonemata ofFunaria hygrometrica were exposed to the herbicidal MT inhibitor oryzalin. A reduction of the growth rate together with a disturbance of oriented polar growth is observed. Both effects are reversible. Visualization of MT by IFT reveals differential sensitivities of MT. At lower concentrations (10^–6 M) only the cytoplasmic MT are depolymerized causing impairment of the migration of the nucleus and the transport of the plastids. Close association of MT with the surface of the plastids is demonstrated. At higher concentrations of oryzalin spindle and phragmoplast MT are affected as well. They are found in unusual orientations and display a variety of aberrant forms like multipolar spindles or the occurrence of several mini-spindles within one cell. The mode of action of oryzalin is discussed and the necessity of a continuous network of cytoplasmic MT between nucleus and growing tip for the maintenance of polar growth is emphasized.Abbreviations CIPC isopropylN-3-chlorophenyl carbamate - DAPI Diamidino-2-phenylindol - DMSO dimethyl sulfoxide - EGTA ethy-leneglycol-tetraacetic acid - FITC fluorescein isothiocyanate - IFT indirect immunofluorescence technique - IPC isopropylN-phenylcar-bamate - MSB microtubule stabilizing buffer - MT microtubule - MTOC microtubule organizing centre - Ph phragmoplast - PIPES piperazine-diethane sulfonic acid - S spindle Dedicated to Prof. Dr. K. E.Wohlfarth-Bottermann on the occasion of his 65th birthday.     

Accelerated cell death 2 suppresses mitochondrial oxidative bursts and modulates cell death in Arabidopsis

The Arabidopsis ACCELERATED CELL DEATH 2 (ACD2) protein protects cells from programmed cell death (PCD) caused by endogenous porphyrin‐related molecules like red chlorophyll catabolite or exogenous protoporphyrin IX. We previously found that during bacterial infection, ACD2, a chlorophyll breakdown enzyme, localizes to both chloroplasts and mitochondria in leaves. Additionally, acd2 cells show mitochondrial dysfunction. In plants with acd2 and ACD2 ⁺ sectors, ACD2 functions cell autonomously, implicating a pro‐death ACD2 substrate as being cell non‐autonomous in promoting the spread of PCD. ACD2 targeted solely to mitochondria can reduce the accumulation of an ACD2 substrate that originates in chloroplasts, indicating that ACD2 substrate molecules are likely to be mobile within cells. Two different light‐dependent reactive oxygen bursts in mitochondria play prominent and causal roles in the acd2 PCD phenotype. Finally, ACD2 can complement acd2 when targeted to mitochondria or chloroplasts, respectively, as long as it is catalytically active: the ability to bind substrate is not sufficient for ACD2 to function in vitro or in vivo. Together, the data suggest that ACD2 localizes dynamically during infection to protect cells from pro‐death mobile substrate molecules, some of which may originate in chloroplasts, but have major effects on mitochondria.     

Co-localization of mitochondria with chloroplasts is a light-dependent reversible response

Co-localization of mitochondria with chloroplasts in plant cells has long been noticed as beneficial interactions of the organelles to active photosynthesis. Recently, we have found that mitochondria in mesophyll cells of Arabidopsis thaliana expressing mitochondrion-targeted green fluorescent protein (GFP) change their distribution in a light-dependent manner. Mitochondria occupy the periclinal and anticlinal regions of palisade cells under weak and strong blue light, respectively. Redistributed mitochondria seem to be rendered static through co-localization with chloroplasts. Here we further demonstrated that distribution patterns of mitochondria, together with chloroplasts, returned back to those of dark-adapted state during dark incubation after blue-light illumination. Reversible association of the two organelles may underlie flexible adaptation of plants to environmental fluctuations.Key words: Arabidopsis thaliana, blue light, chloroplast, green fluorescent protein, mesophyll cell, mitochondrion, organelle positioningHighly dynamic cell organelles, mitochondria, are responsible not only for energy production, but also for cellular metabolism, cell growth and survival as well as gene regulations.^1,2 Appropriate intracellular positioning and distribution of mitochondria contribute to proper organelle functions and are essential for cell signaling.^3,4 In plant cells operating photosynthesis, the co-localization of mitochondria with chloroplasts has been a well known phenomenon for a long period of time.^5,6,7 Physical contact of mitochondria with chloroplasts may provide a means to transfer genetic information from the organelle genome,⁸ as well as to exchange metabolite components; a process required for the maintenance of efficient photosynthesis.^9,10,11Using Arabidopsis thaliana stably expressing mitochondrion-targeted GFP,¹² we have recently examined a different aspect of mitochondria positioning. Although mitochondria in leaf mesophyll cells are highly motile under dark condition, mitochondria change their intracellular positions in response to light illumination.¹³ The pattern of light-dependent positioning of mitochondria seems to be essentially identical to that of chloroplasts.¹⁴ Mitochondria occupy the periclinal regions under weak blue light (wBL; 470 nm, 4 µmol m⁻²s⁻¹) and the anticlinal regions under strong blue light (sBL; 100 µmol m⁻²s⁻¹), respectively. A gradual increase in the number of static mitochondria located in the vicinity of chloroplasts in the periclinal regions with time period of wBL illumination clearly demonstrates that the co-localization of these two organelles is a light-induced phenomenon.¹³In the present study, to ask whether the light-dependent positioning of mitochondria is reversible or not, a time course of mitochondria redistribution was examined transferring the sample leaves from light to dark conditions. The representative results (Fig. 1) clearly show that mitochondria re-changed their positions within several hours of dark treatment. Immediately after dark adaptation, mitochondria in the palisade mesophyll cells were distributed randomly throughout the cytoplasm (Fig. 1A and ^{ref. 13}). Chloroplasts were distributed along the inner periclinal walls and the lower half of the anticlinal walls. On the contrary, mitochondria accumulated along the outer (Fig. 1B) and inner periclinal walls when illuminated with wBL. Chloroplast position was also along the outer and inner periclinal walls. Many of the mitochondria located near the chloroplasts lost their motility. When wBL-illuminated leaves were transferred back to dark condition, the numbers of mitochondria and chloroplasts present on the periclinal regions began to decrease within several hours (Fig. 1C). After 10 h dark treatment, distribution patterns of mitochondria as well as chloroplasts almost recovered to those of dark-adapted cells (Fig. 1D).Open in a separate windowFigure 1Distribution of mitochondria and chloroplasts on the outer periclinal regions of palisade mesophyll cells of A. thaliana under different light conditions. Mitochondria (green; GFP) and chloroplasts (red; chlorophyll autofluorescence) were visualized with confocal microscopy after dark adaptation (A), immediately after wBL (470 nm, 4 µmol m⁻²s⁻¹) illumination for 4 h (B), after dark treatment for 6 h (C) and 10 h (D) following the 4-h wBL illumination, respectively. Bar = 50 µm.To our knowledge, this may be the first report that directly demonstrates that wBL regulates mitochondria and chloroplast positioning in a reversible manner, though the nuclei in A. thaliana leaf cells were also found to reverse their positions when transferred from sBL to dark conditions.¹⁵ Reversible regulation of organelle positioning in leaf cells should play critical roles in adaptation of plants to highly fluctuating light conditions in the nature. Since distribution patterns of mitochondria under wBL and sBL are identical to those of chloroplasts, we can assume that phototropins, the BL receptors for chloroplast photo-relocation movement,¹⁶ may have some role in the redistribution of mitochondria. On the other hand, we also found that red light exhibited a significant effect on mitochondria positioning (Islam et al. 2009), suggesting an involvement of photosynthesis. These possibilities are now under investigation.