Intracellular Localization of 3H-IAA in the Apical Bud of Lycopersicon esculentum

Driss-Ecole, D. and Perbal, G. 1987. Intracellular localizationof ³H-IAA in the apical bud of Lycopersicon esculentum.—J.exp. Bot. 38: 1362–1372. High resolution autoradiography of ³H-IAA was performed on ultra-thinsections of the apical bud of Lycopersicon esculentum treatedby DCC or glutaraldehyde. A quantitative study of the localizationof the labelling in the compartments (cell wall, cytoplasm,vacuoles and nucleus) of four types of cells (cells of the lateralzone of the apex, cells of the pith meristem, proximal and distalcells of the upper part of the pith) was made. The statisticalanalysis of the results has proved that the variability of thelabelling of the cell compartments in the four cell types wassimilar after treatment by DCC or glutaraldehyde. The densityof labelling is higher in each compartment of the meristematiccells compared with those of the differentiating cells. Thecells of the pith meristem can be distinguished from the meristematiccells of the lateral zone of the apex by a greater density oflabelling in the cytoplasm and the nucleus. These two cellulartypes contain a high density of radioactivity in vacuoles. Byconsidering the percentage of labelling of each compartmentrelative to the total labelling in the cell, it can be shownthat the meristematic cells are characterized by a high percentagein the nucleus whereas the vacuoles of the differentiating cellscontain the highest percentage of radioactivity. Key words: ³H-IAA, tomato shoot     

The plant-endogenous Fe(II)-chelator nicotianamine restricts the ferrochelatase activity of tomato chloroplasts

Stripped chloroplasts were prepared from young leaves of a tomatowild type (Lycopersicon esculentum Mill.) and its mutant chloronerva.Several morphological and biochemical abnormalities of thismutant are caused by the total lack of the plant-endogenousFe²⁺ chelator nicotianamine (NA). The ferrochelatase activitywas estimated by determination of ⁵⁹Fe incorporated into haem.A mercaptoethanol concentration of 250 mM was necessary to maintainfull enzyme activity. The reducing agent supported the reducedstate of the active site of the enzyme more than that of theiron as revealed by use of ferrous and ferric ionproviding compoundsas substrates. Chloroplasts of both genotypes exhibited a similar enzyme activity.NA inhibited this activity by nearly 100% depending on the concentrationapplied. On the basis of the formation constant of the Fe(ll)–NAcomplex and the concentrations of iron and NA in the enzymeassay as well as in the tomato shoot apex region it is proposedthat ferrochelatase acts in vivo with an iron level at the attomolarrange which is provided by NA. Key words: Ferrochelatase activity, ferrous ion concentration, nicotianamine, tomato chloroplasts, substrate limitation     

Translocation of copper and other micronutrients in tomato plants (Lycopersicon esculentum Mill.): nicotianamine-stimulated copper transport in the xylem

The influence of the endogenous micronutrient chelator, nicotianamine(NA), and of Cu nutrition on the distribution of Cu, Fe, Mn,Zn, and NA was investigated in eight different shoot organs,roots, and in xylem exudates of the NA-containing tomato wildtype Lycopersicon esculentum Mill. cv. Bonner Beste and itsNA-less mutant chloronerva. Contrary to the other heavy metals, copper transport in thexylem was inefficient in the mutant and was enhanced by an applicationof NA to the roots or leaves in proportion to the applied NAconcentration. Also, with NA application, the Cu concentrationin mutant roots decreased significantly, and increased in theshoot. Fe and Mn transport in the xylem was greater in the mutantthan in the wild type, and was decreased in the mutant by theapplication of NA to the leaves. Zn transport in the xylem wasthe same in both genotypes and was unaffected by NA application.After application of NA to leaves and roots of the mutant itwas possible to detect NA in the xylem exudate (up to 2nmolNA(g^–1 root FWh^–1). High Cu supply (3 µM) resulted in higher Cu and Mn concentrationsin all organs of the wild type as compared to mutant organs,but Fe concentrations were not influenced. Under high Cu supply(3µM) the NA concentrations of roots and the three youngestleaves of the wild type were higher than under normal Cu supply(0.3 µM). The highest concentrations were found in theshoot apex under both Cu conditions (up to 361 nmol NAg^–1FW). It is concluded from our experiments and from the high stabilityconstant of the NA-Cu-complex (log K= 18.6) that NA is involvedin Cu translocation whereas for the translocation of Fe, Mn,and Zn, NA is not essential. Key words: Copper transport, micronutrients, mobilization, nicotianamine, xylem     

Cytochemical Studies on Phytochrome-Mediated Changes of Ca2+ Localization in Etiolated Oat Coleoptile Cells

The antimonate-staining procedure and X-ray microanalysis techniquewere used to determine the pattern of Ca₂₊ localization in etiolatedoat (Avena sativa L.) coleoptile parenchyma cells. Precipitatesof calcium antimonate, indicating the presence of Ca₂₊ and confirmedby X-ray microanalysis, were found associated with the outerand inner surfaces of the plasma membrane of cells of dark-grownseedlings. After exposure of seedlings to red light, precipitatesof calcium antimonate were additionally observed in cisternaeof the endoplasmic reticulum. In the cells of oat coleoptilesexposed to red light and then followed immediately by farredlight, Ca₂₊ was observed on the outside of the plasma membrane,in cell walls and in the vacuoles. The results suggest thatphytochrome mediates the regulation of the intracellular Ca₂₊localization. Key words: Antimonate procedure, Avena sativa L., Ca²⁺ (localization), phytochrome, X-ray microanalysis     

Iron-containing particles accumulate in organelles and vacuoles of leaf and root cells in the nicotianamine-free tomato mutantchloronerva

Summary The mutantchloronerva ofLycopersicon esculentum Mill is the only known plant mutation that leads to a complete loss of the endogenous iron chelator nicotianamine. The mutant exhibits several morphological alterations and a permanent activation of the strategy I reactions of iron uptake as well as iron accumulation in roots and leaves. The electron microscopic energy loss technique of energy spectroscopic imaging (ESI) was used to localise the iron accumulated in the organs of wild-type and mutant plants. Iron-containing particles were detected in the chloroplast stroma and in vacuoles of mutant leaves, and in root cells in vacuoles and in mitochondria. In wild-type organs such particles were found at the same sites but they were smaller in size and occurred less frequently. The findings indicate that these compartments are preferential sites of iron storage or deposition in tomato tissues. It is discussed that the iron-containing particles detected are the result of iron release by oxidative stress. Application of nicotianamine to mutant plants, which reverts the mutant phenotype, led to a significant decrease of the iron-containing particles. This is seen as an indication that they may serve as intermediate iron stores and emphasises the crucial role of nicotianamine for the normal iron distribution in cells and organs.Dedicated Prof. Dr. K. Müntz on the occasion of his 65th birthday     

Subcellular Localization and Characterization of Excessive Iron in the Nicotianamine-less Tomato Mutant chloronerva

To understand the function of the Fe2+-complexing compound nicotianamine (NA) in the iron metabolism of plants we have localized iron and other elements in the NA-containing tomato wild type (Lycopersicon esculentum) and its NA-free mutant chloronerva by quantitative x-ray microanalysis. Comparison of element composition of the rhizodermal cell walls indicated that the wild type accumulated considerable amounts of iron and phosphorus in the cell wall, whereas in the mutant iron and phosphorus were detected in the cytoplasm and vacuoles of the rhizodermis. In mutant leaves containing high iron concentrations in the symplast, electron-dense inclusions were detected in chloroplasts and phloem. Such particles, consisting mainly of iron and phosphorus, were never found in the wild type and were very rarely detected in young chlorotic mutant leaves or after treatment of the mutant with NA. For further characterization the electron-dense inclusions in mutant leaves were isolated and compared by sodium dodecyl sulfate-gel electrophoresis and immunoblotting to ferritin from iron-loaded Phaseolus vulgaris leaves. Antibodies raised against purified Phaseolus leaf ferritin were used. Neither in mutant nor in wild type (iron loaded and control) was ferritin protein detected. These results suggest that the electron-dense inclusions in mutant leaves are not identical with ferritin. It is concluded that NA is necessary to complex ferrous iron in a soluble and available form within the cells. In the absence of NA the precipitation of excessive iron in the form of insoluble ferric phosphate compounds could protect the cells from iron overload.     

Ectopic expression of nicotianamine synthase genes results in improved iron accumulation and increased nickel tolerance in transgenic tobacco

Heavy metals are essential for basic cellular processes but toxic in higher concentrations. This requires the precise control of their intracellular concentrations, a process known as homeostasis. The metal-chelating, non-proteinogenous amino acid nicotianamine (NA) is a key component of plant metal assimilation and homeostasis. Its precise function is still unknown. Therefore, this article aims to contribute new information on the in vivo function of NA and to evaluate its potential use for plant nutrition and crop fortification. For this purpose, a nicotianamine synthase gene of Arabidopsis thaliana was ectopically expressed in transgenic tobacco plants. The presence of extra copies of the nicotianamine synthase gene co-segregated with up to 10-fold elevated levels of NA in comparison with wild type. The increased NA level led to: (a) a significantly increased iron level in leaves of adult plants; (b) the accumulation of zinc and manganese, but not copper; (c) an improvement of the iron use efficiency in adult plants grown under iron limitation; and (d) an enhanced tolerance against up to 1 m m nickel. Taken together, the data predict that NA may be a useful tool for improved plant nutrition on adverse soils and possibly for enhanced nutritional value of leaf and seed crops.     

Preparation of tissues for localization of sex hormones by autoradiography

Summary The intracellular localization of ³H-oestradiol in various tissues was examined. For this purpose standard autoradiograms were prepared after freeze drying, osmium vapour fixation and Epon embedding. The radioactive material showed a preferential localization in all the tissues studied, viz. uterus, vagina and anterior pituitary gland. In all tissues a major proportion of the radioactivity was associated with the cell nuclei. Radioactive labelling outside the cell boundaries and in the surrounding connective tissue was almost lacking. Likewise, a very low number of silver grains were found over the Epon in the clefts and at the margin of the tissue sections. The observations appear to indicate that the diffusion and probably also the intracellular dislocation of the radioactive material are very limited.The validity of the results was examined. Pre- and postmortem changes, rapid freezing and osmium vapour fixation were not observed to influence the localization of the radioactivity. Furthermore, the localization was the same whether embedding was carried out at –10°C or at 50°C.It is concluded that freeze drying, osmium vapour fixation and Epon embedding provide a valid method for accurate intracellular localization of steroid sex hormones.This work was supported by grants from The Norwegian Cancer Society and by Nordisk Insulinfond. The skilful assistance of Miss Helga Friedl and Mrs. Jane Larsen is gratefully acknowledged.     

Interaction of Auxin-TIBA in Lycopersicon esculentum

[5-³H] indoleacetic acid (IAA) was applied to the apical budof young tomato plants (Lycopersicon esculentum Mill.) treatedor not with 2,3,5-triiodobenzoic acid (TIBA). Chromatographicanalysis showed that treatment with TIBA increased the quantityof breakdown products in the apical 1.5 mm segment of the stem.Autoradiographs of ultra-thin sections of this segment wereprepared after treatment with glutaraldehyde. The inhibitionof polar auxin transport by TIBA caused an increase in the densityof labelling of all the cellular types in the shoot apex. TheTIBA treatment provoked a modification of the structure of theshoot apex in which the greatest increases in density of labellingwere detected in the superficial tunical layer and in the pithmeristem. The increase in radioactivity of the superficial tunicallayer could be explained by the presence of polyphenols in thevacuoles. For the pith meristem, the TIBA treatment intensifiedthe high labelling previously observed at this level in thecontrol plants. This could indicate a role for auxin in cellularelongation. It could also point to the importance of the pithmeristem in the functioning of the shoot apex. Key words: [³H] IAA, Lycopersicon esculentum, TIBA     

Localization of phytohemagglutinin in the embryonic axis of Phaseolus vulgaris with ultra-thin cryosections embedded in plastic after indirect immunolabeling

We have examined the properties and subcellular localization of phytohemagglutinin (PHA), the major lectin of the common bean (Phaseolus vulgaris.), in the axis cells of nearly mature and imbibed mature seeds. On a protein basis the axis contained about 15% as much PHA as the cotyledons. Localization of PHA was done with an indirect immunolabeling method (rabbit antibodies against PHA, followed by colloidal gold particles coated with goat antibodies against rabbit immunoglobulins) on ultra-thin cryosections which were embedded in plastic on the grids after the immunolabeling procedure. The embedding greatly improved the visualization of the subcellular structures. The small (4 nm) collodial gold particles, localized with the electron microscope, were found exclusively over small vacuoles or protein bodies in all the cell types examined (cortical parenchyma cells, vascular-bundle cells, epidermal cells). The matrix of these vacuoles-protein bodies appears considerably less dense than that of the protein bodies in the cotyledons, but the results confirm that in all parts of the embryo PHA is localized in similar structures.Abbreviations IgG immunoglobulin G - M_r relative molecular weight - PBS phosphate-buffered saline - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Fe homeostasis in plant cells: Does nicotianamine play multiple roles in the regulation of cytoplasmic Fe concentration?

The cellular and intracellular localization of the non-proteogenic amino acid nicotianamine (NA) in leaves and root elongation zones was immunochemically investigated in pea (Pisum sativum L.) and tomato (Lycopersicon esculentum Mill.) plants grown under various iron regimes and in three mutants defective in the regulation of iron uptake. Strongest immunostaining was observed in the over-accumulating pea mutants brz and dgl, and in iron-loaded wild-type plants. Fe concentration and NA level paralleled staining intensity, indicating that NA synthesis is induced by high iron availability. While label was mainly present in the cytoplasm under normal (10 microM) Fe supply and under Fe deprivation, most of the labeling was present in the vacuole in iron-loaded plants. This pattern resembled the distribution of NA in Fe over-accumulating mutants, indicating the possible importance of vacuolar sequestration in the detoxification of excess Fe. Based on the dependence of the cellular distribution of NA on the iron nutritional status of the plant, a possible role of NA in buffering free Fe in root and leaf cells was inferred. We show here for the first time that the NA concentration is increased in response to iron overload, indicating that, besides other classes of intracellular metal-binding ligands, NA may play an essential role in iron tolerance.     

Immunoassay and ultrastructural localization of isopentenyladenine and related cytokinins using monoclonal antibodies

Two hybridoma cell lines, J40-IV-A1 and J40-IV-C4 were obtained from a fusion of spleen cells of Balb/c mice immunized against an isopentenyladenosine-bovine serum albumin conjugate with X63. Ag 8.653 myeloma cells. These hybrids secrete monoclonal antibodies of the immunoglobulin G (IgG) class and share high affinities and specificities to isopentenyladenine and isopentenyladenosine suitable for the detection of femtomole amounts of these cytokinins in plant extracts by enzyme-linked immunosorbent assay (ELISA). One of the monoclonal antibodies (J40-IV-C4) has been employed to localize isopentenyladenine immunoreactivity in a cytokinin-over-producing mutant of the moss, Physcomitrella patens. After fixation and embedding at low temperature, immunoreactivity was visualized in protonemal filaments of the moss mutant by the use of indirect immunogold labelling. In the mutant, the labelling was predominantly in the wall of the protonemal cells. Neither the wild-type nor control treatments showed any labelling. The signficance of these observations is discussed with respect to the applicability of immunocytochemical techniques for the localization of low-molecular-weight compounds in plant tissue.Abbreviations ELISA enzyme linked immunosorbent assay - HPLC high-performance liquid chromatography - IP isopentenyladenine - IPA isopentenyladenosine - mAB monoclonal antibody - OVE cytokinin-over-producing mutant - RIA radioimmunoassay     

Tomato (Lycopersicon esculentum cv. Pik-Red) Leaf Carboxypeptidase: Identification, N-terminal Sequence, Stress-Regulation, and Specific Localization in the Paraveinal Mesophyll Vacuoles

Wounding of tomato (Lycopersicon esculentum L.) leaves causessystemic induction of a serine-type carboxypeptidase activity.We find this activity to be present in several isoforms. Antibodiesraised against the leaf carboxypeptidase inhibited the enzymeactivity and the immunoprecipitates were resolved into a 69-kDapolypeptide and a doublet of 35/37-kDa proteins on SDS-PAGE.Immunoblot analysis of the leaf proteins also immunodecoratedthe 69-kDa and 35/37-kDa proteins. Amino acid sequence analysisof the amino-terminus of the tomato leaf 69-kDa carboxypeptidaseshowed it to be similar to the barley A-chain carboxypeptidaseI [Sorenson et al. (1986) Carlsberg Res. Commun. 51: 475], sharingAla as the N-terminus and the sequences, AlaProGln and LeuProGlyPhe.Superimposition of a chemical stress (copper treatment) on woundingapparently lowered wound-induced carboxypeptidase activity inthe leaf, suggesting that cupric ions might interact with thewound signal. Immunogold electron microscopy indicated thatthe leaf carboxypeptidase was specifically localized withinthe inclusions of vacuoles of vascular parenchyma cells. Incupric ion-treated tissues, carboxypeptidase was found redistributedto other parts of the cell, indicating that this treatment,but not wounding, causes general vacuolar membrane damage. ⁴Deceased.     

Metabolic engineering of bread wheat improves grain iron concentration and bioavailability

Bread wheat (Triticum aestivum L.) is cultivated on more land than any other crop and produces a fifth of the calories consumed by humans. Wheat endosperm is rich in starch yet contains low concentrations of dietary iron (Fe) and zinc (Zn). Biofortification is a micronutrient intervention aimed at increasing the density and bioavailability of essential vitamins and minerals in staple crops; Fe biofortification of wheat has proved challenging. In this study we employed constitutive expression (CE) of the rice (Oryza sativa L.) nicotianamine synthase 2 (OsNAS2) gene in bread wheat to up‐regulate biosynthesis of two low molecular weight metal chelators – nicotianamine (NA) and 2′‐deoxymugineic acid (DMA) – that play key roles in metal transport and nutrition. The CE‐OsNAS2 plants accumulated higher concentrations of grain Fe, Zn, NA and DMA and synchrotron X‐ray fluorescence microscopy (XFM) revealed enhanced localization of Fe and Zn in endosperm and crease tissues, respectively. Iron bioavailability was increased in white flour milled from field‐grown CE‐OsNAS2 grain and positively correlated with NA and DMA concentrations.     

Postembedding α-tubulin immunolabelling of isolated centrosomes

Summary Accurate ultrastructural localization of the components of centrosomes is an important step toward the determination of their function. We have used an electron microscopy procedure to preserve centrosome-associated antigens which enables their high-resolution localization. The unique part of our procedure is the application of a post-sectioning fixation step which overcomes the poor section contrast and morphological appearance that limits the use of low-temperature processing and Lowicryl embedding. The efficacy of our approach is demonstrated by the efficient labelling of α-tubulin in the well-preserved and contrasted microtubule barrels of the centrides of isolated mammalian centrosomes.     

Response to Phosphate Deficiency in Bean (Phaseolus vulgarisL.) Roots. Respiratory Metabolism, Sugar Localization and Changes in Ultrastructure of Bean Root Cells

Bean plants (Phaseolus vulgarisL. ‘Zlota Saxa’)were cultured on complete (+P) or phosphate-deficient (-P) nutrientmedium. A large increase in glucose concentration was foundin the meristematic zone of -P roots compared to control roots.The increased glucose concentration in the meristematic zonedid not influence total respiration rate. Glucose or uncoupler(carbonyl cyanide m-chlorophenylhydrazone) failed to increasethe respiration rate in -P root segments, but stimulated respirationin +P roots. The ultrastructure of cortical cells from the meristematicroot zone showed marked differences between +P and -P plants.Large vacuoles, invaginations of the plasmalemma and condensedforms of mitochondria were dominating features in cortical cellsof -P roots. Analysis of extracts after treating roots withdimethylsulfoxide (DMSO) indicated different localization ofsugars in the cell compartments. In roots of -P plants, mostof the reducing sugars were detected in the cytoplasm fractionwhile most sucrose was in the vacuole. Observations of the effectof 10% DMSO on cell ultrastructure indicated partial destructionof the plasmalemma but not the tonoplast. The localization ofreducing sugars in secondary vacuoles or plasmalemma invaginationsin the cells from the meristematic region of -P roots is discussed.Copyright1998 Annals of Botany Company. Bean (Phaseolus vulgarisL.), roots, Pi deficiency, respiration, meristematic zone, ultrastructure, sugar efflux, reducing sugars and sucrose localization.     

Immunohistochemical localization of caffeine in young Camellia sinensis (L.) O. Kuntze (tea) leaves

The anatomical localization of caffeine within young Camellia sinensis leaves was investigated using immunohistochemical methods and confocal scanning laser microscopy. Preliminary fixation experiments were conducted with young C. sinensis leaves to determine which fixation procedure retained caffeine the best as determined by high-performance liquid chromatography analysis. High pressure freezing, freeze substitution, and embedding in resin was deemed the best protocol as it retained most of the caffeine and allowed for the samples to be sectioned with ease. Immunohistochemical localization with primary anti-caffeine antibodies and conjugated secondary antibodies on leaf sections proved at the tissue level that caffeine was localized and accumulated within vascular bundles, mainly the precursor phloem. With the use of a pressure bomb, xylem sap was collected using a micro syringe. The xylem sap was analyzed by thin-layer chromatography and the presence of caffeine was determined. We hypothesize that caffeine is synthesized in the chloroplasts of photosynthetic cells and transported to vascular bundles where it acts as a chemical defense against various pathogens and predators. Complex formation of caffeine with chlorogenic acid is also discussed as this may also help explain caffeine’s localization.     

Localization of Phenolic Compounds in Tannin-secreting Cells from Sambucus racemosa L. Shoots

The localization of phenolic compounds was investigated in twokinds of idioblastic tannin cells: (1) mononucleate tannin cellsclose to the promeristem in which they are initiated, and comparedwith the surrounding tissue, and (2) the very long coenocytes(i.e. almost as long as the whole internode of Sambucus) andproducing large amounts of phenolics. One likely pathway forthe appearance of these compounds in the central vacuole ispostulated from their localization: phenolic precursors occuroutside the ER cisternae and enter the interior of parts ofthe ER cisternae which undergo fragmentation and dilatation.Many small vacuoles so formed fuse and lead to the formationof a large central vacuole. Phenolic compounds, tannin cells, idioblasts, Sambucus racemosa L., shoots     

A Comparative Study of the Structure-Function Relationship of Cross Veins in Leaves of Digitaria eriantha and Zea mays

Ultrastructural and functional differences between the crossveins of Digitaria eriantha and Zea mays were investigated.Cross veins of both genera possess similar conducting tissues,namely one tracheary element and one sieve cell. In Digitariaeriantha these conducting elements are associated with onlytwo parenchyma cells, and, those in Zea mays are completelysurrounded by chJorenchyma cells. The protein ribulose 1,5-bisphosphatecarboxylase/oxygenase (rubisco) was used as a probe for CO₂fixation sites by comparing its distribution in the varioustissue types in the leaves of the two genera. The protein wasfound to be equally and uniformly distributed in the stromalregions of the chlorenchyma sheath cell chloroplasts of longitudinalveins of both genera. The chlorenchyma sheath cells in crossveins of Zea mays show a similar distribution of the enzymeas the longitudinal bundles. However, this enzyme was shownto be absent in the cross vein parenchyma cells of Digitariaeriantha and in the mesophyll cells of both genera. Cross veins, immuno-gold labelling, ribulose 1,5-bisphosphate carboxylase/oxygenase, Digitaria eriantha, Zea mays