Tor-Henning Iversen

Immunocytochemical localization of myrosinase in Brassica napus L.

The cytological and intracellular localization of myrosinase (EC 3.2.3.1) has been studied by immunochemical techniques using paraffin-embedded sections of radicles and cotyledons from seeds of Brassica napus L. cv. Niklas. For immunolabelling, sections were sequentially incubated with a monoclonal anti-myrosinase antibody and with peroxidase-and fluorescein-isothiocyanate-conjugated secondary antibodies. Enzyme and fluorescence label was present in typical myrosin cells both in radicles and in cotyledons. With higher magnification, fluorescence label revealed that the intracellular localization of myrosinase was associated with the tonoplast-like membrane surrounding the myrosin grains in the myrosin cells. The results also indicate that a large proportion of the positive myrosin cells are located in the second-outermost cell layer of the peripheral cortex region of the radicles.

MYROSIN CELLS AND MYROSINASE

ABSTRACT The occurrence and distribution of myrosin cells and myrosinases has been followed in different organs during development of 7 species of the Brassicaceae. Using light and electron microscopy the myrosin cells were characterized morphologically and compared to other idioblasts in the tissue. Myrosin grains stain specifically with toluidine blue, methylene blue azur-II, lactophenol aniline blue and fuchsin. At the ultrastructural level myrosin grains were seen to contain proteins, and oleosomes occupy most of the myrosin cells at an early stage of development in young seedlings, together with precursors for mitochondria, plastids and microbodies. Changes in ultrastructure take place during development and light has a negative effect on the extent of cellular differentiation. Myrosin cells are generally found distributed in differentiated tissue, from the imbibition stage up to 192 h after germination. The relative area occupied by the myrosin cells has been calculated by morphometrical analyses an...

Gravity amplifies and microgravity decreases circumnutations in Arabidopsis thaliana stems: results from a space experiment.

In a microgravity experiment onboard the International Space Station, circumnutations of Arabidopsis thaliana were studied. Plants were cultivated on rotors under a light:dark (LD) cycle of 16 : 8 h, and it was possible to apply controlled centrifugation pulses. Time-lapse images of inflorescence stems (primary, primary axillary and lateral inflorescences) documented the effect of microgravity on the circumnutations. Self-sustained circumnutations of side stems were present in microgravity but amplitudes were mostly very small. In darkness, centrifugation at 0.8 g increased the amplitude by a factor of five to ten. The period at 0.8 g was c. 85 min, in microgravity roughly of the same magnitude. In white light the period decreased to c. 60 min at 0.8 g (microgravity value not measurable). Three-dimensional data showed that under 0.8 g side stems rotated in both clockwise and counter-clockwise directions. Circumnutation data for the main stem in light showed a doubling of the amplitude and a longer period at 0.8 g than in microgravity (c. 80 vs 60 min). For the first time, the importance of gravity in amplifying minute oscillatory movements in microgravity into high-amplitude circumnutations was unequivocally demonstrated. The importance of these findings for the modelling of gravity effects on self-sustained oscillatory movements is discussed.

Embryo-specific Proteins in Cyclamen persicum Analyzed with 2-D DIGE

Somatic embryogenesis can be used to produce artificial seeds of Cyclamen persicum, one of the most important ornamental plants for the European market, both as a potted plant in northern Europe and a bedding plant in the cool winters in southern Europe. The aim of this study was to obtain new insights into the molecular biology of somatic embryogenesis, which in turn can be useful for the improvement of tissue culture methodology. Total proteins were characterized from two isogenic cell lines of Cyclamen persicum, one that was embryogenic and one that never has shown any embryogenic capacity. The extracted proteins were separated by two-dimensional differential gel electrophoresis (2-D DIGE) and selected proteins were treated using the ETTAN Dalt Spot Handling Workstation. Protein identification was performed using MALDI-TOF-MS. More than 1200 Cyclamen proteins were detected; 943 proteins were common to both lines. The different protein patterns of the embryogenic and non-embryogenic cell lines were obvious: One hundred eight proteins were more abundant in the embryogenic cells, and 97 proteins in the non-embryogenic cells. Among the differentially expressed proteins, 128 were identified. MALDI-TOF-MS analysis enabled 27 spots to be proposed as candidates for embryo-specific proteins, as they were unique to the embryogenic cell line. The proteins identified are involved in a variety of cellular processes, including cell proliferation, protein processing, signal transduction, stress response, metabolism, and energy state, but the majority are involved in protein processing and metabolism. The main functions of the putative embryo-specific proteins have been discussed in proportion to their role in the somatic embryogenesis process.

Gravitropism and starch statoliths in an Arabidopsis mutant

The mutant TC 7 of Arabidopsis thaliana (L.) Heynh. has been reported to be starch-free and still exhibit root gravitropism (T. Caspar and B. G. Pickard 1989, Planta 177, 185–197). This is not consistent with the hypothesis that plastid starch has a statolith function in gravity perception. In the present study, initial light microscopy using the same mutant showed apparently starch-free statocytes. However, ultrastructural examination detected residues of amyloplast starch grains in addition to the starch-depleted amyloplasts. Applying a point-counting morphometric method, the starch grains in the individual amyloplasts in the mutant were generally found to occupy more than 20% and in a few cases up to 60% of the amyloplast area. In the wild type (WT) the starch occupied on average 98 % of the amyloplast area and appeared as densely packed grains. The amyloplasts occupied 13.9% of the area of the statocyte in the mutant and 23.3% of the statocyte area in the WT. Sedimentation of starch-depleted amyloplasts in the mutant was not detected after 40 min of inversion while in the WT the amyloplasts sedimented at a speed of 6 μm · h-1. The gravitropic reactivity and the curvature pattern were also examined in the WT and the mutant. The time-courses of root curvature in the WT and the mutant showed that when cultivated under standard conditions for 60 h in darkness, the curvatures were 83° and 44°, respectively, after 25 h of continuous stimulation in the horizontal position. The WT roots curved significantly more rapidly and with a more normal gravitropic pattern than those of the mutant. These results are discussed in relation to the results previously obtained with the mutant and with respect to the starch-statolith hypothesis.

Ethylene production during clinostat rotation and effect on root geotropism.

Summary Ethylene production from 24 h old seedlings of Lepidium sativum L. (garden cress) was followed for 20 h both when the roots were growing normally and luring rotation on a horizontal clinostat at 2 rpm. While the ethylene production in both groups was only traceable during the first 2 h, the production of ethylene was afterwards found to be higher in the rotated seedlings. By the use of an «internal standard» gas (propane) the absolute amounts of ethylene on a fresh weight basis were estimated to 2.5 and 8.6 nl · g -1 f.w. for the control and rotated group, respectively, after 5 h. Parallel to the production of ethylene a decrease in root elongation was observed. After 5 h the root length of the rotated seedlings was only 81.4 % of that of the control roots. The application of exogenous ethylene at 0.2 ppm had a dramatic decreasing effect on the development of the geotropic curvatures when the treatment period extended a threshold limit in the range of 5 to 6 h from the injection of the gas in the plant chamber. The inhibitory effect of ethylene on the curvature was caused by decreased root elongation. Elongation ceased completely after 6 h. The present results are interpreted in terms of the possible interaction of IAA and ethylene and the possible participation of auxin in root getropism.

Exploration of plant growth and development using the European Modular Cultivation System facility on the International Space Station

Space experiments provide a unique opportunity to advance our knowledge of how plants respond to the space environment, and specifically to the absence of gravity. The European Modular Cultivation System (EMCS) has been designed as a dedicated facility to improve and standardise plant growth in the International Space Station (ISS). The EMCS is equipped with two centrifuges to perform experiments in microgravity and with variable gravity levels up to 2.0 g. Seven experiments have been performed since the EMCS was operational on the ISS. The objectives of these experiments aimed to elucidate phototropic responses (experiments TROPI-1 and -2), root gravitropic sensing (GRAVI-1), circumnutation (MULTIGEN-1), cell wall dynamics and gravity resistance (Cell wall/Resist wall), proteomic identification of signalling players (GENARA-A) and mechanism of InsP3 signalling (Plant signalling). The role of light in cell proliferation and plant development in the absence of gravity is being analysed in an on-going experiment (Seedling growth). Based on the lessons learned from the acquired experience, three preselected ISS experiments have been merged and implemented as a single project (Plant development) to study early phases of seedling development. A Topical Team initiated by European Space Agency (ESA), involving experienced scientists on Arabidopsis space research experiments, aims at establishing a coordinated, long-term scientific strategy to understand the role of gravity in Arabidopsis growth and development using already existing or planned new hardware.

Acetylenic thiophenes from the roots of Echinops ellenbeckii from Ethiopia

The hexane fraction from the roots of Echinops ellenbeckii O. Hoffm. from Ethiopia yielded seven acetylenic thiophenes of which five compounds (1, 3, 4, 6, 7) are reported for the first time in this species: the monothiophenes 5-(penta-1,3-diynyl)-2-(but-3-en-1-ynyl)-thiophene (1), 5-(penta-1,3-diynyl)-2-(4-acetoxy-but-1-ynyl)-thiophene (2), 5-(penta-1,3-diynyl)-2-(3-hydroxy-4-acetoxy-but-1-ynyl)-thiophene (3), 5-(penta-1,3-diynyl)-2-(3,4-diacetoxy-but-1-ynyl)-thiophene (4), 5-(penta-1,3-diynyl)-2-(3-chloro-4-acetoxy-but-1-ynyl)-thiophene (5), 5-(penta-1,3-diynyl)-2-(3,4-epoxy-but-1-ynyl)-thiophene (6) and the dithiophene 5-[(5-acetoxymethyl-2-thienyl)-2-(but-3-en-1-ynyl)]-thiophene (7). Additionally, four fatty acids (C14, C15, C16 and C18), seven fatty acid esters and three long-chain hydrocarbons could be identified. All the structures were elucidated on the basis of spectral data by GC-MS, HRMS and the NMR techniques.

Ultradian rhythms in Arabidopsis thaliana leaves in microgravity.

Ultradian movements of Arabidopsis thaliana rosette leaves were discovered and studied under microgravity conditions in space. Weightlessness revealed new facets of these movements. The European Modular Cultivation System (EMCS) was used in a long-term white-light, light-darkness (LD; 16 : 8 h) experiment on the International Space Station (ISS). Leaves reacted with slow up or down movement (time constant several hours) after transitions to darkness or light, respectively. Superimposed movements with periods of c. 80-90 min and small-amplitude pulsed movements of 45 min were present in the light. Signal analysis (fast Fourier transform (FFT) analysis) revealed several types and frequencies of movements. Identical phase coupling was observed between the 45-min movements of the leaves of one plant. In darkness, movements of c. 120-min period were recorded. The EMCS allowed 0-g to 1-g transitions to be created. Leaves on plants germinated in microgravity started a negative gravitropic reaction after a delay of c. 30 min. Leaves grown on a 1-g centrifuge reacted to the same transition with an equal delay but had a weaker gravitropic response. The experiments provide unequivocal demonstrations of ultradian, self-sustained rhythmic movements in A. thaliana rosette leaves in the absence of the effect of gravity.

CHARACTERIZATION AND DISTRIBUTION OF DILATED CISTERNAE OF THE ENDOPLASMIC RETICULUM IN INTACT PLANTS, PROTOPLASTS, AND CALLI OF BRASSICACEAE

ABSTRACT The characteristics, occurrence, arid distribution of dilated cisternae (DC) of the rough endoplasmic reticulum (r-ER) in different plant organs of 24 species from three Rhoeadales families were examined by transmission electron microscopy. A typical cisterna originates from a dilation of the r-ER and contains a homogeneous, moderately dense material. The appearance of the cisternal content varies with the development of the cell and ranges from an electron-dense homogeneous matrix, via accumulations consisting of tightly packed longitudinally oriented rods or tubules, to vacuolar-like inclusions which eventually lose the attached ribosomes. The occurrence of DC was followed up in detail in different parts of the seedlings of Chinese cabbage (Brassica chinensis L.) 24 h, 48 h, and 72 h after germination. DC were not found in meristematic cells or cells undergoing differentiation, but appeared in more mature cells. Their number and size increased with the age and the development of the cell. DC we...