Low-Temperature Scanning Electron Microscope Observations of the Meloidogyne incognita Egg Mass: The Gelatinous Matrix and Embryo Development

The root-knot nematode Meloidogyne incognita was cultured monoxenically on excised tomato roots. Galls and egg masses were observed daily using a light microscope. Two phases were distinguished in the gelatinous matrix of the egg mass: a translucent, amorphous material on the surface of the egg mass and a denser, layered phase in which nematode eggs were deposited. Egg masses were also cryofixed, fractured, and observed as frozen, hydrated specimens on a cold stage in a scanning electron microscope (SEM). In the SEM, the layered phase appeared as a meshwork of fibrils that became more loosely associated as the gelatinous matrix aged: Small pearl-like bodies were observed along the fibers of gelatinous matrix. The egg shell surface and several stages of embryo development, including the one-cell stage, initial cleavages, blastula, gastrula, tadpole stage, elongation, and molt of the first-stage juvenile within the egg shell, were observed and photographed with this technique. The developmental events observed were consistent with those described in other nematode species with different techniques.     

Effects of Temperature and Dietary Lipids on Phospholipid Fatty Acids and Membrane Fluidity in Steinernema carpocapsae

The phospholipid composition of Steinernema carpocapsae was studied in relation to diet and culture temperature. When reared at 18 and 27.5 C on Galleria mellonella or on an artificial diet supplemented with lard, linseed oil, or fish oil as lipid sources, nematode phospholipids contained an abundance of 20-carbon polyunsaturated fatty acids, with eicosapentaenoic acid (20:5(n - 3)) predominant, regardless of the fatty acid composition of the diet. Because the level of linolenic acid (18:3(n - 3)) in nematode phospholipids was very low and because eicosapentaenoic acid was present even when its precursor (linolenic acid) was undetectable in the diet, S. carpocapsae likely produces n - 3 polyunsaturated fatty acids by de novo biosynthesis, a pathway seldom reported in eukaryotic animals. Reduction of growth temperature from 25 to 18 C increased the proportion of 20:5(n - 3) but not other polyunsaturated fatty acids. A fluorescence polarization technique revealed that vesicles produced from phospholipids of nematodes reared at 18 C were less ordered than those from nematodes reared at 27.5 C, especially in the outermost region of the bilayer. Dietary fish oil increased fluidity in the outermost region but increased rigidity in deeper regions. Therefore, S. carpocapsae appears to modify its membrane physical state in response to temperature, and eicosapentaenoic acid may be involved in this response. The results also indicate that nematode membrane physical state can be modified dietarily, possibly to the benefit of host-finding or survival of S. carpocapsae at low temperatures.     

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography-mass spectrometry. GA₁ GA₁₉ GA₂₀ and GA₂₉ were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA₁. GA₁₉. GA₂₀ and putative GA₂₉ and GA₅₃ were quantified in the apical buds, while GA₄. GA₈. GA₉ and GA₄₄ were shown to be either absent or present at very low levels. From the cambial region. GA₁ and GA₂₀ were quantified at levels of 0.30 ng (g fresh weight)-¹ and 8.8 ng (g fresh weight)-¹ respectively. These data suggest that the early 13-hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus .     

Gravitropic response growth angle and vertical distribution of roots of wheat (Triticum aestivum L.)

Recent work on root distribution, growth angles and gravitropic responses in Japanese cultivars of winter wheat are reviewed. Vertical distribution of roots, which influences the environmental stress tolerance of plants, was observed in the 12 Japanese cultivars in the field. The root depth index (RDI: the depth at which 50% of the root length has been reached) differed among the cultivars at the stem elongation stage. Since the RDI was closely related to the growth angle of seminal roots obtained in a pot experiment, it was assumed that growth angle is useful for predicting vertical root distribution among wheat genotypes. Gravitropic responses of the primary seminal root of 133 Japanese wheat cultivars assessed by measuring the growth angle in agar medium, were larger in the northern Japanese cultivars and smaller in the southern ones. It was also found that the geographical variation resulted from the wheat breeding process, i.e. genotypes with limited gravitropic responses of roots had been selected in the southern part of Japan where excessive soil moisture is one of the most serious problems.     

Uptake,accumulation and metabolism of auxins in tobacco leaf protoplasts

Uptake and metabolism of exogenous naphthalene-1-acetic acid (NAA) and indole-3-acetic acid (IAA) have been studied in tobacco (Nicotiana tabacum L. cv. Xanthi) mesophyll protoplasts. Both auxins entered protoplasts by diffusion under the action of the transmembrane pH gradient without any detectable participation of an influx carrier. Molecules were accumulated by an anion-trapping mechanism and most of them were metabolized within hours, essentially as glucose-ester and amino-acid conjugates. Protoplasts were equipped with a functional auxin-efflux carrier as evidenced by the inhibitory effect of naphthylphtalamic acid on IAA efflux. Basically, similar mechanisms of NAA and IAA uptake occurred in protoplasts. However, the two auxins differed in their levels of accumulation, due to different membrane-transport characteristics, and the nature of the metabolites produced. This shows the need to estimate the accumulation and the metabolism of auxins when analyzing their effects in a given cell system. The internal auxin concentration could be modulated by changing the transmembrane pH gradient, giving an interesting perspective for discriminating between the effects of intra- and extracellular auxin on physiological processes.Abbreviations BA benzoic acid - C_i/C_e accumulation ratio of auxin - IAAasp N-[3-indolylacetyl]-dl-aspartic acid - NAA naphthalene-1-acetic acid - NAAasp N-[1-naphthylacetyl]-l-aspartic acid - NPA N-1-naphthylphthalamic acid The authors thank Dr. M. Caboche (I.N.R.A, Versailles, France) for his generous gifts of some amide derivatives of 1-NAA, Mr. P. Varennes and Dr. B. Das (I.C.S.N., C.N.R.S., Gif-sur-Yvette, France) for recording and interpreting the mass spectra of NAA glucose ester, and Prof. P. Manigault (Institut des Sciences Végétales, Gif-sur-Yvette) for microscopy measurements of protoplast dimensions. This work was supported by funds from the C.N.R.S, I.N.R.A, and E.E.C.     

Tissue and subcellular immunolocalisation of enzymes of lignin synthesis in differentiating and wounded hypocotyl tissue of French bean (Phaseolus vulgaris L.)

Antisera raised againstl-phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), and a cationic cell-wall peroxidase, which had all been purified from suspension-cultured cells of French bean, have been used to carry out immunogold localisations in the growing plant. Immunoglobulin-G fractions were prepared from each antiserum and used to study the distribution of the enzymes in differentiating and wounded hypocotyls by immunogold techniques and visualisation by both light and electron microscopy. Following silver enhancement to amplify the signal, proteins were detected by confocal microscopy in both developing (pre-xylem/ phloem) and later metaxylem stelar tissue.l-Phenylalanine ammonia-lyase and C4H also accumulated in cells adjacent to metaxylem, presumably involved in maintaining a supply of phenylpropanoid precursors to the enucleated xylem for further lignin synthesis. In these cells, PAL subunits were cytosolic although some were associated with endomembrane. Cinnamate-4-hydroxylase was wholly associated with membrane and particularly high concentrations were found in the Golgi bodies. The cationic peroxidase accumulated in xylem at sites of secondary thickening and in the middle lamella. The three proteins are also involved in defensive lignification. Thus when visualised by light microscopy, PAL and C4H were seen to accumulate to high levels throughout the cell types in wound sites and especially in the epidermal cells. An even more intense general distribution was found upon hyperinduction of wounded cells with-aminooxy--phenylpropionic acid. At the subcellular level, PAL was found to be localised in the cytosol in the wounded cells; however, because of the loss of membrane through mechanical damage, association with membrane structures, particularly endoplasmic reticulum, in unwounded cells is not entirely ruled out. Cinnamate-4-hydroxylase was associated with membranes when these were preserved. In wounded tissue, the peroxidase was found at the growing edges of tylose-like structures in the vascular xylem.Abbreviations AOPP -aminooxy--phenylpropionic acid - C4H cinnamic acid-4-hydroxylase - CHS chalcone synthase - GRP glycine-rich glycoprotein - HRGP hydroxyproline-rich glycoprotein - Ig immunoglobulin - PAL phenylalanine ammonia-lyase G.P.B. thanks the Agicultural and Food Research Council for support.     

The Casparian strip in pea epicotyls: Effects of light on its development

The Casparian strip, which is specific to roots, was studied in the epicotyls of dark-grown seedlings of pea (Pisum sativum L.) where it was found to have the same morphology and properties as the strip in roots. In dark-grown seedlings, the distance between the upper-most position of the Casparian strip and the bending point of the hook (about 37 mm) did not change during growth of the seedlings. In the uppermost 0.5-mm region of the region in which the Casparian strip could be detected by fluorescence microscopy, the plasma membrane was not firmly attached to the cell wall. The development of the Casparian strip continued for about 42 h after dark-grown seedlings were transferred to the light, indicating that (i) the cells that have been determined to form the Casparian strip in darkness form the strip in the light, and that (ii) it takes about 42 h for the cells to complete formation of the strip. Cells in the hook of dark-grown seedlings did not form a Casparian strip when such seedlings were transferred to the light. The Casparian strip was formed in rapidly elongating internodes of light-grown seedlings when the seedlings were transferred to darkness. Light did not control the formation of the Casparian strip in roots.Abbreviation PBS phosphate-buffered saline     

Phenyllactic acid but not tropic acid is an intermediate in the biosynthesis of tropane alkaloids in Datura and Brugmansia transformed root cultures

(S)-(-)-Tropic acid is the acidic moiety of the tropane ester alkaloids, hyoscyamine and scopolamine (hyoscine). When tropic acid is fed to transformed root cultures of Datura stramonium L. or a Brugmansia (Datura) Candida x B. aurea hybrid, the formation of these alkaloids is inhibited. Phenyllactic acid, from which the tropoyl moiety is derived, is considerably less inhibitory. Label from (RS)-phenyl[1,3-¹³C₂]lactic acid is incorporated at high levels into apoatropine, littorine, aposcopolamine, hyoscyamine, 7-hydroxyapoatropine, scopolamine and 7-hydroxyhyoscyamine when fed to these cultures. The presence of an excess concentration of unlabelled tropic acid has little influence on the specific incorporation into these products. It is concluded that free tropic acid is not an intermediate in hyoscyamine biosynthesis but rather that the rearrangement of phenyllactic acid occurs subsequent to its esterification.Abbreviations FM fresh mass - NMR nuclear magnetic resonance spectroscopy We are grateful to Drs. N.J. Walton, A.J. Parr, M.J.C. Rhodes (Institue of Food Research, Norwich) and B. Dräger (Münster, Germany) for helpful and critical discussions. We also wish to thank Dr. P. Bachmann (Braunschweig, Germany) for suggesting the use of the DB-17 column to separate littorine from hyoscyamine and for the modified Excel programme used to calculate the specific incorporations, Yannick Ford (AFRC Co-operative Award Studentship, University of Oxford) and Abigael Peerless for their able assistance, Dr. I. Colquhoun for assistance with some of the NMR spectroscopy and Drs. K. Shimomura (Tsukuba, Japan) and T. Hashimoto (Kyoto, Japan) for pure samples of 7-hydroxyhyoscyamine. J.G.W, gratefully acknowledges support from the Nuffield Foundation under the Small Grants Scheme to promote collaborative experimentation and M.A. is grateful to the Ministry of Education, Iran for a research scholarship.     

The soil fauna: the other last biotic frontier

Different approaches to biodiversity yield global totals as small as 3 million or as large as 80 million species. Erwin's calculation and estimation leads to an estimate of ca 30 million species and relies on four assumptions of which one concerns the ratio between the number of canopy insects and those found elsewhere, especially in the soil. A short survey of the microarthropods living in coastal sand dunes and collected with a new flotation method yielded amazing results. In spite of the severity of the habitat (low organic matter content and extreme dryness), the density of microarthropods varied between 175 000 and 1 400 000 individuals per square metre, i.e., densities 3 to 10 times higher than densities usually observed in any other type of soil. A total of 31 species was recorded, most undescribed and smaller than 200 m. The consequences of these findings on the estimation of the number of species are discussed. It is suggested that the soil, including the deepest horizons and the rhizosphere, might constitute a huge reservoir for biodiversity.