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Summary: Embryo development is a very key phase in the life cycle of seed plants. At maturity, the embryo contains the complete machinery to elaborate the entire plant body. While the embryogenic process is an innate feature of the zygote, gametic and somatic cells can undergo embryogenesis under the appropriate culture conditions. Embryogenesis is a highly regulated process and the use of mutants, especially in Arabidopsis, has allowed the identification of genes regulating pattern formation during this process. The use of such mutants has revealed the eritical roles of auxin levels and transport in the establishment of embryo axis. Root and shoot apical meristem function and integrity, have been defined by examination of genes involved in their identity and function. Further knowledge of the molecular and biochemical aspects of zygotic embryogenesis should contribute to our understanding of the underlying regulatory pathways and networks and also provide critical insights into unique totipotent features of the plant cell.

CYP84 is a recently identified family of cytochrome P450-dependent mono-oxygenases defined by a putative ferulate-5-hydroxylase (F5H) from Arabidopsis. Until recently F5H has been thought to catalyze the hydroxylation of ferulate to 5-OH ferulate en route to sinapic acid. Sinapine, a sinapate-derived ester in the seeds, is antinutritional and a target for elimination in canola meal. We have isolated three F5H-like genes (BNF5H1-3) from a cultivated Brassica napus, whose amphidiploid progenitor is considered to have arisen from a fusion of the diploids Brassica rapa and Brassica oleracea. Two cultivated varieties of the diploids were also found to contain BNF5H3 and additionally either BNF5H1 or BNF5H2, respectively. Whereas all three are >90% identical in their coding sequence, BNF5H1 and BNF5H2 are closer to each other than to BNF5H3. This and additional data suggest that the two groups of genes have diverged in an ancestor of the diploids. B. napus showed maximal F5H expression in the stems, least in the seeds, and subtle differences among the expression profiles of the three genes elsewhere. Transgenic B. napus with cauliflower mosaic virus 35S-antisense BNF5H contained up to 40% less sinapine, from 9.0 +/- 0.3 mg in the controls to 5.3 +/- 0.3 mg g(-1) seed. F5H from Arabidopsis and a similar enzyme from sweetgum (Liquidamber styraciflua) has recently been shown to have coniferaldehyde hydroxylase activity instead of F5H activity. Thus the supply of 5-OH coniferaldehyde or 5-OH ferulate has a bearing on sinapine accumulation in canola seeds.

Myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97) catalyzes the co-translational addition of myristic acid to the amino-terminal glycine residue of a number of important proteins of diverse functions. We have isolated a full-length Arabidopsis thaliana cDNA encoding NMT (AtNMT1), the first described from a higher plant. This AtNMT1 cDNA clone has an open reading frame of 434 amino acids and a predicted molecular mass of 48,706 Da. The primary structure is 50% identical to the mammalian NMTs. Analyses of Southern blots, genomic clones, and database sequences suggested that the A. thaliana genome contains two copies of NMT gene, which are present on different chromosomes and have distinct genomic organizations. The recombinant AtNMT1 expressed in Escherichia coli exhibited a high catalytic efficiency for the peptides derived from putative plant myristoylated proteins AtCDPK6 and Fen kinase. The AtNMT was similar to the mammalian NMTs with respect to a relative specificity for myristoyl CoA among the acyl CoA donors and also inhibition by the bovine brain NMT inhibitor NIP(71). The AtNMT1 expression profile indicated ubiquity in roots, stem, leaves, flowers, and siliques (approximately 1.7 kb transcript and approximately 50 kDa immunoreactive polypeptide) but a greater level in the younger tissue, which are developmentally very active. NMT activity was also evident in all these tissues. Subcellular distribution studies indicated that, in leaf extracts, approximately 60% of AtNMT activity was associated with the ribosomal fractions, whereas approximately 30% of the activity was observed in the cytosolic fractions. The NMT is biologically important to plants, as noted from the stunted development when the AtNMT1 was down-regulated in transgenic Arabidopsis under the control of an enhanced CaMV 35S promoter. The results presented in this study provide the first direct molecular evidence for plant protein N-myristoylation and a mechanistic basis for understanding the role of this protein modification in plants.

A facility for measuring the optical properties of solid materials at cryogenic temperatures is being developed at the National Institute of Standards and Technology. A cryostat that houses four ur bolometric detectors and a six-position sample holder was designed and built. The bolometers operate near 5 K, and the sample temperature can he varied from 6 to 100 K. The beam from a Fourier transform spectrometer is directed to the cryostat by reflective optical components. The measurable wavelengths extend from 1µm to 1 mm, with appropriate sources and beamsplitters in the spectrometer as well as windows and detectors in the cryostat. The angle of incidence on the sample ranges from 7.5 to 60. The mechanical electrical, and optical designs are described in this paper. Initial measurement results at wavelengths from 2 to 30µm and a sample temperature of 10 K are presented.

This work consists of a detailed thermal modeling of two different radiometers operated at cryogenic temperatures. Both employ a temperature sensor and an electrical-substitution technique to determine the absolute radiant power entering the aperture of a receiver. Their sensing elements are different: One is a germanium resistance thermometer, and the other is a superconducting kinetic-inductance thermometer. The finite element method is used to predict the transient and steady-state temperature distribution in the receiver. The nonequivalence between the radiant power and the electrical power due to the temperature gradient in the receiver is shown to be small and is minimized by placing the thermometer near the thermal impedance. In the radiometer with a germanium resistance thermometer, the random noise dominates the uncertainty for small incident powers and limits the ultimate sensitivity. At high power levels, the measurement accuracy is limited by the uncertainty of the absorptance of the cavity. Recommendations are given based on the modeling for future improvement of the dynamic response of both radiometers.

We have developed a facility for measuring the transmittance of optical filters at a wavelength of 1064 nm, using a Nd:YAG laser, a power stabilizer, and linear photodiode detectors. A direct measurement method was used for filters with optical densities (OD’s) less than or equal to 4, and a reference substitution technique was used for filters with OD’s as great as 10. The apparatus and data-acquisition system are described. Measurement results for a set of filters are presented. The expanded uncertainties for the measured OD and deduced absorption coefficient are determined through a detailed analysis of all the uncertainty components.

We have investigated the ordinate scale accuracy of ambient temperature transmittance measurements made with a Fourier transform infrared (FT-IR) spectrophotometer over the wavelength range of 2–10 µm. Two approaches are used: (1) measurements of Si wafers whose index of refraction are well known from 2 to 5 µm, in which case the FT-IR result is compared with calculated values; (2) comparison of FT-IR and laser transmittance measurements at 3.39 and 10.6 µm on nominally neutral-density filters that are free of etaloning effects. Various schemes are employed to estimate and reduce systematic error sources in both the FT-IR and laser measurements, and quantitative uncertainty analyses are performed.

This work consists of a detailed thermal modeling of two different radiometers operated at cryogenic temperatures. Both employ a temperature sensor and an electrical-substitution technique to determine the absolute radiant power entering the aperture of a receiver. Their sensing elements are different: One is a germanium resistance thermometer, and the other is a superconducting kinetic-inductance thermometer. The finite element method is used to predict the transient and steady-state temperature distribution in the receiver. The nonequivalence between the radiant power and the electrical power due to the temperature gradient in the receiver is shown to be small and is minimized by placing the thermometer near the thermal impedance. In the radiometer with a germanium resistance thermometer, the random noise dominates the uncertainty for small incident powers and limits the ultimate sensitivity. At high power levels, the measurement accuracy is limited by the uncertainty of the absorptance of the cavity. Recommendations are given based on the modeling for future improvement of the dynamic response of both radiometers.

An active cavity radiometer of the electrical substitution type with a cone receiver that operates at 2–4 K has been developed for measuring radiant fluxes in the dynamic range of 20 nW to 100 μW within an uncertainty of ±1% (2σ level). It is a broadband absolute detector with a flat overall absorption efficiency that is > 99% for radiation from the visible to long-wavelength IR. The system is designed based on thermal modeling and experimental measurements of concepts. It has been installed in the cryogenic chamber for low-background infrared radiation calibrations at the National Institute of Standards and Technology (NIST) for testing cryogenic blackbody sources, detectors, and optical components. Its time constant, responsivity, and nonequivalence error have been measured. They are in agreement with design predictions. Radiant power measurements of an amplitude-stabilized He–Ne laser beam with the radiometer and an industry standard photodiode detector, QED-200, have been intercompared and found to be in agreement. The intercomparison ratio of the measurements with the absolute cryogenic radiometer and QED-200 was 1.004 in the 75–100-μW range with an uncertainty of 0.5% (the 3σ level).

Abstract A 647-bp 5′-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed in Brassica napus pollen was fused to the β-glucuronidase (GUS) marker gene. This fusion construct was introduced into B. napus plants via Agrobacterium tumefaciens transformation. Analysis of the transgenic B. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.

Abstract: Shoot organogenesis and plant regeneration were readily achieved from cotyledonary petioles and hypocotyls of Brassica carinata. These explants were used for Agrobacterium-mediated transformation. A construct containing the selectable marker genes, neomycin phosphotransferase II, phosphinothricin acetyl transferase and the reporter gene β-glucuronidase, under the control of a tandem 35S promoter, was used for transformation. Although transformation was achieved with both cotyledonary petioles and hypocotyls, cotyledonary petioles responded best, with 30–50% of the explants producing GUS-positive shoots after selection on 25 mg/l kanamycin. Direct selection on L-phosphinothricin also produced resistant shoots but at a lower frequency (1–2%).

Summary Protoplasts from suspension cultures of somatic embryos of white spruce (Picea glauca Moench Voss) were electroporated with plasmids containing the chimeric genes for chloramphenicol acetyl transferase (CAT) or ß-glucuronidase (GUS), under control of one of three promoters. Transient CAT gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) promoter. When the CAT gene was fused to a tandem repeat CaMV 35S promoter (pPBI-363), CAT enzyme activity compared to NOS or 35S promoters increased up to eightfold (cell line WS-34), and were up to 100-fold greater than control (electroporated without plasmid). Comparatively, protoplasts of black spruce (Picea mariana Mill) and jack pine (Pinus banksiana Lamb.), electroporated with pPBI-363, produced increases in CAT activity compared to control of 90-fold and 70-fold, respectively. White spruce (WS-34) protoplasts were subsequently electroporated with the GUS gene fused to the tandem repeat CaMV 35S promoter. Comparatively, GUS enzyme activity increased up to tenfold compared to GUS fused to a CaMV 35S promoter. The results indicated that transient expression of the CAT and GUS genes was influenced by the type of promoter and cell line used, as well as by electroporation conditions.

Methods were developed for transient gene expression in protoplasts of black spruce (Picea mariana) and jack pine (Pinus banksiana). Protoplasts were isolated from embryogenic suspension cultures of black spruce and from non-embryogenic suspensions of jack pine. Using electroporation, transient expression of the chloramphenicol acetyltransferase (CAT) gene was assayed and shown to be affected by the cell line used, by voltage, temperature, and by the plasmid concentration and conformation. Increasing the plasmid DNA concentration (0–150µg ml-1) resulted in higher levels of transient CAT expression. In jack pine, linearized plasmid gave 2.5 times higher levels of CAT enzyme activity than circular. Optimal voltage varied for each cell line of the two species within the range 200–350 V cm-1 (960 µF). A heat shock treatment of protoplasts for 5 min at 45 °C resulted in enhanced CAT gene expression for both species.

Abstract: Cotyledonary somatic embryos of white spruce [Picea glauca (Moench) Voss] were subjected to microprojectile bombardment with a gene construct containing a gus::nptll fusion gene. Somatic embryos were used to re-induce the embryogenic tissue after bombardments. Histochemical assay using X-gluc as a substrate showed that all the embryos (100%) were GUS positive 48 h after bombardment. However, only thirteen out of 605 embryos (2.2%) remained GUS positive after two months in culture. Three of those thirteen (23%) embryo-derived tissues consistently showed GUS activity for eight months in culture. These putatively transfomed embryogenic tissues were subjected to Southern blot analysis and the results suggested integration of the gus::nptll gene expression cassette in the white spruce genome.

Population inversions on the 5g–4f and 4f–3d transitions of C iv at 253.0 and 116.9 nm have been observed in the recombination phase of a theta-pinch plasma initially containing 14 mTorr of acetylene and 0.5 mTorr of hydrogen gas. Population ratios of 17:11:1 for the 5g, 4f, and 3d levels were deduced from measured absolute line intensities of the 5g–4f and 4f–3d, and 3d–2p transitions at 253.0, 116.9, and 38.4 nm. The resulting gain–length product for a single pass through the 23-cm-long plasma column, based on the measured Doppler-broadened linewidth, is 2.3% at 116.9 nm and 5.8% at 253.0 nm.

We have measured the out-of-band transmittance (attenuation) for several narrow-band filters using two Fourier-transform infrared spectrometers in the wavelength range of 2–25 μm. Band-rejection filters are used to eliminate the power transmitted in the main band of the filter under investigation. Neutral-density filters are used for the reference measurement to reduce the effect of nonlinearity. Results from different optical arrangements are compared and discussed. This study demonstrates the feasibility of using Fourier-transform infrared instruments to measure a spectral transmittance of 10^−6.

Population inversions on the 5g-4ƒ and 4ƒ-3d transitions of C IV at 253.0 and 116.9 nm have been observed in the recombination phase of a theta-pinch plasma initially containing 14 mTorr of acetylene and 0.5 mTorr of hydrogen gas. Population ratios of 17:11:1 for the 5g, 4ƒ, and 3d levels were deduced from measured absolute line intensities of the 5g-4ƒ and 4ƒ-3d, and 3d-2p transitions at 253.0, 116.9, and 38.4 nm. The resulting gain—length product for a single pass through the 23-cm-long plasma column, based on the measured Doppler-broadened linewidth, is 2.3% at 116.9 nm and 5.8% at 253.0 nm.

The magnetic-dipole transition between the ^3P_1 and ^3P_2 levels of 2s^22p^4 ground configuration in Co XX has been identified from the emission of a cobalt-seeded plasma in the Texas Experimental Tokamak. The wavelength for the transition is 930.2 ± 0.3 Å.

The magnetic-dipole transition between the ^3P_1 and ^3P_2 levels of 2s^22p^4 ground configuration in Co xx has been identified from the emission of a cobalt-seeded plasma in the Texas Experimental Tokamak. The wavelength for the transition is 930.2 ± 0.3 Å.