We have developed commercially viable phytoremediation/phytomining technologies employing Alyssum Ni-hyperaccumulator species to quantitatively extract Ni from soils. The majority of Ni is stored either in Alyssum leaf epidermal cell vacuoles or in the basal portions only of the numerous stellate trichomes. Here, we report simultaneous and region-specific localization of high levels of Ni, Mn, and Ca within Alyssum trichomes as determined by scanning electron microscopy/energy-dispersive X-ray analysis (SEM/EDX). Plants were grown in high Ni soil, achieving up to 48 400 microg g(-1) Ni in total leaf concentration; however, Ca and Mn were not enriched in the experimental soils. The region-specific localization of hyperaccumulated Ca, Mn, and Ni occurred in three soil types, five Alyssum species/ecotypes, and over a wide range of soil Ni concentrations. The metal concentration in the trichome basal compartment was approximately 15-20% dry weight, the highest ever reported for healthy vascular plant tissue.

We have previously developed phytoremediation and phytomining technologies employing Alyssum Ni hyperaccumulators to quantitatively extract Ni from soils. Implementation of these technologies requires knowledge of Ni localization patterns for the Alyssum species/ecotypes of interest under realistic growth conditions. We investigated Ni uptake and localization in mature Alyssum murale 'Kotodesh' and 'AJ9' leaves. Seedlings were grown in potting mix with an increasing series of NiSO 4 addition (0, 5, 10, 20, 40, 80 mmol Ni kg −1 ), NiC 4 H 6 O 4 addition (0, 5, 10, 30, 60, 90 mmol Ni kg −1 ), in Ni-contaminated soil from metal refining operations, and serpentine soil. Plants at Ni levels 0, 5, 10, 20 mmolkg −1 and in native soils grew normally. Plants at 40 mmolkg −1 exhibited the onset of phytotoxicity, and 60, 80, and 90 mmolkg −1 were demonstrably phytotoxic, but symptoms of phytotoxicity abated within 6 months. Cryogenic complement fractures were made from frozen hydrated samples. High-resolution scanning electron microscope (SEM) images were taken of one half. The other half was freeze-dried and examined with SEM and semi-quantitative energy dispersive x-ray analysis. Ni was highly concentrated in epidermal cell vacuoles and Ni and S counts showed a positive correlation. Trichome pedicles and the epidermal tissue from which the trichome grows were primary Ni compartments, but Ni was not distributed throughout trichomes. Palisade and spongy mesophyll and guard/substomatal cells contained lesser Ni concentrations but palisade mesophyll was an increasingly important compartment as Ni soil levels increased. Ni was virtually excluded from vascular tissue and trichome rays.

To elucidate the means by which polymer solutions protect cells from freezing injury, we cooled human monocytes to -80 degrees C or below in the presence of various polymers. Differential scanning calorimetric studies showed that those polymers which protect cells best have a limiting glass transition temperature (T'g) of approximately -20 degrees C; those with a T'g significantly higher or lower did not protect. Freeze-etch electron micrographs indicated that intracellular ice crystals had formed during this freezing procedure, but remained smaller than approximately 300 nm in the same proportion of cells as survived rapid thawing. We propose that cryoprotection of slowly frozen monocytes by polymers is a consequence of a T'g of -20 degrees C in the extracellular solution. In our hypothesis, the initial concentration and viscosity of protective polymer solutions reduce the extent and rate of cell water loss to extracellular ice and limit the injurious osmotic stress, which cells face during freezing at moderate rates to -20 degrees C. Below -20 degrees C, glass formation prevents further osmotic stress by isolating cells from extracellular ice crystals, virtually eliminating cell water loss at lower temperatures. On the other hand, the protective polymer solutions will allow some diffusion of water away from cells at temperatures above T'g. If conditions are correct, cells will concentrate the cytoplasm sufficiently during the initial cooling to T'g to avoid lethal intracellular freezing between T'g and the intracellular Tg, which has been depressed to low temperatures by that concentration. Thus, when polymers are used as cryoprotective agents, cell survival is contingent upon maintenance of osmotic stress within narrow limits.

Gas chromatographic-electroantennographic detection (G3C-EAD) experiments showed that antennae of males and females of the goldeneyed lacewing, Chrysopa oculata Say (Co. = Chrysopa), consistently responded to four compounds extracted from the abdominal cuticle of males:nonanal, nonanol, nonanoic acid, and (1R*,2S*,5R*,8R*)-iridodial. These compounds were not detected from abdominal cuticle of females. Thoracic extracts of both sexes contained antennal-stimulatory 1-tridecene and EAD-inactive skatole. Chrysopa oculata adults were most sensitive to (1R,2S,5R,8R)-iridodial standard at an EAD-response threshold between 0.1 and 1 pg, which was 10-100 times lower than thresholds for nonanal and nonanoic acid, and up to 10,000 times lower than thresholds for other compounds tested. A similar EAD response pattern was also found in another Chrysopa sp. (Co. quadripunctata Burmeister). In field-trapping experiments, (1R,2S,5R,8R)-iridodial was the only male-specific compound that attracted Co. oculata males. Males also were weakly attracted to (1R,4aS,7S,7aR)-nepetalactol (an aphid sex pheromone component), probably due to the 5% (1R,2S,5R,8R)-iridodial present in the synthetic sample as an impurity. A herbivore-induced plant volatile, methyl salicylate, increased attraction of males to (1R,2S,5R,8R)-iridodial, whereas 1-tridecene was antagonistic. No females were caught in the entire study. Scanning electron micrographs revealed numerous male-specific, elliptical epidermal glands on the 3rd-8th abdominal sternites of Co. oculata, which are likely the pheromone glands. Another lacewing species, Chrysoperla rufilabris (Burmeister) (Cl. = Chrysoperla), did not produce male-specific volatiles or possess the type of gland presumed to produce pheromone in Co. oculata males, but (Z)-4-tridecene was identified as a major antennal-stimulatory compound from thoracic extracts of both sexes of Cl. rufilabris. Thus, (1R,2S,5R,8R)-iridodial (or its enantiomer) is now identified as a male-produced male aggregation pheromone for Co. oculata, the first pheromone identified for lacewings.