Engineering a catabolic pathway in plants for the degradation of 1,2-dichloroethane

Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds that include 1,2-dichloroethane (1,2-DCA). Although plants lack the enzymatic activity required to metabolize this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics, including 1,2-DCA. We have engineered the dhlA and dhlB genes into tobacco (Nicotiana tabacum 'Xanthi') plants and used 1,2-DCA as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-DCA to 2-chloroethanol, which is then metabolized to the phytotoxic 2-chloroacetaldehyde, then chloroacetic acid, by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities, respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing only DhlA produced phytotoxic levels of chlorinated intermediates and died, while plants expressing DhlA together with DhlB thrived at levels of 1,2-DCA that were toxic to DhlA-expressing plants. This represents a significant advance in the development of a low-cost phytoremediation approach toward the clean-up of halogenated organic pollutants from contaminated soil and groundwater.     

Welfare and performance of yearling dairy heifers out-wintered on a wood-chip pad or housed indoors on two levels of nutrition

Wood-chip pads represent a low-cost alternative to housing for cattle during the winter. Considering the negative welfare implications associated with housing indoors on concrete, they may also offer welfare benefits to replacement dairy heifers. However, these animals may not be able to withstand winter weather conditions on a grass silage diet. The aim of this experiment was to evaluate behaviour, limb injuries, dirtiness scores, performance and climatic energy demand (CED) of yearling dairy heifers on two levels of nutrition kept outdoors on a wood-chip pad or indoors in cubicles during the winter. Ninety-six 10-month-old heifers were blocked and assigned in groups of eight, to one of the following four treatments in a 2 × 2 factorial design: (a) indoors, silage only; (b) indoors, silage plus concentrate; (c) outdoors, silage only; and (d) outdoors, silage plus concentrate. There were three replicate groups per treatment. All animals were inspected for skin lesions and were weighed and body condition scored (BCS) at the beginning and end of the trial. Instantaneous scan sampling and continuous all-occurrence behaviour sampling were used to collect behaviour data during two 24-h periods. Animals were also dirtiness scored and group feed intakes were recorded during the trial. Significantly more comfort, social and play behaviours were recorded outdoors (P < 0.05) while trips, slips and falls were only recorded indoors (P < 0.001). Groups outdoors had significantly lower limb lesion scores at the end of the experiment (P < 0.05) and fewer groups outdoors were affected by all categories of limb lesions. However, they were consistently dirtier than animals indoors (P < 0.001). Low-nutrition animals had lower feed intakes, smaller BCS changes and lower average daily weight gains than high-nutrition animals (P < 0.01). Heifers outdoors had significantly lower average daily weight gains and BCS changes (P < 0.05) explained by lower feed intakes (P < 0.01). However, outdoor heifers on both the high- and low-nutrition diets and indoor animals on the low-nutrition diet had lower UFL (feed unit for maintenance and lactation (Irish Republic)) intakes (-0.36, -0.35 and -0.22, respectively) than that required to meet the daily live-weight gains they achieved. Heifers indoors on the high-nutrition diet gained 0.98 kg per day but consumed 0.17 UFL more than what would be recommended to achieve a daily weight gain of 1.0 kg. The CED for outdoor heifers was higher than that of indoor heifers (6.18 v. 5.47 MJ/day per m2 body surface area; P < 0.001, s.e.d. 0.044). However, CED did not exceed heat production in any treatment. Although animal performance was reduced outdoors, the wood-chip pad was associated with welfare benefits compared with cubicle housing.     

The effect of out-wintering pad design on dirtiness score, somatic cell score and mastitis incidence in dairy cows

This study aimed to compare three woodchip out-wintering pad (OWP) designs, and indoor cubicle housing with regard to cow dirtiness scores during the winter housing period, and udder health during both the winter period and the following lactation, for spring-calving dairy cows. The treatments were: an uncovered (UP) and covered (CP) OWP with a concrete feed apron; an uncovered OWP with self-feed silage pit provided directly on the woodchips (SP); and indoor cubicle housing (IC). Data were compared during 2 years: year 1 was a case study while year 2 was an experimental study. In year 1, treatments were UP (space allowance = 12 m2/cow), CP (6 m2/cow) and IC. In year 2, all three OWP designs (12 m2/cow) were compared with IC. Animals were assigned to treatments at the end of lactation in the autumn, and remained there while dry until calving the following spring. Subsequently, all cows were at pasture during lactation. Outcome measures for analysis were cow dirtiness score, somatic cell score (SCS) and incidence of clinical mastitis during the dry period and during lactation. Quarter milk samples were also taken at drying off, calving and 3 weeks post partum both years, and at approximately 113 days in milk in year 2. Samples were analysed for presence of mastitis-causing agents and SCS was determined. Sub-clinical mastitis was diagnosed when cows had an SCS greater than 200 000, or California mastitis test greater than 1 in at least one quarter. In year 1, cows in CP were dirtier than cows in the other two treatments. These animals also had the highest SCS during lactation and tended to have more mastitis-causing agents isolated from quarter milk samples. In year 2, when all cows were stocked at the same density, cows in the sheltered OWP (i.e. CP) had similar dirtiness scores to cows in cubicles and significantly lower dirtiness scores than cows in the unsheltered OWP designs, i.e. UP and SP. However, there were no effects on SCS or quarter sample results. Cleaning of OWP's stocked at 12 m2/cow reduced cow dirtiness scores. However, cleaning of CP in year 1 when cows were stocked at 6 m2/cow had no effect on dirtiness scores. We conclude that dry cows stocked at 12 m2/cow on OWP's are unlikely to have udder health problems in the subsequent lactation. Furthermore, provision of shelter and cleaning of the woodchips are management factors that help to keep cows clean on OWP's.     

Fluorescence lifetime optical projection tomography

We describe a quantitative fluorescence projection tomography technique which measures the 3‐D fluorescence lifetime distribution in optically cleared specimens up 1 cm in diameter. This is achieved by acquiring a series of wide‐field time‐gated images at different relative time delays with respect to a train of excitation pulses, at a number of projection angles. For each time delay, the 3‐D time‐gated intensity distribution is reconstructed using a filtered back projection algorithm and the fluorescence lifetime subsequently determined for each reconstructed horizontal plane by iterative fitting to a mono‐exponential decay. Due to its inherently ratiometric nature, fluorescence lifetime is robust against intensity based artefacts as well as producing a quantitative measure of the fluorescence signal. We present a 3‐D fluorescence lifetime reconstruction of a mouse embryo labelled with an alexa‐488 conjugated antibody targeted to the neurofilament, which clearly differentiates between the extrinsic label and the autofluorescence, particularly from the heart and dorsal aorta. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Oestrogenic activity of CPRG (chlorophenol red-β-D-galactopyranoside), a β-galactosidase substrate commonly used in recombinant yeast oestrogenic assays

The finding that a variety of chemicals display oestrogenic activity has resulted in the development of in vitro and in vivo assays to assess oestrogenic activity. One such assay, the yeast oestrogen assay (YES) makes use of recombinant yeast cells that harbour an oestrogen receptor expression cassette and a reporter construct, coding for bgalactosidase. The induction mechanism starts with the binding of oestrogenic compounds to the oestrogen receptor. This complex activates the production of β-galactosidase. The β-galactosidase activity is thus a measure of the oestrogenic activity of chemical compounds. In the YES assay, the β-galactosidase activity may be quantified with the chromogenic substrate chlorophenol red-β-d-galactopyranoside (CPRG). In the present study it is reported that CPRG or its β-galactosidase degradation product chlorophenol red act in the YES as an oestrogenic compound itself. The implications of this finding are described. It is especially argued that chlorophenol red production after prolonged incubation of the assay might be misinterpreted as an oestrogenic effect of the test compound.     

Effect of an exotic Acacia (Fabaceae) on ant assemblages in South African fynbos

Ant assemblages in South African fynbos invaded by Acacia saligna were compared with ant assemblages in undisturbed fynbos to determine whether ant assemblages change under exotic plants that produce ant‐dispersed seeds. Overall, no differences in the species richness of ants were found between weed‐infested and native sites but there were differences in both ant abundance and the composition of the ant assemblage. Ants were much less abundant in weed‐infested sites. To investigate whether changes in ant assemblages in weed‐infested areas could be due to a preference for native seeds over exotic seeds, seeds of a range of species were offered to ants and ants that handled seeds were identified. Thirteen species of ants handled A. saligna seeds and there was no evidence to suggest that the ant assemblage as a whole preferred native seeds to A. saligna seeds. Hypotheses that may account for this pattern are discussed.     

High lipids accumulation in <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> by applying single and multiple nutrients limitation in a simple chemically defined medium

Microbial oil produced by the oleaginous yeast Rhodosporidium toruloides ATCC 204091 (formerly referred to as Rhodotorula glutinis) has a similar fatty acid composition to the vegetable oils and represents a potential alternative for biodiesel production. Finding strategies to improve the oil production by this yeast is desirable, as it is one of this nutrient’s limitations during the accumulation phase, as well as one of the main factors influencing the process. Therefore, the effect of single or combined nutrient limitation on lipid accumulation by R. toruloides was investigated. Biomass production and lipid accumulation by R. toruloides was improved using experimental designs in a two-step batch culture on a chemically-defined culture medium with high initial glucose concentration. For the first culture step, a Box–Behnken design was applied to optimize the main medium components’ concentrations, while maintaining a high biomass production. A biomass concentration of 44.3 g/L was reached with a medium composed of (g/L): glucose, 100; KH₂PO₄, 4.6; NaNO₃, 13.4; MgSO₄ ^.7H₂O, 0.2; and CaCl₂ ^.2H₂O, 0.11. For the second culture step, the biomass was transferred to lipid accumulation media. A 2³ factorial experimental design was conducted to investigate the effect of N, P and S limitations (individually or jointly) on lipid production from glucose (100 g/L). Lipid accumulation on dry cell mass was 77.04, 65.42, 70.13 and 69.84% for N, P, S and simultaneous nutrients’ limitations, respectively.     

Strigolactones,karrikins and beyond

The plant hormones strigolactones are synthesized from carotenoids and signal via the α/β hydrolase DWARF 14 (D14) and the F‐box protein MORE AXILLARY GROWTH 2 (MAX2). Karrikins, molecules produced upon fire, share MAX2 for signalling, but depend on the D14 paralog KARRIKIN INSENSITIVE 2 (KAI2) for perception with strong evidence that the MAX2–KAI2 protein complex might also recognize so far unknown plant‐made karrikin‐like molecules. Thus, the phenotypes of the max2 mutants are the complex consequence of a loss of both D14‐dependent and KAI2‐dependent signalling, hence, the reason why some biological roles, attributed to strigolactones based on max2 phenotypes, could never be observed in d14 or in the strigolactone‐deficient max3 and max4 mutants. Moreover, the broadly used synthetic strigolactone analog rac‐GR24 has been shown to mimic strigolactone as well as karrikin(‐like) signals, providing an extra level of complexity in the distinction of the unique and common roles of both molecules in plant biology. Here, a critical overview is provided of the diverse biological processes regulated by strigolactones and/or karrikins. These two growth regulators are considered beyond their boundaries, and the importance of the yet unknown karrikin‐like molecules is discussed as well.     

Microsite and elevation zone effects on seed pilferage,germination, and seedling survival during early whitebark pine recruitment

Tree recruitment is a spatially structured process that may undergo change over time because of variation in postdispersal processes. We examined seed pilferage, seed germination, and seedling survival in whitebark pine to determine whether 1) microsite type alters the initial spatial pattern of seed caches, 2) higher abiotic stress (i.e. higher elevations) exacerbates spatial distribution changes, and 3) these postdispersal processes are spatially clustered. At two study areas, we created a seed distribution pattern by burying seed caches in microsite types frequently used by whitebark pine's avian seed disperser (Clark's nutcracker) in upper subalpine forest and at treeline, the latter characterized by high abiotic environmental stress. We monitored caches for two years for pilferage, germination, and seedling survival. Odds of pilferage (both study areas), germination (northern study area), and survival (southern study area) were higher at treeline relative to subalpine forest. At the southern study area, we found higher odds of 1) pilferage near rocks and trees relative to no object in subalpine forest, 2) germination near rocks relative to trees within both elevation zones, and 3) seedling survival near rocks and trees relative to no object at treeline. No microsite effects were detected at the northern study area. Findings indicated that the microsite distribution of seed caches changes with seed/seedling stage. Higher odds of seedling survival near rocks and trees were observed at treeline, suggesting abiotic stress may limit safe site availability, thereby shifting the spatial distribution toward protective microsites. Higher odds of pilferage at treeline, however, suggest rodents may limit treeline recruitment. Further, odds of pilferage were higher near rocks and trees relative to no object in subalpine forest but did not differ among microsites at treeline, suggesting pilferage can modulate the spatial structure of regeneration, a finding supported by limited clustering of postdispersal processes.