A Study of GUS Expression in Arabidopsis as a Tool for the Evaluation of Gene Evolution, Function and the Role of Expression Derived from Gene Duplication.

Gene duplication played a fundamental role in eukaryote evolution and different copies of a given gene can be present in extant species, often with expressions and functions differentiated during evolution. We assume that, when such differentiation occurs in a gene copy, this may be indicated by its maintenance in all the derived species. To verify this hypothesis, we compared the histological expression domains of the three ß-glucuronidase genes (AtGUS) present in Arabidopsis thaliana with the GUS evolutionary tree in angiosperms. We found that AtGUS gene expression overlaps in the shoot apex, the floral bud and the root hairs. In the root apex, AtGUS3 expression differs completely from AtGUS1 and AtGUS2, whose transcripts are present in the root cap meristem and columella, in the staminal cell niche, in the epidermis and in the proximal cortex. Conversely, AtGUS3 transcripts are limited to the old border-like cells of calyptra and those found along the protodermal cell line. The GUS evolutionary tree reveals that the two main clusters (named GUS1 and GUS3) originate from a duplication event predating angiosperm radiation. AtGUS3 belongs to the GUS3 cluster, while AtGUS1 and AtGUS2, which originate from a duplication event that occurred in an ancestor of the Brassicaceae family, are found together in the GUS1 cluster. There is another, previously undescribed cluster, called GUS4, originating from a very ancient duplication event. While the copy of GUS4 has been lost in many species, copies of GUS3 and GUS1 have been conserved in all species examined.

Analysis of AtGUS1 and AtGUS2 in Arabidopsis root apex by a highly sensitive TSA-MISH method.

A new highly sensitive whole-mount in situ hybridization method, based on tyramide signal amplification (TSA-MISH) was developed and a combined GFP detection and TSA-MISH procedure was applied for the first time in plants, to precisely define the spatial pattern of AtGUS1 and AtGUS2 expression in the root apex. ß-glucuronidases (GUSs) belonging to the glycosyl hydrolases (GHs) 79 family, are widely distributed in plants, but their functional role has not yet been fully investigated. In the model system Arabidopsis Thaliana, three different AtGUS genes have been identified which encode proteins with putative different fates. Endogenous GUS expression has been detected in different organs and tissues, but the cyto-histological domains of gene expression remain unclear. The results here reported show co-expression of AtGUS1 and AtGUS2 in different functional zones of the root apex (the cap central zone, the root cap meristem, the staminal cell niche and the cortical cell layers of the proximal meristem), while AtGUS2 is exclusively expressed in the cap peripheral layer and in the epidermis in the elongation zone. Interestingly, both genes are not expressed in the stelar portion of the proximal meristem. A spatial (cortex vs. stele) and temporal (proximal meristem vs. transition zone) regulation of AtGUS1 and AtGUS2 expression is therefore active in the root apex. This expression pattern, although globally consistent with the involvement of GUS activity in both cell proliferation and elongation, clearly indicates that AtGUS1 and AtGUS2 could control distinct downstream process depending on the developmental context and the interaction with other players of root growth control. In the future, the newly developed approaches may well be very useful to dissect such interactions.

Palladium uptake by Pisum sativum: partitioning and effects on growth and reproduction.

Environmental palladium levels are increasing because of anthropogenic activities. The considerable mobility of the metal, due to solubilisation phenomena, and its known bioavailability may indicate interactions with higher organisms. The aim of the study was to determine the Pd uptake and distribution in the various organs of the higher plant Pisum sativum and the metal-induced effects on its growth and reproduction. P. sativum was grown in vermiculite with a modified Hoagland's solution of nutrients in the presence of Pd at concentrations ranging 0.10-25 mg/L. After 8-10 weeks in a controlled environment room, plants were harvested and dissected to isolate the roots, stems, leaves, pods and peas. The samples were analysed for Pd content using AAS and SEM-EDX. P. sativum absorbed Pd, supplied as K2PdCl4, beginning at seed germination and continuing throughout its life. Minimal doses (0.10-1.0 mg Pd/L) severely inhibited pea reproductive processes while showing a peculiar hormetic effect on root development. Pd concentrations ≥1 mg/L induced developmental delay, with late growth resumption, increased leaf biomass (up to 25%) and a 15-20% reduction of root mass. Unsuccessful repeated blossoming efforts led to misshapen pods and no seed production. Photosynthesis was also disrupted. The absorbed Pd (ca. 0.5 % of the supplied metal) was primarily fixed in the root, specifically in the cortex, reaching concentrations up to 200 µg/g. The metal moved through the stem (up to 1 µg/g) to the leaves (2 µg/g) and pods (0.3 µg/g). The presence of Pd in the pea fruits, together with established evidence of environmental Pd accumulation and bioavailability, suggests possible contamination of food plants and propagation in the food chain and must be the cause for concern.

Platinum, palladium, and rhodium deposition to the Prunus laurus cerasus leaf surface as an indicator of the vehicular traffic pollution in the city of Varese area: an easy and reliable method to detect PGEs released from automobile catalytic converters.

BACKGROUND, AIM, AND SCOPE: The widespread use of some platinum group elements as catalysts to minimize emission of pollutants from combustion engines produced a constantly growing increase of the concentration of these elements in the environment; their potential toxicological properties explain the increasing interest in routine easy monitoring. We have found that leaves of Prunus laurus cerasus are efficient collectors of particulate with a dimension <60-80 mum, and a simple and reliable procedure was developed to reveal traces of platinum, palladium, and rhodium released from automotive catalysts. The analysis of the dust deposited on the foliage is a direct indicator of traffic pollution. MATERIALS AND METHODS: Leaves of P. laurus cerasus were washed by sonication in a mixture of water and 2-propanol and the washings, to be discarded, were separated by centrifugation to yield typically 0.05-1.2 g of dust that, after mineralization, was directly submitted for atomic absorption analysis. RESULTS: Comparison of the 2007 and 2004-2005 results showed a dramatic reduction of the platinum levels and revealed that palladium is now the main component of this traffic-related pollution. DISCUSSION: The results are consistent with the increasing diffusion of cars with a diesel engine whose catalysts are made up of Pt and/or Pd alone, and gives a significant insight into the recent evolution in catalyst design that replaces platinum for palladium. CONCLUSIONS: The proposed analytical procedure is simple, with short preparation times, and greatly reduces matrix effects so that atomic absorption spectroscopy can easily detect the three noble metals at the ng/g level in the dust. RECOMMENDATION AND PERSPECTIVES: The results clearly show that Pd concentrations have increased over time, and must be cause for concern.