A rapid method for promoter exchange in Aspergillus nidulans using recombinant PCR

Recombinant PCR has been used to generate linear fragments for promoter replacement by transformation in Aspergillus nidulans. A cassette vector carrying the pyr-4 non-homologous selectable marker and conditional promoter Pr-alcA was constructed for use as a template for PCR, and is suitable for testing the function of essential genes. Two genes involved in polar growth, cotA and bemA, were used to assess the system. Efficient targeting was possible with both genes using approximately 500bp of flanking homologous sequence. Depending on yield, the linear PCR product could be used directly for transformation, or after first cloning into a suitable vector. bemA, a putative homologue of the Saccharomyces cerevisiae BEM1 gene was identified through sequence comparison. In A. nidulans, this protein appears to have a similar role to the yeast Bem1p, which acts as a scaffold protein involved in the establishment of cell polarity.     

5-Aminolevulinic Acid Ameliorates the Growth, Photosynthetic Gas Exchange Capacity, and Ultrastructural Changes Under Cadmium Stress in Brassica napus L.

Heavy-metal toxicity in soil is one of the major constraints for oilseed rape (Brassica napus L.) production. One of the best ways to overcome this constraint is the use of growth regulators to induce plant tolerance. Response to cadmium (Cd) toxicity in combination with a growth regulator, 5-aminolevulinic acid (ALA), was investigated in oilseed rape grown hydroponically in greenhouse conditions under three levels of Cd (0, 100, and 500 μM) and three levels of foliar application of ALA (0, 12.5, and 25 mg l^?1). Cd decreased plant growth and the chlorophyll concentration in leaves. Foliar application of ALA improved plant growth and increased the chlorophyll concentration in the leaves of Cd-stressed plants. Significant reductions in photosynthetic parameters were observed by the addition of Cd alone. Application of ALA improved the net photosynthetic and gas exchange capacity of plants under Cd stress. ALA also reduced the Cd content in shoots and roots, which was elevated by high concentrations of Cd. The microscopic studies of leaf mesophyll cells under different Cd and ALA concentrations showed that foliar application of ALA significantly ameliorated the Cd effect and improved the structure of leaf mesophyll cells. However, the higher Cd concentration (500 μM) could totally damage leaf structure, and at this level the nucleus and intercellular spaces were not established as well; the cell membrane and cell wall were fused to each other. Chloroplasts were totally damaged and contained starch grains. However, foliar application of ALA improved cell structure under Cd stress and the visible cell structure had a nucleus, cell wall, and cell membrane. These results suggest that under 15-day Cd-induced stress, application of ALA helped improve plant growth, chlorophyll content, photosynthetic gas exchange capacity, and ultrastructural changes in leaf mesophyll cells of the rape plant.     

Regulation of Cadmium-Induced Proteomic and Metabolic Changes by 5-Aminolevulinic Acid in Leaves of Brassica napus L.

It is evident from previous reports that 5-aminolevulinic acid (ALA), like other known plant growth regulators, is effective in countering the injurious effects of heavy metal-stress in oilseed rape (Brassica napus L.). The present study was carried out to explore the capability of ALA to improve cadmium (Cd²⁺) tolerance in B. napus through physiological, molecular, and proteomic analytical approaches. Results showed that application of ALA helped the plants to adjust Cd²⁺-induced metabolic and photosynthetic fluorescence changes in the leaves of B. napus under Cd²⁺ stress. The data revealed that ALA treatment enhanced the gene expressions of antioxidant enzyme activities substantially and could increase the expression to a certain degree under Cd²⁺ stress conditions. In the present study, 34 protein spots were identified that differentially regulated due to Cd²⁺ and/or ALA treatments. Among them, 18 proteins were significantly regulated by ALA, including the proteins associated with stress related, carbohydrate metabolism, catalysis, dehydration of damaged protein, CO2 assimilation/photosynthesis and protein synthesis/regulation. From these 18 ALA-regulated proteins, 12 proteins were significantly down-regulated and 6 proteins were up-regulated. Interestingly, it was observed that ALA-induced the up-regulation of dihydrolipoyl dehydrogenase, light harvesting complex photo-system II subunit 6 and 30S ribosomal proteins in the presence of Cd²⁺ stress. In addition, it was also observed that ALA-induced the down-regulation in thioredoxin-like protein, 2, 3-bisphosphoglycerate, proteasome and thiamine thiazole synthase proteins under Cd²⁺ stress. Taken together, the present study sheds light on molecular mechanisms involved in ALA-induced Cd²⁺ tolerance in B. napus leaves and suggests a more active involvement of ALA in plant physiological processes than previously proposed.     

Effects of open grazing and livestock exclusion on floristic composition and diversity in natural ecosystem of Western Saudi Arabia

Livestock grazing is one of the main causes of rangeland degradation in Saudi Arabia. Fencing to exclude grazers is one of the main management practices used to restore vegetation and conserve biodiversity. The main objectives of this study were to investigate the changes in plant diversity and abundance, floristic composition and plant groups of the major life forms in response to thirty-five years of grazing exclosure in western Saudi Arabia. These vegetation attributes and palatability were compared in 30 sampling stands located in the excluded and grazed sites. Our results showed that livestock exclusion significantly increased covers, density and species richness of annuals, grasses, perennial forbs, shrubs and trees. Exclosure enhanced the abundance and richness of palatable species and depressed the development of weedy species. About 66.7% of the recorded species at the excluded site were highly palatable compared to 34.5% at the grazed site. In contrary, about 55.2% unpalatable species were found in the grazed site compared to 25.8% in the protected site. Jaccard’s similarity index between the excluded and grazed sites showed lower values of 0.39%, 0.40% and 0.31% at levels of families, genus and species, respectively. The results suggest that establishing livestock exclusion may be a useful sustainable management tool for vegetation restoration and conservation of plant diversity in degraded rangelands of arid regions.     

Dietary and toxicity exposure of emerging persistent organic pollutants to human health through consumption of cereal crops from Pakistan

There is a lack of scientific literature regarding the bioaccumulation, dietary, and toxicity exposure of emerging persistent organic pollutants through food crops. The current mini-review presents the dietary intake, spatial distribution pattern, and screening-levels risk assessment of persistent organic pollutants (POPs) in the cereal crops and environmental compartments from Punjab Province, Pakistan. Results of congener specific analysis were in accordance to the previously reported pattern of detected POPs across the globe. Spatial distribution was influenced by the industrial and urban fraction and trend of spatial distribution pattern was observed as follows: industrial/urban areas > industrial/peri-urban areas > agricultural/rural areas. Dietary intake of Organochlorine pesticides (OCPs) via consumption of cereal crops was observed higher and was in accordance to the previously reported levels while human health was at marginal risk to cancer. The results of dietary and toxicity exposure of detected POPs warrant auxiliary devotion in future, to this group of contaminants.     

Evolutionarily conserved paired immunoglobulin-like receptor α (PILRα) domain mediates its interaction with diverse sialylated ligands

Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ～22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.