Metallothionein isoform 2A expression is inducible and protects against ROS-mediated cell death in rotenone-treated HeLa cells

The role of MT (metallothionein) gene expression was investigated in rotenone-treated HeLa cells to induce a deficiency of NADH:ubiquinone oxidoreductase (complex I). Complex I deficiency leads to a diversity of cellular consequences, including production of ROS (reactive oxygen species) and apoptosis. HeLa cells were titrated with rotenone, resulting in dose-dependent decrease in complex I activity and elevated ROS production at activities lower than 33%. Expression of MT2A (MT isoform 2A), but not MT1A or MT1B RNA, was significantly inducible by rotenone (up to 7-fold), t-BHP (t-butyl hydroperoxide; 5-fold) and CdCl2 (50-fold), but not ZnCl2. Myxothiazol treatment did not elevate either ROS or MT2A levels, which supports a ROS-related mechanism for rotenone-induced MT2A expression. To evaluate the role of MT2A expression, MT2A and MT1B were overexpressed in HeLa cells and treated with rotenone. Compared with control and MT1B-overexpressing cells, ROS production was significantly lower and cell viability higher in MT2A-overexpressing HeLa cells when ROS production was enhanced by treatment with t-BHP. Mitochondrial membrane potential was noticeably less reduced in both MT-overexpressing cell lines. MT2A overexpression in rotenone-treated cells also significantly reduced or delayed apoptosis induction, as measured by caspase 3/7 activity and cytosolic nucleosome enrichment. We conclude that MT2A offers significant protection against the main death-causing consequences of rotenone-induced complex I deficiency in HeLa cells. Our results are in support of the protective role against oxidative stress ascribed to MTs and provide evidence that MT2A expression may be a beneficial downstream adaptive response in complex I-deficient cells.     

Alteration of copper physiology in mice overexpressing the human Menkes protein ATP7A

The Menkes protein (ATP7A) is defective in the Cu deficiency disorder Menkes disease and is an important contributor to the maintenance of physiological Cu homeostasis. To investigate more fully the role of ATP7A, transgenic mice expressing the human Menkes gene ATP7A from chicken beta-actin composite promoter (CAG) were produced. The transgenic mice expressed ATP7A in lung, heart, liver, kidney, small intestine, and brain but displayed no overt phenotype resulting from expression of the human protein. Immunohistochemical analysis revealed that ATP7A was found primarily in the cardiac muscle, smooth muscle of the lung, distal tubules of the kidney, intestinal enterocytes, and patches of hepatocytes, as well as in the hippocampus, cerebellum, and choroid plexus of the brain. In 60-day- and 300-day-old mice, Cu concentrations were reduced in most tissues, consistent with ATP7A playing a role in Cu efflux. The reduction in Cu was most pronounced in the hearts of older T22#2 females (24%), T22#2 males (18%), and T25#5 females (23%), as well as in the brains of 60-day-old T22#2 females and males (23% and 30%, respectively).     

Root, mycorrhiza and earthworm interactions: their effects on soil structuring processes, plant and soil nutrient concentration and plant biomass

Earthworms, arbuscular mycorrhiza fungi (AMF) and roots are important components of the belowground part of terrestrial ecosystem. However, their interacting effects on soil properties and plant growth are still poorly understood. A compartmental experimental design was used in a climate chamber in order to investigate, without phosphorus (P) addition, the single and combined effects of earthworms (Allolobophora chlorotica), AMF (Glomus intraradices) and roots (Allium porrum) on soil structure, nutrient concentration and plant growth. In our experimental conditions, plant roots improved soil structure stability (at the level of macroaggregates) whereas earthworms decreased it. AMF had no effect on soil structure stability but increased P transfer from the soil to the plant and significantly increased plant biomass. Earthworms had no direct influence on P uptake or plant biomass, and the N/P ratio measured in the shoots indicated that P was limiting. Interactions between AMF and earthworms were also observed on total C and N content in the soil and on total root biomass. Their effects varied temporally and between the different soil compartments (bulk soil, rhizosphere and drilosphere). After comparison with other similar studies, we suggest that effects of earthworms and AMF on plant production may depend on the limiting factors in the soil, mainly N or P. Our experiment highlights the importance of measuring physical and chemical soil parameters when studying soil organism interactions and their influence on plant performance.     

Production of biologically active human lymphotactin (XCL1) by <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Lymphotactin-XCL1 is a chemokine produced mainly by activated CD8+ T-cells and directs migration of CD4+ and CD8+ lymphocytes and natural killer (NK) cells. We expressed human lymphotactin (LTN) by the lactic-acid bacterium Lactococcus lactis. Biological activity of LTN was confirmed by chemo-attraction of human T-cells by chemotaxis demonstrating, for the first time, how this chemokine secreted by a food-grade prokaryote retains biological activity and chemoattracts T lymphocytes. This strain thus represents a feasible well-tolerated vector to deliver active LTN at a mucosal level. Laura M. Zavala-Flores and Julio Villatoro-Hernandez have contributed equally to this work.     

Cellular Pathways Regulating Responses to Compatible and Self-Incompatible Pollen in Brassica and Arabidopsis Stigmas Intersect at Exo70A1, a Putative Component of the Exocyst Complex

In the Brassicaceae, compatible pollen–pistil interactions result in pollen adhesion to the stigma, while pollen grains from unrelated plant species are largely ignored. There can also be an additional layer of recognition to prevent self-fertilization, the self-incompatibility response, whereby self pollen grains are distinguished from nonself pollen grains and rejected. This pathway is activated in the stigma and involves the ARM repeat–containing 1 (ARC1) protein, an E3 ubiquitin ligase. In a screen for ARC1-interacting proteins, we have identified Brassica napus Exo70A1, a putative component of the exocyst complex that is known to regulate polarized secretion. We show through transgenic studies that loss of Exo70A1 in Brassica and Arabidopsis thaliana stigmas leads to the rejection of compatible pollen at the same stage as the self-incompatibility response. A red fluorescent protein:Exo70A1 fusion rescues this stigmatic defect in Arabidopsis and is found to be mobilized to the plasma membrane concomitant with flowers opening. By contrast, increased expression of Exo70A1 in self-incompatible Brassica partially overcomes the self pollen rejection response. Thus, our data show that the Exo70A1 protein functions at the intersection of two cellular pathways, where it is required in the stigma for the acceptance of compatible pollen in both Brassica and Arabidopsis and is negatively regulated by Brassica self-incompatibility.     

Multiplication and cryopreservation of vanilla (<Emphasis Type="Italic">Vanilla planifolia</Emphasis> ‘Andrews’)

A simple and efficient method for multiplication of vanilla (Vanilla planifolia) was developed using in vitro fragmented explants (IFEs) as propagules. IFEs were obtained after dissecting apices from in vitro propagated clusters of plantlets, by cutting the remaining base of these plant clusters into segments of about 1 cm in length. After 4 months of culture on multiplication medium, 100% of IFEs produced up to 15 new shoots per explant, providing an efficient additional method for in vitro propagation of vanilla that maximizes the use of available material. Cryopreservation of apices from in vitro grown plants was achieved using the droplet vitrification protocol. Maximum survival (30%) and further regeneration (10%) of new shoots were obtained for apices derived from clusters of in vitro plantlets produced from microcuttings through a three-step droplet vitrification protocol: 1-d preculture of apices on solid MS medium with 0.3 M sucrose; loading with a 0.4 M sucrose + 2 M glycerol solution for 20–30 min; and exposure to plant vitrification solution PVS3 for 30 min at room temperature. Even though the cryogenic protocol needs to be optimized to improve results, this work represents the first successful report of cryopreservation of vanilla apices.     

Comparing Direct Abiotic Amelioration and Facilitation as Tools for Restoration of Semiarid Grasslands

Desertification can be an irreversible process due to positive feedback among degraded plant and soil dynamics. The recovery of semiarid degraded ecosystems may need human intervention. In restoration practices, the abiotic conditions often need to be improved to overcome the positive plant–soil feedback loops. Using nurse‐plants to improve abiotic conditions for introduced individuals (facilitation) has been suggested as an alternative to direct abiotic amelioration. Here, we compared direct abiotic amelioration and facilitation as tools for restoration of semiarid grasslands in Spain. Seedlings and seeds of Lygeum spartum and Salsola vermiculata were planted and sown in a stably degraded semiarid area in Northeast Spain. Two levels of direct abiotic amelioration (ploughing and damming) and indirect abiotic amelioration through facilitation by Suaeda vera nurse shrubs were compared with a control with no amelioration treatment. The control treatment showed low plant establishment, confirming the practical irreversibility of the degraded state. Plant establishment was significantly higher in the three treatments with interventions than in the control treatment. The best treatment depended on the plant trait considered, but damming was in most cases better than plant facilitation. However, facilitation maintained the nutrient‐rich topsoil layer. Given the relative success of facilitation, revegetation using the facilitative effect of nurse‐plants would, in principle, be recommended for restoring semiarid grasslands. Direct abiotic amelioration would be needed under extreme degradation or harsh climatic conditions.     

Post-colonization temporal genetic variation of an introduced fly, Rhagoletis completa

Evolutionary biologists have been puzzled by the success of introduced species: despite founder effects that reduce genetic variability, invasive species are still successful at colonizing new environments. It is possible that the evolutionary processes during the post-colonization period may increase the genetic diversity and gene flow among invasive populations over time, facilitating their long-term success. Therefore, genetic diversity and population structure would be expected to show greater temporal variation for successful introduced populations than for native populations. We studied the population genetics of the walnut husk fly, Rhagoletis completa, which was introduced into California from the Midwestern US in the early 1900s. We used microsatellites and allozymes to genotype current and historic fly populations, providing a rare perspective on temporal variability in population genetic parameters. We found that introduced populations showed greater temporal fluctuations in allele frequencies than native populations. Some introduced populations also showed an increase in genetic diversity over time, indicating multiple introductions had occurred. Population genetic structure decreased in both native and introduced populations over time. Our study demonstrates that introduced species are not at equilibrium and post-colonization processes may be important in ameliorating the loss of genetic diversity associated with biological invasions.