The identification of aluminium-resistance genes provides opportunities for enhancing crop production on acid soils.

Acid soils restrict plant production around the world. One of the major limitations to plant growth on acid soils is the prevalence of soluble aluminium (Al(3+)) ions which can inhibit root growth at micromolar concentrations. Species that show a natural resistance to Al(3+) toxicity perform better on acid soils. Our understanding of the physiology of Al(3+) resistance in important crop plants has increased greatly over the past 20 years, largely due to the application of genetics and molecular biology. Fourteen genes from seven different species are known to contribute to Al(3+) tolerance and resistance and several additional candidates have been identified. Some of these genes account for genotypic variation within species and others do not. One mechanism of resistance which has now been identified in a range of species relies on the efflux of organic anions such as malate and citrate from roots. The genes controlling this trait are members of the ALMT and MATE families which encode membrane proteins that facilitate organic anion efflux across the plasma membrane. Identification of these and other resistance genes provides opportunities for enhancing the Al(3+) resistance of plants by marker-assisted breeding and through biotechnology. Most attempts to enhance Al(3+) resistance in plants with genetic engineering have targeted genes that are induced by Al(3+) stress or that are likely to increase organic anion efflux. In the latter case, studies have either enhanced organic anion synthesis or increased organic anion transport across the plasma membrane. Recent developments in this area are summarized and the structure-function of the TaALMT1 protein from wheat is discussed.

Assessment of theatre shoe contamination in an orthopaedic theatre.

Prosthetic joint infection (PJI) is a devastating complication of arthroplasty. Numerous protocols reduce potential risk for PJI peri-operatively, but none exist for the management of theatre shoes. Our aim was to assess for bacteria known to cause prosthetic infection on theatre shoes. Forty theatre shoes were analysed; there were coagulase-negative staphylococci on 65% (N = 25), meticillin-susceptible Staphylococcus aureus on 40% (N = 16), and meticillin-resistant S. aureus on 25% (N = 10). Amount of blood spatter correlated poorly with microbial contamination. Shoes harbouring Gram-positive bacteria, including antibiotic-resistant strains, provide a potential route of transmission to the theatre environment.

Aluminium tolerance in plants and the complexing role of organic acids.

The aluminium cation Al(3+) is toxic to many plants at micromolar concentrations. A range of plant species has evolved mechanisms that enable them to grow on acid soils where toxic concentrations of Al(3+) can limit plant growth. Organic acids play a central role in these aluminium tolerance mechanisms. Some plants detoxify aluminium in the rhizosphere by releasing organic acids that chelate aluminium. In at least two species, wheat and maize, the transport of organic acid anions out of the root cells is mediated by aluminium-activated anion channels in the plasma membrane. Other plants, including species that accumulate aluminium in their leaves, detoxify aluminium internally by forming complexes with organic acids.

Interaction between Aluminum Toxicity and Calcium Uptake at the Root Apex in Near-Isogenic Lines of Wheat (Triticum aestivum L.) Differing in Aluminum Tolerance.

Aluminum (Al) is toxic to plants at pH < 5.0 and can begin to inhibit root growth within 3 h in solution experiments. The mechanism by which this occurs is unclear. Disruption of calcium (Ca) uptake by Al has long been considered a possible cause of toxicity, and recent work with wheat (Triticum aestivum L. Thell) has demonstrated that Ca uptake at the root apex in an Al-sensitive cultivar (Scout 66) was inhibited more than in a tolerant cultivar (Atlas 66) (J.W. Huang, J.E. Shaff, D.L. Grunes, L.V. Kochian [1992] Plant Physiol 98: 230-237). We investigated this interaction further in wheat by measuring root growth and Ca uptake in three separate pairs of near-isogenic lines within which plants exhibit differential sensitivity to Al. The vibrating calcium-selective microelectrode technique was used to estimate net Ca uptake at the root apex of 6-d-old seedlings. Following the addition of 20 or 50 [mu]M AlCl3, exchange of Ca for Al in the root apoplasm caused a net Ca efflux from the root for up to 10 min. After 40 min of exposure to 50 [mu]M Al, cell wall exchange had ceased, and Ca uptake in the Al-sensitive plants of the near-isogenic lines was inhibited, whereas in the tolerant plants it was either unaffected or stimulated. This provides a general correlation between the inhibition of growth by Al and the reduction in Ca influx and adds some support to the hypothesis that a Ca/Al interaction may be involved in the primary mechanism of Al toxicity in roots. In some treatments, however, Al was able to inhibit root growth significantly without affecting net Ca influx. This suggests that the correlation between inhibition of Ca uptake and the reduction in root growth may not be a mechanistic association. The inhibition of Ca uptake by Al is discussed, and we speculate about possible mechanisms of tolerance.

Aluminum Tolerance in Wheat (Triticum aestivum L.) (II. Aluminum-Stimulated Excretion of Malic Acid from Root Apices).

We investigated the role of organic acids in conferring Al tolerance in near-isogenic wheat (Triticum aestivum L.) lines differing in Al tolerance at the Al tolerance locus (Alt1). Addition of Al to nutrient solutions stimulated excretion of malic and succinic acids from roots of wheat seedlings, and Al-tolerant genotypes excreted 5- to 10-fold more malic acid than Al-sensitive genotypes. Malic acid excretion was detectable after 15 min of exposure to 200 [mu]M Al, and the amount excreted increased linearly over 24 h. The amount of malic acid excreted was dependent on the external Al concentration, and excretion was stimulated by as little as 10 [mu]M Al. Malic acid added to nutrient solutions was able to protect Al-sensitive seedlings from normally phytotoxic Al concentrations. Root apices (terminal 3-5 mm of root) were the primary source of the malic acid excreted. Root apices of Al-tolerant and Al-sensitive seedlings contained similar amounts of malic acid before and after a 2-h exposure to 200 [mu]M Al. During this treatment, Al-tolerant seedlings excreted about four times the total amount of malic acid initially present within root apices, indicating that continual synthesis of malic acid was occurring. Malic acid excretion was specifically stimulated by Al, and neither La, Fe, nor the absence of Pi was able to elicit this response. There was a consistent correlation of Al tolerance with high rates of malic acid excretion stimulated by Al in a population of seedlings segregating for Al tolerance. These data are consistent with the hypothesis that the Alt1 locus in wheat encodes an Al tolerance mechanism based on Al-stimulated excretion of malic acid.

Direct Evaluation of the Ca2+-Displacement Hypothesis for Al Toxicity.

One explanation for Al toxicity in plants suggests that Al displaces Ca2+ from critical sites in the apoplasm. We evaluated the Ca2+-displacement hypothesis directly using near-isogenic lines of wheat (Triticum aestivum L.) that differ in Al tolerance at a single locus. We measured both the growth and total accumulation (apoplasmic plus symplasmic) of 45Ca and Al into roots that had been exposed to Al alone or to Al with other cations. Root growth in the Al-sensitive line was found to be severely inhibited by low activities of Al, even though Ca2+ accumulation was relatively unaffected. In solutions containing the same activity of the Al3+ and Ca2+ ions as above, but also including either 3.0 mM Mg2+, 3.0 mM Sr2+, or 30 mM Na+, growth improved, whereas 45Ca2+ accumulation was significantly decreased. Since most of the 45Ca2+ accumulated by roots during short-term treatments will reside in the apoplasm, these results indicate that displacement of Ca2+ from the apoplasm by Al cannot account for the Al-induced inhibition of root growth and, therefore, do not support the Ca2+-displacement hypothesis for Al toxicity. We also show that total accumulation of Al by root apices is greater in the Al-sensitive genotype than the Al-tolerant genotype and suggest that cation amelioration of Al toxicity is caused by the reduction in Al accumulation.

Expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco is not associated with either enhanced citrate accumulation or efflux.

Aluminum (Al) toxicity and poor phosphorus (P) availability are factors that limit plant growth on many agricultural soils. Previous work reported that expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco (Nicotiana tabacum; CSb lines) resulted in improved Al tolerance (J.M. de la Fuente, V. Ramírez-Rodríguez, J.L. Cabrera-Ponce, L. Herrera-Estrella [1997] Science 276: 1566-1568) and an enhanced ability to acquire P from alkaline soils (J. López-Bucio, O. Martínez de la Vega, A. Guevara-García, L. Herrera-Estrella [2000] Nat Biotechnol 18: 450-453). These effects were attributed to the P. aeruginosa citrate synthase increasing the biosynthesis and efflux of citrate from roots. To verify these findings we: (a) characterized citrate efflux from roots of wild-type tobacco; (b) generated tobacco lines expressing the citrate synthase gene from P. aeruginosa; and (c) analyzed selected CSb lines described above. Al stimulated citrate efflux from intact roots of wild-type tobacco and root apices were found to be responsible for most of the efflux. Despite generating transgenic tobacco lines that expressed the citrate synthase protein at up to a 100-fold greater level than the previously described CSb lines, these lines did not show increased accumulation of citrate in roots or increased Al-activated efflux of citrate from roots. Selected CSb lines, similarly, failed to show differences compared with controls in either citrate accumulation or efflux. We conclude that expression of the P. aeruginosa citrate synthase gene in plants is unlikely to be a robust and easily reproducible strategy for enhancing the Al tolerance and P-nutrition of crop and pasture species.

Malate-permeable channels and cation channels activated by aluminum in the apical cells of wheat roots.

Aluminum (Al(3+))-dependent efflux of malate from root apices is a mechanism for Al(3+) tolerance in wheat (Triticum aestivum). The malate anions protect the sensitive root tips by chelating the toxic Al(3+) cations in the rhizosphere to form non-toxic complexes. Activation of malate-permeable channels in the plasma membrane could be critical in regulating this malate efflux. We examined this by investigating Al(3+)-activated channels in protoplasts from root apices of near-isogenic wheat differing in Al(3+) tolerance at a single locus. Using whole-cell patch clamp we found that Al(3+) stimulated an electrical current carried by anion efflux across the plasma membrane in the Al(3+)-tolerant (ET8) and Al(3+)-sensitive (ES8) genotypes. This current occurred more frequently, had a greater current density, and remained active for longer in ET8 protoplasts than for ES8 protoplasts. The Al(3+)-activated current exhibited higher permeability to malate(2-) than to Cl(-) (P(mal)/P(Cl) > or = 2.6) and was inhibited by anion channel antagonists, niflumate and diphenylamine-2-carboxylic acid. In ET8, but not ES8, protoplasts an outward-rectifying K(+) current was activated in the presence of Al(3+) when cAMP was included in the pipette solution. These findings provide evidence that the difference in Al(3+)-induced malate efflux between Al(3+)-tolerant and Al(3+)-sensitive genotypes lies in the differing capacity for Al(3+) to activate malate permeable channels and cation channels for sustained malate release.

Aluminum Toxicity in Roots : Correlation among Ionic Currents, Ion Fluxes, and Root Elongation in Aluminum-Sensitive and Aluminum-Tolerant Wheat Cultivars.

The inhibition of root growth by aluminum (Al) is well established, yet a unifying mechanism for Al toxicity remains unclear. The association between cell growth and endogenously generated ionic currents measured in many different systems, including plant roots, suggests that these currents may be directing growth. A vibrating voltage microelectrode system was used to measure the net ionic currents at the apex of wheat (Triticum aestivum L.) roots from Al-tolerant and Al-sensitive cultivars. We examined the relationship between these currents and Al-induced inhibition of root growth. In the Al-sensitive cultivar, Scout 66, 10 micromolar Al (pH 4.5) began to inhibit the net current and root elongation within 1 to 3 hours. These changes occurred concurrently in 75% of experiments. A significant correlation was found between current magnitude and the rate of root growth when data were pooled. No changes in either current magnitude or growth rate were observed in similar experiments using the Al-tolerant cultivar Atlas 66. Measurements with ion-selective microelectrodes suggested that H(+) influx was responsible for most of the current at the apex, with smaller contributions from Ca(2+) and Cl(-) fluxes. In 50% of experiments, Al began to inhibit the net H(+) influx in Scott 66 roots at the same time that growth was affected. However, in more than 25% of cases, Al-induced inhibition of growth rate occurred before any sustained decrease in the current or H(+) flux. Although showing a correlation between growth and current or H(+) fluxes, these data do not suggest a mechanistic association between these processes. We conclude that the inhibition of root growth by Al is not caused by the reduction in current or H(+) influx at the root apex.

Interactive effects of Al, h, and other cations on root elongation considered in terms of cell-surface electrical potential.

The rhizotoxicities of Al(3+) and of La(3+) to wheat (Triticum aestivum L.) were similarly ameliorated by cations in the following order of effectiveness: H(+) approximately C(3+) > C(2+) > C(1+). Among tested cations of a given charge, ameliorative effectiveness was similar except that Ca(2+) was slightly more effective than other divalent cations and H(+) was much more effective than other monovalent cations. H(+) rhizotoxicity was also ameliorated by cations in the order C(3+) > C(2+) > C(1+). These results suggest a role for cell-surface electrical potential in the rhizotoxicity of Al(3+), La(3+), H(+), and other toxic cations: negatively charged cell surfaces of the root accumulate the toxic cations, and amelioration is effected by treatments that reduce the negativity of the cell-surface electrical potential by charge screening or cation binding. Membrane-surface activities of free Al(3+) or La(3+) computed according to a Gouy-Chapman-Stern model correlated well with growth inhibition, which correlated only poorly with Al(3+) or La(3+) activities in the external medium. The similar responses of Al-intoxicated and La-intoxicated roots to ameliorative treatments provide evidence that Al(3+), rather than AlOH(2+) or Al(OH)(2) (+), is the principal toxic species of mononuclear Al. Comparisons of the responses of Al-sensitive and Al-tolerant wheats to Al(3+) and to La(3+) did not support the hypothesis that varietal sensitivity to Al(3+) is based upon differences in cell-surface electrical potential.

Aluminum activates an anion channel in the apical cells of wheat roots.

We describe an anion channel in the plasmalemma of protoplasts isolated from wheat (Triticum aestivum L.) roots that is activated by aluminum (Al3+). In the whole-cell configuration, addition of 20-50 microM AlCl3 to the external solution depolarized the membrane and activated an inward current that could remain active for more than 60 min. The activation by Al3+ was rapid in 20% of protoplasts examined, whereas in another 30% a delay of more than 10 min occurred after Al3+ was added. Once the current was activated, changing the external Cl- concentration shifted the membrane reversal potential with ECl, showing that the channel is more selective for anions than cations (Ca2+, K+, tetraethylammonium+). The channel could be activated by Al3+, but not by La3+, and was observed in protoplasts isolated from the root apex but not in protoplasts isolated from mature root tissue. The anion channel antagonist niflumate inhibited the current in whole cell measurements by 83% at 100 microM. Outside-out patch recordings revealed a multistate channel with single-channel conductances of between 27 and 66 pS.

Quantification of Plasmodium malariae infection in mosquito vectors.

Plasmodium malariae occurs in various tropical regions throughout the world and causes low, yet significant, levels of morbidity in human populations. One means of studying the ecology and frequency of this parasite is by measuring sporozoite loads in the salivary glands of infected mosquitoes. An effective, species-specific test that can be used to detect the presence of sporozoites in mosquitoes is the circumsporozoite ELISA. The aim of the present study was to standardize the circumsporozoite ELISA for P.malariae, by setting quantification parameters using, as antigen, either a synthetic peptide or extracts of whole sporozoites. The standard quantification curves produced indicated that the assay had a lower threshold of sensitivity of 250 sporozoites in a 50-microl sample, equivalent to about 1250 sporozoites in a mosquito.

Cloning and expression of a wheat (Triticum aestivum L.) phosphatidylserine synthase cDNA. Overexpression in plants alters the composition of phospholipids.

We describe the cloning of a wheat cDNA (TaPSS1) that encodes a phosphatidylserine synthase (PSS) and provides the first strong evidence for the existence of this enzyme in a higher eukaryotic cell. The cDNA was isolated on its ability to confer increased resistance to aluminum toxicity when expressed in yeast. The sequence of the predicted protein encoded by TaPSS1 shows homology to PSS from both yeast and bacteria but is distinct from the animal PSS enzymes that catalyze base-exchange reactions. In wheat, Southern blot analysis identified the presence of a small family of genes that cross-hybridized to TaPSS1, and Northern blots showed that aluminum induced TaPSS1 expression in root apices. Expression of TaPSS1 complemented the yeast cho1 mutant that lacks PSS activity and altered the phospholipid composition of wild type yeast, with the most marked effect being increased abundance of phosphatidylserine (PS). Arabidopsis thaliana leaves overexpressing TaPSS1 showed a marked enhancement in PSS activity, which was associated with increased biosynthesis of PS at the expense of both phosphatidylinositol and phosphatidylglycerol. Unlike mammalian cells where PS accumulation is tightly regulated even when the capacity for PS biosynthesis is increased, plant cells accumulated large amounts of PS when TaPSS1 was overexpressed. High levels of TaPSS1 expression in Arabidopsis and tobacco (Nicotiana tabacum) led to the appearance of necrotic lesions on leaves, which may have resulted from the excessive accumulation of PS. The cloning of TaPSS1 now provides evidence that the yeast pathway for PS synthesis exists in some plant tissues and provides a tool for understanding the pathways of phospholipid biosynthesis and their regulation in plants.

Quantitative determination of sporozoites and circumsporozoite antigen in mosquitoes infected with Plasmodium falciparum or P. vivax.

It is essential for malariologists and researchers to have simple and accurate means of assessing the threat of Plasmodium parasites. An attempt was therefore made to re-standardize one of the circumsporozoite (CS) ELISA that can be used to detect and quantify the circumsporozoite antigens of P. falciparum and P. vivax. A two-site, 'sandwich' ELISA based on a monoclonal antibody was used to test for the CS antigen and sporozoites of each Plasmodium species simultaneously. Using the resultant optical-density values, standard curves, that permit the number of sporozoites in an infected mosquito to be estimated from the quantification of the CS antigen, were constructed. Using these plots and the CS ELISA, the presence of just 12.5 sporozoites (i.e. 0.8 pg CS antigen) of P. falciparum, four sporozoites (3.2 pg antigen) of P. vivax-210 or 12.5 sporozoites (32.0 pg antigen) of P. vivax-247 could be demonstrated.