Simultaneous flux and current measurement from single plant protoplasts reveals a strong link between K+ fluxes and current, but no link between Ca2+ fluxes and current

We present a thorough calibration and verification of a combined non-invasive self-referencing microelectrode-based ion-flux measurement and whole-cell patch clamp system as a novel and powerful tool for the study of ion transport. The system is shown to be capable of revealing the movement of multiple ions across the plasma membrane of a single protoplast at multiple voltages and in complex physiologically relevant solutions. Wheat root protoplasts are patch clamped in the whole-cell configuration and current-voltage relations obtained whilst monitoring net K+ and Ca2+ flux adjacent to the membrane with ion-selective electrodes. At each voltage, net ion flux (nmol m(-2) sec(-1)) is converted to an equivalent current density (mA m(-2)) taking into account geometry and electrode efficiency, and compared with the net current density measured with the patch clamp system. Using this technique, it is demonstrated that the K+-permeable outwardly rectifying conductance (KORC) is responsible for net outward K+ movement across the plasma membrane [1:1 flux-to-current ratio (1.21 +/- 0.14 SEM, n = 15)]. Variation in the K+ flux-to-current ratio among single protoplasts suggests a heterogeneous distribution of KORC channels on the membrane surface. As a demonstration of the power of the technique we show that despite a significant Ca2+ permeability being associated with KORC (analysis of tail current reversal potentials), there is no correlation between Ca2+ flux and KORC activity. A very significant observation is that large Ca2+ fluxes are electrically silent and probably tightly coupled to compensatory charge movements. This analysis demonstrates that it is mandatory to measure flux and currents simultaneously to investigate properly Ca2+ transport mechanisms and selectivity of ion channels in general.     

Calcium delivery and storage in plant leaves: exploring the link with water flow

Calcium (Ca) is a unique macronutrient with diverse but fundamental physiological roles in plant structure and signalling. In the majority of crops the largest proportion of long-distance calcium ion (Ca(2+)) transport through plant tissues has been demonstrated to follow apoplastic pathways, although this paradigm is being increasingly challenged. Similarly, under certain conditions, apoplastic pathways can dominate the proportion of water flow through plants. Therefore, tissue Ca supply is often found to be tightly linked to transpiration. Once Ca is deposited in vacuoles it is rarely redistributed, which results in highly transpiring organs amassing large concentrations of Ca ([Ca]). Meanwhile, the nutritional flow of Ca(2+) must be regulated so it does not interfere with signalling events. However, water flow through plants is itself regulated by Ca(2+), both in the apoplast via effects on cell wall structure and stomatal aperture, and within the symplast via Ca(2+)-mediated gating of aquaporins which regulates flow across membranes. In this review, an integrated model of water and Ca(2+) movement through plants is developed and how this affects [Ca] distribution and water flow within tissues is discussed, with particular emphasis on the role of aquaporins.     

Development of a new quantitative gas permeability method for dental implant-abutment connection tightness assessment

Background  

Most dental implant systems are presently made of two pieces: the implant itself and the abutment. The connection tightness between those two pieces is a key point to prevent bacterial proliferation, tissue inflammation and bone loss. The leak has been previously estimated by microbial, color tracer and endotoxin percolation.     

<Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>: a model to monitor bacterial air quality

Background

Low environmental air quality is a significant cause of mortality and morbidity and this question is now emerging as a main concern of governmental authorities. Airborne pollution results from the combination of chemicals, fine particles, and micro-organisms quantitatively or qualitatively dangerous for health or for the environment. Increasing regulations and limitations for outdoor air quality have been decreed in regards to chemicals and particles contrary to micro-organisms. Indeed, pertinent and reliable tests to evaluate this biohazard are scarce. In this work, our purpose was to evaluate the Caenorhaditis elegans killing test, a model considered as an equivalent to the mouse acute toxicity test in pharmaceutical industry, in order to monitor air bacterial quality.

Findings

The present study investigates the bacterial population in dust clouds generated during crop ship loading in harbor installations (Rouen harbor, Normandy, France). With a biocollector, airborne bacteria were impacted onto the surface of agar medium. After incubation, a replicate of the colonies on a fresh agar medium was done using a velvet. All the replicated colonies were pooled creating the "Total Air Sample". Meanwhile, all the colonies on the original plate were isolated. Among which, five representative bacterial strains were chosen. The virulence of these representatives was compared to that of the "Total Air Sample" using the Caenorhaditis elegans killing test. The survival kinetic of nematodes fed with the "Total Air Sample" is consistent with the kinetics obtained using the five different representatives strains.

Conclusions

Bacterial air quality can now be monitored in a one shot test using the Caenorhaditis elegans killing test.

     

Roles of Morphology, Anatomy, and Aquaporins in Determining Contrasting Hydraulic Behavior of Roots

The contrasting hydraulic properties of wheat (Triticum aestivum), narrow-leafed lupin (Lupinus angustifolius), and yellow lupin (Lupinus luteus) roots were identified by integrating measurements of water flow across different structural levels of organization with anatomy and modeling. Anatomy played a major role in root hydraulics, influencing axial conductance (L_ax) and the distribution of water uptake along the root, with a more localized role for aquaporins (AQPs). Lupin roots had greater L_ax than wheat roots, due to greater xylem development. L_ax and root hydraulic conductance (L_r) were related to each other, such that both variables increased with distance from the root tip in lupin roots. L_ax and L_r were constant with distance from the tip in wheat roots. Despite these contrasting behaviors, the hydraulic conductivity of root cells (Lp_c) was similar for all species and increased from the root surface toward the endodermis. Lp_c was largely controlled by AQPs, as demonstrated by dramatic reductions in Lp_c by the AQP blocker mercury. Modeling the root as a series of concentric, cylindrical membranes, and the inhibition of AQP activity at the root level, indicated that water flow in lupin roots occurred primarily through the apoplast, without crossing membranes and without the involvement of AQPs. In contrast, water flow across wheat roots crossed mercury-sensitive AQPs in the endodermis, which significantly influenced L_r. This study demonstrates the importance of examining root morphology and anatomy in assessing the role of AQPs in root hydraulics.     

Wheat grain yield on saline soils is improved by an ancestral Na⁺ transporter gene

The ability of wheat to maintain a low sodium concentration ([Na(+)]) in leaves correlates with improved growth under saline conditions. This trait, termed Na(+) exclusion, contributes to the greater salt tolerance of bread wheat relative to durum wheat. To improve the salt tolerance of durum wheat, we explored natural diversity in shoot Na(+) exclusion within ancestral wheat germplasm. Previously, we showed that crossing of Nax2, a gene locus in the wheat relative Triticum monococcum into a commercial durum wheat (Triticum turgidum ssp. durum var. Tamaroi) reduced its leaf [Na(+)] (ref. 5). Here we show that a gene in the Nax2 locus, TmHKT1;5-A, encodes a Na(+)-selective transporter located on the plasma membrane of root cells surrounding xylem vessels, which is therefore ideally localized to withdraw Na(+) from the xylem and reduce transport of Na(+) to leaves. Field trials on saline soils demonstrate that the presence of TmHKT1;5-A significantly reduces leaf [Na(+)] and increases durum wheat grain yield by 25% compared to near-isogenic lines without the Nax2 locus.     

Voltage-dependent cation channels permeable to NH(+)(4), K(+), and Ca(2+) in the symbiosome membrane of the model legume Lotus japonicus

The symbiosome of nitrogen fixing root nodules mediates metabolite exchange between endosymbiotic rhizobia bacteria and the legume host. In the present study, the ion currents of the symbiosome membrane of the model legume Lotus japonicus were analyzed by patch-clamp recording. Both excised and symbiosome-attached patches exhibited a large inward (toward the cytosolic side of the membrane) current that is activated in a time-dependent manner by negative (on the cytosolic side) potentials. Based on reversal potential determinations and recordings with the impermeant cation N-methyl-glucamine, this current shows a high permeability for monovalent cations with no apparent permeability for anions. The current also showed a finite Ca(2+) permeability. However, the currents were predominantly carried by univalent cations with a slightly greater selectivity for NH(4)(+) over K(+). Increased Ca(2+) concentration inhibited the current with a K(0.5) for inhibition of 0.317 mM. The current showed strong rectification that is mediated by divalent cations (either Mg(2+) or Ca(2+)). The influence of divalent cations is symmetrical in nature, because rectification can be exerted in either direction depending upon which side of the membrane has the highest concentration of divalent cations. However, based on observations with symbiosome-attached patches, the direction of the current in vivo is proposed to be toward the cytosol with cytosolic Mg(2+) acting as the putative gating regulator. The findings suggest that L. japonicus possesses a voltage-dependent cation efflux channel that is capable of exporting fixed NH(4)(+), and may also play an additional role in Ca(2+) transport.     

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.