The defense substance allicin from garlic permeabilizes membranes of Beta vulgaris, Rhoeo discolor, Chara corallina and artificial lipid bilayers.

BACKGROUND: Allicin (diallylthiosulfinate) is the major volatile- and antimicrobial substance produced by garlic cells upon wounding. We tested the hypothesis that allicin affects membrane function and investigated 1) betanine pigment leakage from beetroot (Beta vulgaris) tissue, 2) the semipermeability of the vacuolar membrane of Rhoeo discolor cells, 3) the electrophysiology of plasmalemma and tonoplast of Chara corallina and 4) electrical conductivity of artificial lipid bilayers. METHODS: Garlic juice and chemically synthesized allicin were used and betanine loss into the medium was monitored spectrophotometrically. Rhoeo cells were studied microscopically and Chara- and artificial membranes were patch clamped. RESULTS: Beet cell membranes were approximately 200-fold more sensitive to allicin on a mol-for-mol basis than to dimethyl sulfoxide (DMSO) and approximately 400-fold more sensitive to allicin than to ethanol. Allicin-treated Rhoeo discolor cells lost the ability to plasmolyse in an osmoticum, confirming that their membranes had lost semipermeability after allicin treatment. Furthermore, allicin and garlic juice diluted in artificial pond water caused an immediate strong depolarization, and a decrease in membrane resistance at the plasmalemma of Chara, and caused pore formation in the tonoplast and artificial lipid bilayers. CONCLUSIONS: Allicin increases the permeability of membranes. GENERAL SIGNIFICANCE: Since garlic is a common foodstuff the physiological effects of its constituents are important. Allicin's ability to permeabilize cell membranes may contribute to its antimicrobial activity independently of its activity as a thiol reagent.

Pectate chemistry links cell expansion to wall deposition in Chara corallina.

Pectate (polygalacturonic acid) acts as a chelator to bind calcium and form cross-links that hold adjacent pectate polymers and thus plant cell walls together. When under tension from turgor pressure in the cell, the cross-links appear to distort and weaken. New pectate supplied by the cytoplasm is undistorted and removes wall calcium preferentially from the weakened bonds, loosening the wall and accelerating cell expansion. The new pectate now containing the removed calcium can bind to the wall, strengthening it and linking expansion to wall deposition. But new calcium needs to be added as well to replenish the calcium lost from the vacated wall pectate.  A recent report demonstrated that growth was disrupted if new calcium was unavailable.  The present addendum highlights this conclusion by reviewing an experiment from before the chelation chemistry was understood. Using cell wall labeling, a direct link appeared between wall expansion and wall deposition. Together, these experiments support the concept that newly supplied pectate has growth activity on its way to deposition in the wall. Growth rate is thus controlled by signals affecting the rate of pectate release. After release, the coordination of expansion and deposition arises naturally from chelation chemistry when polymers are under tension from turgor pressure. 

Calcium deprivation disrupts enlargement of Chara corallina cells: further evidence for the calcium pectate cycle.

Pectin is a normal constituent of cell walls of green plants. When supplied externally to live cells or walls isolated from the large-celled green alga Chara corallina, pectin removes calcium from load-bearing cross-links in the wall, loosening the structure and allowing it to deform more rapidly under the action of turgor pressure. New Ca(2+) enters the vacated positions in the wall and the externally supplied pectin binds to the wall, depositing new wall material that strengthens the wall. A calcium pectate cycle has been proposed for these sub-reactions. In the present work, the cycle was tested in C. corallina by depriving the wall of external Ca(2+) while allowing the cycle to run. The prediction is that growth would eventually be disrupted by a lack of adequate deposition of new wall. The test involved adding pectate or the calcium chelator EGTA to the Ca(2+)-containing culture medium to bind the calcium while the cycle ran in live cells. After growth accelerated, turgor and growth eventually decreased, followed by an abrupt turgor loss and growth cessation. The same experiment with isolated walls suggested the walls of live cells became unable to support the plasma membrane. If instead the pectate or EGTA was replaced with fresh Ca(2+)-containing culture medium during the initial acceleration in live cells, growth was not disrupted and returned to the original rates. The operation of the cycle was thus confirmed, providing further evidence that growth rates and wall biosynthesis are controlled by these sub-reactions in plant cell walls.

Petroleum coke and soft tailings sediment in constructed wetlands may contribute to the uptake of trace metals by algae and aquatic invertebrates.

The fate of trace metals in pore water collected from wetland sediments and organisms exposed to petroleum coke were evaluated within in situ aquatic microcosms. Oil sands operators of Fort McMurray, Alberta, Canada produced 60 million tonnes of petroleum coke by 2008, containing elevated concentrations of sulphur and several trace metals commonly seen in oil sands materials. This material may be included in the construction of reclaimed wetlands. Microcosms were filled with a surface layer of petroleum coke over mine-waste sediments and embedded in a constructed wetland for three years to determine how these materials would affect the metal concentrations in the sediment pore water, colonizing wetland plants and benthic invertebrates. Petroleum coke treatments produced significantly elevated levels of Ni. We also found unexpectedly higher concentrations of metals in "consolidated tailings" waste materials, potentially due to the use of oil sands-produced gypsum, and higher background concentration of elements in the sediment used in the controls. A trend of higher concentrations of V, Ni, La, and Y was present in the tissues of the colonizing macrophytic alga Chara spp. Aeshnid dragonflies may also be accumulating V. These results indicate that the trace metals present in some oil sands waste materials could be taken up by aquatic macro-algae and some wetland invertebrates if these materials are included in reclaimed wetlands.

Fluorescent phosphocholine--a specific marker for the endoplasmic reticulum and for lipid droplets in Chara internodal cells.

The staining pattern of 1,2-bis(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine (Bodipy PC) was investigated in internodal cells of the green alga Chara corallina. Ten minutes after dye addition, Bodipy-PC-derived fluorescence appeared in lipid droplets and after 1 h in the cortical endoplasmic reticulum (ER) and in the inner ER tubes. Staining of the ER required energy but was independent of an intact actin or microtubule cytoskeleton and independent of vesicular endocytosis. The size of the lipid droplets varied between 0.25 microm in elongating cells and 3.2 microm in senescent internodes. They moved together with or along the cortical ER cisternae in a cytoskeleton-independent manner or remained immobile up to several minutes. Detachment of lipid droplets from the cortical ER or fusion of lipid droplets was never observed. The results of this study suggest that Bodipy PC is a valuable, less toxic alternative to 3,3'-dihexyloxacarbocyanine iodide (DiOC6) staining of the ER in Chara. They confirm an earlier report about microtubule-dependent cortical ER morphology and dynamics in elongating internodes and offer new perspectives for the study of organelle interactions.

Anti-tropomyosin antibodies co-localise with actin microfilaments and label plasmodesmata.

The actin cytoskeleton and associated actin-binding proteins form a complex network involved in a number of fundamental cellular processes including intracellular trafficking. In plants, both actin and myosin have been localised to plasmodesmata, and thus it is likely that other actin-binding proteins are also associated with plasmodesmata structure or function. A 75-kDa protein, enriched in plasmodesmata-rich cell wall extracts from the green alga Chara corallina, was sequenced and found to contain three peptides with similarity to the animal actin-binding protein tropomyosin. Western blot analysis with anti-tropomyosin antibodies confirmed the identity of this 75-kDa protein as a tropomyosin-like protein and further identified an additional 55-kDa protein, while immunofluorescence microscopy localised the antibodies to plasmodesmata and to the subcortical actin bundles and associated structures. The anti-tropomyosin antibodies detected a single protein at 42.5 kDa in Arabidopsis thaliana extracts and two proteins at 58.5 and 54 kDa in leek extracts, and these localised to plasmodesmata and the cell plate in A. thaliana and to plasmodesmata in leek tissue. Tropomyosin is an actin-binding protein thought to be involved in a range of functions associated with the actin cytoskeleton, including the regulation of myosin binding to actin filaments, but to date no tropomyosin-like proteins have been conclusively identified in plant genomes. Our data suggests that a tropomyosin-like protein is associated with plasmodesmata.

Tension required for pectate chemistry to control growth in Chara corallina.

Recent work showed that polygalacturonate (pectate) chemistry controlled the growth rate of the large-celled alga Chara corallina when turgor pressure (P) was normal (about 0.5 MPa). The mechanism involved calcium withdrawal from the wall by newly supplied pectate acting as a chelator. But P itself can affect growth rate. Therefore, pectate chemistry was investigated at various P. A pressure probe varied P in isolated walls, varying the tension on the calcium pectate cross-links bearing the load of P. When soluble pectate was newly supplied, the wall grew irreversibly but the pectate was inactive below a P of 0.2 MPa, indicating that tension was required in the existing wall before new pectate acted. It was suggested that the tension distorted some of the wall pectate (the dominant pectin), weakening its calcium cross-links and causing the calcium to be preferentially lost to the new pectate, which was not distorted. The preferential loss provided a molecular mechanism for loosening the wall structure, resulting in faster growth. However, the resulting relaxation of the vacated wall pectate would cause calcium to be exchanged with load-bearing calcium pectate nearby, auto-propagating throughout the wall for long periods. There is evidence for this effect in isolated walls. In live cells, there is also evidence that auto-propagation is controlled by binding the newly supplied pectate (now calcium pectate) to the wall and/or by additional Ca(2+) entering the wall structure. A tension-dependent cycle of pectate chemistry thus appeared to control growth while new wall was deposited as a consequence.

Calcium pectate chemistry controls growth rate of Chara corallina.

Pectin, a normal constituent of cell walls, caused growth rates to accelerate to the rates in living cells when supplied externally to isolated cell walls of Chara corallina. Because this activity was not reported previously, the activity was investigated. Turgor pressure (P) was maintained in isolated walls or living cells using a pressure probe in culture medium. Pectin from various sources was supplied to the medium. Ca and Mg were the dominant inorganic elements in the wall. EGTA or pectin in the culture medium extracted moderate amounts of wall Ca and essentially all the wall Mg, and wall growth accelerated. Removing the external EGTA or pectin and replacing with fresh medium returned growth to the original rate. A high concentration of Ca2+ quenched the accelerating activity of EGTA or pectin and caused gelling of the pectin, physically inhibiting wall growth. Low pH had little effect. After the Mg had been removed, Ca-pectate in the wall bore the longitudinal load imposed by P. Removal of this Ca caused the wall to burst. Live cells and isolated walls reacted similarly. It was concluded that Ca cross-links between neighbouring pectin molecules were strong wall bonds that controlled wall growth rates. The central role of Ca-pectate chemistry was illustrated by removing Ca cross-links with new pectin (wall "loosening"), replacing vacated cross-links with new Ca2+ ("Ca2+-tightening"), or adding new cross-links with new Ca-pectate that gelled ("gel tightening"). These findings establish a molecular model for growth that includes wall deposition and assembly for sustained growth activity.

Identifying cytoplasmic input to the cell wall of growing Chara corallina.

Plants enlarge mostly because the walls of certain cells enlarge, with accompanying input of wall constituents and other factors from the cytoplasm. However, the enlargement can occur without input, suggesting an uncertain relationship between cytoplasmic input and plant growth. Therefore, the role of the input was investigated by quantitatively comparing growth in isolated walls (no input) with that in living cells (input occurring). Cell walls were isolated from growing internodes of Chara corallina and filled with pressurized oil to control turgor pressure while elongation was monitored. Turgor pressure in living cells was similarly controlled and monitored by adding/removing cell solution. Temperature was varied in some experiments. At all pressures and temperatures, isolated walls displayed turgor-driven growth indistinguishable in every respect from that in living cells, except the rate decelerated in the isolated walls while the living cells grew rapidly. The growth in the isolated walls was highly responsive to temperature, in contrast to the elastic extension that has been shown to be insensitive to similar temperatures. Consequently, strong intermolecular bonds were responsible for growth and weak bonds for elastic extension. Boiling the walls gave the same results, indicating that enzyme activities were not controlling these bonds. However, pectin added to isolated walls reversed their growth deceleration and returned the rate to that in the living cells. The pectin was similar to that normally produced by the cytoplasm and deposited in the wall, suggesting that continued cytoplasmic input of pectin may play a role in sustaining turgor-driven growth in Chara.