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Studies on aluminum toxicity in an aluminum-sensitive cultivar of barley: impact on ion fluxes and calcium distribution

University of British Columbia

The effects of aluminum (Al) on root growth and development in the aluminum-sensitive cultivar of barley, Hordeum vulgare Lemend. Lam. var. Kearney was studied using low (1 AM) and high concentrations (50 AM) of Al. To analyze the impact of the aluminum treatments, changes in the anatomy, respiration, weight and growth rate of roots were analyzed. Aluminum-induced changes in cell division and cell elongation were also measured in order to determine the importance of each one of these factors in root growth inhibition. These studies were correlated with measurements of the root surface area to determine the impact of aluminum treatment on apoplasmic ion absorption (i.e. potassium(K)) and distribution visualized by dispersive X-raymicro analysis. Both levels of Al completely inhibited the initiation of secondary roots and inhibited primary root growthbut had no effect on germination in Hordeum vulgare var Kearney. The growth rate was progressively reduced as the aluminum concentration increased. Growth was essentially arrested after 4days treatment with 0.1 gM Al, while growth ceased after 2 days treatment with 50 AM Al. However, respiration was found to be unaffected by treatment with 50 AM Al for 2 days. Aluminum at 50gM and 1 AM dramatically reduced cell elongation. However, the same Al treatments appeared to stimulate cell division. The reduction in root length was thus due to an Al-induced inhibition of root cell elongation and not cell division. Root width was increased by Al treatment as was the average width of root cells. Aluminum dramatically reduced K in the apical region of the root(90% inhibition) as shown by energy dispersive X-ray microanalysis and reduced the [P] concentration in the root tip by 20-30%. The effects of short (10 minutes) and long term (5 day) treatments with Al on the influx and efflux of K (86Rb) in Hordeum vulgare var. Kearney were studied. The speed at which inhibition occurred and the persistence of any inhibition in the absence of aluminum was also investigated. The influx of K was inhibited by 55% when treated for 5 days with 5 AM Al at pH 4.2.Plants treated with 50 gM Al for 30 seconds showed a significant reduction in K influx. The Al-induced inhibition of K influx was found to be reversible since plants treated for 10 minutes with50 AM Al returned to control influx levels when transferred toAl-free medium. The K inhibition from long term (5 days)treatment with 5 gM Al was also reversible since K influx returned immediately to control levels when Al was not present. The half times for K efflux from plants treated with 5 AM Al for5 days was found to be similar to controls. When K influx was measured with increasing potassium in solution, it was found that K appear to competitively reduce the impact of Al. The mechanism by which aluminum interferes with ion influx is not known. In this study, the effects of aluminum on the influx of the cations, calcium, potassium and ammonium and the anions, nitrate and phosphate was measured in an aluminum sensitive cultivar of barley. Aluminum (100 AM) was found to inhibit the influx of the cations, calcium (69%), ammonium (40%) and potassium (13%) while enhancing the influx of the anions nitrate (44%) and phosphate (17%). Aluminum interfered with the binding of the cations in the cell wall by the same order of magnitude as influxes were reduced, while phosphate binding was strongly enhanced. Aluminum was found to have a direct effect on cation transport since the treatment time of less than 20 minutes would not probably be long enough to cause indirect effects due to energy-related inhibition. The results could not be caused byAl-induced inhibition in proton pumping and proton motive force (pmf), since nitrate transport, which is believed to be directly coupled to the H-1--ATPase was not inhibited by Al. These results are consistent with a mechanism where Al binds to plasma membrane phospholipids, forming a positive charge layer influencing ion movement to the binding sites of the transport proteins. A positive charge layer could retard the movement of cations to the plasma membrane proportionally to the charge carried by the ion and increase the movement of anions again in proportion to the charge they carried. Chlorotetracycline (CTC) and Fluo-3 were used to study the distribution and concentration of membrane-bound and cytosolic free Ca in the Al-sensitive var. Kearney of Hordeum vulgare. Both Ca-indicator dyes revealed substantial increases influorescence in both root apices and root hair cells after Al-treatment. Mn was used to quench the Fluo-3 fluorescence and ensure that the signal was located in the cytosol and not the cell wall. Confocal scanning laser microscopic observations were used to ascertain that the Fluo-3 signal was restricted to the cytoplasm and it was not the result of the compartmentalization of the indicator within organelles, such as the vacuole. Confocal line plots were utilized to determine the relative concentration of Ca throughout the different regions of the root and root hairs. The effects of Al on the viability of the cells was monitored by differential contrast microscopy and neutral red accumulation within the vacuoles. The results indicate that Al interferes with both Ca distribution and concentration, with some regions of the root being particularly sensitive to its effects. Aluminum increased the CTC and Fluo-3 fluorescence in root apexcells and in the cortical cytoplasm of root hairs. This Al-induced disturbance in cellular calcium could interfere with Ca-dependent processes involved in cell elongation and division andplay a major role in the establishment of well known Al-induced toxicity symptoms.

Thesis/Dissertation

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2008-09-15

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http://hdl.handle.net/2429/1954

Biomedical Engineering

University of British Columbia

UBCV

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