Hydrogen fluoride effects on plasma membrane composition,ATPase activity and cell structure in needles of eastern white pine (Pinus strobus) seedlings

Eastern white pine (Pinus strobus L.) seedlings were grown in controlled environment growth cabinets and fumigated with 0.4 and 1.6 g m^–3 hydrogen fluoride for 2–28 days. Plasma membranes were isolated from needles of treated and control seedlings and their chemical composition and ATPase activity examined to determine early effects of hydrogen fluoride action. In plants treated for 2 days with both fluoride levels, ratios of plasma membrane free sterols:phospholipids and sterols:proteins were drastically higher than ratios in control plants. Seedlings treated with hydrogen fluoride for 8 days contained plasma membranes with elevated phospholipid:protein and sterol:protein ratios and their plasma membrane ATPase activity was higher than that of control plants. Prolonged, 28-day hydrogen fluoride treatment with 1.6 g m^–3 level was the only treatment which produced a drastic inhibition of plasma membrane ATPase activity. During the initial stages of hydrogen fluoride treatment, treated cells did not show alterations of ultrastructure which were previously shown in cells of plants treated with soil applied sodium fluoride. The results of the present study indicate that the plasma membranes may be among the initial sites of hydrogen fluoride injury to plants as well as initial sites of defense reaction.     

A calcium-binding protein with similarity to serum albumin localized to the ER-Golgi network and cell walls of spinach (Spinacia oleracea)

Using polyclonal antibodies raised against human serum albumin (HSA), a 70-kDa microsomal protein with an isoelectric point of approximately 6.5 was detected in spinach ( Spinacia oleracea L.). The protein was purified by selective ammonium sulfate precipitation and anion exchange HPLC. The protein shared 100% identity with the first 15 amino acids at the NH₂ terminus of HSA, including the X-X-H amino acid region, which was identified in HSA as being responsible for binding of copper, zinc, indole derivatives and calcium. Blue staining of the protein with the cationic carbocyanine dye 'Stains-all' and ⁴⁵Ca overlay following SDS-PAGE also suggest that the 70-kDa plant protein binds calcium. The protein reacted positively with carbohydrate specific thymol stain, and the carbohydrates associated with the protein were identified by gas chromatography-mass spectrometry (GC-MS) as galactose and galacturonic acid. The 70-kDa plant protein was present in the detergent-poor phase following Triton X-114 extraction of the microsomal proteins. Cell fractionation using continuous sucrose gradients showed that the protein is present in membrane fractions with high activity of endoplasmic reticulum (ER) and Golgi marker enzymes. Using nitrocellulose tissue prints probed with anti-HSA antibodies, we demonstrated that the protein is present in the apoplastic space of petioles, suggesting that the protein is secreted to the apoplast of cortex cells in plants. Localization and binding properties suggest that the plant protein identified in the present study may participate in secretion processes, possibly involved with the transport of precursors required for cell-wall synthesis.     

Laccaria bicolor aquaporin LbAQP1 is required for Hartig net development in trembling aspen (Populus tremuloides)

The development of ectomycorrhizal associations is crucial for growth of many forest trees. However, the signals that are exchanged between the fungus and the host plant during the colonization process are still poorly understood. In this study, we have identified the relationship between expression patterns of Laccaria bicolor aquaporin LbAQP1 and the development of ectomycorrhizal structures in trembling aspen (Populus tremuloides) seedlings. The peak expression of LbAQP1 was 700‐fold higher in the hyphae within the root than in the free‐living mycelium after 24 h of direct interaction with the roots. Moreover, in LbAQP1 knock‐down strains, a non‐mycorrhizal phenotype was developed without the Hartig net and the expression of the mycorrhizal effector protein MiSSP7 quickly declined after an initial peak on day 5 of interaction of the fungal hyphae with the roots. The increase in the expression of LbAQP1 required a direct contact of the fungus with the root and it modulated the expression of MiSSP7. We have also determined that LbAQP1 facilitated NO, H₂O₂ and CO₂ transport when heterologously expressed in yeast. The report demonstrates that the L. bicolor aquaporin LbAQP1 acts as a molecular signalling channel, which is fundamental for the development of Hartig net in root tips of P. tremuloides.     

Diurnal and seasonal changes of leaf lamina hydraulic conductance in bur oak (Quercus macrocarpa) and trembling aspen (Populus tremuloides)

The aim of this study was to examine the diurnal and seasonal variations in the sensitivity of leaf lamina (K _lam) hydraulic conductance to irradiance in bur oak (Quercus macrocarpa Michx.) and trembling aspen (Populus tremuloides Michx.), which vary in their responses of K _lam to irradiance. K _lam was determined using the high-pressure method and the measurements were carried out in June, August and September. The irradiance response of K _lam in bur oak was present throughout the day and declined in senescing leaves. In trembling aspen, K _lam declined from morning to late afternoon and drastically decreased before the onset of leaf senescence, but it was not sensitive to irradiance. In both tree species, the capacity of the petioles to supply water to leaf lamina changed during the day in accordance with the ability of the leaf lamina to transport water. Petiole hydraulic conductivity (K _pet) declined during the season in bur oak leaves, while it tended to increase in trembling aspen leaves. There was no correlation between the K _lam values and air temperature or light intensity at the time of leaf collection. For trembling aspen, K _pet was negatively correlated with the air temperature suggesting sensitivity to drought. We conclude that the water transport properties of petioles and leaf lamina in the two studied tree species reflect their ecological adaptations. Trembling aspen leaves have high hydraulic conductivity and high stomatal conductance regardless of the irradiance level, consistent with the rapid growth and high demand for water. In contrast, the increased lamina hydraulic conductivity and stomatal conductance under high irradiance in bur oak trees reflect a water conservation strategy.     

Effects of NaCl on responses of ectomycorrhizal black spruce (Picea mariana), white spruce (Picea glauca) and jack pine (Pinus banksiana) to fluoride

Black spruce ( Picea mariana ), white spruce ( Picea glauca ) and jack pine ( Pinus banksiana ) were inoculated with Suillus tomentosus and subjected to potassium fluoride (1 m M KF and 5 m M KF) in the presence and absence of 60 m M NaCl. The NaCl and KF treatments reduced total dry weights in jack pine and black spruce seedlings, but they did not affect total dry weights in white spruce seedlings. The addition of 60 m M NaCl to KF treatment solutions alleviated fluoride-induced needle injury in ectomycorrhizal (ECM) black spruce and white spruce, but had little effect in jack pine seedlings. Both KF and 60 m M NaCl treatments reduced E values compared with non-treated control seedlings. However, with the exception of small reductions of K_r by NaCl treatments in black spruce, the applied KF and NaCl treatments had little effect on K_r in ECM plants. Chloride tissue concentrations in NaCl-treated plants were not affected by the presence of KF in treatment solutions. However, shoot F concentrations in ECM black spruce and white spruce treated with 5 m M KF + 60 m M NaCl were significantly reduced compared with the 5 m M KF treatment. The results point to a possible competitive inhibition of F transport by Cl. We also suggest that the possibility that aquaporins may be involved in the transmembrane transport of F should be further investigated.     

Hebeloma crustuliniforme modifies root hydraulic responses of trembling aspen (Populus tremuloides) seedlings to changes in external pH

The main objective of the study was to compare the effects of short-duration pH treatments on root hydraulic properties in trembling aspen (Populus tremuloides) seedlings that were either inoculated with the ectomycorrhizal fungus Hebeloma crustuliniforme or remained non-inoculated (control). Inoculated and non-inoculated plants were exposed in solution culture to the root zone pH ranging from 4 to 9 and their root hydraulic conductivity was examined using the hydrostatic method and after subjecting the plants to treatments with 100 ??M HgCl₂ (aquaporin blocker) and 0.02% trisodium 3-hydroxy-5,8,10-pyrenetrisulfonic acid (apoplastic transport tracer). In a separate experiment, pure cultures of H. crustuliniforme were also grown on a slid medium with the pH ranging from 4 to 9 to determine their pH growth optimum and changes in medium pH over time in the presence and absence of 8 mM NH₄NO₃. When grown in pure culture, H. crustuliniforme demonstrated maximum growth at pH 7?C8 and was capable of modifying the pH of its growth media, especially in the presence of NH₄NO₃. The plants that were inoculated with H. crustuliniforme had a maximum root hydraulic conductivity at pH 7. At this pH, root hydraulic conductivity was significantly higher compared with non-inoculated plants and showed greater sensitivity of root water transport to pH changes relative to non-inoculated seedlings. Relative apoplastic flux was largely unaffected by pH in inoculated seedlings. Fungal inoculation modified the response of root hydraulic conductivity to pH. The increased root hydraulic conductivity in inoculated seedlings was likely due to an increase in aquaporin-mediated cell-to-cell water transport, particularly at the higher pH. A possible role of fungal aquaporins in the root hydraulic conductivity responses of mycorrhizzal plants should be examined.     

Early effects of triadimefon on water relations, sterol composition and plasma membrane ATPase in white spruce (Picea glauca) seedlings

Seedlings of white spruce ( Picea glauca [Moench] Voss.) were treated with triadimefon solution applied to the soil, and their early responses studied from 12 h to 7 days after treatment. Transpiration rates declined and respiration rates increased immediately after the commencement of triadimefon treatment. Photosynthetic rates declined less than transpiration rates, resulting in an increase in water use efficiency, whereas root and shoot water potentials remained unchanged during the first 5 days of triadimefon treatment. Triadimefon decreased root hydraulic conductivity and inhibited the activity of the plasma membrane ATPase. In addition, triadimefon-treated roots drastically increased the ratios between free sterols and sterol esters and decreased the ratios between sterol esters and acylated sterol glycosides.     

Effects of preconditioning on electrolyte leakasge and lipid composition in black spruce (Picea mariand) stressed with polyethylene giycol

Black spruce ( Picea mariana Mill. B. S. P.) rooted cuttings were grown in solution culture and preconditioned by osmotically stressing plants with polyethylene glycol. After relief from preconditioning stress, water relations, membrane leakiness, and the composition of lipids and fatty acids were compared in preconditioned and control, unconditioned plants. Both groups of plants were subsequently subjected to a severe osmotic stress with polyethylene glycol and examined again. Osmotic stress decreased shoot water potentials and increased the leakage of electrolytes from shoots of stressed, compared with unstressed, plants. However, both unstressed and stressed preconditioned plants leaked less electrolytes compared with unconditioned plants. Changes in sterol, phospholipid and glycolipid composition were observed in preconditioned unstressed and stressed plants. Sterol and phospholipid levels de clined as a result of stress, and preconditioning resulted in a decline in sterol: phospholipid ratios in plants.