Mycorrhizae and Rhizobacteria on Precambrian Rocky Gold Mine Tailings: II. Mine-Adapted Symbionts Alleviate Soil Element Imbalance for a Better Nutritional Status of White Spruce Seedlings

In the context of a phytorestoration project, the purpose of this study was to assess the respective contribution to the nutritional status of Picea glauca seedlings of ectomycorrhizae and rhizobacteria native or not to the Sigma-Lamaque gold mine wastes in northern Quebec, Canada. In a glasshouse experiment, inoculated plants were grown for 32 weeks on coarse waste rocks or fine tailings obtained from the mining site. The survival, health, growth, and nutritional status of plants were better on coarse waste rocks than on fine tailings. Fe and Ca were especially found at high levels in plant tissues but at much lower concentrations on waste rocks. Interestingly, inoculation of microsymbionts had only minimal effects on N, P, K, and Mg plant status that were indeed close or within the concentration range encountered in healthy seedlings. However, both fungal and bacterial treatments improved Fe and Ca concentrations in plant tissues. Fe concentration in the foliage of plants inoculated with the fungi Tricholoma scalpturatum Tri. scalp. MBN0213 GenBank #KC840613 and Cadophora finlandia Cad. fin. MBN0213 GenBank #KC840625 was reduced by >50%. Both fungi were isolated from the mining site. The rhizobacteria, Azotobacter chroococcum, also improved plant Fe level in some cases. Regarding Ca nutritional status, the native bacterial strain Pseudomonas putida MBN0213 GenBank #AY391278 was the only symbiont that reduced foliar content by up to 23%. Ca concentration was negatively correlated with the fungal mycorrhization rate of seedling roots. This relation was especially strong (r = -0.66, p-value ≤ 0.0001) in the case of C. finlandia. Also, a similar relationship existed with root Fe concentration (r = -0.44, p-value ≤ 0.0001). In fact, results showed that seedling performance was more correlated with elevated Ca and Fe concentration in planta than with nutrient deficiency. Also, native microsymbionts were capable of regulating seedling nutrition in the poor substrate of the Sigma-Lamaque gold mine tailings

Aquaporins Respond to Chilling in the Phloem by Altering Protein and mRNA Expression

Previous experiments using heat exchangers (liquid cooled blocks) to chill a portion of plant stem have shown a transient stoppage in phloem translocation and an increase in measured phloem pressure. Although a chilled-induced stoppage of phloem transport has been known for over 100 years, the mechanism of this phenomenon is still poorly understood. Recently, work has highlighted that aquaporins occur within the plasma membrane of the sieve tubes along the entire source-to-sink pathway, and that isoforms of these water channel proteins may change dynamically. Aquaporins show regulatory roles in controlling tissue and cellular water status in response to environmental hardships. Thus, we tested if protein localization and mRNA transcript abundance changes occur in response to chilling in balsam poplar (Populus balsamifera) using immunohistochemistry and qrtPCR. The results of the immunolocalization experiments show that the labeling intensity of the sieve elements treated for only 2 min of chill time significantly increased for PIP2. After 10 min of chilling, this signal declined significantly to lower than that of the pre-chilled sieve elements. Overall, the abundance of mRNA transcript increased for the tested PIP2s following cold application. We discuss the implication that aquaporins are responsible for the alleviation of sieve tube pressure and the resumption of flow following a cold-induced blockage event

The Role of Water Channel Proteins in Facilitating Recovery of Leaf Hydraulic Conductance from Water Stress in <i>Populus trichocarpa</i>

<div><p>Gas exchange is constrained by the whole-plant hydraulic conductance (<i>K</i>_plant). Leaves account for an important fraction of <i>K</i>_plant and may therefore represent a major determinant of plant productivity. Leaf hydraulic conductance (<i>K</i>_leaf) decreases with increasing water stress, which is due to xylem embolism in leaf veins and/or the properties of the extra-xylary pathway. Water flow through living tissues is facilitated and regulated by water channel proteins called aquaporins (AQPs). Here we assessed changes in the hydraulic conductance of <i>Populus trichocarpa</i> leaves during a dehydration-rewatering episode. While leaves were highly sensitive to drought, <i>K</i>_leaf recovered only 2 hours after plants were rewatered. Recovery of <i>K</i>_leaf was absent when excised leaves were bench-dried and subsequently xylem-perfused with a solution containing AQP inhibitors. We examined the expression patterns of 12 highly expressed AQP genes during a dehydration-rehydration episode to identify isoforms that may be involved in leaf hydraulic adjustments. Among the AQPs tested, several genes encoding tonoplast intrinsic proteins (TIPs) showed large increases in expression in rehydrated leaves, suggesting that TIPs contribute to reversing drought-induced reductions in <i>K</i>_leaf. TIPs were localized in xylem parenchyma, consistent with a role in facilitating water exchange between xylem vessels and adjacent living cells. Dye uptake experiments suggested that reversible embolism formation in minor leaf veins contributed to the observed changes in <i>K</i>_leaf.</p></div

Typical images of transpiring <i>P. trichocarpa</i> leaves that were allowed to take up safranin solution.

<p>(A) A control leaf was excised from a well-watered plant, and the petiole was immersed for 2 h in safranin solution. Transpiration during dye uptake was promoted by placing the leaf near a fan at ∼1,000 µmol m⁻² s⁻¹ photosynthetic active radiation. Most leaf veins were stained indicating minimal xylem embolism. (B) Dye uptake in a bench-dried leaf that was subsequently perfused with safranin solution for 2 h. Minor veins exhibited incomplete staining indicating the presence of embolized xylem conduits in minor veins. (C) Dye uptake of a bench-dried leaf subsequently perfused with safranin + HgCl₂ solution for 2 h. Mercury is an aquaporin inhibitor. Staining remained even more incomplete than in (B).</p

Relative expression of 12 aquaporin genes in response to dehydration (y-axis) and dehydration + perfusion with abscisic acid (x-axis).

<p>Detached leaves were either dehydrated on the bench top for 1 h or dehydrated for 1 h and subsequently perfused for 1 h with 50 µM abscisic solution (ABA). Data from fully hydrated detached leaves (perfused for 3 h with 20 mM KCl+1 mM CaCl₂ solution) were used as the control group, and their expression refers to a value of 1. Pearson’s r = 0.725; <i>P</i>≤0.01. Data are means ± SE of three biological replicates.</p

Immunolocalization of AQP proteins in leaves of <i>P. trichocarpa</i> saplings.

<p>Confocal laser scanning micrographs showing the localization of PIP1, PIP2, TIP2 proteins in minor veins of leaf transverse sections (A, B, C respectively). Controls with no primary antibody indicate minimal background fluorescence (D, E, F respectively). Images were taken at an identical setting and were color-coded with an intensity look-up-table (LUT; displayed in A), in which black was used to encode background, and blue, green, yellow, red and white to encode increasing signal intensities. Ph, phloem; PP, palisade parenchyma; Xyl, xylem. Scale bars = 20 µm.</p

Response of leaf hydraulic conductance (A) and stomatal aperture (B) to different perfusion solutions.

<p>Control conditions refer to the <i>K</i>_leaf that was measured after leaves were xylem perfused with filtered (0.2 µm) 20 mM KCl+1 mM CaCl₂ solution (subsequently referred to as ‘artificial xylem sap’, AXS) for 2 h. <i>K</i>_leaf was also measured on leaves that were bench-dried for 1 h (Dehydrated) and on leaves that were bench-dried for 1 h and subsequently perfused for 2 h with AXS (RW AXS), AXS+0.2 mM HgCl₂ (RW HgCl₂), AXS+50 mM H₂O₂ (RW H₂O₂) or AXS+50 µM ABA (RW ABA). Values are means ± SE (n = 6). Different letters denote statistically significant differences by one-way ANOVA with Tukey’s test.</p

Relative expression of aquaporin genes in leaves of plants exposed to a drying-rewatering cycle.

<p>Gene expression was measured in leaves of well-watered control plants (C), drought-stressed plants (D), and 3 h after drought-stressed plants were rewatered (RW). The geometric mean of the expression levels of four reference genes (<i>ACT2</i>, <i>CYC063</i>, <i>TIP41-like</i>, <i>UBQ7</i>) was used to normalize the results. Asterisks denote significant differences in expression level compared to control levels (one-way ANOVA, followed by Bonferroni’s post test, *<i>P</i>≤0.05; **<i>P</i>≤0.01***<i>P</i>≤0.001). Data are means ± SE of three biological replicates.</p