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Sugar analysis
The quantitative determination of soluble and non-soluble sugars was done on extracts from leaf blades. Sugars were extracted using three successive incubations in ethanol (80% v/v) at 85_C. Extraction was assumed to be totally effective after three incubations because a fourth ethanol extraction of the pellets did not contain detectable amounts of glucose. The fraction of soluble sugars was determined spectrophotometrically using an enzyme-linked assay, as described in Sokolov et al. (1998). All enzymes used in this assay were purchased from Roche Diagnostics (Hermans et al., 2004).
Root gravity response assay
Seeds were surface sterilized, stratified in the dark at 4°C for 72h, and germinated vertically on 0.5× strength MS (0.5× MS) salt-containing agar with constant illumination at 22 °C for 5 d. Seedlings were then transferred to agar containing 0.5× MS salt with or without 10–7 mol l–11-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D), and grown horizontally (at 90° rotation) under the same growth conditions. The root gravity response was scored by measuring the angles formed 24h after the gravity change with use of ImageJ (NIH) (Yu and
Wen, 2013).
Auxin transport assay
Arabidopsis seedlings were grown for 6 d and a 10mm segment to the root tip was excised. [3H]-labelled IAA was applied to the cut and the root segments were incubated in the dark for 6h. After incubation, a 5mm segment to the tip was excised and washed with 0.5× MS salt. The washed root tips (15 tips for each measurement) were incubated in scintillation liquid and scintillation counting was carried out (PerkinElmer 1450 Microbeta scintillation counter) for [3H]IAA measurement(Yu and Wen, 2013).
β-Glucuronidase (GUS) staining
Histochemical staining for GUS activity in transgenic plants was performed as described previously (s="nolink">Jefferson et al., 1987). Seedlings were grown under light on MS salt-containing agar for 4 d and transferred to MS salt-containing agar with or without auxin (100nM NAA) in an air-tight chamber with or without ethylene treatment for 2 d. The seedlings were harvested, immersed in the reaction solution (1mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid, 100mM sodium phosphate, 0.1mM EDTA, 0.5mM ferricyanide, 0.5mM ferrocyanide, and 0.1% Triton X-100, pH 7.0) and incubated at 37 °C for 16h(Yu and
Wen, 2013).
Histochemical staining
β-glucuronidase activity in transgenic marker lines was visualized by incubating tissues for 1 h (35S::GUSlines), 2 h (ARR5::GUS lines) or 18 h (DR5::GUS and CYCB1::GUS lines) in darkness at 37 °C in a buffer containing 0.5 mg mL−1 X-gluc (5-bromo-4-chloro-3-indolyl-beta-d-glucuronic acid, cyclohexylammonium salt) dissolved in N-dimethyl-formamide, 0.2 M NaH2PO4, 0.2 M Na2HPO4, 0.5 M EDTA (pH = 8) and 100% Triton X-100.
Mineral analysis
Leaves and roots of plants growing in hydroponic solution were harvested at day 1, 7 and 14 after the CuSO4addition in the nutrient solution. Roots were rinsed with deionized water for 1 min. Fresh material was then dried at 60 °C during 48 h before being crushed into a powder. Dried material was digested with 6 M nitric acid for 2 h at 60 °C and 6 h at 120 °C. Digested samples were assayed by ICP-MS (Purdue Ionomics Information Management Systems, IN, USA). Three protocols of root washing were tested to study their impact on the Cu content in plants treated with 2.5 μM Cu2+ for 7 days (Table IS). The Cu contents after root washing with Pb(NO3)2, which is a way to remove extra-cellular copper [12], [63] and [69], were statistically similar to those washed with water.
Ethylene measurements
Thirty seeds were sown and allowed to grow for 9 days in vertical plates containing MS/2 medium supplemented with 0, 25 or 50 μM CuSO4. Before subjecting plantlets to ethylene measurements, the plates were kept in the growth chamber used for measurements for 1 day to acclimatize the plants (150 μmol m−2 s−1 continuous light and constant temperature of 21 °C). For ethylene measurements, the bottom part of the dish with the agar was covered with a glass plate with an inlet and outlet for gas flow. The system was tided together with two metal pieces and was connected to a sensitive laser-based ethylene detector in combination with a gas flow-through system developed at the Department of Molecular and Laser Physics, University of Nijmegen, the Netherlands. The cuvettes, fitted with inlet and outlet ports, were alternatively flushed with compressed air as carrier gas at a flow rate of 3 l h−1 for 12 h. The flow from each cuvette was directed into a photoacoustic cell where ethylene was quantified. A detailed description of the system has been given previously [10]. For each Cu2+ concentration tested (25 and 50 μM), measurements were done in parallel with control plates and were repeated at least 3 times. The ethylene levels from a cuvette containing an agar plate without seedlings were also measured and subtracted from the emission rates obtained.
Determination of lateral root formation
Length and number of lateral roots developed on the plates were recorded following 6 days of seedling growth. Experiments were repeated four times; each treatment within each experiment included three replicates, four germinated seedlings per replicate (n = 48). For the DR5::GUS line, grown on plates for 144 h, seedlings were harvested for GUS staining (Jefferson et al. 1987). Following GUS staining lateral root primordia were counted and lateral root primordial were photographed with a Leica DMLB light microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a Nikon DS-Fi1 camera. Primordium developmental stage was categorized according to Malamy and Benfey’s (1997) definition of lateral root primordium development. Experiments were repeated twice; each treatment within each experiment included two replicates, four germinated seedlings per replicate (n = 16). Means of replicates were subjected to statistical analysis of two-way ANOVA by multiple-range Tukey–Kramer test (P ≤ 0.05) using the JMP statistical package.
For determination of lateral root density under concentrations of 1 × 10−6 M GR24 and lower, the primary root length of the seedlings grown for 12 days was measured, and the lateral root density was determined under a Leica RZ 75 stereomicroscope. The experiment was repeated three times (n > 20 seedlings). Means of replicates were subjected to statistical analysis according to the Kruskal–Wallis test and box-plot analysis.
Intracellular Mg2+ measurements
The Mg-sensitive fluorescent dye Magnesium Green™-AM (Molecular Probes) {glycine, N-[2-(carboxymethoxy)-4-[[(2′,7′-dichloro-3′,6′-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9′-[9H]xanthen]-5-yl)carbonyl]amino]phenyl]-N-carboxymethyl-, acetoxymethyl ester} was dissolved in dimethylsulfoxide (DMSO) (Sigma) and diluted with a loading solution (0.2 mM CaCl2 and 50 mM mannitol, pH 4.2) to a final concentration of 1.5 µM. Preliminary experiments indicated that the 1.5 µM concentration was sufficient for measuring intracellular Mg2+ while being sufficiently low to avoid damage to root cells induced by laser scanning (data not shown). The final concentration of DMSO in the loading solution was 1% (v/v). Preliminary experiments with different recovery times and temperatures revealed that the minimum 10 h recovery period at 25°C was essential for Magnesium Green™-AM loading into the root tissue (data not shown). Therefore, the dye was loaded into the intact Arabidopsis roots for 1 h on ice (Guo et al. 2009), followed by recovery in BSM for 12 h at 25°C (Bose et al., 2013).
细胞生物反应器-细胞计数仪-细胞培养计数-实验室仪器采购-苏州阿尔法生物实验器材 |
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