Paclobutrazol-induced Drought Tolerance in Containerized English and Evergreen Oak

Plant Material

Two plant species were selected for experimental purposes: evergreen oak (Quercus ilex) and English oak (Q. robur). Both species are commonly planted into UK urban environments and provide forms with contrasting drought capabilities. Four-year-old cell-grown stock approximately 90 cm (36 in) high ± 8 cm (3.2 in) were obtained from a commercial supplier (Alba Trees, Lower Winton, East Lothian, UK). Six months before experiments, trees were potted into 4.5 L (1.17 gal) plastic pots filled with soil (loamy texture, 24% clay, 45% silt, 31% sand, 3.1% organic carbon, pH 6.2) supplemented with the controlled-release nitrogen-based N:P:K (29: 7:9) fertilizer, Bartlett BOOST (The Doggett Corp., Lebanon, NJ) at a rate of 1 g/kg soil. After potting, trees remained outdoors subject to natural environmental conditions and watered as required. In early April 2005, trees were moved to a polythene tunnel to protect against possible spring frosts. As soon as the initial symptoms of bud burst were observed, i.e., leaf emergence (late April), trees were placed under glass-house conditions (22°C ± 2°C [71.6°F ± 35.6°F]) supplemented with 400 W high-pressure sodium lamps providing a photoperiod of 16hr light/8hr dark and minimum 250 μmol/m⁻²/s⁻¹ photosynthetically active radiation (PAR) at the tree crown until PBZ treatments began.

Paclobutrazol Application

Paclobutrazol treatments were applied when both tree species were in full leaf (2 May 2005). Foliar sprays of PBZ were applied until runoff using a handheld sprayer. As a root drench, PBZ was applied to the substrate surface until fully saturated, i.e., when liquid was seen emerging from the base of the pots. Drenching with distilled water served as the control. Concentrations of PBZ applied were: 1) control (no application of PBZ); 2) 2 mL (0.06 fl oz) PBZ per liter water applied as spray until runoff; 3) 4 mL (0.12 fl oz) PBZ per liter water applied as spray until runoff; 4) 5 mL (0.15 fl oz) PBZ per 250 mL (7.5 fl oz) water applied as root drench to achieve a concentration of 0.25 g (0.01 oz) PBZ per 2.5 cm (1 in) girth, approximately 50 mL (1.5 fl oz) per pot; and 5) 10 mL (0.3 fl oz) PBZ per 250 mL (7.5 fl oz) water applied as root drench to achieve a concentration of 0.50 g (0.02 oz) PBZ per 2.5 cm (1 in) girth, approximately 50 mL (1.5 fl oz) per pot.

Plants were left for 2 weeks under glasshouse conditions to permit absorption and uptake of PBZ. During this period, plants were watered as required, generally every 3 days. At week 2, after PBZ treatment (16 May 2005), measurements of plant physiology were made.

Drought Treatment and Experimental Design

Plants were subjected to drought by the cessation of watering for 3 weeks under glasshouse conditions. At week 3 (6 June 2005), a number of physiological measurements were made on leaf tissue as measures of tree vitality. Immediately after measurements, trees were watered as required and placed outdoors subject to natural weather conditions and recovery rates measured over the next 9 weeks at three weekly intervals (27 June, 18 July, 8 August). The experimental design used was a completely randomized block design in which pots were rerandomized on a weekly basis. Ten trees per treatment were used at 0.75 m (0.83 ft) spacings to prevent competition for light.

Tree Vitality Measurements

All tree vitality measurements were taken on leaf material present on the plant at the initiation of the experiment (existing leaves). During recovery from drought, new leaf formation was observed at approximately weeks 6 to 7 on both control and PBZ-treated surviving plants. No tree vitality measurements were taken on any newly formed leaf tissue, i.e., new leaves not present at the time of PBZ application. At each sampling date, five leaves per tree were used for chlorophyll fluorescence and chlorophyll content measurements randomly selected throughout the crown. Leaves were then tagged to ensure the same leaf was measured throughout the study.

Chlorophyll Fluorescence

Chlorophyll fluorescence was used as a measure of damage to the leaf photosynthetic system. Leaves were adapted to darkness for 30 min by attaching light exclusion clips to the leaf surface and chlorophyll fluorescence was measured using a HandyPEA portable fluorescence spectrometer (Hansatech Instruments Ltd., King’s Lynn, UK). Measurements were recorded up to 1 sec with a data acquisition rate of 10 μs for the first 2 ms and 1 ms thereafter. The fluorescence responses were induced by a red (peak at 650 nm) light of 1500 μmol/m⁻²/s⁻¹ PAR intensity provided by an array of six light-emitting diodes. A performance index (PI) based on an equation that combines the relationship of calculated relative number of reaction centers (RC) per energy absorbed (ABS) and then multiplied by two expressions describing the yields of light trapping (φpo) and subsequent electron transport (Ψ₀), i.e., RC/ABS × φpo/(1 − φpo) × Ψ₀/(1 − Ψ₀; Clark et al. 1998, 2000; Percival and Fraser 2001) was used to quantify any effects on leaf tissue. PI values have been shown to be a highly sensitive measure of leaf photosynthetic activities and provide an indirect measure of plant vitality. PI values were automatically calculated by the HandyPEA.

Leaf Chlorophyll Content

A Minolta chlorophyll meter SPAD-502 (Spectrum Laboratories, Inc., Plainfield, IL) was used. Chlorophyll was measured at the midpoint of the leaf next to the main leaf vein. Calibration was obtained by measurement of absorbance at 663 and 645 nm in a spectrophotometer (PU8800 Pye Unicam, Cambridge, UK) after extraction with 80% v/v aqueous acetone (regression equation = 6.01 + 0.055x; r² adjusted = 0.90, P < 0.01) (Lichtenthaler and Wellburn 1983).

Photosynthetic CO₂ Fixation

Light-induced CO₂ fixation (Pn) was measured in two pre-darkened (20 min) fully expanded leaves per tree near the top of the canopy (generally approximately the fourth leaf from the apex) using an Infra Red Gas Analyser (LCA-2 ADC; ADC Ltd, Hoddesdon, Herts, UK). The irradiance on the leaves was 700 to 800 μmol/m⁻2 PAR saturating with respect to Pn; the velocity of the airflow was 1 mL/s⁻¹/cm⁻² leaf area. Calculation of the photosynthetic rates was carried out according to Von Caemmerer and Farquhar (1981). All photosynthetic measurements were taken in the early morning between 8:00 a.m. and 10:00 a.m. on clear or partly cloudy days.

Leaf Necrosis

Leaf necrosis was assessed visually. Each tree was rated on a 0 to 5 rating scale using a visual indexing technique and ratings on the scale: 0 = no necrosis observed; 1 = less than 5% of leaves affected and no aesthetic impact; 2 = 5% to 20% of leaves affected with some yellowing but little or no defoliation; 3 = 21% to 50% of leaves affected, significant defoliation and/or leaf yellowing; 4 = 51% to 80% of leaves affected, severe foliar discoloration; and 5 = 81% to 100% of foliage affected with 90% to 100% defoliation.

Leaf Electrolyte Leakage

Quantitative drought damage to leaf tissue was assessed by measuring electrolyte leakage of entire leaves (two leaves per tree) at each sampling date. Excised leaves were placed in 50 mL (1.5 fl oz) universal bottles containing 30 mL (0.9 fl oz) distilled water and gently shaken by hand. Samples were stored at 22°C (71.6°F) for 24 hr in darkness before conductivity measurements using a Jenway conductivity probe and M4070 m (BDH, Leicestershire, Loughborough, UK). Total solute leakage was obtained by autoclaving for 1 hr at 121°C (249.8°F) and 0.103 MPa. Results are presented as percent solute leakage after 24 hr (McKay 1992)

Carotenoids

Carotenoids (lutein, β-carotene, neoxanthin, α-carotene, zeaxanthin, antheraxanthin, violaxanthin) were analyzed using a high-performance liquid chromatography (HPLC) system (Schindler and Lichtenthaler 1994). Leaf samples were excised from trees and immediately placed in liquid nitrogen. Fifty leaf samples per treatment, i.e., five leaves per tree, were taken. Pigment analysis was performed the next day. Pigments were extracted from leaves with 100% aqueous acetone (quartz sand mortar) and separated and quantitatively determined by reverse-phase HPLC applying a Nucleosil C₁₈ column (particle size 4 μm). The solvent systems for the combined isocratic and gradient separation and quantification were acetonitrile–methanol–0.2 M tris/HCL buffer and methanol–hexane (Lichtenthaler et al. 1992).

Superoxide Dismutase Activity

Superoxide dismutase (SOD) activity was based on the method described by Kraus and Fletcher (1994). In summary, extracts were prepared at 4°C (39.2°F) by homogenizing 0.2 g (0.07 oz) of leaf tissue (generally three leaves per plant) in 4 mL (0.12 fl oz) of 0.1 M Na₂HPO₄/NaH₂PO₄ buffer (pH 7) with a mortar and pestle and centrifuged at 16,000 × g for 10 min. The supernatant was filtered through Watman paper (No. 1) (Sigma-Aldrich, Poole, UK) and 1 mL (0.03 fl oz) was applied to a Pharmacia PD-10 chromatography column (Waters HPLC Solutions, Centennial Park, Elstree, Hertfordshire, UK) containing Sephadex G-250 (Waters HPLC Solutions) equilibrated with 50 mM Na₂CO₃ buffer (pH 10.2) to remove low-molecular-weight components and exchange the buffer. The assay was performed using 0.1 mM diethylenetriaminepentaacetic acid in the reaction mixture and the subsequent increase in absorbance at 550 nm followed on a spectrophotometer (PU8800 Pye Unicam) equipped with a kinetics module for 6 min at 24°C (75.2°F).

Catalase Activity

A total of 0.3 g (0.11 oz) of foliar tissue (generally five leaves per plant) were homogenized in 5 mL (0.15 fl oz) of 0.2 M Tris-Cl⁻ (buffer pH 7.8) containing 0.13 mM EDTA and 80 μM soluble polyvinylpyrrolidone with a Polytron homogenizer (Glen Mills, Clifton, NJ) for 30 sec on ice and centrifuged at 3000 × g for 10 min. Catalase activity was determined by following the consumption of hydrogen peroxide (extinction coefficient 39.4 mm⁻¹/cm⁻¹) at 240 nm for 2 min. The reaction mixture contained 2 mL of 100 mM Na₂HPO₄/NaH₂PO₄ buffer (pH 6.5) and 50 μL of plant extract and the reaction was initiated by adding 10 μL of 30% (w/v) hydrogen peroxide.

Proline

Leaf samples (1 g [0.035 oz]) were extracted with 3% sulphosalicylic acid. Extracts (2 mL [0.06 fl oz]) were held for 1 hr in boiling water by adding 2 mL (0.06 fl oz) ninhydrin and 2 mL (0.06 fl oz) glacial acetic acid after which cold toluene (4 mL [0.12 fl oz]) was added. Proline content was measured by a spectrophotometer (Shimadzu UV 1601; Shimadzu UK, Milton Keynes, Buckinghamshire, England) at 520 nm and calculated as μg/g dry leaf weight against standard proline.

Plant Height and Leaf Mean Leaf Area

At the cessation of the experiment, trees were destructively harvested and height recorded by measuring the distance from the tip of the leading apical shoot to the soil surface. Mean leaf size was calculated by quantifying total leaf area per tree using a Delta-T leaf area meter (Delta-T Devices, Cambridge, UK) and dividing by the total number of leaves per tree.

Statistical Analysis

Data for each species was analyzed independently by one-way analysis of variance using the Genstat V program (Committee of the Statistics Department, Rothamsted Experimental Station, Harpenden, Hertfordshire, UK) for Windows. Before the analysis, data were examined for normality and homogeneity of variance (Levene 1960) and data were transformed [log (y + 0.5)] when necessary. When significant differences occurred, the means were separated by least significant difference test (P < 0.05). Survival recorded as percentage and visual leaf necrosis were analyzed after appropriate statistical transformation (arcsin[x^0.5]). Because measurements over time were obtained from the same plant, the relationship between chlorophyll fluorescence and SPAD readings over time after PBZ treatments was quantified using repeated measures analysis (quadratic regression).