Tree Physiology 34, 343–354 doi:10.1093/treephys/tpu025

Research paper

Sexually different physiological responses of Populus cathayana to nitrogen and phosphorus deficiencies Sheng Zhang1, Hao Jiang1, Hongxia Zhao1, Helena Korpelainen2 and Chunyang Li1,3 Laboratory of Mountain Surface Processes and Ecological Regulation, Institute of Mountain Hazards and Environment, Chinese Academy of Sciences, Chengdu 610041, China; 2Department of Agricultural Sciences, University of Helsinki, PO Box 27, Helsinki FI-00014, Finland; 3Corresponding author ([email protected])

Received December 8, 2013; accepted March 3, 2014; published online April 16, 2014; handling Editor Jörg-Peter Schnitzler

Previous studies have shown that there are significant sexual differences in the morphological and physiological responses of Populus cathayana Rehder under stressful conditions. However, little is known about sex-specific differences in responses to nutrient deficiencies. In this study, the effects of nitrogen (N) and phosphorus (P) deficiencies on the morphological, physiological and chloroplast ultrastructural traits of P. cathayana males and females were investigated. The results showed that N and P deficiencies significantly decreased plant growth, foliar N and P contents, chlorophyll content, photosynthesis, and instantaneous photosynthetic N- and P-use efficiencies (PNUE and PPUE) in both sexes. Males had higher photosynthesis, higher PNUE and PPUE rates, and a lower accumulation of plastoglobules in chloroplasts than did females when exposed to N- and P-deficiency conditions. Nitrogen-deficient males had higher glutamate dehydrogenase and peroxidase activities, and a more intact chloroplast ultrastructure, but less starch accumulation than did N-deficient females. Phosphorus-deficient males had higher nitrate reductase, glutamine synthetase and acid phosphatase activities, but a lower foliar N : P ratio and less PSII damage than did P-deficient females. These results suggest that N and P deficiencies cause greater negative effects on females than on males, and that the different sexes of P. cathayana may employ different strategies to cope with N and P deficiencies. Keywords: dioecy, nutrient deficiency, photosynthesis, PNUE, PPUE.

Introduction Dioecy is reported in 7.5% of flowering plant genera and ~6% of angiosperm species (Renner and Ricklefs 1995). Sexspecific functional differences have evolved in dioecious plants (Dawson and Bliss 1989). Generally, sexual differences have been explained as being consequences of different reproductive functions (Obeso 2002, Sanchez-Vilas and Retuerto 2012). However, sexual differences have been observed also before reproductive maturity, and these differences are typically greater under stressful conditions (Xu et al. 2008, Zhang et al. 2011, Montesinos et al. 2012). Therefore, it is essential to investigate sex-related stress reactions at non-flowering life stages (Dudley and Galen 2007). Many previous studies have

shown that young male and female plants possess sexually different responses to drought, light and temperature (Dawson et al. 2004, Li et al. 2007, Xu et al. 2008, Zhao et al. 2009, Zhang et al. 2011). However, studies on sex-specific responses to nutrient deficiencies are scarce. With increasing anthropogenic activity and soil erosion, plant growth limitation caused by nitrogen (N) and phosphorus (P) deficiencies has increased, especially in alpine forests (Körner 1999). Nitrogen is often limited in young soils of the temperate zone, while P causes a primary limitation in acid, calcareous/alkaline and highly weathered soils. Previous studies have shown that N deficiency decreases plant growth and photosynthesis, and increases the root-to-shoot ratio and

© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

Downloaded from http://treephys.oxfordjournals.org/ at University of Utah on December 2, 2014

1Key

344  Zhang et al.

Materials and methods Plant materials and experimental design The experiment was a completely randomized design with eight factorial combinations of two levels of sex (male and female), N (deficiency and control) and P (deficiency and control). The mother trees were collected from Qinghai Province, China (LeDu, 36°31′N, 102°28′E). The F1 individuals used in the experiments were produced by hybridizing P. cathayana male and female branches in the laboratory using a water culture method. Before treatments, 160 collected cuttings (10 cm long) were planted in a liquid medium, which was a modified Hoagland solution containing 1.25 mM KNO3, 1.25 mM Ca(NO3)2 ⋅ 4H2O, 0.5 mM MgSO4 ⋅ 7H2O, 0.25 mM KH2(PO4), 11.6 µM H3BO3, 4.6 µM MnCl2 ⋅ 4H2O, 0.19 µM ZnSO4 ⋅ 7H2O, Tree Physiology Volume 34, 2014

0.12 µM Na2MoO4 ⋅ 2H2O, 0.08 µM CuSO4 ⋅ 5H2O and 10 µM Fe supplied as Fe(III)-EDTA (Fodor et al. 2005). Every day, pH was adjusted to 6.5 ± 0.2 with 0.1 M HCl and 0.1 M NaOH. When the acclimated sprouts of the cuttings had developed ~15 nodes and were ~30 cm in height (60 days after transplanting), 40 male and 40 female cuttings with an approximately equal root length and shoot height were selected for the nutrient-deficiency experiments. There were four treatments for each sex, namely control, N deficiency, P deficiency and combined N and P deficiency. For the N-deficiency treatment, NO3− was replaced by Cl−, and for the P-deficiency treatment, (PO4 )3− was replaced by (SO4 )2− . Each plant was grown in a separate plastic pot containing 5 l of nutrient solution. The nutrition solution was changed every 3 days. The cuttings were grown in a naturally lit greenhouse under ambient conditions with a daytime temperature of 19–28 °C, a night-time temperature of 12–18 °C and a relative humidity of 40–85% during the treatment period at the Chengdu Institute of Biology, the Chinese Academy of Sciences. The treatments lasted for 2  months. At the end of the experiment, the fourth and fifth fully expanded leaves (counted from the top of the plants) were collected to be used for the analyses.

Growth measurements Height growth measurements were based on the length of the stem from the collar to the apex. The total leaf area was determined by the Portable Laser Area Meter (CI-203; CID, Inc., Camas, WA, USA). Each cutting was harvested at the end of the experiment and partitioned into leaves, stems and roots. All samples were dried separately at 80 °C to constant weight and weighed. The dried leaves (fourth to sixth leaves) were ground and used for the element analysis. The leaf mass per area (LMA) was calculated as the leaf dry weight divided by the leaf area.

Chlorophyll fluorescence measurements The fourth fully expanded leaves were selected for chlorophyll fluorescence measurements using the PAM chlorophyll fluorometer (PAM 2100, Walz, Effeltrich, Germany). First, the leaf samples were placed in the dark for 30 min using an aluminum foil cover, and minimum fluorescence (Fo) and maximum fluorescence (Fm) were measured. Then, the leaves were illuminated with actinic light at an intensity of 250 µmol m−2 s−1, which was the light intensity inside the greenhouse at the time of the measurements. The actinic light was removed, and minimum fluorescence (Fo′) and maximum fluorescence (Fm′) were measured by illuminating the leaves with far-red light for 3 s. A saturating white light pulse of 8000 µmol m−2 s−1 was applied for 0.8 s, and Fm and Fm′ were measured. Measurements were carried out from 08:00 to 09:30. Chlorophyll fluorescence kinetic parameters were measured and calculated as described by Vankooten and Snel (1990).

Downloaded from http://treephys.oxfordjournals.org/ at University of Utah on December 2, 2014

starch content (Boyce et al. 2006, Boussadia et al. 2010, Kant et al. 2011). Nitrogen deficiency also affects plants’ metabolic processes and gene expression. Glutamate dehydrogenase (GDH), nitrate reductase (NR) and glutamine synthetase (GS) participate in N metabolism. These enzymes determine N-use efficiency in various plant species and their activities can change significantly depending on soil N status (Duff et al. 1991, Paczek et al. 2002). Phosphorus deficiency decreases leaf area, root growth and photosynthetic rate (Turnbull et al. 2007, Warren 2011). Phosphorus deficiency also causes leaf senescence and changes in metabolic processes, for instance, through reductions in phosphate and phosphorylated intermediates (Huang et al. 2008, Warren 2011). Among these effects, a decrease in photosynthesis is the most common and important effect on plants caused by N and P deficiencies. The rate of photosynthesis at light saturation accomplished per unit N or P in a leaf, called instantaneous photosynthetic N- or P-use efficiency (PNUE or PPUE), has been considered to be an important functional trait of leaf physiology (Bown et al. 2007, Hidaka and Kitayama 2009). Moreover, N and P deficiencies cause the production of reactive oxygen species (ROS) and consequent antioxidant responses in plants (Shin et al. 2005, Tewari et al. 2007). Populus cathayana Rehder, a dioecious, fast-growing tree species, was employed as a model species to assess sex-­ specific responses to nutrient deficiencies. Previous studies have indicated that male and female poplars have different physiological responses to N deposition, and males show a higher photosynthetic ability than do females (Chen et al. 2011, Zhao et al. 2011). In this study, it is hypothesized that there are sexually different responses to N and P deficiencies, and that P. cathayana males have a higher tolerance to nutrient deficiencies than do females. We will answer the following questions: are there differences in nutrient-deficiency tolerance traits between the sexes, and do males have a higher photosynthetic ability when compared with females under N and P deficiencies?

Sexual differences of poplar under nutrient deficiency  345

Gas exchange measurements

Chlorophyll pigment measurements Three cuttings (the fourth and fifth leaves), randomly selected from each sex and treatment, were used for the measurements of pigments and other parameters. The pigment extraction was conducted according to Lichtenthaler (1987). Leaf discs (1.0 cm2) were cut from each leaf immediately after gas exchange measurements and extracted in 80% chilled acetone (v/v) after weighing. The absorbance of extracts was measured using a spectrophotometer (Unicam UV-330; Unicam, Cambridge, UK) at 470, 646 and 663 nm. Chlorophyll concentrations and carotenoid contents (Caro) were calculated from equations derived from Porra et al. (1989). The total chlorophyll content (Tchl) was the sum of chlorophyll a (Chl a) and chlorophyll b (Chl b).

Foliar N and P content measurements Dried samples (0.2 g) were used for N and P content measurements. Nitrogen was determined by the semi-micro Kjeldahl method (Mitchell 1998) and P by induced plasma emission spectroscopy (Hötscher and Hay 1997). Photosynthetic N-use efficiency is expressed as the rate of photosynthesis at light saturation divided by the foliar N content per area. Photosynthetic P-use efficiency is expressed as photosynthesis at light saturation divided by the foliar P content per area. In this study, the net photosynthetic rate (Pn) was measured under the light saturation condition, and it was then used to calculate PNUE and PPUE.

Hydrogen peroxide content measurements The hydrogen peroxide (H2O2) content was determined as a H2O2–titanium complex resulting from the reaction of tissueH2O2 with titanium tetrachloride at 410 nm using a spectrophotometer (Brennan and Frenkel 1977). Absorbance values were calibrated with a standard curve generated using known concentrations of H2O2. Control or blank H2O2 measurements

Ascorbic acid and reduced glutathione content measurements Reduced ASA was determined as described by Lei et al. (2006). Fresh leaves (0.2 g) were homogenized in 5 ml of cold 5% (w/v) m-phosphoric acid and centrifuged at 10,000 g for 15 min. Approximately 300 ml of supernatant was incubated for 5 min in a total volume of 700 ml containing 100 mM KH2PO4 and 3.6 mM EDTA. The color was developed with 400 ml of 10% (w/v) trichloroacetic acid (TCA), 400 ml of 44% o-phosphoric acid, 400 ml of 65 mM α,α′-dipyridyl in 70% ethanol and 200 ml of 110 mM FeCl3. The reaction mixtures were incubated at 40 °C for 1 h and quantified at 525 nm. Reduced glutathione (GSH) was assayed according to Guri (1983) with minor modifications. Briefly, a total of 0.3 g of leaves was homogenized in ice-cold 5% TCA (containing 5 mM EDTA). The homogenate was centrifuged at 10,000 g for 10 min. The reaction mixture contained 0.5 ml of distilled water, 1.0 ml of leaf homogenate, 1.0 ml of 0.2 M potassium phosphate buffer (pH 7.5) and 0.5 ml of the reagent dithiobis2-nitrobenzoic acid (DTNB). Glutathione was determined at 412 nm using a spectrophotometer. A standard curve in the range of 0–100 µM GSH was used.

Enzyme activity assays Samples (0.5 g of leaves) were ground in liquid N and extracted with 50 mM potassium phosphate buffer (pH 7.8) containing 0.1 mM EDTA, 1% (w/v) PVP, 0.1 mM PMSF and 0.2% (v/v) Triton X100 for the peroxidase (POD) measurements. Peroxidase activity was assayed in 2 ml of 100 mM potassium phosphate buffer containing 40 mM guaiacol, 10 mM H2O2 and the enzyme extract including 100 µg proteins at 25 °C using a spectrophotometer at 436 nm. The activity was based on the rate of tetraguaiacol production using an extinction coefficient of 25.5 mM−1 cm−1. The protein concentration was determined according to Bradford (1976). Nitrate reductase activity was measured in vitro according to Scheible et al. (1997) with some modifications. Briefly, 0.2 g of leaves were homogenized with an ice-cold extraction buffer (0.1 M Tris–HCl (pH 8.0), 2 mM MgSO4 ⋅ 7H2O, 5 mM EDTA, 2 mM dithiothreitol (DTT), 1% polyvinylpyrolidone, 2 mM monosodium glutamate and 2 mM cysteine). The homogenate was centrifuged at 12,000 g for 10 min. Then, 0.2 ml of the extract was mixed with 1 ml of assay buffer (0.1 M Tris–HCl (pH 8.0), 5 mM KNO3, 0.25 mM NADH) at 25 °C for 30 min. The reaction was stopped by adding 0.5 ml of 1% (w/v) hydroxylamine hydrochloride, and the color was developed by adding 0.5 ml of 0.2% (w/v) α-naphthylamine. Tubes were allowed to stand for 20 min at room temperature, and then the absorbance was measured at 540 nm

Tree Physiology Online at http://www.treephys.oxfordjournals.org

Downloaded from http://treephys.oxfordjournals.org/ at University of Utah on December 2, 2014

Net photosynthesis rate (Pn), stomatal conductance (gs) and intercellular CO2 concentration (Ci) were measured using a portable photosynthesis measuring system, LI-COR 6400 (LI-COR, Lincoln, NE, USA) between 08:00 and 11:30 h. The saturated photosynthetic photon flux density (PPFD) was determined by preliminary experiments. A carbon dioxide gas cylinder (LI-COR) was used to provide the constant and stable CO2. Prior to measurements, the samples were illuminated with saturated PPFD provided by the LED light source of the equipment for 10 min to achieve a full photosynthetic induction. A standard LI-COR leaf chamber (2 × 3 cm2) was used. The optimal parameters were as follows: leaf temperature 28 °C, leaf air vapor pressure deficit 1.5 ± 0.5 kPa, PPFD 1400 mmol m−2 s−1 and CO2 concentration 350 ± 5 µmol mol−1, which was near the ambient CO2 concentration.

were performed in the presence of 10 mM ascorbic acid (ASA) in the sample buffer.

346  Zhang et al.

Transmission electron microscopy Small leaf sections (2 mm in length), from the middle part of a leaf avoiding the midrib, were selected for the transmission electron microscope analysis (Zhao et al. 2009). The sections were fixed in 2.5% (v/v) glutaral pentanedial in 0.2 M phosphate-­ buffered saline (sodium phosphate buffer, pH 7.0) for 3 h at 22 °C and post-fixed in 2% osmium tetraoxide (OsO4) for 2 h. Then, the leaves were sequentially dehydrated in 30, 50, 70 and 90% acetone, and embedded in Epon 812 for 2 h. Ultrathin sections (80 nm) were sliced, stained with uranyl acetate and lead citrate, and mounted on copper grids for viewing in the H-600IV TEM (Hitachi, Tokyo, Japan) at an accelerating voltage of 60.0 kV.

Tree Physiology Volume 34, 2014

Statistical analysis All data were analyzed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). To assess differences in total physiological and biochemical properties between the sexes, a multivariate analysis of variance (MANOVA) was used. In MANOVA, the overall mean of the groups (partitioned to a series of sums of squares) was compared by test statistics (Pillai’s trace, Wilks’ lambda, Lawley–Hotelling and Roy’s largest root), and the between-group variance was expressed as F-statistics. Once the MANOVA tests were established, post hoc comparisons (ANOVA) were conducted using the Tukey’s test for means of individual parameters at a significance level of P 

Sexually different physiological responses of Populus cathayana to nitrogen and phosphorus deficiencies.

Previous studies have shown that there are significant sexual differences in the morphological and physiological responses of Populus cathayana Rehder...
2MB Sizes 0 Downloads 3 Views