Quantitative Proteomic Analysis Reveals Populus cathayana Females

Article pubs.acs.org/jpr

Quantitative Proteomic Analysis Reveals Populus cathayana Females Are More Sensitive and Respond More Sophisticatedly to Iron Deficiency than Males Sheng Zhang,*,† Yunxiang Zhang,†,‡ Yanchun Cao,§ Yanbao Lei,† and Hao Jiang† †

Key Laboratory of Mountain Surface Processes and Ecological Regulation, Institute of Mountain Hazards and Environment, Chinese Academy of Sciences, Chengdu 610041, China ‡ University of Chinese Academy of Sciences, Beijing 100039, China § Henan Polytechnic, Zhengzhou 450046, China S Supporting Information *

ABSTRACT: Previous studies have shown that there are significant sexual differences in the morphological and physiological responses of Populus cathayana Rehder to nitrogen and phosphorus deficiencies, but little is known about the sex-specific differences in responses to iron deficiency. In this study, the effects of iron deficiency on the morphology, physiology, and proteome of P. cathayana males and females were investigated. The results showed that iron deficiency (25 days) significantly decreased height growth, photosynthetic rate, chlorophyll content, and tissue iron concentration in both sexes. A comparison between the sexes indicated that iron-deficient males had less height inhibition and photosynthesis system II or chloroplast ultrastructural damage than iron-deficient females. iTRAQ-based quantitative proteomic analysis revealed that 144 and 68 proteins were decreased in abundance (e.g., proteins involved in photosynthesis, carbohydrate and energy metabolism, and gene expression regulation) and 78 and 39 proteins were increased in abundance (e.g., proteins involved in amino acid metabolism and stress response) according to the criterion of ratio ≥1.5 in females and males, respectively. A comparison between the sexes indicated that iron-deficient females exhibited a greater change in the proteins involved in photosynthesis, carbon and energy metabolism, the redox system, and stress responsive proteins. This study reveals females are more sensitive and have a more sophisticated response to iron deficiency compared with males and provides new insights into differential sexual responses to nutrient deficiency. KEYWORDS: dioecious, plant proteomics, iron deficiency, sexual difference, woody plant

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INTRODUCTION Iron is an essential element in most living plants and plays a key role in many physiological and metabolic processes, including photosynthesis and respiration. Iron is an irreplaceable cofactor in many redox and electron-transport reactions occurring within plant cells;1 however, its chemical properties in oxygenated environments limit its availability. Iron is only slightly soluble under alkaline and aerobic conditions. Plants with low iron availability often suffer from iron limitation. Iron deficiency is a major nutritional disorder that causes a decrease in vegetative growth and marked reductions in yield and quality.2 Iron deficiency results in a set of morphological, physiological, and metabolic changes in living organisms. In higher plants, the most obvious symptoms of iron deficiency are the development of intercostal chlorosis decrease in total protein and chloroplast protein contents in young leaves,3 the inhibition of root elongation, and increase in the diameter of apical root zones.4 It is commonly known that iron deficiency induces leaf chlorosis due to the decrease in chlorophyll pigment contents.5 Consequently, a pronounced iron deficiency results in a reduction of the amount of light harvesting complexes in both photosynthetic system I (PS I) and PS II.6,7 In iron-deficient © XXXX American Chemical Society

leaves, however, not all photosynthetic pigments and components of electron transport chain are decreased to the same extent. For example, the activity of PS I is more depressed than PS II in sugar beet, pear, and peach under iron deficiency, probably due to a higher amount of iron concentration per PS I than PS II.8 Iron deficiency also leads to a change of many metabolic profile,9 decrease in ribulose bisphosphate carboxylase (RuBisCO) content,10 and suppression of the ironcontaining gene expression, for example, iron−sulfur (Fe−S) cluster protein gene;11 however, the exact molecular mechanism through which iron deficiency affects photosynthetic activity is not well known. Recently, the applications of proteomic techniques have resulted in more sophisticated insights into adaptive mechanisms of plants, especially the response of roots to iron deficiency. Using gel-based 2-DE approaches, studies had shown that many proteins were commonly and species-specifically changed among different plant roots. The commonly changed proteins were involved in oxidative stress and defense and carbon and nitrogen Received: August 12, 2015

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DOI: 10.1021/acs.jproteome.5b00750 J. Proteome Res. XXXX, XXX, XXX−XXX

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Journal of Proteome Research

ZnSO4·7H2O, 0.12 μM Na2MoO4·2H2O, 0.08 μM CuSO4· 5H2O, and 10 μM Fe supplied as Fe(III)-EDTA.23 The pH was adjusted to 5.5 ± 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), 50 male and 50 female cuttings with an approximately equal root length and shoot height were selected for the experiments. The iron-deficient individuals grew in Hoagland solution without Fe(III)-EDTA. The control individuals grew in the complete Hoagland solution. Therefore, there were a total of four treatments, namely, control males and females and irondeficient males and females. Each treatment included 25 cuttings. These 25 cuttings were separated into five groups (each group included five cuttings). Each plant grew in a separate plastic pot containing 10 L of nutrient solution. The nutrition solution was changed every 3 days. The cuttings grew 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. According to a pre-experiment (n = 20), it was expected that most of the cuttings would be dead after 35 days of iron deficiency. Therefore, in this study, the treatments lasted for 25 days. At the end of the experiment, the fourth fully expanded leaves (counted from the top of the plants) were collected for analyses.

metabolisms, while the species-specific proteins were mainly involved in cell-wall biosynthesis, secondary metabolism, energy metabolism, and protein metabolism.12 Also, thylakoid membrane proteome had been performed in iron-deficient plants.13,14 With the advances in high-throughput proteomic technology, a quantitative proteomic approach (iTRAQ) has been developed. Using this method, studies have been performed on the roots and shoots of Arabidopsis responding to iron deficiency.15−17 For example, Zargar et al. (2015) reported that Fe deficiency might lead to the disruption of sugar synthesis and utilization in shoots.15 Pan et al. (2015) reported that post-transcriptional coordination was partially dependent on the E3 ligases ring domain ligase 1 (RGLG1) and RGLG2 in iron-deficient Arabidopsis roots.17 Therefore, understanding how plants respond to iron deficiency at the leaf proteomic level will provide a new level of information on iron homeostasis processes that can ultimately help to reduce the effects of iron deficiency in plants. Previous studies on iron deficiency focused on graminoids and crops, whereas little work has been conducted on woody plants, especially dioecious woody plants. Populus cathayana Rehd. is a dioecious and fast-growing tree species that is widely distributed in the Northern, Central, and Southwestern regions of China. It is generally accepted that a higher investment in reproduction may be detrimental to females and is more common in high-resource microsites, leading to reduced stress tolerance.18 Many previous studies have shown that young P. cathayana males and females possess sexually different responsive mechanisms to environmental stresses.19−22 For P. cathayana, drought-stressed males had higher photosynthetic ability than females and could be contributed to a higher water use efficiency and better protective system.19,20 Chillingstressed males exhibited a better chloroplast structure and higher osmotic adjustment substance contents (e.g., proline and soluble sugar) than females, suggesting that males possess a better self-regulation mechanism.21,22 Zhang et al. (2014)23 reported that P. cathayana females were more sensitive than males to nitrogen and phosphorus deficiencies. Nitrogen or phosphorus deficiency caused more negative effects in females than in males in photosynthesis, organelle ultrastructures, and nitrogen metabolic processes. We speculate that P. cathayana females may be more sensitive to iron deficiency than males; however, the exact molecular mechanism of sex-specific responses to iron deficiency is unknown. In this study, a quantitative proteomic approach was used. The objectives are to evaluate the sex-different changes in the leaf proteome under iron-deficient conditions and provide insights into the molecular mechanism of photosynthetic differences between P. cathayana males and females responding to nutrient deficiency.

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Gas-Exchange Measurements

Five cuttings (the fourth fully expanded leaves) of each sex per treatment were randomly selected for gas exchange, chlorophyll fluorescence, and pigment measurements. Net photosynthesis rate (A), stomatal conductance (gs), and intercellular CO2 concentration (Ci) were measured using a portable photosynthesis measuring system, LI-COR 6400 (LI-COR, Lincoln, NE), 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 for providing a constant and stable CO2. Prior to conducting the measurements, the samples were illuminated with saturated PPFD provided by the LED light source of the equipment for 10 min to achieve full photosynthetic induction. A standard LICOR 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; CO2 concentration, 400 ± 5 μmol mol−1; and photosynthetic photon flux density, 1400 mmol m−2 s−1 (for controls) and 600 mmol m−2 s−1 (for iron-deficient individuals), which were the light saturation points according to preliminary experiments. Chlorophyll Fluorescence and Pigment Measurements

Chlorophyll fluorescence was measured using a 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 the minimal fluorescence (Fo) and the maximal 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 the minimal fluorescence (Fo′) and the maximal 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. The measurements were carried out from 08:00 to 09:30. Chlorophyll fluorescence kinetics parameters were

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The F1 individuals of P. cathayana cuttings were used as the materials and the mother trees were collected from Qinghai Province, China. The mean altitude, annual rainfall, and annual temperature in the area are 3160 m, 335 mm, and 6.9 °C, respectively. Prior to treatment initiation, the 200 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 B


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iron-deficient males were in set 1, and control females and irondeficient females were in set 2. The sample of MC1 (113) was labeling in both sets as a check group. The detailed labeling condition is listed in Table 1.

measured and calculated, as described by Vankooten and Snel.24 The pigment extraction was conducted according to Lichtenthaler.25 Leaf discs (1.0 cm2) were cut from each leaf immediately after chlorophyll fluorescence measurements and extracted in 80% chilled acetone (v/v) after weighting. The absorbance of the extracts was measured using a spectrophotometer (Unicam UV-330; Unicam, Cambridge, U.K.) at 470, 646, and 663 nm. Chlorophyll concentrations and carotenoid contents (Caro) were calculated from equations derived from Porra et al. (1989).26 The total chlorophyll content (Tchl) was the sum of chlorophyll a (Chl a) and chlorophyll b (Chl b).

Table 1. Labeling Strategy Used for iTRAQa Set 1 labeling treatment 113 114 115 115 117 118 119

Tissue Iron Concentration Measurements and Leaf Transmission Electron Microscopy

Iron elemental concentration was measured as described by Takkar and Kaur.27 Roots, stems, and leaves were dried at 70 °C for 48 h and ground. 2 g of materials was used. Iron concentration was determined by atomic absorption spectroscopy (GBC932, GBC, Melbourne, Australia) after HNO3 digestion. Small leaf sections (2 mm in length), obtained from the middle position of a leaf while avoiding the midrib, were selected for transmission electron microscopy analysis.21 Five biological replicates were used. The sections were fixed in 2.5% (v/v) glutaral pentanedial in 0.2 M of PBS (sodium phosphate buffer, pH 7.0) for 3 h at 22 °C and postfixed 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 a H-600IV TEM (Hitachi, Tokyo, Japan) at an accelerating voltage of 60.0 kV.

MC1 MC2 MC3 MT1 MT2 MT3

Set 2

protein content (mg g−1 Fw) 30.43 28.34 31.21 21.45 15.43 18.23

treatment

protein content (mg g−1 Fw)

MC1 FC1 FC2 FC3 FT1 FT2 FT3

30.43 34.65 31.64 36.76 16.32 11.35 15.43

a

FC, control female; MC, control male; FT, iron-deficient females; MT, iron-deficient males; Fw, fresh weight.

Separation of Peptides by Strong Cation Exchange and ESI Mass Spectrometric Analysis

The labeled samples were fractionated using a LC-20AB highperformance liquid chromatography (HPLC) system (Shimazu, Kyoto, Japan) using a 4.6 mm × 250 mm Ultremex strong cation exchange (SCX) column (Phenomenex, Torrance, CA). After reconstitution of the labeled peptide mixtures with 4 mL of buffer A (10 mM NaH2PO4 in 25% ACN, pH 2.6), SCX separation was performed at a flow rate of 1 mL min−1 using elution buffer A for 10 min, followed by a linear gradient of 5−60% buffer B (25 mM NaH2PO4, 1 M KCl in 25% ACN, pH 2.7) for 20 min and 100% buffer B for 2 min before equilibrating with buffer A for 10 min prior to the next injection. The eluted fractions were monitored by measuring the absorbance at 214 nm, desalted with a Strata X C18 column (Phenomenex), and finally vacuum-dried. Each fraction was resuspended in buffer A (5% ACN, 0.1% FA) and centrifuged at 20 000g for 10 min, and the final concentration of peptide was ∼0.5 μg μL−1 on average. Ten μL supernatant was loaded on a LC-20 AD nanoHPLC (Shimadzu, Kyoto, Japan) by the autosampler onto a 2 cm C18 trap column. Then, the peptides were eluted onto a 10 cm analytical C18 column (inner diameter 75 μm) packed in-house. The samples were loaded at 8 μL min−1 for 4 min; then, the 35 min gradient was run at 300 nL min−1 starting from 2 to 35% B (95% ACN, 0.1% FA), followed by 5 min of linear gradient to 60%, followed by 2 min linear gradient to 80%, maintenance at 80% B for 4 min, and finally returning to 5% in 1 min. The MS analysis was performed using a Triple TOF 5600 System (AB SCIEX, Concord, ON) fitted with a Nanospray III source (AB SCIEX) and a pulled quartz tip as the emitter (New Objectives, Woburn, MA). Data were acquired using an ion spray voltage of 2.5 kV, curtain gas of 30 psi, nebulizer gas of 15 psi, and an interface heater temperature of 150 °C. The MS was operated with a RP of ≥30 000 fwhm for TOF MS scans. For IDA, survey scans were acquired in 250 ms and as many as 30 product ion scans were collected if exceeding a threshold of 120 counts per second (counts/s) and with a 2+ to 5+ charge state. Total cycle time was fixed to 3.3 s. Q2 transmission window was 100 Da for 100%. Four time bins were summed for each scan at a pulser frequency value of 11 kHz through monitoring of the 40 GHz multichannel TDC detector with

Protein Extraction, Digestion, and iTRAQ Labeling

Proteins were extracted using acetone methods.28 Three biological replicates (three cuttings) were used for each treatment. The leaves were ground to a fine powder and suspended in 0.5 M triethylammonium bicarbonate (TEAB) buffer with 1 mM phenylmethyl sulfonyl fluoride and 0.1% SDS, followed by sonication for 5 min and centrifugation at 25 000g for 20 min. The supernatant was transferred to another tube, 0.5 M TEAB buffer was added to the pellet to repeat the protein extraction, and the sample was again centrifuged at 25 000g for 20 min. The proteins in the combined supernatant were reduced (10 mM DTT, 56 °C for 60 min), alkylated (55 mM iodoacetamide, dark room temperature for 45 min), precipitated by precooled acetone at −20 °C for 2 h, and then centrifuged at 25 000g for 20 min. The pellet was washed twice with acetone, and the final pellet was dissolved in 0.5 M TEAB buffer with 0.1% SDS, sonicated for 15 min, and centrifuged at 25 000g for 20 min. The supernatant was used for liquid digestion, and the protein concentration was determined using the Bradford assay.29 The processed protein (100 μg) was removed from each sample solution and digested with Trypsin Gold (Promega, Madison, WI) at a ratio of protein/trypsin 20:1 at 37 °C for 12 h. After trypsin digestion, peptides were dried by vacuum centrifugation. The peptides were reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for 8-plex iTRAQ (Applied Biosystems). In this study, three biological replicates in each treatment and sex were used for iTRAQ. Therefore, there was a total of 12 samples that were divided into two sets for analysis. Control males and C


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young leaves had lost their green color and had become chlorotic in both sexes; furthermore, the petiole of irondeficient females turned red. 25 days later, the old leaves had also lost their green color and sexual differences in morphology were obvious, in which leaves were yellower in females than males (Figure 1). Height growth was inhibited more in females

four-anode channel detect ion. A sweeping collision energy setting of 35 ± 5 eV coupled to iTRAQ adjusted rolling collision energy was applied to all precursor ions for collisioninduced dissociation. In this experiment, full MS scans were acquired in a mass range of m/z 350 to 1500 in a scan time of 250 ms. Fragment ion spectra were acquired in the mass range of m/z 100−2000 and excluded for further fragmentation during 15 s. Protein Identification and Quantitation

The original MS/MS file data (*.wiff) were transferred to the *.mgf format and then searched against a local poplar database (ftp://ftp.jgi-psf.org/pub/compgen/phytozome/v9.0/ Ptrichocarpa/annotation/) using the MASCOT server (version 2.3.02, Matrix Science, Boston, MA). The data were downloaded from Populus trichocarpa V 3.0 (73013 protein-coding transcripts) (http://phytozome.jgi.doe.gov/pz/portal.html). The Mascot search settings were as follows: one missed cleavage site by trypsin allowed with fixed modification of carbamidomethyl (C), iTRAQ8plex (N-term), and iTRAQ8plex (K) and variable modifications of Gln → pyro-Glu (N-term Q), Oxidation (M), and iTRAQ8plex (Y). The fragment and peptide mass tolerance values were ±0.05 Da and ±10 ppm, respectively. To estimate the false discovery rate (FDR) for a measure of identification certainty in each replicate set, we employed the target-decoy strategy of Elias and Gygi (2007).30 It was required that each confident protein identification involved at least two unique peptide identifications with a high degree of confidence (FDR 1%) indicated in Mascot. The ratio of treatments to controls over ±1.5 and P value ≤0.05 were considered to indicate differential expressed proteins (DEPs). Proteins identified within a family were grouped in the Mascot protein family summary.31 Sample of MC1 was used as reference (the check group), based on the weighted average of the intensity of report ions in each identified peptide. The final ratios of protein were then normalized by the median average protein ratio for the mixes of different labeled samples. This normalization corrects the systematic error.32 Only DEPs that were identified in all three biological replicates were further used to analysis for sexually differential expression. The sexually differential expressed proteins were conducted using Student’s t test.

Figure 1. Morphology of P. cathayana females (A) and males (B) as affected by 25 days of iron deficiency. Photograph courtesy of Sheng Zhang. Copyright 2015.

Figure 2. Height growth of P. cathayana females and males as affected by 25 days of iron deficiency. * denotes significant difference according to t test at P < 0.05.

Statistical Analysis

For physiological parameters, the effects of iron deficiency, sex, and their interaction were analyzed by analysis of variance (ANOVA) using a randomized complete-block design in SPSS 16.0 (SPSS, Chicago, IL). Prior to analysis, the data were checked for normality and the homogeneity of variances. Posthoc comparisons were tested using the Tukey’s test at a significance level of P < 0.05. Mean values and standard errors were determined for each variable. For different expressed proteins, Student’s t test and a significance level of P ≤ 0.05 were used. We used the hierarchical clustering software PermutMatrix v1.9.3 (http://www.lirmm.fr/~caraux/PermutMatrix/) to cluster the different expressional proteins;33 Ward’s algorithm was used.

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than in males, and this sexual difference was significant (Figure 2); however, 35 days later, more than half of the leaves that had lost their green color had died and abscised in both sexes (data not shown), and the photosynthetic parameters could not be measured. Therefore, a time point of 25 days was selected to investigate the different sexual responses to iron deficiency. To estimate the physiological conditions of the plants under iron deficiency, we measured gas-exchange and chlorophyll fluorescence parameters. As shown in Table 2, compared with the controls, the A, gs, transpiration (E), maximum efficiency of photosystem II (PS II, Fv/Fm), maximum effective quantum yield of PS II (yield), and photochemical quenching coefficient (qP) decreased, but Ci and the nonphotochemical quenching coefficient (qN) increased under iron-deficient conditions in both sexes. A comparison between the sexes indicated that irondeficient males had higher values of Fv/Fm, yield, qP, qN, gs, and E than iron-deficient females. Additionally, iron deficiency significantly decreased the Chl a, Chl b, Caro, and Tchl contents in both sexes; however, the sexual differences were not significant.

RESULTS

Morphological and Photosynthetic Characteristics of Male and Female Poplars in Response to Iron Deficiency

Morphologically, the apex leaf veins of P. cathayana females were a slight pale yellow after 5 days of iron deficiency, compared with 7 days for males. 10 days later, all iron-deficient D


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Each value is the mean ± SE (n = 5). FC, control female; MC, control male; FT, iron-deficient females; MT, iron-deficient males; Fsex, sex effect; FFe, iron deficiency effect; Fsex×Fe, sex and iron deficiency interaction effect; Fw, fresh weight. Within a column, values followed by different letters are significantly different at P < 0.05 according to Tukey’s test.

Changes in Ultrastructural Morphology and Iron Elemental Concentration in Male and Female Poplars in Response to Iron Deficiency

As shown in Figure 3, the chloroplasts of the control males and females were ellipsoidal in shape with 15−30 well-arranged thylakoids (on average 21) in each granum and unbroken cellular membranes (Figure 3A,B); however, under iron-deficient

Figure 3. Transmission electron micrographs of mesophyll cells of P. cathayana males and females exposed to iron deficiencies. (A) Control males, (B) control females, (C) 25 days iron-deficient males, (D) 25 days iron-deficient females, (E) the thylakoid number per granum, and (F) the percentage of starch grain size in chloroplast. Ch, chloroplast; CW, cell wall; M, mitochondrion; N, nucleolus; Ps, plastoglobule; SG, starch grain; V, vacuole; Pm, plasma membrane.

conditions, the chloroplast shapes were smaller, subcircular, and irregular (Figure 3C,D). The structure of the thylakoids and mitochondria became considerably swollen and misshapen and plastoglobules accumulated in the chloroplasts. Iron deficiency caused a significant decrease in the thylakoid number per granum (47.78% in males and 68.38% in females, Figure 3E) and the starch grain size (22.21% in males and 55.34% in females, Figure 3F). A comparison between the sexes indicated that there were smaller chloroplasts, fewer grana, more swollen mitochondria, and more invaginations of the plasma membrane in irondeficient females than in iron-deficient males. Iron deficiency significantly changed the total iron concentration of the roots, stems, leaves, and whole plants in both sexes (Table 3). An overall tissue comparison indicated that the highest iron concentration was in the roots. Under control

a

0.02b 0.03b 0.00a 0.00a 0.69 ± 0.66 ± 0.14 ± 0.17 ± 0.981 0.000 0.218 0.15b 0.09b 0.00a 0.00a 3.76 ± 3.84 ± 0.19 ± 0.24 ± 0.550 0.000 0.506 0.07b 0.01b 0.00a 0.00a 1.06 ± 1.16 ± 0.02 ± 0.03 ± 0.404 0.000 0.557 0.09b 0.05b 0.00a 0.00a 2.70 ± 2.68 ± 0.17 ± 0.21 ± 0.969 0.000 0.628 0.03b 0.01a 0.01c 0.01d 0.52 ± 0.39 ± 0.63 ± 0.74 ± 0.542 0.000 0.000 0.03c 0.01c 0.01a 0.02b 0.83 ± 0.90 ± 0.62 ± 0.71 ± 0.001 0.000 0.359 0.02c 0.01c 0.03a 0.01b 0.61 ± 0.66 ± 0.16 ± 0.25 ± 0.001 0.000 0.252 0.01c 0.00c 0.03a 0.01b 0.78 ± 0.81 ± 0.30 ± 0.45 ± 0.000 0.000 0.000 0.17c 0.26c 0.40a 0.36b 8.60 ± 9.41 ± 2.97 ± 4.41 ± 0.003 0.000 0.296 1.63a 2.13a 3.82b 3.07b 0.22b 0.64c 0.30a 0.64a

0.02bc 0.06c 0.04a 0.05b 0.55 ± 0.69 ± 0.24 ± 0.41 ± 0.006 0.000 0.779 19.57 ± 23.34 ± 3.60 ± 4.71 ± 0.000 0.000 0.023 FC MC FT MT Fsex FFe Fsex×Fe

294.18 ± 290.46 ± 357.73 ± 360.09 ± 0.815 0.000 0.308

Caro (mg g−1 Fw) Tchl (mg g−1 Fw) Chl b (mg g−1 Fw) Chl a (mg g−1 Fw) qN qP yield Fv/Fm E (mmol m−2 s−1) Ci (μmol mol−1) gs (mol m−2 s−1) A (μmol m−2 s−1) treatments

Table 2. Gas-Exchange Parameters, Chlorophyll Fluorescence Parameters, and Chlorophyll Pigment Contents of P. cathayana Males and Females as Affected by Iron Deficiencya


E


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Table 3. Iron Concentration in Roots, Stems, Leaves and Whole Plant Levels in P. cathayana Males and Females as Affected by 25 Days of Iron Deficiencya roots (g g−1 Dw)

treatments

1.97 ± 1.16 ± 1.22 ± 0.97 ± 0.000 0.000 0.000

FC MC FT MT Fsex FFe Fsex×Fe

0.03c 0.04ab 0.06b 0.02a

stems (g g−1 Dw) 0.31 ± 0.29 ± 0.37 ± 0.33 ± 0.000 0.000 0.149

leaves (g g−1 Dw) 0.41 ± 0.41 ± 0.33 ± 0.37 ± 0.000 0.003 0.000

0.01ab 0.01a 0.00c 0.00b

whole plants (g g−1 Dw) 2.70 ± 1.85 ± 1.67 ± 1.93 ± 0.000 0.000 0.000

0.01c 0.00c 0.01a 0.00b

0.05c 0.04ab 0.02a 0.07b

a

FC, control female; MC, control male; FT, iron-deficient females; MT, iron-deficient males; Fsex, sex effect; FFe, iron deficiency effect; Fsex×Fe, sex and iron deficiency interaction effect; Dw, dried weight. Values are means ± SE (n = 5). Within a column, values followed by different letters are significantly different at P < 0.05 according to Tukey’s test.

Table 4. Overall nanoLC−MS/MS Identification Results of Three Biological Replicate Samples of Each Treatment in Two Sets

a

group

total identified spectra

matched spectra

matched unique spectra

identified peptide

identified unique peptide

identified protein

Set_1a Set_2b

300542 417964

39247 34710

29108 25997

13897 11603

11374 9502

4083 3413

Set_1 included MC 1, 2, and 3 and MT 1, 2, and 3. bSet_2 included MC 1, FC 1, 2, and 3, and FT 1, 2, and 3.

conditions, female roots had higher iron concentration than males’, but there was no significant sexual difference in stems and leaves. Under iron-deficient conditions, females had higher iron concentration in the roots and stems but lower concentration in the leaves compared with males. In addition, the extent of the iron decrease in female roots was greater than that measured in males.

increased proteins in abundance were mainly classified into six functional categories and the differentially decreased proteins in abundance were mainly classified into five functional categories (Figures S-3 and S-4). Hierarchical cluster analysis according to Pearson’s distance indicated two groups with clearly different expressional patterns among the differentially expressed proteins (Figure 4). There were two distinct groups: One

Detection of Iron Deficiency Caused Alterations in Protein Abundance in Poplars

To investigate the changes in the protein profiles in response to 25 days of iron deficiency, we extracted proteins from poplar leaves and analyzed using an iTRAQ-based shotgun proteomics strategy. Table 1 showed an overview of the extraction results of total protein content in both sexes. The change in relative concentration of any given protein after 25 days of irondeficiency was obtained from the iTRAQ 8-plex reporter ion ratios by a weighted average of all confidently identified peptides, requiring that at least two were uniquely assigned to any given protein for it to be classified as “identified”. The coefficients of variation (CV value) of biological replicates were 50% in both sexes. Iron is involved in chlorophyll biosynthesis, thylakoid synthesis, and chloroplast development. Chlorophylls are often directly related to the iron fraction in leaves and the quantification of chlorophylls is a good means to estimate the consequential effects of iron deficiency on plant metabolism.1 These results are in agreement with numerous reports that iron starvation leads to a concomitant reduction of chlorophylls, carotenoids, and plastidial proteins.34−36 All of these perturbations may occur either because iron is directly involved in the biosynthesis of these components or because reduced levels of iron concentration result in a stoichiometric decrease in other elements. Regardless of the cause, the ultimate consequence is a considerable decrease in photosynthetic capacity;1 however, in this study, we found that female morphological and physiological responses to iron deficiency are faster than those of males. Chlorophyll fluorescence, an indicator of the photochemical efficiency of PS II, can provide insights into the ability of plants to tolerate stresses and the extent to which these stresses damage the photosynthetic apparatus.37 In this study, Fv/Fm decreased considerably under iron-deficient conditions, suggesting that there was a decrease in the potential activities and an increase in the disorder of the electron transport chain of PS II. A comparison between the sexes indicated that irondeficient females had smaller Fv/Fm, yield, qP, and qN values than iron-deficient males, indicating that P. cathayana males had less damage to PS II and a better ability to dissipate excessive light energy through radiative and nonradiative pathways. The effects of iron deficiency are often associated with cytological alterations that mainly affect the chloroplast G


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increased in abundance in iron-deficient females, for example, cystathionine beta-lyase, cysteine synthase, methylthioribose-1phosphate isomerase, and lactoylglutathione lyase. The higher abundance of these proteins indicates certain amino acid metabolic processes may be enhanced by iron deficiency in females, such as L-methionine, L-cysteine, and glutathione biosynthetic processes. Therefore, to a certain extent, the metabolic substrates of the TCA cycle may participate in amino acid metabolism. A protein of gamma aminobutyrate transaminase 3 (GABAT) was increased in abundance in iron-deficient females. GABA-T catalyzes the first step of the catabolism of gammaaminobutyric acid (GABA), a nonprotein amino acid wellknown to accumulate in plants in response to environmental stimuli.55 Increasing evidence shows that GABA plays a role in carbon or nitrogen metabolism and acts as a signaling molecule in developmental processes. This study is the first to report that GABA-T is increased in abundance in iron-deficient plants. Additionally, S-adenosyl-L-homocysteinase (AHCY) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) and functions as a regulator of biological transmethylation by controlling the concentration of AdoHcy, a potent competitive inhibitor of all S-adenosyl-L-methionine methyltransferases. This is also the first finding that AHCY increases in abundance in iron-deficient female poplars. Redox System. Ferredoxin is the most well-known nonheme Fe−S protein, which acts as an electron transmitter in a number of metabolic processes, such as photosynthesis and nitrogen reduction. In iron-deficient females, several ferredoxin proteins were decreased in abundance. A decrease in ferredoxin concentration is correlated with lower nitrate reductase activity under iron-deficient conditions. Because of the involvement of iron in various steps of nitrate reduction, positive correlations between iron supply, ferredoxin concentration, and nitrate reduction are easy to understand.56 Other important Fe−S proteins are the isozymes of superoxide dismutase (SOD), which contain Cu, Zn, Mn, or Fe as metal components during the detoxification of superoxide anion-free radicals through the formation of H2O2. Evidence shows that in iron-deficient leaves Fe-SOD activity is decreased while Cu/Zn-SOD activity is increased. In this study, we did not find a significant change of Fe-SOD, but Cu/Zn-SOD was increased in abundance in irondeficient males. On the contrary, although iron-deficient plants reduced the abundance of antioxidative enzymes and increased H2O2 concentration, an enhanced oxidative cell damage was not apparent, which may be due to the very low concentrations of active iron required for ROS generation through the Haber− Weiss or Fenton reactions.56 In addition to heme and Fe−S proteins, the abundance of non-iron-containing redox proteins (e.g., thioredoxin) was also changed in iron-deficient females, but they exhibited different expression patterns. Therefore, we cannot obtain a clear understanding of the role of the thioredoxin system under iron-deficient conditions from the current proteome data. Stress-Induced Proteins. Heat shock protein (HSP) genes can be induced expression by not only heat stimulation but also other stresses, including of iron deficiency.57 HSPs perform a chaperone function by stabilizing new proteins to ensure correct folding or by helping to refold proteins that were damaged by cell stress. Interestingly, large-molecular-weight HSPs were increased in abundance in iron-deficient females, for example, 90 and 70 kDa, compared with small HSPs in males, for example, 17.9 and 22 kDa. Although the exact function of

conditions. Cyt is a constituent of the redox systems in chloroplasts and mitochondria and is also a component of the redox chain in nitrate reductase. Other heme enzymes are catalase and peroxidase, which are susceptible to low iron supply. Evidence shows that under iron-deficient conditions, the activity of both enzymes rapidly decreases in plant tissues.47 Similarly, in this study, many isozymes of catalase and peroxidase were decreased in abundance in both sexes, such as peroxidase 4, 15, 51, 64 and L-ascorbate oxidase. Therefore, the hinderance of electron transport chain and the decrease in peroxidase may contribute to an increase in reactive oxygen species in plant cells to iron deficiency.46,48 Additionally, iron deficiency increased the abundance of several proteins involved in gene expression and regulation in both sexes, for example, peptidyl-prolyl cis−trans isomerases (PPIase) and chaperonin 60 subunit beta 2 (CPN 60−2). The two types of proteins are involved in protein folding. PPIase catalyzes the cis−trans isomerization of proline imidic peptide bonds in oligopeptides, and this is the slowest step in protein folding.49 Therefore, iron deficiency also changed the process of gene expression. Studies of transcriptome and posttranslational modification may provide new insights into the future. Sex-Specific Changes to Iron Deficiency in Proteome

Photosynthesis. Except for the common changed proteins, iron deficiency induced a greater decrease in females than in males with respect to the abundance of proteins related to photosynthesis, including cytochrome bf (Cyt bf) complex, PS I P700 chlorophyll a apoprotein A1 (psaA), and PsbP domaincontaining proteins and RuBisCO chains. Cyt bf and psaA are involved in the primary electron donor of PS I. In the thylakoid membranes, ∼20 iron atoms are involved in electron transport; thus there is a higher iron requirement for the structural and functional integrity of the thylakoid membranes. In P. cathayana females, there was greater leaf number and total leaf area50−52 and greater thylakoid number.19,21 Moreover, the foliar iron concentration and some thylakoid membrane proteins in abundance were decreased more in iron-deficient females, for example, thylakoid formation protein 1 and 14 kD of the thylakoid membrane phosphoprotein, which reduced photosynthetic electron transport. Therefore, females may suffer fast and more serious affects to iron deficiency than males, which may subsequently lead to serious damage to PS II. PsbP proteins also decreased in abundance, which are essential for the regulation and stabilization of PS II.53 Therefore, the greater decrease in these proteins may be attributed to the greater reduction in female photosynthesis. Additionally, the decrease in RuBisCO abundance may be another important reason for the lower photosynthesis in iron-deficient females. These results are similar to those reported for spinach suffering from iron deficiency.5 Carbohydrate, Amino Acid, and Secondary Metabolic Processes. Two key enzymes involved in sugar and starch synthesis (fructose-bisphosphate aldolase and glucose-1-phosphate adenylyltransferase large subunit) were decreased in abundance in iron-deficient females, indicating lower carbohydrate accumulation in leaves. Studies have shown that irondeficient plants are characterized by low concentrations of starch and sugar.54 The chloroplast ultrastructure also confirmed this phenomenon in this study. This may be due to the impairment of photosynthetic electron transport and the decreased photosynthetic rate previously discussed; however, several proteins involved in amino acid metabolism were H


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Journal of Proteome Research Notes

these HSPs is unknown, evidence showed that chloroplast stromal HSP 70 is involved in protection against photoinhibition58−60 and mitochondrial HSP 70 is thought to bind the incoming precursor and through ATP hydrolysis and provide the driving force for the translocation reaction.61 Small HSPs were considered to function as molecular chaperones, preventing undesired protein−protein interactions and assisting in refolding of denatured proteins.62 Therefore, different molecular weights of HSPs play different function in plant cells, and the abundant change of these HSPs may depend on sex. Additionally, in iron-deficient males, a universal stress protein A-like protein was increased in abundance in irondeficient females, while a hypersensitive-induced response protein 2 was increased in abundance in iron-deficient males. Although these proteins were responsive to a wide range of abiotic and biotic stresses, including to nutrient starvation,63−66 the exact function is unknown.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Excellent Young Scientist Program of the National Natural Science Foundation of China (no. 31322014), the National Key Basic Research Program of China (no. 2012CB416901), and Young Talent Team Program of the Institute of Mountain Hazards and Environment (SDSQB-2012-02).

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(1) Thoiron, S.; Pascal, N.; Briat, J. F. Impact of iron deficiency and iron re-supply during the early stages of vegetative development in maize (Zea mays L.). Plant, Cell Environ. 1997, 20, 1051−1060. (2) Abadía, J.; Vázquez, S.; Rellán-Á lvarez, R.; El-Jendoubi, H.; Abadía, A.; Á lvarez-Fernández, A.; et al. Towards a knowledge-based correction of iron chlorosis. Plant Physiol. Biochem. 2011, 49, 471−482. (3) Santos, C. S.; Roriz, M.; Carvalho, S. M. P.; Vasconcelos, M. W. Iron partitioning at an early growth stage impacts iron deficiency responses in soybean plants (Glycine max L.). Front. Front. Plant Sci. 2015, 6, 325−325. (4) de la Guardia, M. D.; Alcántara, E. A comparison of ferric-chelate reductase and chlorophyll and growth ratios as indices of selection of quince, pear and olive genotypes under iron deficiency stress. Plant Soil 2002, 241, 49−56. (5) Timperio, A. M.; D’Amici, G. M.; Barta, C.; Loreto, F.; Zolla, L. Proteomics, pigment composition, and organization of thylakoid membranes in iron-deficient spinach leaves. J. Exp. Bot. 2007, 58, 3695−3710. (6) Chauhan, D.; Folea, M.; Jolley, C. C.; Kouril, R.; Lubner, C. E.; Lin, S.; et al. Novel photosynthetic strategy for adaptation to low-iron aquatic environments. Biochemistry 2011, 50, 686−92. (7) Msilini, N.; Essemine, J.; Zaghdoudi, M.; Harnois, J.; Lachaâl, M.; Ouerghi, Z.; et al. How does iron deficiency disrupt the electron flow in photosystem I of lettuce leaves? J. Plant Physiol. 2013, 170, 1400− 1406. (8) Pushnik, J. C.; Miller, G. W. Iron regulation of chloroplast photosynthetic function: mediation of PS I development. J. Plant Nutr. 1989, 12, 407−421. (9) López-Millán, A. F.; Morales, F.; Gogorcena, Y.; Abadía, A.; Abadía, J. Metabolic responses in iron deficient tomato plants. J. Plant Physiol. 2009, 166, 375−384. (10) Winder, T. L.; Nishio, J. N. Early iron-deficiency stress-response in leaves of sugar-beet. Plant Physiol. 1995, 108, 1487−1494. (11) Song, Z. Z.; Yang, Y.; Xu, J. L.; Ma, R. J.; Yu, M. L. Physiological and transcriptional responses in the iron-sulphur cluster assembly pathway under abiotic stress in peach (Prunus persica L.) seedlings. Plant Cell, Tissue Organ Cult. 2014, 117, 419−430. (12) López-Millán, A. F.; Grusak, M. A.; Abadía, A.; Abadía, J. Iron deficiency in plants: an insight from proteomic approaches. Front. Plant Sci. 2013, 4, 1−7. (13) Andaluz, S.; López-Millán, A. F.; De las Rivas, J. D.; Aro, E. M.; Abadiá, J.; Abadiá, A. Proteomic profiles of thylakoid membranes and changes in response to iron deficiency. Photosynth. Res. 2006, 89, 141− 155. (14) Laganowsky, A.; Gómez, S. M.; Whitelegge, J. P.; Nishio, J. N. Hydroponics on a chip: Analysis of the Fe deficient Arabidopsis thylakoid membrane proteome. J. Proteomics 2009, 72, 397−415. (15) Zargar, S. M.; Kurata, R.; Inaba, S.; Oikawa, A.; Fukui, R.; Ogata, Y.; et al. Quantitative proteomics of Arabidopsis shoot microsomal proteins reveals a cross-talk between excess zinc and iron deficiency. Proteomics 2015, 15, 1196−1201. (16) Zargar, S. M.; Fujiwara, M.; Inaba, S.; Kobayashi, M.; Kurata, R.; Ogata, Y.; et al. Correlation analysis of proteins responsive to Zn, Mn, or Fe deficiency in Arabidopsis roots based on iTRAQ analysis. Plant Cell Rep. 2015, 34, 157−166.

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CONCLUSIONS Our results showed that there are common and sexual differences in the response of P. cathayana females and males to iron deficiency in terms of physiology and proteome dynamics. A 25 day iron deficiency induced common responsive proteins in two sexes, mainly involved in photosynthesis, energy metabolism, and gene expression regulation. The sexually different responsive proteins are involved in photosynthesis, carbohydrate, amino acid and secondary metabolic processes, the redox system, and stress-induced proteins. P. cathayana females showed greater growth inhibition, photosynthetic decline, and larger protein changes than males under iron-deficient conditions, suggesting that females are more sensitive and have a more sophisticated response to iron deficiency compared with males. Additionally, in this study, many identified sexually differential proteins are chloroplast proteins. Therefore, the next interesting topic of research could be the chloroplast proteome when plants are suffering from iron deficiency

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ASSOCIATED CONTENT

S Supporting Information *

Table S-1. Functional classifications and identified proteins with significantly common changes in both P. cathayana males and females exposed to iron deficiency. Table S-2. Functional classifications and identified proteins with significant changes in P. cathayana females only after 25 days of iron deficiency. Table S-3. Functional classifications and identified proteins with significant changes in P. cathayana males only after 25 days of iron deficiency. Figure S-1. The coefficients of variation (CV value) of biological replicates in P. cathayana males and females. Figure S-2. The error distribution of all matched peptides between the theoretical values and real values. Figure S-4. Category and number of decreased protein abundance caused by iron deficiency in P. cathayana males and females. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b00750.

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REFERENCES

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Quantitative Proteomic Analysis Reveals Populus cathayana Females

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