Hydroponics on a Chip: Analysis of the Fe Deficient Arabidopsis Thylakoid Membrane Proteome

JOURNAL OF PROTEOMICS 72 (2009) 397– 415

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Hydroponics on a chip: Analysis of the Fe deficient Arabidopsis thylakoid membrane proteome

Arthur Laganowskya,1,2, Stephen M. Gómezb, Julian P. Whiteleggec, John N. Nishiod,⁎ aDepartment of Biological Sciences, California State University, Chico, United States bSchool of Math, Science and Engineering, Central New Mexico Community College, Albuquerque, United States cThe Pasarow Mass Spectrometry Laboratory, The Jane & Terry Semel Institute for Neuroscience and Human Behavior, The Molecular Biology Institute and The Brain Research Institute, University of California, Los Angeles, United States dBiocompatible Plant Research Institute, College of Natural Sciences, California State University, Chico, CA 95929-0555, United States

ARTICLE DATA ABSTRACT

Keywords: The model plant Arabidopsis thaliana was used to evaluate the thylakoid membrane Intact mass proteomics proteome under Fe-deficient conditions. Plants were cultivated using a novel hydroponic Proteomics system, called “hydroponics on a chip”, which yields highly reproducible plant tissue Iron deficiency samples for physiological analyses, and can be easily used for in vivo stable isotope labeling. Arabidopsis thaliana The thylakoid membrane proteome, from intact chloroplasts isolated from Fe-sufficient and Chloroplast Fe-deficient plants grown with hydroponics on a chip, was analyzed using liquid Thylakoids chromatography coupled to mass spectrometry. Intact masses of thylakoid membrane Hydroponics proteins were measured, many for the first time, and several proteins were identified with Hydroponics on a chip post-translational modifications that were altered by Fe deficiency; for example, the doubly phosphorylated form of the photosystem II oxygen evolving complex, PSBH, increased under Fe-deficiency. Increased levels of photosystem II protein subunit PSBS were detected in the Fe-deficient samples. Antioxidant enzymes, including ascorbate peroxidase and peroxiredoxin Q, were only detected in the Fe-deficient samples. We present the first biochemical evidence that the two major LHC IIb proteins (LHCB1 and LHCB2) may have significantly different functions in the thylakoid membrane. The study illustrates the utility of intact mass proteomics as an indispensable tool for functional genomics. “Hydroponics on a chip” provides the ability to grow A. thaliana under defined conditions that will be useful for systems biology. © 2009 Elsevier B.V. All rights reserved.

Abbreviations: Ant, antheraxanthin; β-car, β-carotene; BPDS, bathophenanthrolinedisulfonate; BSA, bovine serum albumen; Chl, chlorophyll; Cyt, cytochrome; HEPES, 4-2-hydroxyethyl-1-piperazineethanesulfonic acid; HILEP, hydroponic isotope labeling of entire plants; IFD, intermediate Fe-deficiency; LCMS+, liquid chromatography-mass spectrometry plus; LHC, Light harvesting chlorophyll (protein); Lut, lutein; MES, 2-(N-morpholine) ethanesulfonic acid; Neo, neoxanthin; NPQ, non-photochemical quenching; ρBQ, para- benzoquinone; PTM, post-transcriptional modification; RT, retention time; SFD, severe Fe-deficiency; TIC, total ion chromatogram; Vio, violaxanthin; Zea, zeaxanthin. ⁎ Corresponding author. Tel.: +1 530 898 4589; fax: +1 530 898 4363. E-mail address: [email protected] (J.N. Nishio). 1 Present address: Department of Chemistry and Biochemistry, University of California, Los Angeles, United States. 2 Supported by NIH Chemistry Biology Interface Training Program Grant 5T32GM008496.

nutrient deficient symptoms. Hoagland's solution is fashioned 1. Introduction after the Hanford soil from California's Central Valley, and it provides a lush nutrient environment for most plants. Knowledge of growth conditions and plant status at time of Optimization of nutrient schemes for all cultured plants is a harvest and tissue preparation is imperative for critical reasonable pursuit. Table 1 shows that certain elements have analyses of data collected from plants. As the magnitude of been used at significantly higher rates than that used in 1/4- systematic molecular data uploaded to public servers strength Hoagland's solution. We have not made a direct increases, it is important to consider uniformity and consis- comparison, but the general health of our plants suggests that tency in cultural conditions among scientific laboratories. such additional nutrition is not required. Soil-grown plants from different laboratories and/or gardens Arabidopsis has been grown hydroponically for plant may lack uniformity and consistency; however, comparable proteomic, intact organelle isolation, and physiological stu- plants are necessary in today's post-genomic era. The model dies. For example, Bindschedler et al. [8] used hydroponic plant, Arabidopsis thaliana cv. Columbia, is routinely cultured isotope labeling of entire plants (HILEP) to label whole plants in soil. Individual laboratories and even different growth with the stable isotope of nitrogen, 15N, to study differential chambers in individual laboratories may yield inconsistent expression of apoplastic protein by 14N/15N peptide pairs in results. Growing plants hydroponically will not eliminate response to oxidative stress [8]. Huttlin et al. [9] analyzed ten growth chamber differences or internal differences within a day old-plants grown in liquid culture containing 6% (partial), growth chamber, but hydroponics can be useful in generating and 98% (full) 15N for a quantitative proteomic study of full more uniform plants overall. versus partial metabolic labeling [9]. Others have also recently The nature and size of A. thaliana make hydroponic used hydroponically grown Arabidopsis [10–12]. cultivation a challenge. A. thaliana has been grown on different In the present paper, we provide information on the soil-less medium substitutes such as rockwool [1], perlite construction and development of a robust growing system vermiculite mix [2], stainless steel wire cloth [3], sponge [4], suitable for today's systematic approaches for studying A. and polyurethane raft [4,5]. Rockwool, due to its water thaliana. A benefit of growing the plants with the described saturating nature, resulted in unhealthy plants and an overall system is that all parts of the plant can be readily harvested low percentage of surviving plants, as also noted by Tocquin and the plants are reasonably uniform. et al. 2003 [6]. Additionally, root tissue may be difficult to The technique outlined here for growing A. thaliana harvest in some rockwool systems. The polyurethane raft provides ample tissue for most biochemical studies, and system [5] cultured plants in peat for 30 d before plants were under our growth conditions, it will provide plant material up transferred to a hydroponic system. to seven weeks, before the onset of flowering. At all stages of Several nutrient formulations (Table 1) have been utilized plant development, plants can be readily selected for uni- for hydroponic culture of A. thaliana. In our years of growing A. formity or phenotype of shoots and/or roots. The design of the thaliana on 1/4-strength Hoagland's solution [7], symptoms of system was intended for minimal maintenance. The floating nutrient deficiency do not develop. Many other plants grown growth system provided consistent root immersion at all on 1/2-strength Hoagland's solution also do not exhibit growth stages. The system can also be used for stable isotope, root-fed labeling of whole plants for plant proteomic experiments (Supplementary Fig. 1). Though the outlined system Table 1 – Atomic and chemical composition of Arabidopsis requires precision, the reproducibility and uniformity of plants hydroponic mediums. grown using the system improves comparative analyses. Atomic Hoagland Somerville Tocquin Gibeaut Generally, nutrient studies are easier to conduct using element/ and Arnon and Orgen et al. [6] et al. [1] hydroponic systems. The utility of the developed system was chemical 1950 1982 (0.25×) component (0.25×) demonstrated by a top down proteomic examination of thylakoid membrane proteins in response to Fe-deficiency. μ μ μ μ B 11.5 M 17.5 M 9.68 M50M The Arabidopsis plants grown using the described hydroponic Ca+2 1.25 mM 0.5 mM 1.01 mM 1.5 mM − system exhibited Fe-deficiency symptoms that matched Cl 4.5 μM 9.505 μM 4.06 μM50μM Cu+2 0.0775 μM 0.125 μM 0.21 μM 1.5 μM previous work with plants such as sugarbeet, sunflower, Fe+2 22.375 μM 42.5 μM 22.4 μM72μM soybean, tomato, and spinach. Plants exhibiting intermediate K+ 1.25 mM 1.25 mM 5.1 mM 1.305 mM and severe Fe-deficiency symptoms were examined using Mg+ 0.5 mM 0.5 mM 0.498 mM 0.75 mM liquid chromatography-mass spectrometry plus (LCMS+) + μ μ μ μ Mn 2.25 M 3.5 M 2.03 M10M (Supplementary Fig. 2) [13]. Mo+ 0.025 μM 0.05 μM 0.0139 μM 0.525 μM The present proteomic study focused on the thylakoid Na+ 0.025 μM 2.55 μM 31.3 μM 200 μM + –– μ membrane proteome. Four major protein complexes are NH4 0.159 mM 0.45 M − associated with thylakoid membranes — PS II, Cyt b f complex, NO3 3.75 mM 2.25 mM 7.149 mM 4.25 mM 6 − PO4 0.25 mM 0.625 mM 0.13 mM 0.5 mM PS I, and ATP Synthase. The core of PS II is composed of PSBA − 2 SO4 0.501 mM 0.500 mM 0.521 mM 0.764 mM (D1) and PSBD (D2), that is surrounded by PSBC (CP43) and PSBB +2 Zn 0.193 μM 0.25 μM 0.314 μM 2.0 μM (CP47), and these four components interact with the oxygen- μ – μ – EDTA 22.375 M 22.3 M evolving complex proteins PSBO1-2, small intrinsic membrane EDDHA – 42.5 μM –– proteins (PSBE, PSBF, PSBI, PSBJ, PSBH, PSBK, PSBL, PSBM, PSBN, DTPA –––72 μM Si –––0.1 mM PSBT, PSBX, PSBY, PSBZ), and other proteins [For review see; 14]. Photosystem II of higher plants is a native dimer, where JOURNAL OF PROTEOMICS 72 (2009) 397– 415 399

each monomer contains one heme Fe, Cyt b559,located transferred into a modified one mL pipette box (Fisher brand, between PSBE/PSBF, and one non-heme Fe in the D1/D2 core for example). The sides of 1 mL pipette tip holders were cut to [15]. PSAA and PSAB create the core of PS I that interacts with form short plastic trays with 101 evenly spaced holes. The the stromal hump composed of PSAC, PSAD, and PSAE, and modified tip holder was floated on top of 500 mL of 1/16-strength small intrinsic membrane proteins (PSAF, PSAG, PSAH, PSAI, Hoagland's solution. Circles (0.713 cm2) were cut out of 0.318 cm PSAJ, PSAK, PSAL, PSAN, PSAO) [For review see; 14]. Photo- thick foam (purchased from a local mailing store). A single slit system I contains three 4Fe4S centers designated Fx,FA, and FB; along the radius, to hold the one-week old A. thaliana plant, was where Fx is located in the PSAA/B core, and FA and FB are cut with a razor blade. Tweezers were used to gently peel the located in PSAC subunit of the stromal hump [16]. The Cyt b6f one-week old plants off the agar plates and to insert the plants complex is a native dimer with each monomer containing four into the slit of the foam plugs, with leaves resting on the top of large subunits; Cyt b6 (PETB), Cyt f (PETA), subunit IV (PETD), the foam plug. The root of the plant was inserted into the hole of and Rieske Fe-S protein (PETC); the dimer is surrounded by the floating pipette tip holder, and the foam plug was inserted small membrane proteins PETG, PETL, PETM, and PETN, and in into the hole or rested (floated) above the hole (Fig. 1). The plants addition a ninth subunit in higher plants, the ferredoxin: were cultured in the pipette box for two-weeks (Fig. 1). At this + NADP reductase (PETH) [For review see; 17]. The Cyt b6f early stage of growth, mild aeration is recommended, but complex contains three hemes and the Rieske Fe–S center, a optional, as also noted by Arteca and Arteca 2000 [25]. 2Fe2S cluster [18]. The chloroplast ATP-synthase can be After two weeks the plants were transferred to standard divided into two components, the membrane proton channel, commercial black plant trays (0.28 m×0.56 m) purchased from a CF(0), and the catalytic core, CF(1); Alpha (ATPA), beta (ATPB), local hardware store. Three-quarter inch rigid insulation foam gamma (ATPC), delta (ATPD) and epsilon (ATPE) make up CF(0) (Formular 250, Owens Corning®) was cut to fit inside the plant in a 3:3:1:1:1 stoichiometry, while a or subunit IV(ATPI), b or tray and to serve as a floating support for the plants. Twenty- subunit I (ATPF), b' or subunit II (ATPG), and c or subunit III one equally spaced holes with a 2.54 cm diameter were drilled in (ATPH) make up CF(1) in a 1:1:1:14 stoichiometry (For review the foam and a 0.32 cm depth counter sink with a diameter of see; [19,20]). Three of the four protein complexes require Fe 3.81 cm was cut to snugly accommodate a poker chip of similar (Supplementary Fig. 1). diameter and provide an overall flush appearance. The plastic Previous studies have measured intact masses of thylakoid poker chips, purchased from a local super store, were center membrane proteins from Arabidopsis and other plants. Gómez drilled with a 0.635 cm drill bit. Black plastic (152.4 μmthick)was et al. [21] identified around 40 intact protein masses for pea and attached to the sides of the tray to block light entry between the tobacco thylakoid membrane proteins [21]. In another study by floating foam and plant tray. The plant tray was filled with 6 L of Gómez et al. [22], a set of 58 thylakoid membrane proteins from 1/4-strength Hoagland's solution (constant aeration), and three four plant species, including Arabidopsis, were measured [22]. week old plants from the pipette trays were transferred into the Zolla et al. [23] analyzed the proteome of PS I components from center hole of the poker chips. The poker chips containing the different plant species and measured intact masses for several plantlets were then placed into the countersunk holes in the Arabidopsis thylakoid membrane proteins [23]. Intact mass tags foam base. The completed apparatus is shown in Fig. 1. were measured for protein subunits from Cyt b6f isolated from After two weeks of growth in the Hydroponics on a Chip Arabidopsis, grown using the hydroponics on a chip system [24]. system, the nutrient medium was replaced with fresh 1/4- The present study measured many intact mass tags, some strength Hoagland's solution, and the nutrient deficiency was for the first time, for Arabidopsis thylakoid membrane proteins initiated [26]. The control and minus-Fe solutions were and extends our knowledge of the intact mass thylakoid supplemented with 1 mM sodium bicarbonate to buffer the membrane proteome. While most previous intact thylakoid solution [26]. Plants, six weeks from sow date, were healthy protein mass studies relied upon analysis of isolated com- and relatively uniform in size (Fig. 1). plexes or discreet membrane domains; in the present study, we investigate for the first time the utility of LCMS+ as a tool 2.2. Fe-reductase Activity for examining whole thylakoid membrane proteome expression in response to a physiological perturbation, Fe deficiency. One gram of blotted dry roots was added to 10 mL of nutrient solution, buffered with 5 mM 2-(N-morpholine) ethanesulfonic acid (MES), pH 6.0. Bathophenanthrolinedisulfonate (BPDS) 2. Materials and methods and Fe(III)-EDTA were added to final concentrations of 0.3 mM and 0.2 mM, respectively. The concentration of reduced Fe was 2.1. Hydroponics on a chip determined spectroscopically by absorbancy measurements ε − 1 −1 at 535 nm ( 535 =22.14 mmol cm ). A. thaliana cv. Columbia plants were grown under a 10 h photoperiod with a photon flux of 150–200 μmol photons 2.3. Isolation of intact chloroplasts − − m 2·s 1 (photons measured as “photosynthetically active” radiation (Li-Cor PAR sensor)); the temperature was 21 °C in Approximately equal amounts of tissue were harvested from the light and 19 °C in the dark. Plants were cultured as follows. chlorotic, Fe-deficient plants or green, control plants. Each Sterilized seeds were placed on 1.25% agar containing 1/16- preparation was prepared from 36 to 72 individual hydro- strength Hoagland's solution and left horizontally overnight. ponically grown plants. Older leaf tissue was not used. The following day the agar plates were set vertically for one Chloroplasts were isolated as previously described [27]. Leaf week. One week after sowing the A. thaliana plants were samples were suspended in ice cold extraction buffer (330 mM 400 JOURNAL OF PROTEOMICS 72 (2009) 397– 415

Fig. 1 – Arabidopsis thaliana ‘Hydroponics on a Chip' system. A. Seeds are sown onto agar plates containing 1/16× Hoagland's and allowed to germinate with agar plates vertically supported. B. Plants one week after sowing. After one week of growth on agar plates the plants were delicately inserted into foam disc slits and placed into the pipette box apparatus containing 1/8× Hoagland's solution. C. Enlarged view of one week old seedlings. D. Two week old plants. Two week old plantlets were gently transferred to poker chips; roots are carefully fed through a hole in the center of a poker chip. The chips are placed on the foam tray for use in the Hydroponics on a Chip system, containing 1/4× Hoagland's solution. E. Four week old plants (two weeks after transfer to Hydroponics on a Chip) were transferred into Fe-sufficient or Fe-deficient 1/4× Hoagland's solution. F. Five week old Fe deficient (left) and sufficient (right) plants ready for harvest.

Sorbitol, 10 mM Sodium Pyrophosphate, 5 mM MgCl2,2mM 330 mM Sorbitol, 0.2% BSA, pH at 4 °C was adjusted to 7.6 with NaAscorbate, 0.1% bovine serum albumin (BSA), pH at 4 °C was KOH). The resuspended pellet was layered onto either a 40% adjusted to 6.5 with HCl) and homogenized with a Brinkman Percoll pad (40% Percoll, 2 mM Disodium EDTA, 1 mM MgCl2, polytron. The homogenate was filtered through 8 layers of 50 mM HEPES, 0.33 mM sorbitol, 0.2% BSA, pH 7.6 at 4 °C) or a 36% diaper liners (Gerber) and spun in a centrifuge at 1200× g for Optiprep pad (Axis-shield, Olso, Norway), 0.27 mM Sucrose,

10 min at 4 °C. The pellet was resuspended in Resuspension 2mMNa2EDTA, 1 mM MgCl2,50mMHEPES,0.2%BSA,pH7.6)

Buffer (2 mM Disodium EDTA, 5 mM MgCl2,1mMMnCl2,50mM before spinning at 1200× g for 10 min at 4 °C. The pellets from 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), Percoll and Optiprep were resuspended in Resuspension Buffer. JOURNAL OF PROTEOMICS 72 (2009) 397– 415 401

Table 2 – Leaf Chl content of control and Fe-deficient linear gradient from 5 to 25% Buffer B between 5 and 10 min plants after one week of treatment. after injection, 25 to 75% Buffer B between 10 to 130 min and − Sample Chl a/b Leaf Chl (nmol cm 2) 75 to 100% Buffer B between 130 and 150 min. Mass spectra were recorded on a PerkinElmer Life Sciences Sciex API III+ Control 1.26±0.3 14.9±2 triple-quadrupole mass spectrometer with Ionspray™ source Iron deficient 2.04±0.4 5.15±4 (Applied Biosystems, Foster City, CA) as described [21,22,31]. Chlorophyll measured by acetone extraction of representative leaf The instrument was scanned from 600–2300 m/z with a dwell plugs from leaves prior to intact chloroplast isolation (n=3). timeof1msgivingatotalscantimeof6s.Anorificepotential of 65 V was used in all experiments. The measured protein molecular mass reconstructions were computed using Bio- MultiView 1.3.1 software (Applied Biosystems, Foster City, − − 2.4. Pigment concentration CA) or empirically using formula M=(mn +1 1.008/ mn mn +1) − (mn 1.008), where m is the mass to charge. Calculated and Intact chloroplasts, thylakoids, or leaf plugs (ground after experimental average molecular masses were generated freezing with liquid nitrogen) were extracted in 80% acetone/ from published protein sequence databases and online water and pigment concentrations were measured spectro- proteomic tools (PIR, TAIR, Swiss-Prot, and TagIdent (ca. photometrically (8452A Diode Array Spectrophotometer, Hewlett expasy.org/tools/tagident.html) under ARATH). Packard) using absorbancies at 663 nm and 647 nm, minus the 730 nm background value [28]. 2.8. Trypsin cleavage

2.5. Protein determination Trypsin cleavage was performed as described by [21]. For LCMS+, the HPLC eluent containing separated proteins is split before ESI

Intact chloroplasts were lysed in dH2O, and the resultant mass spectrometry, and the deflected stream is collected with a thylakoids and soluble proteins separated by centrifugation. fraction collector. The collected fractions can be stored for Protein content was measured by a modified Lowry Assay [29] further manipulation and analyses (the “plus” of LCMS+). and measured spectrophotometically at 730 nm and 660 nm. Selected fractions (10 μL aliquots) were reduced with dithio- Protein concentration was estimated using a standard curve of threitol (10 mM in 50 mM ammonium bicarbonate; 30 min, BSA. 24 °C), alkylated with 15 μL iodoacetamide (55 mM in 50 mM ammonium bicarbonate; 20 min, 24 °C), and treated with 12.5 μL 2.6. Oxygen evolution trypsin (Promega sequencing grade modified by reductive methylation) (6 ng trypsin/μl in 50 mM ammonium bicarbonate; Oxygen evolution was measured polarographically with a 3 h, 37 °C). After incubation, samples were dried by centrifugal Rank oxygen electrode in a temperature-controlled, water- evaporation and stored at −20 °C prior to high pressure micro- jacketed cuvette. Intact chloroplasts were lysed in hypo- liquid chromatography-MSMS. osmostic shocking buffer (10 mM Tris–HCl pH=8) and used for each assay. PSI was assayed as ascorbate reduced 2,6- 2.9. Analysis of trypsin digested samples by tandem mass dichlorophenolindophenol (DCPIPH2)tomethylviologen spectrometry

(15 mM Na-pyrophosphate, 2.5 mM MgCl2,5mMNaCl, μ 300 mM sorbitol, 1 mM NaAscorbate, 2.5 mM NH4Cl, 0.2 mM Dried tryptic digests were redissolved in 30 l buffer C (water/ methyl viologen, 0.1 mM DCPIP, pH 8). Whole chain electron acetonitrile/formate, 95/5/0.1, v/v/v), and 5 μlwereinjectedonto transport was assayed as water to methyl viologen (15 mM Na- a microbore reverse-phase column (200 μm×10 cm; PLRP/S pyrophosphate, 2.5 mM MgCl2, 5 mM NaCl, 300 mM sorbitol,

1 mM NaAscorbate, 2.5 mM NH4Cl, 0.2 mM methyl viologen, pH 8); and PSII was assayed as water to para-benzoquinone ρ ρ ( BQ) (15 mM HEPES, 2.5 mM MgCl2, 0.2 mM BQ, 5 mM NaCl,

300 mM sorbitol, 2.5 mM NH4Cl, pH 7).

2.7. Reverse phase liquid chromatography coupled to electro-spray ionization mass spectromety

Primary reverse-phase chromatography was performed as described previously [21,22,30]. Reverse-phase chromatography performed using a polystyrene/divinylbenzene copolymer (Polymer Labs PLRP/S; 5 μm×300 Å; 2.1×150 mm) stationary- phase column eluted at a flow rate of 100 μL/min at 40 °C. The primary gradient (Buffer A, 0.1% triflouroacetic acid/ Fig. 2 – Ferric reductase activity. Representative trace of water; Buffer B, 0.05% triflouroacetic acid in 1:1 acetonitrile/ control (●) and intermediately iron deficient (■) plant roots. 2-propanol, v/v) eluted extrinsic polypeptides and predomi- The concentration of reduced Fe, based on root fresh weight, nantly small to moderately sized intrinsic proteins. The was measured spectroscopically by absorbancy ɛ − 1 −1 column was equilibrated in 5% Buffer B followed by a stepped measurements at 535 nm ( 535 =22.14 mmol cm ). 402 JOURNAL OF PROTEOMICS 72 (2009) 397– 415

Table 3 – Putative and sequence dependent protein assignments of Fe-deficient and Fe-sufficient whole thylakoid membrane proteins from LCMS and MS/MS analysis. Protein Locus Intact Calc. Cont. Control, Int. -Fe, Int. -Fe, -Fe, -Fe, MW Type Description of mass gravy RT MW RT MW RT Protein

APX4⁎ At4g09010 31,804.9 −0.373 59 31,791? PLS l-ascorbate peroxidase ATPA⁎ AtCg00120 55,328 −0.047 83 ? 82 55,326 PSS ATP synthase alpha chain ATPB⁎ AtCg00480 53,934 −0.089 64.54 53,885; 64.1 53,878;53920 66.02 53,897;+30; PSS a ATP synthase beta +34;+93 +72;+114 chain ATPC1 At4g04640 35,709.19 −0.105 61.9 35,708 62.37 35,707;+31 62.67 35,708;+20; PSS ATP synthase gamma +73 chain 1 ATPD At4g09650 20,570.59 −0.049 61.9 20,570; 62.37 20,571;+29 62.67 20,570 PSS ATP synthase delta +27 chain ATPG At4g32260.1 16,352.87 −0.107 45.47 16,350; 45.5 16,350 46.46 16,343;+18 I b ATP synthase b chain +27;+53 precursor ATPH AtCg00140 7977 0.998 103.64 8003 103.49 8004;+16; –– I ATP synthase C chain +30 ATPE⁎ AtCg00470 14,498 −0.034 58 14,249;+160 PSS ATP synthase epsilon chain DRT112⁎ At1g20340 10,451.65 ––– 43.01 10,452 –– –PC isoform, ct precursor K12B20⁎ At5g37720 19,404 −0.768 –– 41 19,414 –– C RNA export factor/ binding F4B12.1⁎ At3g15190 21,813 −0.341 –– 42 21,806 –– –Chloroplast 30S Riboprotein F2N1.18⁎ At4g01150 17,686 0.200 81 ? PG Expressed Protein F2N1.21⁎ At4g01050 49,357 −0.172 80 48,582? T Hydroxyproline-rich glycoprotein F13|12.21⁎ At3g47070 10,554 −0.527 40 ? –– –– PSS Expressed Protein F16M2.10⁎ At3g63160 7252 −0.157 58 7125? – Expressed Protein F21B7.22⁎ At1g03610 18,823 −0.252 43 18,828 –– –– –Expressed Protein F21.18⁎ At3g61870 29,567 0.093 81 29,596? –– –– C Expressed Protein F2P9.6⁎ At1g74070 18,665 −0.171 –– 45 18,642 –– –Peptidyl-prolyl cis- trans isomerase F23J3.7⁎ At4g09040 34,011 −0.603 –– 45 34,008 –– –RNA recognition motif F4F15.26⁎ At3g52150 27,731 −0.258 –– 47 27,733 –– RNA recognition motif F9L11.15⁎ At1g32990 16,698.7 0.101 53 16,694 – 50s RiboProtein, L11 family HCF136⁎ At5g23120 35,843 −0.362 52 36,850? PLS PS II stability/ assembly K2A18.27⁎ At5g66190 40,301 −0.364 57 40,353? PSS Ferredoxin NADP+ reductase LHCA-3⁎^ At1g61520 25,056.64 −0.013 66.27 25,056 66.22 25,061;+19; 66.27 25,056 I Chl A-B binding +31 protein LHCA-4 At3g47470 22,255.36 −0.156 65.46 22,255; 65.51 22,256 64.75 22,253 I Chl A-B binding +28 protein 4 LHCB-1.1⁎^ At1g29910 24,905.18 0.022 69.42 24,905; 69.88 24,904;+25; 69.53 24,903;24918; I Chl A-B binding +27;+52 +48 24965 protein 2 LHCB-1.2 At1g29920 24,905.18 0.022 69.42 24,905; 69.88 24,904;+25; 69.53 24,903;24918; I Chl A-B binding 165/ +27;+53 +49 24966 180 LHCB-1.3 At1g29930 24,905.18 0.02 69.42 24,905; 69.88 24,904;+25; 69.53 24,903;24918; I Chl A-B binding +27;+54 +50 24967 protein 2 LHCB-1.5⁎^ At2g34420 24,916.2 −0.113 70.8 24,914; 71.4 24,905 72.27 24,915 I Chl A-B binding +24;+46 protein LHCB-2.1⁎^ At2g05100 24,930.18 −0.117 69.27 24,932 69.88 24,929 71.66 24,932 I Chl A-B binding protein LHCB-2.2 At2g05070 24,944.25 −0.085 76.35 24,941; 75.66 24,946 75.42 24,941 I Chl A-B binding +33 protein LHCB-2.4 At3g27690 24,932.26 −0.121 69.42 24,932 69.88 24,929 71.66 24,932 I Chl A-B binding protein LHCB-3 At5g54270 24,280.65 0.033 72.12 24,280; 72.61 24,280 73.23 24,279;+20; I Chl A-B binding +27;+52 +41 protein LHCB-4.1⁎^ At5g01530 28,212.07 −0.02 70.8 28,212 71.4 28,210;+29 72.7 28,212 I Chl A-B binding CP29 JOURNAL OF PROTEOMICS 72 (2009) 397– 415 403

Table 3 (continued) Protein Locus Intact Calc. Cont. Control, Int. -Fe, Int. -Fe, -Fe, -Fe, MW Type Description of mass gravy RT MW RT MW RT Protein

LHCB-4.2⁎^ At3g61870 29,585.3 0.093 81 29,596 –– –– I Chl A-B binding protein LHCB-5⁎^ At4g10340 26,447.35 −0.025 73.24 26,447; 73.53 26,447;+25 74.66 26,443;+28; I Chl A-B binding CP26 +31 +44 LHCB-6⁎^ At1g15820 23,106.32 0.033 69.42 23,105 69.88 23,105 70.04 23,112 I Chl A-B binding protein MAA21.12⁎ At3g63490 37,609 −0.224 49 36,958; –– –– –Riboprotein L1 family +32 MLM24.13⁎ At3g23400 30,436 −0.194 60 ? PSS Plastid Lipid Assoc. protein PAP MNC6.3⁎ At5g53490 17,427.69 −0.093 –– 49 17,442;+15 –– L Thylakoid Lumen 17.4kD MPE11.21⁎ At3g26060 23,663 −0.402 40.26 23,660 –– –– –Peroxiredoxin NDPK3⁎ At4g11010 17,126.55 ––– 48.44 17,129 –– –Nucleoside diphosphate kinase PAP1⁎ At4g04020 28,776.2 −0.267 59 28,776; +40 –– – – PETB AtCg00720 24,153 0.571 69.53 22,159 I Cyt b6 c PETC⁎ At4g03280 19,000.68 −0.106 50.74 19,001; 50.39 19,000 51.13 19,004 PLS Cyt b6-f Fe–S subunit +34;+44 –– PETD AtCg00730 17,431 0.551 67.74 17,291; 67.44 17,292 I Cyt b6-f subunit 4 +30 PETF At1g60950 10,277.2 −0.033 –– – – 42.1 10,288 PSS Fd, ct precursor

PETG AtCg00600 4204 1.086 96.77 4196;4237 97 4196;4237 97.28 4195;+41 I Cyt b6-f subunit V

PETM At2g26500.1 4188.89 0.233 83.42 4188 83.71 4189 84.58 4188 I Cyt b6-f subunit (petM)

PETN AtCg00210 3213.82 1.544 101.99 3205;3218 101.31 3209 101.85 3206 I Cyt b6-f subunit VIII PLAS-1⁎ At1g76100 17,564 0.221 –– 42 17,646 –– PLS Plastocyanin PSAB⁎ AtCg00340 82,475 0.120 90 82,426? –– –– I PS I D1 subunit PSAC⁎ AtCg01060 8901 −0.076 40.26 8906,+19 –– –– PSS PS I Subunit VII PSAD1⁎ At4g02770 17,847.39 −0.368 41.07 17,848; 41.94 17,848;+27; 42.1 17,848 PSS PS I RC subunit II +29;+112 +112 PSAD2⁎ At1g03130 17,662.2 −0.387 41.07 17,663 41.94 17,658 –– PSS PS I RC subunitII PSAE1 At4g28750 10,469.76 −0.174 33.44 10,472; –– 34.62 10,468 PSS PS I RC subunit IV +21;+76 PSAG At1g55670 10,991.37 0.019 66.27 10,992 66.22 10,991 67.75 10,992;+21 I PS I RC V, precursor PSAH1⁎ At3g16140 10,355.82 −0.064 50.74 10,356; 50.39 10,355;+28; 51.18 10,355 I PS I RC subunit VI +27 +58 PSAH2 At1g52230 10,354.79 −0.106 49.72 10,355; 50.39 10,355;+28; 51.18 10,355 I PS I RC subunit VI +28;+58 +57 PSAI ATCG00510 4134 1.07 38.63 4131;+27 39.45 4126 39.55 4134;4127 I psaI PSI I protein PSAK At1g30380 8461.8 0.346 60.48 8462 60.45 8462 60.48 8462 I PS I RC subunit PSAL⁎^ At4g12800 17,969.81 0.31 64.49 17,970 64.45 17,968 66.02 17,965;+24 I PS I RC subunit XI PSAN⁎ At5g64040 9705.03 −0.159 38.63 9701;+28 39.45 9702;+26 39.81 9703 PLS PS I RC subunit PSI-N PSAO At1g08380.1 10,104.7 0.316 78.18 10,105 78.31 10,104 79.75 10,104 I expressed protein PSBB⁎ AtCg00680 51,871.1 0.067 86 ? 87 ? 87 52,804? I CP47 PSBC AtCg00280 50,042.63 0.252 100.37 50072; 100.55 50,088;+30 101.9 50,036;+98 I PS II 44 kDa RC +14;+74 precursor PSBE⁎ AtCg00580 9265 0.046 78.18 9255;+28 78.31 9255 79.44 9255;+28 I Cyt b559 a subunit PSBF AtCg00570 4424 0.646 57.78 4422 58.01 4424 –– I Cyt b559 b subunit PSBH AtCg00710 7570 0.35 91.39 7570;+29; 92.13 7570;+80; 92.1 7571;+28; I phosphoprotein, PS II +80;+160 +160 +78;+160 OEC PSBI AtCg00080 4168 0.677 79.65 4161 79.65 4161 –– –PS II RC I protein PSBK AtCg00070 4237.16 1.388 94.54 4238 95.86 4237 95.35 4236 I PS II K protein PSBM AtCg00220 3783 1.429 105.3 3750 105.3 3747 105.41 3747 I PS II RC M protein PSBO1⁎ At3g50820 26,565.7 −0.347 42.19 26,564; 43.36 26,563;+27; 42.1 26,564;+33; PLS OEE protein +28:+54; +54 +53;+79 +79 PSBO2⁎ At5g66570 26,571.7 −0.326 41.07 26,571: 41.94 26,568;+28; 42.1 26,573;+23; PLS OEE protein 1-1 +26;+52 +56 +45 PSBP1⁎ At1g06680 20,212.41 −0.343 43.16 20,213; 43.36 20,214;+25 43.26 20,212 PLS PS II OEC 23 +26;+52

(continued(continued on on next next page) page) 404 JOURNAL OF PROTEOMICS 72 (2009) 397– 415

Table 3 (continued) Protein Locus Intact Calc. Cont. Control, Int. -Fe, Int. -Fe, -Fe, -Fe, MW Type Description of mass gravy RT MW RT MW RT Protein

PSBP2⁎ At2g30790 28,441 −0.327 44 28,467 –– –– –OEE protein 2-2 PSBQ⁎ At4g21280 16,309.57 −0.319 44.38 16,310; 44.68 16,309;+14 44.53 16,309;+25; PLS OEE protein 3 +29;+55 +39;+54 PSBQ2⁎ At4g05180 16,349 −0.239 46 13,450, 46 16,345 46 16,350,+85 S OEE protein 3-2 +27 PSBR At1g79040 10,341.64 −0.001 63.53 10,342; 64.1 10,342;+17; 64.75 10,341;+16; IPSII +28 +30 +22;+34 PSBS⁎ At1g44575 22,457.23 0.282 88.14 22,455 88.68 22,456;+16 88.14 22,455;+27 I PS II PSBT AtCg0690 3822 103.64 3826 103.49 3826 103.68 3827 PLS PS II 5 kDa, ct precursor PSBW At4g28660.1 15,109.29 −0.271 33.44 15,174; –– –– PLS PS II RC W +463; +514; +577 PSBX At2g06520 4183.94 0.519 76.35 4182;+30 75.66 4184 76.44 4183 I Membrane protein PSBY-A2 At1g67740 4893.68 0.361 65.46 4892;+5; 65.51 4894 67.75 4893;+18; I PS II core complex +9 +27 psbY RK16⁎ Atcg00790 15,284 −0.486 –– 42 15,291 –– –Chloroplast 50s Riboprotein RPL3A⁎ At2g43030 24,178.3 −0.351 44 24190 44 24,181 –– –Riboprotein L3 family RPL4⁎ At1g07320 25,511 −0.395 –– 48 25,521 –– S 50S Riboprotein L4 RRF⁎ At3g63190 21,722.88 ––– 47.02 21,723 –– –Ribosome recycling factor, chloroplast TCTP At3g16640 18,910.34 – 56.25 18911 57.23 18,908 –– –Translationally controlled tumor protein homolog T10F20.8⁎ At1g18070 49,519 −0.328 –– 48 49,510 –– –EF-1-alpha related GTP binding T20H2.20⁎ At1g20020 41,142 −0.379 57 41,050? PSS Ferredoxin-NADP+ reductase T20O10.24⁎ At3g63140 49,519 −0.17 49 41,414, –– –– –MRNA binding, +15 Epimerase T22H22.12⁎ At1g032280 14,774 −0.251 –– 50 –– C Histone H2A T26I12.21⁎ At3g55330 17752 −0.221 49 17738 –– –– PLS PSII OEC 23 T31J12.6⁎ At1g09340 42,592 −0.365 –– 51 ? –– UK Expressed protein T28K15⁎ At1g12250 30,046 −0.333 –– 45 30,051 –– L Thylakiod Lumen Protein T30F21.4⁎ At1g78630 26,772 −0.47 –– 46 26,721 –– –Riboprotein L13 family YUP8H12⁎ At1g05190 24,690 −0.435 –– 44 24,950 –– –Riboprotein L6 family

Calculated intact masses of proteins are derived from Tagident (ca.expasy.org/tools/tagident.html) searching ARATH, Uniprot (http://www.pir. uniprot.org), ExPasy Peptide Mass (http://www.expasy.org/tools/peptide-mass.html), tair (http://www.arabidopsis.org) and from [21,22,30,104]. Intact mass tags calculated by BioMultiView. Calculated grand average of hydropathicity (GRAVY) index, curated location in thylakoid, and description of protein are from [104]. ⁎Digested protein sequence verified by tandem MS/MS ( Supplementary Table 2): ?Unable to confirm by intact mass or by MS/MS; 0TPS: thylakoid-peripheral-stromal-side. a PSS: Peripheral-stromal side. b I: Integral. c PLS: Peripheral-lumenal side.

5 μm, 300 Å; Michrom Biosciences, San Jose, CA) equilibrated in (m/z 400–1500), data-dependent MSMS on the two most buffer C and eluted (2 μl/min) with an increasing concentration abundant ions with exclusion after two MSMS experiments. of buffer D (acetonitrile/formate, 100/0.1, v/v: time/% buffer D; 0/ Individual sequencing experiments were matched to an A. 0, 3/0, 8/20, 13/35, 23/75). The effluent was directed through a thaliana sequence database using Mascot software (Matrix stainless steel nanoelectrospray emitter (ES301; Proxeon, Sciences, London, UK). To consider all possible peptide Odense, Denmark) at 2.4 kV for ionization without nebulizer sequence permutations, the “no enzyme” option was used as gas, interfaced to a hybrid quadrupole/TOF mass analyzer (Q one of the search parameters. The tryptic peptide sequence STAR XL; Applied Biosystems, Foster City, CA) operated in the chemical formula was determined by Protein Prospector soft- information-dependent acquisition mode with a survey scan ware (http://prospector.ucsf.edu/). JOURNAL OF PROTEOMICS 72 (2009) 397– 415 405

3. Results

3.1. Iron deficiency

Plants grown using “Hydroponics on a Chip”, either in Fe- sufficient or Fe-deficient medium, provided homogenous plants for intact chloroplast isolation (Fig. 1). There are many hydroponic solution formulations (Table 1) for growing Arabidopsis. When 1/4-x modified Hoagland's solution was used, there were no observable phenotypical affects or any characteristic nutrient deficiency symptoms. We did not observe any difficulties in isolation of intact chloroplasts from Fe-deficient or Fe-sufficient plants. However, it was imperative to isolate chloroplasts from plants before the onset of the daylight cycle to reduce the concentration of starch in the isolates. When several isolations were conducted, the growth chamber lights were turned off, until after harvesting. After one week of Fe-deficiency treatment, the total Chl content per leaf area of Arabidopsis leaves decreased three- fold compared to Fe-sufficient leaves, while the Chl a/b ratio increased slightly (Table 2). The ferric reductase assay was Fig. 4 – Total ion chromatogram of thylakoid membrane performed after one week of Fe-deficiency treatment. Com- proteins isolated from A. thaliana plants one week after Fe pared to Fe-sufficient plants, the activity was almost thirty- deficiency treatment (intermediate Fe deficiency). fold higher in roots of Fe-deficient plants after 30 min (Fig. 2). The Fe-deficient plants exhibited ferric reductase activity − − rates around 12 nmol g 1 root fresh wt min 1, in the range of Oxygen evolution data for PS I, PS II, and whole chain electron other studies of Fe-deficient Arabidopsis [32,33]. The root transport showed no significant differences between the biomass of Fe-deficient plants was elevated, and roots were different separation techniques (data not shown). swollen. Roots from Fe-deficient plants were markedly yellow, likely due to flavins, as previously noted in other studies [34]. 3.3. Thylakoid intact mass proteome 3.2. Electron transport Mass spectra, collected from LCMS+ of intact thylakoid membrane proteins, were reconstructed using Biomultiview The effect of Percoll and Optiprep, which is considered to be a software to determine the experimental mass. The list of highly inert, on chloroplasts in density gradients was com- experimental masses was then matched to a list of possible or pared by measuring oxygen evolution of thylakoids from lysed known calculated masses of thylakoid membrane proteins to intact chloroplasts isolated in either Percoll or Optiprep. provide a putative identification (Table 3). Further identification of proteins detected in the TIC was achieved by examining appropriate fractions of the HPLC eluent by LC-

Fig. 3 – Total ion chromatogram of Fe-sufficient A. thaliana Fig. 5 – Total ion chromatogram of thylakoid membrane thylakoid membrane proteins, tentative and verified proteins isolated from A. thaliana plants two weeks after Fe identifications. deficiency treatment (severe Fe deficiency). 406 JOURNAL OF PROTEOMICS 72 (2009) 397– 415

Table 4 – Mass reconstruction (MR) intensity values of Fe-deficient and Fe-sufficient whole thylakoid membrane proteins from LCMS analysis. Protein Cont. RT Control, MW MR Intensity Int. -Fe, RT Int. -Fe, MW MR Intensity

ATPB⁎ 64.54 53,885;+34;+93 9260 64.1 53,878;53,920 13,700 ATPC1 61.9 35,708 10,556 62.37 35,707;+31 15,546 ATPD 61.9 20,570;+27 13,552 62.37 20,571;+29 55,196 ATPG 45.47 16,350;+27;+53 36,250 45.5 16,350 7247 ATPH 103.64 8003 117,234 103.49 8004;+16;+30 374,326 LHCA-3 ⁎^ 66.27 25,056 15,809 66.22 25061;+19;+31 3933 LHCA-4 65.46 22,255; +28 19,843 65.51 22,256 22,821 LHCB-1.1⁎^ 69.42 24,905;+27;+52 40,731 69.88 24,904;+25;+48 30,405 LHCB-1.2 69.42 24,905;+27;+53 40,731 69.88 24,904;+25;+49 30,405 LHCB-1.3 69.42 24,905;+27;+54 40,731 69.88 24,904;+25;+50 30,405 LHCB-1.5⁎^ 70.8 24,914;+24;+46 56,701 71.4 24,905 30,405 LHCB-2.1⁎^ 69.27 24,932 13,072 69.88 24,929 25,628 LHCB-2.2 76.35 24,941;+33 8696 75.66 24,946 23,257 LHCB-2.4 69.42 24,932 13,072 69.88 24,929 25,628 LHCB-3 72.12 24,280;+27;+52 56,625 72.61 24,280 38,598 LHCB-4.1⁎^ 70.8 28,212 8822 71.4 28,210;+29 11,307 LHCB-5⁎^ 73.24 26,447;+31 63,148 73.53 26,447;+25 10,198 LHCB-6⁎^ 69.42 23,105 16,299 69.88 23,105 9988 PETC⁎ 50.74 19,001;+34;+44 9537 50.39 19,000 20 PETD 67.74 17,291;+30 13,180 67.44 17,292 6236 PETG 96.77 4196;4237 304,591 97 4196;4237 294,031 PETM 83.42 4188 66,972 83.71 4189 57,388 PETN 101.99 3205;3218 2260 101.31 3209 1346 PSAD1⁎ 41.07 17,848;+29;+112 20,900 41.94 17,848;+27;+112 12,729 PSAD2⁎ 41.07 17,663 7360 41.94 17,658 1166 PSAG 66.27 10,992 47,630 66.22 10,991 31,320 PSAH1⁎ 50.74 10,356;+27 53,590 50.39 10,355;+28;+58 27,688 PSAH2 49.72 10,355;+28;+58 21,467 50.39 10,355;+28;+57 24,201 PSAI 38.63 4131;+27 187 39.45 4126 18 PSAK 60.48 8462 83,021 60.45 8462 63 PSAL⁎^ 64.49 17,970 21 64.45 17,968 78 PSAN⁎ 38.63 9701;+28 25,200 39.45 9702;+26 1991 PSAO 78.18 10,105 98,863 78.31 10,104 15,192 PSBC 100.37 50,072;+14;+74 2133 100.55 50,088;+30 2972 PSBE⁎ 78.18 9255;+28 40,156 78.31 9255 109,938 PSBH 91.39 7570;+29;+80;+160 4000 92.13 7570;+80;+160 9500 PSBI 79.65 4161 19,232 79.65 4161 728 PSBK 94.54 4238 29,755 95.86 4237 573,912 PSBM 105.3 3750 2359 105.3 3747 120 PSBO1⁎ 42.19 26,564;+28:+54;+79 32,311 43.36 26563;+27;+54 41,464 PSBO2⁎ 41.07 26,571:+26;+52 12,692 41.94 26,568;+28;+56 10,148 PSBP1⁎ 43.16 20,213;+26;+52 14,577 43.36 20,214;+25 5698 PSBQ⁎ 44.38 16,310;+29;+55 47,439 44.68 16,309;+14 48,088 PSBR 63.53 10,342;+28 69,518 64.1 10,342;+17;+30 44,401 PSBS⁎ 88.14 22,455 43,281 88.68 22,456;+16 76,591 PSBT 103.64 3826 60 103.49 3826 51918 PSBX 76.35 4182;+30 102,095 75.66 4184 385,454 SBY 65.46 P4892;+5;+9 205,692 65.51 4894 210,204

Intensity values are from the average of 8 scans around reported retention time value and 12 iterations of reconstruction using BioMultiView 1.3.1 software.

MSMS for sequence dependent identification (Table 3). The TIC's from control and Fe-deficient samples were clearly sample variability is illustrated in Supplementary Figs. 3 and 4. different, which showed that the protein composition of The putative protein identifications from intact mass and Arabidopsis thylakoids was significantly altered by Fe- unequivocal identifications by sequence dependent identifi- deficiency. The experimental mass reconstruction intensity cation were labeled on the TIC (Fig. 3). values for several proteins identified in both IFD and control Plants after one-week of Fe-deficiency (intermediate Fe- were determined (Table 4). As discussed below, significant deficiency, IFD) and two-weeks after treatment (severe Fe- changes in thylakoid membrane proteins were observed deficiency, SFD) were analyzed by LCMS+ (Figs. 4 and 5). The (Figs. 6 and 7). TIC's provide a qualitative assessment of the thylakoid Interestingly, proteins from the ATPase appeared relatively membrane proteome based on an equal protein basis. The constant, as previously noted in other studies [35,36]. The JOURNAL OF PROTEOMICS 72 (2009) 397– 415 407

synthase gamma chain (ATPC) revealed global changes in the thylakoid membrane proteome (Fig. 8). Notably, there was an increase in the PS II 22 kDa protein (PSBS) intensity detected by the mass spectrometer (Table 4; Figs. 3, 5–8). PSBS is associated with PS II protection from photodamage in Arabidopsis, where it is directly involved in re-association of PS II and LHCII [37]. PSBS has also been associated with PSII protection in the single-celled alga, Chlamydomonas reinhardtii [38]. LCMS+ of intact thylakoid membrane proteins provided experimental masses allowing us to identify or deduce post- transcriptional modifications. The phosphorylation pattern of a 7.5 kDa phosphoprotein, identified as PS II oxygen evolving complex protein (PSBH) was altered by Fe-deficiency (Fig. 9). PSBH has two phosphorylation sites (Thr-3, Thr-5), and we measured three forms of the protein—no modification, single phosphorylation (+80 Da), and double phosphorylation (+160 Da). The majority of the protein observed in the control was in the non-phosphorylated state, while the majority of PSBH in the IFD sample was in the doubly phosphorylated form. The total abundance of the non-phosphorylated form in the two samples, based on ion intensity, was similar; so there was an increase in total PSBH (Fig. 7), as well as an increase in the phosphorylated forms in the IFD sample (Fig. 9). In the SFD sample, the PSBH was mainly in the non-phosphorylated state, but with half the intensity compared to the other two samples. Little singly- or doubly-phosphorylated forms were observed in the SFD sample; additionally, there was a modified form (+28), most likely due to formylation of the protein. Photosystem II reaction center protein K (PSBK) and reaction center protein T (PSBT) showed a dramatic increase in the IFD sample. The change is difficult to observe in the TIC's normalized to ATPC (Fig. 8), but revealed when comparing mass reconstruction intensity values (Table 4; Figs. 6 and 7). PSBK is part of the PSII reaction center. PSBK co-purifies with the core antenna complex (CP43/PSBC), required for PSII accumulation, and may be involved in assembly and stability of PSII complex [39]. The small hydrophobic protein PSBT is associated with the D1/D2 (PSBA/PSBD) heterodimer. PSBT is required for post- translational repair of photodamaged PSII. It appears to stabilize

the structure of the QA-binding region on D2 (PSBD), enhancing

efficient recovery of QA photoreduction, required for the efficient biogenesis of PSII complex [40,41].AChlamydomonas knockout of psbT showed decreased electron transfer and reduced amounts of atomic manganese in PSII [41]. Fig. 6 – Matrix plot of mass reconstruction normalized (0–1; Most of the LHC protein masses were reconstructed and – min max) intensity values, from table 4. The control and observed under Fe-sufficient and Fe-deficient conditions intermediately Fe-deficient samples were normalized so that (Table 3). Compared to control thylakoids, the overall abun- the total ion counts of each sample were equal. The first dance or intensity level was decreased in the IFD and SFD column represents control and the second column represents samples (Figs. 6 and 7). Based on our proteomic study, we were the intermediately Fe-deficient sample. The third column, Δ, unable to detect a possible candidate that would have a contains the Fe-deficient minus control values. A positive homologous function to the isi gene family in Cyanobacteria difference indicates more protein observed in iron deficient [42]. Timperio et al. [43] reported that in Fe-deficient spinach sample than control (blue), while a negative difference LCHB4, LHCB6, and LHCB2 were reduced, while LHCB3 indicates more protein in control sample (red). Data plotted increased [43]. In our study with Arabidopsis, we did not using python pylab module. observe similar findings for LHCII proteins. LHCB2.1, LHCB2.2, and LHCB2.3 slightly increased, while the remaining LHCII and all LHCI proteins decreased (Figs. 6 and 7). Note that LHCB1.1, protein intensity difference and relative change observed for LHCB1.2, and LHCB1.3 cannot be distinguished because the ATPB and ATPC were similar in the IFD sample and control amino acid sequences of the processed proteins are identical. (Figs. 6 and 7). Normalizing TIC's to the intensity of ATP The total ion count under the peak was averaged for the three 408 JOURNAL OF PROTEOMICS 72 (2009) 397– 415

Fig. 7 – The relative change between control and intermediately Fe deficient Arabidopsis thaliana thylakoid membrane proteins. The ratio of intermediately Fe deficient to control intensity values (Table 4) are plotted. A positive value indicates an increase in intermediately iron deficient proteins, while a negative value indicates a decrease in control. The absolute value of 1 represents no change. Values greater than 4 are labeled with separate data point labels.

samples in the control; in the IFD LHCB1.5 was averaged with the three (total counts divided by four). More PS I protein subunit intact masses were measurable in the control sample than in the Fe deficient samples. PS I subunit XI protein (PSAL) was deficient in the IFD samples (Fig. 4) and very little was detected in the SFD sample (Fig. 5). PS I reaction-center subunit D1 protein (PSAB) and PSI subunit VII protein (PSAC) intact masses were only detectable in the control sample. However, PS I reaction-center subunit II protein (PSAD2) was found in both control and the IFD. In control and IFD samples, PS I reaction center protein subunit VI (PSAH2) intact mass reconstruction revealed the parent protein, and the parent with a plus 28 Da and a 57 Da adduct. The modifications were not observed in the SFD sample. Protective enzymes against oxidative stress were identified. Enzymes that reduce hydrogen peroxide, ascorbate peroxidase (APX4) and peroxiredoxin Q (PRXQ), were found in the IFD sample. Other soluble anti-oxidant enzymes were not identified or found in any of the preparations. Fig. 8 – Control and intermediately iron deficient TIC's In the IFD samples, we identified many proteins involved in normalized to ATPB mass reconstruction intensity value. protein synthesis (Table 3). Large and small chloroplast Intensity values were divided by the mass reconstructed ribosomal proteins, and even the elongation factor (EF-1) intensity value for ATPB from BioMultiView 1.3.1 software were detected. Interestingly, ribosomal recycling factor (RRF) (Table 4). TIC normalization provides an assessment of global was detected in the IFD sample. changes in the thylakoid membrane proteome under Fe-deficiency. For example, the peaks containing light harvesting complex proteins (LHC's; retention time (RT) 4. Discussion 70 min) and photosystem II oxygen-evolving complex proteins (RT 42 min) are dramatically reduced when In the present work, a hydroponic system was developed for compared to control. Peaks for protective proteins, such as Arabidopsis that was simple to construct and resulted in PSBS, increased. A more reliable comparison based on excellent plant growth and uniformity. Plants grown with deconvoluted data is illustrated in Figs. 6 and 7. the system were used for a proteomic analyses of Fe deficient JOURNAL OF PROTEOMICS 72 (2009) 397– 415 409

Overall, the TIC's corroborated earlier work on Fe-deficient thylakoids, in that the observed difference amongst the TIC's were similar to differences observed in thylakoids separated by LiDS-PAGE [29]. One of the major benefits of the intact protein mass analyses is that we can now readily observe post-transcriptional modifications in proteins from physiologically different plants. In the IFD sample we identified a dramatic increase in the doubly phosphorylated form of PSBH (Fig. 9). PSBH has been suggested to be involved in the stable assembly of native

dimeric PSII complexes [44], involved in electron flow from QA

to QB [45], disruption of the psbH gene in C. reinhardtii led to inactive PSII complexes [46], and phosphorylation is regulated by redox and is light-dependent [47]. Gómez et al. [21] observed the doubly phosphorylated form of PSBH under elevated light conditions compared to low light grown plants [21]. The phosphorylation status of PSBH and its effect on PS II stability is uncertain, but from our proteomic study it suggests that double phosphorylation may be involved in maintaining and/ or enhancing stability under Fe-deficient stress-induced conditions. Other possibilities exist, for example, it is possible that a different redox state exists in the IFD sample, which alters the phosphorylation state. Under extreme Fe-deficiency, the plants were quite senescent, and little or no phosphorylations were observed. More PSBS, a pigment chaperonin [48], was detected in Fe- Fig. 9 – Reconstructed molecular mass of A. thaliana thylakoid deficient samples than control. PsbS knockout plants exhibit a membrane protein PSBH (calc. avg. mass 7.5 kDa). severe decrease in NPQ [49]. Arabidopsis plants over expressing Post-transcriptional modifications of PSBH were PSBS had a 2-fold increase in feedback de-excitation, more detected—single phosphorylation (avg. mass 7650 Da) and resistance to photoinhibition, and greater protection from doubly phosphorylated (avg. mass 7730 Da). The small peak over reduction of PSII electron acceptors [50]. Under Fe- in C (avg. mass 7566 Da), that is also detected in A, is deficiency Chl levels decrease and chaperonins may provide presumed to be due to a formylation (+28 Da). A: Severely stability to remaining pigments, as well as maintain photo- iron deficient; B: Intermediately Fe deficient; C: Fe sufficient system activity. control. The dashed line in B and C delineates the ion counts Post translational modifications of PSAH2 were observed. for the parent molecule in Fe sufficient plants. In control and IFD samples, two modifications of +28 and +57 adducts were observed and none in the SFD sample. Arabi- dopsis plants devoid (expressing protein levels less than 3%) of PSAH have PSI containing 50% PSAL, and a decrease in NADP+ thylakoids. Proteomic studies of nutrient deficient tissue at photoreduction activity [51]. Interestingly, we also observed a various developmental stages and/or different stages of decrease in detectable PSAL in Fe-deficient samples. Whereas nutrient deficiency will be greatly enhanced by utilization of PSBH was formylated under severe Fe-deficiency, PSAH2 the system described. Additionally, other types of stress exhibited the opposite post-translational modification, with conditions can be easily applied. the Fe-sufficient and IFD samples with the +28 mass. Taken Fig. 1 shows that uniform Fe-deficiency was induced, and together, the formylations may have a physiological role, but that sufficient chlorotic tissue was generated to readily make they also may be an artifact of preparation. More study is measurements on the chlorotic tissue (see for example the required. TIC's from LCMS (Figs. 3–5); Supplementary Fig. 3). The system PSAL is necessary for trimerization of PS I and may also provided consistent Fe-deficient plants from batch to constitute the trimer-forming domain in the structure of PS I batch. [52]. PSAL does not bind any Chl a, but it forms hydrophobic Though the tissue samples were heterogeneous (whole interactions with carotenoid molecules [53]. Notably, PSI has a leaves of different size and development), the similarity of high Fe content, and it is one of the first protein complexes plants from each treatment allows for excellent statistical affected by Fe deficiency, and it is extremely deficient in Fe sampling on a “whole” plant basis; and plenty of tissue was chlorotic leaves. Cross-linking studies in plants suggested available for biochemical analyses. From our thylakoid close contact between external light harvesting complexes membrane proteome study, we have identified many post- LHCI and PSAL, supporting the idea that the function of PSAL transcriptional modifications, as well as potential protein is to facilitate the “input" of excitation energy from the candidates, involved in Fe homeostasis within the chloroplast. external antenna complexes in plants [53].Aproteomic As expected, many proteins involved in oxidative stress, study of Fe-deficient spinach thylakoid membrane proteins photosystem stability and maintenance were identified. showed a large reduction in PSI proteins, but the antennae 410 JOURNAL OF PROTEOMICS 72 (2009) 397– 415 composition of PSI was not compromised [43]. The presence of idase show increased resistance to Paraquat, a superoxide PSAL and PSAH, in Fe-deficient tissue may be related to generating herbicide; however, no benefit was observed for stabilizing the few remaining PS I complexes. Also, it may be a photoinhibitory treatments even under Fe or Cu overload [65]. dynamic relationship between the levels of the two proteins to Photosystem II can produce superoxide [66]. The presence of maintain PS I in a “standby”-state for Fe resupply (Fe efficient APX4 in IFD plants suggests the potential role for increasing plants can alter the rhizosphere to make Fe available). superoxide resistance under Fe-deficiency, especially for PS II. PSAD2 was observed in control and IFD thylakoids, but not Conversely, Arabidopsis plants overexpressing glycolate oxi- in SFD samples. PSAD is exposed on the stromal side of PS I dase in chloroplasts accumulate hydrogen peroxide and show and provides the docking site for ferredoxin [54]. In etiolated retarded growth, yellowish leaves, and increased photosensi- Aradidopsis seedlings, PSAD could not be detected [55], and tivity [67]. Also identified in the IFD sample was peroxiredoxin protein levels decreased in Fe deficient Chlamydomonas [56]. Q or PRXQ (MPE11.21). PRXQ reduces hydrogen peroxide using The knock-out of psaD exhibits a phenotype with a decrease in two cysteines, which form an intramolecular disulfide, and it growth rate, light-green leaf coloration, and increased photo- is re-reduced by thioredoxin. PRXQ attaches to the thylakoid sensitivity [57]. Under Fe-deficiency, ferredoxin is reduced, so membrane, is detected in preparations of PS II, mediates psaD expression may be decreased under Fe-deficiency or the plastid peroxide detoxification, and is linked to NAD(P)H lack of ferredoxin or unstable PSI itself may result in improper metabolism [68,69]. A five-fold increase in PRXQ protein was incorporation into the PSI complex. PSAD may degrade under observed in a proteomic study of Fe-deficient Chlamydomonas such conditions. [56]. Overexpression of a homologous PrxQ gene, from Suaeda Many proteins associated with chloroplast protein synth- salsa,inArabidopsis led to increased salt and low-temperature esis were observed in the IFD plants. In the IFD sample we tolerance [70]. Lamkemeyer et al. [69] suggest that PRXQ has a identified the ribosome recycling factor (RRF). RRF is essential specific function in protecting photosynthesis. In rice, the 2- for protein synthesis termination and suggested to be Cys peroxiredoxin, BAS1, is involved in plastid detoxification involved in chloroplast mRNA translation [58]. Interestingly, during darkness [71]. The identification of both antioxidant in Fe-deficient sugar beets 16S and 25S rRNA and ctDNA levels proteins in the IFD preparations suggests a higher level of were unaffected, but RNA synthesis was reduced by 50% [59]. protection may be required to maintain the PS II complex. The decrease in mRNA synthesis may require an increase in The comparison between Fe-deficient and control samples efficacy and stability of the chloroplast ribosome under Fe- was performed on an equal protein basis. Under Fe-deficiency deficiency. Also, the eukaryotic polypeptide elongation factor total protein per leaf area and Chl per leaf area decreases EF-1-α was identified in the IFD sample. EF-1 is essential for [36,72]. Generally, there are four strategies to examine protein protein synthesis, as it is involved in accurate amino acid profiles in relation to the physiological state of plants—equal selection and aminoacyl-tRNA binding during the elongation leaf area, equal protein, equal weight, or equal Chl. The phase [60,61]. Many of the chloroplast ribosomal proteins were fundamental physiological consideration, of course, is specific identified in the preparations, as well. The proteins, RRF and activity. Furthermore, protein in relation to a metabolic EF-1, may play a critical role in maintaining and stabilizing the activity, such as electron transport or carbon fixation can protein synthesis machinery under Fe-deficiency or other provide clues about systematic changes that may be physio- environmental stresses. logically relevant. For example, an analysis of thylakoid Many proteins from the chloroplast ATPase were consis- protein profiles based on equal electron transport would tent between control and Fe-deficient plants on an equal allow the evaluation of the photosynthetic machinery still protein basis, as seen in other studies [29,62]. Studies of Fe- operable and provide information on how such minimal deficient leaves from sugar beet ATP/ADP and NADPH/NADP+ machinery is maintained. The current experimental regime ratios showed no appreciable decrease compared to controls provides an assessment of protein population, but lacks [63]. In sugar beet, PS II is present but inactive under Fe- kinetic information on protein turnover or expression. Analy- deficiency [62]. Our analysis indicates that the majority of sis using an equal protein basis may introduce unintended ATPase subunits is retained under Fe-deficiency (Fig. 9), biases, since many photosynthetic proteins decrease (like suggesting that the ATPase may remain operable and partly light harvesting proteins), and ultimately the remaining accounts for the unchanged charge status of leaves. A proteins take on more significance, though they may have proteomic study of Fe deficient sugar beet identified three changed little. Proteomic analysis based on equal protein polypeptides from the ATPase complex that did not change in provides insight into general changes and patterns in protein signal intensity [64]. In contrast, a proteomic study of Fe- expression profiles. More specifically, it provides an excellent deficient spinach thylakoid membrane proteins showed a basis for understanding Fe mediated metabolic activities. decrease in ATPase proteins [43]. Nevertheless, any number of Follow up experiments might examine the protein expression questions arises. What is the efficiency of ATPase under Fe- patterns under Fe-resupply or the combined effect of Fe- deficiency? Is the pump maintained or in disrepair? Does it deficiency and an abiotic perturbation, such as drought, or a work with altered efficiency? Why keep these proteins at an biotic perturbation that causes disease. Studies with 15N will abundant level if the photosynthetic machinery is not work- provide kinetic insight into turnover and expression. ing? Similar type questions, framed on the basis of the present An analysis based on equal leaf area would provide study, can be posed for many of the other proteins observed. information related to protein per leaf area, as in Fe-deficient Many organisms use oxidative signals during environmen- plants the total protein per leaf area decreases. In plants the tal stress. We identified APX in the IFD sample. Arabidopsis leaf area is significant, because it is the basis of light plants overexpressing the thylakoid-bound ascorbate perox- interception. The dynamic range required for comparing Fe- JOURNAL OF PROTEOMICS 72 (2009) 397– 415 411 sufficient and extremely Fe chlorotic tissue on an equal protein IIa, LHCB5 or CP26/Lhc IIc, and LHCB6 or CP24/Lhc IId), as well basis is limiting, so a mathematical prediction of equal area (or as the minor Lhc IIb protein encoded by lhcb3, are present in equal Chl) protein profiles based on the real equal protein set is the membrane in reduced amounts [79–82].TheminorLHC an appropriate strategy. For Fe-deficiency, an equal Chl proteins (LHCB4/5/6) have higher Chl a/b ratios than LHC IIb analysis could evaluate the pattern or set of photosynthetic (LHCB1/2/3), and the LHCB4/5/6 proteins have either a lower proteins that change in relation to Chl during the onset of Fe- requirement for Chl b for proper assembly, or they have a deficiency or upon recovery during Fe-resupply. higher affinity for the small amount of Chl b that may be The complex nature of the thylakoid membrane proteome present. still provides challenges, but our analysis under Fe-deficiency Chloroplast greening during development is an ordered allowed us to identify key proteins involved in Fe-stress. It is process as the pigment proteins assemble in the membrane is not surprising that many proteins found in the Fe-deficient an ordered process with the assembly of the LHC apoproteins samples, that couldn't be found in the control, were directly dependent upon the presence of Chl b [78,83–85].The involved in maintenance, stability, and organization of the apoproteins of LHC II appear before the apoproteins of LHC I photosynthetic machinery. As discussed above, the proteins during chloroplast maturation [84,85].Duringchloroplast in Fe-deficient samples were detected, in part because our greening the LHC II proteins appear in the membrane in the analysis is based on an equal protein basis in samples with order LHCB4/5→ LHCB6/3→ LHCB1/2, assembling first at significantly lowered light-harvesting proteins (and others) monomeric pigment-protein complexes, then forming the due to the Fe-deficiency. Although, many questions remain mature trimeric light-harvesting complexes [84]. The LHC I unanswered, the present study illustrates how intact mass proteins appear in the order LHCA1/4 (LHCI-730)→LHCA2/3 proteomics can provide unique insight into functional aspects (LHCI-680) [85]. The minor LHC II apoproteins (LHCB4/5/6) are of thylakoid protein expression levels under Fe deficiency. Of proposed to form a “connector” between the core complex of particular interest are the PTM's, which require further PS II and the major Lhc IIb antenna [81]. The order of assembly investigation. in the membrane is believed to be an indication of the During the on-set of Fe deficiency, Chl levels decrease, proximity of each LHC apoprotein to the core complex and which is coupled with a decrease in LHC's and other reflect its position on the assembly hierarchy. photosystem complex proteins [62,72]. Previous Fe deficiency Two of the LHC proteins that are resistant to Fe-deficiency studies show that thylakoid proteins do not respond in stress are those that appear earliest in the chloroplast green- synchrony. The following proteins are listed by their general ing. In addition, LHCA4 and LHCB4 are the most stable in Chl sensitivities to Fe deficiency, with the first being most b-less mutants, which is consistent with the proposed location sensitive (that is it decreases most rapidly) LHC's, Cyt b6f,PS I of these proteins as the antenna most closely associated with (Cyt f and P700 tend to change in concert), PS II, and ATP the core complexes of each photosystem. The model of synthase [27,35,43,56,59,62,72]. greening chloroplasts is not applicable to the Fe-deficient The Chl content in IFD Arabidopsis plants decreased sig- chloroplast due to the early stage of growth, where the plants nificantly, while the Chl a/b ratio increased (Table 2). As the total were grown in complete media and fully green before being Chl content decreased, the amount of Chl b decreased faster switched to Fe-deficient media. The development of chlorosis than that of Chl a. Chlorophyll b is found only in the LHC during the onset of Fe deficiency is more a senescent proteins, and these proteins disappear as the Chl b content phenomenon, rather than developmental. decreases. Our data showed that the LHC-I and LHC-II proteins Senescence of the leaf involves initial degradation of were reduced in the Fe-deficient plants, but the amount varied stromal proteins followed by catabolism of Chl and then depending upon the antenna protein. The LHC's detected in Fe- degradation of thylakoid-bound proteins [86]. Dark-induced deficient plants ranged in sensitivity from very sensitive (LHCB5 senescence of the LHC II proteins in barley occurs at different and LHCA3), to moderately sensitive (LHCB1/3/6), to insensitive rates in order, from least stable to most stable; LHCB2.1/ (LHCA4, LHCB4/2). In contrast, LHCB3 increased in Fe deficient 3→LHCB6/4/1→LHCB2.2/5 [87], while the LHC I proteins are spinach [43].InChlamydomonas Fe deficiency increases LHCBM1, stable for up to 6 days in the dark [88]. Stay-green mutants in LHCBM3, and LHCSR3 more than 4-fold [46].LHCSR3isnotfound chloroplast senescence have been used to show that Chl b in land plants, and it is upregulated under stress in organisms must be converted to Chl a before catabolism of Chl b occurs like diatoms and others that exhibit high NPQ capacities [73]. [89].Instay-green mutants the LHC proteins are protected Chlorophyll b-less mutants have a similar pigment profile from proteolysis as long as they are bound with Chl b in the with high Chl a/b ratios and very little assembled LHC in the membrane. thylakoid membrane. The chlorina mutants lack the major LHC The differential rates of degradation of the LHC proteins protein complex of PSII, LHC IIb, which is assembled from the may be to preserve those LHC proteins involved in protecting gene products of lhcb1, lhcb2 and lhcb3; however, they have the remaining PS I and PS II core complexes from photo- normal electron transport rates for PSI and PSII [74–76]. The damage [90]. The observation that both gene expression and LHC proteins are greatly reduced in the mutants, but the protein accumulation of LHCB4 and the LHC-I proteins expressed mRNA levels of the genes for these proteins are at increase when senescent barley plants are moved from high wild-type levels [77,78]. The results from Chl b-less mutants light to low light [91] is in potential conflict with the role of suggest that there is a requirement for Chl b for stable these proteins in photoprotection. assembly of LHC proteins into the membrane. Pigment loss during senescence does not occur at equivalent While the major LHC protein complex is missing in Chl rates. Chl a,Chlb, neoxanthin (Neo), and β-carotene (β-car) all b-less mutants, the minor LHC proteins (LHCB4 or CP29/Lhc disappear at similar rates; while the xanthophylls, lutein (Lut), 412 JOURNAL OF PROTEOMICS 72 (2009) 397– 415 zeaxanthin (Zea), antheraxanthin (Ant), and violaxanthin (Vio) was elevated in the IFD sample. As a major component of the do not appreciably decrease during senescence [90]. A similar membrane bound Fo, it too appears to be quite stable under change in pigment ratios (large decreases in Chl a,Chlb,Neo Fe deficiency. The ATPB precursor, ATPG, was decreased, and β-car, but little change in Lut, Zea, Ant, and Vio) have been which suggests that synthesis of the ATPase may decrease. observed in Fe-deficient sugar beet leaves [92]. The similar Since the other proteins were present in the Fe-deficient pigment profile in scenescent chloroplasts and Fe-deficient sample, this may indicate a certain degree of stability of the chloroplasts suggest that similar degradation mechanisms ATPase, not necessarily increased synthesis. Proteins may be operating in both cases. Our results are consistent with involved in ROS protection, APX4 and PRXQ, increased the observation that photoprotection of the remaining PS I and under Fe deficiency. PS II core complexes may be the main function of the LHC A complete understanding of the regulation and mechan- proteins that are unchanged between control and Fe-deficient ism of the Fe deficiency response in plants remains elusive plants. [5,105–107]. Several components involved in the Fe deficiency All LHC proteins likely contain 2 Lut per LHC apoprotein response have been identified (for review see; [105,107]). [93]. The xanthophyll cycle pigments, Zea, Ant, and Vio, are Interestingly, many of the components identified are primar- involved in the main mechanism of non-photochemical ily localized to the root tissues, but several have been localized quenching (NPQ) of excess excitation energy [94]. The majority to the shoot tissue. Little is known about chloroplast Fe of the xanthophylls are found in the minor LHC II (LHCB4/5/6) uptake, but studies show the process is light-dependent, and and the LHC I proteins [93]. The minor LHC II apoproteins bind requires Fe(III) chelate reductase activity [105]. Our study only 15% of the Chl in PS II, but bind up to 80% of the provides insight into the reorganization of the thylakoid xanthophyll cycle pigments in PS II [94]. membrane proteome under Fe deficiency. The remaining xanthophyll cycle pigments in PS II appear The methodology presented in the present paper illus- to be bound by PSBS, a member of the LHC superfamily that trates the usefulness of intact mass thylakoid studies for has four transmembrane helices instead of three [95,96]. PSBS functional genomics. The TIC's were useful for examining binds both Chl and zea [48,97]. PSBS-less mutants in Arabi- overall changes in protein expression (Figs. 3–5). Fig. 7 dopsis illustrate that the pigment-protein is probably the site of illustrates how individual protein comparisons can be made much of the NPQ in leaves[50,98]. A model has been proposed between two physiologically different samples. The metho- where PSBS binds Zea when the lumen becomes acidic and is dology also demonstrates certain limitations. The differen- located in between the major LHC II antenna and the core tial abundance of proteins and complexity (heterogeneity) of complex [99]. The LCMS+ data showed that PSBS is a stable proteins samples within peaks can result in signal suppres- thylakoid membrane protein during Fe-deficient growth. sion. The problem is related to resolution of the chromato- Our LCMS+ experiments also demonstrated that LHCB2 graphy. Thus, one of the more useful strategies for apoproteins were more stable under Fe-deficient conditions improving our investigations will be to use isolated com- than the other LHC proteins (Fig. 7). It is a surprising and plexes from the thylakoids. Simplification of the sample will significant result suggesting that LHCB2 apoproteins are more allow better resolution and quantification. Such improve- resistant to degradation once fully assembled in the mem- ments in resolution combined with subtle modification of brane. The observation contrasts with the Chl b-less mutant isotope ratios will provide useful information about pool and greening studies, where the LHCB1 and LHCB2 have sizes and turnover. similar or identical profiles. There has been little, if any, biochemical evidence that the LHCB1 and LHCB2 apoproteins have different functions. The two light-harvesting isoforms have very similar primary sequence and putative assembled Appendix A. Supplementary data mature structure in the membrane [100–102]. The LHCB2 proteins and PSBS protein are both stable in Fe-deficient Supplementary data associated with this article can be found, thylakoid membranes. Although we have no direct evidence in the online version, at doi:10.1016/j.jprot.2009.01.024. that LHCB2 and PSBS interact, the similar change in ratio between control and Fe-deficient suggests that the LHCB2 proteins could be the attachment for the major light- harvesting antenna to PSBS. 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