Breakthrough Technologies

Bioorthogonal Noncanonical Amino Acid Tagging(BONCAT) Enables Time-Resolved Analysis of ProteinSynthesis in Native Plant Tissue1[OPEN]

Weslee S. Glenn, Shannon E. Stone, Samuel H. Ho, Michael J. Sweredoski, Annie Moradian, Sonja Hess,Julia Bailey-Serres, and David A. Tirrell*

Division of Chemistry and Chemical Engineering (W.S.G., S.E.S., S.H.H., D.A.T.), and Proteome ExplorationLaboratory (M.J.S., A.M., S.H.), California Institute of Technology, Pasadena, California 91125; and Center forPlant Cell Biology, University of California, Riverside, California 92521 (J.B.-S.)

ORCID IDs: 0000-0001-7733-7291 (W.S.G.); 0000-0002-6617-3874 (S.E.S.); 0000-0001-7647-0752 (S.H.H.); 0000-0003-0878-3831 (M.J.S.);0000-0002-5904-9816 (S.H.); 0000-0002-8568-7125 (J.B.-S.); 0000-0003-3175-4596 (D.A.T.).

Proteomic plasticity undergirds stress responses in plants, and understanding such responses requires accurate measurement ofthe extent to which proteins levels are adjusted to counter external stimuli. Here, we adapt bioorthogonal noncanonical aminoacid tagging (BONCAT) to interrogate protein synthesis in vegetative Arabidopsis (Arabidopsis thaliana) seedlings. BONCATrelies on the translational incorporation of a noncanonical amino acid probe into cellular proteins. In this study, the probe is theMet surrogate azidohomoalanine (Aha), which carries a reactive azide moiety in its amino acid side chain. The azide handle inAha can be selectively conjugated to dyes and functionalized beads to enable visualization and enrichment of newly synthesizedproteins. We show that BONCAT is sensitive enough to detect Arabidopsis proteins synthesized within a 30-min intervaldefined by an Aha pulse and that the method can be used to detect proteins made under conditions of light stress, osmoticshock, salt stress, heat stress, and recovery from heat stress. We further establish that BONCAT can be coupled to tandem liquidchromatography-mass spectrometry to identify and quantify proteins synthesized during heat stress and recovery from heatstress. Our results are consistent with a model in which, upon the onset of heat stress, translation is rapidly reprogrammed toenhance the synthesis of stress mitigators and is again altered during recovery. All experiments were carried out withcommercially available reagents, highlighting the accessibility of the BONCAT method to researchers interested in stressresponses as well as translational and posttranslational regulation in plants.

Elevated temperatures, limited water resources, andhigh salt concentrations in arable soils are expected toprofoundly and increasingly affect the productivity ofcrops in the coming years (Mickelbart et al., 2015).

Several promising marker-assisted breeding and ge-netic engineering strategies have been employed tohelp address this global challenge (Kasuga et al., 1999;Lopes and Reynolds, 2010; Mickelbart et al., 2015). Forexample, HVA1, a Late Embryogenesis Abundant genefrom barley (Hordeum vulgare) shown to delay leafwilting, was expressed in wheat (Triticum aestivum;Sivamani et al., 2000), rice (Oryza sativa; Xu et al., 1996),and corn (Zea mays; Nguyen and Sticklen, 2013) toproduce salt-tolerant lines with higher water usage ef-ficiency. Despite these early and promising successes,target selection remains a critical challenge associatedwith genetic engineering and marker-assisted breeding(Bita and Garets, 2013). Gaining further insight into thephysiological mechanisms that govern stress toleranceand adaptation will improve our ability to rationallyengineer crops.

Proteomic plasticity is a hallmark of the stress re-sponse in plants and was first shown over 35 years agoby incorporating radiolabeled amino acids into proteinssynthesized during anaerobic stress in maize (Sachset al., 1980) and heat stress in soybean (Glycine max; Keyet al., 1981). With advances inmass spectrometry-basedpeptide identification, new strategies have been devel-oped to measure the extent to which protein levels areadjusted in response to environmental stimuli (Huot

1 Thisworkwasfinancially supported by theGordon andBettyMooreFoundation through grant GBMF2809. W.S.G. was supported by a Na-tional Research Council Ford Foundation Post-Doctoral Fellowship and aUnited Negro College Fund/ Merck Foundation Post-Doctoral Fellow-ship. The Proteome Exploration Laboratory is supported by the Gordonand Betty Moore Foundation, through grant GBMF775, the BeckmanInstitute, and the NIH through grant 1S10RR029594-01A1.

* Address correspondence to tirrell@caltech.edu.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:David A. Tirrell (tirrell@caltech.edu).

W.S.G. conceived of and implemented the project and performedmost of the experiments with contributions from all other authors;S.H.H., J.B.-S., and W.S.G. designed and conducted gel imaging andimmunoblotting experiments; A.M., S.H., and W.S.G. designed andran the mass spectrometry experiments; S.E.S., M.J.S., S.H., J.B.-S.,and W.S.G. conducted all bioinformatic analyses of the mass spec-trometry data; S.E.S., M.J.S., and W.S.G. constructed figures with in-put from all other authors;W.S.G., J.B.-S., and D.A.T. wrote the articlewith valuable contributions from all authors.

[OPEN] Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.16.01762

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et al., 2014; Fristedt et al., 2015). But there are limitationsto these technologies, which for the most part yieldinformation about steady-state protein abundances.Moreover, traditional shotgun proteomics strategiesprovide reduced coverage in samples where a fewhighly abundant proteins predominate. For example,Rubisco comprises 30% to 60% of the leaf proteome andobstructs detection of less abundant proteins in leafsamples (Kim et al., 2013). To counter this problemspecifically, Kim et al. (2013) developed a protaminesulfate precipitation method to selectively depleteRubisco and thus enrich for less abundant proteins.Pulsed stable isotope labeling by amino acids in cellculture was introduced to monitor de novo proteinsynthesis, but poor label incorporation has prevented itfrom being widely adopted in plant systems. Otherdifferential proteomics techniques, including stableisotope labeling by amino acids in cell culture(Lewandowska et al., 2013), hydroponic isotope label-ing of entire plants (Bindschedler et al., 2008), 13CO2labeling (Chen et al., 2011), and isobaric tags for relativeand absolute quantitation (Ge et al., 2013), haveemerged in recent years as tools to probe in vivo proteinsynthesis in plants. Notably, multiplexed quantifica-tions with techniques such as isobaric tags for relativeand absolute quantitation are inherently problematicdue to known interference problems when using iontrap sources (McAlister et al., 2014). Modern instru-ments with MultiNotch MS3 capabilities can overcomethese limitations but impose significant sensitivitypenalties (McAlister et al., 2014). The instrumentationrequired for such sophisticated experiments is expen-sive and limits widespread use.

Identifying proteins that are translated within brieftime intervals remains challenging, because such pro-teins constitute a small fraction of the total proteome.While transcriptomic approaches offer good time res-olution (on the minute time scale; Kreps et al., 2002;Preston et al., 2009), transcript abundances and proteinlevels are frequently discordant (Vélez-Bermúdez andSchmidt, 2014; Fukao, 2015). Translating ribosome af-finity purification (Zanetti et al., 2005; Mustroph et al.,2009; Jiao and Meyerowitz, 2010), followed by mRNA

quantitation or ribosome footprint mapping (Juntawonget al., 2014), provides proxies for protein synthesis atspecific time points in environmental or developmentalprocesses. However, measurements of mRNA associ-ation with ribosomes and footprinting techniques maystill fail to provide accurate information on changesin the proteome because of artifacts arising from har-vesting and lysis steps (Ingolia, 2014) and turnoverfollowing protein synthesis. To complement the existingchemical biology toolkit for plant proteomics, we intro-duce bioorthogonal noncanonical amino acid tagging(BONCAT), which enables sensitive detection andidentification of proteins synthesized within definedtime intervals.

In the BONCAT method, a noncanonical amino acidis pulsed into the cells of interest, where it is incorpo-rated into newly synthesized proteins. Here weemployed the Met surrogate azidohomoalanine (Aha),which carries an azide moiety that is amenable to bio-orthogonal click chemistry (Fig. 1). Bioorthogonal clickchemistry refers to a set of reactions that are fast andhighly selective in complex biological settings and thatcan be carried out under mild conditions (Sletten andBertozzi, 2009; McKay and Finn, 2014). Pulse-labelingwith Aha allows the investigator to exploit the selec-tivity of the copper-catalyzed azide-alkyne cycloaddi-tion (CuAAC) and the strain-promoted azide–alkynecycloaddition (SPAAC) to visualize and enrich newlysynthesized proteins (Fig. 1).

BONCAT has been used to study proteome-wideresponses in mammalian and microbial culture(Dieterich et al., 2006; Zhang et al., 2014; Bagert et al.,2014) and to analyze subpopulations of cells in complexmulticellular eukaryotes (Erdmann et al., 2015;Genheden et al., 2015; Yuet et al., 2015), but has yet to beemployed in plant systems. Here, we applied BONCATto Arabidopsis (Arabidopsis thaliana) to visualize pro-teins synthesized within 3 h after imposition of foursignificant abiotic perturbations: light stress (shift todarkness), high temperature, salt, and osmotic stress.We further coupled BONCAT-assisted enrichment totandem liquid chromatography mass spectrometry(LC-MS/MS) to identify proteins synthesized de novo

Figure 1. Scheme of BONCAT in native planttissues. Aha is pulsed into aerial tissues whereit can be incorporated into nascent proteins.The azide enables conjugation to fluorophoresor beads for visualization or enrichment, re-spectively. CuAAC was used to conjugateTAMRA alkyne to nascent proteins. SPAAC (abiocompatible “click reaction”) was employedto conjugate nascent proteins to beads for en-richment.

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during transient heat stress and a recovery period. Theresults from our screen are consistent with a model inwhich cellular energy resources are rapidly channeledto optimize a protective response at the onset of stress.We suggest that BONCAT should be broadly useful inthe investigation of plant physiology and develop-mental plasticity.

RESULTS

BONCAT Can Be Used to Label Newly SynthesizedProteins in Arabidopsis Seedlings

We pulsed Aha (1 mM) into 5-d-old seedlings (see“Methods”) and allowed labeling to proceed for up to3 h (Fig. 2). To determine whether Aha is taken up andincorporated into newly synthesized proteins, we firstmacerated aerial tissues of Aha-exposed seedlingsgrown under nonstress conditions with liquid nitrogenand then resuspended the resulting powder in a simpleTris buffer containing SDS. Total protein was isolatedusing chloroform-methanol precipitation, resuspendedin a Tris buffer, and treatedwith TAMRA-alkyne (Fig. 2)under CuAAC conditions to conjugate the fluorophoreto Aha-labeled proteins (Fig. 1). We then resolved theproteins via one-dimensional SDS-PAGE and measuredin-gel fluorescence (Fig. 2B). Only Aha-labeled proteinsare tagged with the fluorophore (Fig. 2, B and C). Weobserved light labeling in aerial tissues in periods asshort as 30 min (Fig. 2B). These results confirm that Ahawas both transported into intact tissues and activated bythe endogenous methionyl-tRNA synthetase.To test the applicability of the BONCAT method to

the study of stress responses, we used the Aha-labelingprotocol described above, but also exposed the seedlings

to short-term acute abiotic stresses for 3 h, including ashift to darkness, osmotic shock (200 mM mannitol, aproxy for drought), high salt (300 mM NaCl), and heatshock (37°C; Fig. 3). Only the salt stress caused a changein phenotype, where leaves collapsed slightly upon ex-posure (Fig. 3B). Importantly, the seedlings exhibited nodetectable difference in phenotype resulting from Ahaexposure over the course of the experiment (Fig. 3). Weobserved significant Aha labeling under all conditions incomparison with negative controls, where seedlingswere grown under normal conditions but no Aha wasadded (Fig. 3C). Equal amounts of total protein wereloaded into each lane (Fig. 3D) to allow labeling levels tobe compared between conditions (Fig. 3C).

Aha Incorporation Is 2-Fold Higher at an AmbientTemperature of 37°C vs 22°C

While we observed protein labeling under each of thetested conditions (Fig. 3C), we note that labeling in-tensity was approximately 2-fold stronger (2.1 6 0.4)under heat shock versus other tested conditions, sug-gesting that either translation rates are faster or Ahauptake is greater at higher temperatures. Several stud-ies with cell suspension cultures and seedlings haveshown decreases in protein synthesis rates at elevatedtemperatures (Barnett et al., 1979; Matsuura et al., 2010).We note here that although the ambient temperature inour experiment was 37°C, aerial tissues reached a peaktemperature of only approximately 31°C (measured byinfrared thermometer at the end of the stress period justprior to harvest), likely owing to evaporative coolingeffects. Notably, Ishihara et al. (2015) report higher pro-tein synthesis rates at 28°C than at 21°C in Arabidopsis

Figure 2. A, Probes used in this study. Aha is aMet surrogate repletewith an azidemoiety, whichrenders labeled proteins amenable to cycloaddi-tion with fluorescent alkynyl probes (5,6-TAMRAalkyne) and strained cyclooctyne reagents(DBCO-agarose). B, Time course of Aha incor-poration into nascent proteins of aerial tissues inArabidopsis seedlings. TAMRA-alkyne was con-jugated to newly synthesized Aha-tagged proteinsto render them fluorescent. The gel was visualizedwith an excitation laser at 532 nm and an emis-sion band pass filter at 580 nm. C, Loading con-trol. After measuring fluorescence, the gel wasstained with colloidal blue to confirm equalloading.

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seedlings using 13CO2 labeling. These results are consis-tent with ours, where the tissues experience a tempera-ture ramp from 22°C to a maximum of 31°C.

Separately, to rule out the possibility that increasedincorporation of Aha was a result of greater uptake, weperformed pulses with Aha concentrations up to 4 mM

and labeled for 3 h at room temperature (SupplementalFig. 1). Incorporation was measured by in-gel fluores-cence. Under these conditions, Aha labeling appears tobe saturated by 1 mM (Supplemental Fig. 1). These re-sults demonstrate that increased incorporation athigher temperatures is likely not due to greater Ahauptake, but to increased protein synthesis.

BONCAT Can Be Used to Enrich Newly SynthesizedProteins in Arabidopsis Seedlings

We tested our ability to enrich newly synthesizedproteins through a dibenzoazacyclooctyne (DBCO)-agarose bead pull-down method (Figs. 1 and 2A).Specifically, we pulsed Aha into Arabidopsis seedlingsand allowed nascent proteins to be labeled for 3 h atroom temperature. To remove nonprotein contami-nants and obtain better quality samples for mass spec-trometry, we isolated total protein from seedlings viatrichloroacetic acid-acetone precipitation followed byphenol extraction (Wang et al., 2006; Wu et al., 2014).

We resuspended samples in phosphate-buffered saline(PBS) supplemented with 1% SDS and checked for la-beling by subjecting total protein to TAMRA-alkyneconjugation (Fig. 4B). A separate aliquot of total pro-tein was subjected to SPAAC conditions to conjugatethe Aha-labeled proteins to DBCO-agarose beads. Theresin was washed extensively to remove nonspecificallybound proteins. Finally, Aha-labeled proteins wereeluted from the resin via trypsin digestion and subjectedto detergent removal and desalting prior to LC-MS/MS(Orbitap Elite) and analysis via MaxQuant software.

To quantify enrichment, we performed parallel pulldowns from seedlings at 22°C labeled with Aha for 3 hand seedlings unexposed to Aha. We subjected eachpull down to cleanup and LC-MS/MS and quantifiedthe total sum of all Arabidopsis MS1 peptide extracted-ion chromatogram areas for both enriched and unla-beled mock samples. We found enrichments of at least44-fold across three biological replicates.

Identifying Candidate Proteins Involved inThermotolerance and Recovery from Heat Stress

After demonstrating the feasibility of labeling aerialtissues under stress conditions and developing an en-richment protocol, we sought to identify the de novosynthesized proteins that were involved in heat stress

Figure 3. Labeling five separate stress conditions in Arabidopsis. A, Arabidopsis seedlings prior to Aha pulse and stress exposure.B, Seedlings 3 h after Aha pulse (1 mM) and constant exposure to stress conditions 1 to 5 (defined at left of figure). C, In-gelfluorescence assay to demonstrate labeling under stress conditions. Newly synthesized proteins incorporate Aha; TAMRA-alkyneis conjugated to proteins containing Aha. Labeling is observed under each condition. Little background signal is observed in anegative control, where plants were not exposed to Aha. D, Colloidal Blue loading control to demonstrate equal loading acrosslanes. Gels are representative examples of at least three biological replicates.

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and in recovery from heat stress by using a combinationof BONCAT-assisted enrichment and LC-MS/MS. Weopted to study heat stress because it has been wellcharacterized in Arabidopsis at the level of translation(Matsuura et al., 2010, 2013; Mittler et al., 2012), andbecause thermotolerance has been shown to be inducedby heating seedlings to 38°C for 90 min (Larkindale andVierling, 2008), a timescale that is easily accessible toBONCAT analysis (Fig. 2B). Further, heat stress can bealleviated easily to monitor protein synthesis duringrecovery from stress (McLoughlin et al., 2016), an aspectof stress physiology that is relatively understudied atthe level of protein synthesis.Seedlings were treated with Aha and exposed to ei-

ther heat shock (37°C) or room temperature control(22°C) conditions for 3 h. To study recovery, seedlingswere first exposed to ambient heat shock conditions(37°C) for 3 h, allowed to recover for 4 h at room tem-perature (22°C), then treated with Aha at room tem-perature for 3 h. Experiments were carried out inbiological triplicate for each condition (Fig. 4A).Aha-labeled proteins in experimental and control

samples were conjugated to DBCO-agarose beads andsubjected to the enrichment protocol. In total, weidentified and quantified 3,341 proteins across the fourconditions [1 mM Aha control at room temperature(RT), 2 mM Aha control at RT, heat stress (37°C), andrecovery followingheat stress (37°C to 22°C; SupplementalTable S1)]. We identified 2,973 proteins across the roomtemperature control series 2 (2 mM Aha) and heat shocksamples alone (Fig. 4C).To assess enrichment of known heat-responsive

proteins, we first filtered our dataset for proteins thatwere either significantly up-regulated in response toheat (P value, 0.01) or that were found in all three heatshock replicates and in none of the control series 2 (2mM

Aha) replicates [Supplemental Table S1; Label-FreeQuantitation (LFQ) value = 0, Columns DV - DX)].These criteria identified a total of 189 proteins as heat-responsive BONCAT-enriched proteins (SupplementalTable S1; Column EL). Proteins with a gene ontogeny(GO) annotation of “Response to Heat” were found tobe significantly overrepresented (P value 8 x 10222;Fisher’s exact test) in the population of heat-responsiveBONCAT-enriched proteins. These results clearlydemonstrate that the BONCAT method detects en-richment of de novo synthesized “heat-responsiveproteins” in response to 3 h of heat stress. We notethat this assay detected proteins encoded by nuclear,mitochondrial, and plastid genes, showing that Aha isincorporated into proteins synthesized in different cel-lular compartments.

We constructed a volcano plot of proteins sharedbetween heat shock samples and control series twosamples to visualize proteins with statistically signifi-cant fold-changes (Fig. 4D). Our list of up-regulatedproteins contains many known heat stress markers,including ClpB1, Hsp90-1, probable mediator of RNApolymerase II transcription subunit 37c, and HeatShock Protein (HSP)70-5 (Queitsch et al., 2000; Lin et al.,2001; Sung et al., 2001; Takahashi et al., 2003; Yamadaand Nishimura, 2008). Our analysis also identifiedproteins with statistically significant fold-changes thathave not been annotated previously.

We performed principal component analysis on thebasis of normalized LFQ values for each protein (Fig. 5).We found three distinct clusters: control samples, heatshock samples, and recovery samples. These results il-lustrate the repeatability of biological replicates inBONCAT analysis, and the clustering of the two controlseries (1 mM and 2 mM Aha) suggests that Aha does notcause significant perturbation of protein synthesis at

Figure 4. Enrichment of newly synthe-sized proteins for proteomics. A, Treat-ments used for this study. Arrowheadsindicate time of introduction of Aha. B,Labeling under various conditions. C,Venn diagram of proteins identified incontrol conditions versus heat shock con-ditions. D, Volcano plot of ratios of ex-pression levels of proteins shared betweenheat shock and control conditions. Pro-teins with higher average expression in RTsamples fall on the left side of the plot,whereas proteins with higher average ex-pression in HS samples are displayed onthe right. To construct the plot, LFQ valueswere averaged for each condition. Then,the HS average was divided by the RT av-erage and the Log2 value was taken. Eachpoint represents a protein. Proteins shownin red have the GO annotation “Responseto Heat.” amb, ambient; C1, control series1; C2, control series 2; HS, heat shock; R,recovery.

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these concentrations. Furthermore, the separate clus-tering of the control and recovery samples shows thatmetabolism does not simply return to the preimpositionstate following heat stress.

Next, we constructed heat maps to compare proteinlevels across conditions (Fig. 6; Supplemental Table S1;Supplemental Fig. S2). This analysis demonstrates thedistinction in BONCAT-identified proteins under thethree conditions, including themarked up-regulation ofheat response proteins under heat shock. Notably,many BONCAT-labeled proteins highly expressedduring heat shock are synthesized at lower levels dur-ing the recovery period than under control conditions,clearly demonstrating that seedlings rapidly adjust tochanging conditions in part by altering the synthesis ofproteins.

To validate our BONCAT results, we performedimmunoblot detection of two up-regulated proteins:ClpB1 (HSP101) and HSP 70-5. For this purpose, 5-d-old seedlings were grown identically to those in theBONCAT screen, then exposed to 22°C for 3 h (control),37°C for 3 h (heat shock), or 37°C for 3 h then 22°C for7 h (recovery; these conditions mimic the 4-h “rest”period plus the 3-h labeling period in the BONCATexperiment). We then extracted total protein in a pro-cedure identical to protein extraction for analysis byLC-MS/MS. As anticipated, we observed strong in-duction for both ClpB1 and HSP 70-5 under heat stress(Fig. 7). Importantly, immunoblotting detected differ-ences in abundance across treatment samples, irre-spective of time of synthesis. In contrast, BONCATmeasures protein synthesized within specified timeframes.

Most of the proteins highly up-regulated during heatshock are down-regulated during the recovery periodaccording to BONCAT detection (Fig. 6; SupplementalFig. S2). We hypothesized that these highly up-regulatedproteins would not be degraded rapidly during the re-covery period because thermal priming, a thermotolerance

mechanism, has been shown to occur under similar heatstress conditions on similar timescales (Larkindale andVierling, 2008; Mittler et al., 2012). The results of our im-munoblotting support the hypothesis. Specifically, ClpB1andHSP70-5were not degraded during the recovery fromheat shock (Fig. 7), although their de novo synthesis wassignificantly reduced (Supplemental Table S1). Therefore,ClpB1 and HSP70-5 are stable over the course of ourexperiment.

BONCAT As a Hypothesis Generator

Gratifyingly, the BONCAT screen revealed 80 vali-dated biomarkers (GO molecular function “response toheat”) of the heat stress response in Arabidopsis, in-cluding ClpB1 (HSP101) and HSP70-5. Notably, otherproteins without an annotation of “response to heat”were also up-regulated during heat stress, suggesting apossible role in thermotolerance. Measuring proteinssynthesized during recovery from heat stress yielded

Figure 5. Principal component analysis of mass spectrometry resultsbased on LFQ values. This analysis shows clear separation of controlsamples, heat shock samples, and recovery samples. Inset showszoom-in of controls cluster.

Figure 6. Partial heat map of proteins with GO annotation “response toheat” found in this study. Significance of each fold change was calcu-lated using the R package limma. Heat maps were created usingGENE-E, where the sample clustering was performed using the averagelinkage and Euclidean distance and the gene clustering was performedusing the average linkage and 1-Pearson correlation coefficient. For heatmap visualization, proteins had to be quantified in at least two controlsamples and two “treated” samples (either heat shock or recovery).Relative protein expression was normalized individually for each pro-tein so that the average control expression was zero.

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new information. For example, during recovery weobserved induction (2.75-fold) of zeaxanthin epoxidase,which catalyzes the first committed step in the bio-synthesis of abscisic acid (Xiong and Zhu, 2003), ahormone known to promote stomatal closure (Morillonand Chispeels, 2001; Park et al., 2015). Stomatal clo-sure, in turn, minimizes water loss due to evaporativecooling (Xiong and Zhu, 2003). Therefore, we hy-pothesize that under these conditions zeaxanthinepoxidase is down-regulated during heat stress topromote evaporative cooling, then up-regulated dur-ing recovery to promote stomatal closure and preventdesiccation. While detailed assignment of functionalroles to heat-responsive proteins in the context ofimposition and recovery is beyond the scope of thisstudy, the observed changes in protein synthesissuggest mechanistic hypotheses that merit furtherevaluation.

DISCUSSION

Proteins are cellular workhorses that carry out tightlyorchestrated developmental and adaptive programs.As sessile organisms, plants must retain a high degree

of proteomic plasticity to enable rapid responses to abarrage of environmental cues (Huot et al., 2014;Fristedt et al., 2015; Mickelbart et al., 2015). In plants, asin most eukaryotes, mRNA abundance is often a poorproxy for protein levels (Ingolia, 2014; Vélez-Bermúdezand Schmidt, 2014; Fukao, 2015), as indicated by thediscordance between total mRNA abundance andpolyribosome-associated mRNAs under control andabiotic stress conditions (reviewed by Roy and vonArnim, 2013). To address this issue, we used BONCATin intact Arabidopsis seedlings to identify and quantifyproteins synthesized under conditions of abiotic stress.As implemented here, BONCAT enables identificationof proteins synthesized over periods of a few hours andrequires no manipulation of the translational machin-ery or presumption that a ribosome-associated mRNAwill produce a stable protein (Fig. 2). Furthermore, en-dogenousMet levels do not need to be depleted for Ahato be incorporated into newly synthesized proteins.

In an initial test of the method, we found evidencethat newly synthesized proteins can be labeled inseedlings subjected to a variety of abiotic stresses, in-cluding osmotic shock, high salt, and heat shock (Fig.3). We then compared the populations of Arabidopsisproteins made under normal growth conditions tothose made under conditions of mild heat stress andduring recovery from heat stress (Fig. 4). Changes inprotein synthesis in response to heat stress were readilyobserved in periods as short as 3 h. We used bio-informatic (Figs. 4–6) and immunoblotting analyses(Fig. 7; Supplemental Figs. S3 and S4) to validate theBONCAT-labeled proteins under three conditions.Unsurprisingly, the GO annotation “Response to Heat”was overrepresented among proteins up-regulated inresponse to heat shock. This result is congruent withprevious studies showing selective induction of stressmitigators during mild heat stress. Our identification ofde novo synthesized markers of the heat shock re-sponse provides validation of the BONCAT method asa tool for the study of proteome dynamics in plants. Atthe same time, we found many proteins, includingpresumably low abundance transcription factors likeBIM1, that have not previously been associated withmitigation of heat stress, illustrating the potential valueof time-resolved proteomic studies as a source of newmechanistic hypotheses.

Furthermore, we assessed protein synthesis duringrecovery from heat stress and found proteins mosthighly up-regulated in response to heat stress weresynthesized during recovery at levels similar to orlower than pre-heat shock levels (Fig. 6). This resultshowcases a mechanism by which plants avoid syn-thesizing a surfeit of stress-associated proteins duringrapidly changing conditions. Based on reduced de novosynthesis and maintained abundance, neither ClpB1nor HSP 70-5 was rapidly degraded during the recov-ery period, consistent with previous work showing thatplants can develop “thermal memory” (Larkindale andVierling, 2008) that protects seedlings exposed to mildheat stress from subsequent assaults. This observation

Figure 7. Immunoblotting analysis of select proteins shown inBONCAT screen to be up-regulated in response to heat stress. A, ClpB1(HSP101) and B, HSP70-5 were found to be highly up-regulated inresponse to heat stress. These proteins are not synthesized at high levelsduring the recovery period. Neither are they rapidly degraded duringthe recovery period. Steady-state ClpB1 levels during recovery are0.95 6 0.08 when the fluorescent signal of heat shock samples is nor-malized to 1.00. Relative fluorescence values are provided for thecontrol (room temperature), heat shock, and recovery for HSP70-5. C,Loading control. All fluorescence signals were normalized to ColloidalBlue staining over the entire lane. Cont, control (room temperature),Rel. Fl, relative fluorescence; Rec, recovery.

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suggests that the BONCATmethodmay also be appliedto consider dynamics in protein turnover, as the proteindetected will reflect both the synthesis and turnoverwithin the time-period of labeling. Importantly, turno-ver and synthesis may be differentially regulated. Thus,the BONCAT method is unlikely to detect highly un-stable proteins.

It is worth noting that our model employed vegeta-tive seedlings. While understanding stress responses atall developmental stages is important to the engineer-ing of more robust crops, previous studies have shownthat yield losses are caused most prominently by as-saults to reproductive tissues (Young et al., 2004; Zinnet al., 2010). The BONCATmethod should prove usefulacross many developmental stages and tissues andunder a wide variety of conditions, and can help un-ravel the genetic basis of traits involved in tolerance toboth abiotic and biotic stresses. Notably, all reagentsand probes used in this study are commercially avail-able, further underscoring the ease by which thismethodology can be readily adopted by laboratorieswith access to proteomic facilities.

MATERIALS AND METHODS

Plant Growth Conditions

Arabidopsis (Arabidopsis thaliana) seeds (Col-0 accession) were subjected tovapor phase sterilization (100 mL of 6% [v/v] commercial bleach and 3 mL of37%HCl [v/v]) for approximately 3 h. Seedlings were then dispensed on 40 mLsolid media (100- x 15-mm petri dishes) containing 0.53 strength Murashigeand Skoog (MS) salts (Sigma-Aldrich), 13 MS vitamins (Sigma-Aldrich), 0.3%(w/v) Suc, and 0.9% (w/v) Phytagel (Sigma-Aldrich). Plates were inverted at4°C in darkness for 2 d to break seed dormancy. Plates were then placed at 22°Cunder 24-h daylight and positioned so that roots would grow into the medium.After germination, plants were allowed to grow for 5 d under constant light.

Seedling Flood Technique to Deliver NoncanonicalAmino Acid

The protocol was adapted from the study of Ishiga et al. (2011). Aha labelingmedium containing 0.53MS salts (Sigma-Aldrich), 5% Suc, 0.0025% Silwet L-77(Lehle Seeds), 1 mM Aha in 10 mM citrate buffer (pH 5.6) was freshly preparedprior to running the experiment. Seedlings were fully submerged in the Ahalabeling medium for 2 min. After the medium was decanted, the petri dishcovers were replaced and the plates were wrapped in foil. The seedlings werethen exposed to short-term acute abiotic stresses for 3 h. Shift to darkness wasaccomplished by covering plates with foil. To approximate osmotic shock, Ahalabeling medium was supplemented with 200 mM mannitol and pulsed ontoseedlings during delivery of Aha. High salt stress was accomplished by supple-menting Aha labeling medium with 300 mM NaCl. To heat shock samples, plateswere placed in an oven at an ambient temperature of 37°C immediately followingthe 2-min submersion in Aha labelingmedium. All proteomics experiments wereinitiated on the morning of day 5 (germination is defined as day 0).

In-Gel Fluorescence to Monitor Protein Labeling

After labeling with Aha, aerial tissues were immediately harvested, flashfrozen with liquid nitrogen, and stored at 280°C until subsequent workupsteps. Frozen tissues were macerated in liquid nitrogen. Frozen powder wasimmediately transferred into an Eppendorf tube containing 1 mL of lysis buffer(100 mM Tris, pH 8 containing 4% w/v SDS). Lysates were subjected to soni-cation at 80°C for 40 min, then subjected to further heating at 95°C for 30 min.Cellular debris was removed by centrifugation at 16,100 relative centrifugalforce (RCF) RCF for 5 min. The conditions for labeling the lysate by CuAAC forin-gel fluorescence were based on a previously published protocol (Hong et al.,

2009). Protein concentrations in the cleared lysate weremeasured via bicinchoninicacid assay. Forty micrograms of protein lysate was added to PBS to a final totalvolume of 221 mL. In a separate tube, the alkynyl dye, copper (II) sulfate, andtris(3-hydroxypropyltriazolylmethyl)amine were premixed and allowed to reactfor 3 min in the dark. Then, aminoguanidine HCl and sodium ascorbate wereadded to the lysate-PBSmixture. Final concentrations were as follows: alkynyl dye(2.5 mM), CuSO4 (1 mM), tris(3-hydroxypropyltriazolylmethyl)amine (5 mM), ami-noguanidine HCl (5 mM), and sodium ascorbate (5 mM). All components weregently mixed (one inversion) and allowed to react for 15 min in the dark at roomtemperature without shaking. Proteins were then extracted by methanol/chloroform/water precipitation. Pellets were washed extensively (at least threetimes). The pellet was then dissolved in 23 SDS sample loading buffer and soni-cated for 30 min at 80°C. Samples were heated to 95°C for 5 min and electro-phoresed in precast 4% to 12% Bis-Tris polyacrylamide gels. The gel was washedwith a fixing solution (50% water, 40% ethanol, 10% acetic acid) in the dark for10 min and rinsed twice with water (23 10 min) prior to being subjected to fluo-rescence imaging with an excitation laser at 532 nm and an emission band passfilter at 580 nm (Typhoon GE Healthcare). After fluorescence imagining, the gelwas stained with Colloidal Blue (InstantBlue, Expedeon) for 1 h and imaged toensure equal protein loading among all lanes. To obtain relative fluorescencevalues, signal intensity was measured for the entire lane in both the TAMRA(fluorescence) channel and the colloidal blue channel. Next, the ratio of fluores-cence intensity to colloidal blue intensity was calculated and normalized to thecontrol lane. Calculating the relative fluorescence values as ratios in this mannerallows for comparison between lanes even if lane loading varies slightly.

Cell Lysis and Protein Extraction for Enrichment

Sampleswere prepared in biological triplicatewith one plate (approximately0.5–0.7 g tissue fresh weight) representing one biological replicate. Sampleswere prepared with protocols adapted from Wang et al. (2006) and Wu et al.(2014). Frozen aerial tissues were macerated in liquid nitrogen with a mortarand pestle. The resultant powder (0.1–0.3g) was transferred into a 1.7-mL tube(typically tissues from one plate were divided into six separate 1.7-mL tubes).The pellets were washed with 10% trichloroacetic acid in acetone by filling thetubes, mixing themwell by vortexing then centrifuging at 16,000 RCF for 3 minat 4°C. The supernatant was removed. The pellet was washed with 80%methanol containing 0.1 M ammonium acetate then with 80% acetone. Thepellets were allowed to dry at room temperature for 15 min to remove residualacetone. Next, 0.8 mL of liquefied phenol (pH 8.0, Sigma-Aldrich, catalog no.P4557) and 0.8 mL of SDS dense buffer [30% Suc, 2% SDS, 0.15% sodium azide(to prevent bacterial growth), 0.1 M Tris-HCl, pH 8.0, and 2-mercaptoethanol toa final concentration of 5% added fresh] were added to the tube. The contentswere mixed thoroughly and incubated for 5 min at room temperature in a fumehood. Next the tubeswere centrifuged at 16,000 RCF for 3min at 4°C. The upperphenol phase was transferred into a new 1.7-mL tube while taking precautionsnot to disturb the middle SDS interface. The new 1.7-mL Eppendorf tube wasfilled with methanol containing 0.1 M ammonium acetate and stored at 220°Covernight to precipitate the protein. The tubes were then centrifuged at 16,000RCF for 5 min at 4°C. The pellet was washed once with 100% methanol, thenwith 80% acetone. Next, the protein pellets were resuspended in PBS [pH 7.4,1% SDS, 100 mM chloroacetamide, and 13 cOmplete EDTA-free protease in-hibitors (Sigma Aldrich, catalog no. 11873580001)] then pooled. Protein con-centrations were measured via bicinchoninic acid assay. Approximately 0.3 mgtotal protein was carried through the enrichment procedure.

Enrichment Procedure and LC-MS/MS Sample Preparation

Samples were adjusted to a consistent volume (approximately 500 mL) with1% SDS in PBS. Then, samples were diluted 23 with 8 M urea containing 13EDTA-free protease inhibitor (Sigma Aldrich, catalog no. 11873580001) and0.85 M NaCl.

Separately, DBCO-agarose beads (50 mL of 23 slurry; Click ChemistryTools, catalog no. 1034-2) were washed 33with 0.8% SDS in PBS. Then, proteinsamples were added to the resin and shaken at 1,200 rpm at room temperatureovernight (for at least 16 h). The resin was washed with 1 mL water. Then,0.5 mL DTT (1 mM in 0.8% SDS in PBS) was added to the resin and incubatedwith shaking (1,200 rpm) for 15min at 70°C. The supernatant was removed, andfree thiols were blockedwith iodoacetamide (0.5 mL of a solution at 7.4 mg/mLdissolved in PBS with 0.8% SDS) for 30 min in the dark at 1,200 rpm.

The resin was transferred to a spin column (Poly-prep chromatographycolumns, Bio-Rad, catalog no. 731-1550) and subjected to the following washes:

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83 5 mL 0.8% SDS in PBS, 83 5 mL 8 M urea, 83 5mL 20% acetonitrile (ACN).For the secondwashwith each solution, the columnwas capped and allowed tosit for 10 min prior to draining. After the washes, beads were transferred to anEppendorf tube with 10% ACN in 50 mM ammonium bicarbonate. Tubes werecentrifuged for 5 min at 2,000 g and the liquid was removed. Then, 100 mL 10%ACN in 50 mM ammonium bicarbonate was added to the beads and 100 ngtrypsin was added. The beads were shaken at 1,200 rpm and 37°C overnight.The supernatant was collected, and beads were washed twice with 150 mL 20%ACN. Supernatants from the 20% ACN washes were pooled with the super-natant from the overnight tryptic digest. Pooled supernatants were filtered(Pierce centrifuge columns 0.8 mL, ThermoFisher Scientific, catalog no. 89868)to remove any beads that carried through, then dried on a speedvac.

Digestedpeptideswere redissolved in 100mL50mMammoniumbicarbonateand treated with HiPPR detergent removal resins (ThermoFisher Scientific,catalog no. 88306). Finally, digested peptides were subjected to desaltingclean-up step with a ZipTip (C18).

LC-MS/MS Analysis

Trypsin-digested samples were subjected to LC-MS/MS analysis on ananoflow LC system, EASY-nLC II, (Thermo Fisher Scientific) coupled to aOrbitrap Elite Hybrid Ion Trap-Orbitrap Mass Spectrometer (Thermo FisherScientific) equipped with a nanospray Flex ion source, essentially as describedpreviously (Kalli et al., 2013).

Briefly, for the EASY-nLC II system, solvent A consisted of 97.8% H2O, 2%ACN, and 0.2% formic acid and solvent B consisted of 19.8% H2O, 80% ACN,and 0.2% formic acid. A 50-mm self-pack Picofrit column (New Objective, Inc.)with coated tip was packed in-house with 3.0 mm ReproSil-Pur C18AQ resin(120-Å pore size, Dr. Maisch) to 16 cm. Samples were directly loaded onto thecolumn resin, and the columnwas heated to 65°C. The peptides were separatedwith a 120-min gradient at a flow rate of 3220 nL/min. The gradient was asfollows: 2% to 30% Solvent B (150 min), 30% to 100% B (1 min), and 100% B(9 min). Eluted peptides were then ionized using a standard coated silica tip(New Objective) as an electrospray emitter and introduced into the massspectrometer. The Orbitrap Elite was operated in a data-dependent mode, au-tomatically alternating between a full-scan (m/z 400–1,600) in the Orbitrap andsubsequent MS/MS scans of the 20 most abundant peaks in the linear ion trap(Top20 method). Data acquisition was controlled by Xcalibur 2.2 and Tune 2.7software (Thermo Fisher Scientific). Raw mass spectrometry files have beenuploaded to the Japan Proteome Standard Repository (jPOSTrepo), a publiclyavailable data repository (ProteomeXchange ID: PXD005577).

Bioinformatic Analysis

Rawfileswere processed and searchedusingMaxQuant (v. 1.5.3.30; Cox andMann, 2008; Cox et al. 2011). Spectra were searched against the UniProt Ara-bidopsis database (33,353 sequences) and a contaminant database (245 se-quences). A decoy database was generated by MaxQuant using a reversedsequence strategy to estimate the false discovery rate. Trypsin was specified asthe digestion enzyme with up to two missed cleavages allowed. Oxidation ofMet (+15.9949), protein N-terminal acetylation (+42.0106), and replacement ofMet with Aha (24.9863) were allowed as variable modifications. Carbamido-methylation of Cys (+57.0215) was specified as a fixed modification. Precursorion tolerance was less than 4.5 ppm after recalibration and fragment ion tol-erance was 0.5 D. “Match Between Runs” and LFQwere enabled in MaxQuant.Protein and peptide identifications had an estimated false discovery rate ,1%.

Significance of fold changes was calculated using the R package limma(Benjamini and Hochberg 1995; Smyth GK 2004; Ritchie et al., 2015). Heatmapswere created using GENE-E where the sample clustering was performed usingthe average linkage and Euclidean distance and the gene clustering was per-formed using the average linkage and 1-Pearson correlation coefficient. Forheatmap visualization, proteins had to be quantified in at least two controlsamples and two “treated” samples (either heat shock or recovery). Relativeprotein expression was normalized individually for each protein so that theaverage control expression was zero.

Immunoblotting

Unconjugated primary antibodies against ClpB1 and HSP70-5 were pur-chased from Abcam. Goat antimouse IgG (H+L) (A-21235) and goat antirabbitIgG (H+L) (A-21429) secondary antibodies with conjugated Alexa Fluor werepurchased fromLife Technologies (Thermo Fisher Scientific). Lysate (10mg)was

electrophoresed in 4-12% precast Bis-Tris polyacrylamide gels for all westernBlots. The 0.2-mm nitrocellulose membranes were blocked with 3% w/v nonfatdry milk in TBST for 1 h prior to incubating with antibodies in TBST. Primaryantibodies [anti-ClpB1 (Ab80121, 1:5,000) and anti-HSP70-5 (ab5439, 1:5,000)]were incubatedwith their respective membranes at 4°C overnight with 3%w/vnonfat dry milk prior to staining with the secondary antibody (1:5,000) for 1 hand imaging (PMT 400 V; 50 mm pixel size). Fluorescence signals were nor-malized to total signal from Colloidal Blue staining for each sample. To ensurethat samples were measured in the linear range for quantitation, 1.5 mg, 10 mg,and 15 mg total protein were loaded for each heat shock replicate, and fluo-rescence was measured after incubation with the anti-HSP70-5 antibody andthe secondary antibody as described above (Supplemental Fig. S4A). Fluores-cence intensity was plotted against total protein concentration, which showsthat signals were in the linear range for accurate quantitation (r2 = 0.994;Supplemental Fig. S4B).

Accession Numbers

Raw mass spectrometry files have been uploaded to the Japan Proteomestandard Repository under accession number PXD005577.

Supplemental Data

The following supplemental materials are available.

Supplemental Table S1. Protein groups.

Supplemental Figure S1. Concentration series.

Supplemental Figure S2A. Heat map – all.

Supplemental Figure S2B. Heat map – ANOVA.

Supplemental Figure S2C. Heat map – GO: response to heat.

Supplemental Figure S2D. Heat map – Proteins regulated by heat stress.

Supplemental Figure S2E. Heat map – GO: stress response.

Supplemental Figure S3. Full image of ClpB1 (HSP101) and HSP70-5 west-ern blots.

Supplemental Figure S4. HSP70-5 fluorescence measurements lie in linearrange.

ACKNOWLEDGMENTS

We thank the laboratory of Professor Elliot Meyerowitz, especially ArnavazGarda and Dr. Paul Tarr, for seeds, space in growth chambers, and helpfuldiscussions. We also thank Roxana Eggleston-Rangel for helpful discussions onsample preparation for mass spectrometry.

Received November 17, 2016; accepted January 14, 2017; published January 19,2017.

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