Plant Science 221–222 (2014) 48–58

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Chaperone function of two small heat shock proteins from maize Roger D. Klein a , Tamutenda Chidawanyika a , Hannah S. Tims a,1 , Tea Meulia b , Robert A. Bouchard c , Virginia B. Pett a,∗ a b c

Department of Chemistry, The College of Wooster, Wooster, OH 44691, USA Molecular and Cellular Imaging Center, Ohio Agricultural Research and Development Center, Wooster, OH 44691, USA Horticulture and Crop Science, Ohio Agricultural Research and Development Center, Wooster, OH 44691, USA

a r t i c l e

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Article history: Received 23 August 2013 Received in revised form 27 January 2014 Accepted 29 January 2014 Available online 6 February 2014 Keywords: Small heat shock protein Chaperone Zea mays ␣-Crystallin domain Oligomerization Transmission electron microscopy

a b s t r a c t Small heat shock proteins (sHsps) are molecular chaperones that protect cells from the effect of heat and other stresses. Some sHsps are also expressed at specific stages of development. In plants different classes of sHsps are expressed in the various cellular compartments. While the Class I (cytosolic) sHsps in wheat and pea have been studied extensively, there are fewer experimental data on Class II (cytosolic) sHsps, especially in maize. Here we report the expression and purification of two Class II sHsps from Zea mays ssp. mays L. (cv. Oh43). The two proteins have almost identical sequences, with the significant exception of an additional nine-amino-acid intervening sequence near the beginning of the N-terminus in one of them. Both ZmHsp17.0-CII and ZmHsp17.8-CII oligomerize to form dodecamers at temperatures below heat shock, and we were able to visualize these dodecamers with TEM. There are significant differences between the two sHsps during heat shock at 43 ◦ C: ZmHsp17.8-CII dissociates into smaller oligomers than ZmHsp17.0-CII, and ZmHsp17.8-CII is a more efficient chaperone with target protein citrate synthase. Together with the previous observation that ZmHsp17.0-CII but not ZmHsp17.8-CII is expressed during development, we propose different roles in the cell for these two sHsps. © 2014 Elsevier Ireland Ltd. All rights reserved.

Introduction Small heat shock proteins play many roles in the cell Small heat shock proteins (sHsps) play important roles in the cells of almost every organism in all three kingdoms of life [1–4]. Especially diverse in plants, sHsps are expressed in large quantities in response to heat shock. For instance, the sHsps constitute more than 1% of total protein in heat-stressed pea leaves [5]. First characterized as molecular chaperones that bind to cellular proteins and prevent heat-induced aggregation [6,7], sHsps also protect against many other stresses such as cold, drought, and metal ions [8–10]. sHsps are now recognized as crucial “protein stability sensors” [11], ATP-independent “holdases” [12,13] that bind to hydrophobic residues of partly unfolded proteins, then pass the target proteins along to either ATP-dependent folding systems such as HSP70 or degradation by ubiquination or macroautophagy [14]. In addition to stress-induced expression some sHsps are expressed during

∗ Corresponding author. Tel.: +1 330 461 0629. E-mail addresses: [email protected] (R.D. Klein), [email protected] (T. Chidawanyika), [email protected] (H.S. Tims), [email protected] (T. Meulia), [email protected] (R.A. Bouchard), [email protected] (V.B. Pett). 1 Current address: Messiah College, Mechanicsburg, PA 17055, USA. 0168-9452/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2014.01.012

specific developmental stages in many organisms [15]. Furthermore, sHsps are expressed constitutively in mammalian muscle, nervous tissue, and lens of the eye. Because their function is of such fundamental importance, it is not surprising that mutations in sHsps result in a variety of disease states such as cataract, neuropathy, and myopathies [16–19], and that regulation of sHsp expression is important in protein mis-folding diseases such as Alzheimer’s and Parkinson’s [16,20,21]. sHsps share a conserved ˛-crystallin domain, variable N-terminus and C-terminal extension The ␣-crystallin domain (ACD), named for the crystallin proteins that are responsible for the refractive power of the vertebrate lens [22], is the shared motif among the sHsps. This highly conserved C-terminal domain consists of 90–100 amino acids that form a stable ␤-sheet sandwich structure. In the crystal structures of the sHsps from hyperthermophilic archaeon Methanococcus jannaschii (reclassified as Methanocaldococcus jannaschii) and wheat (Triticum aestivum), dimers are formed by ␤6-strand exchange between the ACDs of the paired monomers [23,24]. In Xanthomonas citri pv. citri [25] and vertebrate crystal structures dimers are stabilized by antiparallel interactions between ␤-strands from each monomer, which form an extended ␤-sheet [22,26,27]. With some exceptions [28] the dimers further associate into large oligomeric

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complexes that vary from 12 to more than 48 monomers in different organisms [29]. Below heat-shock temperature the oligomers are storage structures; as the temperature rises, there is extensive subunit exchange and formation of both smaller and larger assemblies [30–34]. Various experimental approaches, notably nanoelectrospray mass spectrometry, have shown that smaller oligomers are the effective chaperones that stabilize target proteins [35]. In contrast to the genetic and structural stability of the central ACD, the N-terminus and the C-terminal extensions of sHsps have highly variable sequences. In the full-length sHsps that have been successfully crystallized the N-terminus is either partly or fully disordered [23–25]; thus far only truncated ACDs have been crystallized from vertebrate sHsps [22,26,27,36]. Even when the sHsps form stable complexes in solution with target proteins, the N-terminal arm continues to be locally dynamic [12]. Although the N-terminus appears to be intrinsically disordered, there is convincing evidence that this part of the sequence provides the variability needed for association into oligomers as well as recognition and binding to multiple target proteins [14,15,28,37,38]. Plant sHsps exhibit remarkable variety Perhaps because of their inability to move away from stressors, land plants express a remarkable range of sHsps. There are 12 gene subfamilies of plant sHsps; each family is expressed in a particular cellular compartment and may include several members [39]. Class I (CI) and Class II (CII) sHsps, which are found in the cytoplasmic/nuclear compartment, are the most studied sHsps. These two subfamilies share sHsp consensus I and plant sHsp consensus II in the ACD; they are differentiated by class-specific consensus regions in the N-terminus. (See Fig. 1 for sequence comparisons of CI and CII sHsps.) Experimental studies of sHsps from pea, tomato, and rice have shown that CI and CII sHsps stabilize target proteins during heating both in vitro and in vivo [40–42]. Likewise, both classes are expressed during seed development/germination in pea [43], during tomato ripening in fruit and seeds [40] and in varied tissues of the rice plant, especially in seeds [44]. However, CI and CII demonstrate differences in oligomerization behavior with heating, efficiency of target protein protection, and characteristics of heatshock complexes [42,45–48], leading to the conclusion that the differences in N-terminal sequence affect expression and function of CI and CII sHsps. Two Class II sHsps from maize have intriguing differences in function The two CII sHsps from maize examined in this study, ZmHsp17.0-CII and ZmHsp17.8-CII differ only slightly in sequence: ZmHsp17.8-CII has a glycine- and proline-rich intervening sequence of nine amino acids in the N-terminus that is missing in ZmHsp17.0-CII. With the exception of this intervening sequence, 93% of the amino acids in the two proteins are identical; 97% are closely similar when conserved or semi-conserved substitutions are included. In Fig. 1 sequences of some plant CI and CII sHsps are compared, showing the N-terminal CI and CII-specific sequences, plant-specific consensus II, eukaryotic sHsp consensus I, and the proline-rich C-terminal extension with I/VxI/V motif in the tail. Another distinction between CI and CII sequences is the abundance of methionine residues in the N-terminus of CII sHsps. Translation is initiated by these internal AUGs as well as the initial AUG in maize during heat shock and microsporogenesis [53], giving rise to isoforms that may respond to specific protein targets, stresses, or developmental signals. These methionines may also interact with exposed aromatic residues in unfolding proteins [54], thus contributing to the region’s role in target recognition and binding.

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We note that intervening sequences similar to that which differentiates Zm17.8-CII from Zm17.0-CII is present in some other sHsps in both classes I and II. Both of these CII sHsps are expressed during heat shock, but only ZmHsp17.0-CII is expressed early in anthers during microsporogenesis and in radicles under metal-ion stress [8,51,55]. The difference in developmental expression suggested a possible difference in function for the two sHsps. Intrigued by these differences that correlate with a relatively small difference in N-terminal primary structure, we expressed and purified the two proteins in order to investigate the oligomerization behavior and efficiency of these two sHsps in protecting a target protein, citrate synthase (CS), from thermal stress.

Material and methods Engineering the vector construct A uniform strategy was employed in cloning the three ZmHsp17-CII coding regions for expression of their polypeptides in Escherichia coli using the IMPACT-CNTM system (NEB). The Nterminal primer for amplifying each coding region comprised six spacer bases followed by CAT (so that the resulting amplicon contained an NdeI restriction site) followed by a segment matching the coding sequence from its natural ATG start codon and extending through enough bases to give a Tm in the upper 50s ◦ C. The C-terminal primer had a 3 segment matching the proteincoding segment of the sequence over its final anti-codons extending through enough bases to give a Tm in the mid-60s ◦ C. The region 5 to this comprised a cysteine codon replacing the stop codon of the coding region and an inverted SapI restriction site plus seven spacer bases. The restriction site was positioned so that cleavage of the amplicon would expose the three bases of the cysteine codon as the 3 extension of the coding region, allowing in-frame fusion to the intein sequence of the vector as discussed below. Amplification was performed using the cDNA clone templates for ZmHsp17.8-CII and ZmHsp17.6-CII (formerly designated 18-3 and 18-9 [50]) and ZmHsp17.0-CII (formerly designated 18-1 [51]) and with their respective primer pairs using the FailSafeTM system (Epicenter) as follows. Reaction Mix: 10 ␮M Forward and Reverse primers, 1.2 ␮L each; 500 pg/mL cDNA plasmid, 0.8 ␮L; 2× D-mix, 6.0 ␮L; FailSafe polymerase mix, 0.1 ␮L; dH2 O, 2.7 ␮L. PCR conditions: (1) 94 ◦ C, 5 min; (2) 94 ◦ C, 30 s; (3) 64 ◦ C, 30 s; (4) 68 ◦ C, 2 min; (5) go to #2 4×; (6) 90 ◦ C, 30 s; (7) 68 ◦ C, 30 s; (8) 68 ◦ C, 2 min; (9) go to #6 29×; (10) 10 ◦ C, hold. The amplicons were initially cloned into the TOPOTM vector pCRII (Invitrogen) and transformed into E. coli host strain DH5␣. Colonies with inserts of the expected size were identified by colonyPCR performed using the universal M13 primers for the pCRII vector using the Green TaqTM system (Promega) as follows. Reaction Mix: 10 ␮M Forward primer and Reverse primer, 0.3 ␮L each; pick of colony in 25 ␮L dH2 O, 0.4 ␮L; Green Taq mix, 3.0 ␮L; dH2 O, 2.0 ␮L. PCR conditions: (1) 95 ◦ C, 5 min; (2) 95 ◦ C, 30 s; (3) 54 ◦ C, 30 s; (4) 72 ◦ C, 2 min; (5) go to #2 29×; (6) 72 ◦ C, 4 min; (7) 10 ◦ C, hold. Plasmid DNA of isolates was prepared from 4-mL liquid cultures of positive colonies and DNA sequencing performed using the same universal M13 primers for the pCRII vector in order to identify a completely accurate copy for each ZmHsp17-CII. Once identified, these were excised by a double-digest with NdeI and SapI, and then gel-purified. They were then ligated into IMPACTTM expression vector pTYB1 (NEB) that had been double-digested with NdeI and SapI. The pTYB1 vector contains an IPTG-inducible T7 promoter. Once made, the ligations were re-transformed into E. coli host strain DH5␣. Insert-positive clone colonies were identified by PCR using the Impact T7 forward and Intein reverse primers and the Green

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Fig. 1. Sequence alignment of ZmHsp17.0-CII and ZmHsp17.8-CII (bold) with Class I and Class II sHsps from plants, showing an intervening hydrophilic sequence of three or more amino acids (light gray highlight) in the N-terminal region of some sHsps. Rectangles show positions of the first two ␣-helices in the crystal structure of TaHsp16.9-CI [23]; the dotted outline for the ␣-2 helix indicates that the glycine- and proline-rich intervening sequence in some sHsps, including ZmHsp17.8-CII, may interrupt the secondary structure, while ZmHsp17.0-CII and other Class II sHsps may have the same secondary structure in this region as the wheat Class I sHsp. Internal start codons in Class II sHsps are shown in dark gray highlight. N-terminal Class I specific consensus, with conserved sequences SXXFD and WDPF, italic; Class II specific consensus, italic. In the ␣-crystallin domain, the plant consensus, underline; eukaryote consensus, underline; I/VXI/V C-terminal tail motif, light gray font [22,23,26,49]. Sequence information: TaHsp16.9-CI, Q9H0H5:Q41560 (with one change T7S [23]); ZmHsp17.2-CI, Q9H0H5:Q43701; PsHsp18.1-CI, Q9H0H5:P19243 (with one change P37L [23]); ZmHsp17.4-CI, Q9H0H5:B4F976; OsHsp19.0-CII, Q9H0H5:Q6Z6L5; AtHsp17.7-CII, Q9H0H5:O81822; OsHsp18.0-CII, Q9H0H5:Q5VRY1; ZmHsp17.6-CII, Q9H0H5:P24631 (formerly called 18-9 [50], with one change A45R); ZmHsp17.8-CII (formerly called 18-3 [50]), Q9H0H5:P24632; ZmHsp17.5-CII, Q9H0H5:B6T339; ZmHsp17.0-CII (formerly called 18-1 [51]), Q9H0H5:Q08275; TaHsp17.3-CII, Q9H0H5:Q43660; TaHsp17.3-CII, Q9H0H5:Q94KM0; PsHsp17.7-CII [52]; SpHsp17.3-CII, Q9H0H5:O82013; GmHsp17.0-CII, Q9H0H5:P05477.

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TaqTM system as above. Amplicons were bound to ZymoTM columns and cleared of unincorporated nucleotides and salts by two washes. They were then eluted in dH2 O and samples were sequenced using the same primers to check that the sequence was still correct and the insertion in-frame. Plasmid DNA was prepared from liquid cultures of confirmed positives and transformed into E. coli host strain ER2566 for expression. Immediately following the SapI site the expression vector contains the coding sequence for a 454-amino acid intein from the Saccharomyces cerevisiae VMA1 gene modified in two ways. First, a 52 amino acid chitin-binding domain has been inserted allowing for affinity purification of the fusion precursor product on a chitin column. Second, the intein has been modified so that it undergoes only N-terminal thio-cleavage at the cysteine in the SapI site 3base extension. Expression cultures in this host can be induced to synthesize and accumulate the respective ZmHsp-CII/intein/chitinbinding-domain fusion proteins by induction with IPTG at mid-log phase. The fusion protein can then be isolated and substantially purified by passing the lysate of confluent cells over the chitin column, followed by several washes. Finally, after introduction of DTT and incubation, the intein self-cleaves, releasing the desired protein into the void volume of the column while the intein and chitin-binding domain remain attached. The released protein may then be washed from the column. The thiol residue that remains at the carboxyl terminus after intein cleavage is hydrolyzed during subsequent buffer exchange, yielding the native ZmHsp-CII protein sequence. To prepare for this, stock cultures were grown for each positive isolate, made up as 15% glycerol stocks, sub-divided into 1 mL aliquots, and frozen at −80 ◦ C.

Expression and purification Starter culture, consisting of 10 mL sterile LB (Fisher), 100 ␮g/mL ampicillin (rpi), 50 ␮L glycerinated stock ER2566 host cells with the engineered pTYB1 vector construct as described above, was grown overnight, and added to 1 L sterile LB, 100 ␮g/mL ampicillin, in a 2800-mL culture flask, shaken at 37 ◦ C. IPTG was added (1 mM) to induce expression of the desired fusion protein at mid-log phase (∼3.5 h). Expression conditions were 48 h at 15 ◦ C. Pelleted cells were resuspended in ice-cold 20 mM 2-amino-2-methyl-1,3propanediol (AMPD, Sigma) pH 8.5, 1 M NaCl, 0.5% Triton X-100 (Sigma), 10 ␮g/mL RNase A (Sigma), protease inhibitor cocktail (rpi) and sonicated with eight 30-s bursts alternated with 30-s icing. Although DNase I cleaved the target protein, RNase A removed nucleic acid impurity. The expected band for the fusion protein (63 kDa) was visible on the SDS-PAGE of cleared lysate. Cleared lysate was applied to affinity column consisting of 20 mL chitin beads (NEB) equilibrated in cold 20 mM AMPD, pH 8.5, 1 M NaCl, 0.5% Triton X-100, 1 mM tris-(2-carboxyethyl)phosphine (TCEP), 1 mM EDTA. The slurry was rocked 30 min allowing the fusion protein to bind to the column. With column upright, flowthrough was collected. The column was washed with 10 column volumes (CV) of the equilibration buffer to remove cellular proteins not bound to the resin and let stand 1 h at ambient temperature. To achieve on-column refolding of the protein, the detergent was removed while the protein was bound to the column by washing with 10 CV of column buffer (CB, 20 mM AMPD, pH 8.5, 0.5 M NaCl, 1 mM EDTA) plus 5 mM ␤-cyclodextrin (Sigma). After 1 h at ambient temperature the column was washed with 10 CV of CB to remove ␤-cyclodextrin. The column was flushed with 60 mL CB plus 50 mM DTT (rpi) and after 48 h at ambient temperature the column was rocked 30 min with 20 mL storage buffer (SB, 50 mM NH4 OAc, pH 7.0, 500 mM NaCl or 50 mM NaCl) and the flowthrough containing the target protein was collected. Hydrolysis of the thiol residue

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remaining after cleavage was achieved by alternate concentration and washing with SB in Amicon Ultra centrifugal filter device at 4 ◦ C. Leaving the solution on the filter overnight (refrigerated) allowed the adsorbed protein to solubilize, increasing the yield. The desired native protein was removed and frozen at −80 ◦ C. Molecular weights of the monomer sHsps were verified by SDS-PAGE. Native C II small heat shock proteins obtained: ZmHsp17.0-CII (154 amino acids, 17,046.7 kDa, theoretical pI, 7.84, computed extinction coefficient 4470 M−1 cm−1 ) and ZmHsp17.8-CII (164 amino acids, 17,813.4 kDa, theoretical pI 5.33, computed extinction coefficient 4470 M−1 cm−1 ). Correct folding and oligomer formation was confirmed by native PAGE and/or blue native PAGE. The estimated absorptivity values of these sHsps, which do not have Trp residues, can have error as much as 10%; furthermore, impurities with considerably higher absorptivity values, even though present at low concentrations, may artificially increase the measured absorbance. Therefore, concentrations were estimated by comparison of SDSPAGE band for the native monomer with standard lysozyme bands. SDS-PAGE, native PAGE, blue native PAGE SDS-PAGE 4–20% Tris–HCl ready gel (Bio-Rad) or NuPAGE 4–12% Tris/glycine SDS-PAGE gel (Invitrogen) with Tris–glycine SDS running buffer; 6× Laemmli sample buffer (Boston BioProducts) with 10% added ␤-mercaptoethanol; Coomassie stain or GelCode Blue stain (Thermo Scientific); Precision Plus All Blue protein standard (Bio-Rad). Native PAGE Native PAGE omits SDS, which would break down quaternary structure; therefore, the protein itself must be negatively charged in order to migrate properly down the gel. 4× sample buffer [56] (240 mM Tris pH 8.5 at 4 ◦ C, 20 mM ␧-aminocaproic acid, 4 mM benzamidine, 0.04% bromophenol blue, 60% sucrose); High Molecular Weight Calibration Kit for Native Electrophoresis (Amersham); running buffer Tris–glycine (Bio-Rad), with additional Tris base to bring the pH of the buffer to 8.5 at 4 ◦ C, above the pI for the protein. At 4 ◦ C a 4–20% polyacrylamide gel (Bio-Rad 161-1177) was run overnight at constant 105 mV. At 9, 24, and 36 ◦ C the gels were run overnight at constant 3 mA (to avoid heating and distortion of the gel), with alterations in the buffer compositions to take into account temperatures higher than 4 ◦ C and to maintain pH > pI. Gels were soaked 30 min in solution of 0.05% SDS, 7.5% acetic acid, rinsed in H2 O, soaked with 10 ␮L Sypro Orange stain (Sigma) in 50 mL 7.5% acetic acid for 30 min, rinsed 30 s in 7.5% acetic acid, photographed with the Kodak EDAS 290 electrophoresis documentation and analysis system in UV light and Rf values calculated. Blue native PAGE In this system Coomassie G-250 binds to the protein and ensures a net negative charge without denaturing the protein [57]. Blue native PAGE cathode and anode buffers were purchased from Invitrogen; samples were loaded on a Novex 4–16% Bis-Tris blue-native gel (Invitrogen BN1002) and run at 150 V for 2.5 h at 4 ◦ C. At higher temperatures (9, 24, 36, 43 ◦ C) the gels were run at constant 3–5 mA. Coomassie-stained bands were fixed with 40% methanol/10% acetic acid (microwaved on high for 45 s, rocked for 15 min at ambient temperature), and destained with 8% acetic acid (microwaved on high for 45 s, rocked for 2 h). NativeMark unstained protein standard (Invitrogen) was used to calculate Rf values. Cross-linking the subunits in the oligomer Dialdehydes cross-link proteins by reacting with amine groups (lysine residues of proteins) in solution; thus, the reaction is

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quenched by adding amines at high concentration. In our experiments glutaraldehyde (GA) cross-linked most discriminatively between 0.01% and 0.10% (1–10 mM) [58,59]. Small heat shock protein subunits were chemically cross-linked in a solution that contained 4 ␮M sHsp monomers with 0.01, 0.05 or 0.10% GA (Sigma–Aldrich) in 10 mM Tris, 1 mM EDTA pH 8.0. (Safety note: concentrated solutions of cross-linking agents should be handled with care in the hood; pipette tips and tubes should be disposed in a hazard container.) The solutions were incubated at 0, 22 and 42 ◦ C for 0, 1 or 10 min. The cross-linking reaction was quenched by addition of a concentrated Tris/glycine solution (final concentrations 1 M Tris, 0.5 M glycine, pH 9.0). Cross-linked species were resolved on SDS-PAGE. The molecular weight of resolved bands was determined by measuring Rf values and comparing these to the protein molecular weight standard with a Ferguson plot. To compare the overall oligomer size of the cross-linked sHsps to the native sHsp oligomer, cross-linked samples were run alongside non-cross-linked samples on native PAGE and on blue native PAGE. Chaperone experiments Capability of the sHsps to prevent denaturation of the client protein CS was measured by light scattering experiments using a Cary Eclipse fluorescence spectrophotometer with Cary single-cell Peltier accessory. CS (Sigma) was dialyzed at 4 ◦ C into 50 mM Tris, 2 mM EDTA, pH 8 and centrifuged; using the measured absorbance at 280 nm and molar absorptivity 1.78 mL mg−1 cm−1 , the concentration was adjusted to 170 nM (monomer, 48.969 kDa) with 40 mM HEPES/KOH buffer, pH 7.6 and 3-mL aliquots were frozen at −80 ◦ C. A 3-mL quartz cuvette was cleaned with 2 M nitric acid to remove aggregated protein, rinsed 10 times, dried with methanol. A 350 ␮L sample solution in 50 mM NH4 OAc, pH 7.0, 50 mM NaCl was added to the cuvette followed by 2650 ␮L CS, thereby holding the [CS monomer] constant at 150 nM in every experiment. Fluorescence excitation and emission was set to 500 nm, slits 5 nm, averaging time 10 s, cycle time 0.032 min, at 525 V. Temperature was set at 25 ◦ C with slow stirring. After 2 min, temperature was increased to 43 ◦ C and the fluorescence emission monitored for 50 min. Control experiments with CS alone, with chaperone alone, and with bovine serum albumin (BSA, Sigma) and CS were performed. The maximum absorbance of CS alone (average of 4 runs) was observed at 23.25 min with 14% SD; the subsequent decrease in absorbance is attributed to the formation of large aggregates. With chaperone alone, the absorbance at 23.25 min was 5% of the CS maximum. CS with BSA instead of chaperone had 96% of the CS maximum absorbance, thus eliminating the possibility that protein concentration alone prevents CS aggregation (see Supplemental Figure 1). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci. 2014.01.012.

Fig. 2. BN-PAGE for ZmHsp17.0-CII (17.0) and ZmHsp17.8 (17.8) showing an increase in smaller oligomers at higher temperature. Panel A: 36 ◦ C, lane 1, standards, lane 2, ZmHsp17.0-CII with faint, sharp band running at 595 kDa, intense, broad band at 310 kDa representing the dodecamer, and less intense bands representing oligomers running at 185, 120, and faint band at 60 kDa; Panel B: 43 ◦ C; lane 1, ZmHsp17.0-CII with bands running at 308, 180, 125 kDa; lanes 2 and 3, two concentrations of ZmHsp17.8-CII with bands running at 329 (dodecamer), 242, 194, 125 kDa; lane 4, standards.

QuantiFire-Model S99835 digital camera (image size 2048 square pixels, 150 pixel/inch). sHsp oligomer size was estimated by measuring 20 particles using the NIH ImageJ software.

Results Purification and refolding of Zea mays sHsps In producing these native sHsps for experimental study we found that the IMPACT-CNTM system was more effective than the polyhistidine tag method we previously tried. We modified the typical IMPACT-CNTM procedure to include washing the fusion protein while it was bound to the affinity column with nonionic detergent and a relatively high salt concentration to reduce nonspecific binding of other proteins. The detergent was removed by washing to refold the protein while the protein remained bound to the column. These modifications, especially the oncolumn refolding, resulted in greater purity. In comparison to literature reports and our experience with a different target protein, these sHsp fusion proteins were more resistant to the final cleavage reaction that removes the intein tag. Increasing the pH, concentration of DTT, cleavage temperature, and cleavage time facilitated the cleavage reaction and increased the yield of native sHsps.

Native PAGE and blue native PAGE up to 36 ◦ C indicate one major form with only minor amounts of smaller and larger oligomers

Electron microscopy Purified ZmHsp17.8-CII (50 mM NH4 OAc, pH 7.0, 50 mM NaCl buffer) at 0.3 mg/mL or 3:1 mixtures with CS (40 mM HEPES/KOH, pH 7.6) were heated at 43 ◦ C for 30 min then put on ice. Samples were diluted with buffer as necessary for best imaging. A 5 ␮L droplet was immediately absorbed onto a formovar and carbon coated copper grid for 1 min. Grids were then washed by floating them briefly on a drop of distilled water and then were stained with 2% aqueous uranyl acetate for 2 min. Samples were observed at ambient temperature on a Hitachi H-7500 transmission electron microscope and images taken with an Optronics

The oligomeric state of ZmHsp17.0-CII at various temperatures was of particular interest, since this sHsp is active during development as well as during heat shock. Native PAGE and blue native PAGE experiments at temperatures from 4 to 36 ◦ C showed only one major species, which ran as a 17- or 18-mer. As shown in Fig. 2A at 36 ◦ C it was possible to discern a faint band representing low amounts of a ∼34-mer, a broad band for the ∼18-mer, but only faint bands for smaller oligomers. Likewise, native PAGE and BN-PAGE for ZmHsp17.8-CII at 4 ◦ C (results not shown) revealed only one major oligomeric form with the same apparent degree of oligomerization as ZmHsp17.0-CII.

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in approximately equal concentration. Samples of ZmHsp17.8CII had four tighter bands. Significantly, at 43 ◦ C the band for the smallest oligomer of ZmHsp17.8-CII was the most intense. ZmHsp17.8-CII dissociates into a different subset of oligomers during heat stress than does ZmHsp17.0-CII. TEM images show the effects of prior heat treatment

Fig. 3. Cross-linking ZmHsp17.0-CII with glutaraldehyde (GA), a comparison with two Class I sHsps. Panel A: BN-PAGE of cross-linked ZmHsp17.0-CII: lane 1, standards; lanes 2-3 ZmHsp17.0-CII cross-linked with 0.1% GA for 10 and 20 min, nominal MWs 290 (major band) and 530 kDa (minor band). Panel B: SDS-PAGE of ZmHsp17.0-CII cross-linked at two concentrations of GA: lane 1, 0.01% GA 1 h with bands at 17, 33, 52, 73, 98 kDa; lane 2, uncross-linked ZmHsp17.0-CII, 17 kDa; lane 3, standards; lane 4, 0.1% GA 1 min, bands represent monomer (17, faint), dodecamer (150–250, broad) and two dodecamers (350 kDa). Panel C: SDS-PAGE showing monomer and dodecamer for ZmHsp17.0-CII, TaHsp16.9-CI, and PsHsp18.1-CI: lane 1, cross-linked ZmHsp17.0-CII, 200 kDa; lane 2, uncross-linked ZmHsp17.0-CII, 17 kDa; lane 3, standards; lane 4, cross-linked TaHsp16.9-CI; lane 5, uncross-linked TaHsp16.9-CI, lane 6, cross-linked PsHsp18.1-CI, lane 7, uncross-linked PsHsp18.1CI. The proteins were cross-linked with 0.1% GA for the same length of time.

These Class II sHsps are dodecamers below heat-shock temperature Heuberger et al. [60] pointed out that for membrane proteins the mass of Coomassie dye molecules increases the apparent mass of the oligomers in BN-PAGE; they scaled the molecular weight values using independently determined molecular weight information. Considering that plant C I sHsps typically oligomerize as dodecamers but that C II sHsps may have a different oligomeric structure [41,45–48] we investigated the degree of oligomerization of the Zea mays C II sHsps by a complementary method. After cross-linking with GA, the oligomers were sized by electrophoresis. When samples of ZmHsp17.0-CII cross-linked with GA were run on BN-PAGE at ambient temperature (Fig. 3A), the nominal oligomer sizes were the same as the native sHsp in Fig. 2A: an 18mer in high concentration with a small amount of 32-mer. Thus, the cross-linking reaction preserved the native oligomeric structure. In Fig. 3B, the cross-linked sHsp, run on SDS-PAGE, clearly corresponds to a dodecamer (∼200 kDa) rather than an 18-mer. There is an additional, faint band of approximately 24 subunits, which could be two dodecamers linked together. Therefore, we conclude that the mass of these sHsps appears larger on BN-PAGE, and scaling of the molecular weight is appropriate. In Fig. 3C, there is a comparison of cross-linked ZmHsp17.0-CII with cross-linked TaHsp16.9-CI and PsHsp18.1-CI. These two C I sHsps from wheat and pea have been shown by various methods including X-ray crystallography [23,47] to form dodecamers. The SDS-PAGE gel reveals that the major oligomers of all three sHsps are the same in size and correspond to a dodecamer. At heat shock temperature there is a significant increase in smaller oligomers At heat-shock temperature (43 ◦ C) the oligomerization pattern changed dramatically from that observed at 36 ◦ C. As shown in Fig. 2, for both ZmHsp17.0-CII and ZmHsp17.8-CII the broad, intense band at 310 kDa attributed to the dodecamer decreased significantly in intensity and the bands representing smaller oligomers increased in intensity. At 43 ◦ C ZmHsp17.0-CII had three, broad, diffuse bands of similar intensity, representing three oligomeric forms

Fig. 4 compares TEM images of ZmHsp17.8-CII before and after heat treatment at 43 ◦ C. Prior to heat treatment the images of the two sHsps consisted of a heterogeneous mixture of large objects, like raspberries, and small objects, like round beads. At higher magnification in Fig. 4A, one can see that the smaller objects, like doughnuts, have a central hole and round bumps around the ˚ are perimeter. The raspberries, varying in size from 25 to 38 A, clumps of doughnuts in various orientations. The variable size of these clumps indicates that these raspberries are loose associations rather than stable structures. It is not surprising, therefore, that these associations do not survive passage through the BN-PAGE gel. In contrast to the image in Fig. 4A, the image after preheating ZmHsp17.8-CII at 43 ◦ C shown in Fig. 4B is strikingly different, more homogeneous, showing individual round objects with a central hole. The diameter of these single doughnuts ranges from 6 to 11 nm, with mean 8.6 ± 1.4 nm, very similar to the size of TaHsp17.8-CII oligomers (9 nm) in TEM [41]. Apparently dynamic association-dissociation in the preheated sample favors the discrete dodecamer-sized oligomers rather than the larger heterogeneous clumps observed without preheating in Fig. 4A. There are a few edge-on views of the doughnuts in Fig. 4, showing that the objects are wider than they are high. The oligomers are squat cylinders with a central hole and they assume a preferred orientation with the larger face parallel to the grid. When we preheated mixtures of ZmHsp17.0-CII or ZmHsp17.8-CII with CS, the TEM images (not shown) were quite similar to that in Fig. 4B and the diameter of the doughnuts was similar also. We did not see evidence of larger heat-shock complexes. These two sHsps have different chaperone capability in light-scattering experiments The chaperone efficiency of these two sHsps was tested in a series of experiments with CS as the target protein. At 43 ◦ C, the heat-shock temperature for maize, CS denatures and forms aggregates that scatter light. In control experiments with CS alone there is a rapid rise in light scattering over the first 25 min of heating (Supplemental Figure 1), but in the presence of ZmHsp17.0-CII or ZmHsp17.8-CII the rise in light scattering is delayed or prevented in a ratio-dependent manner, showing that these two sHsps prevent aggregation and are chaperones for CS. As shown in Fig. 5 in the presence of ZmHsp17.0-CII at a ratio of 0.8:1 sHsp monomer to CS monomer, the light scattering of CS is decreased by 51% after 25 min compared to CS alone. Increasing the ratio to 1.6:1 and 3.2:1 decreases the light scattering by 71% and 76% after 25 min. At all three of these ratios the light scattering continues to increase until the end of the experiment (50 min). Either the heat shock complex dissociates and allows CS to aggregate, or the heat-shock complex itself aggregates and scatters light. At ratio 3.2:1 variability was observed in the light-scattering curves; one trial had light scattering 44% higher than the mean of the other two trials. Rather than averaging disparate results, two curves that closely agreed were averaged and are shown. At higher ratio (4.8:1) of ZmHsp17.0-CII the light scattering curves were not reproducible and the light scattering was in one experiment higher than that of CS alone. At the same concentration of pure ZmHsp17.0-CII the light scattering curve was essentially flat (Supplemental Figure 1). Thus,

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Fig. 4. Transmission electron micrographs of negatively stained ZmHsp17.8-CII. Panel A: ZmHsp17.8 at ambient temperature showing raspberry-like clumps of doughnutshaped objects. The doughnuts are 6.4 ± 0.8 nm in diameter. Scale bar 10 nm. Panel B: ZmHsp17.8 after 30 min preheating at 43 ◦ C. The image is more homogeneous, a sheet of doughnut-shaped objects 8.6 ± 1.4 nm in diameter. Some representative particles (also indicated by arrows in the image), which were used to estimate the diameter, are displayed in the bottom panel. Scale bar 10 nm.

although the pure ZmHsp17.0-CII does not aggregate during heat shock, it seems that the heat shock complex between ZmHsp17.0CII and CS does aggregate at higher ratios of ZmHsp17.0-CII. In the presence of ZmHsp17.8-CII at the lowest ratio (0.75:1 sHsp monomer to CS monomer) there is only an 8% decrease in light scattering, but at ratios of 1.5:1 and 3:1 the effect of added sHsp is pronounced, decreasing the light scattering by 64% and 84% after 25 min. Increasing the ratio to 4.5:1 produces negligible additional protection, indicating stoichiometric saturation. In contrast to ZmHsp17.0-CII, the light scattering curves for ZmHsp17.8-CII level off after 25 min, indicating that ZmHsp17.8-CII continues to prevent further CS aggregation until the end of the experiment. There was no indication that the heat shock complex between ZmHsp17.8-CII and CS aggregates, even at the highest ratio. Clearly both of these C II sHsps protect CS from the deleterious effects of heating, and they do so with greater effect at higher ratios of sHsp. At the lowest ratio of sHsp:CS, ZmHsp17.0-CII is the more effective chaperone, decreasing the absorbance at 25 min by 51%

compared with 8% for ZmHsp17.8-CII. At higher ratios of sHsp:CS, ZmHsp17.8-CII is more effective in decreasing the light scattering at 25 min. Significantly, there is a consistent difference in the shape of the light scattering curves for the two sHsps. While the light scattering curves for ZmHsp17.0-CII continue to increase even when the greatest amount of the chaperone is present, the curves for ZmHsp17.8-CII tend to level off after 30 min of heating. In summary, ZmHsp17.8-CII effectively protects CS from aggregation at a ratio of 3:1 sHsp:CS over the 50 min of the experiment, while ZmHsp17.0CII provides the same protective effect at the same ratio for less than 15 min. Analysis of supernatant solutions and pellets after heat shock verify that ZmHsp17.8-CII is significantly more protective Solutions with varying ratios of sHsp:CS were heated at 44 ◦ C for 15 min (ZmHsp17.0-CII) and 50 min (ZmHsp17.8-CII), then centrifuged. The supernatant solutions contained large amounts of the

Fig. 5. Light-scattering plots for 150 nM CS monomer with varying ratios of sHsp monomer: CS monomer at 43 ◦ C. The dotted line shows the increase in light scattering over time when CS denatured in the absence of sHsp. Experiments with ZmHsp17.8-CII (17.8) are indicated by solid lines with increasing weight at higher ratios of ZmHsp17.8-CII. Dashed lines denote experiments with ZmHsp17.0-CII (17.0). Each line represents the average of at least three experiments, except ZmHsp17.8-CII, 4.5:1 and ZmHsp17.0-CII 3.2:1, two experiments. %SD for runs with three experiments ranged from 2 to 13%.

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Fig. 6. SDS-PAGE of supernatants and pellets after heating solutions of sHsp and CS at 44 ◦ C. Both ZmHsp17.8-CII (17.8) and ZmHsp17.0-CII (17.0) solubilize CS at heat-shock temperatures, but there is significantly more precipitation of both chaperone and target protein with ZmHsp17.0-CII. The ZmHsp17.8-CII mixtures were heated for 50 min; the ZmHsp17.0-CII mixtures were heated for 15 min. The absolute amount of sHsp decreases left to right; the absolute amount of CS increases left to right. Panel A: supernatants: lane 1–3, molar ratio ZmHsp17.8-CII:CS 4.5, 3.0, 0.75, mass ratio 1.6, 1:1, 0.3; lanes 4–5, molar ratio ZmHsp17.0-CII:CS 6.7, 1.7, mass ratio 2.3, 0.6; lane 6, molecular-weight standards; lane 7, CS alone unheated, lane 8, ZmHsp17.8-CII alone unheated, lane 9, ZmHsp17.0-CII alone unheated. Panel B: pellets from the experiments shown in Panel A: lanes 1–3, ZmHsp17.8-CII, lanes 4–5, ZmHsp17.0-CII, lane 6, molecular-weight standards. Masses of the standards are shown in the center column.

sHsp monomer and the CS monomer at ∼50 kDa, as shown by the intense bands in the SDS-PAGE gel (Fig. 6A, lanes 1–5). Substantial amounts of CS remained in the solution, even at the lowest concentration ratio of sHsp:CS for both ZmHsp17.0-CII and ZmHsp17.8-CII. Particularly in lane 3, where the concentration ratio for ZmHsp17.8CII was only 0.75:1 the intense band for CS monomer at ∼50 kDa demonstrates the capability of this chaperone to stabilize the CS in solution during heating. After centrifugation of the heated solutions, the pellets were solubilized and run on SDS-PAGE. There was some precipitation of sHsp and CS in all cases. The pellet bands in the SDS-PAGE gel for ZmHsp17.8-CII heated with CS for 50 min are very weak (Fig. 6B, lanes 1–3), but even though the solutions of ZmHsp17.0-CII with CS were heated for only 15 min, there was far more precipitation of both sHsp and target protein with these samples (Fig. 6B, lanes 4–5). The most intense CS band was in lane 5, the sample that had a visible precipitate, where the ratio of ZmHsp17.0-CII to CS was 1.7:1. The comparison between lanes 5 and 3 is striking. Although the ratio of ZmHsp17.0-CII to CS in the lane 5 sample was more than double the ratio of ZmHsp17.8-CII to CS in lane 3, and the ZmHsp17.0-CII sample was heated only one-third as long, a much larger proportion of CS precipitated in lane 5. Even for the sample in lane 4, where the ratio of ZmHsp17.0-CII to CS was increased to 6.7:1, there is more CS precipitation than with ZmHsp17.8-CII in lane 3 (ratio of 0.75:1). These results confirm that both of these sHsps act as chaperones for CS during heat shock, but that ZmHsp17.8-CII is a more efficient chaperone for CS than ZmHsp17.0-CII. BN-PAGE run at 44 ◦ C with these mixtures of CS and sHsp immediately after the heating experiments described above failed to show high-molecular weight heat-shock complexes between CS and sHsps (results not shown). Discussion Stable storage configuration for ZmHsp17.0-CII and ZmHsp17.8-CII is dodecamer In plants, the CI and CII storage oligomers are typically dodecameric [31,47], and in this study cross-linking followed by SDS-PAGE showed this to be the case with the two sHsps from Z.

mays. The crystal structure of TaHsp16.9-CI disclosed a dodecamer composed of two hexameric disks measuring 9.5 nm in diameter and 5.5 nm high with a 2.5 nm central hole [23]. Likewise, the TEM images of both ZmHsp17.0-CII and ZmHsp17.8-CII reveal many ring-shaped or doughnut-shaped objects 8.6 ± 1.4 nm wide (Fig. 4B) with a central depression or hole; the edge-on views show that the objects are wider than they are high. The range of the measurements, from 6 to 11 nm, however, indicates some heterogeneity in the oligomers. This is consistent with BN-PAGE evidence at temperatures below and above heat shock temperature (Fig. 2) showing both larger and smaller oligomers than the dodecamer. On the quaternary level the structure of these sHsps from Z. mays are similar to each other and similar to that of TaHsp16.9-CI. We conclude that the TEM image in Fig. 4B shows the dodecameric storage structure that we had surmised from the cross-linking data. Oligomers smaller than the dodecamer are the likely active chaperones At heat shock temperature higher oligomers of ZmHsp17.0-CII and ZmHsp17.8-CII dissociate to smaller forms (Fig. 2). As others have concluded for CI sHsps [30,31,35] the functional oligomer for CII chaperone binding to its CS target is probably smaller than the dodecamer. Although a CI sHsp (PsHsp18.1-CI) dissociates to dimers and monomers at heat-shock temperature [30], like other investigators [47] we found no evidence of dimer formation for these CII sHsps. Compared to ZmHsp17.0-CII, ZmHsp17.8-CII dissociates into smaller species at heat-shock temperature; this difference is presumably a consequence of the nine additional amino acids in the N-terminus of ZmHsp17.8-CII. When the two Z. mays sHsps are heated with substrate protein CS, the TEM image is very similar to Fig. 4B, a sheet of doughnuts, with no larger objects of regular form that might correspond to heat-shock complexes. Likewise, we were not able to observe heatshock complexes by BN-PAGE, perhaps because the complexes dissociated during migration through the gel even though it was held at 43 ◦ C. These complexes are apparently dynamic as others have found [12,29,30], and the lability is probably necessary for refolding by other higher-molecular-weight Hsps, by analogy to observations with HSP70 co-chaperones [63].

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These two Class II sHsps from Zea mays are specialized chaperones The cytosolic CI and CII sHsps in plants originated before mosses diverged from seed plants [64], and they have evolved to a diverse group of chaperones that exhibit significant differences in expression, location in the cell, and function [43,49,65]. It was previously shown [51,55] that ZmHsp17.0-CII is expressed at different times and varying levels during anther development but ZmHsp17.8-CII was not expressed above background; both sHsps were expressed during heat shock. Furthermore, previous investigators [8] found that the expression of the two sHsps in radicles responds differently to the stress of heavy metal ions. ZmHsp17.0-CII RNA transcripts accumulate when stressed by either Cd2+ or Zn2+ , but there is little or no change in the level of transcripts of ZmHsp17.8-CII. Although both of these sHsps are expressed during the emergency situation triggered by heat shock, it seems that ZmHsp17.0-CII is optimized for challenges to client proteins that occur when heat is not an issue. On the primary level the N-terminal intervening sequence that differentiates ZmHsp17.8-CII from ZmHsp17.0-CII is found in some other CI and CII sHsps, but not in TaHsp16.9-CI. In Fig. 1 the intervening sequence corresponds to the center of the ␣-2 helix in the TaHsp16.9-CI crystal structure [23]. In some sHsps this glycinerich intervening sequence also includes proline residues; these two amino acids have the lowest potential for ␣-helix secondary structure and high propensity for coil [61,62]. It seems likely from the sequence information that the secondary structure of ZmHsp17.8CII is different from and less structured than TaHsp16.9-CI or ZmHsp17.0-CII in this section of the N-terminus, although as we have seen from the TEM images the quaternary structures are similar. In our light-scattering experiments ZmHsp17.0-CII delays aggregation in a concentration-dependent manner, but ZmHsp17.8-CII at a ratio of 3:1 solubilizes CS over the entire 50 min of the experiment. That ZmHsp17.8-CII gives significantly better protection for CS against heat shock is especially clear from the precipitation experiments. More CS precipitated with ZmHsp17.0-CII, after a shorter heating time, even when there was over eight times as much ZmHsp17.0-CII compared to ZmHsp17.8CII. Therefore, it appears that ZmHsp17.0-CII is best adapted to its developmental role, while ZmHsp17.8-CII is more efficient at protecting cellular proteins from heat shock. Furthermore, our results highlight the importance of the N-terminal portion of the sHsp, which seems to be intrinsically disordered yet crucial in recognition, binding and solubilizing of target proteins. Our knowledge about these CII sHsps from Z. mays is unique in the sense that there is previously published information on the genes and their expression [8,51,55,66] and now in the present work we have detailed information on the behavior of the corresponding proteins in solution. We do not have similar information on other CII sHsps from Z. mays to compare to our results; previous studies of cytosolic sHsps from Z. mays dealt with the genes and the expression of those genes [65,67] and focused on CI sHsps [68,69]. We can compare the solution studies here with other plant CII sHsps, and we can point out some tantalizing evidence that the lack of the N-terminal intervening sequence that differentiates ZmHsp17.0-CII from ZmHsp17.8-CII lessens CII sHsp chaperone capability during heat shock in some other plant species. (See Fig. 1 for sequence comparisons.) In a heating experiment similar to the one shown in Fig. 6 PsHsp17.7-CII and AtHsp17.7-CII (which lack the intervening sequence) were less effective than TaHsp17.8-CII (which has the intervening sequence) in stabilizing luciferase (luc) in solution, although all three were more effective than the CI sHsps that also lack the sequence [47]. PsHsp17.7-CII (but not AtHsp17.7CII) precipitated significantly more with target proteins luc and

MDH, and it formed a larger heat-shock complex with luc than the other five CII and CI sHsps in the study. Although ZmHsp17.0CII alone did not aggregate when heated (Supplemental Figure 1) it precipitated significantly more than ZmHsp17.8-CII in the presence of target protein CS. It is possible that the chaperone-target complex for sHsps that lack the N-terminal hydrophilic sequence is less soluble and may aggregate during heating. If both the heatshock complex and the unchaperoned target protein aggregate during the experiment, this would explain why the light-scattering curve continues to increase with ZmHsp17.0-CII over time (Fig. 5) even though ZmHsp17.0-CII solubilizes substantial amounts of CS (Fig. 6). In our experimental work the biochemical evidence from native PAGE and from the light-scattering data are sensitive to the sequence-level differences in the two sHsps and provide evidence for separate roles of these two sHsps. Our blue-native PAGE results at high temperature suggest that the small (probably functional) oligomers are different for ZmHsp17.0-CII compared to ZmHsp17.8-CII. The light-scattering data show that ZmHsp17.8-CII is a more efficient chaperone during heat shock. Taken together, these biochemical results imply that ZmHsp17.8-CII is specialized for dealing with heat-disrupted client proteins while ZmHsp17.0CII is optimized for developmental tasks. Significance Studies like these investigating functional differences among sHsps have practical applications in developing heat-resistant plants. In a warming world, it is necessary to design and engineer effective sHsps for food crops, and this process has already begun [70,71]. Also, since sHsps impact a wide range of human diseases [72], especially neurodegenerative diseases [73–75], increasing our knowledge of their mechanism of action may lead to therapeutic benefit. Acknowledgements Thanks to the many Senior Independent Study students and summer research students at The College of Wooster who laid the foundation for the work reported here. Thanks to Eman Basha and Elizabeth Vierling for samples provided. References [1] Y. Sun, T.H. MacRae, Small heat shock proteins: molecular structure and chaperone function, Cell. Mol. Life Sci. 62 (2005) 2460–2476. [2] M. Haslbeck, T. Franzmann, D. Weinfurtner, J. Buchner, Some like it hot: the structure and function of small heat-shock proteins, Nat. Struct. Mol. Biol. 12 (2005) 842–846. [3] P. Van den, I. Jssel, D.G. Norman, R.A. Quinlan, Molecular chaperones: small heat shock proteins in the limelight, Curr. Biol. 9 (1999) R103–R105. [4] R. Mittler, A. Finka, P. Goloubinoff, How do plants feel the heat? Trends Biochem. Sci. 37 (2012) 118–125. [5] A.E. DeRocher, K.W. Helm, L.M. Lauzon, E. Vierling, Expression of a conserved family of cytoplasmic low molecular weight heat shock proteins during heat stress and recovery, Plant Physiol. 96 (1991) 1038–1047. [6] J. Horwitz, Alpha-crystallin can function as a molecular chaperone, Proc. Natl. Acad. Sci. U S A 89 (1992) 10449–10453. [7] U. Jakob, M. Gaestel, K. Engel, J. Buchner, Small heat shock proteins are molecular chaperones, J. Biol. Chem. 268 (1993) 1517–1520. [8] R.A. Bouchard, Z. Yang, R.I. Greyson, D.B. Walden, Zinc and cadmium induction of small heat-shock gene transcripts in maize seedlings, Maydica 49 (2004) 105–114. [9] N. Iqbal, S. Farooq, R. Arshad, A. Hameed, Differential accumulation of high and low molecular weight heat shock proteins in Basmati rice (Oryza sativa L.) cultivars, Genet. Resour. Crop Evol. 57 (2009) 65–70. [10] B. Grigorova, I.I. Vaseva, K. Demirevska, U. Feller, Expression of selected heat shock proteins after individually applied and combined drought and heat stress, Acta Physiol. Plant. 33 (2011) 2041–2049. [11] H.S. Mchaourab, J.A. Godar, P.L. Stewart, Structure and mechanism of protein stability sensors: chaperone activity of small heat shock proteins, Biochemistry 48 (2009) 3828–3837.

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