ClpB/Hsp100 proteins and heat stress tolerance in plants

http://informahealthcare.com/bty ISSN: 0738-8551 (print), 1549-7801 (electronic) Crit Rev Biotechnol, Early Online: 1–13 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2015.1051942

REVIEW ARTICLE

ClpB/Hsp100 proteins and heat stress tolerance in plants Ratnesh Chandra Mishra and Anil Grover

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Yeungnam University on 06/30/15 For personal use only.

Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India

Abstract

Keywords

High-temperature stress can disrupt cellular proteostasis, resulting in the accumulation of insoluble protein aggregates. For survival under stressful conditions, it is important for cells to maintain a pool of native soluble proteins by preventing and/or dissociating these aggregates. Chaperones such as GroEL/GroES (Hsp60/Hsp10) and DnaK/DnaJ/GrpE (Hsp70/Hsp40/nucleotide exchange factor) help cells minimize protein aggregation. Protein disaggregation is accomplished by chaperones belonging to the Caseinolytic Protease (Clp) family of proteins. ClpB/Hsp100 proteins are strikingly ubiquitous and are found in bacteria, yeast and multicellular plants. The expression of these proteins is regulated by heat stress (HS) and developmental cues. Bacteria and yeast contain one and two forms of ClpB proteins, respectively. Plants possess multiple forms of these proteins that are localized to different cellular compartments (i.e. cytoplasm/nucleus, chloroplast or mitochondria). Overwhelming evidence suggests that ClpB/Hsp100 proteins play decisive roles in cell adaptation to HS. Mutant bacteria and yeast cells lacking active ClpB/Hsp100 proteins are critically sensitive to high-temperature stress. Likewise, Arabidopsis, maize and rice mutants lacking cytoplasmic ClpB proteins are very sensitive to heat. In this study, we present the structural and functional attributes of plant ClpB forms.

Bacteria, chaperone, heat stress, heat shock protein, Hsp100, plants, proteostasis, rice, thermotolerance, yeast

Introduction Because of their sessile lifestyle, plants are highly vulnerable to heat stress (HS). The reproductive processes (i.e. flowering and seed setting) are particularly susceptible to HS and can directly lead to crop productivity loss (Bita & Gerats, 2013). The need to produce transgenic crops with enhanced heat tolerance to meet the growing food demand of the 21st century is indeed a pressing issue. It has emerged from the last two decades of research that ClpB/Hsp100 proteins are critical in governing plant thermotolerance. Arabidopsis mutants with defects in ClpB/Hsp100 protein expression are extremely sensitive to heat. Likewise, maize and rice mutant plants defective in ClpB/Hsp100 synthesis have impaired thermotolerance. Hsp100 genes from plant species can complement thermotolerance defects in yeast Hsp100 mutant strains (Lee et al., 1994; Schirmer et al., 1994; Singh et al., 2010). In this review, we discuss the current understanding of the plant ClpB/Hsp100 proteins.

Proteostasis and molecular chaperones Living cells contain thousands of properly folded and functionally active proteins. The term proteostasis includes

Address for correspondence: Prof. Anil Grover, Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi – 110021, India. Tel: +91-11-24115097. Fax: +91-11-24115270. E-mail: [email protected]

History Received 22 April 2014 Revised 26 August 2014 Accepted 29 April 2015 Published online 30 June 2015

processes associated with biogenesis, folding, unfolding, trafficking and turnover of proteins. While being liberated as nascent polypeptides from ribosomes under native cellular conditions, proteins are folded into biologically active, threedimensional (3D), tertiary and quaternary structures. Any deviation from this general scheme, either spontaneously or under cellular stress, may result in misfolded or partially folded proteins. Such contorted proteins have exposed hydrophobic residues that have a high affinity for aggregation. Proteostasis is of the utmost importance for proper cellular functions to be maintained. While protein folding is primarily governed by a ‘‘blueprint’’ in the form of an amino acid sequence, this process is accomplished by a plethora of molecular chaperones to avoid misfolding and intracellular aggregation. The chaperones GroEL/GroES (Hsp60/Hsp10) and DnaK/DnaJ/GrpE [Hsp70/ Hsp40/nucleotide exchange factor (NEF)] recognize and refold misfolded proteins into their biologically active conformations. These chaperones can prevent protein aggregation. However, they are limited in their ability to disaggregate clumps of misfolded proteins. Caseinolytic Protease (Clp) proteins play crucial roles in protein aggregate removal. Peptides scavenged from these aggregates can either be solubilized into their native folded state or directed to proteolytic degradation. Clp proteins, along with other molecular chaperones, constitute the cellular molecular arsenal to combat protein damage induced by physiological, chemical and pathological stresses.

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R. C. Mishra & A. Grover

Crit Rev Biotechnol, Early Online: 1–13

Diversity of Clps Clp proteins belong to the large AAA (ATPases associated with diverse cellular activities) superfamily of proteins. The AAA+ protein family consists of eight different subfamilies (ClpA, B, C, D, M, N, X and Y) identified across different species (Butler et al., 2006; Dougan et al., 2003; Gottesman, 2003; Schlieker et al., 2005). Clps are marked by the presence of one to two nucleotide binding domains (NBDs) (Mogk et al., 2008). Based on the number of NBDs, Clps are broadly divided into two major classes. Class I proteins (e.g. ClpA, ClpB, ClpC and ClpD) are relatively large with sizes ranging from 68 to 110 kDa and have two NBDs. Class II proteins (e.g. ClpM, ClpN, ClpX and ClpY/HslU) are smaller in size (40–50 kDa) and have one NBD (Figure 1). Clps are widely distributed in living organisms. ClpA has been found in Gram-negative bacteria. ClpC is more widespread and has been found in Gram-positive bacteria, cyanobacteria and in the chloroplasts of algae and higher plants. ClpD, identified first in Arabidopsis (called Erd1), is localized to chloroplasts and restricted to only higher plants. ClpM and ClpN are more rare, and the former has been found in Mus musculus (a mammal) and Plasmodium falciparum (a protozoan) and the latter in Pseudomonas aeruginosa. ClpX and ClpY were discovered in bacteria, with the former also identified in humans and higher plants (Hillier et al., 1996; Shanklin et al., 1995). The phylogenetic relatedness of different Clp forms is presented in Supplementary Figure 1. Class I forms (ClpA, ClpB, ClpC and ClpD) are grouped

together on the left, while class II forms (ClpX and ClpY) are on the right in the phylogenetic tree (Supplementary Figure 1). The Clp ATPase system was discovered as a complex capable of hydrolyzing casein in vitro (Hwang et al., 1987; Katayama-Fujimura et al., 1987). It was found to be a twocomponent protease system composed of a ClpA regulatory ATPase module and a ClpP proteolytic component. ClpP resembles the cytosolic 26S proteasome (Schelin et al., 2002). The ClpAP complex forms a general protein chopper, along with the Lon protease machinery, that degrades SsrA-tagged substrates (Wang et al., 2007). Because of the threading property of ClpA, this complex state is especially useful for the degradation of large protein aggregates. The ClpAP complex gains specificity towards aggregated proteins through association with the ClpS adapter, which inhibits the general activity of ClpAP enabling it to specifically bind and degrade aggregated proteins (Dougan et al., 2002). Subsequent to ClpA discovery in Escherichia coli, ClpB proteins sharing close homology with ClpA were identified (Gottesman et al., 1990). Additionally, ClpB was also identified in yeast (Sanchez & Lindquist, 1990). In the early 1990s, reports that plants accumulate high-molecular weight Hsps of 100–110 kDa started appearing (Schirmer et al., 1994; Singla & Grover, 1993). Singla and Grover (1993) noted that a heat-induced, 100 kDa protein in rice cross-reacts to anti-yeast ClpB antisera, heralding the beginning of plant ClpB research. It was initially believed that

Class Clp type

Class I

Figure 1. The organization and distribution of Clps in diverse living forms. The structure and organization of Clp domains is largely conserved in bacteria, fungi, plants and other eukaryotes. The biological species containing the respective Clp members are shown in the figure. Supplementary Figure 1 can be referenced to see the phylogenetic relatedness among diverse Clp subfamilies. Clp proteins are marked by the presence of nucleotide binding domains (NBD) with two NBDs in class I and one NBD in class II Clp proteins. NBDs (NBD1, dark green; NBD2, blue) are highly conserved domains. The two NBDs in the ClpB subfamily of class I protein are separated by a long M-domain. The Mdomain is not conserved and varies in length and homology. ClpA lacks the M-domain, but a small M-domain is present in ClpC and ClpD. N-domain sequences (purple) are relatively less conserved. The class II HslU and ClpN proteins lack an N-domain. A small spacer interrupts the only NBD of HslU, which is different from the spacer of other ClpB proteins. Hsp104 (yeast ClpB) has an additional unique C-terminal domain (black), which is a signature of fungal ClpBs. [See the online article for color version of this figure]

Class II


+

Domain architecture

Distribuon

ScHsp104

Budding yeast

Plant ClpB

Higher plants

Bacterial ClpB

Bacteria

ScHsp78

Budding yeast (mitochondria)

ClpA

Gram-negave bacteria

Bacterial ClpC

Gram-posive bacteria, cyanobacteria

Plant ClpC

Chloroplast of algae and higher plants

ClpD

Chloroplast of higher plants

Clp M

Mammals and protozoa

Clp N

Rare in few bacteria eg. Pseudomonas aeruginosa Prokaryotes, eukaryotes Including Plants and mammals (in mitochondria) Bacteria

ClpX ClpY/HslU

N-terminal

M-domain

Mitochondrial transit pepde

Spacer

Chloroplasc transit pepde

NBD2

NBD1

C-terminal extension


DOI: 10.3109/07388551.2015.1051942

ClpB is a functional ortholog of ClpA because of the close homology of these two proteins. However, it was soon found to be an untenable notion as ClpB cannot replace ClpA in casein degradation by ClpP (Zolkiewski, 2006). ClpB lacks the tripeptide IGF/L responsible for ClpP association, but other Clps do contain this motif. Clps, in general, are involved in the removal of stress-related protein aggregates. Importantly, ClpB functions solely in a non-degradative pathway of protein renaturation to liberate native functional proteins from aggregates. The yeast and bacterial ClpB forms that had been experimentally engineered to interact with ClpP, when co-expressed with ClpP in deletion strains, failed to be thermotolerant (Tessarz et al., 2008; Weibezahn et al., 2004). Thus, ClpB proteins are important to reactivate heat aggregated proteins under demanding, stressful conditions. The ClpB subfamily is characteristically induced by HS and is different from other Clp members in this respect. As plant ClpBs have a molecular weight of approximately 100 kDa and HS inducible expression, they are also referred to as Hsp100 proteins (Kim et al., 2012; Singh et al., 2010). We will restrict our discussion to only ClpB/Hsp100 proteins for the remainder of this review.

Distribution and phylogenetic relatedness of plant ClpBs ClpBs have been found in prokaryotes (referred to as ClpB), yeast (Hsp104) and plants. Plants are uniquely endowed with separate organelle and cytoplasmic forms of ClpB. These forms are referred to as ClpB-C (cytoplasmic), ClpB-M (mitochondrial) and ClpB-P (chloroplastic) (Mishra & Grover, 2014). Phylogenetic analysis of the amino acid sequence of the different ClpB forms in different biological species is shown in Figure 2. This analysis shows that besides being found in autotrophs, ClpBs are present in protozoa, cellular slime molds and choanoflagellates (free living, colonial, unicellular flagellate that are a close living relative of animals). Importantly, there is no reported ClpB-like protein in animals. From the phylogenetic analysis, it appears that ClpBs have three different evolutionary lineages, two comprising prokaryotes, fungi, protists and slime molds and one representing the plant kingdom. In the plant lineage, there is a defined hierarchy in the evolutionary pattern of these proteins. Moving from algae through pteridophytes to flowering plants, ClpBs have diverged into specific forms in dicot and monocot groups. In recent years, comprehensive details of genes coding for ClpB proteins in Arabidopsis thaliana and Oryza sativa have been unveiled. Singh et al. (2010) showed that both Arabidopsis and rice contain three ClpB proteins localized to different cellular compartments: cytoplasm/nucleus [At1g74310 (AtClpB-C) and Os05g44340 (OsClpB-C)], chloroplast [At5g15450 (AtClpB-P) and Os03g31300 (OsClpB-P)] and mitochondria [At2g25140 (AtClpB-M) and Os02g08490 (OsClpB-M)]. We identified ClpB-C, ClpB-P and ClpB-M proteins in over 30 plant species by performing a blast analysis using the three rice ClpB homologues on the plant sequence database ‘‘Phytozome’’. Figure 3 shows the phylogenetic tree generated using ClpB sequences from diverse dicot and monocot species. Figure 3 highlights that

ClpB/Hsp100 proteins and HS tolerance in plants

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the presence of three ClpB homologs is a hallmark of plant cells. Figure 4 shows a phylogenetic tree constructed using ClpB protein sequences from selected species across the evolutionary spectrum, including plant ClpB-C, ClpB-M and ClpB-P forms, to decipher the relatedness of plant ClpB homologs with the ClpBs from other species. From this detailed phylogenetic analysis, the following important observations are accrued: (a) a plant cell has multiple ClpB proteins (cytoplasmic, plastidial and mitochondrial) as opposed to the solitary cytoplasmic ClpB form in prokaryotes, (b) ClpB proteins in plant species exhibit three specific lineages, two that are more closely related (ClpB-M and ClpB-P) and a third (ClpB-C) that arose independently, and the ClpB-C lineage contains ClpB proteins of eukaryotic members such as protists (Dictyostelium), algae, lower plants (bryophytes and pteridophytes) and higher plants (dicots and monocots), (c) ClpB proteins, regardless of either organelle or cytoplasmic localization, tend to clump together in different dicot as well as monocot species (Figures 3 and 4); hence, ClpB proteins corroborate the taxonomic classification of monocots and dicots species based on a plethora of other traits, (d) yeast Hsp104 (ScHsp104) forms a separate clade altogether, suggesting that fungal ClpB may have evolved separately and is distinct from prokaryotic and plant ClpBs and (e) the organelle proteins form a separate lineage that is composed of ClpBs from bacteria, cyanobacteria, unicellular algae and diverse plant species, suggesting that organelle and cytoplasmic ClpBs of plants have significantly diverged (Figure 4). The endosymbiotic theory proposes that organelles of eukaryotes have originated as a result of symbioses between separate single-celled organisms (Gould et al., 2008). The data in Figure 4 support the idea that the origin of plant

OsClpB-C Monocots Eudicots Club-mosses Mosses Green algae Apicomplexans kinetoplasds Red algae Cyanobacteria Cellular slime moulds Choanoflagellates Chytrids Diatoms Oomycetes

Figure 2. Phylogenetic analysis of ClpB genes across diverse genera. The OsClpB-C protein sequence was blasted in the Blast-P program of NCBI against all identified ClpB-like proteins in diverse genera to generate an unrooted force tree (similar to a radial tree, where nodes are pushed away for better presentation). The tree was constructed using a method based on fast minimum evolution with a maximum sequence difference of 0.85 using the Grishin distance model. This representation is a diagrammatic imitation of the same tree.


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Figure 3. The phylogenetic relatedness of cytoplasmic and organelle ClpB forms from various plant species. Sequences were obtained from the Phytozome database and aligned using the global alignment tool (with free end gaps) of the geneious software (www.geneious.com), and a tree was generated using the default parameters. The phylogenetic tree (unrooted radial) was viewed and modified for better presentation using Fig Tree v1.4.0 software (http://tree.bio.ed.ac.uk/software/figtree). Details of the protein sequences used to generate this phylogenetic tree are shown in Supplementary Table 1. The three lineages consist of proteins from both monocots and dicots and form separate clades. Cytoplasmic (ClpB-C), mitochondrial (ClpBM) and chloroplastic (ClpB-P) lineages are marked separately (monocots species, blue fonts; dicots species, red fonts). [See the online article for color version of this figure]

organelle ClpB proteins is from endosymbionts. The organelle ClpB lineage as a whole is more related to the cyanobacterial lineage than to any other bacterial species; this suggests that the plastidial homologues may have originated from a cyanobacterial endosymbiont possibly from a unicellular green alga host. Furthermore, it appears that mitochondrial ClpB homologues may have originated not from an endosymbiont of mitochondrion, but from chloroplastic ClpB homologues.

ClpB protein domains ClpB proteins contain a remarkably similar modular architecture across species. Broadly, a ClpB protein protomer has three distinct regions: NBD1, NBD2 and a long M-domain that separates the two NBDs, with flanking N- and C-termini (Figure 5). The monomers undergo oligomerization to form a functional ClpB hexamer. A typical 3D arrangement of the different ClpB domains in a functional hexamer is shown in Figure 6.


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DOI: 10.3109/07388551.2015.1051942

Figure 4. The evolutionary relationships between plant ClpB homologs and ClpB proteins from various organisms. Details of the protein sequences used to generate this phylogenetic tree are shown in Supplementary Table 1. Sequences were aligned using geneious software (www.geneious.com) with the default parameters (Global alignment with free end gaps) to generate a tree. The Fig tree software was used to visualize and edit the tree (http:// tree.bio.ed.ac.uk/software/figtree).

The N-terminal domain of the ClpB protomer precedes NBD1 and is connected by a glycine-rich long unstructured linker leading to its high motility. Considering its mobility and interaction with different substrates in vivo, the N-domain has been associated with substrate recognition and binding activity. Increasing or decreasing the flexibility of the linker

lowers the reactivation efficiency of the aggregated substrates (Mizuno et al., 2012; Zhang et al., 2012). NBDs take part in the binding and hydrolysis of ATP, which provides energy critical for the oligomerization and translocation of threaded polypeptides across the central channel of the ClpB hexamer (Mogk et al., 2008). NBDs comprised several

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N

NBD2

NBD1 M-domain

(A)

1

2

3

4

5

6

1


NBD1

(B)

C

1. 2. 3. 4. 5. 6.

Walker A Channel Loop 1(with Y residue) Walker B1 Sensor 1 Mof Arginine Finger Walker B2

2

1

2

3 4

M-domain

NBD2

1. 2.

1. 2. 3. 4. 5. 6.

Spacer Signature II Spacer Signature III

Amino acid posions

5

6

Walker A Channel Loop 2 (with Y residue) Walker B Sensor 1 Mof Signature IV with R finger mof Signature V with Sensor 2 Mof Conserved Glutamate residue

Conserved Tyrosine residue

NBD1

1 Consensus

206 212 207 209

E. Coli Yeast Arabidopsis Rice

213 219 214 216

243 249 244 246

257 263 258 260

Walker A

264 270 265 267

279 285 280 282 Walker B1

Channel Loop 1 Conserved Lysine residue

Consensus E. Coli Yeast Arabidopsis Rice

309 311 310 312

318 320 319 321

384 386 385 387

332 334 333 335

396 398 397 399 Walker B2

Arginine Finger

Sensor 1 Mof

M-domain Consensus E. Coli Yeast Arabidopsis Rice

491 495 492 494

542 552 543 544

511 515 512 514

551 561 552 553 Spacer Signature III

Spacer Signature II

Conserved Tyrosine residue

NBD2

Conserved Glutamate residue


Channel Loop 2

Walker A

683 692 684 685

674 683 675 676

659 668 660 661

648 657 649 650

612 621 613 614

605 614 606 607

Walker B

Conserved Lysine residue


711 720 712 713

722 731 723 724 Sensor 1 Mof

748 757 750 751

766 775 768 769 Signature IV with R finger mof

813 824 815 816

821 832 823 824

857 908 911 912

Signature V with Sensor 2 Mof

Figure 5. Domain organization of a typical ClpB protomer with insights into the various conserved motifs, residues and signature sequences. (A) The diagrammatic sketch shows the whole protein broadly divided into domains arranged in the following sequence: N-domain, NBD1, M-domain, NBD2 and C-terminal domain. The extent of these domains is shown by the line diagram above the diagrammatic sketch (N, NBD1, M-domain, NBD2 and C). The M-domain is intercalated at the C-terminus of NBD1 and thereby separates the two NBDs. Furthermore, the NBDs and M-domain have several consensus motifs with structural and functional relevance (numbered within each domain and indicated below the schematic representation). Walker A is an ATP binding site and Walker B takes part in ATP hydrolysis. (B) Global alignment with free end gaps of ClpB proteins from E. coli, S. cerevisiae, O. sativa (monocot) and A. thaliana (dicot) using the default settings of the geneious software (www.geneious.com). The consensus amino acid sequences (comprised the above schematic representation in A) are shown in three panels representing NBD1 (blue), M-domain (pink) and NBD2 (green). The conserved amino acid residues critical for NBD function, lysine (K) in Walker A and glutamate (E) in Walker B are highlighted. The location of the conserved aromatic amino acid tyrosine that lines the axial channel of the ClpB hexamer in both the NBDs is also shown. The position of the conserved arginine residue that forms the arginine finger motif is shown. The M-domain, which is characteristic of ClpB proteins, is shown, and the two signature sequences present in this domain are highlighted. Spacer signature II is important for the disaggregation of substrates. The amino acid positions of different motifs for respective species are indicated. [See the online article for color version of this figure]


DOI: 10.3109/07388551.2015.1051942

N-domain

Channel loop (Y) Glycine rich linker NBD1

NBD2

NBD1

NBD2

M-domain


Axial Channel

Figure 6. Diagrammatic representation of the 3D architecture of a typical ClpB hexamer. Various domains with an axial channel through which the threaded substrate passes are shown. The N-domain is free moving, by virtue of the long glycine-rich (structureless) linker, which assists in efficient substrate recognition and binding. The organization of both NBDs along with the locations of the conserved aromatic amino acid tyrosine, which constitutes the channel loop (Y), is shown. The positions of the tyrosine residues line the central pore, which assists in the binding and translocation of substrates through the axial channel. The long M-domain that differentiates ClpB from other Clp subfamilies extrudes out of the ClpB body forming a molecular toggle that can freely make contact with NBDs of parent and/or neighboring protomers is also shown. More information on conserved motifs, residues and signature sequences in the respective ClpB domains is provided in Figure 5.

motifs, as shown in Figure 5. The Walker A motif provides the site of ATP binding with highly conserved Lys residues (K-212, NBD1 and K-611, NBD2 of E. coli ClpB) contacting the phosphate groups of the b and g nucleotides. Walker B takes part in ATP hydrolysis and has conserved Glu residues (E-279, NBD1 and E-678, NBD2 of E. coli ClpB) proposed to be a catalytic base. It has been suggested that: (a) Walker A, and not Walker B, is required for the ATP binding of both NBDs, (b) Walker B in both NBDs is required for ATPase activity, (c) ATP binding to Walker A of NBD1, and not hydrolysis, is essential and sufficient for oligomerization and (d) a mutation in the Walker A motif in each of the NBDs will abolish its chaperone function (Watanabe et al., 2002). An arginine finger motif contacts the g-phosphate of the ATP bound to a neighbored subunit, an action that is critical to bring about oligomerization (Figure 5). Tyrosine (Y) residues of the channel loop line the central pore and play a crucial role in substrate binding with a possible translocation mechanism (Figures 5 and 6) (Lum et al., 2004). The long M-domain, which protrudes laterally from the ClpB ring, makes ClpB unique among members of the Clp family (Figure 6). The long M-domain comprised coiled-coil-like heptads forming four helices, each of which is essential for the oligomerization and chaperone activity of ClpB (Desantis & Shorter, 2012; Kedzierska et al., 2003). Recent studies have suggested that this coiled-coil structure forms two wings (with motif 1 and motif 2) and that both of these contribute to maintaining a repressed ClpB activity state. While motif 2 docks intra-molecularly to NBD1 (regulating ClpB unfolding power), motif 1 interacts with a neighboring NBD1 domain. Furthermore, stabilizing motif 2 docking suppresses ClpB

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activity but destabilization results in enhanced activity with increased unfolding power in vitro (Oguchi et al., 2012). The C-terminal domain is essential for oligomerization, ATPase and chaperone activity (Barnett et al., 2000). The C-terminal domain of Hsp104 is decisive for the nuclear localization of this protein. Hsp104 has a longer C-terminal extension that is largely acidic and includes tetrapeptide DDLD and VLPNH residues that are conserved in fungal ClpBs but not in other orthologs (Mackay et al., 2008). The complexity in structure of ClpB proteins reveals that each of the domains is essential and contributes towards the overall disaggregation activity of ClpB.

Mutant forms of plant ClpB-C proteins A comprehensive account of the mutational analysis of different ClpB domains and motifs of bacterial and yeast ClpBs is provided in Supplementary Table 2 (Barnett & Zolkiewski, 2002; Barnett et al., 2000; Bosl et al., 2005; Cashikar et al., 2002; Doyle et al., 2007; Ferreira et al., 2001; Hattendorf & Lindquist, 2002; Jung et al., 2002; Kurahashi & Nakamura, 2007; Li & Sha, 2003; Liu et al., 2002; Lum et al., 2004; Mogk et al., 2003; Schirmer et al., 1998, 2001, 2004; Schlieker et al., 2004; Parsell et al., 1991, 1994; Schaupp et al., 2007; Tkach & Glover, 2008; Wendler et al., 2007). In this section, a mutational analysis of plant ClpB-C is provided. A great deal of information on plant ClpB-C biology has emerged from Arabidopsis Hot1 (locus encoding Arabidopsis ClpB-C) mutants (Hong & Vierling, 2000). In Hot1-1, a conserved Glu residue in the second ATP binding domain of ClpB-C is converted to a Lys residue. This mutant is defective in its acquired thermotolerance response and has complete arrest of hypocotyl growth during HS. In addition, the naturally highbasal thermotolerance of Arabidopsis seeds was also compromised (Queitsch et al., 2000). A similar phenotype was obtained in case of the ClpB-C null mutant, Hot1-3 (a T-DNA is inserted in the first exon). These data suggest that either the loss of function or the absence of ClpB-C has a similar effect towards the sensitivity of Arabidopsis plants to HS. The Hot1-2 mutant (a T-DNA inserted in the promoter region of the AtClpB-C gene, 101 bases upstream of ATG) accumulates 10fold less protein in seeds and shows severely compromised basal seed thermotolerance (Hong & Vierling, 2001). Another gain-of-function mutant Hot1-4, with a mutation in the coiledcoil domain (M-domain), displays a more severe phenotype and is sensitive even to 38 C, which is a permissive temperature for the Hot1-3 mutant (Lee et al., 2005). Lee et al. (2005) obtained 43 suppressor mutants that restored the 38 C sensitivity of the Hot1-4 mutant. Among these mutants, 7 were extragenic and 33 were intragenic, with a second mutation within the ClpB-C, resulting in 13 different amino acid substitutions that were located in the NBD1, NBD2 and C-terminus. The strong suppressors of Hot1-4 were all located in NBD1, highlighting a functional interaction between NBD1 and the coiled-coil domains. In maize, four independent homozygous mutants that contain the mutator (Mu) transposable element in the ClpB-C gene were obtained (Nieto-Sotelo et al., 2002). All four mutants were null for ClpB-C protein and were sensitive in the extreme to high temperatures. A reduction in the seed ClpB-C

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levels in the rice Hsa32 (HS associated 32 kDa protein) knock out mutant is associated with retarded seed germination upon HS (Lin et al., 2014). The decay of heat-induced ClpBC was faster in the Hsa32 mutant, and, as a consequence, long-term acquired thermotolerance (LAT) was adversely affected.


Stress-regulated expression profiling of plant ClpB-Cs The cellular expression of ClpB genes is tightly regulated within cells. The ScHsp104 gene is induced after exposure to HS, ethanol and arsenite (Sanchez et al., 1992). ClpB-C expression is characteristically heat-inducible in all plant species analyzed so far including Arabidopsis, maize, soybean, tobacco, wheat and rice (Agarwal et al., 2003; Campbell et al., 2001; Lee et al., 1994, 2006; Nieto-Sotelo et al., 2002; Singh et al., 2010; Wells et al., 1998; Young et al., 2001). Plant ClpB-Cs show induction under several other stresses apart from HS. In wheat, ClpB-C is induced by dehydration and ABA treatment (Campbell et al., 2001). In rice, ClpB-C shows an induction in response to oxidative and heavy metal stress (Singh et al., 2010, 2012). AtClpB-C transcripts are induced significantly by oilseed rape mosaic virus infection (Carr et al., 2006). The bulk of published work has been devoted to the HS-induced expression of ClpB-Cs. Development-regulated expression profiling of plant ClpB-Cs ClpB genes are developmentally regulated apart from stressregulation. ScHsp104 is not detected during the exponential growth at normal growing temperatures but is induced during the early stages of sporulation and upon transition from the exponential to stationary growth phases (Sanchez & Lindquist, 1990). Plant ClpB-C genes are constitutively expressed during reproductive stages. Seeds of Oryza sativa, Triticum aestivum, Zea mays and Brassica juncea constitutively accumulate high levels of ClpB-C proteins (Singla et al., 1998). In rice, the immature embryos (7th day of anthesis) accumulate low levels of ClpB-C that increase linearly with the maturation process, reaching its maximum in mature embryos (35th day of anthesis). In maize embryonic cells, ClpB-C accumulates predominantly in the nuclei and disappears within 3 days of imbibition (Nieto-Sotelo et al., 2002). In maize, higher levels of ClpBC protein is noted in male floral meristematic regions such as tassel. In addition, detectable low levels of ClpB-C were noted in pre-anthesis anther, mature pollen and silk tissues (Young et al., 2001). Singh et al. (2012) observed that the rice ClpB-C promoter drives Gus expression (used as a reporter system) in anther, style and ovary tissues along with significantly higher expression in the embryonic half of the seeds. OsClpB-C has high expression, especially in the milk and dough stages of grain development. Tonsor et al. (2008) have reported that knocking down AtClpB-C decreases fruit production, days to germination and bolting, number of florets and total dry mass, which is what ultimately compromises plant fitness. Thus, ClpB-C may be involved in specific development-related functions besides HS tolerance.


Genetic regulation of ClpB-C gene expression The genetic regulation of ClpB involves a heat responsive promoter in E. coli that is specifically controlled by a heat shock sigma factor. ScHsp104 expression during HS and metal toxicity is independently mediated by the heat shock factor 1 (Hsf1) and Msn2/Msn4 transcriptional activators, respectively. Hsf1 binds to two heat shock elements (HSEs) and Msn2/Msn4 binds to three stress responsive elements (STREs) present in the ScHsp104 promoter (Singh & Grover, 2010). In maize, five HSEs were reported within the proximal 289 bases of the ZmClpB-C promoter region (NietoSotelo et al., 1999). Six putative HSEs were reported in the AtClpB-C promoter (Hong & Vierling, 2001). HSEs direct the binding of Hsfs, and this binding is considered critical for inducing transcription. In rice, the heat-inducible expression of OsClpB-C is mediated specifically by OsHsfA2C. This Hsf binds to the only HSE (with configuration of nnnnnnGAAnnTTC) present in the rice ClpB-C promoter (Singh et al., 2012). In maize, it was noted that ZmClpB-C transcripts are also regulated post-transcriptionally, in which the 50 UTR plays an important role. In the latter work, it was reported that an internal ribosome entry site (IRES) element presents in the 50 UTR aids in the uninterrupted translation of ZmClpB-C protein during HS (Dinkova et al., 2005). While we analyzed the HS-mediated expression of plant ClpB-Cs above, possible cis- and trans-acting factors involved in the expression of ClpB-Cs during reproductive stages remain largely unexplored.

Biochemical activities of plant ClpB-C proteins ClpBs are implicated in the resolubilization of protein aggregates formed under cellular stress. Both wild-type and ScHsp104 mutant (DScHsp104) cells accumulate heat-induced protein aggregates. However, only the wild-type cells resolve the aggregates during the recovery phase (Parsell et al., 1994). Agarwal et al. (2003) expressed OsClpB-C in DScHsp104 cells and observed clearing of stress-associated aggregates during the recovery period, suggesting a possible protein disaggregation activity of OsClpB-C protein. In Arabidopsis, wild-type and Hot1 mutant plants accumulated high levels of proteins in the insoluble fraction during HS, which was greatly reduced only in the case of wild-type plants after 3 h of recovery (Lee et al., 2005). The analysis of Hot1-4 mutants revealed that after recovery from HS, the concentration of insoluble protein was high in mutants and was reduced in Hot1-4 suppressor mutants (the strongest suppressor had the least insoluble protein and vice versa). These observations suggest that ClpB-C proteins contain classical protein chaperone activity. In E. coli, Pontis et al. (1991) found that ClpB interacts with anaerobic reductase. It has been further shown that ClpB converts inactive dimers of TrfA (plasmid RK2 replication initiation protein) to an active monomer, thereby leading to the activation of replication (Konieczny & Liberek, 2002). However, the inventory of plant ClpB-C substrates is largely empty. Owing to the strong thermosensitive phenotype displayed by the plant ClpB-C mutants, it is speculated that the inventory of ClpB-C substrates may include a whole range of cellular proteins. It remains to be studied whether the selection of substrates by ClpB-C disaggregase is generalized or specific.


DOI: 10.3109/07388551.2015.1051942

ClpB-C has been implicated in translational activation. Wells et al. (1998) identified the 102 kDa, TMV 50 leader (V) RNA-binding protein as ClpB-C. This study suggests that ClpB-C facilitates translational activation activity: a 50-fold up-regulation in the luciferase (luc) reporter activity was noted from V-luc (and not from luc alone) in yeast cells expressing tobacco ClpB-C. There was no such up-regulation in control yeast cells lacking ClpB-C. It was proposed that ClpB-C and the V sequence form a two-component translation regulatory complex. In a separate study, ClpB-C was shown to bind an internal light regulatory element (iLRE) of ferredoxin (Fed1) mRNA. This binding enhanced the translational activity of the downstream transcript (Ling et al., 2000). Furthermore, Chang et al. (2007) generated transgenic tobacco expressing V-luc and rice ClpB-C, and reported the trans-activation of luciferase expression in the transgenics (Chang et al., 2007). Furthermore, ClpB-Cs are considered important in seed biology. In maize, ClpB-C proteins are found in dry seeds but are lost during the seed germination process (Lazaro-Mixteco et al., 2012). Mass spectrometry analysis of proteins from cap-binding complexes of maize seeds revealed that ClpB-C is a part of cap-binding complexes. This study proposed that ClpB-C interacts with the translational machinery during early seed germination (Lazaro-Mixteco et al., 2012). It also suggested that ClpB-C might act as a switch; as such, its presence in cap-binding complexes guarantees the translational activation of specific mRNAs during early germination. This group proposed that ClpB-C has a role both in translation regulation and in the disaggregation of unwanted protein complexes during embryo maturation and germination. This study has opened up a novel aspect of ClpB-C function in plants. ScHsp104 shuttles between the cytosol and the nucleus during HS (Tkach & Glover, 2008). Employing yeast twohybrid and bimolecular fluorescent complementation techniques in transient assays, Singh et al. (2012) observed that OsClpB-C is localized in the nucleus. Nieto-Sotelo et al. (2002) showed that ClpB-C is predominantly located within the nucleus in maize embryonic tissues. While the above studies suggest the nuclear localization of ClpB-Cs, definite evidence that HS triggers the preferential shuttling of these proteins from the cytoplasm to the nucleus is lacking. It has been noted that ClpB-Cs physically interact with specific Hsfs (Singh et al., 2012). This raises the possibility that ClpB-C may regulate transcription. The interaction of ClpB-C with Hsfs may suggest a possible feedback loop in ClpB-C regulation. The cellular significance of this interaction is left to be unveiled. Altogether, it can be interpreted that plant ClpB-Cs shuttle between the cytoplasm and nucleus, and that their functions may involve a complex interplay of transcriptional control, translational control and chaperone activity.

Organelle ClpB forms of plants As discussed previously, plants contain both organelle (ClpBP and ClpB-M) and cytoplasmic (ClpB-C) ClpB forms. However, the biology of organelle ClpB forms is poorly analyzed compared to cytoplasmic ClpB. The transcripts of the AtClpB-P and AtClpB-M genes show that high


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temperature induces up-regulation (Lee et al., 2006). In addition, AtClpB-P transcripts have a constitutive basal expression. High-temperature-induced expression of the rice organelle ClpB forms has also been noted (Singh et al., 2010). In addition, OsClpB-M and OsClpB-P have low levels of constitutive expression. In lima beans, ClpB-P protein is expressed constitutively but shows high up-regulation post HS (Keeler et al., 2000). Genevestigator analysis has revealed that all of the ClpB isoforms in rice show significant transcript abundance in seeds (Singh et al., 2010). It was noted that the AtClpB-P mutant is seedling lethal, in that it grows and develops several pair of leaves (though chlorotic in appearance) and eventually dies. This mutant has several anomalies in chloroplast development, although the number of chloroplast remained the same as in the wild type (Lee et al., 2006; Myouga et al., 2006; Zybailov et al., 2009). These data project that AtClpB-P is essentially a housekeeping gene and thus differs from AtClpB-C, which essentially functions post-HS. T-DNA mutants of both the AtClpB-P and AtClpB-M do not have any defect in thermotolerance, as noted for AtClpB-C gene defective mutants (Lee et al., 2006). In contrast, transgenic tomato plants antisensed for the ClpBP gene were impaired in their acquired thermotolerance response (Yang et al., 2006). These reports corroborate that the organelle ClpBs may have some housekeeping functions apart from their overall activity required during HS. The detailed biology of organelle ClpBs, with respect to their precise functions, needs to be established. As with ClpB-C, it is not known whether plants ClpB-P and ClpB-M can potentially perform chaperone activities. It would be useful to swap the above proteins among the cellular locations to seek answer to this question. Furthermore, it may be possible that similar to how ClpB-C functions in the cytoplasm, organelle ClpB forms functions specifically for their respective compartments after HS. There is definitely a need to focus on these issues to a greater depth. ClpB interacting co-chaperones There are several leads from bacterial and yeast systems which show that ClpB interaction with the Hsp70 system is of paramount importance for the disaggregase function of ClpB/ Hsp104/Hsp78. Mogk et al. (1999) showed that ClpB forms a bi-chaperone system with DnaK (Hsp70) chaperone assembly (that includes DnaK/DnaJ/GrpE) to solubilize aggregates efficiently in bacteria. This bi-chaperone system liberates non-native, chaperone recognizable folding intermediates that then undergo proper folding to attain a native structure with the assistance of sHsps (Watanabe et al., 2000). ScHsp104 function requires collaboration with the Hsp70/Hsp40/NEF system in yeast. The yeast mitochondrial ClpB (ScHsp78) interacts with the matrix Hsp70 (Ssc1p/Mdj1p/Mge1p) system. Escherichia coli ClpB can complement the thermotolerance of Synechococcus sp. ClpB null mutant but not that of DScHsp104 cells (Eriksson & Clarke, 2000; Miot et al., 2011). This observation has been explained as a speciesspecific interaction between the ClpB and cognate Hsp70 co-chaperones. Glover & Lindquist (1998) noted that ScHsp104 acts efficiently in concert with its cognate Hsp70 co-chaperones and that this function cannot be substituted by


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the E. coli DnaK system. ScHsp78 reactivates heat aggregated firefly luciferase in collaboration with the Ssc1p/Mdj1p/ Mge1p machinery, which cannot be substituted with the E. coli DnaK/DnaJ/GrpE machinery (Krzewska et al., 2001). Evidence suggests that the M-domain of ClpB interacts with the DnaK chaperone and that this interaction is responsible for imparting species-specificity to the core ClpB/Hsp104 disaggregase (Mogk et al., 2008). Replacing the bacterial ClpB M-domain with ScHsp104 M-domain and vice versa enabled chimeras to cooperate with the noncognate Hsp70/DnaK chaperone (Sielaff & Tsai, 2010). Importantly, E. coli ClpB can complement DScHsp104 thermotolerance if it is modified to interact with ScHsp70 or if DnaK and a cognate nucleotide exchange factor are provided (Reidy et al., 2012). The above account advocates that the species-specificity of ClpBs lies in their co-chaperone requirement and not in terms of its basic activity, which is more likely to be common throughout species. While bacterial and yeast ClpB collaborating Hsp70/ Hsp40 chaperone machinery is being debated, information on the association of plant ClpBs and the Hsp70 chaperone systems is lacking (Doyle et al., 2007; Glover & Lindquist, 1998; Goloubinoff et al., 1999; Motohashi et al., 1999). In Arabidopsis, genetic analysis employing Hot1-4 and its various suppressor mutants suggests an interaction between ClpB and sHsps to resolubilize denatured proteins produced after HS (Lee et al., 2005). From the above discussion, it becomes clear that the specific functional interaction of the chaperone proteins is critical. A model representing the current understanding of ClpB function is shown in Supplementary Figure 2. This model highlights that the ClpB chaperone is aided by Hsp70/Hsp40/NEF proteins in its chaperoning action. The lacuna in our knowledge of partner proteins functioning in collaboration of plant ClpBs is also projected in Supplementary Figure 2.

Global warming and heat tolerant transgenic crops In the present situation where global warming and the associated increase in ambient temperature is a threat to plant ecosystems, there is an urgent need to genetically engineer crop plants to sustain as well as to increase their yields to meet the conspicuous need of global food requirements (Grover et al., 2013). The resurrection property of ClpBs (i.e. their ability to extract native, soluble proteins from the protein aggregates) makes them unique and distinct from other Clps and conventional molecular chaperones. Under extreme high-temperature stress, where the production of new proteins almost stops and 80–90% of already existing cellular proteins form toxic aggregates, ClpBs provide necessary machinery to liberate entangled proteins to their functional state, which is critical for the survival of the plant. The heatinduced expression of these proteins is a conserved phenomenon in the course of evolution from prokaryotic microbes to higher plants. On the evolutionary scale, animal cells lack ClpBs and the reason for this could be the fact that animals have the ability to physically move away from HS. ClpB mutations in different bacteria (e.g. Helicobacter pylori, E. coli and T. thermophilus), cyanobacteria (Synechococcus sp.), yeast and plants result in a thermosensitive phenotype


(Allan et al., 1998; Porankiewicz & Clarke, 1997; Squires et al., 1991). Furthermore, the constitutive overexpression of ClpB-Cs has been shown to increase the basal thermotolerance of plants in several studies (Chang et al., 2007; KatiyarAgarwal et al., 2003; Queitsch et al., 2000). Chang et al. (2007) overexpressed rice ClpB-C in tobacco and reported that the thermotolerance corresponded with the titer of the accumulated protein: overexpressing transgenics performed remarkably better compared to the non-transformed and/or vector transformed plants. Plants, being sessile, are more vulnerable to HS than other life forms, and this may explain the basis for more complex organization of ClpB genes in plants as opposed to bacteria or yeast. Although housekeeping organelle plant ClpBs are also HS induced, the constitutive overexpression of AtClpB-P in transgenic plants inhibits chloroplast development with a mild pale green phenotype (Myouga et al., 2006). Thus, among the three ClpB forms, ClpB-Cs appear to be suitable candidate genes for the development of heat tolerant transgenics.

Strategies to develop more effective ClpB-C machinery From the above discussion, ClpB-Cs appear to be strong candidate genes for improving plant heat tolerance using a plant genetic engineering approach. To take this issue further, two questions need immediate attention: (a) will higher levels of ClpB-C be achieved by strong promoters driving ClpB-C transgene expression and improve cellular heat tolerance and (b) will it be possible to generate more efficient forms of ClpB-C proteins? Recently, it was noted that engineering ScHsp104 to make it more efficient was successful in vitro; however, cells overexpressing the efficient forms were increasingly sensitive to HS under in vivo conditions (Lipinska et al., 2013). The authors of this study argued that the overexpression of the efficient form over the native form with optimal activity under conditions when it is not needed, may actually affect proteostasis negatively. An attempt to constitutively overexpress AtClpB-C in Arabidopsis resulted in the co-suppression of the endogenous protein (Queitsch et al., 2000). It thus appears that nature has purposely designed ClpB-C genes to remain almost ‘‘silent’’ in vegetative tissues unless it is required during HS conditions. This may be necessitated because: (a) the unnecessary function of this protein may pose a potential risk to the selective protein turnover required to maintain harmony in the cellular protein environment and a regulatory state of different metabolic pathways and (b) it requires enormous energy for its function, and thus, its activity is undesirable under control conditions. Considering the phenotype enhancement with the increased titer and the actual conditions of fieldcultivation of crops where temperature changes take place gradually, it may be prudent to aim at inducible overexpression over constitutive overexpression in genetic engineering experiments. This also needs to be looked at with the view that inducible expression will not come in the way of the native gene expressing constitutively in reproductive tissues.

Conclusion The production of transgenic plants with enhanced genetic capacity for heat tolerance is greatly needed in the wake of

DOI: 10.3109/07388551.2015.1051942

global warming and climate change. The tools of plant biotechnology are being used to produce transgenic plants with high-heat tolerance to address this need. Considering that: (a) ClpB-C proteins have critical roles in heat response, (b) these are highly conserved in several plant species and (c) mutant plants lacking these proteins are highly heat sensitive, and there is a definite need to expand our understanding of ClpB-C protein functions, particularly in relation to their application for enhancing plant heat tolerance.


Declaration of interest The authors report no declarations of interest. RCM acknowledges the Council of Scientific and Industrial Research (CSIR) and the Government of India, New Delhi, for the Fellowship award. AG is thankful to the Centre for Advanced Research and Innovation on Plant Stress and Development Biology, the Department of Biotechnology (DBT), the Government of India and J.C.Bose Fellowship award grant, the Department of Science and Technology (DST), and the Government of India for financial support.

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DOI: 10.3109/07388551.2015.1051942


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Supplementary material available online Supplementary Tables S1–S2 and Figures S1–S2.