# Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

Nathalie Banzet, Christiane Richaud, Yves Deveaux, Michaël Kazmaier, Jean Gagnon, Christian Triantaphylidès

► To cite this version:

Nathalie Banzet, Christiane Richaud, Yves Deveaux, Michaël Kazmaier, Jean Gagnon, et al.. Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells. The Plant Journal, 1998, 13 (4), pp.519-527. 10.1046/j.1365-313x.1998.00056.x. hal-04346599

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# Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

Nathalie Banzet<fnref n="1" />, Christiane Richaud<fnref n="1" />, Yves Deveaux<fnref n="1" />, Michaël Kazmaier<fnref n="1" />, Jean Gagnon<fnref n="2" /> and Christian Triantaphylidès<fnref n="1" />,*

## Summary

Changes in gene expression, by application of H₂O₂, O₂⁻- generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H₂O₂ and gamma irradiation, but not to O₂⁻- generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I–IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H₂O₂ and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H₂O₂ action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with *Chenopodium rubrum* mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H₂O₂ pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential.

Received 3 May 1997; revised 7 October 1997; accepted 4 November 1997.

## Introduction

In both procaryotic and eukaryotic cells, oxygen activation is a general phenomenon leading to the production of O₂⁻, H₂O₂ and °OH (active oxygen species; AOS) by a step-wise one-electron transfer to molecular oxygen (Halliwell and Gutteridge, 1989). AOS are formed in normal cell metabolism and their level increases under stress conditions. As other organisms, plants have developed protective systems, either constitutive or inducible, to counteract oxidative stress (OS), and have also gained the ability to control and to utilize the production of AOS (Inzé and Van Montagu, 1995). Several lines of evidence have revealed, for instance, that the oxidative burst occurring during plant pathogen interactions is related to signal transduction pathways (Doke, 1997).

Changes in plant gene expression in response to various types of OS have been investigated. Photo-oxidative and other environmental stresses lead to the regulation of anti-oxydant enzymes such as superoxide dismutases (Scandalios, 1997). Besides plant pathogen interactions, the synthesis of pathogenesis-related proteins is also enhanced by OS and ozone action (Kangasjärvi *et al.*, 1994), as well as UV irradiation (Green and Fluhr, 1995). Surprisingly little attention has been paid to the induction of heat shock proteins (HSP) by OS in plants. In bacteria, partial overlapping of OS and heat shock (HS) responses has been observed (Storz *et al.*, 1990). Oxidative agents also induce the synthesis of HSP in cyanobacteria (Mittler and Tel-Or, 1991), yeast (Jamieson *et al.*, 1994), *Drosophila* cells (Courgeon *et al.*, 1990) and mammalian cells (Storz and Polla, 1996). Reports on plants are generally not conclusive and concern the induction by UV-B radiation of 'heat shock-like proteins' in *Vigna sinensis* seedlings (Nedunchezhian *et al.*, 1992), the accumulation by gamma irradiation of putative small HSP (smHSP) in tomato fruits (Ferullo *et al.*, 1994), and the very recent characterization of a parsley smHSP gene induced by ozone (Eckey-Kaltenbach *et al.*, 1997).

Small HSP represent the major family of HSP induced by heat stress in plants (reviewed by Waters *et al.*, 1996). All plant smHSP genes are nuclear-encoded and are divided in different classes according to their subcellular localization. Six classes are currently described. Class I and II smHSP are cytosolic proteins, and two other classes are characterized by their localization in chloroplasts and in the endoplasmic reticulum (Vierling, 1991). In addition, a mitochondrial class as well as another endomembrane class are proposed (Lafayette *et al.*, 1996). The function of plant smHSP is not

<footnote n="1">¹CEA-Cadarache, Direction des Sciences du Vivant, Département d'Ecophysiologie Végétale et de Microbiologie, Laboratoire de Radiobiologie Végétale, 13108 Saint-Paul Lez Durance Cedex, France, and</footnote>

<footnote n="2">²Institut de Biologie Structurale, CEA/CNRS, 41 avenue des Martyrs, 38027 Grenoble Cedex 1, France</footnote>

<footnote>*For correspondence (fax +33 04 42 25 62 86; e-mail trianta@dsvcad.cea.fr).</footnote>
well understood. Eukaryotic smHSP are considered to be
molecular chaperones (Arrigo and Landry, 1994). Chaperone
activity for plant smHSP was demonstrated *in vitro* (for
example Lee *et al.*, 1997) and suggested *in vivo* (reviewed
by Waters *et al.*, 1996). Great differences exist, however,
between mammalian and plant smHSP with respect to the
number of expressed proteins and their localization, and
their ability to be regulated by phosphorylation.

Expression of stress proteins generally confers adaptive
advantages to cells. A mild HS is sufficient to induce the
synthesis of HSP, and leads to the acquisition of thermotoler-
ance in all organisms (Nover, 1991). Similarly, pre-exposure
at low concentrations of H₂O₂, O₂⁻ generating reagents,
ethanol or heavy metals induces, in most cases, a tolerance
towards a lethal concentration of the same reagent
(reviewed by Crawford and Davies, 1994; Nover, 1991). It has
also been demonstrated, in bacteria, yeast and mammalian
cells, that pre-exposure of cells to HS renders cells resistant
to OS (Crawford and Davies, 1994). Direct evidence for the
existence of a relationship between HSP expression and
protection against OS has been provided by expressing
human or *Drosophila* small HSP27 in murine cells (Mehlen
et al., 1995). Therefore, adaptation to OS appears to be the
result of complex mechanisms in which various classes of
stress proteins, including not only antioxidant enzymes but
also HSP, could be involved.

The object of the present work was to characterize and
compare the early effects of various oxidative stresses, such
as H₂O₂ application, the action of O₂⁻ generating agents
and gamma irradiation, on tomato suspension cultures
(*Lycopersicon esculentum*), with special reference to smHSP
synthesis. Analyses at the protein level by 2-dimensional
PAGE were carried out in order to compare global responses.
HSP expression by oxidative stresses was then assessed at
RNA level using cDNA probes from pea class I-IV smHSP.
In addition, the unknown main HSP induced by oxidative
treatments was purified for microsequencing purposes. Its
identification by sequence analysis and immunodetection
strongly indicates that this protein belongs to the mitochon-
drial smHSP class. Finally, experimental data supporting the
induction of cell resistance against lethal OS by HS or a mild
OS are provided. The role of stress proteins in terms of
cell adaptation to oxidative injury, with special emphasis on
mitochondrial protection, is then discussed.

Results

Comparison of protein synthesis in response to oxidative
and heat shock treatments

Short-term changes in protein synthesis were analysed
using 2-dimensional polyacrylamide gel electrophoresis (2-
D PAGE) of [³⁵S]-labelled proteins (Figure 1). Compared to
control experiments, HS (2 h at 40°C) and gamma irradiation

(3 kGy) reduced the synthesis of most proteins with simul-
taneous induction of stress proteins. Application of H₂O₂
(5 mM), methyl viologen (MV) (5 mM) or digitonin (150 µM)
apparently did not decrease protein synthesis. Some stress
proteins were, however, newly synthesized. Concentrations
ranging from 50 to 500 µM for MV, and from 60 to 100 µM
for digitonin, led to similar results (data not shown). With
reference to the HS response, the three main gamma-
induced proteins corresponded to HSP22, HSP17 and HSP15
(Figure 1b,c; proteins 1, 2 and 3, respectively), according
to their location on 2-D patterns, Among the nine proteins
induced in H₂O₂-treated cells, seven could be identified as
HSP, three of them being the previously mentioned proteins
(Figure 1d). Following MV or digitonin application, eight and
four proteins, respectively, were weakly induced (Figure 1e-
f). With the exception of the weakly induced proteins, HSP17
(protein 2) under both conditions and HSP15 (protein 3) in
response to MV action, no other stress protein could be
identified as smHSP.

Time-course of smHSP mRNA induction by oxidative
stresses

The steady-state level of mRNA encoding for several classes
of smHSP was analysed by Northern blot experiments at
different times following gamma irradiation (3 kGy) and
H₂O₂ application (5 mM). The four heterologous cDNA
probes from pea smHSP, corresponding to chloroplastic
HSP21, class I cytosolic HSP18.8, class II cytosolic HSP17.1
and endoplasmic reticulum HSP22.7, hybridized to the cor-
responding mRNA in heat-shocked tomato cells. Only class
I and II cytosolic mRNA were induced by gamma irradiation
and H₂O₂ application (Figure 2). This result confirmed our
prior observation at protein level, that both OS induced only
a partial HS response. The specificity of class I and II cytosolic
HSP mRNA induction was demonstrated by rehybridization
of the same filter with a 25S rRNA probe (Figure 2c,f). Indeed,
the 25S rRNA level remained unchanged after OS, but was
notably reduced in RNA preparations after HS (3 h at 40°C;
Bell *et al.*, 1988). Therefore, it is difficult to quantify the level
of HSP mRNA induction by gamma irradiation and H₂O₂
with respect to HS. The response to gamma irradiation was
immediate and reduced in time, with an apparent maximum
observed 1 h after the treatment, followed by a rapid decay
in mRNA level. In contrast, the response to H₂O₂ application
was delayed, with an apparent maximum 6 h after the treat-
ment, followed by a slow decay in mRNA level.

Characterization of a mitochondrial HSP22 by sequence
analysis and immunodetection

The major protein spot common to HS, H₂O₂ action and
gamma irradiation (Figure 1; protein 1) was purified from
heat-shocked material and microsequenced for charac-
<figure><img src="image_3.png" /><figcaption>**Figure 1.** 2-D PAGE analyses of stressed tomato cell proteins.</figcaption></figure>

2-D PAGE autoradiography of phenol-extracted proteins from cultured cells after 6 h incubation in the presence of ²⁵S methionine/cysteine. (a) control experiment; (b) HS treatment (2 h at 40°C); (c) gamma irradiation (3 kGy); (d) H₂O₂ application (5 mM); (e) methyl viologen application (0.5 mM); (f) digitonin application (150 µM). Arrow heads indicate HSP and OS induced potential HSP; OS induced proteins are identified by (c) a circle for gamma-irradiated cells, (d) a square for H₂O₂-treated cells, (e) a reversed triangle for MV treated cells, and (f) a triangle for digitonin-treated cells. Main proteins showing similar electrophoretic migration between HS and OS are numbered.

terization. Purification was performed, according to gel mobility characteristics (MW 22 kDa and pI 5.5), by preparative IEF followed by semi-preparative 2-D PAGE (see the Experimental procedures). Four sequences (i.e. N-terminal and internal sequences) were obtained and analysed using the program MegAlign from the DNASTar software package, the search being focused on HSP. The four tomato sequences showed significant homology with the known plant mitochondrial smHSP from *Pisum sativum* (HSP22), *Chenopodium rubrum* (HSP23) and *Glycine max* (HSP24)

(Figure 3). Two N-terminal sequences were obtained, differing by a single amino acid, showing sequence heterogeneity for the N-terminus of the mature protein. The N-terminal sequence was closely related to *Chenopodium rubrum* mitochondrial HSP23, as revealed by amino acid identity (57%) and similarity (71%). The two internal sequences were obtained from peptides separated by HPLC after cyanide bromine cleavage of the protein. Peptide 3 presented 50% sequence identity (94% similarity) in an internal segment, and peptide 4 showed 71% identity
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Time-course of smHSP mRNA induction by oxidative stress. Northern blot analysis, using pea smHSP cDNA, of mRNA from cells submitted to heat shock, to H₂O₂ action and to gamma irradiation. C, control cells; HS, heat-shocked cells (2 h at 40°C); γ, irradiated cells (3 kGy); H₂O₂-treated cells (5 mM), immediately after the treatment, or incubated for 15 min, 1, 2, 6 or 10 h prior to RNA extraction. Hybridations with (a, d) class I cytosolic, (b, e) class II cytosolic pea smHSP cDNA, and (c, f) with *Arabidopsis* th. 25S rRNA probe.</figcaption></figure>

(100% similarity) with the C-terminal region of mitochondrial smHSP.

In Western blot experiments, the antibody directed against mitochondrial HSP22 from *Pisum sativum* gave only a faint signal with a 22 kDa protein from heat-shocked tomato cells (data not shown). Conversely, the antibody raised against *Chenopodium rubrum* mitochondrial HSP23 recognized a protein of 22 kDa, which was absent in untreated cells, accumulated after heat shock, gamma irradiation and H₂O₂ action, but not after MV or digitonin application (Figure 4a). Further evidence stemmed from the comparison of autoradiography and immunodetection of the same membrane of radiolabelled proteins separated by 2-D PAGE. The radiolabelled protein spot, which had been previously sequenced, was clearly recognized by the anti-

body in protein extracts from heat-shocked cells (Figure 4b,c), from cells irradiated at 3 kGy or treated by 5 mM H₂O₂ (data not shown).

### HSP22 accumulation by oxidative treatment and heat shock

The dose-dependent accumulation of HSP22 in response to oxidative stresses was investigated by Western blot experiments using *Chenopodium rubrum* anti-HSP23. Increasing doses of H₂O₂ (0.5–20 mM) and gamma irradiation (0.1–5 kGy) were applied and protein accumulation was analysed after 6 h of recovery (maximum accumulation, see below). The results indicated that HSP22 accumulated at doses above 1 mM for H₂O₂ application and 2 kGy for gamma irradiation (Figure 5a,b).

The accumulation of tomato HSP22 was also similarly investigated as a function of time following a mild (30 min) or a severe (3 h) HS at 40°C (data not shown), and after 5 mM H₂O₂ or 3 kGy irradiation (Figure 5c). HSP22 was synthesized early after HS and was detected 30 min after the beginning of the stress application. The accumulation reached maximum levels 3 h after treatment in both cases (mild or severe HS), stabilized and decreased after 24 h under mild conditions, and remained constant for at least 48 h under severe conditions. Following OS, HSP22 accumulated later and slower than after HS, and at lower levels (Figure 5c). For H₂O₂ action, the protein was detected after 2 h, the maximum level of accumulation (40%, compared to 3 h HS) was reached after 6 h, and the decrease occurred between 24 h and 48 h, as observed for mild HS. For gamma irradiation the maximum of accumulation (28%, compared to 3 h HS) was reached earlier, within 4 h, and appeared stable at least for 48 h, as for severe HS.

### Cell adaptation to oxidative stress

The capacity of tomato cells to develop an adaptive response to a lethal H₂O₂ dose was examined. (Figure 6). It was observed that packed cell volume is a critical factor to investigate the lethal effects of H₂O₂. Cultured cells were treated at the beginning of the exponential phase of growth (8–10% of packed cell volume) and growth was estimated and compared to controls. Whereas a mild HS (30 min, 40°C) or a mild H₂O₂ treatment (2 mM) could be considered as non-lethal, application of 10 mM H₂O₂ immediately stopped cell growth. Both mild pretreatments and incubation for 6 h before application of the lethal H₂O₂ dose induced smHSP and, as assessed by cell growth, were effective in cell protection.
<figure><img src="image_5.png" /><figcaption>**Figure 4.** Immunoblot characterization of tomato HSP22.</figcaption></figure>

Western blot analyses of tomato cell protein extracts using an antibody raised against *Chenopodium rubrum* mitochondrial HSP23.

(a) Compared immunodetection of proteins extracted, unless stated, 6 h after stress application; C, control; γ, 3 kGy-irradiation; HS, HS at 40°C for 3 h and extraction; H₂O₂, 5 mM H₂O₂; MV, 0.5 mM MV; Dig, 150 µM digitonin.

(b, c) 2-D analyses of ³⁵S-labelled protein extracts from heat-shocked cells;

(b) autoradiography and (c) immunodetection of the same membrane.

strongly suggest the accumulation of a mitochondrial smHSP by OS, the production of O₂⁻ not being required for that gene expression.

The synthesis of smHSP in tomato cells by OS seems to be at least related to the chemical nature of the AOS involved. On one hand, as is described for other eukaryotes (Jamieson et al., 1994; Storz and Polla, 1996), H₂O₂ induces a partial HS response in tomato cells, whereas O₂⁻ does not seem to play this role. It is now established that H₂O₂ leads, for instance, to the activation of the human transcription factor (HSF) and its DNA-binding activity (Storz and Polla, 1996). Several HSF have been identified in plants (reviewed by Nover and Scharf, 1997), and it cannot be excluded that some of them might be sensitive to H₂O₂ and regulated by oxidation. On the other hand, gamma irradiation generates a burst of AOS that can be estimated (Riley, 1994). At a 3 kGy dose, for instance, the highly reactive °OH and the relatively stable O₂⁻ radicals are generated at levels of 0.8-0.9 µmol g⁻¹, respectively (Ferullo et al., 1994). Oxidative damage to pro-

<figure><img src="image_6.png" /><figcaption>**Figure 5.** Accumulation of HSP22 in tomato cells submitted to oxidative stress.</figcaption></figure>

Western blots of protein extracts using the *Chenopodium rubrum* mitochondrial HSP23 antibody with reference to heat-shocked cells (40°C for 3 h). Dose-dependent accumulation after 6 h of cell recovery following (a) H₂O₂ action, and (b) gamma irradiation. (c) Kinetics of HSP22 accumulation after a 5 mM H₂O₂ treatment or a 3 kGy gamma irradiation; bands corresponding to HSP22 were integrated and results are expressed as means with error bars from three independent experiments.

teins appears sufficient to activate HSP gene transcription (Voellmy, 1996). Thus, the synthesis of smHSP by massive doses of gamma rays could be explained, to some extent, as the result of protein oxidation by °OH production.

*Does HSP expression lead to cellular adaptation and mitochondrial protection in plants?*

Tomato cells pretreated with a mild HS or 2 mM H₂O₂ acquire resistance towards a subsequent lethal concentration of H₂O₂ (Figure 6). In both cases, adaptation is related to the synthesis of stress proteins, particularly smHSP. Our work provides the first evidence for HS-mediated adaptation of plant cells against oxidative injury. Since HS similarly induces plant cell cross-resistance to salt shock (Harrington and Alm, 1988), toxic metals (Wollgiehn and Neumann, 1995) and chilling injury (Sabehat et al., 1996), these results, taken together, suggest that HSP must play an important role in plant adaptation to various environmental stress conditions.

In response to H₂O₂ action and to gamma irradiation,
<figure><img src="image_7.png" /><figcaption>**Figure 6.** Cell adaptation to lethal OS by HS or H₂O₂ pretreatments. Cell growth, expressed in dry weight, as a function of time following stress application at the beginning of the exponential phase of growth. Open symbols, control experiments; HS was performed at 40°C for 30 min; H₂O₂ was applied at a final concentration of 2 mM or 10 mM. Closed symbols, adaptive experiments; the lethal H₂O₂ treatment (10 mM) was applied after non-lethal pretreatment and 6 h of cell recovery; (a) HS and (b) H₂O₂ pretreatments. Results are expressed as means with error bars from five independent cultures carried out simultaneously.</figcaption></figure>

one of the main accumulated tomato proteins is mitochondrial HSP22. It is presently known that mitochondrial electron transport chain components (Zhang *et al.*, 1990), as well as Fe-S cluster enzymes, such as aconitase (Verniquet *et al.*, 1991), are very sensitive to AOS. Therefore, protection of mitochondria appears essential to OS tolerance. A protective role against cell oxidative injury has been demonstrated for mitochondrial superoxide dismutase by complementation in yeast (Balzan *et al.*, 1995). In plants, seedling protection against chilling injury can be achieved by acclimation or H₂O₂ application, and is correlated with the induction of mitochondrial antioxydant enzymes (Prasad *et al.*, 1994, 1995). Besides, the first evidence for OS protection of mitochondria by HSP was recently provided in human premonocytic cells (Polla *et al.*, 1996). Thus, our results together with the observation that chilling tolerance acquired by HS in tomato fruit is at least correlated to the synthesis of

HSP22 (Sabehat *et al.*, 1996), suggest a protective role for plant mitochondrial smHSP against OS.

## Conclusions

The accumulation of smHSP observed in this work, together with the recent characterization of an ozone-induced smHSP gene in parsley cells (Eckey-Kaltenbach *et al.*, 1997), are some of the first examples of OS-induced HSP synthesis in plants. As similarly described in microorganisms and mammalian cells: (i) some HSP genes, smHSP genes in plants at least, are expressed by OS; (ii) H₂O₂ application gives a greater response; and (iii) O₂⁻ production does not seem either sufficient, or necessary, for OS accumulation of smHSP. Furthermore, one of the main OS-induced HSP in plants is a mitochondrial smHSP. Finally, protection by HS, or mild OS, against lethal OS is also shown and can be related to HSP accumulation. In many cases, over-expression of antioxidant enzymes in transgenic plants leads to enhanced protection against OS (for example Inzé and Van Montagu, 1995). Since it has been demonstrated recently that derepression of the HS response by genetic engineering is possible and increases basic thermotolerance in *Arabidopsis thaliana* (Lee *et al.*, 1995), it can therefore be suggested that constitutive expression of elevated levels of HSP is an alternative strategy for protection against OS.

## Experimental procedures

### Plant material and treatments

Calli were initiated from tomato plant stems (*Lycopersicon esculentum* Mill., cv. sweet 100, Vilmorin, France) on an agar-plated MS medium (Murashige and Skoog, 1962). Cell suspension cultures were then adapted from the calli in MS medium, containing 30 g l⁻¹ sucrose, supplemented with 1 mg l⁻¹ benzylamino purine, 10 mg l⁻¹ 2,4 dichlorophenoxy acetic acid and adjusted at pH 5.8. Typically, cells were inoculated at a final 2% packed cell volume (PCV) and cultured at 25°C under continuous light.

Chemical or physical treatments were applied at the beginning of the exponential phase of growth, corresponding to 8–12% PCV (i.e. 2–3 g l⁻¹ dry weight of plant material). H₂O₂ (0.5–20 mM), methyl viologen (0.5–5 mM) or digitonin (80–150 µM) was applied to the culture medium. In order to avoid side-effects due to radiolysis of the culture medium, cells were collected, gamma-irradiated (0.1–5 kGy, at a dose rate of 35 or 155 Gy min⁻¹; ⁶⁰Co gamma-irradiator, 2200 Tbq; CIS Bio International, France) and resuspended in the medium. Heat shock was achieved by incubating cell suspensions at 40°C (30 min or 3 h). At the end of the treatment, cells were harvested, frozen and stored at –80°C. For the protein-labelling experiments, a mixture of [³⁵S] Met/Cys (Amersham Life Sci., UK; 18.5 MBq) was added to the suspension culture, at the same time as treatment or just after gamma irradiation.

Experiments for cell adaptation were carried out similarly. The protective HS (30 min at 40°C) and H₂O₂ (2 mM) pretreatments were applied 6 h before the H₂O₂ lethal treatment (10 mM).
## Protein extraction and gel electrophoreses

Proteins were phenol-extracted as described previously (Ferullo *et al.*, 1994), and quantified using the Coomassie Plus Kit (Pierce Europe BV, the Netherlands). When necessary, specific radioactivity was determined. For SDS-PAGE analyses, proteins were diluted in Laemmli buffer, loaded onto a 15% polyacrylamide gel (40 µg per well) and separated. 2-D PAGE analyses were carried out with the Multiphor II system using the manufacturer's standard procedures (Pharmacia LKB Biotechnology, Sweden). Proteins (60–100 µg per well) were first separated by isoelectrofocusing (IEF; Immobilin DryStrip NL 3–10, 110 or 180 mm), and then the second dimension was performed on precast polyacrylamide gels (ExcelGel SDS 12–14, 245 × 180 × 0.5 mm, or 8–18, 245 × 110 × 0.5 mm). Gels were silver-stained, incubated with the Amplify solution (Amersham Life Sci., UK), desiccated (Gel Dryer 583, Bio-Rad SA, France) and autoradiographed on β-max Hyperfilms (Amersham Life Sci., UK).

## Immunoblotting analyses

After SDS-PAGE or 2-D PAGE separation, proteins were electrotransferred onto nitrocellulose membranes (0.45 µm, Bio-Rad SA, France). Membranes were washed (1 h, 37°C) in TBS-BSA [Tris-HCl 20 mm, pH 7.6, 500 mm NaCl, 3% (w/v) bovine serum albumin] and first incubated overnight at room temperature with the goat antiserum directed against HSP23 from *Chenopodium rubrum* (a gift from K. Kloppstech, University of Hannover, Germany), diluted 1/1000 in TBS-BSA. Membranes were washed with TBS-BSA (37°C; 3 × 10 min, and 1 h), incubated with rabbit anti-goat IgG-conjugated peroxidase (1/8000 in TBS-BSA; 25°C; 1.5 h; Sigma Chimie, France), and washed again in TBS-BSA (37°C; 1 h). Peroxidase activity was revealed using the ECL kit (Amersham Life Sci., UK). Antibodies directed against *Pisum sativum* mitochondrial HSP22 (a gift from C. Lenne and R. Douce, CEA, Grenoble, France) and against tomato chloroplastic HSP22 (a gift from L. Nover, University of Frankfurt, Germany) were similarly tested.

## Isolation of mRNA and Northern hybridization

PolyA⁺ RNA were prepared, as described by Pötter and Kloppstech (1993), with the following modifications. The extracts were thawed (37°C) and proteinase K was added for digestion (10 min). The mRNA were eluted from oligo(dT)-cellulose at 60°C (3 ml of Tris-HCl 10 mM, pH 7.4) and precipitated overnight at -20°C (in 0.3 M Na-acetate, 75% ethanol). After centrifugation (4°C; 14 500 g; 30 min) the pellet was washed (ethanol 70%) and resuspended in 400 µl of TE (Tris-HCl 10 mM, EDTA 1 mM, pH 8). Aliquots of 5 µg per well were loaded in a 1.2% agarose gel. Electrophoresis and the transfer of mRNA onto a nylon Hybond N™ membranes (Amersham Life Sci., UK) were carried out as described by Maniatis *et al.* (1982). Labelling of the probes was performed with a Megaprime kit (Amersham Life Sci., UK), using [α-³²P]dCTP. Hybridization and hybridization wash were carried out at moderate stringency, as described by Maniatis *et al.* (1982). An autoradiograph was taken on a MP film (Amersham Life Sci., UK) exposed overnight. The cDNA inserts of pea smHSP corresponding to chloroplastic HSP21 (pAZ48), cytosolic class I HSP18.1 (pAZ53), endoplasmic reticulum HSP22.7 (pAZ63) and cytosolic class II HSP17.1 (pAZ64), were gifts from E. Vierling (University of Tucson, Arizona, USA), and the *Arabidopsis* th. 25S rRNA insert used as constitutive probe was a gift from T. Desprez (INRA Versailles, France).

## Purification of HSP22 for N-terminal and internal amino acid sequence determination

Total phenol-extracted proteins (up to 100 mg) were separated by preparative IEF, using the Rotofor Cell (Bio-Rad SA, France), under denaturing conditions [Tris-HCl 1 mM, pH 8, urea 8 M, CHAPS 8 mM, ampholytes 3–10 2% (v/v)]. The IEF separation was repeated on the fractions containing HSP22 (pH c. 5.5) and then HSP22 was purified by preparative 2-D PAGE (Coomassie Blue R250 staining). For N-terminal amino acid sequence determination, proteins were transferred onto PVDF membrane (0.2 µm, Bio-Rad SA, France) and HSP22 spots excised. For CNBr digestion, polyacrylamide fragments containing HSP22 were excised, and the protein concentrated by SDS-PAGE and then transferred onto PVDF membrane.

CNBr digestion was performed by incubating dried PVDF membrane fragments (37°C; 24 h) under N₂ atmosphere in the dark, with 150 µl formic acid 70% (v/v) and CNBr 25 mM. Peptides were extracted from the membrane fragments with 100 µl 40% (v/v) acetonitrile (37°C; 3 h), then with 100 µl 40% (v/v) acetonitrile, 0.05% (v/v) TFA (37°C; 20 min). The supernatants were pooled and evaporated. Membrane fragments were finally washed with 250 µl of 6 M guanidine hydrochloride (37°C; 1 h) and the supernatant pooled with the preceding extract. The mixture was submitted to HPLC on a C₄ Vydac 'Narrowbore' column (Touzart et Matignon, France) using solvents A [0.1% (v/v) TFA in water] and B [0.1% (v/v) TFA in acetonitrile] under the following conditions: 5% (v/v) B for 5 min, then a linear gradient from 5% to 65% (v/v) B for 45 min, and finally 65% (v/v) B for 5 min at a 200 µl. min⁻¹ flow rate. Small peptides were eluted and collected for microsequencing. N-terminal and internal amino acid sequences were determined by automated Edman degradation, using an Applied Biosystem model 477 A protein sequensor, amino acid phenylthiohydantoin derivatives being identified on-line with a model 120 A HPLC system, as recommended by the manufacturer (Perkin Elmer, France).

## Acknowledgements

The authors wish to thank Y. Lienart, M.-H. Montané and J.-L. Montillet for discussions and help, and S. Cuiné for the HPLC peptide separation. We thank E. Vierling for the four pea cDNA, T. Desprez for the *Arabidopsis* th. 25S rRNA probe, and C. Lenne and R. Douce for the pea HSP22 antibody. We are indebted to K. Kloppstech for the *Chenopodium* r. HSP23 antibody, and for early communication of research results and discussions. We are grateful to L. Nover and B. Polla for many stimulating discussions.

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SWISS-PROT accession number P81161 (mitochondrial HSP22 in *Lycopersicon esculentum*.