Brassinosteroid functions to protect the translational machinery and heat-shock protein synthesis...

Brassinosteroid functions to protect the translationalmachinery and heat-shock protein synthesis followingthermal stress

Sangeeta Dhaubhadel1, Karen S. Browning2, Daniel R. Gallie3 and Priti Krishna1,*

1Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada N6A 5B7,2Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712, USA, and3Department of Biochemistry, University of California, Riverside, CA 92521-0129, USA

Received 20 August 2001; revised 26 November 2001; accepted 17 December 2001.*For correspondence (fax +1 519 661 3935; e-mail [email protected]).

Summary

In addition to their essential role in plant development, brassinosteroids have the ability to protect plants

from various environmental stresses. Currently it is not understood how brassinosteroids control plant

stress responses at the molecular level. We have begun an investigation into the molecular mechanisms

underlying 24-epibrassinolide (EBR)-mediated stress resistance. Earlier we found that treatment of

Brassica napus seedlings with EBR leads to a signi®cant increase in their basic thermotolerance, and

results in higher accumulation of four major classes of heat-shock proteins (hsps) as compared to

untreated seedlings. Surprisingly, previous studies have shown that while hsp levels were signi®cantly

higher in treated seedlings during the recovery period, transcripts corresponding to these hsps were

present at higher levels in untreated seedlings. To understand mechanisms controlling hsp synthesis in

EBR-treated and untreated seedlings, we studied protein synthesis in vivo as well as in vitro, and assessed

the levels of components of the translational machinery in these seedlings. We report here that increased

accumulation of hsps in EBR-treated seedlings results from higher hsp synthesis, even when the mRNA

levels are lower than in untreated seedlings, and that several translation initiation and elongation factors

are present at signi®cantly higher levels in EBR-treated seedlings as compared to untreated seedlings.

These results suggest that EBR treatment limits the loss of some of the components of the translational

apparatus during prolonged heat stress, and increases the level of expression of some of the components

of the translational machinery during recovery, which correlates with a more rapid resumption of cellular

protein synthesis following heat stress and a higher survival rate.

Keywords: brassinosteroid, thermotolerance, protein synthesis, heat-shock proteins, eEFs, eIFs.

Introduction

Brassinosteroids (BRs) are natural plant steroidal com-

pounds that promote growth and affect a broad spectrum

of physiological responses at nanomolar to micromolar

concentrations (reviewed by Mandava, 1988; Mussig and

Altmann, 1999). These properties, together with the

®ndings that BRs have an essential role in plant develop-

ment, have given these compounds the status of plant

hormones. Rapid advance has been made through studies

of BR-de®cient and BR-insensitive mutants (reviewed by

Clouse and Sasse, 1998; Li and Chory, 1999). BR-de®cient

mutants have provided a better understanding of BR

biosynthesis, and BR-insensitive mutants are helping in

the dissection of BR signal-transduction pathways. The

cloning of the BRI1 gene identi®ed a leucine-rich repeat

(LRR) receptor-like kinase as a putative receptor of BR (Li

and Chory, 1997), and recently, direct binding studies of

BRI1 and brassinolide have provided convincing evidence

that BRI1 is at least one component of the BR receptor

(Wang et al., 2001).

In addition to their role in plant development, BRs have

the ability to protect plants from various environmental

stresses, including chilling, drought, salinity and herbicidal

injury (reviewed by Khripach et al., 2000), and heat stress

(Dhaubhadel et al., 1999; Kulaeva et al., 1991; Wilen et al.,

The Plant Journal (2002) 29(6), 681±691

ã 2002 Blackwell Science Ltd 681

1995). Currently the understanding of this aspect of BRs at

the molecular level is very limited. We have established a

system with which to study the anti-stress effect of 24-

epibrassinolide (EBR) in a reproducible and consistent

manner, and have begun an investigation into the

molecular mechanisms underlying EBR-mediated stress

resistance. We have found that treatment of Brassica

napus seedlings with 1 mM EBR leads to a signi®cant

increase in their basic thermotolerance (Dhaubhadel et al.,

1999). As a ®rst step towards understanding how treat-

ment with EBR induces heat tolerance in B. napus, we

analyzed the accumulation of four major classes of heat-

shock proteins (hsps) in untreated and treated seedlings

before, during and after heat stress. Our results demon-

strated that compared to untreated seedlings, EBR-treated

seedlings accumulated higher levels of all the hsps

examined, both during and after exposure to high tem-

perature stress. Surprisingly, while the hsp levels were

signi®cantly higher in treated versus untreated seedlings

during the recovery period, transcripts corresponding to

these hsps were found to be at higher levels in untreated

seedlings. This pattern of expression suggests that, in

addition to transcriptional regulation, hsp synthesis in

EBR-treated seedlings is also controlled at the post-

transcriptional level.

Most eukaryotes respond to heat shock by increasing the

rate of transcription of hsp genes to attain high levels of

hsps. However, the heat-shock response is not always

controlled at the level of transcription. In Xenopus oocytes

it is mediated at the translational level by activation of

preformed hsp mRNA (Bienz and Gurdon, 1982), whereas

in Leishmania infantum the accumulation of hsp70 in

response to heat shock occurs at the post-transcriptional

level by mechanisms involving speci®c sequences of the

3¢-untranslated regions (Quijada et al., 1997). Expression of

hsps in plants is regulated primarily at the transcriptional

level, but there is a growing appreciation of control

mechanisms that in¯uence translational ef®ciency (Gallie

et al., 1997). Translational selection of hsp mRNA does not

always correlate with rapid synthesis and accumulation of

hsp mRNA. For instance, in response to heat shock,

undifferentiated carrot callus cells synthesize higher levels

of hsp mRNAs than do globular embryos. However, both

cell types synthesize comparable levels of hsps (Apuya

and Zimmerman, 1992).

Changes in the translational machinery itself can also

affect protein synthesis following an environmental stress.

Following heat shock in wheat, the expression and phos-

phorylation status of speci®c initiation factors is altered,

which may be important for the selective translation of

certain mRNAs under this condition (Gallie et al., 1997,

1998b). Further, some mRNAs can be associated with

polysomes, but arrested in their translation. Translational

arrest of polysome-associated mRNAs has been observed

in plants following transition from light to dark (Berry et al.,

1988), or exposure to environmental stresses such as

onset of hypoxia (Crosby and Vayda, 1991), or wounding

(Davies, 1993). Thus it appears that regulation by transla-

tional control is common during plant responses to

various stresses (Kirk and Kirk, 1985; Taliercio and

Chourey, 1989).

To understand mechanisms controlling hsp synthesis in

EBR-treated and untreated seedlings, we have studied

protein synthesis in vivo as well as in vitro, and assessed

the levels of components of the translational machinery in

these seedlings. Here we report that increased accumula-

tion of hsps in EBR-treated compared to untreated seed-

lings results from higher hsp synthesis in EBR-treated

seedlings, even when the mRNA levels are lower than in

untreated seedlings. Signi®cant differences in the levels of

several translation initiation factors (eIFs) and elongation

factors (eEFs) were detected between EBR-treated and

untreated seedlings. These results suggest that differences

in the translational machinery of the two sets of seedlings

are responsible for differential hsp synthesis.

Results

Hsp synthesis in untreated and EBR-treated Brassica

napus seedlings

In our earlier study we had detected higher steady-state

levels of hsps and their corresponding mRNAs in EBR-

treated seedlings than in untreated seedlings during heat

stress. However, during recovery hsp mRNA levels in EBR-

treated seedlings were lower than in untreated seedlings,

although hsp levels remained higher in treated seedlings

(Dhaubhadel et al., 1999). To further investigate this para-

dox, we studied protein synthesis by labeling newly

synthesized polypeptides with [35S]methionine and

[35S]cysteine before, during and after heat stress.

Figure 1 shows that during heat stress, synthesis of most

proteins was shut off in untreated seedlings and that the

synthesis of hsps was induced, as would be expected. The

expression of hsp70 was maximally induced in response to

heat stress, followed by the induction of hsp90. The levels

of [35S]-labeled hsp70 and hsp90 during heat stress were

approximately 29- and 21-fold higher, respectively, in

EBR-treated than in untreated seedlings. Furthermore,

during this period the synthesis of several other proteins

appeared to be induced in EBR-treated seedlings.

Following exposure to 45°C, the seedlings were allowed

to recover at 20°C, and at selected time points proteins

were labeled with [35S]. Although, at 6 h of recovery,

synthesis of hsps had slowed relative to their synthesis

during heat stress, the substantial difference in hsp levels

between the two sets of seedlings was obvious (Figure 1,

compare lanes 2.5 h C, E with 6 h C, E). By 19 h, synthesis

682 Sangeeta Dhaubhadel et al.

ã Blackwell Science Ltd, The Plant Journal, (2002), 29, 681±691

of hsp70 and hsp90 in EBR-treated seedlings had returned

to control levels (20°C). By comparison, protein synthesis

was undetectable in untreated seedlings during the period

of recovery examined, despite the lack of apparent heat-

induced physical damage at these time points. These

results, and those of our earlier study (Dhaubhadel et al.,

1999), indicate that during recovery from heat stress, hsp

synthesis is not correlated with hsp mRNA levels, and

therefore it must be under post-transcriptional control.

Polysome-associated hsp mRNAs during heat stress and

recovery

The presence of a speci®c mRNA species in the cell does

not necessarily correlate with active translation or trans-

lation in accordance with its levels. Thus it is possible that

hsp mRNAs observed in untreated seedlings are not

translated ef®ciently during the recovery period. In order

to examine the pattern of recruitment of hsp mRNAs into

polysomes in treated and untreated seedlings, a protocol

developed by Bailey-Serres and Freeling (1990) was used

to isolate polysomes. The pellet fraction obtained by this

method represents polysome-bound mRNA, whereas the

supernatant fraction contains free RNA.

Examination of hsp transcripts demonstrated that a class

II shsp (small hsp) and hsp90 mRNAs were present in the

polysome fraction from seedlings exposed for 3 or 4 h to

45°C (Figure 2). The level of polysome-associated hsp90

mRNA was approximately twofold higher in EBR-treated

seedlings relative to untreated seedlings exposed to a 3 h

heat stress, but the difference was much less dramatic

than that observed for hsp synthesis (21-fold) following a

2.5 h heat shock (Figure 1). The level of polysome-associ-

ated and free hsp mRNAs decreased signi®cantly during

recovery at 20°C from the levels observed during stress.

However, in accordance with the higher steady-state

mRNA levels in untreated seedlings during recovery

(Dhaubhadel et al., 1999), more hsp mRNA was detected

in the polysome fraction of untreated seedlings during this

period. Hsp90 and class II shsp transcripts representing

free mRNA species, and possibly mRNAs bound to a

single ribosome (monosomes), were also detected in the

Figure 2. Analysis of hsp transcripts associated with polysomes inBrassica napus seedlings during heat stress and recovery.Seedlings grown in the absence (C) or presence (E) of 1 mM EBR wereexposed to 45°C for 3 or 4 h. Seedlings exposed to 45°C for 4 h wereallowed to recover at 20°C for 6 or 19 h. RNA was isolated fromresuspended polysome pellet (P) and supernatant (S). RNA (10 mg) wasseparated on a denaturing formaldehyde agarose gel, blotted onto amembrane, and hybridized separately with [32P]-labeled gene fragmentsencoding a class II shsp, hsp90 and small subunit of rubisco. Aphotograph of the ethidium bromide-stained gel is shown below theblot.

Figure 1. Protein synthesis in untreated and EBR-treated Brassica napusseedlings.A set of seedlings grown in the absence (C) or presence (E) of 1 mM EBRwere labeled for 1 h with [35S]methionine and [35S]cysteine at 20°C. Asecond set of seedlings were exposed to 45°C for 1.5 h then labeled at45°C for 1 h. A third set were exposed to 45°C for 4 h then allowed torecover at 20°C for 5 or 18 h followed by labeling for 1 h at 20°C. Totalproteins (30 mg) were separated on a 10% polyacrylamide gel and¯uorographed. Numbers on the left indicate molecular mass markers inkDa. Arrows indicate the positions of hsp70 and hsp90. A photograph ofthe dried Coomassie blue-stained gel is shown below.

Brassinosteroid and thermotolerance 683


supernatant fraction. If all factors were considered to be

constant in the two sets of seedlings, then the level of hsp

synthesis does not correlate with the level of hsp mRNAs

present in the polysomal fraction of these seedlings. Thus

other factors must exist that contribute to higher synthesis

of hsps in treated seedlings.

Actin and ribulose 1,5 bisphosphate carboxylase

(rubisco) small-subunit mRNAs were used as control

mRNAs that are not induced by heat stress. Actin mRNA

levels also served as a loading control. Actin transcripts

were detected at approximately equivalent levels in all

samples with the exception of recovery time points, where

the levels were higher for EBR-treated seedlings in the

supernatant fraction (data not shown). Rubisco mRNA

was undetectable in untreated seedlings following 19 h

recovery (Figure 2), presumably indicating the onset of

their death despite the lack of visible symptoms. By

contrast, in EBR-treated seedlings the level of rubisco

mRNA was highest following 19 h of recovery, indicating

good recovery of seedlings from heat stress.

Translational analysis of polysome-associated mRNAs

It is possible that hsp mRNAs recruited onto polysomes in

untreated seedlings were arrested in their translation, and/

or mRNAs recruited onto polysomes in EBR-treated seed-

lings were translated in a highly ef®cient manner. To

further assess translational activity under each set of

conditions, polysomes isolated for the analysis described

in Figure 2 were translated in vitro using wheatgerm

lysate. The ef®ciency of the in vitro translation system was

examined in several ways: (i) translation of brome mosaic

virus (BMV) RNA or total RNA isolated from EBR-treated

B. napus seedlings resulted in different translation pro-

ducts (Figure 3a, lanes 1 and 2); (ii) when RNA was

omitted, no translation products were obtained (lane 3).

Total RNA (deproteinized) isolated from untreated and

treated seedlings that had been exposed to 45°C for 3 h

was ef®ciently translated in vitro in the absence of 7-

methylguanosine monophosphate (m7GMP), a speci®c

inhibitor of translation initiation (Figure 3b). However, in

the presence of m7GMP protein synthesis was completely

inhibited, as expected.

The translation products of polysomes isolated from

EBR-treated and untreated seedlings are shown in Figure

3(c). No signi®cant difference in the levels of hsp90 and

hsp70 following 3 h heat stress was detected between the

two sets of seedlings, but higher levels of both hsps were

consistently observed in EBR-treated seedlings following

4 h heat stress. Addition of m7GMP to the wheatgerm

Figure 3. Translational analysis in vitro ofpolysome-associated mRNAs.(a) BMV RNA (lane 1), deproteinizedBrassica napus RNA (lane 2) and water (lane3) were incubated for translation in vitro inthe presence of [35S]methionine. Thetranslation products were separated on a10% SDS±polyacrylamide gel and detectedby ¯uorography.(b) Deproteinized total RNA from untreated(C) and EBR-treated (E) B. napus seedlingsthat had been exposed to 45°C for 3 h wereincubated for translation in vitro in thepresence of [35S]methionine, but in theabsence (±) or presence (+) of m7GMP. Thetranslation products were separated on a7.5% SDS±polyacrylmide gel and detectedby ¯uorography.(c) Polysomes were isolated from untreated(C) and EBR-treated (E) seedlings that hadbeen exposed to 45°C for 3 or 4 h, as wellas from seedlings exposed to 45°C for 4 hthat had recovered at 20°C for 6 or 19 h.Resuspended polysome pellets (8 mg) wereincubated for translation in vitro in thepresence of [35S]methionine, but in theabsence (±) or presence (+) of m7GMP. Thetranslation products were analyzed asdescribed in (a).Numbers on the left indicate molecularmass markers in kDa; arrows indicate thepositions of hsp70 and hsp90.



system greatly reduced polysome-directed protein synthe-

sis. Signi®cant differences in the translational competence

of polysomes were observed between the two sets of

seedlings during the recovery period, in both the presence

and absence of m7GMP (Figure 3c). The higher amounts,

as well as greater number, of products synthesized in EBR-

treated seedlings during the recovery period are in agree-

ment with the results of protein synthesis in vivo (Figure 1).

Although some in vivo events were not duplicated

in vitro (compare Figure 1, lanes 2.5 h HS, C, E with

Figure 3c, lanes 3 h HS, C, E), overall, the results of run-off

translation of polysomes in vitro appear to further con®rm

the hypothesis that hsp synthesis is more ef®cient in EBR-

treated seedlings.

Analysis of translation factors in EBR-treated and

untreated seedlings

We determined whether the levels of translation factors in

untreated and EBR-treated seedlings were different and, if

so, whether they could be correlated with the levels of hsp

synthesis in these seedlings. Previously, antibodies raised

against wheat eIFs and eEFs have been used in determin-

ing the levels of these factors by Western blotting

(Browning et al., 1990; Gallie et al., 1998a; Gallie et al.,

1998b). Antibodies to wheat proteins that cross-reacted

speci®cally with B. napus polypeptides of sizes consistent

with those of wheat factors were used. In each case,

puri®ed wheat protein was included as a reference for the

B. napus proteins, although signals representing the wheat

factor and B. napus factor were obtained from different

®lm exposures due to the less intense response of the

wheat antibodies to B. napus proteins (Figure 4). The anti-

eEF1Bg antibody detected two extra bands in B. napus, one

of which was also detected in the wheat sample. The

identity of the bands is not known, but is likely to represent

a degradation product. eEF1A, eEF2 and eEF1Bg accumu-

lated to approximately the same levels in untreated and

EBR-treated seedlings at the normal growth temperature

(20°C). Following 1 h temperature stress at 45°C, the level

of eEF1A was slightly higher in treated than in untreated

seedlings. The difference was more pronounced following

4 h heat stress (8-fold higher in EBR-treated seedlings), but

the most signi®cant difference in the level of eEF1A in the

two sets of seedlings was following 19 and 24 h of

recovery. During this time, the level of eEF1A was 16-

and 18-fold higher, respectively, in EBR-treated seedlings

than in untreated seedlings. Similarly, the levels of eEF2

and eEF1Bg were also much higher in treated seedlings

during recovery as compared to untreated seedlings.

Furthermore, following heat stress the levels of eEF1A

and eEF2 were signi®cantly higher than the levels present

under control conditions (20°C) in EBR-treated seedlings.

eEF1A is highly conserved in evolution (Pokalsky et al.,

1989). A cDNA encoding eEF1A was isolated from B. napus

by RT±PCR and its identity con®rmed by sequence analysis

(data not shown). The cDNA was used as a probe in

Northern blotting. An approximately 2-fold higher level of

eEF1A transcripts was detected in EBR-treated seedlings

relative to untreated seedlings at 20°C (Figure 5). eEF1A

mRNA levels declined in both sets of seedlings as a

function of the length of exposure to heat stress, but

were restored to higher levels during recovery only in EBR-

treated seedlings. The pattern of eEF1A transcript

expression did not precisely correlate with eEF1A protein

expression (Figure 4), but was consistent with it in that

there was higher expression in EBR-treated seedlings.

Among the initiation factors, only the antisera raised

against wheat eIFiso4E and eIF4A recognized similarly

sized proteins in B. napus. Expression of eIFiso4E was

higher in EBR-treated than in untreated seedlings at all

times, including at 20°C (Figure 6). The difference between

the two sets of seedlings was most pronounced during

recovery from heat stress. eIF4A also accumulated to

higher levels in EBR-treated seedlings, but only during the

recovery period. The levels of both proteins were much

higher in EBR-treated seedlings during recovery than

under non-stress condition (20°C). Because expression of

chaperonin 60-b (cpn60-b) does not change signi®cantly in

response to heat stress (Parsell and Lindquist, 1993), it was

used as a control. As expected, cpn60-b was present at

approximately the same level in both untreated and EBR-

Figure 4. Accumulation of translation-elongation factors in untreated andEBR-treated Brassica napus seedlings during heat stress and recovery.Seedlings grown in the absence (C) or presence (E) of 1 mM EBR wereexposed to 45°C for 1, 2, 3 or 4 h. Seedlings exposed to 45°C for 4 hwere allowed to recover at 20°C for 6, 19 or 24 h. Total proteins (30 mg)were separated on a 10% SDS±polyacrylmide gel and transferred ontonitrocellulose membrane by electroblotting. The eEFs were detected bysequential incubation of the membrane with anti-eEF1A, eEF1Bg andeEF2 antibodies and peroxidase-conjugated anti-rabbit IgG followed bythe chemiluminescent reaction. Arrows on the right indicate puri®edwheat proteins (P) against which antibodies were raised. An anti-cpn60-bantibody was used to detect cpn60-b as a loading control.



treated seedlings. Thus the high levels of eIFs and eEFs in

EBR-treated seedlings are not a result of global changes to

cellular proteins in these seedlings, but rather are the

result of a speci®c event(s) occurring during the heat-

stress response in the presence of EBR.

Discussion

The results of this study demonstrate that, in comparison

to untreated seedlings, EBR-treated B. napus seedlings

have signi®cantly higher translational activity during and

following exposure to heat stress, and that this difference

in protein synthetic activity is accompanied by dramatic

differences in the expression level of several translation

factors in the two sets of seedlings. Earlier we had

observed that the transcript levels of hsp100, hsp90,

hsp70 and low molecular weight hsps are higher in heat-

stressed EBR-treated seedlings than in untreated seed-

lings, but during recovery the opposite is observed.

Nevertheless, hsps accumulate to higher levels in treated

than in untreated seedlings during and after exposure to

heat stress (Dhaubhadel et al., 1999). The higher level of

hsps in EBR-treated seedlings may be due to increased

synthesis and/or increased stability.

Hsp synthesis in vivo does not correlate with the level of

polysome-associated hsp mRNAs

To understand the regulation of hsp synthesis in EBR-

treated seedlings, newly synthesized proteins were labeled

with [35S]. Figure 1 shows that hsp synthesis in treated

seedlings was signi®cantly higher than in untreated seed-

lings, not only during stress but also during recovery. The

apparent difference in hsp synthesis between the two sets

of seedlings during the recovery period may have resulted

from either preferential recruitment of hsp mRNAs onto

polysomes in EBR-treated seedlings, the translational

arrest of polysome-associated hsp mRNAs in untreated

seedlings, or differences in the activity of the translational

machinery between the two sets of seedlings. To distin-

guish between these possibilities, the levels of polysome-

associated mRNAs were determined for a class II shsp and

hsp90 during and after heat stress. The overall pattern of

polysome-associated hsp mRNAs (Figure 2) correlated

with steady-state hsp mRNA levels observed in our

previous study (Dhaubhadel et al., 1999). For instance,

hsp mRNA levels were higher in untreated seedlings than

in EBR-treated seedlings during recovery (Dhaubhadel

et al., 1999), and so was their association with polysomes

(Figure 2). Surprisingly, the pattern of hsp synthesis did

not correlate with the level of RNA observed. A consider-

able amount of hsp mRNA was also detected in the

supernatant fraction, indicating that not all hsp mRNA was

loaded onto polysomes during heat stress. This is not

unusual ± it has been noted previously that carrot callus

cells accumulate hsp17.7 mRNA to a high level in response

to heat shock, but recruit only a small fraction of the mRNA

into polysomes (Apuya and Zimmerman, 1992). A charac-

teristic feature of the plant heat-stress response is the

translational competence of hsp mRNAs in a cellular

environment in which non-hsp mRNAs are subject to

translational repression. Non-hsp mRNAs may become

sequestered in heat-shock granules, degraded, or a pro-

portion may remain bound to polysomes (Apuya and

Zimmerman, 1992). The presence of rubisco mRNA in the

polysome fraction indicates that a portion of it remains

bound to ribosomes. The decline in the level of rubisco

mRNA during prolonged heat stress probably results from

its degradation, and its increase during the recovery period

may result from an increase in its stability and/or synthe-

sis. While understanding the mechanism governing

rubisco mRNA sequestration/availability during heat stress

is not the focus of this work, the pattern of rubisco mRNA

Figure 6. Accumulation of translational initiation factors in untreated andEBR-treated Brassica napus seedlings during heat stress and recovery.Seedlings grown in the absence (C) or presence (E) of 1 mM EBR wereexposed to 45°C for 1, 2, 3 or 4 h. Seedlings exposed to 45°C for 4 hwere allowed to recover at 20°C for 6, 19 or 24 h. Total proteins (30 mg)were separated by SDS±polyacrylamide gel and transferred tonitrocellulose membrane by electroblotting. The initiation factors weredetected by sequential incubation of the membrane with anti-eIF4A andeIFiso4E antibodies and peroxidase-conjugated anti-rabbit IgG followedby the chemiluminescent reaction. Arrows on the right indicate puri®edwheat proteins (P) against which antibodies were raised. Cpn60-b wasdetected as a loading control.

Figure 5. Accumulation of eEF1A transcripts in untreated and EBR-treated Brassica napus seedlings during heat stress and recovery.Seedlings grown in the absence (C) or presence (E) of 1 mM EBR wereexposed to 45°C for 1, 2, 3 or 4 h. Seedlings exposed to 45°C for 4 hwere allowed to recover at 20°C for 6, 19 or 24 h. Total RNA (15 mg) wasseparated on a denaturing formaldehyde agarose gel, blotted onto amembrane and hybridized with [32P]-labeled eEF1A gene fragment. Thesame blot, after removal of the probe, was hybridized with [32P]-labeled18S rRNA gene fragment to serve as loading control.



expression and recruitment onto polysomes con®rms

ef®cient recovery of EBR-treated seedlings from heat

stress and damaging effects of heat stress in untreated

seedlings.

Translation is inhibited in untreated seedlings following

heat stress

Translation of isolated polysomes was carried out in vitro

in order to gain further insight into the mechanism

governing hsp synthesis in EBR-treated seedlings.

Translation of intact polysomes isolated from treated and

untreated seedlings during heat shock demonstrated that

hsp mRNAs predominated in the polysomal fraction

(Figure 3c). The failure to observe signi®cant expression

of non-heat-shock protein synthesis during the in vitro

translation of polysomes isolated from heat-stressed

seedlings is consistent with the observation that heat

shock causes the disassembly of most, if not all, non-heat-

shock polysomes and the preferential recruitment of heat-

shock mRNAs to polysomes (Key et al., 1981).

In contrast to the substantially higher level of hsp

expression observed in vivo in EBR-treated seedlings

(Figure 1), the in vitro translation of polysomes isolated

from treated and untreated seedlings during a heat shock

yielded similar levels of hsps, particularly following a 3 h

exposure to the stress (Figure 3c). Translation of poly-

somes in the presence of m7GMP also revealed similar

levels of translation products in both sets of seedlings.

m7GMP inhibits new cap-dependent initiation, but allows

translational run-off from the polysomes using elongation

factors from the wheatgerm lysate. This accounts for the

difference in the level of translation in the absence (re-

initiation and elongation can take place) and presence

(attached ribosomes complete elongation but cannot

reinitiate translation) of m7GMP. Results presented in

Figure 3(c) indicate that polysomes isolated from the two

sets of seedlings during a heat stress were of similar size,

suggesting that the difference between in vivo and in vitro

hsp expression between the two sets of seedlings was not

a consequence of differential ribosome loading that would

occur as a result of differences in translational initiation.

The failure of untreated, heat-stressed seedlings to

synthesize hsps in vivo to the level observed for EBR-

treated, heat-stressed seedlings may be a result of inhibi-

tion of translation elongation. Such inhibition may result

from reduced activity of one or more of the eEFs during a

short exposure to a heat stress or a reduction in their level,

as was observed for a prolonged exposure to the stress

(Figure 4). Under such a scenario, inhibition of elongation

would be reversible, as polysomes isolated from untreated

seedlings yielded a similar level of protein products as

those isolated from EBR-treated seedlings when translated

in wheatgerm lysate (Figure 3c). Inhibition of translation

elongation might also have been achieved through the

association of a factor that reduced the rate of elongation

in the untreated seedlings. An alternative explanation for

the higher level of hsp expression in EBR-treated seedlings

in vivo (Figure 1) might be that hsp70 and hsp90 proteins,

in particular, are more stable in these seedlings during a

heat stress than in untreated seedlings. This possibility is

supported by the ratio of the abundance of these two

proteins relative to other proteins synthesized in vivo

(Figure 1) compared to the same ratio observed when the

polysomes were translated in vitro (Figure 3c).

During recovery, a higher level of protein synthesis was

observed in vivo in EBR-treated seedlings than in

untreated seedlings (Figure 1). When polysomes isolated

from both sets of seedlings during their recovery from a

heat shock were translated in vitro, those isolated from

EBR-treated seedlings yielded a higher level of protein

products than did those isolated from untreated seedlings

(Figure 3c). These observations suggest that polysomes

from treated seedlings were larger (more ribosomes per

polysome) than those of untreated seedlings, resulting in a

higher yield of translational run-off products during in vitro

translation. Larger polysomes may have resulted from a

higher rate of translation initiation in EBR-treated recover-

ing seedlings, consistent with the observation of higher

levels of eIFs in these seedlings (Figure 6). A difference in

polysome size may also have resulted from an acute

inhibition of elongation in untreated seedlings during

recovery, a possibility which is supported by the substan-

tial reduction in the level of elongation factors in the

untreated seedlings (Figure 4). Inhibition of elongation

following formation of the 80S ribosome at an initiation

codon would inhibit further ribosome loading and thus

reduce polysome size. An increase in translational initi-

ation in EBR-treated seedlings and an inhibition of elonga-

tion, as well as re-initiation in untreated seedlings during

recovery, are not mutually exclusive.

EBR treatment leads to an increase in the expression of

translation elongation and initiation factors

The translational machinery in plants is modi®ed in

response to thermal stress through changes in their

abundance and phosphorylation state (Gallie et al., 1997;

Gallie et al., 1998a; Gallie et al., 1998b). To identify factors

involved in translation that may be responsible for the

differential protein synthesis in untreated and EBR-treated

seedlings, we compared the levels of eIFs and eEFs in

untreated and EBR-treated seedlings before, during and

after heat stress. Dramatic differences in the levels of the

eEFs examined were observed between the two sets of

seedlings following exposure to heat stress, although

differences to a lesser extent were also observed before

and during heat stress (Figure 4). The main function of



eEF1A in translation is to bind GTP and aminoacyl-tRNA

and bring them to the elongating 80S ribosome during the

elongation phase, whereas the functions of eEF1Bg and

eEF2 are to recycle the eEF1AGDP complex and catalyze

the GTP-dependent translocation of the peptidyl-tRNA

from the A site to the P site on the ribosome, respectively.

eEF2 also removes deacylated tRNA from the ribosome

(Riis et al., 1990). Among the eEFs, eEF1A has been most

extensively studied. There is evidence to suggest that

increased expression of eEF1A leads to longevity while

decreased expression of eEF1A correlates with senes-

cence. For example, over-expression of eEF1A in

Drosophila melanogaster lengthened the mean lifetime

of the ¯ies by maintaining protein synthesis for a longer

period (Shepard et al., 1989), and high-®delity mutations in

eEF1A increased the life span of Podospora anserina (Silar

and Picard, 1994). An increased level of gene dosage of

eEF1A in yeast was found to correlate with increased

suppression of nonsense mutations (Mahn-Song et al.,

1989). In contrast, decrease in the levels and catalytic

activity of eEF1A were observed in the terminal phases of

cell aging (Cavallius et al., 1986; Webster, 1985). Progres-

sive decline in protein synthesis is an age-related change.

As eEF1A is involved in the synthesis of all proteins, and

the decline in its levels and activity parallel the decline in

protein synthesis during aging, it is considered to play a

crucial role in aging. Together, these studies point to a link

between the levels of eEF1A in the cell with the rate and

®delity of translation. In view of these ®ndings, the higher

levels of this factor in EBR-treated B. napus seedlings

correlates with the greater ability of these seedlings to

survive a severe heat stress. In an earlier study, heat shock

was found to have little effect on the amount of eEF1A and

eEF2 in wheat leaves (Gallie et al., 1998a). In that study, the

duration of heat stress was 90 min versus up to 4 h in our

study. In agreement with this previous study, no major

changes in the levels of eEF1A and eEF2 were detected in

untreated B. napus seedlings up to 2 h. Because the

increase in the abundance of eEFs following 4 h of heat

shock and during recovery was seen only in EBR-

treated seedlings, we conclude that this effect, directly or

indirectly, is EBR-mediated. The eEF1A transcript levels

indicated that EBR can also mediate an effect under normal

growth temperatures (Figure 5).

Alteration in the level or activity of initiation factors may

also regulate translation. We could measure the levels of

only eIF4A and eIFiso4E in B. napus seedlings, due to lack

of cross-reactivity of antibodies raised to other wheat eIFs

(e.g. eIF4B, eIF4G, eIF4E, eIFiso4G). The eIF4A, in conjunc-

tion with eIF4F and eIF4B, functions to remove secondary

structures in the 5¢ untranslated regions of mRNAs to

facilitate binding of the 40S ribosomal subunit (Balasta

et al., 1993). eIFiso4F (in which eIFiso4E is present as the

cap-binding subunit) also exhibits ATP-dependent RNA

unwinding in the presence of eIF4A (Browning, 1996). In an

earlier study, changes in the expression of certain eIFs in

response to heat shock were noted in wheat leaves, but the

levels of both eIF4A and eIFiso4E were observed to remain

unchanged (Gallie et al., 1998b). The duration of heat

stress in that study was 90 min as compared to the

maximum of 4 h in the present study. By 4 h exposure to

45°C, the levels of eIFiso4E had declined in untreated B.

napus seedlings, whereas no signi®cant change was seen

for eIF4A (Figure 6). An increase in the amount of both

factors was detected in treated seedlings during recovery.

EBR treatment also led to an increase in the expression of

eIFiso4E under non-stress conditions. Recently it was

shown that brassinolide treatment increases the transcript

levels of eIF3i (equivalent to mammalian p36 subunit) in

bean cell cultures and Arabidopsis (Jiang and Clouse,

2001). In developing wheat seeds, the highest levels of

eIF4A and eIFiso4E, as well as of several other eIFs, were

correlated with the period of greatest protein synthetic

activity (Gallie et al., 1998b). We extrapolate from this

observation that higher hsp as well as global protein

synthesis during heat stress and recovery in EBR-treated

seedlings may, in part, be the result of higher levels of

translation factors in these seedlings. Thus it appears that

EBR treatment limits the loss of some of the components

of the translational apparatus during a prolonged heat

stress, and increases the level of expression of some

components of the translational machinery during

recovery, which correlates with a more rapid resumption

of cellular protein synthesis following a heat stress.

Summary of the molecular changes in EBR-treated

B. napus seedlings that may contribute to increased

resistance to heat and other stresses

The role of hsps in protecting mRNAs through formation

of heat shock granules and other macromolecules during

heat shock is well documented (Nover et al., 1989; Vierling,

1991). Members of the hsp70 family are known to also

interact with polysomes in both mammalian and yeast

cells (Beckmann et al., 1990; James et al., 1997; Nelson

et al., 1992). In addition to a protective function, hsps can

also regulate translation. Examples include the require-

ment for yeast DnaJ homologue (Brodsky et al., 1998) and

plant Hsp101 (Ling et al., 2000) to aid in the ef®cient

translation of certain mRNAs. Although a speci®c regula-

tory involvement of hsps in translation during heat stress

is not known, the possibility may be suggested from the

above examples. In summary (Figure 7), EBR treatment

results in a higher heat-mediated induction of hsp expres-

sion, which may in turn be responsible for the mainten-

ance of the translational machinery during a prolonged

exposure to stress. Alternatively, EBR treatment may

maintain expression of the translational machinery during



a heat stress, which might result in higher hsp expression,

two possibilities that are not mutually exclusive. Although

higher levels of hsps must contribute to increased

thermotolerance in EBR-treated seedlings, using differen-

tial display we sought to ®nd factors other than hsps that

may directly or indirectly contribute to EBR-mediated

increase in stress tolerance. Four cDNAs characterized

thus far that were upregulated in treated seedlings encode

3-ketoacyl CoA thiolase, myrosinase, glycine-rich protein

22, and a hypothetical protein (S. Dhaubhadel & P.

Krishna, unpublished results). The thiolase transcript

levels were higher in treated seedlings as compared to

untreated seedlings during heat stress, but transcripts of

the other three cDNAs were present at higher levels in

treated seedlings prior to any stress. Higher expression of

3-ketoacyl thiolase, myrosinase and glycine-rich protein 22

can be linked, at least hypothetically, to an increase in the

general stress resistance of plants. The results of the

present study, coupled with increased expression of genes

involved in a variety of physiological responses (S.

Dhaubhadel & P. Krishna, unpublished results) and other

as-yet unidenti®ed factors in treated seedlings, may con-

tribute to increased overall stress tolerance in these

seedlings (Figure 7).

Experimental procedures

Plant material and growth conditions

Plant material, growth conditions and heat stress were asdescribed previously (Dhaubhadel et al., 1999).

In vivo protein labeling

Brassica napus seedlings were grown for 14 days in liquidmedium supplemented with either 1 mM EBR or 0.01% ethanol(control). At appropriate times, Trans [35S] Label (ICN Biomedical,

Aurora, OH, USA) was added to the medium at a concentration of40 mCi ml±1. The labeling was continued for 1 h, following whichplant tissue above the medium was collected and quick-frozen inliquid nitrogen. Protein extraction and quanti®cation were carriedout as described previously (Dhaubhadel et al., 1999). Totalsoluble proteins of each sample were separated on SDS±polyacrylamide gels which were stained with Coomassie blueR-250, destained and ¯uorographed.

Polysome isolation

Polysomes were isolated according to the procedure of Bailey-Serres and Freeling (1990) with some modi®cations. Frozen planttissue was ground to powder under liquid nitrogen using a mortarand pestle. Powdered tissues were immediately suspended inpolysome extraction buffer (0.2 M Tris±HCl pH 9.0, 0.4 M KCl,25 mM EGTA, 35 mM MgCl2, 0.2 M sucrose, 15 mM b-mercapto-ethanol). All manipulations were carried out on ice or at 4°C. Theextract was homogenized for 30 sec using a polytron homo-genizer following the addition of Triton X-100 to a ®nal concen-tration of 1%. The extract was incubated on ice for 15 min withoccasional mixing, then centrifuged at 30 000 g for 20 min. Thesupernatant was layered over a 3 ml sucrose cushion (1.75 M

sucrose, 40 mM Tris±HCl pH 9.0, 0.2 M KCl, 5 mM EGTA,30 mM MgCl2, 7 mM b-mercaptoethanol), and centrifuged at173 000 3 g in a Type 90 Ti rotor (Beckman, Coulter,Mississauga, ON, USA) at 4°C for 4.5 h in an ultracentrifuge.The polysome pellet was resuspended in resuspension buffer(40 mM Tris±HCl pH 8.5) and incubated on ice for 30 min. Theresuspended polysome preparations were clari®ed by centrifuga-tion at 3500 3 g in a microcentrifuge for 1 min at 4°C to discardany undissolved material. The polysomal RNA was quanti®ed bymeasuring the absorbance at 260 nm.

RNA isolation and Northern blotting

The resuspended polysomes were extracted twice with 0.5 volphenol : chloroform, then precipitated with 3 M sodium acetate(pH 5.2) and absolute ethanol. The RNA pellet was recovered bycentrifugation at 16 000 3 g for 20 min at 4°C in a micro-centrifuge. To isolate RNA from supernatant, the supernatantwas diluted 2 : 1 in DEPC-treated water and processed in thesame way as polysomal pellet RNA. Northern blotting (Figure 2)was carried out as described previously (Dhaubhadel et al., 1999).

Total RNA was isolated from frozen plant tissue according toGlisin et al. (1974) and analyzed by Northern blotting (Figure 5).Following hybridization with radiolabeled eEF1A gene fragment,the membrane was washed twice at room temperature for 15 minwith 1 3 SSC, 0.1% SDS, then once with 0.25 3 SSC, 0.1% SDS at55°C for 5 min, followed by autoradiography. The same blot afterstripping was hybridized with an 18S ribosomal DNA fragment toserve as loading control. For generation of eEF1A gene fragment,RT±PCR was carried out using RNA isolated from EBR-treatedseedlings grown at 20°C for 14 days. Reverse transcription wascarried out using random hexamers. Conserved sequences, F (5¢-GCTGAGATGAACAAGAGGTC-3¢) and R (5¢-CAACAGTCTGCC-TCATGTC-3¢), which were identi®ed by alignment of eEF1Agenes from wheat (accession no. M90077), tomato (accessionno. X14449), barley (accession no. Z23130) and Arabidopsis(accession no. X16430), were used as primers for PCR. The PCRconditions were as follows: 95°C for 1 min, 52°C for 30 sec and72°C for 2 min (35 cycles). The PCR product was cloned into theTA cloning vector and sequenced at the Robart SequencingFacility, London, Ontario.

Figure 7. Summary of the molecular changes in EBR-treated Brassicanapus seedlings that may contribute to increased resistance to heat andother stresses.Photograph of the seedlings at 7 days' recovery at 20°C after exposure to45°C for 4 h, reproduced from Dhaubhadel et al. (1999).



In vitro translation of polysomes

For translation of polysomes in vitro, 8 mg resuspended polysomeswere incubated with 12.5 ml wheatgerm extract (Promega,Madison, WI, USA) supplemented with 80 mM amino acid mixtureminus methionine, 67 mM potassium acetate, 40 units RNAsinRibonuclease inhibitor and 1.25 ml [35S]methionine (1000 Cimmol±1, Amersham Biosciences, Baie d'UrfeÂ , QC). The ®nalvolume was made up to 25 ml with sterile distilled water. Whererequired, the reaction mixtures were supplemented either with200 mM m7GMP to block translational initiation, or with an equiva-lent volume of water (controls). The translation reactions wereincubated at 26°C for 60 min, and the results were analyzed byelectrophoresis on a 10% SDS±polyacrylamide gel followed bystaining, destaining and ¯uorography. Control reactions usingBMV RNA, deproteinized B. napus RNA, or no RNA were alsocarried out.

Analysis of translational initiation and elongation factors

by Western blot analysis

Total soluble proteins of each sample were separated on SDS±polyacrylamide gels and transferred to nitrocellulose membranesas described previously (Dhaubhadel et al., 1999). The blots wereprobed separately with anti-eEF1A, eEF1Bg, eEF2, eIF4A, eIFiso4Eand cpn60-b antibodies, each at a dilution of 1 : 1000, followed byincubation with peroxidase-conjugated anti-rabbit IgG (1 : 5000)and chemiluminescent reaction (ECL System, AmershamBiosciences, Baie d'UrfeÂ , QC).

Acknowledgements

This research was supported by research grants to P.K. from DowElanco and the Natural Sciences and Engineering ResearchCouncil of Canada. S.D. was a recipient of the Ontario GraduateScholarship.

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