Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica napus to a range of

abiotic stresses
Author(s): Sateesh Kagale, Uday K. Divi, Joan E. Krochko, Wilfred A. Keller and Priti
Krishna
Source: Planta, Vol. 225, No. 2 (January 2007), pp. 353-364
Published by: Springer
Stable URL: http://www.jstor.org/stable/23389554
Accessed: 20-09-2016 01:24 UTC

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Planta (2007) 225:353-364
DOI 10.1007/s00425-006-0361-6

ORIGINAL ARTICLE

Brassinosteroid confers tolerance in Arabidopsi

and Brassica napus to a range of abiotic stresses
Sateesh Kagale • Uday K. Divi • Joan E. Krochko •
Wilfred A. Keller • Priti Krishna

Received: 27 April 2006 / Accepted: 17 July 2006 / Published online: 12 August 2006
© Springer-Verlag 2006

Abstract In addition to an essential role in plant

although BR augments thermotolerance in plants, it is
development, brassinosteroids (BRs) appear tonothave
necessary for hsp expression during HS.
the ability to protect plants against various environ
mental stresses. However, studies confirming the Keywords
abil Abiotic stress ■ Arabidopsis thaliana •
ity of BRs to modulate plant responses to different Brassica napus • Brassinosteroid • Drought •
environmental stresses are lacking. Earlier we had Stress tolerance

demonstrated that treatment with 24-epibrassinolide

(EBR), a BR, increases the basic thermotolerance of Abbreviations
Brassica napus and tomato seedlings [Plant Mol Biol BR Brassinosteroid
40:333-342, 1999]. Here we demonstrate that EBR DRE Drought-responsive element
treatment enhances seedling tolerance to drought and EBR 24-Epibrassinolide
cold stresses in both Arabidopsis thaliana and B. napus, Hsp Heat shock protein
and helps to overcome a salt-stress-induced inhibition HS Heat stress

of seed germination. The ability of EBR to confer tol

erance in plants to a variety of stresses was confirmed
through analysis of expression of a subset of drought Introduction

and cold stress marker genes. Transcriptional changes

in these genes were more apparent in EBR-treated A. Brassinosteroids (BRs), a class of plant polyhydroxy
thaliana, in particular during earlier time points of steroids that are structurally similar to animal and
stress. To see if BR is essential for the heat stress (HS) insect steroid hormones, control a broad range of
response, we made use of BR-deficient mutants. Both responses in plants such as cell division and expansion,
det2-l and dwf4 mutants still expressed heat shock pro xylem differentiation, seed germination, vegetative
teins (hsps) to high levels during HS, indicating that growth, and apical dominance (Sasse 2003). Though
the effects of BR on plant development were known as
early as the 1970s (Mandava 1988), it is the recent
molecular genetic studies of BR-deficient mutants and
BR-insensitive mutants that have established an essen
S. Kagale ■ U. K. Divi • P. Krishna (El) tial role for BRs in plant growth and development, a
Department of Biology, The University of Western Ontario,
led to the identification of several BR signaling com
London, ON, Canada N6A 5B7
e-mail: pkrishna@uwo.ca nents (Clouse and Sasse 1998; Clouse 2002). Unlike th
steroid hormone receptors in animals that function
J. E. Krochko • W. A. Keller
transcriptional activators, a major BR receptor, BR
Plant Biotechnology Institute,
is a membrane-localized leucine-rich repeat recepto
National Research Council of Canada,
110 Gymnasium Place, like kinase (Li and Chory 1997). BR binding to BR
Saskatoon, SK, Canada S7N 0W9 leads to dimerization of BRI1 with another recepto

Ô Springer

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354 Planta (2007) 225:353-364

kinase BAK1, and kinase activation, which initiates a tance against these stresses (reviewed in Krishna
signaling cascade leading to gene expression (Vert 2003a). For instance, treatment with BR of a wheat
et al. 2005; Wang et al. 2006). The downstream compo variety sensitive to drought stress appeared to increase
nents of this cascade include a glycogen synthase water uptake and membrane stability, as well as reduc
kinase-3/SHAGGY-like kinase BIN2, a serine/threo ing ion leakage (Sairam 1994). Improved salt tolerance
nine phosphatase BSU1, and the transcription factors in response to BR treatment was reported in barley
BES1 and BZR1. BIN2 negatively regulates BR signal (Kulaeva et al. 1991), and improved germination rates
ing by phosphorylating BES1 and inhibiting its binding in the presence of high salt were observed in Eucalyp
to BR target promoters, as well as its transcriptional tus calmaldulensis (Sasse et al. 1995) and rice (Anura
activity (Vert and Chory 2006). Following BR binding, dha and Rao 2001). A stimulatory effect of BR on the
BES1 and BZR1 are rapidly dephosphorylated, proba growth of maize and cucumber seedlings was also seen
bly by BSU1, leading to activation of their transcrip under chilling stress (He et al. 1991; Katsumi 1991).
tional activities (Vert et al. 2005; Wang et al. 2006). While these findings are encouraging, there have been
Many of the genes induced by BR application encode few follow-up experiments in nearly all instances and
cell wall loosening enzymes (Goda et al. 2002; Mussig the results are limited to measurements of plant growth
et al. 2002). Genes involved in cell division, vascular aspects. To confirm the protective properties of BRs to
differentiation, phytohormone interaction, carbon par a broad range of abiotic stresses, we have now studied
titioning, and cell rescue have also been identified as the effects of EBR on Arabidopsis thaliana, a genetic
BR-regulated genes (Mussig and Altmann 2003; Vert model system, and B. napus, an oil crop plant, under
et al. 2005). drought, cold, and high salt conditions. Here we show
In addition to its pivotal role in plant growth and that EBR treatment mediates resistance to these

development, BR appears to protect plants from a stresses, as well as increases the basic thermotoler
variety of environmental stresses, including high and of A. thaliana seedlings. Through studies of BR-d
low temperature stress, drought, salinity, herbicidal cient mutants, we also show that although BR can
injury, and pathogen attack (Khripach et al. 2000; ment tolerance to HS, it is not essential for
Krishna 2003a). However, studies confirming the abil expression during HS treatments.
ity of BR to modulate plant stress responses, as well as
providing a framework under which such effects of BR
can be studied in a reproducible manner are lacking, Materials and methods

despite such effects being suggested in several prelimi

nary reports. Our previous studies focused on the abil Plant material and growth conditions
ity of 24-epibrassinolide (EBR), a BR, to confer
tolerance in plants to high temperature stress. Our Arabidopsis thaliana ecotype Columbia wild type
results established that treatment with EBR increases (WT), BR-deficient A. thaliana mutants det2-l (pro
the basic thermotolerance of Brassica napus and vided by Dr J. Li) and dwf4 (obtained from Arabidop
sis Biological Resource Center, stock no. CS374), and
tomato seedlings, resulting in higher accumulation of
the major classes of heat shock proteins (hsps) in spring B. napus (cv. Westar) were used in this study. A.
EBR-treated seedlings as compared to untreated thaliana seeds were surface-sterilized by sequentially
seedlings (Dhaubhadel et al. 1999). Further investiga
soaking seeds in 75% ethanol for 1 min, rinsing twice in
tion into how hsps accumulate to higher levels insterile-distilled water, soaking in 1.05% sodium hypo
EBR-treated seedlings revealed that EBR treatmentchlorite for 20 min with stirring, and rinsing 4-5 times
modulates the translational machinery, leading towith sterile-distilled water. The seeds were then trans
higher hsp synthesis during heat stress (HS) and more ferred to sterile Whatman No. 3 filter paper and par
rapid resumption of cellular protein synthesis follow tially air-dried under laminar flow. A. thaliana seeds
ing HS (Dhaubhadel et al. 2002). In addition to hsps, were grown in Petri-plates at 22°C under a 16 h day
we found genes involved in a variety of other physio length (80 |xE rrf2 s"1) on a nutrient medium [Murash
logical responses to also be up-regulated in EBR ige and Skoog (1962) salts, B5 vitamins, 1% sucrose]
treated seedlings (S. Dhaubhadel and P. Krishna, solidified with 1 % agar and amended with either 1 |iM
unpublished results). EBR or 0.01% ethanol (solvent for EBR). Seeds were
Drought, cold, and soil salinity are the other majorstratified (4°C) in the dark for 3 days immediately after
environmental stress factors that adversely affect plant plating to ensure uniform germination. B. napus seed
growth and productivity. Earlier studies have suglings were grown as described by Dhaubhadel et al.
gested the potential of BRs for increasing plant resis (1999).

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Planta (2007) 225:353-364 355

Stress treatments various time points during drought stress and quick
frozen. Cold treatments were given by transferring
21-day-old A. thaliana and 14-day-old B. napus seed
All experiments involving drought, cold, and HS were
lings to a growth chamber set at 2°C for a maximum
repeated three times. For drought stress, 21- and 14
day-old A. thaliana and B. napus seedlings, respecof 3 days. Plant tissue was collected at various time
points during the cold treatment. For HS, 21-day-old
tively, were transplanted into pots containing coarse
sand. Seedlings were allowed to reestablish growthA. thaliana seedlings grown at 22°C were exposed to
for 5 (A. thaliana) or 3 days (B. napus) and then 43°C
sub for varying lengths of time (1, 2, 3, or 4 h), and
jected to drought stress by withholding water forthen
up returned to 22°C to recover for a period of
to 96 h (A. thaliana) or 60 h (B. napus). Following
7 days. At this time, seedlings exposed to 3 h of HS
were scored as alive, dead, or recovering. Values in
drought stress, the seedlings were allowed to recover
Fig. 5b represent average percentages of 135 seed
by watering them regularly for the next 2 days. Seed
lings that survived and continued to grow werelings under the three categories. When required, plant
counted. Values represent average percentage oftissue
85 above the medium was collected immediately
A. thaliana (Fig. la) and 104 B. napus (Fig. lb) seed
after HS and recovery period and quick-frozen for
lings. Plant tissue above the sand was collected atRNA and protein isolation.

watered drought stress

J|#EBR

□ Control
■ EBR

72h 80h
■ 36h 48h

Hours after drought

Hours stress
after d

Fig. 1 Epibrassinolide enhances drought stress tolerance in A.80 h of drought stress, b B. napus seedlings grown in the ab
and
sence
thaliana and B. napus seedlings, a A. thaliana seedlings grown in (C) or presence of 1 (xM EBR (EBR) were transplanted
the absence (C) or presence of 1 |_iM EBR (EBR) were trans into pots containing sand. After 3 days, drought stress was given
planted into pots containing sand. After 5 days, drought stress
by withholding water for up to 48 h. Photograph of the plants was
was given by withholding water for up to 80 h. Photograph oftaken
the at 36 h of drought stress. The graph below indicates propor
plants was taken at 72 h of drought stress. The graph belowtion
indi of plants recovering after being watered at 36 and 48 h of
cates proportion of plants recovering after being watered at 72
drought stress

■ö Springer

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356 Planta (2007) 225:353-364

RNA isolation and northern blot analysis BNCBF5, 28 cycles for BNDREB, and 23 cycles for
actin (internal control). The following primers were
Total RNA was isolated from frozen plant tissue using used: DREB2A-F, 5'TGACGGTACTACTGTGGC
TRIzol reagent (Life Technologies, Burlington, ON, TGAG3'; DREB2A-R, 5'GTCGCCATTTAGGTC
USA). For northern blot analysis, 10 (ig of total RNA ACGTAG3'; BNCBF5-F, 5'CTTCTCTGAAATGAT
was separated on a denaturing formaldehyde agarose GGGCTC3'; BNCBF5-R, 5'CTCGTCCATATAA
gel and blotted onto a Biotrans+ membrane (ICN Bio AACCCATC3'; BNDREB-F, 5'GAGTATGAGTCT
medical, Aurora, OH, USA). Blots were hybridized CCGGTTACG3'; BNDREB-R, 5'GTTATGTCCG
with 32P-labelled cDNA fragments under the condi AAATGTACGG3'; Atactin-F, 5'TGCTCTTCCTCA
tions described previously (Krishna et al. 1995). Fol TGCTAT3'; Atactin-R, 5'ATCCTCCGATCCAGAC
lowing hybridization, the membranes were washed ACTG3'; Bnactin-F, 5'ATCACCATCGGAGCTGA
twice in 2x SSC with 1 % SDS at room temperature for G3'; Bnactin-R, 5'GAAGCATTTCCTGTGGACG3'.
10 min, twice in 0.5 x SSC with 0.5% SDS at 52°C for
15 min, and if required, twice in 0.1 x SSC with 0.1% Western blot analysis
SDS at 65°C for 10 min, and autoradiographed. The B.
napus hsp90-l (Krishna et al. 1995), SCE70 (Ko et al. Total cellular proteins from WT and mutant A. thaliana
1992), A. thaliana hsplOl (Schirmer et al. 1994), and seedlings were isolated as described previously (Dha
class II hspl7.6 cDNAs (provided by Dr E. Vierling) ubhadel et al. 1999). Proteins (20 ng) were separated on
were used to detect hsp90, hsp70, hsplOl, and class II 10% SDS-polyacrylamide gels and transferred onto
shsp transcripts, respectively. The blots were stripped nitrocellulose membranes by electroblotting using the
and re-hybridized with an 18S ribosomal DNA frag Trans-Blot Semi-Dry Electrophoretic Transfer Cell
ment to indicate RNA loading. The BN115 and BN28 (BioRad, Hercules, CA, USA). Hsps were detected by
cDNA clones (provided by Dr J. Singh) were used for sequential incubation with polyclonal antisera against
the detection of BN115 and BN28 transcripts, respec plant hsplOl at a dilution of 1:2,500 (provided by Dr E.
tively. cDNAs for BnPIPl (accession no. AFI 18382), Vierling), or antisera against hsp90 at a dilution of
BnD22 (accession no. X65637), Btg-26 (accession no. 1:5,000 (Krishna et al. 1997), followed by peroxidase
S77096), BNDREB (accession no. AF084185), and conjugated anti-rabbit IgG at a dilution of 1:5,000 and
BNCBF5 (accession no. AF499031) genes were chemiluminescent detection (ECL System, Amersham,
obtained by RT-PCR using RNA isolated from Baie d'Urfe, QC, Canada).
drought-stressed B. napus seedlings. Similarly, cDNAs
for rd29A (accession no. D13044), ERD10 (accession Germination assay
no. NM_180616), rd22 (accession no. D10703), dehy
drin (accession no. NM_127721), COR47 (accession Brassica napus seeds were germinated on the solidified
no. AB004872), DREB2A (accession no. NM_120623), nutrient medium described above, amended with
and CBF1 (accession no. U77378) genes were obtained either 1 or 2 |aM EBR, and 0, 50,100, 150, 200, 250, or
by RT-PCR using RNA isolated from drought- and 300 mM NaCl. Germination counts were taken at 24,
cold-stressed A. thaliana seedlings. The cDNAs were 48,72, and 96 h after imbibition. Nutrient medium con
cloned into a TA cloning vector and verified by taining 0.01% ethanol and various concentrations of
sequencing. NaCl served as control. Germination experiments were
repeated three times. Values in Fig. 4b represent the
RT-PCR analysis average germination percentages of 80 seedlings.

Seven micrograms of total RNA isolated from

drought-stressed A. thaliana, and B. napus seedlings Results

were reverse transcribed using the oligo (dT)18 primer

and Super Script First Strand Synthesis System for EBR increases drought tolerance in Arabidopsis
RT-PCR (Invitrogen, Carlsbad, CA, USA). PCR was thaliana and Brassica napus seedlings
carried out with an initial denaturation step of 94°C
for 4 min followed by various cycles of denaturation Arabidopsis thaliana and B. napus seedlings grown on
(30 s at 94°C), annealing (45 s at 56°C), and extension EBR and then transplanted to sand were subjected to
(1 min at 72°C). After the last cycle, a final extension drought stress as described in Sect. "Materials and
was carried out for 5 min at 72°C. PCR was per methods." These experiments were repeated three
formed for 30 cycles for DREB2A, 30 cycles for times. Visible morphological changes in response to

•Ö Spring er

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Planta (2007) 225:353-364 357

drought stress, such as leaf wilting, reduction in showed a circadian rhythm expression pattern at early
growth, and complete drying of some seedlings, were time points of stress when transcript levels were rela
frequently observed in untreated A. thaliana and B. tively lower. This was overcome by high expression of
napus seedlings, but were considerably reduced in transcripts at later time points. These experiments
EBR-treated seedlings (Fig. 1). In accordance with this were repeated at least two times and the changes
observation, the survival rate of EBR-treated seedlings described were reproducible.
was noticeably higher than the survival rate of In B. napus, transcripts of an aquaporin gene
untreated seedlings. About 80 and 28% of EBR BNPIP1, encoding a plasma membrane intrinsic pro
treated A. thaliana seedlings watered at 72 and 80 h tein (Gao et al. 1999), and a Kunitz proteinase inhibi
after drought stress, respectively, survived and contin tor gene BnD22 (Downing et al. 1992) were present at
ued to grow. In comparison, only 35 and 7% of the higher levels in EBR-treated seedlings, but transcripts
untreated seedlings survived (Fig. la). All of the of Btg-26, a gene encoding a putative aldehyde dehy
untreated seedlings were killed by 80-82 h of drought drogenase (Stroeher et al. 1995) and a dehydrin homo
stress, whereas EBR-treated seedlings remained alive log were at higher levels in untreated seedlings
past 90 h of drought treatment. (Fig. 2b). BN PI PI and Btg-26 were also expressed in
Brassica napus seedlings grown in the presence of the absence of drought stress, albeit at lower levels
EBR showed similar resilience to drought stress. About than during stress.
90 and 60% of EBR-treated B. napus seedlings watered Transcription factors of the CBF/DREB family in A.
at 36 and 48 h after drought stress, respectively, sur thaliana bind to ds-acting drought-responsive elements
vived. However, only 20 and 6% of the untreated seed (DRE) and regulate gene expression in response to cold,
lings survived (Fig. lb). All of the untreated seedlings drought, and salt stress (Shinozaki and Yamaguchi
were killed by 50-52 h of drought stress, whereas EBR Shinozaki 2000). We analyzed by RT-PCR the mRNA
treated seedlings remained alive past 60 h of drought levels of DREB2A, a transcription factor involved in
treatment. Together, these results demonstrate that drought-responsive gene expression. The expression of
EBR treatment increases tolerance to drought stress in DREB2A transcripts was comparable between
both A. thaliana, and B. napus, seedlings. untreated and EBR-treated A. thaliana seedlings during
drought (data not shown). Orthologues of the A. thali
EBR affects the expression of drought-responsive ana CBF/DREB genes have also been isolated from B.
genes in Arabidopsis thaliana and Brassica napus napus (Gao et al. 2002). BNCBF5 is highly related to A.
thaliana CBFl/DREBlb (Gao et al. 2002), and
Response to drought stress is a relatively well-charac BNDREB (accession no. AF084185) is closely related to
terized phenomenon in plants, which results in major BNCBF5 (89% amino acid identity). The transcript lev
reprogramming of gene expression (Ramanjulu and els of BNCBF5 and BNDREB were higher in EBR
Bartels 2002; Seki et al. 2002). We therefore compared treated B. napus seedlings than in untreated seedlings,
the expression of a subset of genes known to be regu both in the absence of stress and during drought stress
lated in response to drought stress, in EBR-treated and (five- and twofold, respectively, at 36 h; Fig. 2c).
untreated seedlings. Transcripts of rd29A and ERD10,
encoding late embryogenesis abundant proteins (Yam EBR affects the expression of cold-responsive genes
aguchi-Shinozaki and Shinozaki 1993; Kiyosue et al. in Arabidopsis thaliana and Brassica napus
1994), clearly accumulated to higher levels (two- to
threefold) in EBR-treated A. thaliana seedlings at ear The cold acclimation process is stimulated primarily by
lier time points of stress (up to approximately 60 h of exposure of plants to low nonfreezing temperatures,
stress), but at later time points the levels were compa resulting in several biochemical and physiological altera
rable between untreated and treated seedlings tions that allow plants to survive subsequent freezing
(Fig. 2a). Transcript levels of rd22, an ABA-dependent temperatures (Guy 1990; Thomashow 1993). During
dehydration-responsive gene (Iwasaki et al. 1995), cold acclimation, regulatory proteins and transcriptional
switched from being slightly elevated in EBR-treated activators, such as members of the CBF/DREB family,
seedlings at earlier time points to being higher in are rapidly induced in response to low temperature, fol
untreated seedlings at the later time point(s). The lowed by expression of target genes encoding enzymes
differences in the expression of these genes at 72 and or structural components that participate in direct pro
84 h, as well as of a dehydrin gene (accession no. tection of cells against cold stress (Thomashow 1998,
NM_127721) in the two sets of seedlings did not appear 1999). To study the effects of EBR on gene expression
significant. Interestingly, rd29A, ERD10, and rd22 during cold acclimation of A. thaliana and B. napus,

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 358 Planta (2007) 225:353-364

Fig. 2 Expression profiles of

drought-responsive
Oh 12 h 24 h 36 h 48 h 60 h 72genes
h 84 h in
untreated and EBR-treated
CECECECECECECECE
A. thaliana (a) and B. napus
(b, c) seedlings, a A. thaliana mm m 1WHNI rd29A
seedlings were exposed to
drought stress (described in .

m m <* • rd22
the legend of Fig. la) for vary
ing lengths of time. Transcript « mm •
ERD10
levels of rd29A, rd22, ERD10
and a dehydrin were analyzed
by northern blotting. The
■ mm Dehydrin

expression of 18S rRNA was

18s rRNA
analyzed to serve as a loading
control, b B. napus seedlings
were exposed to drought
stress (described in the legend
Oh 12 h 24 h 36 h Oh 12 h 24 h 36 h
of Fig. lb) for varying lengths
CECECECE CECECECE
of time. Transcript levels of
BNPIP1, BnD22, Btg-26 and
a dehydrin homolog were ana
lyzed by northern blotting.
The levels of 18S rRNA

served as a loading control,

c Transcript levels of the tran
scription factors BNCBF5 and actin
Dehydrin
BNDREB were analyzed by
homolog
RT-PCR. Actin transcript lev
els were assayed as controls 18s rRNA

seedlings growing in the absence and presence of EBR

were subjected to 2°C for up to 3 days. Transcripts of the
three structural genes tested—rd29A (Yamaguchi CBF1

Shinozaki and Shinozaki 1993), a BN115 (Weretilnyk rd29A

et al. 1993) homolog, and COR47 (Gilmour et al. 1992),
BN115
were observed at much higher levels in EBR-treated A. homolog
thaliana seedlings as compared with untreated seedlings COR47
• • • •
(Fig. 3). A slight difference in the transcript size of the
BN115 homolog between treated and untreated seed hsp90

lings was also consistently noted in replicate experi 18s rRNA

ments. In line with the expression patterns of the
structural genes, transcripts of CBF1, a cold-responsive Fig. 3 Expression profiles of cold-responsive genes in untreated
transcription factor (Stockinger et al. 1997), were and EBR-treated A. thaliana seedlings. Seedlings grown in the
expressed at slightly higher levels (up to twofold) in absence (C) or presence of 1 (iM EBR (E) were exposed to 2°C
for up to 3 days. Transcript levels of CBF1, rd29A, BN115 homo
EBR-treated A. thaliana seedlings at earlier time points
log, COR47 and hsp90 in A. thaliana were analyzed by northern
(3, 6, and 9 h) of cold treatment. Interestingly, there blotting. Expression of 18S rRNA served as a loading control
were no noticeable differences between untreated and
treated B. napus seedlings in the expression of regula
tory genes (BNCBF5 and BNDREB) and structural on saline soils is frequently observed. To determine the
genes [BN115 (Weretilnyk et al. 1993), BN28 (Boothe influence of EBR on salt-stress-induced inhibition of
et al. 1995), and hsp90 (Krishna et al. 1995)] that seed
weregermination, B. napus seeds were allowed to ger
studied (data not shown). minate on a nutrient medium containing 1 or 2 |iM
EBR and either 50, 100, 150, 200, 250, or 300 mM
EBR helps to overcome low germination rates NaCl. Germination counts were taken at 24,48, 72, and
of Brassica napus under high salt conditions 96 h after imbibition. Presence of EBR in the medium,
in particular at a concentration of 2 |xM, considerably
Since seed germination is a highly sensitive phase in
reduced the inhibitory effect of high salt on seed germi
nation as evidenced by increase in germination and
the life cycle of plants to salt stress, germination failure

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 Planta (2007) 225:353-364 359

early seedling etgrowth

al. 1999) and promotes (Fig. 4h
NaCl in the (Dhaubhadel et lacking
medium al. 2002). To d
tion and cotyledon for hsp synthesis, formation we extend
yledons were ana well WT seedlings formed and BR-def w
the medium. A At stress 96 treatment
h inthat the wou
age germination A. thaliana rate seedlingsof was 86, ide
in treatments lings in the absence or20
containing pre
respectively (Fig.
ing them 4b). to 43°C for In 1, com
2, 3, a
mination rate in the absence of EBR was 56, 25, the seedlings to recover at 2
and 3%, respectively. Since we do not aim to pursue seedlings exposed to 2, 3, and 4 h of HS exhibited
the effect of EBR on germination at the molecular increasing levels of bleaching, whereas in EBR-treated
seedlings, mild to severe bleaching was observed only
level, we restricted our observations to the crop plant
B. napus. with 4 h of HS (Fig. 5a). After the recovery period,
seedlings that had been subjected to 3 h of HS were
EBR increases the basic thermotolerance scored as dead, alive, or recovering. A markedly higher
of Arabidopsis thaliana seedlings proportion (>90%) of EBR-treated seedlings survived
(alive + recovering) the 3 h heat treatment than did the
We have shown previously that EBR enhances theuntreated
basic seedlings (22%; Fig. 5b). These results dem
thermotolerance of B. napus seedlings (Dhaubhadel onstrate that EBR enhances the basic thermotolerance

of A. thaliana seedlings, similar to its effect on B. napus

(Dhaubhadel et al. 1999).
a Control 1 nM 2 nM

Hsp synthesis is not compromised in BR-deficient

mutants

The expression of hsps was examined in untreated

EBR-treated WT as well as BR-deficient (det2-l
dwf4) seedlings before, during, and after heat tre
ment. In contrast to B. napus where a considerabl
increase in hsp accumulation occurs in EBR-trea
seedlings (Dhaubhadel et al. 1999), transcripts co
sponding to hsplOl, hsp90, hsp70, and class II s
were present at comparable levels in untreated an
EBR-treated WT A. thaliana seedlings (data no
shown). Similarly, the steady-state levels of hsplOl
hsp90 proteins, with the exception of 20 h recove
time point, were also comparable in both sets of W
seedlings (data not shown). Thus, the differences
the expression of hsps in untreated and EBR-treat
WT A. thaliana seedlings are subtle as compared to
B. napus.
Contrary to the expectation that BR-deficient
mutants may be compromised in hsp expression, tran
scripts of all four classes of hsps were present at high
levels in untreated det2-l and dwf4 mutant seedlings
Salt concentration (mM)
(Fig. 6a). Notably, untreated det2-l seedlings accumu
Fig. 4 Epibrassinolide helps to overcome low germination rates
lated hsp transcripts, including the heat-induced hsplOl
of B. napus under high salt conditions, a B. napus seeds were a
lowed to germinate on a nutrientand shsp, even in the absence of
medium any HS. HsplOl pro 250 mM
containing
NaCl and either 0.01% ethanoltein was also present in
(Control) or det2-l
1seedlings
or 2in [iMthe absence
EBR. Pho
tograph of the germinating seedlings
of HS (Fig. 6b).was
Overall,taken at
there was no 96 hthat
indication after imb
bition. b Percentage of seeds germinated on the nutrient medium
hsp expression was impaired in BR-deficient mutants
containing 0, 100, 150, 200, 250, and 300 mM NaCl and eithe
0.01% ethanol (Control) or 2 during
|iMHSEBR and recovery.
(EBR).EBR-treated mutant seedlings
Germinated seed
lings were counted at 96 h afterhad imbibition
lower levels of hsps as compared to their untreated

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 360 Planta (2007) 225:353-364

Control EBR

Fig. 5 Epibrassinolide treatment

graph incr
of the plants
b Seedlings
erance of A. thaliana exposed
seedlings, a A. th
nutrient medium recovering
containing either
after 0
7 da
1 |iM EBR (EBR) were exposed to 43°C

counterparts. This stress, as well likely derives

as confirming the ability of BR to protect
ence in the phenotypes against high salt stress. of untrea
(dwarf) and EBR-treated Marker genes with known responses mutant
to drought and
type restored by cold stress,exogenous
as well as genes believed or demonstrated
EBR
to enhance drought and cold tolerance of plants, were
studied for their expression patterns in EBR-treated
Discussion and untreated seedlings. We observed two- to fivefold
increases in the expression of some of these genes in
Though BRs have been implicated in drought, cold, response to EBR. It is to be noted that BR-regulated
and salt stress responses (reviewed in Krishna 2003a), genes on average show expression changes of less than
experimental conditions under which the stress allevi (Vert et al. 2005). Thus, although BR-induced
twofold
ating effects of BR can be studied in a reproducible changes in transcript levels are modest, BR-induced
manner at the morphological level have not been changes in plant phenotypes are clear, implying that
described in literature nor have there been any moleceither BR controls gene expression at other levels or
ular studies in this direction. Thus, there is a lacuna of that the changes affected by BR are together sufficient
convincing evidence of the ability of BR in modulatingfor inducing the phenotypes.
plant responses to a variety of environmental stresses. It was consistently noted that rd29A, ERD10, and
Our previous studies have established the role of BR inrd22 mRNAs accumulated to higher levels in EBR
modulating plant response to high temperature stresstreated A. thaliana seedlings at early time points of
(Dhaubhadel et al. 1999, 2002). In the present study we drought stress, but were either comparable in the two
provide the first molecular evidence confirming the sets seedlings or higher in untreated seedlings during
role of BR in plant responses to drought and cold later time points (Fig. 2a). The rd29A and ERD10

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 Planta (2007) 225:353-364 361

22°C HEAT SHOCK RECOVERY expression at the post-transcriptional level (Dhaubha

1 h 2 h 3 h 4 h 6h
del et al. 2002), it is possible that this effect is com
pounded with further increases at the protein level, at
CE CECECECECE
least, for some genes (this could not be tested due to
det2-1 * hsp101 unavailability of antibodies). The higher expression of
BNPIP1 and BnD22 transcripts in EBR-treated B.
• m • • • • • m » hsp90 napus seedlings (Fig. 2b) also correlated with increased
drought tolerance of these seedlings, which, in part,
m m m • ** m ««. hsp70
may derive from the proposed functions of these genes,
such as better distribution of water, and efficient
mmmm- - #
class II
shsp
defense against the large number of proteases pro
18S rRNA duced during stress conditions, respectively. A possible
role for BR in controlling aquaporin activities was pre
viously suggested in an indirect study (Morillon et al.
dwf4 - • hsp101
2001). Though further investigation is required, our
results establish a correlation between EBR and the
» 9 • m • • » hsp90
expression of one aquaporin gene BN PIP 1. In cont
itmHi • • « to BNPIP1 and BnD22, transcripts of Btg-26 and a
class II
dehydrin homolog clearly accumulated to higher levels
• #ili shsp in untreated B. napus seedlings. While at a first
instance the higher expression of a subset of genes in
18S rRNA
EBR-treated seedlings and that of another set of genes
in untreated seedlings may seem incongruous, the
22°C HEAT SHOCK RECOVERY
rationale likely lies in the functions of these genes, as
1 h 2 h 3 h 4 h 6h well as their induction pathways. If the untreated seed
lings are not as robust as EBR-treated seedlings to
CECECECE CECE
begin with and are unable to mount as rapid a stress
det2-1 hsp101 response as EBR-treated seedlings, then it is possible
that some stress-responsive genes, induced via another
hsp90
pathway, eventually accumulate to higher levels in
untreated seedlings, indicating a higher degree of stress
dwf4 hsp101
in these seedlings. Indeed, the early drought-induced
hsp90 phenotype in untreated seedlings and the delayed
drought-induced phenotype in EBR-treated seedlings,
Rubisco
correlate with the higher expression of Btg-26 and the
dehydrin homolog in the untreated seedlings.
Fig. 6 Expression profiles of hsp transcripts and proteins in A.
In response to low temperature, with the exception
thaliana mutant (det2-l and dwf4) seedlings during HS and recov
of hsp90, transcripts of all genes analyzed clearly accu
ery. a Seedlings grown in the absence (C) or presence of EBR (E)
mulated to higher levels in EBR-treated A. thaliana
were exposed to 43°C for 1,2,3, or 4 h. Seedlings heat stressed for
3 h were allowed to recover at 22°C for 6 h. Transcripts of hsplOl,
seedlings (Fig. 3). Transcripts of the BN115 homolog
hsp90, hsp70, and a class II shsp were analyzed by northern blot
and COR47 repeatedly showed slightly different mobil
ting. Expression of 18S rRNA was determined to serve as a load
ity in the two sets of seedlings. The exact difference in
ing control, b HsplOl and hsp90 levels in the aforementioned
the processing of the transcripts in the two sets of seed
seedlings were determined by western blotting. Coomassie blue
staining of ribulose-l,5-bisphosphate carboxylase/oxygenase
lings, and whether one type is more stable or better
(Rubisco) was used as a loading control
translated than the other, remains to be determined.
Flowever, the likelihood that EBR, directly or indi
genes encode a class of proteins that have molecularrectly, could affect gene expression through differential
chaperone-like functions, preventing protein aggregaprocessing of transcripts should be noted. Unlike in A.
tion during water stress (Goyal et al. 2005). The thaliana, the cold-regulated genes tested in B. napus
increase in the transcript levels of these genes at earlierwere expressed at more or less similar levels in
time points suggests that EBR-treated seedlings are untreated and EBR-treated seedlings (data not
likely positioned to tolerate stress better right from theshown). There could be several reasons for these
results. The effect of EBR on these genes may be
beginning. Due to the positive influence of BR on gene

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 362 Planta (2007) 225:353-364

apparent at the protein

seedlings level,
expressed hsp transcripts and proteins during or
cold tolerant than A.
HS and recovery, thaliana,
and in det2-l these were present even a
treatment may in the be
absence of needed to
any HS. Our results, together with a as
napus seedlings, previousor similar observation
a largerin the BR-deficient gen
studied. mutant cpd (Szekeres et al. 1996), suggest that BR defi
The expression of the structural genes in response to ciency itself is a form of cellular stress or a develop
stress must result from a combination of factors—the mental state that leads to activation of the hsp genes.
degree of stress experienced by the seedling depending Since hsps are also expressed in response to develop
on its fitness, and the effectiveness by which the mentalseed and other stress signals (Krishna 2003b), their
ling can respond to stress. Transcript levels of tran activation in the absence of stress in BR-deficient
scription factors can be taken as an indirect measure of
mutants versus in response to HS in WT as well as BR
the degree by which the stress response will deficient be mutants, must occur via different pathway
launched. In compliance with the higher stress toler What may be the role of BR then? Our data coll
ance of EBR-treated B. napus seedlings, transcriptstively of suggest that the effect of EBR on plant stre
the transcription factors BNCBF5 and BNDREB were response is not comparable to a switch being turned o
present at higher levels in these seedlings, both inor theoff, but rather that EBR augments plant response
absence of drought stress and under drought condi to heat and other stresses, resulting in higher plant t
tions (Fig. 2c). Similarly, the expression of CBF1 erance to these stresses. In BR-deficient mutants, phy
mRNA in response to cold stress was higher in EBR iological or developmental conditions arising from BR
treated A. thaliana seedlings (Fig. 3). Higher expres deficiency may trigger, via other hormones and sign
sion of transcription factors involved in activatinging thecomponents, the expression of the same category of
CBF regulon, which in turn protects plants from
stress genes whose expression in WT plants is au
drought and cold stresses, supports the idea that mented
EBR by BR. It would be interesting to know wh
treated seedlings are better primed than untreated other stress genes are expressed in BR-deficient
seedlings to respond to stress. Transcript levels of mutants
one under normal growing conditions.
transcription factor included in our study, DREB2A, In conclusion, the results of the present study dem
were comparable between EBR-treated and untreated onstrate that EBR enhances tolerance of seedlings to
A. thaliana seedlings with, or without, drought stress
variety of abiotic stresses, and that this effect involv
(data not shown). This is not surprising given that changes in the expression of genes encoding both str
DREB2 proteins require post-translational activation tural and regulatory proteins. Future characterizatio
(Liu et al. 1998). of global gene expression changes will extend ou
Continuing evidence that EBR-treated WT A. thali understanding of how EBR improves tolerance
ana (present study) and B. napus (Dhaubhadel et al.
plants to a wide range of environmental stresses.
1999) seedlings are more thermotolerant than
untreated seedlings unequivocally establishes the role Acknowledgments This research was supported by the Natur
of EBR in HS responses of these plants. However, Sciences and Engineering Research Council of Canada and C
unlike B. napus where considerable differences were tech Inc. We thank Drs J. Li and E. Vierling for providing det2-
mutant seeds and anti-hsplOl antibody, respectively, and Debb
detected between the two sets of seedlings (Dhaubha Dong for excellent technical help.
del et al. 1999), hsp transcript and protein levels in
EBR-treated WT A. thaliana seedlings during HS
were, for the most part, comparable with the levels in
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