Mol Breeding (2016)36:76
DOI 10.1007/s11032-016-0495-6
Stress-inducible overexpression of glyoxalase I is preferable
to its constitutive overexpression for abiotic stress tolerance
in transgenic Brassica juncea
Ravi Rajwanshi . Deepak Kumar .
Mohd Aslam Yusuf . Suchandra DebRoy .
Neera Bhalla Sarin
Received: 5 May 2015 / Accepted: 23 May 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract The glyoxalase system catalyzes the conversion of cytotoxic methylglyoxal to D-lactate via the
intermediate S-D-lactoylglutathione. It comprises two
enzymes, Glyoxalase I (Gly I) and Glyoxalase II (Gly
II), and reduced glutathione which acts as a cofactor
by anchoring the substrates in the active sites of the
two enzymes. The overexpression of both Gly I and
Gly II, either alone or in combination, has earlier been
reported to confer tolerance to multiple abiotic
stresses. In the present study, we sought to evaluate
the consequences of constitutive and stress-induced
overexpression of Gly I on the performance and
Electronic supplementary material The online version of
this article (doi:10.1007/s11032-016-0495-6) contains supplementary material, which is available to authorized users.
R. Rajwanshi D. Kumar M. A. Yusuf S. DebRoy N. B. Sarin (&)
School of Life Sciences, Jawaharlal Nehru University,
New Delhi 110067, India
e-mail: [email protected]
R. Rajwanshi
Department of Biotechnology, Assam University, Silchar,
Assam 788011, India
D. Kumar
Department of Bioscience and Biotechnology, Banasthali
University, Banasthali, Rajasthan 304022, India
M. A. Yusuf
Department of Bioengineering, Integral University,
Dasauli, Kursi Road, Lucknow 226026, India
productivity of plants. Towards this end, several Gly I
transgenic Brassica juncea lines (designated as R and
S lines) were generated in which the glyoxalase I (gly
I) gene was expressed under the control of either a
stress-inducible rd29A promoter or a constitutive
CaMV 35S promoter. Both the R and S lines showed
enhanced tolerance to salinity, heavy metal, and
drought stress when compared to untransformed
control plants. However, the S lines showed yield
penalty under non-stress conditions while no such
negative effect was observed in the R lines. Our results
indicate that the overexpression of the gly I gene under
the control of stress-inducible rd29A promoter is a
better option for improving salt, drought and heavy
metal stress tolerance in transgenic plants.
Keywords Brassica juncea Glyoxalase I CaMV
35S promoter rd29A promoter Abiotic stress Transgenic
Introduction
Brassica juncea (Indian mustard) is an important
oilseed crop belonging to the Cruciferae family. It is a
high biomass crop and is also helpful to phytoremediate heavy metals in polluted soils (Ebbs and Kochian
1998; Lin et al. 2004). Amongst the various Brassica
species, amphidiploids (B. napus, B. juncea, and B.
carinata) have been categorized as relatively salt
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tolerant in comparison with the diploids (B. campestris, and B. nigra) species. This inter- and intraspecific variation in Brassicas can be exploited
through selection and breeding or genetic transformation by a desired gene of interest for enhancing the
stress tolerance of the crops. Out of the few options
available, the Glyoxalase system is one of the potential
candidates that can play a crucial role in providing
enhanced abiotic stress tolerance in genetically modified crops (Kaur et al. 2014). The Glyoxalase system
is vital for many biological functions and has long
been known in animal systems (Thornalley 1990). It is
ubiquitous in nature and plays an important role
throughout the biological life cycle such as in the
regulation of cell division and proliferation, protection
against oxoaldehyde toxicity, and microtubule
assembly.
Glyoxalase I (EC 4.4.1.5) catalyzes the isomerization of hemithioacetal, which is formed by a nonenzymatic reaction between reduced glutathione
(GSH) and methylglyoxal (MG), to S-D-lactoylglutathione, whereas Glyoxalase II (EC 3.1.2.6) catalyses
the hydrolysis of S-2-hydroxyacylglutathione derivatives to GSH and D-lactate (Jagt 1988; Uotila 1989;
Thornalley 1990). Earlier studies have shown dosedependent upregulation of B. juncea Gly I activity in
response to certain abiotic stresses (Veena et al. 1999).
Esparteo et al. (1995) reported that in tomato, Gly I
was upregulated in response to salt stress. Previous
studies have shown enhanced tolerance to various
abiotic stresses viz. NaCl, methylglyoxal (MG),
mannitol, and H2O2 by overexpression of either gly I
in tobacco, Arabidopsis, rice, and blackgram or gly II
in tobacco, rice, and B. juncea (for a recent review, see
Kaur et al. 2014). Gene pyramiding of complete
glyoxalase pathway enzymes further enhanced the
tolerance against salinity and heavy metal in tobacco
and tomato (Singla-Pareek et al. 2003; Alvarez
Viveros et al. 2013). Recently, Mustafiz et al. (2014)
reported the heterologous expression of Gly I
(OsGLYI-11.2) from O. sativa which requires Ni2?
as a cofactor for its activity showed improved
adaptation to various abiotic stresses caused by
increased scavenging of MG, lower Na?/K? ratio,
and maintenance of reduced glutathione levels in
Nicotiana tabacum.
Apart from choosing a right candidate gene, the use
of an appropriate promoter regulating the expression
of a transgene is necessary to generate transgenic
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Mol Breeding (2016)36:76
plants with greater tolerance to abiotic stress without
compromising on the yield. The cauliflower mosaic
virus (CaMV) 35S promoter is the most commonly
used promoter to drive expression of transgenes in
plants (Odell et al. 1985). With special reference to
glyoxalase pathway, most of the studies were performed using the constitutive CaMV 35S promoter
regulating the expression of glyoxalase gene(s) for
development of transgenic plants (Singla-Pareek et al.
2003, 2006; Yadav et al. 2005a, b; Saxena et al. 2011;
Mustafiz et al. 2014). Bhomkar et al. (2008) had shown
the efficacy of Bj gly I expression under the control of
constitutive cestrum yellow leaf curling virus
(CmYLCV) promoter in blackgram. Recently, Mustafiz et al. (2014) reported that the OsGLYI-11.2 under
the influence of CaMV 35S promoter improves the
adaptation to various abiotic stresses due to increased
scavenging of MG, maintenance of reduced glutathione levels, and lower Na?/K? ratio in tobacco.
Although previous studies have reported the best
performance of constitutive promoters in regulating
the glyoxalase pathway gene(s) during abiotic stress
conditions, data regarding the performance of the
same during unstressed conditions are missing. A few
studies have been conducted using stress-inducible
‘‘responsive to desiccation’’ (rd29A) gene promoter to
regulate the expression of gly I gene in transgenic
plants (Rajwanshi et al. 2007; Roy et al. 2008).
Previous studies have shown that rd29A and rd29B
genes are induced under high temperature, high salt,
and drought or with exogenous abscisic acid (ABA)
treatment due to the presence of drought-responsive
element (DRE) and ABA-responsive element (ABRE)
(Yamaguchi-Shinozaki and Shinozaki 1993, 1994;
Bihmidine et al. 2013). Kasuga et al. (2004) showed
superior performance of rd29A as compared to CaMV
35S promoter in driving the expression of Arabidopsis
DREB1 gene for salt tolerance in tobacco. Also, the
use of rd29A promoter for the overexpression of the
dehydration responsive binding factor protein
(DREB1A) in transgenic Arabidopsis reduced the
negative effects on plant development, which included
stunted growth and delayed flowering (Kasuga et al.
1999). Previous studies have shown adverse effects
and undesirable phenotypes like stunted growth,
smaller leaves, delayed flowering, and reduction or
lack of tuber production under normal growth conditions due to constitutive overexpression of CBF genes
in plants (Gilmour et al. 2000; Hsieh et al. 2002a, b;
Mol Breeding (2016)36:76
Kasuga et al. 2004; Benedict et al. 2006). According to
Wang et al. (2005), constitutive overexpression of the
CBF gene may reduce the pool of necessary transcriptional machinery needed for tuberization and,
thereby, may result in stunted growth and delayed
tuberization in transgenic potato. On the contrary,
stress-inducible rd29A promoter had shown better
regulation and expression of the transgenes by significantly introducing the desired stress tolerance without
negatively effecting the agronomically important
traits in wheat (Pellegrineschi et al. 2004), potato
(Celebi-Toprak et al. 2005; Pino et al. 2007; Behnam
et al. 2007), and mulberry (Das et al. 2011). However,
leaky and improper timing of expression of the
transgene regulated by rd29A promoter may not be
beneficial and might result in agronomical penalties
(Karim et al. 2007). In the present study, we evaluated
the efficacy of gly I gene expression driven by CaMV
35S and rd29A promoters by comparative analysis of
the performance of transgenic B. juncea plants under
non-stressed or abiotic stress conditions.
Materials and methods
Vector constructs used for B. juncea
transformation
The CaMV 35S:gly I construct was made using the gly
I cDNA from B. juncea (Bj gly I, Accession No.
Y13239; Veena et al. 1999) which was cloned in a
pBI121 binary vector (Clontech) to give rise to pBI-S1,
where both the gly I and the reporter genes gfp:gusA
were under the control of separate CaMV 35S
promoters and hpt II was the selectable marker
(Fig. 1a). For rd29A:gly I construct, binary plasmid
pCAMBIA2301 (pC2301) was used as the cloning
backbone. It contains the CaMV 35S promoter-driven
neomycin phosphotransferase II (npt II) gene, conferring resistance against kanamycin to the plants. The
cloning strategy for the constructs has been described
in a previous report (Roy et al. 2008; Fig. 1b).
Plant material and B. juncea transformation
Healthy seeds of B. juncea L. cv. Varuna were used as
the source of explants for transformation experiments.
The recombinant plasmids were introduced into
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Agrobacterium tumefaciens (GV 3101) by freeze–
thaw method (H}ofgen and Willmitzer 1988). Fiveday-old hypocotyl explants were transformed with
either CaMV 35S:gly I or rd29A:gly I gene constructs.
The transformants were selected on MS medium
containing the appropriate antibiotics, i.e., hygromycin (20 mg l-1) or kanamycin (40 mg l-1), respectively. Regenerated plantlets were put on semi-solid
MS I2 (MS supplemented with 2 mg l-1 IBA) rooting
medium. The plantlets were later transferred to
agropeat for hardening and finally transferred to the
green house (Pental et al. 1993; Rajwanshi and Sarin
2013). The transgenic lines derived from transformation with CaMV 35S:gly I and rd29A:gly I gene
constructs were designated as S and R, respectively.
PCR and Southern blot analysis
Putative transformants and UC plants were screened
for the presence of the transgene by PCR using their
genomic DNA as template. PCR amplifications of gly
I, CaMV 35S:gly I, rd29A:gly I, and npt II genes were
performed using respective gene-specific primer sets
(Supplementary Table 1). The stable integration of
transgene in the PCR positive lines was confirmed by
Southern blot analysis. About 20 lg of genomic DNA
was digested with Xba I enzyme, blotted onto a
positively charged nylon membrane using the capillary transfer method (Sambrook et al. 1989), and
probed using a P32 radiolabeled gly I cDNA according
to the standard protocol.
Northern blot analysis
Total RNA from leaf tissue was isolated using TRIzol
reagent (Ambion, CA, USA) as per the vendor’s
protocol. About 20 lg of total RNA was fractionated
on 1.5 % formaldehyde denaturing agarose gel
according to Sambrook et al. (1989). The RNA was
transferred onto a positively charged nylon membrane
by capillary transfer in 20X SSC buffer for 18–20 h
and cross-linked by UV irradiation at 254 nm for
2 min. The RNA blots were probed with radiolabeled
gly I cDNA. The blots were scanned using a
phosphorimager, and the relative transcript abundance
was calculated using the Image Gauge software (Fuji
Photo film Co. Ltd., Japan).
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Mol Breeding (2016)36:76
LB
a
Pst I
35S Term.
hpt II
CaMV 35S
CaMV 35S
RB
Pst I
Xba I
Kpn I
gly I
35S Term.
GFP-GUS
CaMV 35S
35S Term.
580 bp
1.2 Kb
Eco RI
LB
b
35S Term. lox npt II
CaMV 35S
hsp
cre
RB
Eco RI Xba I
Bam HI
lox
35S Term.
gly I
rd29A
Nos
4006 bp (4.0 Kb)
M UC S-10 S-12 S-15 S-18 S-22 S-24 S-33 S-36 S-42
M
UC
R-6
R-12
R-14 R-16
R-17
R-18 R-19 R-27
23130 bp
9416 bp
4361 bp
2027 bp
564 bp
d
c
Fig. 1 Diagrammatic representation of construct CaMV
35S:gly I (a) and rd29A:gly I (b) used for the transformation
of B. juncea. Southern blot of DNA isolated from T1 transgenic
lines of B. juncea transformed with CaMV 35S:gly I (c) and
rd29A:gly I (d) using radiolabeled gly I cDNA probe M denotes
DNA molecular weight marker. The lane UC is for digested
DNA from the untransformed B. juncea
Quantitative real-time reverse transcriptase PCR
analysis
using a QuantiFast SYBR Green PCR master mix
(Qiagen GmbH) according to manufacturer’s instruction. Reverse transcription reaction was carried out at
44 °C for 60 min followed by 92 °C for 10 min. The
subsequent PCRs were carried out at 95 °C for 5 min
followed by 40 cycles of 95 °C for 15 s and 60 °C for
30 s each, according to the method described previously by Katiyar et al. (2012). Comparative threshold
(Ct) values were normalized to actin control and
compared to obtain the relative expression levels as
explained by Katiyar et al. (2012).
Total RNA from leaf tissue was treated with DNase I
(Fermentas, USA). Random hexamer primers and
Superscript III Kit (Invitrogen) were used to generate
first-strand cDNA according to manufacturer’s protocol. This cDNA (5 ng) was used as template in a
reverse transcriptase reaction mixture (20 ll). Genespecific primers were designed using IDT PrimerQuest (http://www.idtdna.com/scitools/applications/
primerquest/default.aspx). Bj actin primers were
used as an internal control having the amplicon size of
100 bp. Bj gly I primers showing the amplicon size of
128 bp were used to test the samples using an ABI
Prism 7700 sequence detector (Applied Biosystems,
USA). The sequences of specific primers used for
amplification of Bj gly I and Bj actin are given in
Supplementary Table 1. The PCR was performed
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Western blot analysis
The polyclonal Gly I antibody has been raised,
following the protocol of Harlow and Lane (1988).
Total protein was extracted from the leaf tissue
following the procedure detailed in Kumar et al.
(2013). Estimation of protein concentration was done
Mol Breeding (2016)36:76
according to Bradford method (Bradford 1976).
Western blotting was done according to the method
described by Towbin et al. (1979). The immunoblot
was probed with HRP-labelled anti-rabbit secondary
antibody and developed using diaminobenzidine.
Leaf disc senescence assay for tolerance against
methylglyoxal (MG), heavy metal (ZnCl2),
mannitol, and salinity stress (NaCl)
Healthy and fully expanded fourth leaves from T1
generation of UC and transgenic plants (60-day-old)
were briefly washed in deionized water. Leaf discs
(1 cm diameter) were punched out and floated on
10 ml solution of MG (5 or 10 mM for 3 days), ZnCl2
(20 mM for 5 days), mannitol, or NaCl (400, 600 or
800 mM for 5 days). Discs floated on sterile distilled
water served as the experimental control (Fan et al.
1997). The treatment was carried out under continuous
fluorescent white light (fluence density of
50 lmol m-2 s-1) at 25 ± 2 °C. The effects of the
treatment on leaf discs was assessed by observing
phenotypic changes and measurement of chlorophyll
content as described by Arnon (1949).
Measurement of glyoxalase I activity
The specific activity of Gly I was assayed in healthy
and fully expanded leaves from UC and transgenic
plants (60 days old) of similar age according to the
protocol described by Ramaswamy et al. (1983). The
standard enzyme assay mixture contained 0.1 M
phosphate-buffered saline (pH *7.0), 3.5 mM
methylglyoxal, 1.7 mM GSH, 16 mM MgS04, and
crude protein extract in a final volume of 1 ml. The
assay mixture prior to addition of the crude extract was
incubated for 7 min at room temperature to allow nonenzymatic formation of hemithioacetal from methylglyoxal and GSH. The Gly I activity was measured
spectrophotometrically as a function of thioester (S-Dlactoylglutathione) by measuring the rate of change in
absorbance at 240 nm (UV-260, Shimadzu). The
molar absorption coefficient of the thioester (S-Dlactoylglutathione) at 240 nm is 3370 m-1 cm-1. The
specific activity of the enzyme was expressed in units
mg-1 of protein.
Page 5 of 15
76
Measurement of MG content
MG was extracted from leaf tissue (0.3 g) of UC and
transgenic plants (60 days old) by homogenizing in
3 ml of 0.5 M perchloric acid. MG levels were
measured in this extract following the protocol
described by Yadav et al. (2005a). The assay mixture
(1 ml) contained 100 ll of 5 M perchloric acid,
250 ll of 7.2 mM 1,2-diaminobenzene, and 650 ll
of the sample extract (which was added last), and the
absorbance of the derivative was read at 336 nm. The
concentration of MG was calculated from a standard
curve and expressed in terms of lM g-1 fresh weight
(FW).
Measurement of MDA content for lipid
peroxidation
Lipid peroxidation was measured in terms of malondialdehyde (MDA) content by the reaction with
thiobarbituric acid (TBA) according to Heath and
Packer (1968). The Brassica leaves of UC and
transgenic plants (60 days old) were ground to a fine
powder in liquid nitrogen. Three millilitre of 10 %
trichloroacetic acid was added to 0.2 g of the powder
and left at 4 °C overnight. After centrifugation at
10009g for 20 min, the supernatant was transferred to
a new tube for measurements. To 2 ml of the
supernatant, 2 ml of 0.6 % TBA was added. The
mixture was vortexed thoroughly, heated in boiling
water for 15 min, cooled immediately, and centrifuged. Absorbance values of the supernatant were
detected at wavelengths of 532 and 450 nm. The
formula for the calculation of MDA content was:
MDA content (lmol/l) = 6.45 9 OD532 - 0.56 9
OD450.
Measurements of the fast chlorophyll
a fluorescence transients
Chlorophyll a fluorescence measurements were taken
as explained by Yusuf et al. (2010) on intact young
leaves of transgenic as well as UC plants (60 days old)
adapted to dark for 1 h. Three plants from each plant
type and treatment were used, and six measurements
per plant were taken (3 biological replicates and 6
technical replicates). Photosynthetic activity was
measured as photochemical yield (Fv/Fm), which
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represents the maximum quantum yield of photosystem II, by recording the chlorophyll fluorescence using
a portable Handy-PEA fluorimeter (Plant Efficiency
Analyser, Hansatech Instruments Ltd., King’s Lynn
Norfolk, PE 30 4NE, UK). Measurements were taken
at room temperature (25 °C) using saturating pulse of
white light (8000 lmol/m2 s-1 for 0.8 s).
Relative water content (RWC) measurement
The RWC was measured in the leaves of the UC and
transgenic plants (60 days old) in the same developmental stage (Schonfeld et al. 1988). The fresh weight
(FW) of the leaves was taken immediately after
excision. Turgid weight (TW) was measured by soaking
the fresh leaves in distilled water for 12 h at 25 °C until
they were fully saturated and quickly blotting them dry
before weighing. The dry weight (DW) was obtained
after oven drying the leaf samples for 72 h at 80 °C. The
RWC was determined using the equation:
RWC = (FW - DW) 9 100/(TW - DW).
Assessment of transgenic plants for abiotic stress
tolerance
For salt, heavy metal, and drought stress treatment,
21-day-old seedlings from UC and transgenic B. juncea
plants (T1 generation) were transferred to earthen pots
and subjected to different abiotic stresses by irrigating
them with NaCl (200 mM), ZnCl2 (5 mM), or mannitol
(200 mM) starting 2 weeks after transfer until the
flowering stage was reached. The plants were grown in
1:1 mixture of garden soil and agropeat in the green house
under controlled temperature of 25 ± 2 °C, 60 % relative humidity and 16-h light/8-h dark photoperiod with
light intensity of 150 lmol/m2 s-1. Phenotypic changes
were determined by the measurement of shoot/root
length, seed weight, and relative water content (RWC) in
the transgenic as well as the UC plants. Various
physiological parameters were measured for both
stressed and unstressed plants before harvesting.
Statistical analysis
The data collected were expressed as average ± standard deviation of the mean of three independent
replicates for every data set and analysed using
Student’s t test. Significance was defined as p \ 0.05.
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Mol Breeding (2016)36:76
Results
Generation of transgenic B. juncea plants
and confirmation of transgene integration
Nine putative transgenic lines of CaMV 35S:gly I (S10, S-12, S-15, S-18, S-22, S-24, S-33, S-36, and S-42)
and eight of rd29A:gly I (R-6, R-12, R-14, R-16, R-17,
R-18, R-19, and R-27) transgenic B. juncea were
found to be PCR positive using forward primer of
CaMV 35S or rd29A promoter and reverse primer of
gly I, respectively (data not shown). All S and R lines
were PCR positive for gly I gene, whereas the latter
were also PCR positive for npt II gene when tested
with gene-specific primers (Supplementary Table 1).
Nine and 8 independent transgenic lines, respectively,
of B. juncea transformed with CaMV 35S:gly I (S-10,
S-12, S-15, S-18, S-22, S-24, S-33, S-36, and S-42)
and rd29A:gly I (R-6, R-12, R-14, R-16, R-17, R-18,
R-19, and R-27) were confirmed by Southern blot
analysis. Multiple (2–3) copies of gly I gene were
found to be integrated in both CaMV 35S:gly I
(Fig. 1c) and rd29A:gly I (Fig. 1d) transgenic
plants.
Expression analysis of the gly I gene in rd29A:gly I
and CaMV 35S:gly I transgenic B. juncea
under abiotic stress and non-stress condition
Six independent lines of rd29A:gly I (R-6, R-12,
R-16, R-17, R-18, and R-27) and CaMV 35S:gly I
(S-12, S-22, S-24, S-33, S-36, and S-42) transgenic
B. juncea were analysed to study the expression of
gly I gene under the influence of stress-inducible
and constitutive promoters under non-stress condition. Results from Northern blot analysis showed
the overexpression of the gly I gene under the
influence of constitutive CaMV 35S promoter under
non-stressed condition (Fig. 2a). On the contrary,
lack of expression or very low expression of the gly
I gene driven by the stress-inducible rd29A promoter was seen under non-stressed condition
(Fig. 2a). qRT-PCR analysis showed the expression
pattern of rd29A:gly I and CaMV 35S:gly I (data
for respective representative lines R-16 and S-42
shown in the figure)-transformed B. juncea under
different abiotic stress conditions. Results confirmed
the induction of the gly I gene driven by the rd29A
Mol Breeding (2016)36:76
R-27
R-18
R-17 R-16
R-12
R-6
S-12
UC
S-22
S-24
76
S-33
S-36
S-42
Gly I
rRNA
1
Gly I Transcript Level
a
2
9.5
8.5
7.5
6.5
5.5
4.5
3.5
2.5
1.5
0.5
1
3
2
4
3
6
5
4
7
5
9
8
6
7
8
10
9
10
11
11
12
13
12
13
rd29A gly I/CONTROL/35S gly I
b
7
UC
S-42
R-16
6
Gly I expression level
(Fold Change)
Fig. 2 a (Upper panel)
Northern blot showing the
glyoxalase I transcript
abundance in the total RNA
of untransformed control
(UC), CaMV 35S:gly I and
rd29A:gly I transformed B.
juncea under non-stress
condition. a (Lower panel)
Quantitation of the relative
glyoxalase I transcript
abundance based on the
densitometry of the signals
obtained in the Northern blot
(the numbers on the X-axis
correspond to the lanes
shown in upper panel of a).
b qRT-PCR analysis
showing fold change in gly I
expression in different B.
juncea lines under nonstress and stress conditions
Page 7 of 15
5
4
3
2
1
0
Water
promoter during NaCl (200 mM)-, mannitol
(200 mM)-, and ZnCl2 (5 mM)-induced stress conditions. The constitutive expression of gly I gene
was also observed in S-42 line under non-stressed
condition (Fig. 2b). Western blot analysis of the
representative lines of CaMV 35S:gly I (S-12, S-22,
S-24, S-33, S-36 and S-42) employing polyclonal
antibodies raised against BjGly I showed the presence of a single prominent band of *27 kDa,
corresponding to the expected size of the transgene,
indicating that the transgene was being expressed
constitutively in CaMV 35S:gly I-transformed B.
juncea (Fig. 3a). Induced expression of Gly I protein
was observed under NaCl stress condition in the
rd29A:gly I transgenic plants (Fig. 3b). Very low
expression was also observed in the UC plants under
NaCl stress (Fig. 3a, b). No Gly I protein was
expressed in the rd29A-gly I-transformed B. juncea
lines (R-6, R-12, R-16, R-17, R-18 and R-27) under
non-stress conditions (Fig. 3c).
NaCl (200mM)
UC
S-12
Mannitol (200 mM)
S-22
S-24
S-33
ZnCl2 (5mM)
S-36
S-42
a
BjGly I (27 kDa)
Ponceau
UC
R-6
R-12
R-16
R-17
R-18
R-27
b
BjGly I (27 kDa)
Ponceau
UC
c
R-6
R-12
R-16
R-17
R-18
R-27
BjGly I (27 kDa)
Ponceau
Fig. 3 Western blot analysis of Gly I expression in untransformed control (UC), transgenic lines of B. juncea transformed
with CaMV 35S:gly I (a) and rd29A:gly I grown under stress
condition (b) rd29A:gly I transgenics grown under non-stress
condition. c Blots were hybridized with BjGly I polyclonal
antibodies followed by horseradish peroxidase-conjugated antirabbit secondary antibody and detected chromogenically using
diaminobenzidine. Lower panel in each figure shows the
ponceau-stained membrane after protein transfer for checking
the equal loading
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Mol Breeding (2016)36:76
b
a
Glyoxalase I activity
(U/mg protein)
14
UC
12
S-12
S-42
R-16
R-18
MG (µmol/g FW)
76
10
8
6
4
2
0
Water
200 mM NaCl
200 mM
Mannitol
S-12
S-42
UC
12
S-12
S-42
R-16
R-18
10
8
6
4
2
0
Water
e
200 mM NaCl 200 mM Mannitol
UC
100
S-12
S-42
R-16
5 mM ZnCl2
R-18
250
200
150
100
50
200 mM NaCl
d
14
R-16
300
Water
Fv/Fm
MDA content (µmol/g FW)
UC
350
0
5 mM ZnCl2
c
400
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
UC
Water
S-12
200 mM NaCl
200 mM
Mannitol
S-42
R-16
200 mM
Mannitol
5 mM ZnCl2
R-18
5 mM ZnCl2
R-18
RWC (%)
80
60
40
20
0
Water
200 mM NaCl
200 mM
Mannitol
5 mM ZnCl2
Fig. 4 Gly I activity (a), methylglyoxal content (b), malondialdehyde content (c), photosynthetic efficiency (measured as Fv/
Fm) (d), and relative water content (e) in transgenic B. juncea
transformed with CaMV 35S:gly I (lines S-12 and S-42),
rd29A:gly I (lines R-16 and R-18), and untransformed control
(UC) plants. The standard deviation (±SD) is indicated by
vertical bars in each graph (n = 3)
Transgenic B. juncea plants transformed
with rd29A:gly I and CaMV 35S:gly I resist toxic
levels of MG and tolerate NaCl, mannitol,
and ZnCl2 stress
plants tolerated stress up to 10 mM of MG (as
observed after 3 days) and 20 mM ZnCl2, 800 mM
NaCl and mannitol stress (as observed after 5 days),
while those from the UC bleached on the day of
observation even at lower concentration of stress
causing agents. The leaf discs from all the transgenic plants were greener as compared to the leaf
discs from the UC plants. Measurement of chlorophyll content in the leaf discs of these plants after 3
and 5 days (as per the treatment) further confirmed
the observed delay in senescence (Fig. 4a–d, right
panels). The chlorophyll content in the leaf discs
from transgenic plants was higher than UC by 1.14-,
1.96-, and 5.32-fold (line S-42), and by 1.16-, 1.99-,
and 5.4-fold (line R-16) at 400, 600, and 800 mM
NaCl, respectively (Supplementary Figure 1a, right
panel). On exposing the leaf discs to 400, 600, and
Leaf disc senescence assays
Leaf disc senescence assays were performed to
assess the effect of overexpression of the gly I gene
under the influence of the CaMV 35S promoter as
well as rd29A promoter in conferring tolerance
against abiotic stresses induced by NaCl, mannitol,
MG, and ZnCl2. Delay in senescence was observed
in the leaf discs of T1 transgenic plants as compared
to the UC plants at different concentrations of NaCl,
mannitol, MG, and ZnCl2 (Supplementary Figure 1a–d, left panels). The discs from the transgenic
123
Mol Breeding (2016)36:76
800 mM mannitol solution, the chlorophyll content
in the leaf discs from the transgenic plants was
higher than UC by 1.06-, 1.11-, and 1.2-fold (line
S-42), and by 1.15-, 1.57-, and 4.08-fold (line R-16;
Supplementary Figure 1b, right panel). On floating
the leaf discs on 20 mM ZnCl2, line S-42 and R-16
showed 4.5- and 5.0 fold higher chlorophyll content
compared to the UC (Supplementary Figure 1c,
right panel). On 5 and 10 mM MG, chlorophyll
content in the former case was 2.73- and 2.8-fold
higher in the transgenic lines S-42 and R-16,
respectively, as compared to the UC, whereas in
the latter treatment it was 3.05- and 3.75-fold higher
in transgenic lines S-42 and R-16 compared to the
UC plants (Supplementary Figure 1d, right panel).
Enhancement of glyoxalase I activity in Gly I
overexpressing transgenic plants corresponds
with decreased methylglyoxal content and lipid
peroxidation as well as improved photosynthetic
efficiency
To gain more insight into the role of Gly I in providing
tolerance to stress, Gly I enzyme activity was
measured in mature leaves of UC and transgenic
plants. The enzyme activity in the transgenic plants
transformed with CaMV 35S:gly I was 5.0- and 4.0fold higher (lines S-12 and S-42), whereas in the plants
transformed with rd29A:gly I it was 1.42- and 1.2-fold
higher (lines R-16 and R-18) relative to the UC plants
when grown under non-stress condition. Under NaCl
(200 mM) stress, a 5.3- and 4.5-fold increase in the
Gly I activity was detected in the CaMV 35S:gly I
plants (lines S-12 and S-42) and 6.2- and 5.6-fold
increase in the rd29A:gly I plants (lines R-16 and
R-18) as compared to UC plants (Fig. 4a). However,
the plants growing under mannitol (200 mM) stress
had an increase of 5.8- and 5.2-fold in the CaMV
35S:gly I transgenic plants (lines S-12 and S-42) and
6.3- and 5.5-fold in the rd29A:gly I plants (lines R-16
and R-18) (Fig. 4a). Similar results were obtained
under ZnCl2 stress (5 mM), where the CaMV 35S:gly I
transgenic plants (lines S-12 and S-42) showed 5.6and 5.3-fold enhancement in activity and the
rd29A:gly I plants showed 6.8- and 6.5-fold increase
as compared to the UC plants under similar stress
(Fig. 4a).
The level of MG was almost similar in UC and
transgenic plants under non-stress condition.
Page 9 of 15
76
However, in response to salt (200 mM NaCl), drought
(200 mM mannitol), and heavy metal (5 mM ZnCl2)
stress, the UC plants exhibited 51.7, 45.6, and 49.1 %
increase in MG concentration, respectively, whereas
this increase was only 37.8, 28.4, and 39.6 % (line
S-12) and 39, 28.8, and 40.2 % (line S-42) in CaMV
35S:gly I transgenic plants and 29.3, 19.1, and 34.4 %
(line R-16) and 32.3, 21.9, and 36.2 % (line R-18) in
rd29A:gly I plants (Fig. 4b).
The MDA level, an indicator of membrane damage
due to lipid peroxidation, was measured in UC and
transgenic plants under salt (NaCl), drought (mannitol), and heavy metal (ZnCl2) stress conditions. The
MDA content in the UC plants increased by 2.7-fold
under NaCl condition, 2.5-fold under mannitol stress,
and by 2.84-fold under ZnCl2 stress compared to that
in the UC plants grown under normal non-stress
condition. However, the increase in MDA content was
only 1.5- and 1.6-fold under NaCl, 1.5- and 1.6-fold
under mannitol stress, and 1.6- and 1.7-fold under
ZnCl2 stress in the CaMV 35S:gly I transgenic lines
S-12 and S-42, respectively, and was only 1.35- and
1.36-fold under NaCl, 1.32- and 1.31-fold under
mannitol stress, and 1.43- and 1.44-fold under ZnCl2
stress in the rd29A:gly I transgenic lines R-16 and
R-18, respectively (Fig. 4c), thus implicating a role for
constitutive and inducible overexpression of Gly I in
mitigation of stress-induced damage in the transgenic
plants.
The photosynthetic efficiency (Fv/Fm) of the
transgenic and the UC plants was determined using
the fluorescence measurements made with HandyPEA (see ‘‘Materials and Methods’’). It was observed
that under non-stress conditions, the transgenic plants
had slightly higher photosynthetic efficiency as compared to UC plants. After exposure of plants to salt
(NaCl)-, drought (mannitol)-, and heavy metal
(ZnCl2)-induced stress, the Fv/Fm ratio of UC plants
decreased by 54.8, 51.2, and 59.7 %, respectively,
while in CaMV 35S:gly I transgenic plants (lines S-12
and S-42) the corresponding reduction was only 22.8
and 25.3 % under NaCl, 21.6 and 22.8 % under
mannitol, and 25.3 and 27.7 % under ZnCl2 stress.
Similarly, the Fv/Fm reduction in the rd29A:gly I
plants (lines R-16 and R-18) was only 15.6 and 15.8 %
under NaCl, 13.2 and 14.6 % under mannitol, and 18.0
and 19.5 % under ZnCl2 stress (Fig. 4d). These results
showed that transgenic plants had more robust photosynthetic machinery.
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76
Page 10 of 15
Transgenic B. juncea plants show better water
retention capacity in comparison with UC plants
To investigate whether the enhanced tolerance of the
transgenic plants corresponded with their increased
ability to hold water, the RWC of two representative
transgenic lines each of CaMV 35S:gly I (S-12 and
S-42) and rd29A:gly I (R-16 and R-18)-transformed B.
juncea was assessed and compared with the UC plants
grown under different abiotic stress conditions such as
salt (NaCl), drought (mannitol), and heavy metal
(ZnCl2) as well as under unstressed condition in the
green house (see Materials and Methods). The RWC in
leaf tissues of UC plants was reduced significantly
from 91 % under non-stress to 54 % under NaCl
stress, 47 % under mannitol stress and 51 % under
ZnCl2 stress condition. Compared to these, decrease in
the RWC content in CaMV 35S:gly I transgenic plants
was from a control value of 92 and 90 % (line S-12 and
S-42) to 77 and 72 % under NaCl stress, 74 and 71 %
under mannitol stress, and 72 and 70 % under ZnCl2
stress condition. However, the decrease in the RWC
content in rd29A:gly I transgenic plants was from a
control value of 93 and 92 % (line R-16 and R-18) to
81 and 79 % under NaCl stress, 79 and 78 % under
mannitol stress, and 78 and 74 % under ZnCl2 stress
conditions, respectively (Fig. 4e). The results suggested that the water holding ability of transgenic plant
was higher than that of UC plants under stress
conditions.
Comparison of growth parameters of UC
and transgenic plants
Twenty-one-day-old seedlings of T1 transgenic plants
of rd29A:gly I and CaMV 35S:gly I each and UC
plants were subjected to different abiotic stresses by
irrigating them with NaCl (200 mM), ZnCl2 (5 mM),
or mannitol (200 mM) for mimicking the salt, heavy
metal, and drought stress, respectively (see materials
and methods, Supplementary Figure 2). Based on
parameters such as the shoot length, root length, and
seed production per plant, it was observed that the
performance of transgenic plants was better than the
UC plants under various stress conditions (Table 1). In
response to salt (200 mM NaCl), drought (200 mM
mannitol), and heavy metal (5 mM ZnCl2) stress, the
UC plants exhibited 86.07, 94.06 and 93.60 %
decrease in average seed weight per plant,
123
Mol Breeding (2016)36:76
respectively, whereas this decrease was only 85.21,
80.0, and 30.43 % (data shown for representative line
S-42) in case of CaMV 35S:gly I transgenic plants and
84.82, 75.25, and 26.19 % (data shown for representative line R-16) in case of rd29A:gly I transgenic
plants when compared with their respective controls
irrigated with water (Table 1). Decrease in average
shoot length per plant was observed in UC plant in
response to NaCl, mannitol, and ZnCl2 stress which
was 39.16-, 40.66-, and 6.29 %, respectively, whereas
this decrease was only 25.37-, 14.40-, and 5.51 % (line
S-42) in case of CaMV 35S:gly I transgenic plants and
23.56-, 13.29-, and 4.60 % (line R-16) in case of
rd29A:gly I transgenic plants when compared with
their respective controls. Similar trend showing
decrease in average root length was also observed in
response to NaCl, mannitol, and ZnCl2 stress in UC,
S-42 and R-16 plants (Table 1). Under unstressed
condition, decrease in average seed weight per plant
was observed in all the five CaMV 35S:gly I-transformed B. juncea lines (S-12, S-18, S-22, S-24, and
S-42) which ranged between 58.44–77.85 % when
compared with UT controls. On the contrary, more
average seed weight per plant was observed in three
rd29A:gly I-transformed transgenic B. juncea lines
R-16, R-18, and R-27 which was 9.81, 32.87, and
39.04 %, respectively, under unstressed condition.
Two rd29A:gly I-transformed transgenic B. juncea
lines R-6 and R-12 also showed low average seed
weight per plant which was 39.49 and 31.50 % under
unstressed condition when compared with UT controls
(Supplementary Table 2). This decrease as well as
increase in the average seed weight in these five
rd29A:gly I lines may be independent of the Gly I
activity as the gly I gene was not induced during
unstressed condition (Fig. 2; Supplementary Table 2).
Results show, amongst the transgenic plants of B.
juncea, the performance of transgenics with rd29A:gly
I was comparatively better than those transformed
with CaMV 35S:gly I line, under both, stress as well as
unstressed conditions (Table 1).
Discussion
Several strategies have been employed for conferring
abiotic stress tolerance in transgenic plants. During
stress conditions, a number of biochemical pathways
work together to maintain the cellular homeostasis.
The letters W, N, M, Z represent water (control), NaCl (200 mM), mannitol (200 mM), and ZnCl2 (5 mM), respectively. Each of values is the mean of three replicates ± SD for
each line (n = 3)
1.19
± 0.01
0.73 ± 0.27
4.81 ± 0.84
0.8 ± 0.14
0.23 ± 0.05
0.17 ± 0.27
1.15 ± 1.11
0.28 ± 0.21
0.26 ± 0.10
4.38 ± 1.0
Seed weight
(g)
0.61 ± 0.19
33.9 ± 1.82
73.1 ± 2.10
35.2 ± 2.34
22.24 ± 1.82
20.1 ± 2.08
46.02 ± 2.1
52.1 ± 1.82
29.5 ± 3.38
84.2 ± 7.2
Root length
(cm)
28.0 ± 2.34
149.5 ± 10.5
195.6 ± 10.79
164.6 ± 11.7
149.1 ± 6.5
130.0 ± 6.5
174.2 ± 16.1
174.2 ± 19.5
110.3 ± 6.5
185.9 ± 16.9
Shoot length
(cm)
113.1 ± 10.4
3.55
± 0.07
67.0
± 2.34
37.4
± 3.12
186.6 ±
5.64
Page 11 of 15
169.6
± 5.2
Z
M
N
W
N
M
Z
N
W
W
M
Z
Transgenic line (R-16)
Transgenic line (S-42)
UC
Parameters
Table 1 Comparison of growth parameters of untransformed control (UC) and transgenic B. juncea plants transformed with CaMV 35S-gly I and rd29A-glyI under different
abiotic stress and non-stress conditions
Mol Breeding (2016)36:76
76
Some of the genes related to these pathways have been
well characterized and have been manipulated to
develop stress tolerant transgenic plants. MG is a byproduct of glycolysis that is produced in bulk amounts
during stress conditions. High accumulation of MG
inhibits cell proliferation, degrades proteins by modifying several amino acid residues, and is, thus, toxic
to the cells (Abordo et al. 1999; Martins et al. 2001).
Glyoxalase pathway enzymes (Gly I and II) have been
shown to detoxify MG in plants (Singla-Pareek et al.
2003, 2006; Alvarez Viveros et al. 2013). Overexpression of Gly I of the glyoxalase pathway results in
improved survival under MG stress, and transgenic
plants overexpressing gly I gene were found to tolerate
higher levels of salinity (Veena et al. 1999; Roy et al.
2008; Bhomkar et al. 2008). Studies carried out by
Garg et al. (2002), Singla-Pareek et al. (2003), and
Alvarez Viveros et al. (2013) suggested that the
metabolic engineering of the whole pathway is better
than engineering a single component of the pathway.
These findings showed the effectiveness of glyoxalase
system in conferring enhanced abiotic stress tolerance.
The overexpression of a particular gene for stress
tolerance is expected to be most beneficial for the plant
when it is only expressed upon the induction of stress
conditions. This would spare the plants of the need to
divert its resources of metabolites and transcription
and translation machinery from growth and developmental pathways towards the overexpression of the
transgene which would not hold much significance
under conditions of non-stress. The use of an appropriate stress-inducible promoter to regulate the gly I
gene expression can, thus, be a better option for the
generation of abiotic stress tolerant plants. In the
present investigation, we evaluated this premise by
overexpressing gly I gene under the control of
constitutive CaMV35S and stress-inducible rd29A
promoters and comparing various performance parameters under conditions of non-stress or abiotic stress.
The preliminary assessment of salt tolerance
potential of the transgenic plants was done using
in vitro leaf disc senescence assay (Jha et al. 2013;
Kumar et al. 2013; Singla-Pareek et al. 2003; Yusuf
et al. 2010). Although this assay provides a useful
index for estimating salt tolerance potential, the
interpretations are limited due to the isolated nature
of the system (Bhaskaran and Savithramma 2011).
The observed response could result from continuous
transport of a high concentration of salt over a long
123
76
Page 12 of 15
time inside the leaf tissue, resulting in the death of the
tissue as indicated by Munns (2005). Therefore,
validation of the potential tolerance at the whole plant
level is necessary. We did the necessary evaluation of
the plants in terms of their growth and productivity
when grown in pots and irrigated with solutions of
stress-inducing agent. The transgenic B. juncea plants
overexpressing gly I gene under the influence of
CaMV 35S promoter showed enhanced tolerance
against NaCl, mannitol, and ZnCl2 stress. Physiological data revealed that the average percentage decrease
in the shoot length, root length, and seed production
per plant was comparatively less in transgenic plants
having CaMV 35S:gly I in comparison with the UC
plants under various stress conditions (Table 1).
Plants experience varied levels of stress conditions
during their entire life cycle due to change in the
climatic conditions. Taking such fluctuating stress
conditions into consideration, the regulation of gly I
gene driven by the constitutive CaMV 35S promoter
was also analysed under non-stress conditions. The
data show that the gly I transgene was constitutively
expressing during stress as well as non-stress conditions (Fig. 2a). This could be linked to the observed
retardation in growth parameters of the transgenic
plants in comparison with the UC plants under nonstress conditions (Supplementary Table 2). The constant presence of excess Gly I under non-stress
conditions could probably compromise the growth
and development of these transgenic plants due to
competition for the building blocks and machinery
needed for RNA and protein synthesis under nonstress conditions (Wang et al. 2005).
The transgenic plants expressing gly I gene under
the influence of stress-inducible rd29A promoter also
showed enhanced tolerance against salinity, heavy
metal, and drought stress in comparison with UC
plants. Experimental data showed that the shoot
length, root length, and seed production per plant
were enhanced in transgenic plants transformed with
rd29A:gly I in comparison with UC plants during
various stress conditions (Table 1). The performance
of transgenic plants with rd29A:gly I was better in
NaCl (200 mM), mannitol (200 mM), and ZnCl2
(5 mM) treatments as compared to those transformed
with CaMV 35S:gly I. Physiological data revealed that
the average percentage decrease in the shoot length,
root length, and seed production per plant was
maximum in UC plants followed by CaMV 35S:gly I
123
Mol Breeding (2016)36:76
transgenic plants and least in case of rd29A:gly I
transgenic plants when compared with their respective
controls (Table 1). Successful induction of gly I gene
was observed after NaCl, mannitol, and ZnCl2 stress
treatments (Fig. 2b). Under non-stress conditions, the
performance of transgenic plants expressing the gly I
gene driven by stress-inducible rd29A promoter
proved better than those with constitutive expression
of same gene as evident by higher shoot length and
higher seed production per plant (Supplementary
Table 2). The gly I gene driven by the stress-inducible
rd29A promoter showed negligible induction of the
gly I gene under non-stress conditions which could
save the metabolic energy of the cells for utilization in
other developmental processes of the plant (Fig. 2a).
Thus, it seems desirable to generate plants with
transgene expression driven by a stress-inducible
promoter, so that the specific mRNA and proteins are
not produced unless the plant faces stress. Benefits of
using stress-inducible promoters over constitutive promoters have been reported by many groups, previously
(Jaglo-Ottosen et al. 1998; Kasuga et al. 1999; Gilmour
et al. 2000; Wen-li et al. 2005 Jaglo et al. 2001; Hsieh
et al. 2002a, b; Kasuga et al. 2004; Pellegrineschi et al.
2004; Benedict et al. 2006; Pino et al. 2007). Use of the
stress-inducible rd29A promoter minimized the negative effects in transgenic Arabidopsis growth in comparison with the constitutive CaMV 35S promoter
overexpressing DREB1A transcription factor (Kasuga
et al. 1999). In another study done by Wen-li et al.
(2005), the use of the strong CaMV 35S promoter to
drive the expression of afp (antifreeze protein) gene
also resulted in growth retardation under normal
growing conditions in transgenic tobacco. In contrast,
the expression of afp by stress-inducible promoter,
Prd29A from Arabidopsis showed minimal effects on
plant growth while providing an increased tolerance to
cold stress condition. Ito et al. (2006) overexpressed the
OsDREB1 (a gene for DREB1 ortholog from Oryza
sativa) or DREB1 genes in Oryza sativa and Arabidopsis that showed growth retardation under normal
conditions but improved tolerance to drought, highsalt, and low-temperature stresses. Similar observations
were reported in transgenic Arabidopsis, tomato,
tobacco, and wheat overexpressing DREB1A/CBF3 or
CBF1/DREB1B (Jaglo-Ottosen et al. 1998; Kasuga
et al. 1999; Gilmour et al. 2000, 2004; Jaglo et al. 2001;
Hsieh et al. 2002a, b; Kasuga et al. 2004; Pellegrineschi
et al. 2004).
Mol Breeding (2016)36:76
Our results are in agreement with those of other
researchers who showed that stress-inducible promoters are a better option for the expression of genes under
irregular stress conditions. Thus, overexpression of
genes of interest under the control of stress-inducible
promoters could be a more desirable alternative for
engineering value addition in transgenic crops.
Acknowledgments We thank Prof. S. K. Sopory, I.C.G.E.B.,
New Delhi, for the gift of the gly I cDNA and CaMV 35S:gly I
construct. We acknowledge the gift of seeds of B. juncea from
Late Prof. Shyam Prakash of Indian Agricultural Research
Institute (IARI), New Delhi. Dr. Mukesh Saxena, Jawaharlal
Nehru University, New Delhi provided the Gly I antibody. This
research project was implemented with financial contributions
from the Swiss Agency for Development and Cooperation,
Government of Switzerland and the Department of
Biotechnology (DBT), Government of India under the IndoSwiss Collaboration in Biotechnology. University Grants
Commission (UGC), Government of India, and DBT (in the
initial phase) are duly acknowledged for the fellowship and Prof.
Thomas Hohn for providing training to RR at the University of
Basel, Switzerland.
Compliance with ethical standards
Conflict of interest
conflict of interest.
All the authors declare that there is no
References
Abordo EA, Minhas HS, Thornalley PJ (1999) Accumulation of
alpha-oxoaldehydes during oxidative stress: a role in
cytotoxicity. Biochem Pharmacol 58:641–648
Alvarez Viveros MF, Inostroza-Blancheteau C, Timmermann T,
González M, Arce-Johnson P (2013) Overexpression of
GlyI and GlyII genes in transgenic tomato (Solanum
lycopersicum Mill.) plants confers salt tolerance by
decreasing
oxidative
stress.
Mol
Biol
Rep
40(4):3281–3290
Arnon DI (1949) Copper enzymes in isolated chloroplasts:
polyphenol oxidase in Beta vulgaris. Plant Physiol 24:1–15
Behnam B, Kikuchi A, Celebi-Toprak F, Kasuga M, Yamaguchi-Shinozaki K, Watanabe KN (2007) Arabidopsis
rd29A:DREB1A enhances freezing tolerance in transgenic
potato. Plant Cell Rep 26:1275–1282
Benedict C, Skinner JS, Meng R, Chang Y, Bhalerao R, Huner
NPA, Finn CE, Chen THH, Hurry V (2006) The CBF1dependent low temperature signalling pathway, regulation,
and increase in freeze tolerance are conserved in Populus
spp. Plant, Cell Environ 29:1259–1272
Bhaskaran S, Savithramma DL (2011) Co-expression of Pennisetum glaucum vacuolar Na?/H? antiporter and Arabidopsis H?-pyrophosphatase enhances salt tolerance in
transgenic tomato. J Exp Bot 62:5561–5570
Bhomkar P, Upadhyay CP, Saxena M, Muthusamy A, Prakash
NS, Pooggin M, Hohn T, Sarin NB (2008) Salt stress
Page 13 of 15
76
alleviation in transgenic Vigna mungo L. Hepper (blackgram) by overexpression of the glyoxalase I gene using a
novel Cestrum yellow leaf curling virus (CmYLCV) promoter. Mol Breed 22:169–181
Bihmidine S, Lin J, Stone JM, Awada T, Specht JE, Clemente
TE (2013) Activity of the Arabidopsis RD29A and RD29B
promoter elements in soybean under water stress. Planta
237:55–64
Bradford MM (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal Biochem
72:248–254
Celebi-Toprak F, Behnam B, Serrano G, Kauga M, YamaguchiShinozaki K, Naka H, Watanabe JA, Yamanaka S,
Watanabe KN (2005) Tolerance to salt stress of the transgenic tetrasomic tetraploid potato, Solanum tuberosum cv.
Desiree appears to be induced by the DREB1A gene and
rd29A promoter of Arabidopsis thaliana. Breed Sci
55:311–319
Das M, Chauhan H, Chhibbar A, Mohd Q, Haq R, Khurana P
(2011) High-efficiency transformation and selective tolerance against biotic and abiotic stress in mulberry, Morus
indica cv. K2, by constitutive and inducible expression of
tobacco osmotin. Transgenic Res 20:231–246
Ebbs SD, Kochian LV (1998) Phytoextraction of Zinc by Oat
(Avena sativa), Barley (Hordeum vulgare), and Indian
Mustard (Brassica juncea). Environ Sci Technol
32(6):802–806
Esparteo J, Sanchez-Aguayo I, Pardo JM (1995) Molecular
characterization of glyoxalase I from a higher plant;
upregulated by stress. Plant Mol Biol 29:1223–1233
Fan L, Xheng S, Xuemin W (1997) Antisense suppression of
phospholipase D-c retards abscisic acid- and ethylenepromoted senescence of postharvest Arabidopsis leaves. Plant
Cell 9:2183–2196
Garg AK, Kim JK, Owens TG, Ranwala AP, Choi YD,
Konchian LV, Wu RJ (2002) Trehalose accumulation in
rice plants confers high tolerance level to different abiotic
stresses. Proc Natl Acad Sci USA 99:15898–15903
Gilmour SJ, Sebolt AM, Salazar MP, Everard JD, Thomashow
MF (2000) Overexpression of the Arabidopsis CBF3
transcriptional activator mimics multiple biochemical
changes associated with cold acclimation. Plant Physiol
124:1854–1865
Gilmour SJ, Fowler SG, Thomashow MF (2004) Arabidopsis
transcriptional activators CBF1, CBF2, and CBF3 have
matching functional activities. Plant Mol Biol 54:767–781
Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor
Heath RL, Packer L (1968) Photoperoxidation in isolated
chloroplasts. I. Kinetics and stoichiometry of fatty acid
peroxidation. Arch Biochem Biophys 125:189–198
H}
ofgen R, Willmitzer L (1988) Storage of competent cells for
Agrobacterium transformation. Nucleic Acids Res
16(20):9877
Hsieh TH, Lee JT, Charng YY, Chan MT (2002a) Tomato plants
ectopically expressing Arabidopsis CBF1 show enhanced
resistance to water deficit stress. Plant Physiol
130:618–626
Hsieh TH, Lee JT, Yang PT, Chiu LH, Charng YY, Wang YC,
Chan MT (2002b) Heterology expression of the
123
76
Page 14 of 15
Arabidopsis C-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and
oxidative stresses in transgenic tomato. Plant Physiol
129:1086–1094
Ito Y, Katsura K, Maruyama K, Taji T, Kobayashi M, Seki M,
Shinozaki K, Yamaguchi-Shinozaki K (2006) Functional
analysis of rice DREB1/CBF-type transcription factors
involved in cold-responsive gene expression in transgenic
rice. Plant Cell Physiol 47:141–153
Jaglo KR, Kleff S, Amundsen KL, Zhang X, Haake V, Zhang JZ,
Deits T, Thomashow MF (2001) Components of the Arabidopsis C-repeat/dehydration-responsive element binding
factor cold-response pathway are conserved in Brassica
napus and other plant species. Plant Physiol 127:910–917
Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O,
Thomashow MF (1998) Arabidpsis CBF1 overexpression
induces COR genes and enhances freezing tolerance. Science 280:104–106
Jagt DL (1988) The glyoxalase system. In: Dolphin D, Poulsen
R, Abramovic O (eds) Glutathione: chemical, biochemical
and medical aspects, vol 2. Academic Press, New York,
pp 597–641
Jha B, Mishra A, Jha A, Joshi M (2013) Developing transgenic
Jatropha using the SbNHX1 gene from an extreme halophyte for cultivation in saline wasteland. PLoS ONE
8(8):e71136
Karim S, Aronsson H, Ericson H, Pirhonen M, Leyman B, Welin
B, Mäntylä E, Palva ET, Van Dijck P, Holmström K-O
(2007) Improved drought tolerance without undesired side
effects in transgenic plants producing trehalose. Plant Mol
Biol 64:371–386
Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki
K (1999) Improving plant drought, salt and freezing tolerance by gene transfer of a single stress inducible transcription factor. Nat Biotechnol 17:287–291
Kasuga M, Miura S, Shinozaki K, Yamaguchi-Shinozaki K
(2004) A combination of the Arabidopsis DREB1A gene
and stress-inducible rd29A promoter improved droughtand
low-temperature stress tolerance in tobacco by gene
transfer. Plant Cell Physiol 45(3):346–350
Katiyar A, Smita S, Lenka SK, Rajwanshi R, Chinnusamy V,
Bansal KC (2012) Genome-wide identification and comparative analysis of MYB transcription factor family in rice
and Arabidopsis. BMC Genom 13(1):544
Kaur C, Ghosh A, Pareek A, Sopory SK, Singla-Pareek SL
(2014) Glyoxalases and stress tolerance in plants. Biochem
Soc Trans 42:485–490
Kumar D, Singh P, Yusuf MA, Upadhyaya CP, Roy SD, Hohn
T, Sarin NB (2013) The Xerophyta viscosa aldose reductase (ALDRXV4) confers enhanced drought and salinity
tolerance to transgenic tobacco plants by scavenging
methylglyoxal and reducing the membrane damage. Mol
Biotechnol 54:292–303
Lin JM, Salido AL, Butcher DJ (2004) Phytoremediation of lead
using Indian mustard (Brassica juncea) with EDTA and
electrodics. Microchemical J 76(1–2):3–9
Martins AM, Cordeiro CA, Ponces Freire AM (2001) In situ
analysis of methylglyoxal metabolism in Saccharomyces
cerevisiae. FEBS Lett 499:41–44
Munns R (2005) Genes and salt tolerance: bringing them together. New Phytol 167:645–663
123
Mol Breeding (2016)36:76
Mustafiz A, Ghosh A, Tripathi AK, Kaur C, Ganguly AK, Bhavesh NS, Tripathi JK, Pareek A, Sopory SK, Singla-Pareek
SL (2014) A unique Ni2?-dependent and methylglyoxal-inducible rice glyoxalase I possesses a single active site and
functions in abiotic stress response. Plant J 78:951–963
Odell JT, Nagy F, Chua NH (1985) Identification of DNA
sequences required for activity of the cauliflower mosaic
virus 35S promoter. Nature 313:810–812
Pellegrineschi A, Reynolds M, Pacheco M, Brito RM, Almeraya
R, Yamaguchi- Shinozaki K, Hoisington D (2004) Stressinduced expression in wheat of the Arabidopsis thaliana
DREB1A gene delays water stress symptoms under
glasshouse conditions. Genome 47:493–500
Pental D, Pradhan AK, Sodhi YS, Mukhopadhayaya A (1993)
Variation amongst Brassica juncea cultivars for regeneration
from hypocotyl explants and optimization of conditions for
Agrobacterium mediated genetic transformation. Plant Cell
Rep 12:462–467
Pino MT, Skinner JS, Park EJ, Jeknic Z, Hayes PM, Thomashow
MF, Chen TH (2007) Use of a stress inducible promoter to
drive ectopic AtCBF expression improves potato freezing
tolerance while minimizing negative effects on tuber yield.
Plant Biotechnol J 5(5):591–604
Rajwanshi R, Sarin NB (2013) Selection of genetically transformed Brassica juncea L. cv. Varuna (Indian mustard)
based on Positech system. J Plant Biochem Biotechnol
22(2):214–221
Rajwanshi R, Roy SD, Pooggin M, Hohm T, Sarin NB (2007)
Marker free approach for developing abiotic stress tolerant
transgenic Brassica juncea (Indian mustard). In: Proceedings
of the 2007 WSEAS international conference on cellular and
molecular biology, biophysics and bioengineering, Athens,
Greece, pp 110–116
Ramaswamy O, Guha-Mukherjee S, Sopory SK (1983) Presence of glyoxalase I in pea. Biochem Int 7:307–318
Roy SD, Saxena M, Bhomkar PS, Pooggin M, Hohn T, Sarin NB
(2008) Generation of marker free salt tolerant transgenic
plants of Arabidopsis thaliana using the gly I gene and cre
gene under inducible promoters. Plant Cell, Tissue Organ
Cult 95:1–11
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning,
vol 1, 2nd edn. Cold Spring Harbour Laboratory, Cold
Spring Harbour
Saxena M, Roy SD, Singla-Pareek SL, Sopory SK, Bhalla-Sarin
N (2011) Overexpression of the glyoxalase II gene leads to
enhanced salinity tolerance in Brassica juncea. Open Plant
Sci J 5:23–28
Schonfeld MP, Richard JC, Carver BF, Mornhi NW (1988)
Water relations in winter wheat as drought resistance
indicators. Crop Sci 28:526–531
Singla-Pareek SL, Reddy M, Sopory SK (2003) Genetic engineering of the glyoxalase pathway in tobacco leads to
enhanced salinity tolerance. Proc Natl Acad Sci USA
100(25):14672–14677
Singla-Pareek SL, Yadav SK, Pareek A, Reddy MK, Sopory SK
(2006) Transgenic tobacco overexpressing glyoxalase
pathway enzymes grow and set viable seeds in zinc-spiked
soils. Plant Physiol 140(2):613–623
Thornalley PJ (1990) Glyoxalase system: new developments
towards functional characterization of metabolic pathways
fundamental to biological life. Biochem J 269(1):1–11
Mol Breeding (2016)36:76
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl
Acad Sci USA 76(9):4350–4354
Uotila L (1989) Glutathione thioesterases in glutathione:
chemical, biochemical and medical aspects, coenzymes
and cofactors. In: Dolphin D, Poulson R, Avramovik O
(eds) Part A, vol III. Wiley-Interscience, New York,
pp 767–804
Veena, Reddy VS, Sopory K (1999) Glyoxalase I from Brassica
juncea: molecular cloning, regulation and its overexpression confer tolerance in transgenic tobacco under stress.
Plant J 17:385–395
Wang Y, Chen B, Hu Y, Li J, Lin Z (2005) Inducible excision of
selectable marker gene from transgenic plants by the Cre/
lox site-specific recombination system. Transgenic Res
14(5):605–614
Wen-li X, Mei-qin L, Xin S, Cun-fu L (2005) Expression of a
carrot 36 kD antifreeze protein gene improves cold stress
tolerance in transgenic tobacco. For Stud China 7(4):11–15
Yadav SK, Singla-Pareek SL, Ray M, Reddy MK, Sopory SK
(2005a) Methylglyoxal levels in plants under salinity stress
Page 15 of 15
76
are dependent on glyoxalase I and glutathione. Biochem
Biophys Res Commun 337:61–67
Yadav SK, Singla-Pareek SL, Ray M, Reddy MK, Sopory SK
(2005b) Transgenic tobacco plants overexpressing glyoxalase enzymes resist an increase in methylglyoxal and
maintain higher reduced glutathione levels under salinity
stress. FEBS Lett 579:6265–6271
Yamaguchi-Shinozaki K, Shinozaki K (1993) Characterization
of the expression of a desiccation-responsive rd29 gene of
Arabidopsis thaliana and analysis of its promoter in
transgenic plants. Mol Gen Genet 236:331–340
Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low temperature or high salt stress.
Plant Cell 6:251–254
Yusuf MA, Kumar D, Rajwanshi R, Strasser RJ, TsimilliMichael M, Govindjee Sarin NB (2010) Overexpression of
c-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress: physiological
and chlorophyll a fluorescence measurements. Biochim
Biophys Acta 1797:1428–1438
123