Overexpression of Arabidopsis Molybdenum Cofactor Sulfurase Gene Confers Drought Tolerance in Maize (Zea mays L.)
et al. (2013) Overexpression of Arabidopsis Molybdenum Cofactor Sulfurase Gene Confers Drought Tolerance in Maize
(Zea mays L.). PLoS ONE 8(1): e52126. doi:10.1371/journal.pone.0052126
Overexpression of Arabidopsis Molybdenum Cofactor Sulfurase Gene Confers Drought Tolerance in Maize (Zea mays L.)
Yao Lu. 0
Yajun Li. 0
Jiachang Zhang 0
Yitao Xiao 0
Yuesen Yue 0
Liusheng Duan 0
Mingcai Zhang 0
Zhaohu Li 0
Lam-Son Phan Tran, RIKEN Plant Science Center, Japan
0 State Key Laboratory of Plant Physiology and Biochemistry, College of Agronomy and Biotechnology, China Agricultural University , Beijing , People's Republic of China
Abscisic acid (ABA) is a key component of the signaling system that integrates plant adaptive responses to abiotic stress. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene (LOS5) in maize markedly enhanced the expression of ZmAO and aldehyde oxidase (AO) activity, leading to ABA accumulation and increased drought tolerance. Transgenic maize (Zea mays L.) exhibited the expected reductions in stomatal aperture, which led to decreased water loss and maintenance of higher relative water content (RWC) and leaf water potential. Also, transgenic maize subjected to drought treatment exhibited lower leaf wilting, electrolyte leakage, malondialdehyde (MDA) and H2O2 content, and higher activities of antioxidative enzymes and proline content compared to wild-type (WT) maize. Moreover, overexpression of LOS5 enhanced the expression of stress-regulated genes such as Rad 17, NCED1, CAT1, and ZmP5CS1 under drought stress conditions, and increased root system development and biomass yield after re-watering. The increased drought tolerance in transgenic plants was associated with ABA accumulation via activated AO and expression of stress-related gene via ABA induction, which sequentially induced a set of favorable stress-related physiological and biochemical responses.
. These authors contributed equally to this work.
Drought stress is one major environmental stress that adversely
affects crop growth and productivity worldwide. Developing
drought-tolerant crops would be the most promising and effective
approach to improving agricultural productivity and water use
efficiency against drought and water shortage. Drought tolerance
in plant involves perception of stress signals and subsequent signal
transduction, resulting in activation of various physiological and
metabolic responses . Up to date, hundreds of genes and their
related signaling pathways have been identified as important for
drought tolerance, and genetic engineering using some of these
genes has increased plant drought tolerance [2,3].
ABA is a key component of the signaling system that integrates
the adaptive response of plants to abiotic stress including drought
and salinity. It is involved in plant responses to regulation of
growth and development, including shoot and root growth, and
leaf transpiration . ABA accumulation in plant cells occurs
quickly as plants respond to drought stress, which promotes
expression of ABA-inducible genes  and stomatal closure to
reduce transpirational water loss . To some extent, putative
high ABA content induced stomatal closure, which is important
for plant tolerance of water stress . Accordingly, an important
strategy for plant drought tolerance is regulation of stomatal
movement by ABA actions.
ABA de novo biosynthesis occurs in leaves, stems, and roots of
most plant species primarily in plastids, but the last two steps occur
in the cytoplasm where xanthoxin is converted to ABA [7,8]. The
9-cis-epoxycarotenoid dioxygenase (NCED) is a rate-limiting
enzyme in ABA biosynthesis which catalyses the cleavage of
9cis-violaxanthin and/or 9-cis-neoxanthin to produce xanthoxin in
plastids [9,10]. Xanthoxin is converted to abscisic aldehyde by
dehydrogenase/reductase in the cytoplasm . Abscisic aldehyde
is oxidized to ABA by aldehyde oxidase (AO) . AO needs the
sulphurylated form of a molybdenum cofactor (MoCo) for its
activity , and the LOS5 gene encodes the MoCo sulfurase
involved in regulation of ABA biosynthesis .
The aforementioned steps show the molecular mechanism of
ABA biosynthesis, and genetic engineering using some of these
genes has improved plant drought tolerance. For example,
AtZEPoverexpressing transgenic Arabidopsis showed smaller stomatal
aperture, enhanced de novo ABA biosynthesis, and higher tolerance
of osmotic stress than WT Arabidopsis . Overexpression of
NCED may increase endogenous ABA levels, trigger stomatal
closure, and lead to higher drought tolerance in transgenic
Arabidopsis , tobacco , creeping bentgrass  and
transgenic tomato [2,18,19].
LOS5 is an important gene that regulates the last step of ABA
biosynthesis and enhanced expression in Arabidopsis is induced by
drought, salt, and ABA treatment . Overexpression of LOS5 in
rice under field conditions resulted in more spikelet fertility and
Error bars denote the standard deviation values, and asterisks indicated
a significant difference (*P,0.05) compared with the corresponding
yield than for non-transgenic plants . Our early work showed
that overexpression of LOS5 in tobacco improved drought
tolerance via reducing water loss and increasing antioxidant
systems . Maize is grown on more than 30 million ha annually
in China, especially in Chinas semi-arid and arid regions where
water shortage limits irrigation. However, maize is especially
sensitive to water stress because of its relatively sparse root system
, and this sensitivity to water stress can lead to dramatic
fluctuations in yield due to frequent drought and poor irrigation
management, as often the case in China. With the functions of
stress-inducible genes well recognized, genetic manipulation is an
effective approach for the enhancement of stress tolerance in
The goal of the current study was to evaluate the effect of
overexpression of Arabidopsis LOS5 in maize subjected to drought
stress. The study also aimed to explore the difference in stress
resistance mechanism between transgenic LOS5 and WT maize,
including their physiological and morphological responses under
drought stress. Also, regulatory networks influenced by LOS5 gene
expression in maize were monitored by quantifying the expression
of known stress-related genes.
Generation of transgenic maize lines overexpressing
The vector pCAMBIA1300-LOS5 (Figure 1A) was introduced
into maize via Agrobacterium tumefacians-mediated transformation.
The primary transformed plants were designated as T0 plants, and
seeds from self-fertilization of T0 plants were used to raise T1
progeny. A total of 16 independent maize transgenic lines were
generated, and the transformations were confirmed by PCR
analysis (Figure 1B). Two dominant lines, M-6 and M-8, were
selected and homozygous T4 transgenic plants were used for
Seedlings 21-d-old of WT and T4 transgenic maize had similar
morphological characteristics under well-watered conditions
(Table 1). The fresh weight of shoots and roots, number of visible
leaves, and total leaf area exhibited similar values among WT and
To investigate the role of overexpressing LOS5 in transgenic
maize under drought stress, RNA blot analysis was used to
monitor LOS5 transcription in leaves under different
droughtstress conditions. Under well-watered conditions, the LOS5 gene
was transcribed in leaves of transgenic plants and not in WT plants
(Figure 1C). Moreover, expression of LOS5 of M-6 and M-8 maize
was 3.3- and 2.8-fold greater than WT maize under drought stress
LOS5 overexpression improves ABA accumulation
The LOS5 gene encoding MoCo sulfurase is involved in
regulation of AO, which regulates the last step of ABA
biosynthesis, so a native protein gel assay was used to analyze
the AO activity of leaf extracts from transgenic (M-6 and M-8) and
WT maize (Figure 2A). Under well-watered conditions, AO
activity of M-6 and M-8 maize was 26 and 9% higher than that of
WT maize (data no shown). Otherwise, the AO activities of
transgenic lines M-6 and M-8 were 112 and 87% higher than
those of WT maize under D1 treatment. However, there was no
difference in AO activities between transgenic and WT plants
under D2 treatment or after re-watering (except for M-6).
To determine whether overexpressing LOS5 increased ABA
levels in transgenic plants under drought stress, M-6 and M-8
maize under D1 treatment exhibited 78 and 90% higher ABA
levels than WT maize (Figure 2B). Also, M-6 and M-8 lines under
D2 treatment had 66 and 79% higher ABA content than WT
maize. It was interesting to see that ABA concentrations were
similar in transgenic and WT plants during normal growth
conditions or after re-watering. Clearly overexpression of the LOS5
gene in transgenic maize was strongly induced by drought stress.
Expression of molybdenum cofactor sulphurase gene (ZmABA3)
in transgenic lines was the same or very similar to WT maize
under well-watered conditions. Although drought markedly
increased the expression of ZmABA3 of transgenic and WT maize,
similar levels were showed in expression of ZmABA3 between
transgenic and WT plants (Figure 2C). However, drought
significantly enhanced the expression of ZmAO1 of transgenic
lines compared with WT maize (Figure 2D). There was no
difference in expression of ZmAO1 between transgenic and WT
plants under well-watered conditions.
LOS5 overexpression decreases stomatal aperture to
reduce water loss
To investigate whether overexpression of LOS5 improved water
stress tolerance in maize, T4 transgenic seedlings were exposed to
drought by withholding water. There were no marked differences
in leaf turgor between WT and transgenic maize prior to drought
stress (Figure 3A). After 2 d of withholding water, leaves of WT
plants showed slight wilting, but transgenic lines M-6 and M-8 had
normal turgid leaves. After 5 d of withholding water, leaves of WT
plants were severely wilted and of transgenic lines M-6 and M-8
were moderately wilted. After 10 d of withholding water, leaves of
M-6 maize showed less wilting than the M-8 line and of the WT
maize completely wilted. After re-watering 2 d, leaves of the
transgenic lines M-6 and M-8 showed less damage than those of
WT maize. Survival rates were determined for WT and transgenic
maize. Only 35% of WT maize recovered, whereas 72 to 88% of
transgenic lines survived (Figure 3B).
Transpirational water loss of 21-d-old seedlings from lines M-6
and M-8 was 28 and 21% less than WT plants (Figure 3C). The
reduced water loss by transgenic maize indicated that stomatal
action was regulated by overexpressing LOS5. Under well-watered
conditions, stomatal apertures of transgenic lines were larger than
WT maize (Figure 3D). However, stomatal apertures under D1
condition of M-6 and M-8 lines were reduced by 15 and 13%
compared with WT maize. Exposed to D2 treatment, stomatal
apertures of M-6 and M-8 lines dropped 38 and 28% compared
with WT maize. After D2 treatment, the plants were re-watered
and recovery was evaluated after 2 d of normal water. Stomatal
apertures of transgenic maize were larger than WT plants.
LOS5 overexpression holds high leaf water potential and
RWC of transgenic maize under drought stress
To further characterize the drought response, the leaf water
potential and RWC of transgenic and WT leaves under water
stress conditions were evaluated. Under well-watered conditions,
leaf water potential of transgenic lines was similar to WT maize
(Figure 4A). Drought stress caused the leaf water potential of
transgenic and WT plants to decline, but that of transgenic lines
was much higher than WT maize. After re-watering, leaf water
potential of transgenic and WT plants was restored, and values of
transgenic lines were higher than those of WT maize.
The RWC under drought stress was maintained at a higher
level in transgenic LOS5 leaves than in WT maize (Figure 4B). For
example, under D1 treatment, the RWC of M-6 and M-8 lines
were 11 and 10% higher than that of WT maize and under D2
treatment it was 19 and 16% higher. The RWC was similar
between transgenic and WT plants under well-watered condition
or after re-watering.
Low cell membrane damage of transgenic maize under
Membrane damage to transgenic and WT maize under water
deficit stress can be assessed by H2O2 accumulation, electrolyte
leakage and MDA content. When exposed to D1 drought stress,
M-6 and M-8 lines produced 30 and 25% less H2O2 than WT
maize, and under D2 treatment produced 27 and 24% less H2O2
than WT maize (Figure 5A). However, for well-watered or
rewatered plants, H2O2 contents were similar in transgenic and WT
Electrolyte leakage and MDA content of transgenic and WT
maize increased gradually with increasing water stress but was
markedly lower in transgenic lines than WT plants under drought
stress (Figure 5B, C). Electrolyte leakage and MDA content were
similar between transgenic and WT maize for well-watered
condition or after re-watering.
Proline accumulation is one positive response to drought that
helps minimize dehydration in many plant species. WT and
transgenic maize under well-watered condition had similar proline
contents, but the contents increased with increasing water stress
(Figure 5D). For example under D1 drought-stress, proline
contents of M-6 and M-8 lines were 49 and 42% higher than
those of WT plants and under D2 treatment were increased by 63
and 53% compared with WT maize. After re-watering, proline
contents of transgenic and WT plants were similar and recovered
to levels similar to well-watered plants that never were exposed to
To assess whether LOS5 overexpression affected activated
oxygen production, which leads to damaged cell structures,
enzymatic antioxidants were measured. Under well-watered
conditions, activities of catalase (CAT), superoxide dismutase
(SOD), and peroxidase (POD) were similar in transgenic and WT
Fresh weight of root (g.plant21)
Number of leaves (No.plant21) Total leaf area (cm2)
The data point are the mean of two independent biological experiments, and each experiment comprised five samples. Error bars denote the standard deviation values.
Figure 2. Aldehyde oxidase (AO) activity, ABA levels and expression of ZmABA3 and ZmAO1 in leaves. (A) Native PAGE assay for AO
activity from transgenic maize (lines M-6 and M-8) and WT leaf extracts. WW, well-watered; D1, moderate drought stress (60% normal water supply)
for 5 d; D2, severe drought stress (40% normal water supply) for 5 d; RW, re-watered for 2 d. (B) Changes in ABA content in transgenic maize exposed
to drought stress. Drought stress treatment was same as (A). Expression of molybdenum cofactor sulphurase ZmABA3 (C) and ZmAO1 (D) under
wellwatered and 20% PEG conditions. The expression level of transgenic lines is shown relative to the expression of WT plants grown under well-watered
condition. Error bars denote the standard deviation values, and asterisks indicated a significant difference (*P,0.05) compared with the
maize (Figure 6). When plants were drought stressed, the activities
of CAT, POD and SOD increased greatly and were higher in
transgenic maize than WT plants. After re-watering, the activities
of CAT, POD and SOD in transgenic and WT plants decreased
rapidly and were similar between transgenic and WT maize.
LOS5 overexpression enhances abiotic stress-related
We evaluated whether overexpression of LOS5, which increased
drought tolerance in maize, lead to phenotypic changes in the
expression patterns of stress-responsive genes in transgenic maize.
In real-time PCR analysis under well-watered conditions, the
expression of abiotic stress-related genes, such as Rad17, NCED1,
CAT1 and ZmP5C1, was similar for transgenic and WT maize.
However, under drought stress, it was markedly higher in
transgenic lines than WT maize (Figure 7). For example under
drought stress, the expression of Rad17 in M-6 and M-8 leaves was
3.5- and 2.1-fold greater than in WT maize (Figure 7A).
Expression of NCED1 (vip14) under drought stress in M-6 and
M-8 lines was by 2.7- and 2.0-fold greater than for WT maize
(Figure 7B). Expression of CAT1 increased under drought stress in
all plants but was much higher in transgenic lines than WT maize
(Figure 7C). Expression of ZmP5CS1 under drought stress in lines
M-6 and M-8 was 3.1- and 2.2-fold higher than for WT maize
Transgenic maize exhibited more robust root systems
Transgenic and WT maize were subjected to D2 treatment for
5 d and then were returned to normal water supply for 5 d. LOS5
expressing maize produced more dry mass than WT, and in both
cases, the difference in root biomass was markedly greater than
shoot biomass. For example, lines M-6 and M-8 produced 19 and
15% more dry shoot mass and 33 and 25% more dry root mass
than WT maize (Table 2). After drought stress, the root/shoot
ratio of M-6 and M-8 plants was 13 and 10% higher than that of
WT maize. Thus, overexpression of LOS5 in maize under water
stress significantly improved their performance, which probably
was primarily due to the increased root system of LOS5-expressing
A feasible strategy for increasing abiotic stress tolerance in
plants is through applied plant biotechnology, which relies on
expression of genes involved in signaling and regulatory pathways
, or genes encoding proteins conferring stress tolerance ,
or enzymes present in pathways . Genetic engineering is
intensively explored to enhance plant stress tolerance, and several
engineered plants have improved stress resistance phenotypes
[3,18]. To test whether this strategy could improve maize
performance under drought conditions, transgenic maize that
overexpressed the Arabidopsis LOS5 gene was generated. When
exposed to drought stress, the expression of LOS5 was greatly
upregulated in transgenic M-6 or M-8 maize although
transcription of the transgene was driven by the constitutive super promoter
(Figure 1C, D). This phenomenon was also observed in rice
expressing SaVHAC1 gene , transgenic betA gene in wheat ,
cotton  and maize . Several studies have reported that
transgene transcript of constitutive expression of a stress response
gene in homologous or heterologous systems is induced by
environmental stresses [10,25,28,29]. The expression of genes at
the cellular level is involved in transcriptional and
posttranscriptional mechanisms, and posttranscriptional control of transcript
accumulation is an important mechanism for gene regulation
under stress [30,31]. The results in this study suggested that a
Figure 4. Leaf water status assay. Changes in leaf water potential (A) and relative water content (B) in WT and transgenic maize (lines M-6 and
M8) subjected to drought stress. Drought stress treatment was imposed as described in Fig. 2 (A) above. Error bars denote the standard deviation
values, and asterisks indicate a significant difference (*P,0.05) compared with the corresponding controls.
posttranscriptional regulation mechanism might be involved in
controlling the stability of LOS5 transcript. These results were in
consistent with the observations that the ABA level in transgenic
plants increased much more than that in WT plants after drought
stress (Figure 2B).
Map-based cloning revealed that LOS5 encoded a molybdenum
cofactor sulphurase, and sulphurase catalyzed production of a
sulfurylated molybdenum cofactor required by aldehyde oxidase
(AO), which functions in the last step of ABA biosynthesis and
functioned indirectly in ABA biosynthesis . In our research
with maize, overexpressing LOS5 evidently increased the AO
activity under two drought stress conditions (Figure 2A). Then,
ABA accumulation was markedly enhanced in M-6 or M-8
transgenic maize subjected to drought stress (Figure 2B). These
observations are supported by the model for stress induction of
ABA biosynthesis , whereby an initial increase in ABA from
overexpression of one ABA biosynthetic gene, such as LOS5/
ABA3, could result in increased expression of other ABA
biosynthetic genes, AAO3  and LOS6/ABA1 . Collectively,
Figure 5. Cell membrane damage and non-enzymatic antioxidants assay. Changes in H2O2 content (A), MDA content (B), electrolyte leakage
(C), and proline content (D) in WT and transgenic maize (lines M-6 and M-8) subjected to drought stress. Drought stress treatment was imposed as
described in Fig. 2 (A) above. Error bars indicate SE, and asterisks indicate a significant difference (*P,0.05) compared with the corresponding
these genes would lead to a sustained increase in de novo ABA
We have shown that it was possible to reliably generate
transgenic maize with high ABA content by manipulating a single
key ABA biosynthetic gene under drought stress. Overexpressing
LOS5 did not enhance the expression of ZmABA3 compared to WT
maize under well-watered or drought conditions (Figure 2C), but
markedly increased the expression of ZmAO1 and AO activity
which led to ABA accumulation in maize leaves under drought
stress (Figure 2). However, under well-watered conditions, the
expression of ZmAO1 and ABA contents in LOS5 transgenic maize
were very similar to those of WT maize, which was inconsistent to
overexpression of NCED in plants [2,10,19,34]. NCED catalyzes
the first specific step in ABA biosynthesis and affects ABA
production when overexpressed or underexprssed , whereas
the LOS5 gene is stress-induced, and its transcript increased in
response to drought, ABA, NaCl, and PEG treatments .
Otherwise, MoCo sulfurase (LOS5) convertion of di-oxygenated
MoCo to mono-oxygenated MoCo is required to activate ABA
aldehyde oxidase and indole-3-acetaldehyde oxidase, which are
involved in ABA and IAA biosynthesis . These observations
suggested that constitutive expression of LOS5 in maize could not
promote ABA accumulation under well-watered or re-watering
conditions, and might regulate indole-3-acetaldehyde oxidase
involving IAA biosynthesis. The effect of LOS5 overexpression
on regulating auxin biosynthesis requires further investigation.
ABA is a key component of the signaling system that integrates
the adaptive response of plants to drought and osmotic stress.
Under drought stress, ABA accumulation was critical for stomatal
closure that led to reduced transpirational water loss and induced
expression of drought- and desiccation-tolerant genes . Under
drought stress, overexpression of LOS5 in maize induced much
higher ABA concentrations than non-transgenic plants.
Transgenic lines had smaller stomatal apertures than WT maize, which
led to less water loss in transgenic plants under drought conditions
Overexpression of LOS5 in maize showed markedly lower
transpiration rates than WT plants, which led to reduce wilting in
transgenic lines under drought conditions (Figure 3). Otherwise,
overexpression of LOS5 in maize leaves under drought stress
resulted in higher RWC and lower leaf water potential than WT
plants (Figure 4), which was an important strategy for transgenic
plants to conserve water capability to reduce wilting. Our results
were consistent with overexpression of NCED that led to increased
ABA production and reduced leaf transpiration under drought
conditions, which consequently increased the drought tolerance of
transgenic plants [2,10,19].
Overexpression of LOS5 under drought stress altered the
expression of ABA-regulated genes (Figure 7). The maize ABA
responsive gene Rab17 is induced by water deficit, ABA, and
desiccation in embryo and vegetative tissues . Expression of
Rab 17 under drought stress in LOS5-overexpressing lines was
2.1to 3.5-fold higher than in WT maize (Figure 7A). ABA is
synthesized from C40-carotenoids, in which the oxidative cleavage
of cis-epoxycarotenoids by NCED is the rate-limiting step of ABA
biosynthesis in higher plants . Expression of NCED is induced
by drought and salt stress [16,38]. Under drought stress, lines M-6
and M-8 overexpressing LOS5 markedly enhanced the expression
of NCED1 compared to WT maize (Figure 7B). These results
suggested that overexpressing LOS5 promoted ABA accumulation
thereby regulating expression of ABA responsive and biosynthetic
Stress-induced production of reactive oxygen species (ROS) is a
common metabolic response to environmental stress in plants .
ROS are signaling molecules that regulate plant-protective stress
responses including ABA-induced activation of stomatal closure
and induction of defense gene expression .
Water-stressinduced ABA accumulation regulated ABA-stress-responsive gene
expression including ROS network genes such as SOD, APX and
CAT [4,41]. Overexpressing LOS5 in maize greatly increased
expression of CAT1 compared to WT plants (Figure 7C), and the
activity of CAT under drought stress in LOS5 -expressing maize
was higher than in WT plants (Figure 6C), which resulted in
reduced H2O2 accumulation (Figure 5A) and cytoplasmic damage
as detected by electrolyte leakage (Figure 5C).
LOS5-expressing maize under drought stress promoted
accumulation of proline (Figure 5D), and the expression of ZmP5SC1
was increased 2- to 3-fold compared to WT plants (Figure 7D).
Stress-induced P5CS1 (a key enzyme in proline biosynthesis) gene
expression under osmotic stress required ABA [14,42]. It is
possible that LOS5-overexpressing plants under drought stress
could accumulate proline by overproducing ABA. Our results
clearly indicate that overexpression of LOS5 enabled maize to
detoxify ROS efficiently (Figure 5) and to enhance drought stress
tolerance via mobilizing ROS-scavenging enzymes and activating
signaling molecules that regulate ROS-scavenging genes. In this
context, plants with a putative high ABA level might be most
tolerant to stressful conditions .
In conclusion, overexpression of LOS5 in maize subjected to
drought stress increased drought tolerance by regulating AO
activity to promote ABA accumulation. ABA accumulation in
transgenic maize exposed to drought stress reduced water loss, and
activated expression of stress-regulated genes that alleviated
membrane damage. These data provide important insights into
application of an ABA-related biosynthesis gene and significantly
furthers our understanding of stress gene regulation and stress
Shoot dry weight (g.plant21)
Root dry weight (g.plant21)
Seedlings 21-d-old WT and transgenic maize (lines M-6 and M-8) were exposed to severe drought stress (D2) by withholding water for 5 d and then restoring it for 5 d.
The data points are the mean of two independent biological experiments, and each experiment comprised ten samples. Error bars denote the standard deviation
values, and asterisks indicate a significant difference (*P,0.05) compared with the corresponding controls.
Materials and Methods
Construction of the binary vector and transformation
A constitutive super promoter, which consists of three copies of
the octopine synthase enhancer in front of the manopine synthase
promoter, was cloned as a SalIXbaI fragment into the pCAMBIA
1300 binary vector containing a hygromycin-resistant selectable
marker (Figure 1A). LOS5 cDNA of Arabidopsis was cloned as an
XbaIKpnI fragment downstream of the super promoter in the
modified pCAMBIA 1300 . The recombinant plasmid was
introduced into the A. tumefaciens strain EHA105, which was used to
Transformation of maize inbred line Zheng 58 immature
embryos was modified as described in Frame et al. . Immature
zygotic embryos (2 mm) were dissected and inoculated in A.
tumefaciens suspension for 5 min. After infection, embryos were
transferred to the surface of cocultivation medium and incubated
in the dark at 20uC for 3 d and then transferred to resting medium
at 25uC for 7 d. Infected embryos were transferred to selection
medium 6 weeks later. Small pieces of Type II callus were
regenerated on regeneration medium for 14 d. Mature somatic
embryos were transferred to shoot induction medium or rooting
medium to form plantlets with fully formed shoots and roots in the
growth chamber at a light intensity of 50 mmol m22 s21.
Transgenic maize with hygromycin resistance plants were
transplanted into the pots (15615620 cm) filled with a mixture
of vermiculite and sand (1:1; v/v) and grown in the greenhouse.
Polymerase chain reaction (PCR) analysis of transgenic
PCR analysis was carried out assaying T0 and T3 maize lines
carrying the LOS5 gene. Genomic DNA was isolated from
expanding leaves of 21-d-old transgenic maize at V2 growth stage
and untransformed WT plants by the cetyltrimethylammonium
bromide method . Equal amounts of 200 ng of total DNA
were amplified in 50 ml reactions using specific primers for LOS5
gene, forward primer
59-CCTGATGGCTCTTGGTTTGGCTAC -39 and reverse primer
59-TTCCACTGACGACGGTTCCATTCC -39 to amplify a 325 bp sequence from the LOS5 gene
coding region. The PCR reactions were conducted for an initial
denaturation at 95uC for 5 min, followed by 35 cycles of 30 s at
94uC, 45 s at 55uC, and 30 s at 72uC, and a final extension at
72uC for 10 min. PCR products were separated by electrophoresis
on a 1% (w/v) agarose gel.
RNA isolation and RNA blot analysis
Total RNA was isolated from fresh leaves of 21-d-old T4
transgenic and WT maize, grown normally or maintaining 60%
normal water supply for 5 d, with the TRIZOL reagent
(Invitrogen GmbH, Karlsruhe, Germany) and RNAeasy columns
(Qiagen, Hilden, Germany) according to the manufacturers
protocols. Leaves from the maize seedlings, 2 g per sample, were
collected and ground into fine powder in liquid nitrogen, and then
100 mg of homogenized powder was added to 1 ml TRIZOL and
incubated at 60uC for 5 min. Samples were centrifuged at
10 000 rpm for 10 min, and the supernatant was transferred to
a new tube. Then 200 ml chloroform were added and incubated at
room temperature for 23 min. Samples were again centrifuged as
described above, and the aqueous supernatant was transferred to
the Qia shredder column and centrifuged for 30 s at 10 000 rpm.
A 350 ml aliquot of RLT buffer (plus b-mercaptoethanol) and
250 ml absolute ethanol were added to the flow-through and
passed through an RNAeasy spin column. The quality of RNA
was checked on a 1% agarose gel. RNA concentration was
calculated using Nanodrop 2000 according to the manufacturers
instructions (Thermo Scientific, Wilmington, DE, USA), and then
RNA samples were transferred onto nylon membranes. The RNAs
were immoblized to the membrane at 1200 mJ/cm2 for 12 s,
airdried and then baked for 2 h at 80uC in a vacuum oven. The
membrane was prehybridized for 1 h at 64uC in the hybridization
solution [1% BSA, 1 mM EDTA (pH 8.0), 0.5 mM Na2HPO4
(pH 7.2), 7% SDS]. Then hybridization at 64uC was performed
overnight with the denatured 32P-labelled probe made by LOS5
gene special primers (forward primer
59-GGGAAAGGGTGGAGGAGT-39 and reverse primer 59-
GTAGCCAAACCAAGAGCC-39). The membrane was washed with solution I [0.5%
BSA, 1 mM EDTA (pH 8.0), 40 mM Na2HPO4 (pH 7.2), 5%
SDS] at 64uC for 5 min, and two times with solution II [0.1 mM
EDTA (pH 8.0), 40 mM Na2HPO4 (pH7.2), 1% SDS] at 64uC for
10 min each. The membrane was wrapped in saran wrap and
exposed to a phosphor screen for 25 h. Radioactivity was
detected by scanning the phosphor screen using a phosphor
Real-time quantitative PCR (RT-qPCR) analysis
Seedlings 7-d-old of WT and T4 transgenic maize were placed
in a box with nutrient solution and grown in a growth chamber.
After solution culture for 14 d, plants were subjected to water
deficit induced by 20% PEG in the nutrient solution, as had been
selected in a preliminary experiment. After 12 h water deficit
stress, expanding leaves of transgenic and WT were collected in
liquid nitrogen before isolation of RNA. Total RNA was isolated
using TRIZOLH reagent (Invitrogen, CA, USA) and purified
using Qiagen RNeasy columns (Qiagen, Hilden, Germany)
according to the instructions of the manufacturer. Reverse
transcription was performed using Moloney murine leukemia
virus (M-MLV; Invitrogen) according to the method described by
Zhang et al. . Primer Express program 3.0 (Applied
Biosystems, Foster, CA, USA) was used to design the primers for
the genes chosen: LOS5, forward 59-TGATGCTGCAAAGGGTT
GTGCTAC-39 and reverse
59-AATTGAAGCAGCAACAGTGCCTCC-39; ZmAO1, forward
59-GGGAGGCTGTGTACGTTGAT -39 and reverse
59-TCTCCACCGCTTGGAATATC-39; Zm ABA3, forward 59-
CGGCAGGTGTACTTTGGGCAAA-39 and reverse 59-CGGGGTCCTGATTC
GGTCACTCAG -39; Rab17, forward
59-CCCATAAGTACAGTGGCTGTGCT-39 and reverse
59-ACGTACAAATTCACCCCACAAGTA-39; NCED1 (Vp14), forward
59-AGTTGTTGTCACCCAG TCCAG-39 and reverse
59-CACGCACCGATAGCCACA39; ZmP5CS1, forward 59-ACTGCAA TGTCCACTTATCC-39
and reverse 59-TAACCTAGACTAGACACAGC-39; CAT1,
forward 59-CTAACAGGCTGTCGTGAGAAGTG-39 and reverse
59-TGTCAGTGCGTCAACCCATC-39; b-actin, forward
59GATTCCTGGGATTGCCGAT-39 and reverse
59-TCTGCTGCTGAAAAG TGCTGAG-39, and the Actin gene was chosen as
an internal control to normalize all data. Real-time quantitative
RT-PCR was performed on a 7500 real-time PCR system
(Applied Biosystems) using SYBRH Premix Ex Taq TM (Perfect
Real Time) (TaKaRa Code: DRR041A). According to the
manufacturers protocol, 1.5 mL cDNA, 0.4 mL PCR forward/
reverse primer (10 mmol), 10 mL 26SYBRH Premix Ex TaqTM
and 0.4 mL ROX Reference Dye II (506) were suspended in a
final volume of 20 mL with ddH2O. RT-qPCR cycling conditions
consisted of an initial polymerase activation step at 95uC for
30 sec, 40 cycles of 5 sec at 95uC, and 35 sec at 60uC. Melt-curve
analysis was performed to monitor primer-dimer formation and
amplification of gene specific products. The relative quantification
measure stomatal apertures on each randomly selected digitized
image from six sections of the abaxial surface.
Plant material and growth conditions
Seeds of WT and T4 transgenic maize (lines M-6 and M-8) were
planted into pots (15615620 cm deep) filled with a mixture of
vermiculite and sand (1:1; v/v) and grown in a growth chamber
with a 14 h photoperiod at a 25/30uC night/day temperature
cycle, 400 mmol m22 s21 irradiance (enhanced with high-pressure
sodium lamps), and a relative humidity of 60%.
Drought stress was induced in 21-d-old seedlings of WT and T4
transgenic maize by completely withholding irrigation for 10 d.
Drought-stress-tolerant phenotypes of transgenic lines were
observed, and the number of wilted plants was scored and
photographed. Then, the wilted plants were re-watered and
resumed growth; drought-stress-tolerant phenotypes of transgenic
maize after 2 d were recorded. Survival rate was recorded after
7 d of recovery from 14 d of drought stress and was defined as the
number of healthy plants divided by the total number (50 plants) of
each lines or WT.
Drought experiments also were conducted with WT and T4
transgenic maize grown in the pots as described above. Plants were
watered to capacity daily by providing about 400 ml water per
pot. After 21 d of growth with normal water supply, uniform
plants were divided into four groups: well-watered group,
moderate drought group (D1, 60% normal water supply), severe
drought group (D2, 40% normal water supply), and re-watered
group (re-watered after D2 treatments). For the drought
treatments, D1 and D2 irrigations were done with 180 ml and
120 ml water per pot daily, for 5 d. Then, the re-watered group
following D2 treatment was supplied with normal water regime for
2 d and some plants were further cultured for another 5 d for
biomass analysis. At each harvest, the plant was separated into
shoots and roots. Shoots were cut at the cotyledon node, and fresh
weight determined. Roots were measured by pulling pots from the
ground and soaking the root mass in water, then manually stirring
and pouring into a sieve (0.25 mm2 mesh). The sieve was
suspended in a large water bath and shaken continuously until
roots were washed free of soil. Soil materials remaining on the
sieve were removed manually. The separated root fractions were
collected to determine fresh weight. Then all samples were cured
at 105uC for 30 min and dried at 70uC to determine the shoot and
root dry weight. Fresh samples of all treatments were used for
immediate assays or frozen in liquid nitrogen and stored at 280uC
for physiological and biochemical analysis (see below).
Water loss and stomatal aperture measurements
T4 transgenic and WT maize were grown in pots under
wellwatered conditions for 21 d and then drought stress was imposed
for 5 d as described above. Leaves of maize were cut and
transpirational water loss was measured as described by Chen et
al. . The uppermost fully expanded leaves of WT and T4
transgenic maize under drought treatments were used in the
experiments. Stomatal bioassay was performed as described by Pei
et al.  with slight modifications. Leaves were carefully cut into
10-mm long and 5-mm wide strips, and the strips were
immediately incubated in FAA fixative liquid (38% formaldehyde,
acetic acid and 50% alcohol, 5:5:90). Stomata were observed
under a scanning electron microscope (S-570; Hitachi, Japan), and
the width and length of stomatal apertures were measured using
image analysis computer software (Scion Image; Scion Corp.,
Frederick, MD; and National lnstitutes of Health, Bethesda, MD).
For each independent measurement, five stomata were selected to
Leaf water potential, RWC, and ABA content
Leaf water potential of the uppermost fully expanded leaves of
WT and T4 transgenic maize was taken on 0 d (21-d-old
seedlings) and 5 d after initiation of different drought treatments
and 2 d after re-watering as described above. A pressure chamber
(Model 3000, Soil Moisture Equipment Corp., Santa Barbara,
CA, USA) was used to measure the leaf water potential, with one
leaf per plant and six plants per treatment. The RWC was
measured as described by Gaxiola et al. . Endogenous ABA
content was measured by an indirect enzyme-linked
immunosorbent assay (ELISA) as described by Yang et al. .
Electrolyte leakage and MDA, proline and H2O2 content
The uppermost fully expanded leaves of WT and T4 transgenic
maize, from 0 d (21-d-old seedlings) and 5 d after initiation of
different drought treatments and 2 d after re-watering, were
washed briefly in deionized water and 5-mm-diam leaf discs were
punched out. Membrane damage was assayed by measuring ion
leakage from leaf discs as described by Shou et al. . The extent
of lipid peroxidation was estimated by measuring the amount of
MDA as described by Quan et al. . Proline content was
measured according to Bates et al. , and H2O2 content was
measured as described by Brennan and Frenkel .
SOD, POD, CAT, and AO enzyme assays
Fresh leaf segments from T4 transgenic and WT maize with
different drought treatment were crushed into fine powder in a
mortar and pestle under liquid nitrogen. Soluble protein content
was determined following the Bradford method  with BSA as
standard. Total SOD activity was assayed according to
Giannopolitis and Ries . POD activity was determined by the guaiacol
oxidation method of Aebi . CAT activity was measured
following Nakano and Asada . AO activity was measured by
Native PAGE as described by Porch et al. . Plant tissue was
ground to a powder with liquid nitrogen and homogenized in
icecold extraction buffer (250 mM TRIS-HCl, pH 7.5, 1 mM
EDTA, 10 mM GSH, and 2 mM DTT,10 uM FAD, 50 uM
leupetin, 80 uM sodium molybdate). A ratio of 1 g leaf tissue to
5 ml buffer (1:5 w/v) was used, and homogenized plant material
was centrifuged at 18 000 g and 4uC for 25 min. The resulting
supernatant was subjected to native polyacrylamide gel
electrophoresis (PAGE) on 7.5% polyacrylamide gels in a Laemmli buffer
system in the absence of SDS at 4uC. Each lane in the gel was
loaded with above 400 ug protein. After electrophoresis, the gel
was immersed in 0.2 M phosphate buffer (pH 7.5) for 10 min,
then AO activity staining was developed at room temperature in a
mixture containing 0.1 M TRIS-HCl, pH 7.5, 0.1 mM phenazine
methosulphate, 0.5 mM MTT (3 [4, 5-dimethylthiazol-2-yl] 2,
5diphenyltetrazolium -bromide), and 1 mM substrate
(1-naphthaldehyde or indole-3-aldehyde) in the dark for about 1 h. After
activity attaining, the gels were scanned to quantify the relative
intensity of formazan bands which were directly proportional to
enzyme activity  using the Quantity One computer software in
Bio-Rad ChemiDoc SRS (Bio-Rad, Hercules, CA, USA). Native
PAGE was carried out with a Protein II xi Cell (JunYi, Beijing,
Results are based on two independent experiments with at least
three replicate tissue samples from three to four transgenic or WT
plants in each treatment. Data were analysed using the Students
ttest (SPSS 13.0 for Windows; SPSS Inc., Chicago, IL, USA).
Significant differences were determined based on P,0.05 or
We thank Dr. Jiankang Zhu (University of California, Riverside, CA, USA)
and Dr. Zhizhong Gong (China Agricultural University, Beijing) for
supplying LOS5 gene and excellent technical assistance. The authors also
thank Dr. Calvin G. Messersmith, Professor Emeritus, Department of Plant
Sciences, North Dakota State University, Fargo, for technical improvement
of the manuscript.
Conceived and designed the experiments: MCZ ZHL. Performed the
experiments: YL YJL JCZ YTX. Analyzed the data: YSY LSD.
Contributed reagents/materials/analysis tools: YL YJL JCZ YTX. Wrote
the paper: MCZ ZHL.
1. Zhu JK ( 2002 ) Salt and drought stress signal transduction in plants . Annu Rev Plant Biol 53 : 247 - 273 .
2. Tung SA , Smeeton R , White CA , Black CR , Taylor IB , et al. ( 2008 ) Overexpression of LeNCED1 in tomato (Solanum lycopersicum L.) with the rbcS-3C promoter allows recovery of lines that accumulate very high levels of abscisic acid and exhibit severe phenotypes . Plant Cell Environ 31 : 968 - 981 .
3. Xiao BZ , Chen X , Xiang CB , Tang N , Zhang QF , et al. ( 2009 ) Evaluation of seven function-known candidate genes for their effects on improving drought resistance of transgenic rice under field conditions . Mol Plant 2 : 73 - 83 .
4. Yamaguchi-Shinozaki K , Shinozaki K ( 2006 ) Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stress . Annu Rev Plant Biol 57 : 781 - 803 .
5. Schroeder JI , Kwak JM , Allen GJ ( 2001 ) Guard cell abscisic acid signalling and engineering drought hardiness in plants . Nature 410 : 327 - 330 .
6. Xiong LM , Zhu JK ( 2003 ) Regulation of abscisic acid biosynthesis . Plant Physiol 133 : 29 - 36 .
7. Tan BC , Schwartz SH , Zeevaart JAD , McCarty DR ( 1997 ) Genetic control of abscisic acid biosynthesis in maize . Proc Natl Acad Sci USA 94 : 12235 - 12240 .
8. Seo M , Koshiba T ( 2002 ) Complex regulation of ABA biosynthesis in plants . Trends Plant Sci 7 : 41 - 48 .
9. Chernys JT , Zeevaart JA ( 2000 ) Characterization of the 9-cis-epoxycarotenoid dioxygenase gene family and the regulation of abscisic acid biosynthesis in avocado . Plant Physiol 124 : 343 - 353 .
10. Iuchi S , Kobayashi M , Taji T , Naramoto M , Seki M , et al. ( 2001 ) Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis . Plant J 27 : 325 - 333 .
11. Gonzalez-Guzman M , Apostolova N , Belles JM , Barrero JM , Piqueras P , et al. ( 2002 ) The short-chain alcohol dehydrogenase ABA2 catalyzes the conversion of xanthoxin to abscisic aldehyde . Plant Cell 14 : 1833 - 1846 .
12. Seo M , Aoki H , Koiwai H , Kamiya Y , Nambara E , et al. ( 2004 ) Comparative studies on the Arabidopsis aldehyde oxidase (AAO) gene family revealed a major role of AAO3 in ABA biosynthesis in seeds . Plant Cell Physiol 45 : 1694 - 1703 .
13. Bittner F , Oreb M , Mendel RR ( 2001 ) ABA3 is a molybdenum cofactor sulfurase required for activation of aldehyde oxidase and xanthine dehydrogenase in Arabidopsis thaliana . J Biol Chem 276 : 40381 - 40384 .
14. Xiong LM , Lee H , Ishitani M , Zhu JK ( 2001 ) The Arabidopsis LOS5/ABA3 locus encodes a molybdenum cofactor sulfurase and modulates cold stress- and osmotic stress-responsive gene expression . Plant Cell 13 : 2063 - 2083 .
15. Park HY , Seok HY , Park BK , Kim SH , Goh CH , et al. ( 2008 ) Overexpression of Arabidopsis ZEP enhances tolerance to osmotic stress . Biochem Bioph Res Co 375 : 80 - 85 .
16. Qin X , Zeevaart JAD ( 2002 ) Overexpression of a 9-cis-epoxycarotenoid dioxygenase gene in Nicotiana plumbaginifolia increases abscisic acid and phaseic acid levels and enhances drought tolerance . Plant Physiol 128 : 544 - 551 .
17. Aswath CR , Kim SH , Mo SY , Kim DH ( 2005 ) Transgenic plants of creeping bentgrass harboring the stress inducible gene, 9-cis-epoxycarotenoid dioxygenase, are highly tolerant to drought and NaCl stress . Plant Growth Regul 47 : 129 - 139 .
18. Thompson AJ , Jackson AC , Symonds RC , Mulholland BJ , Dadswell AR , et al. ( 2000 ) Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes over-production of abscisic acid . Plant J 23 : 363 - 374 .
19. Thompson AJ , Andrews J , Mulholland BJ , McKee JMT , Hilton HW , et al. ( 2007 ) Overproduction of abscisic acid in tomato increases transpiration efficiency and root hydraulic conductivity and influences leaf expansion . Plant Physiol 143 : 1905 - 1917 .
20. Yue Y , Zhang M , Zhang J , Duan L , Li Z ( 2011 ) Arabidopsis LOS5/ABA3 overexpression in transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) results in enhanced drought tolerance . Plant Sci 181 : 405 - 411 .
21. Laboski CAM , Dowdy RH , Allmaras RR , Lamb JA ( 1998 ) Soil strength and water content influences on corn root distribution in a sandy soil . Plant and Soil 203 : 239 - 247 .
22. Shinozaki K , Yamaguchi-Shinozaki K , Seki M ( 2003 ) Regulatory network of gene expression in the drought and cold stress responses . Curr Opin Plant Biol 6 : 410 - 417 .
23. Wang W , Vinocur B , Shoseyov O , Altman A ( 2004 ) Role of plant heat shock proteins and molecular chaperones in the abiotic stress response . Trends Plant Sci 9 : 244 - 252 .
24. Park EJ , Jeknic Z , Sakamoto A , Denoma J , Yuwansiri R , et al. ( 2004 ) Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage . The Plant J 40 : 474 - 487 .
25. Baisakh N , RamanaRao MV , Rajasekaran K , Subudhi P , Janda J , et al. ( 2012 ) Enhanced salt stress tolerance of rice plants expressing a vacuolar H+-ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Loisel . Plant Biotech J 10 : 453 - 464 .
26. He C , Yang A , Zhang W , Gao Q , Zhang J ( 2010 ) Improved salt tolerance of transgenic wheat by introducing beta gene for glycine synthesis . Plant Cell Tiss Organ Cult 101 : 65 - 78 .
27. Lv A , Yang A , Zhang K , Wang L , Zhang J ( 2007 ) Increase of glycinebetaine synthesis improves drought tolerance in cotton . Mol Breeding 20 : 233 - 248 .
28. Wei A , He C , Li B , Li N , Zhang J ( 2011 ) The pyramid of transgenes TsVP and BetA effectively enhances the drought the drought tolerance of maize plants . Plant Biotech J 9 : 216 - 229 .
29. Yang Q , Chen ZZ , Zhou XF , Yin HB , Li X , et al. ( 2009 ) Overexpression of SOS (Salt Overly Sensitive) genes increases salt tolerance in transgenic Arabidopsis . Mol Plant 2 : 22 - 31 .
30. Cohen A , Moses MS , Plant AL , Bray EA ( 1999 ) Multiple mechanisms control the expression of abscisic acid (ABA)-requiring genes in tomato plants ecposed to soil water deficit . Plant Cell Enviorn 22 : 989 - 998 .
31. Shi H , Lee B , Wu S , Zhu J ( 2003 ) Overexpression of a plasma membrane Na+/ H+ antiporter gene improves salt tolerance in Arabidopsis thaliana . Nature Bioteh 21 : 81 - 85 .
32. Xiong LM , Schumaker KS , Zhu JK ( 2002 ) Cell signaling during cold, drought and salt stress . Plant Cell 14 : S165 - S183 .
33. Seo M , Peeters A JM , Koiwai H , Oritani T , Marion-Poll A , et al. ( 2000 ) The Arabidopsis aldehyde oxidase 3 (AAO3) gene product catalyzes the final step in abscisic acid biosynthesis in leaves . Proc Natl Acad Sci USA 97 : 12908 - 12913 .
34. Parent B , Hachez C , Redondo E , Simonneau T , Chaumont F , et al. ( 2009 ) Drought and abscisic acid effects on aquaporin content translate into changes in hydraulic conductivity and leaf growth rates: a trans-scale approach . Plant Physiol 149 : 2000 - 2012 .
35. Porch TG , Tseung C , Schmelz EA , Settles AM ( 2006 ) The maize Viviparous10/ Viviparous13 locus encodes the Cnx1 gene required for molybdenum cofactor biosynthesis . Plant J 45 : 250 - 263 .
36. Kizis D , Page`s M ( 2002 ) Maize DRE-binding proteins DBF1 and DBF2 are involved in rab17 regulation through the drought-responsive element in an ABAdependent pathway . Plant J 30 : 679 - 689 .
37. Nambara E , Marion-Poll A ( 2005 ) Abscisic acid biosynthesis and catabolism . Annu Rev Plant Biol 56 : 165 - 185 .
38. Tan BC , Joseph LM , Deng WT , Liu L , Cline K , et al. ( 2003 ) Molecular characterization of Arabidopsis 9-cis-epoxycarotenoid dioxygenase gene family . Plant J 35 : 44 - 56 .
39. Mittler R ( 2002 ) Oxidative stress, antioxidants and stress tolerance . Trends Plant Sci 7 : 405 - 410 .
40. Desikan R , Mackerness SAH , Hancock JT , Neill SJ ( 2001 ) Regulation of the Arabidopsis transcriptome by oxidative stress . Plant Physiol 127 : 159 - 172 .
41. Jiang MY , Zhang JH ( 2002 ) Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in maize leaves . J Exp Bot 53 : 2401 - 2410 .
42. Strizhov N , Abraham E , Okresz L , Blickling S , Zilberstein A , et al. ( 1997 ) Differential expression of two P5CS genes controlling proline accumulation during salt-stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis . Plant J 12 : 557 - 569 .
43. Frame BR , Shou HX , Chikwamba RK , Zhang Z , Xiang C , et al. ( 2002 ) Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system . Plant Physiol 129 : 13 - 22 .
44. Saghai-Maroof MA , Soliman KM , Jorgensen RA , Allard RW ( 1984 ) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics . Proc Natl Acad Sci USA 81 : 8014 - 8018 .
45. Gong Z , Yamazaki M , Sugiyama M , Tanaka Y , Saito K ( 1997 ) Cloning and molecular analysis of structural genes involved in anthocyanin biosynthesis and expressed in a formaspecific manner in Perilla frutescens . Plant Mol Biol 35 : 915 - 927 .
46. Zhang A , Jiang M , Zhang J , Ding H , Xu S , et al. ( 2007 ) Nitric oxide induced by hydrogen peroxide mediates abscisic acid-induced activation of the mitogenactivated protein kinase cascade involved in antioxidant defense in maize leaves . New Phytol 175 : 36 - 50 .
47. Chen ZZ , Zhang HR , Jablonowski D , Zhou XF , Ren XZ , et al. ( 2006 ) Mutations in ABO1/ELO2, a subunit of holo-elongator, increase abscisic acid sensitivity and drought tolerance in Arabidopsis thaliana . Mol Cell Biol 26 : 6902 - 6912 .
48. Pei ZM , Kuchitsu K , Ward JM , Schwarz M , Schroeder JI ( 1997 ) Differential abscisic acid regulation of guard cell slow anion channels in Arabidopsis wild-type and abi1 and abi2 mutants . Plant Cell 9 : 409 - 423 .
49. Gaxiola RA , Li J , Undurraga S , Dang LM , Allen GJ , et al. ( 2001 ) Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump . Proc Natl Acad Sci USA 98 : 11444 - 11449 .
50. Yang JC , Zhang JH , Wang ZQ , Zhu QS , Wang W ( 2001 ) Hormonal changes in the grains of rice subjected to water stress during grain filling . Plant Physiol 127 : 315 - 323 .
51. Shou HX , Bordallo P , Fan JB , Yeakley JM , Bibikova M , et al. ( 2004 ) Expression of an active tobacco mitogen-activated protein kinase kinase kinase enhances freezing tolerance in transgenic maize . Proc Natl Acad Sci USA 101 : 3298 - 3303 .
52. Quan RD , Shang M , Zhang H , Zhao YX , Zhang JR ( 2004 ) Improved chilling tolerance by transformation with betA gene for the enhancement of glycinebetaine synthesis in maize . Plant Sci 166 : 141 - 149 .
53. Bates LS , Waldren RP , Teare ID ( 1973 ) Rapid determination of free proline for water stress studies . Plant and Soil 39 : 205 - 207 .
54. Brennan T , Frenkel C ( 1977 ) Involvement of hydrogen peroxide in the regulation of senescence in pear . Plant Physiol 59 : 411 - 416 .
55. 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 .
56. Giannopolitis CN , Ries SK ( 1977 ) Superoxide dismutase . I. Occurrence in higher plants . Plant Physiol 59 : 309 - 314 .
57. Aebi H ( 1984 ) Catalase in vitro . Method Enzymol 105 : 121 - 126 .
58. Nakano Y , Asada K ( 1981 ) Hydrogen peroxide is scavenged by ascorbatespecific peroxidase in spinach chloroplasts . Plant Cell Physiol 22 : 867 - 71 .
59. Zdunek E , Lips SH ( 2001 ) Transport and accumulation rates of abscisic acid and aldehyde oxidase activity in Pisum sativum L. in response to suboptimal growth conditions . J Exp Bot 52 : 1269 - 1276 .