Plant Cell Tiss Organ Cult

DOI 10.1007/s11240-014-0684-0

ORIGINAL PAPER

Effect of over-and under-expression of glyceraldehyde

3-phosphate dehydrogenase on tolerance of plants to water-deficit
stress
Sajeesh Kappachery Gangadhar Baniekal-Hiremath
Jae Woong Yu Se Won Park

Received: 30 July 2014 / Accepted: 4 December 2014

Springer Science+Business Media Dordrecht 2014

Abstract While involved in a functional genomics program, we found that the overexpression of potato (Solanum
tuberosum) glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) gene in yeast improves its water-deficit stress
(drought) tolerance. But, the effect of altered (under and
over) expression of GAPDH on water-deficit stress tolerance of higher plants is not yet studied. In this study, we
used a versatile reverse genetics tool called virus-induced
gene silencing and down-regulated the expression of
GAPDH gene in tobacco (Nicotiana benthamiana) to
examine the relevance (effect of underexpression) of
GAPDH on drought tolerance of higher plants. Leaf discs
made from silenced and nonsilenced tobacco plants were
subjected to water-deficit stress. Measure of cell viability
and the content of chlorophyll in stressed and nonstressed
leaf discs were determined to quantify the effect of stress.
Leaf discs made from the gene-silenced plants were found
to be more severely affected by the stress than the leaf discs
made from nonsilenced plants, implying the importance of
GAPDH gene in drought tolerance of plants. Furthermore,
to reiterate the involvement of GAPDH in drought tolerance of plants, potato transgenic plants constitutively
overexpressing the GAPDH gene were generated and their
performance under drought condition was analyzed.
Transgenic potato plants showed improved drought tolerance when compared to wild-type potato. On the whole,

Electronic supplementary material The online version of this

article (doi:10.1007/s11240-014-0684-0) contains supplementary
material, which is available to authorized users.
S. Kappachery  G. Baniekal-Hiremath 
J. W. Yu  S. W. Park (&)
Department of Molecular Biotechnology, Konkuk University,
Seoul 143701, South Korea
e-mail: sewpark@konkuk.ac.kr

our results confirm that the GAPDH gene plays an

important role in drought tolerance of higher plants, and its
constitutive overexpression by genetic engineering can be
used to improve drought tolerance of crop plants like
potato.
Keywords Potato  Drought tolerance  Glyceraldehyde
3-phosphate dehydrogenase  VIGS  Tobacco  Transgenic
potato

Introduction
Finding key genes involved in stress tolerance of crop
plants is important for better understanding of the genetic
basis of their tolerance and thereby the development of
improved cultivars which can perform better in harsh
environments. The importance of a particular gene in stress
tolerance is usually studied by making genetically modified
organisms with regulated (under or over) expression of the
gene under quest. With the advent of new methods, such as
yeast functional screening (Eswaran et al. 2010) and virusinduced gene silencing (VIGS) (Senthil-Kumar et al.
2007), it has become easier to screen candidate stress-tolerance genes to validate their relevance in stress tolerance.
The yeast functional screen was successfully used to screen
stress-enriched cDNA libraries to identify potential stresstolerance genes from various plant species (Eswaran et al.
2010; Priyanka et al. 2010; Sajeesh et al. 2013). This yeastbased forward genetics tool identifies candidate genes from
a plant based on their potential to improve stress tolerance
of yeast when the cDNA coding for the gene is overexpressed in it. Whereas, VIGS is a reverse genetics tool used
for discovery of gene functions. It exploits natural defense
mechanisms in plants against viruses for down-regulation

123

Plant Cell Tiss Organ Cult

of cognate mRNA transcripts of the targeted gene (BurchSmith et al. 2004). Since its advent in the 1990s, the
method is being optimized for use in various plant species,
including crop plants (Demircan 2010; Martin et al. 2013;
Jiang et al. 2014; Tian et al. 2014). Tobacco (Nicotiana
benthamiana) was the primary host for the development of
VIGS technique (Ratcliff et al. 2001; Ruiz et al. 1998).
Standard protocols are available in the literature for VIGS
study in tobacco (Zhu and Dinesh-Kumar 2009), making
this technique easy to use and a popular one to get rapid
confirmation for the relevance of various genes in stress
tolerance of plants. Development and use of VIGS leaf disc
assays (Ku et al. 2011; Ramegowda et al. 2013) further
simplified this functional screening method. These two
functional screening methods viz. Yeast functional
screening and VIGS studies in plants can be used for
preliminary screening of plant genes for their role in stress
tolerance. Once a genes involvement in stress tolerance is
found relevant through these preliminary screening methods, its ability to improve particular stress tolerance of
plants can be tested by making transgenic plants with the
regulated expression of that particular gene, leading to the
discovery of a gene whose expression can be modulated to
improve the stress tolerance of plants/crop plants.
Drought is one of the major abiotic stresses limiting the
growth and yield of many crop plants. Among the food
crops, potato is a major crop affected by drought. Potato
ranks fourth in global production after wheat, maize, and
rice (Gilani and Nasim 2007). Its extreme vulnerability to
drought stress (Iwama and Yamaguchi 2006) makes its
cultivation less profitable in many regions of the world.
Adverse effects of drought stress on potato occurs at all
stages of the crop, leading to reduced plant growth and
yield (Lynch and Tai 1989; Weisz et al. 1994). With the
completion of whole genome sequencing of potato, it is
emerging as a model crop plant for studying the genetic
basis of stress/drought tolerance mechanism of crop plants.
Recent yeast-based functional screening study performed
in our laboratory reported 69 genes from potato as potential
drought-tolerance genes (Sajeesh et al. 2013). Among these
69 genes, 12 were identified as the most important ones
that can be chosen for further study in higher plants. In this
study, we used VIGS (for down-regulation of gene
expression) and transgenic technology (for overexpression
of the gene) for analyzing the relevance of glyceraldehyde
3-phosphate dehydrogenase (GAPDH, one among the
above-mentioned 12 genes) in drought tolerance of higher
plants. Homolog of the GAPDH gene was silenced in
tobacco using VIGS. Leaf discs made from the silenced
and nonsilenced plants were used to test the significance of
GAPDH in water-deficit stress. Furthermore, transgenic
plants (potato) overexpressing the GAPDH gene were
generated and their performance under drought condition

123

was checked to substantiate the findings from the VIGS

study.

Materials and methods

Virus-induced gene silencing in tobacco and drought
stress analysis
Growing tobacco plants
Tobacco (N. benthamiana) seeds were germinated on flat
trays containing soil-less potting mixture (Professional
growing mix, SKU: 505, Sun Gro. Horticulture Ltd, Canada). Two weeks old plants were transplanted into pots
(10 cm height 9 10 cm diameter) containing the same
potting mixture. Plants were grown in culture room under
23 2 C, 16 h light and 8 h dark, and relative humidity
of 6065 %. Pots were irrigated to its maximum capacity
every 3 days; in addition, 50 mL of fertilizer solution
(Hyponex, N:P:K = 20:20:20, 1 g/L) was applied twice in
a week. One-month-old uniformly grown plants were taken
for the experiment.
VIGS fragment isolation and cloning
A 340-bp long fragment of GAPDH gene of N. benthamiana was used as VIGS fragment. It was amplified and
isolated from tobacco (N. benthamiana) first strand cDNA
synthesized from leaf RNA extract. Specific primers used
for the VIGS fragment isolation are shown in Table 1. The
VIGS vectors TRV1 (Liu et al. 2002), TRV2::00 and
TRV2-LIC (Dong et al. 2007) used in this study were
obtained from Dr. Akula Nokaraju (Chonnam National
University, Republic of Korea). A ligation-independent
method described by Dong et al. (2007) was used for
cloning VIGS fragments into the TRV2-LIC vector. The
cloning mixture/product was transformed into Escherichia
coli DH5a competent cells. Transformants were selected
on LB (Duchefa Biocheme, Netherlands), kanamycin
selection media and colony PCR with PF-50 -TGTTACTC
AAGGAAGCACGATGAGCT, PR-50 -CAGGCACGGAT
CTACTTAAAGAACGTAG primers was performed to
select positive transformants. Plasmids were isolated, and
correctness of the sequence of VIGS fragment was confirmed by sequencing with either of the above primers.
Further, the construct was transformed into Agrobacterium
(strain GV2260) and used for agro-infiltration/infection.
Agro-infiltration of tobacco
Agrobacterium carrying TRV1, TRV2::00, and TRV2-LIC
derivative with VIGS fragment were grown at 28 C in LB

Plant Cell Tiss Organ Cult

Table 1 List of primers
Primer name, sequence

Purpose

VFGAPDH_N.bF, 50 -CGACGACAAGACCCTGTTGTGATCTCTGCTCCTAGC

Isolation of VIGS fragment from tobacco for

silencing GAPDH gene

VRGAPDH_N.bR, 50 -GAGGAGAAGAGCCCTCCATTCCAGTCAATTTTCCA
NbEF1a RT_F, 50 -TGGTGTCCTCAAGCCTGGTATGGTTGT

Real time PCR primer for tobacco elongation factor1 alpha gene (internal control)

NbEF1a RT_R, 5 -ACGCTTGAGATCCTTAACCGCAACATTCTT

NbGAPDHRT_F, 50 -ATTCCATGGGCTGAAGCTG

Real time PCR primer for quantification of tobacco

GAPDH gene expression

NbGAPDHRT_R, 50 -TTCTTGGCACCACCCTTCAA
VFPDS_N.bF, 50 -CGACGACAAGACCCTCCACGACCCGAAGATTGACA
VFPDS_N.bR, 50 -GAGGAGAAGAGCCCTAGTTCTCCAAACAGGTTCTGC
StGAPDH_FF, 50 -CACCATGGCTAAGGTTAAGATTGGAATTAACG

Isolation of VIGS fragment from tobacco for

silencing PDS gene

StGAPDH_FR, 5 -TTACTGAACTGATGACATGTGAATAATCAAGT

Isolation of full length cDNA of GAPDH gene from

potato

StGAPDHRT_F, 50 -ATGGACCATCAGCCAAGGATTGGA
StGAPDHRT_R, 50 -ACACATCAACAGTTGGGACTCGGA

Real time PCR primer for quantification of potato

GAPDH gene expression

StACTINRT_F, 50 -GAATGGAAGCAGCTGGAATC

Real time PCR primer for potato actin gene (internal

control)

StACTINRT_R, 50 -CTGGTGGTGCAACAACCTTA
M13 forward (-20), 50 -GTAAAACGACGGCCAG
M13 reverse, 50 -CAGGAAACAGCTATGAC
pMDC32F, 50 -TGTTTGAACGATCGGGGAAATTCGAGCTCC
pMDC32R, 50 -GGATCCCCGGGTACCGGGCC

liquid medium containing antibiotics kanamycin (50 mg/L)

and rifampicin (10 mg/L). Cells were harvested from
overnight-grown cultures, resuspended in infiltration buffer
(10 mM MES and 200 lM acetosyringone), and incubated
for 3 h at room temperature. After incubation, OD600 was
adjusted to 1, Agrobacterium strains carrying TRV1 and
TRV2-LIC derivatives were mixed at 1:1 ratio and 0.5 mL
of the resultant mixture was injected into 2nd and 3rd
leaves of 1-month-old plants using a needleless syringe.
Naming of each set of infiltrated plants was done according
to the specialty of TRV2 vector, mock (vector control) for
TRV2::00, TRV2::GAPDH for plants infiltrated with
TRV2 vector carrying VIGS fragment for silencing GAPDH gene (Fig. 1). Agro-infiltrated plants were maintained
at 19 2 C for a week, and then the temperature was
raised to usual 23 2 C. Photoperiod was maintained as
stated earlier.
Assessing the progression of gene silencing and its
quantification
To visually assess the general success and progression of
gene silencing, phytoene desaturase (PDS) gene was
silenced in tobacco. A 361-bp long VIGS fragment for
silencing PDS gene was isolated from tobacco; primers
used for fragment isolation are given in Table 1. Cloning
and the silencing procedure followed were the same as that
used for GAPDH gene silencing. PDS gene silencing is
commonly used as a marker to test the general success and
progression of gene silencing as its effect can be visually

Amplification of casstte carrying gene from

pENTRTM Directional TOPO vector
PCR confirmation of insertion of gene carrying
cassate into plant genome

confirmed. Silencing of the PDS gene causes photobleaching in plants, especially the newly developing leaves
turn white (Fig. 1). To quantify the silencing of GAPDH
gene in GAPDH-silenced plants, the leaves were collected
from wild-type, mock and GAPDH-silenced plants and
real-time PCR was performed. Expression of GAPDH in
mock and GAPDH-silenced plants was compared to that of
wild-type plants. Expression of a housekeeping gene
Elongation factor-1 alpha was used as an internal control to
normalize the gene expression. Specific primers used for
real-time PCR are listed in Table 1.
Preparation of leaf discs and water-deficit stress treatment
Leaf discs (13 mm in diameter) were excised from the
leaves of gene-silenced, mock and non-inoculated wildtype plants grown under non-stress conditions at 20th day
after agro-infiltration. Well-expanded young leaves of
similar size and maturity same as that of leaf marked with
star sign in the picture of PDS-silenced plant (Fig. 1) were
used for making leaf discs. On average, 10 leaf discs were
made from one leaf; one leaf each from eight plants was
used for the experiment, making a total of 80 leaf discs.
Stress was imposed by incubating leaf discs (five Petri
dishes with nine leaf discs each) in 30 % polyethylene
glycol (PEG-8000, Sigma Aldrich Inc., St. Louis, MO,
USA) solution in water; for control treatment, leaf discs
(five Petri dishes with nine leaf discs each) were floated on
sterile water (Fig. 2). After 60 h, photographs were taken,
and samples were collected to quantify cell viability and

123

Plant Cell Tiss Organ Cult

Fig. 1 Wild type, mock, TRV2::GAPDH, PTRV2::PDS plants after 50 days of growth (20 days after agro-infiltration). Leafs having similar size
and maturity to that of the one marked with star sign in PTRV2::PDS plants is used for making leaf discs

Fig. 2 Drought stress assay. Water deficit stress treatment was imposed by incubating leaf disc on a solution containing 30 % PEG in sterile
water, for control treatment leaf disc was floated on sterile water. Photographs were taken after 60 h of treatment

chlorophyll content. The whole experiment was repeated

twice.
Determination of cell viability and total chlorophyll
content of leaf discs
2,3,5-Triphenyl-2H tetrazolium chloride (TTC) (Alfa Aesar., Heysham, England) assay was performed to assess the
extent of cell viability. TTC solution (0.4 %) was prepared
in sodium phosphate buffer (50 mM, pH 7.4). Three leaf
discs were incubated in 1 mL of TTC solution at room
temperature for 12 h. Later the leaf discs were rinsed twice
in sterile distilled water and boiled for 30 m at 95 C in
1.25 mL of ethanol to extract the formazone. Total volume
was adjusted to 1.25 mL with ethanol, and the absorbance
was measured at 485 nm using a UV-visible spectrophotometer (Shimadzu, UV-2550, Japan). Chlorophyll content

123

in leaf discs was determined by the method of Arnon

(1994). Three leaf discs were grounded in 2 mL of 80 %
acetone, mixed well, and kept at 4 C overnight in the dark.
Supernatant was collected after centrifugation (5,000 rpm),
absorbance was recorded at 663, 645, and 652 nm, and the
amount of total chlorophyll was calculated.
Generation of GAPDH overexpressing potato
transgenic plants and analysis of their drought tolerance
Gene isolation, plasmid construction, plant transformation
and regeneration
Full length cDNA coding for GAPDH was isolated from
specific plasmid carrying the gene from a potato cDNA
expression library prepared in our lab for a previous work
(Sajeesh et al. 2013). Its sequence can be found in

Plant Cell Tiss Organ Cult

GENBANK under the accession number JX845304. See

Table 1 for the specific primers used for gene isolation. A
modified Gateway cloning method described by KamalKumar et al. (2012) was used for cloning. In short, an entry
clone is generated by using pENTRTM Directional TOPO
Cloning Kit (Invitrogen, CA, USA), and then the cassette
carrying the gene was PCR amplified from entry clone
using M13 forward (-20) and M13 reverse primers
(Table 1). This cassette was further mobilized into a plant
overexpression vector PMDC 32 by LR recombination (LR
Clonase II TM enzyme mix, Invitrogen, CA, USA). The
resultant vector carrying the gene was mobilized into the
Agrobactrium (strain GV3101) for plant transformation.
Leaves of 1-month-old potato plants (Solanum tuberosum L. cv. Earlaine) generated from nodal cultures on MS
(Murashige and Skoog 1962) basal medium containing 2 %
sucrose was used for transformation. The procedure used
for transformation was the same as that reported by
Banerjee et al. (2006) except that the antibiotic hygromycin
(20 mg/L) was used instead of kanamycin for selection of
putative transformants. Putative transformants were multiplied by nodal culture, and the insertion of the cassette
carrying the transgene into genomic DNA was confirmed
both by PCR (for primers used, see Table 1) as well as by
Southern blotting. For Southern blotting, genomic DNA
(40 lg) isolated from wild-type and putative transgenic
plants were digested overnight with the restriction enzyme
EcoRI, fractionated on an agarose gel, blotted onto a nylon
membrane, and hybridized with a probe for hygromycin
gene. The probes used were previously labeled using a
Biotin DecaLabel kit (K0652, Fermentas, EU), and the
bands hybridised with the probe was detected by using the
Biotin Chromogenic Detection Kit (K0661, Fermentas,
EU).
Drought treatment of potato and data collection
In vitro drought stress assay by using PEG infused
media For in vitro drought stress treatment, nodal segments of 1-month-old in vitro grown potato plants (T0
generation) were used. Nodal segments were cut from
wild-type and transgenic plants and inoculated into plates
containing MS-20 basal media for 2 days, then the nodes
were subcultured into boxes containing MS-20MES media
with 0, 7, and 10 % PEG for stress study (Table 2). Cultures were grown under standard conditions (25 2 C
and 16 h photoperiod). Observations and sample collection
for quantification of stress was done after 5 weeks. Whole
plant fresh weight and shoot length were measured. For
comparison of the tolerance of wild type and transgenic
plants to drought stress, we calculated the plant tolerance
index (PTI) for plants grown in 7 % PEG containing media

as average of whole plant fresh weight/average of whole

plant fresh weight under drought.
Drought stress assay in plant room using plants grown
in pots containing soil-less potting mix
Three weeks old in vitro grown wild-type and transgenic
(T0 generation) potato plants developed from nodal cultures were transferred to pots (10 cm height 9 10 cm
diameter) containing soil-less potting mixture (Professional
growing mix, SKU: 505, Sun Gro. Horticulture Ltd, Canada). Plants were grown in plant room under 25 2 C,
16 h light and 8 h dark, and relative humidity of 5560 %.
Pots were irrigated to its maximum capacity every 3 days;
in addition, 50 mL of fertilizer solution (Hyponex,
N:P:K = 20:20:20, 1 g/L) was applied twice in a week.
Five weeks old uniformly grown plants were taken for
the drought stress experiment. Twelve plants each of wildtype and two transgenic lines (L1 and L2) were used for the
drought stress experiment. For control treatment, plants
were routinely irrigated, whereas for drought treatment,
water supply was withheld for 21 days. On the 21st day of
withholding water, photographs were taken, shoot length
and shoot fresh weight were measured to quantify the
effects of stress on plant growth. PTIs was calculated as
PTIs = average fresh weight of shoots under stress/average fresh weight of shoots under drought. Expression of
GAPDH gene in leaves of each type of plants, both under
control and drought conditions, was quantified by real-time
PCR.
Statistical analysis
The statistical significance of the data was tested by analysis of variance (AGRES ANOVA package version 7.01;
TNAU, India). The least significance test was used to
detect the differences among treatments.

Results
Accuracy of sequence of VIGS fragments cloned
into TRV2-LIC vector
The VIGS fragments for silencing GAPDH and PDS genes
were isolated and successfully cloned into TRV2-LIC
vector. VIGS fragments were sequenced by using vectorspecific sequencing primers and intactness of the sequences
was confirmed by BLAST analysis. Sequences matched
100 % to the earlier reported tobacco sequences deposited
in GENBANK (JQ256517.1 for GAPDH and EU165355.1
for PDS).

123

Plant Cell Tiss Organ Cult

Table 2 List of different types of media, overlay solutions and their
preparation
Name of the medium

Components and method of preparation

MS-20

4.40519 g/L ready made MS salts and

vitamins (Duchefa Biochemie), 20 g/L
sucrose, 0.7 % plant agar, pH to 5.7,
Autoclave to sterilize

MS-20 MES (control

media)

4.40519 g/L ready made MS salts and

vitamins (Duchefa Biochemie), 20 g/L
sucrose, 4.2 g/L MES Monohydrate
(Duchefa Biochemie), 0.7 % plant agar,
pH to 5.7 Autoclave to sterilize

7 % PEG containing
overlay solution

Add all the components for making one

liter of MS-20 MES media except plant
agar and make up volume to 930 mL
with double distilled water, adjust pH to
5.7 and autoclave. After autoclaving
bring the media to laminar air flow and
add 70 g of PEG-8000

10 % PEG containing
overlay solution

Add all the components for making one

liter of MS-20 MES media except plant
agar and make up volume to 900 mL
with double distilled water, adjust pH to
5.7 and autoclave. After autoclaving
bring the media to laminar air flow and
add 100 g of PEG-8000

MS-20 MES media

containing 7 % PEG
(7 % PEG containing
stress media)

To a culture box containing 50 mL of

solidied MS-20 MES media add 75 mL
of 7 % PEG containing overlay solution
keep for 1618 h and then decant the
over lay solution

MS-20 MES media

containing 10 % PEG
(10 % PEG containing
stress media)

To a culture box containing 50 mL of

solidied MS-20 MES media add 75 mL
of 10 % PEG containing overlay
solution keep for 1618 h and then
decant the over lay solution

Efficiency of down-regulation by VIGS

and studied its effect on drought tolerance using a simple

leaf disc assay. Leaf discs from the gene-silenced plants
were found to be severely affected by drought stress
compared to leaf discs made from wild-type or mock
infiltrated plants (Fig. 2). Under stress conditions, cell
viability in leaf discs is reduced by 18 % in wild-type as
well as mock infiltrated plants as compared to the respective non-stressed controls, whereas it was reduced by
36.6 % in gene-silenced leaf discs (Fig. 3). Under drought
stress, the percentage of reduction in chlorophyll content
was 3436 % for wild and mock infiltrated plants, whereas
it was 64 % in gene-silenced leaf discs (Fig. 4). Overall,
the negative effects of stress were significantly higher for
leaf discs made from GAPDH-silenced plants, implying the
importance GAPDH in drought tolerance of plants.

Fig. 3 Cell viability of wild-type, mock and GAPDH silenced leaf

discs under stress and non-stress. The values are mean SE, n = 6.
The different letters above the columns indicate significant differences at P \ 0.05

PDS gene silencing by VIGS was used as a visible marker

to check the general success and progression of VIGS
procedure used in this study. The method was found
effective in silencing gene, and the newly developed leaves
of the PDS gene-silenced plants turned white (Fig. 1). To
check the efficiency of the VIGS system used here in
down-regulation of GAPDH gene, its expression was
quantified in GAPDH silenced, mock infiltrated and wildtype plants by real-time PCR. Relative to wild-type plants,
there was not much change in GAPDH expression in mock
infiltrated plants, whereas it was reduced by 40 5 % in
GAPDH-silenced plants.
Effect of GAPDH gene silencing on water-deficit stress
tolerance
To infer the significance of GAPDH in drought tolerance of
plants, we down-regulated its gene expression in tobacco

123

Fig. 4 Effect of water deficit on chlorophyll content of wild-type,

mock and GAPDH silenced leaf discs. Total chlorophyll was
estimated from both stressed (after 60 h) and non-stressed leaf discs
and percent reduction over non-stressed was calculated. The values
are mean SE, n = 6. The different letters above the columns
indicate significant differences at P \ 0.05

Plant Cell Tiss Organ Cult

Comparison of the performance of GAPDH

overexpressing transgenic plants with its wild type
under control as well as drought stress conditions
To know whether the overexpression of GAPDH gene can
improve drought tolerance of higher plants, we generated
transgenic potato plants constitutively overexpressing
GAPDH gene. Two lines of transgenic potato plants L1 and
L2 (Fig. 5) each having a single copy of trans-gene were
used for the experiment (electronic supplementary material, Fig. 1). Both transgenic and wild-type plants were
grown on media containing 0, 7, and 10 % PEG to test their
tolerance to drought. Observations were made after
5 weeks of growth, both wild-type and transgenic plants
grew equally well in the control media, whereas in PEGcontaining stress media the growth of transgenics was
better than that of wild type (Fig. 6). Total weight and
shoot length were measured; both the parameters were
almost the same for all the plants grown in control media,
whereas there was a clear difference between wild-type and
transgenic plants when they were grown in 7 and 10 %
PEG-containing media (Figs. 7, 8). Wild-type plants have
an average height of 5 cm in 7 % PEG media, whereas
transgenic plant lines L1 and L2 have an average height of

Fig. 5 Development of putative transgenic plants from co-cultivated

potato leaf and their confirmation by PCR. a, b Development of callus
on callus induction media. c Development of shoot on shoot induction
media. d Shoot elongation and rooting on MS basal media. e Gel

7.3 and 7 cm, respectively. Plant growth was higher for

transgenics over wild-type plants at 7 %. A similar trend of
reduced growth of wild types over transgenics was
observed in 10 % PEG-containing media (Figs. 6, 7, 8). To
compare the effect of drought on plants, plant tolerance
index (PTI = weight under stress/weight under normal
condition) was calculated for plants grown in media containing 7 % PEG, and it was found that PTI for wild-type
plants was 0.122 and for transgenic lines L1 and L2 it was
0.421 and 0.292, respectively. Both the transgenic lines
have more than double the PTI scores of wild-type plants,
confirming improved performance of transgenics over
wild-type plants under drought stress.
Furthermore, drought stress assay was done with plants
grown in soil-less potting mixture. In this case, for droughtstress induction, water supply was withheld for 21 days.
Both the wild-type and transgenic plants grew equally well
under control condition (Shoot length *21 cm and shoot
weight *6.8 g), whereas growth of transgenics under
drought condition was better than that of wild type (Fig. 9).
Wild-type plants have an average height of 12.5 cm under
drought condition, whereas the height of transgenic plant
lines L1 and L2 was 16 and 15.9 cm, respectively
(Fig. 10). Similarly, shoot fresh weight was higher for

picture showing PCR amplification of transgene (Lanes, P? = Positive plasmid, M = 1 kb marker, W = wild type potato plants, L1 and
L2 = two different lines of transgenic potato

123

Plant Cell Tiss Organ Cult

Fig. 6 Morphological aspect of wild-type (W) and transgenic plants (L1 and L2). Picture taken after 5 weeks of growth in control and stress
media (containing 7 and 10 % PEG)

transgenics over wild-type plants under stress conditions

(Fig. 11). Tolerance index-s (PTIs) of wild-type plants was
0.41 and for transgenic lines L1 and L2 it was 0.57, which
is more than 16 % higher than the wild-type plants, confirming improved performance of transgenics over wildtype plants under drought stress. Expression of the GAPDH
gene under the nonstress condition was more than sixfold
higher in transgenics compared to wild-type plants. Even
under drought stress, higher expression of GAPDH was
observed in transgenics compared to wild-type plants
(Fig. 12). Overall, transgenic plants (potato) constitutively
overexpressing GAPDH were found to grow better than the
wild-type plants under drought stress.

stress tolerance mechanism of crop plants (potato) could

help faster development of its stress-tolerant cultivars.
Previous yeast-based functional screening study performed
in our laboratory found that overexpression of potato
GAPDH gene in yeast enhanced the drought tolerance of
yeast cells, and the gene was shortlisted as one of the most
important genes to be considered for a detailed study on its
role in drought tolerance of potato/higher plants (Sajeesh
et al. 2013). Hence, the present study was aimed to educe
the role (relevance) of GAPDH in drought tolerance of
potato/higher plants.

Discussion
Global warming and the consequent shortage of water is a
major threat for profitable cultivation of crop plants in
many parts of the world. Development and supply of
drought-tolerant cultivars of major crops like potato could
help farmers to curb this negative effect of global warming
on world agriculture production. Cultivated potatoes are
extremely sensitive to drought stress. Even a trivial water
shortage can cause a reduction in leaf size and photosynthesis, and consequently affects the number, size, and the
percentage of marketable tubers (Fabeiro et al. 2001; van
Loon 1981). Because of extreme sensitivity to drought
stress and the availability of whole genome sequence
information, potato plant is emerging as a model crop plant
for studying drought tolerance mechanisms working in
crop plants. Identification of key genes involved in the

123

Fig. 7 Total weight of wild type (W) and transgenic (Lines L1 and
L2) potato plants under different treatments. The values are mean of
two independent experiments SE. The different letters above the
columns indicate significant differences at P \ 0.05

Plant Cell Tiss Organ Cult

Fig. 8 Shoot length of wild type (W) and transgenic (Lines L1 and
L2) potato plants under different treatments. The values are mean of
two independent experiments SE. The different letters above the
columns indicate significant differences at P \ 0.05

To get a quick confirmation of the relevance of GAPDH

in drought tolerance of higher plants, its gene expression
was down-regulated in tobacco, and leaf discs made from
silenced and nonsilenced plants were used for the drought
stress assay. VIGS procedure used for down-regulation of
GAPDH in tobacco was successful in significantly reducing the GAPDH gene expression. Leaf discs made from
silenced plants were found to be severely affected by
drought stress when compared to leaf discs made from
nonsilenced plants (Fig. 2). Under stress conditions,
reduction in the amount of viable cells and content of
chlorophyll was significantly higher in leaf discs made
from silenced plants than in the leaf discs made from wildtype and/or mock-infiltrated plants (Figs. 3, 4). These

findings clearly show that when the GAPDH gene

expression is down-regulated, plant cells/tissue became
less tolerant to drought stress, implying the importance of
GAPDH in drought tolerance of plants. Furthermore, to
substantiate the inference from the VIGS study, potato
transgenic plants constitutively overexpressing GAPDH
gene were generated and its performance under drought
stress was studied. It was found that under water-deficit
stress created by adding PEG in culture media, the transgenic plants grew better than the wild-type plants (Fig. 6).
Under stress, growth indices like total plant weight
(Fig. 7), shoot length (Fig. 8), and PTI were higher for
transgenic plants than the wild-type plants, confirming a
clear improvement in drought tolerance up on constitutive
overexpression of GAPDH. Similarly, it was found that
under water-deficit stress created by withholding water
supply to the plants grown in pots containing soil-less
potting mixture, the transgenic plants grew better than the
wild-type plants (Fig. 9). Shoot length (Fig. 10), shoot
weight (Fig. 11), and PTIs were higher for transgenic
plants than the wild-type plants under drought stress,
reconfirming an obvious improvement in drought tolerance
of potato up on constitutive overexpression of GAPDH.
Overall, transgenic overexpression of the GAPDH gene in
yeast improved its drought tolerance (Sajeesh et al. 2013),
its down-regulation in tobacco increased its susceptibility
to drought, and its constitutive overexpression in potato
improved drought tolerance in potato plants. Altogether,
these findings clearly show that the altered expression of
GAPDH can effect drought tolerance of plants and
enhanced expression of GAPDH under stress will help
plants to survive better under water-deficit stress.

Fig. 9 Morphological aspect of wild-type (W) and transgenic plants (L1 and L2). Picture was taken on 21st day after withholding water supply

123

Plant Cell Tiss Organ Cult

Fig. 10 Shoot length of wild-type (W) and transgenic (Lines L1 and

L2) potato plants under different treatments. The values are
mean SE, n = 6. The different letters above the columns indicate
significant differences at P \ 0.05

Fig. 11 Shoot fresh-weight of wild-type (W) and transgenic (Lines

L1 and L2) potato plants under different treatments. The values
are SE, n = 6. The different letters above the columns indicate
significant differences at P \ 0.05

GAPDH is one of the essential enzymes in glycolysis.

Drought stress induces GAPDH expression in potato
(Sajeesh et al. 2013); its increased expression may be one
of the natural mechanisms in potato/crop plants to adapt to
drought stress. GAPDH may act as a mediator of stressinduced metabolic responses and other integrated metabolic changes during stress, and may also act to provide
additional energy needed for the cellular adjustment
required for growth under stress, by channeling carbon
away from glycerol into the pathway, leading to glycolysis
and ATP formation (Jeong et al. 2000). In this study,
constitutive overexpression of GAPDH was found to help
transgenic plants in maintaining a higher level of GAPDH
compared to wild-type plants both under stress and nonstress conditions (Fig. 12). Improved growth of GAPDHoverexpressing transgenic plants under stress, which is

123

Fig. 12 Fold change in expression level of GAPDH genes in potato.

W = wild type, L1 = Transgenic line 1 and L2 = Transgenic line 2.
The values are mean SE, n = 6. The different letters above the
columns indicate significant differences at P \ 0.05

observed in this study, could be due to its additional ability

to channel more energy for growth and development than
the wild-type plants. Another reason for its relatively better
growth under stress could be its added ability to limit the
formation of methyl-glyoxal (MG), a cytotoxic by-product
produced mainly from intermediates of glycolysis called
triose-phosphates (dihydroxyacetone phosphate and glyceraldehyde-3-phosphate). The high rate of glycolysis
under stress could lead to increased formation of triosephosphates resulting in higher production of MG (Sommer
et al. 2001). Relatively higher expression of GAPDH in
transgenic potato might have reduced the pool of triosephosphates in its cells, leading to less accumulation of MG
under stress than in wild-type plants. Higher level of MG
inhibits cell proliferation (Ray et al. 1994); its accumulation in cells results in increased degradation of proteins,
DNA modification, and inactivation of antioxidant defense
system (Martins et al. 2001). Hence, under drought condition, constitutive overexpression of GAPDH might have
helped the cells of transgenic potato plants in reducing the
accumulation of MG, leading to a better cell proliferation
rate and overall plant growth than the wild-type plants.
However, not much is known about the role of GAPDH in
drought tolerance, except that its constitutive overexpression improved stress tolerance of yeast and plants. A more
detailed biochemical as well as transcriptomic analysis of
GAPDH-overexpressing transgenic plants could help us in
deducing its mechanism of action in improving drought
tolerance of plants.
In conclusion, the present study confirmed that GAPDH,
which was identified as a potential drought-tolerance gene
from our previous yeast-based functional screening study, has
an important role to play in drought tolerance of higher plants,
and its constitutive overexpression by genetic engineering can
improve drought tolerance of crop plants like potato.

Plant Cell Tiss Organ Cult

Acknowledgments This work was supported by a Grant from the
Next-Generation BioGreen 21 Program (No. PJ008182), Rural
Development Administration, Republic of Korea.

References
Arnon DI (1994) Copper enzymes in isolated chloroplasts: polyphenoloxidase in beta vulgaris. Plant Physiol 24:15
Banerjee AK, Prat S, Hannapel DJ (2006) Efficient production of
transgenic potato (S. tuberosum L. ssp. andigena) plants via
Agrobacterium tumefaciens-mediated transformation. Plant Sci
170:732738
Burch-Smith TM, Anderson JC, Martin GB, Dinesh-Kumar SP (2004)
Applications and advantages of virus induced gene silencing for
gene function studies in plants. Plant J 39:734746
Demircan T, Akkaya MS (2010) Virus induced gene silencing in
Brachypodium distachyon, a model organism for cereals. Plant
Cell Tissue Org Cult 100:9196
Dong Y, Burch-Smith TM, Liu Y, Mamillapalli P, Dinesh Kumar SP
(2007) A ligation-independent cloning tobacco rattle virus vector
for high-throughput virus-induced gene silencing identifies roles
for NbMADS4-1 and -2 in floral development. Plant Physiol
145:11611170
Eswaran N, Parameswaran S, Sathram B, Anantharam B, Kumar
GRK, Tangirala SJ (2010) Yeats functional screen to identify
genetic determinants capable of conferring abiotic stress tolerance in Jatropha curcas. BMC Biotechnol 10:23
Fabeiro C, Olalla MS, de Juan JA (2001) Yield and size of deficit
irrigated potatoes. Agric Water Manag 48:255266
Gilani GS, Nasim A (2007) Impact of foods nutritionally enhanced
through biotechnology in alleviating malnutrition in developing
countries. J AOAC Int 90:14401444
Iwama K, Yamaguchi J (2006) Abiotic stresses. In: Gopal J, Khurana
SMP (eds) Handbook of potato production, improvement and
post-harvest management. Food Product Press, New York,
pp 231278
Jeong MJ, Park SC, Kwon HB, Byun MO (2000) Isolation and
characterization of the gene encoding glyceraldehyde-3-phosphate dehydrogenase. Biochem Biophys Res Commun
278:192196
Jiang Y, Ye S, Wang L, Duan Y, Lu W, Liu H, Fan D, Zhang F, Luo
K (2014) Heterologous gene silencing induced by tobacco rattle
virus (TRV) is efficient for pursuing functional genomics studies
in woody plants. Plant Cell Tissue Org Cult 116:163174
Kamal-Kumar YS, Purayannur S, Verma PK (2012) An alternative
approach in gateway cloning when the bacterial antibiotic
selection cassettes of the entry clone and destination vector are
the same. Mol Biotechnol. doi:10.1007/s12033-012-9549-0
Ku H-M, Hu CC, Chang HJ, Lin YT, Jan FJ, Chen CT (2011)
Analysis by virus induced gene silencing of the expression of
two proline biosynthetic pathway genes in Nicotiana benthamiana under stress conditions. Plant Physiol Biochem
49(10):11471154

Liu Y, Schiff M, Marathe R, Dinesh-Kumar SP (2002) Tobacco Rar1,

EDS1 and NPR1/NIM1 like genes are required for N mediated
resistance to tobacco mosaic virus. Plant J 30:415429
Lynch DR, Tai GCC (1989) Yield and yield component response of
eight potato genotypes to water stress. Crop Sci 29:12071211
Martin RC, Glover-Cutter K, Martin RR, Dombrowski JE (2013)
Virus induced gene silencing in Lolium temulentum. Plant Cell
Tissue Org Cult 113:163171
Martins AMTBS, Cordeiro CAA, Freire AMJP (2001) In situ analysis
of methylglyoxal metabolism in Saccharomyces cerevisiae.
FEBS Lett 499:4144
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue culture. Physiol Plant 15:473497
Priyanka B, Sekhar K, Reddy VD, Rao KV (2010) Expression of
pigeonpea hybrid-proline-rich protein encoding gene
(CcHyPRP) in yeast and Arabidopsis affords multiple abiotic
stress tolerance. Plant Biotechnol J 8:7687
Ramegowda V, Senthil-kumar M, Udayakumar M, Kirankumar SM
(2013) A high-throughput virus-induced gene silencing protocol
identifies genes involved in multi-stress tolerance. BMC Plant
Biol 13:193
Ratcliff F, Martin-Hernandez AM, Baulcombe DC (2001) Tobacco
rattle virus as a vector for analysis of gene function by silencing.
Plant J 25:237245
Ray S, Dutta S, Halder J, Ray M (1994) Inhibition of electron flow
through complex I of the mitochondrial respiratory chain of
Ehrlich ascites carcinoma cells by methylglyoxal. Biochem J
303:6972
Ruiz MT, Voinnet O, Baulcombe DC (1998) Initiation and maintenance of virus-induced gene silencing. Plant Cell 10:937946
Sajeesh K, Yu JW, Gangadhar BH, Park SW (2013) Rapid
identification of potential drought tolerance genes from Solanum
tuberosum by using a yeast functional screening method. CR
Biol 336:530545
Senthil-Kumar M, Govind G, Kang L, Kirankumar SM, Udayakumar
M (2007) Functional characterization of Nicotiana benthamiana
homologs of peanut water deficit-induced genes by virusinduced gene silencing. Planta 225:523539
Sommer A, Fischer P, Krause K, Boettcher K, Brophy PM, Walter
RD, Liebau E (2001) A stress responsive glyoxalase I from the
parasitic nematode Onchocerca volvulus. Biochem J
353:445452
Tian J, Cheng L, Han Z, Yao Y (2014) Tobacco rattle virus mediated
gene silencing in strawberry plants. Plant Cell Tissue Org Cult.
doi:10.1007/s11240-014-0669-z
van Loon CD (1981) The effect of water stress on potato growth,
development and yield. Am J Potato Res 58:5169
Weisz R, Kaminski J, Smilowitz Z (1994) Water deficit effects on
potato leaf growth and transpiration: utilizing fraction extractable soil water for comparison with other crops. Am J Potato Res
71:829840
Zhu X, Dinesh-Kumar SP (2009) Virus-induced gene silencing
(VIGS) to study gene function in plants. In: Doran T, Helliwell C
(eds) RNA interference: principles and protocols. CABI, Wallingford, pp 2630

123