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DOI 10.1007/s11240-014-0684-0
ORIGINAL PAPER
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,
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
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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
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Purpose
VFGAPDH_N.bF, 50 -CGACGACAAGACCCTGTTGTGATCTCTGCTCCTAGC
VRGAPDH_N.bR, 50 -GAGGAGAAGAGCCCTCCATTCCAGTCAATTTTCCA
NbEF1a RT_F, 50 -TGGTGTCCTCAAGCCTGGTATGGTTGT
Real time PCR primer for tobacco elongation factor1 alpha gene (internal control)
NbGAPDHRT_R, 50 -TTCTTGGCACCACCCTTCAA
VFPDS_N.bF, 50 -CGACGACAAGACCCTCCACGACCCGAAGATTGACA
VFPDS_N.bR, 50 -GAGGAGAAGAGCCCTAGTTCTCCAAACAGGTTCTGC
StGAPDH_FF, 50 -CACCATGGCTAAGGTTAAGATTGGAATTAACG
StGAPDH_FR, 5 -TTACTGAACTGATGACATGTGAATAATCAAGT
StGAPDHRT_F, 50 -ATGGACCATCAGCCAAGGATTGGA
StGAPDHRT_R, 50 -ACACATCAACAGTTGGGACTCGGA
StACTINRT_F, 50 -GAATGGAAGCAGCTGGAATC
StACTINRT_R, 50 -CTGGTGGTGCAACAACCTTA
M13 forward (-20), 50 -GTAAAACGACGGCCAG
M13 reverse, 50 -CAGGAAACAGCTATGAC
pMDC32F, 50 -TGTTTGAACGATCGGGGAAATTCGAGCTCC
pMDC32R, 50 -GGATCCCCGGGTACCGGGCC
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
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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
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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).
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MS-20
7 % PEG containing
overlay solution
10 % PEG containing
overlay solution
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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
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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)
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
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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
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
Fig. 9 Morphological aspect of wild-type (W) and transgenic plants (L1 and L2). Picture was taken on 21st day after withholding water supply
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