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Article history:
Received 24 June 2013
Accepted after revision 28 September 2013
Available online 8 November 2013
Identication of major stress tolerance genes of a crop plant is important for the rapid
development of its stress-tolerant cultivar. Here, we used a yeast functional screen method
to identify potential drought-tolerance genes from a potato plant. A cDNA expression
library was constructed from hyperosmotic stressed potato plants. The yeast transformants expressing different cDNAs were selected for their ability to survive in hyperosmotic
stress conditions. The relative tolerances of the selected yeast transformants to multiple
abiotic stresses were also studied. Specic potato cDNAs expressed in the tolerant yeast
transformants were identied. Sixty-nine genes were found capable of enhancing
hyperosmotic stress tolerance of yeast. Based on the relative tolerance data generated,
12 genes were selected, which could be most effective in imparting higher drought
tolerance to potato with better survival in salt and high-temperature stresses. Orthologues
of few genes identied here are previously known to increase osmotic stress tolerance of
yeast and plants; however, specic studies are needed to conrm their role in the osmotic
stress tolerance of potato.
2013 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.
Keywords:
cDNA library
Drought
Solanum tuberosum
Tolerance genes
Yeast functional screening
1. Introduction
The actual productivity of crops usually falls far short of
its maximum potential, mainly due to various environmental constraints, such as drought, extremes in temperature, salinity, etc. Such constraints are expected to
augment in coming decades due to global warming and
associated changes in climate. Thus, to sustain the present
rate of increase in agriculture production, it is important to
develop improved varieties of crop plants with higher
tolerance to various stresses. Better understanding of the
mechanisms by which a plant can cope with adverse
* Corresponding author.
E-mail address: sewpark@konkuk.ac.kr (S.W. Park).
1
These authors contributed equally to this work.
environments is important for the development of stresstolerant plants. Studies in various plant species, especially
in model plants such as Arabidopsis and tobacco have
revealed various mechanisms involved in stress tolerance
of plants, and those mechanisms were summarized in
various reviews [13]. To make use of accumulating
information on stress tolerance mechanisms of plants for
the development of stress-tolerant varieties of a particular
agricultural crop, it is important to investigate the specic
stress tolerance mechanisms of that particular crop too.
Therefore, it is time for us to strengthen our understanding
of the stress tolerance mechanisms working in agriculturally important plants, such as potato, tomato, rice, etc.
Potato (Solanum tuberosum L.) is highly sensitive to
environmental stress. The effect of environmental constraints on potato cultivation is well reected in the
discrepancy between its average and record yields;
1631-0691/$ see front matter 2013 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.crvi.2013.09.006
531
532
Fig. 1. Different stages of in vitro potato plantlet development through nodal culture. Color online.
533
media [CM media], see Table 1), devoid of uracil, was used
for the selection of yeast transformants. For the selection of
drought stress-tolerant transformants of yeast, a synthetic
dened medium containing sorbitol, galactose and rafnose (drought selection media [DSM], see Table 1) was
used. To set an optimum concentration of sorbitol in the
selection medium for the selection of drought-tolerant
yeast transformants, an empty vector was transformed
into the yeast strain BY4741 to result in a control yeast cell.
A two-day-old single colony of the control yeast cell grown
on a CM selection plate was selected and inoculated in
5 mL of liquid CM medium, which was then grown
overnight; 100 mL of 103 dilution of the culture was
plated on DSM media containing various concentrations of
sorbitol (1.75 M, 2 M, 2.1 M, and 2.2 M) and was grown for
ve days. The minimum concentration (Fig. 3) at which the
control yeast cells failed to grow was selected to be used
for the screening of drought-tolerant yeast transformants.
Unless specied, the yeast cultures were grown at 30 8C
and for liquid broth cultures, the shaker was set to 250
rotations per minute.
2.8. Transformation of potato cDNA library into yeast and
screening for drought tolerance
The transformation of yeast was performed by following the method described by Benatuil et al. [21]. Briey,
300 mL of yeast competent cells were transformed with
5 mL of library plasmid (500 ng of plasmid per mL) by
electro-transformation using a Micro Pulser (Bio-Rad,
California, USA); the transformation product was grown
in half-strength YPD medium (1% yeast extract + 2%
peptone + 2% dextrose) containing 0.5 M of sorbitol for
1 h, and the cells were then collected by centrifugation and
were re-suspended in 1 mL of the CM medium.
To determine the effect of increasing concentrations of
sorbitol on the survival of yeast transformants, we plated
100 mL of the re-suspended transformation products on
DSM plates containing varying concentrations of sorbitol
(0 M, 1 M, 1.5 M, 1.75 M, 2 M, and 2.1 M), and incubated for
ve days. According to the observed trend in growth of the
transformed cells on these media, DSM plates with a 2.1 M
sorbitol medium (2.1 M DSM medium) were used to select
for the drought-tolerant yeast transformants (Fig. 4). After
ve days of incubation, the colonies that had been grown
on a 2.1 M DSM medium were picked and streaked on fresh
plates containing the 2.1 M DSM medium. To compare the
growth of tolerant yeast cells to that of the control yeast
cells, the control yeast cells were streaked on each plate, as
shown in Fig. 5.
2.9. Determination of the relative tolerance of yeast colonies
to drought, salt and high-temperature stresses
To test the relative tolerance of the selected yeast
transformants to drought stress, they were grown in the
liquid CM medium overnight, and the OD at 600 nm
(OD600) was adjusted to 2 in a liquid DSM media containing
2.1 mol of sorbitol per liter. Four dilutions 101, 102, 103,
104 of the culture were made in the same liquid medium,
5 mL of each dilution were drop-plated on DSM plates
534
Table 1
Compositions of the yeast synthetic selection media.
Name of the medium
Components
1.92 g
6.7 g
20 g
100 mL
275 mL
Drought selection
media (DSM)
Salt selection
media (SSM)
1.92 g
6.7 g
Different concentrations
(1, 1.5, 1.75, 2, 2.1, 2.2 M)
20 g
100 mL
275 mL
1.92 g
6.7 g
1.2 M
20 g
100 mL
275 mL
containing 2.1 mol sorbitol per liter, and they were grown
for ve days. Yeast transformants showing growth at 104,
103, 102 and 101 were allocated a relative score of 4, 3, 2
and 1, respectively. The experiment was repeated three
times, and the average of the three readings was calculated
to compare the relative tolerance of yeast transformants to
the stress.
Similarly, to test the relative tolerance of the selected
yeast colonies to the saline stress, the OD600 of the
overnight grown culture was adjusted to 2 in liquid salt
Fig. 3. Growth of control yeast cells on drought selection media containing different concentrations of sorbitol. Color online.
Fig. 4. Growth of yeast cells transformed with the potato cDNA library on drought selection media containing different concentrations of sorbitol (the same
amount of transformation product was plated on each plate). Color online.
535
Fig. 5. Comparison of the growths of control and tolerant yeast cells. (a) Control plate without stress. (b) Drought selection plate contacting 2.1 mol of
sorbitol per liter. Control yeast transformed with empty vector (C), D1 to D11 are drought-resistant transformants of yeast. Color online.
Sr. No.
Clone ID
GenBank
accession
number
Salt
High
temperature
Multiple
stress
StDT3
StDT10
JX845303
JX839744
2
2
1
0.7
2
1.7
5
4.4
3
4
StDT11
StDT15
JX839745
JX683406
1
2
0.7
1
2
3
3.7
6
JX683407
6
7
StDT20, 94,
152, 205
StDT21
StDT22, 122
JX683408
JX683409
N-acetyltransferase
Proteinase inhibitor type 2 CEVI57
1.3
2
0
1
2
2
3.3
5
StDT24
JX683410
JX683417
Thioredoxin H-type 2
1.3
3.3
10
11
12
13
StDT25, 51,
126,166
StDT26
StDT28
StDT29, 150
StDT30, 93
JX683411
JX683412
JX683437
JX683428
2
1
2.3
1
2
0
1
0
2
2
2
2
6
3
5.3
3
14
StDT32, 71
JX683424
1.7
3.7
15
16
StDT33, 135
StDT41
JX683435
JX683414
3
2
2
2
2
2
7
6
17
StDT43
JX845304
18
19
20
21
StDT45
StDT48
StDT49, 179
StDT50
JX905218
JX683415
JX951420
JX839759
2.7
3
2
3
3
1
1
1
2
2
2
2
7.7
6
5
6
22
StDT52
JX683418
Glyceraldehyde-3-phosphate
dehydrogenase
p23 Co-chaperone
Acyl-CoA-binding protein
Hypothetical protein
RNA recognition motif containing
protein
LE25 protein
1.3
3.3
23
24
25
StDT53, 72
StDT55, 78
StDT56
JX683419
JX951424
JX896424
1.7
1.3
3
2
1
3
2
2
2
5.7
4.3
8
26
27
28
29
StDT57
StDT58
StDT59
StDT62, 188
JX683421
JX905211
JX905212
JX839758
2
1.3
3
1
0
2
3
1
2
2
2
2
4
5.3
8
4
Response to stress
Translation
Response to stress
1
2
536
Table 2
List of the identied genes, their relative ability to enhance tolerance of yeast cells to different abiotic stress and biological process in which the genes are involved.
Table 2 (Continued )
Sr. No.
Clone ID
GenBank
accession
number
Salt
High
temperature
Multiple
stress
1
1
1
1.3
0.7
1
2.3
0
0
0
2
1.7
3
1
2
4
5
4
2.3
2.7
Translation
1.3
3.3
Dehydration responsive
1.3
3.3
30
31
32
33
34
StDT63
StDT64
StDT67
StDT74
StDT75
JX839746
JX839747
JX683423
JX683425
JX839748
35
StDT79
JX839749
36
StDT80
JX683426
37
StDT82
JX683427
Proteinase inhibitor I
38
39
StDT85
StDT87
JX905213
JX905214
1.3
1
0
1
2
2
3.3
4
40
41
StDT99, 111
StDT100
JX683433
JX683430
1
1
1
0
2
2
4
3
42
StDT102, 136
JX839750
1.3
4.3
43
44
45
StDT103
StDT105
StDT110
JX845305
JX683431
JX683432
1.3
3
2
1
3
2
2
1
2
4.3
7
6
46
47
48
49
50
StDT112
StDT116
StDT118
StDT120, 174
StDT121
JX905215
JX683434
JX951421
JX683440
JX905216
2.7
1.3
1.3
1
1.3
1
1
1
1
1
2
2
2
2
2
5.7
4.3
4.3
4
4.3
51
52
StDT134
StDT138
JX839752
JX839753
UDP-glucose:glucosyltransferase
ADP, ATP carrier protein
2
1.3
2
0
2
2
6
3.3
53
54
55
StDT141
StDT147
StDT149
JX951422
JX839754
JX683436
2.7
1
1
3
1
1
1
2
2
6.7
4
4
56
StDT151
JX896425
Hypothetical protein
60S ribosomal protein L1
Inducible plastid-lipid associated
protein
Tyramine N-feruloyltransferase 4/11,
partial
1.3
4.3
Drought
538
Table 2 (Continued )
Sr. No.
Clone ID
GenBank
accession
number
Salt
High
temperature
Multiple
stress
StDT154
JX845306
58
StDT160
JX839755
Fructokinase 3
59
60
StDT162
StDT171
JX683438
JX683439
1
1
1
0
2
2
4
3
61
62
StDT181
StDT182
JX683441
JX683442
1.7
2
1
2
2
2
4.7
6
63
64
65
66
StDT184
StDT185
StDT189
StDT190
JX839760
JX839757
JX683443
JX683444
Alpha-1,4-glucan-protein synthase
Lipase, partial
Hypothetical protein
Stress enhanced protein 2
2.3
2
2.3
2
1
0
1
0
1
2
2
2
4.3
4
5.3
4
67
68
StDT200
StDT202
JX951423
JX683445
Hypothetical protein
Putative thioredoxin m2
3
2.3
1
1
2
2
6
5.3
69
StDT213
JX683446
Hypothetical protein
1.7
4.7
57
539
540
Fig. 6. Representative gure of the relative tolerance test of the selected yeast colonies to drought (b), salt (c) and high temperature (d) stresses. Numbers in
the picture represent the clone ID of the cDNA expressed by the particular yeast transformant. (a) Growth under control condition. Color online.
541
Fig. 7. Fold change in expression level of different genes with respect to the corresponding controls (0 h treatment) in whole plant under drought stress (0 h/
control/without treatment, 2 h, 12 h and 24 h, n = 3, values = SE).
542
543
544
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