Running title:
Accepted Article
1
AtCBF3 enhances heat tolerance of potato plants
2 3
Corresponding author:
4
Xinghong Yang
5
College of Life Science, State Key Laboratory of Crop Biology,
6
Shandong Key Laboratory of Crop Biology, Shandong Agricultural University,
7
Taian 271018,
8
People’s Republic of China
9
E-mail: [email protected]
10
Phone: +86 538 8246167
11
Fax:
+86 538 8246167
12 13 14
Title：Potato plants ectopically expressing Arabidopsis thaliana CBF3 exhibit enhanced
15
tolerance to high-temperature stress1
16
Haiou Dou1, Kunpeng Xv1, Qingwei Meng1, Gang Li1, Xinghong Yang1*
17 18
1
19
Shandong Agricultural University, Taian 271018, People’s Republic of China
State Key Laboratory of Crop Biology，Shandong Key Laboratory of Crop Biology,
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/pce.12366 1
This article is protected by copyright. All rights reserved.
Accepted Article
20 21
*Corresponding author:
22
Xinghong Yang
23
Tel: +86 538 8246167;
24
Fax: +86 538 8246167;
25
E–mail address: [email protected]
26 27 28 29 30 31 32 33 34 35
Abstract
36
CBF3, a known cold-inducible gene that encodes a transcription factor, was isolated from
37
Arabidopsis thaliana and introduced into the potato (S. tuberosum cv. ‘luyin NO.1’) under the
38
control of the CaMV35S promoter or the rd29A promoter. Our results revealed that high
39
temperatures of 40 °C or above can significantly induce AtCBF3 expression. After heat stress,
40
the net photosynthetic rate (Pn), the maximal photochemical efficiency of PSII (Fv/Fm) and
41
the accumulation of the D1 protein were higher in the transgenic lines than in the wild-type 2
This article is protected by copyright. All rights reserved.
(WT) line. Moreover, compared to the WT line, O2·– and H2O2 accumulation in the transgenic
43
lines were reduced. A Q-PCR assay of a subset of the genes involved in photosynthesis and
44
antioxidant defense further verified the above results. Interestingly, under heat stress
45
conditions, the accumulation of HSP70 increased in the WT line, but decreased in the
46
transgenic lines. These results suggest that potato plants ectopically expressing AtCBF3
47
exhibited enhanced tolerance to high temperature, which is associated with improved
48
photosynthesis and antioxidant defense via induction of the expression of many
49
stress-inducible genes. However, this mechanism may not depend on the regulatory pathways
50
in which HSP70 is involved.
51
Key words
52
AtCBF3; high temperature; photosynthesis; antioxidant defense; potato
Accepted Article
42
53 54 55 56 57 58
Introduction High temperature is strongly detrimental to plant growth and development. Heat stress is
59
one of the primary abiotic stresses that limit plant biomass production and productivity
60
(Boyer 1982). The early changes caused by high-temperature stress involve the
61
reprogramming of signal transduction components, transcription factors and proteins
62
associated with the metabolism of reactive oxygen species (ROS) under stressful conditions.
63
Photosynthetic organisms are directly exposed to acute temperature alterations and are 3
This article is protected by copyright. All rights reserved.
considered the most heat sensitive. In the temperature region of 20-40 °C, inhibition of
65
photosynthesis in spinach (Spinacia oleracea L.) has been discovered and it is completely
66
reversible by lowering the temperature (Weis 1981). However, prolonged exposures at even
67
higher temperatures (> 40 °C) generally result in an irreversible inhibition of photosynthesis,
68
due to a disruption in thylakoid membrane lipid integrity and concomitant damage to
69
photosystem II (PSII) (Berry & Björkman 1980; Seemann et al. 1984; Bilger, Schreiber &
70
Lange 1987). In other word, a certain degree of heat stress can directly damages the
71
photosynthetic apparatus and decreases the photosynthetic rate, the photochemical efficiency
72
of PSII and the duration of the assimilate supply (Prasad et al. 2008; Prasad et al. 2009).
Accepted Article
64
73
Meanwhile, high-temperature stress can promote the accumulation of ROS in the plant,
74
particularly when the antioxidant capacity to remove ROS is low (Djanaguiraman, Prasad &
75
Seppanen 2010). Under stress conditions, ROS can damage cellular components, including
76
DNA, proteins and membrane lipids, leading to loss of function and cell death (Mittler 2002).
77
The potato (Solanum tuberosum L.) is planted in two-thirds of the countries in the world
78
and is the fourth most important food crop after rice, wheat and corn (Ortiz & Wa-tanabe
79
2004). The potato prefers a cool environment, and the major potato-producing regions are
80
located in the relatively cool climates of the northern temperate zone and Andean tropical
81
highlands. In addition, the potato is a frost-sensitive species, and its normal growth and
82
development are inhibited by high temperature. When the temperature rises above 25 °C,
83
tubers growth is arrested, and when exceeding 39 °C, growth of stems and leaves is arrested.
84
Krauss & Marschner (1984) assumed that high temperature during tuber development might
85
affect tuber yield and starch content via carbohydrate metabolism within the tuber. High 4
This article is protected by copyright. All rights reserved.
temperature not only inhibits tuber growth rate but also alters carbohydrate partitioning in
87
potato plants from tubers to shoots, which reduces overall plant yield (Borah & Milthorpe
88
1962; Ewing 1981; Wolf, Marani & Rudich 1990). Thus, high temperature is one of the most
89
important environmental factors that limit the growth and yield of potatoes.
Accepted Article
86
90
The cold-regulatory C-repeat binding factor (CBF) pathway, which is widely conserved in
91
many plants (Chew & Halliday 2010), is best characterized in Arabidopsis (Hua 2009;
92
Thomashow 2010). Arabidopsis encodes three cold-inducible CBF genes, CBF1, CBF2, and
93
CBF3 (Stockinger, Gilmour & Thomashow 1997; Gilmour et al. 1998; Medina et al. 1999), also
94
referred to as DREB1b, DREB1c, and DREB1a, respectively (Liu et al. 1998). These genes
95
encode transcription factors that are members of the APETALA2 (AP2) /
96
ethylene-responsive factor (ERF) family of DNA-binding proteins (Riechmann &
97
Meyerowitz 1998). CBF proteins bind to the C-repeat (CRT) / dehydration-responsive
98
element (DRE), a regulatory element that is present in the promoters of approximately 100
99
cold-regulated (COR) genes, and induce the expression of COR genes (Maruyama et al. 2004;
100
Fowler & Thomashow 2002; Vogel et al. 2005). Expression of the CBF genes occurs within
101
approximately 15 min during low temperature (4 °C) treatment, followed by induction of the
102
CBF downstream target genes beginning at approximately 2 to 3 h. CBF/DREB1 factors
103
appear to be ubiquitously expressed in plants regardless of their freezing tolerance capacity,
104
and the ability of ectopic CBF/DREB1 transgene activity to increase freezing tolerance has
105
been demonstrated in many plants, such as wheat and barley (Morran et al. 2011), rice (Gutha
106
& Reddy 2008) and poplar (Benedict et al. 2006). In addition, in potatoes, previous
107
examination has established that the plant is responsive to ectopic CBF transgene activity 5
This article is protected by copyright. All rights reserved.
(Pino et al. 2006). Besides, additional studies showed that the Arabidopsis CBF gene
109
(AtCBF1 and AtCBF3) can function in potatoes to increase freezing tolerance (Pino et al.
110
2007; Pino et al. 2008). Moreover, direct CBF transgene expression using a stress-inducible
111
(rd29A) promoter, which induces low background expression under non-stressful conditions,
112
significantly improves freezing tolerance without negatively impacting agronomically
113
important traits in the potato. However, expression of CBF3 under the control of a constitutive
114
promoter (CaMV 35S) might not completely mimic the activation of CBF3 by low
115
temperature (Sarah J et al. 2000).
Accepted Article
108
116
Once CBF transcription factors were confirmed to improve the freezing tolerance of plants,
117
they were successively transferred to various plants in the hope of enhancing the tolerance of
118
these plants to stress. For example, AtCBF1 gene expression vectors have been transfected
119
into tomatoes, which reduced ROS accumulation and increased the proline content,
120
significantly enhancing the resistance of the plants to low temperature and drought conditions
121
(Hsieh et al. 2002). Transfection of rice with OsCBF was found to increase the content of
122
proline and soluble sugar and its resistance to cold, drought and salt stresses (Ito et al. 2006).
123
Similarly, the AtCBF3 gene has been transfected into maize, tobacco and Arabidopsis itself,
124
also improving the cold-, drought- and salt-tolerance of the plants (Gilmour et al. 2000;
125
Al-Abed et al. 2007; Liu et al. 2011).
126
Overall, the expression of CBF genes can not only enhance the resistance of plants to low
127
temperatures but also improve the drought- and salt-tolerance of plants. Based on these results,
128
previous interpretations indicate that similar DREB/CBF genes in different plants may
129
participate in a different cellular signal transduction pathway. Under stress conditions, plants 6
This article is protected by copyright. All rights reserved.
initiate various stress signal transduction pathways, and these pathways are linked together
131
via some common components, thus forming a complex signaling network (Haake et al.
132
2002). For each individual form of DREB/CBF in the same and different plants, the condition-
133
and time-induced expressions are distinct. Moreover, the CRT/DRE element to which
134
DREB/CBF transcription factors bind is located in the promoters of some stress-responsive
135
genes, indicating that DREB/CBF genes can be involved in various stress reactions
136
(Stockinger, Gilmour & Thomashow 1997; Liu et al. 1998).
Accepted Article
130
137
Despite studies on low temperature and other stresses, to our knowledge, no report is
138
available regarding whether the CBF transcription factor functions in response to high
139
temperature to enhance plant thermo-tolerance. To examine whether CBF can improve the
140
heat resistance of plants, we performed the following study.
141
In the present study, we utilized AtCBF3-transgenic potato plants to investigate whether
142
overexpression of AtCBF3 could enhance the tolerance of the potato to high temperature. In
143
addition, we also performed a preliminary examination of the regulatory pathway that plants
144
use to resist high temperature in the presence of AtCBF3 and the mechanism involved in
145
high-temperature-induced expression of AtCBF3.
146 147
Materials and methods
148 149
Plant materials
150 151
The 35S::AtCBF3 and rd29A::AtCBF3 (rd29A, the stress-inducible promoter) constructs 7
This article is protected by copyright. All rights reserved.
were introduced into Agrobacterium tumefaciens strain EHA105 using a previously described
153
transformation method (Pino et al. 2007). Young potato leaf explants (S. tuberosum cv. ‘luyin
154
NO.1’) were utilized for transgenic plant generation. The procedures of potato transformation
155
and tissue culture were performed as previously described (Pino et al. 2007). The
156
pre-cultivation medium consisted of MS-3 % sucrose (pH 5.8) containing 1 mg L–1 of the
157
auxin indole-3-acetic acid (IAA). The co-cultivation medium consisted of MS-3 % sucrose
158
(pH 5.7) containing 1 mg L–1 IAA and 2 mg L–1 trans-Zeatin (ZT). Finally, the callus
159
induction medium consisted of MS-3 % sucrose (pH 5.8) containing 2 mg L–1 ZT, 1 mg L–1
160
IAA, 250 mg L–1 cefotaxime and 50 mg L–1 kanamycin.
Accepted Article
152
161 162
Plants and growth conditions
163 164
T0 potato plants were grown at 25 °C under a 16 h/8 h day/night photoperiod (300-400
165
μmol m−2 s−1) in a greenhouse (Pino et al. 2007). In this study, the potato plants included a
166
wild type (WT) line and two transgenic lines that expressed the A. thaliana CBF3 gene under
167
the control of the CaMV35S promoter (referred to as S1, S2) or the rd29A promoter (referred
168
to as R1, R6). After 6 weeks, a portion of the seedlings divided into five groups were exposed
169
directly to various temperatures (25, 30, 35, 40 or 45 °C) for 2 h in a growth chamber. The
170
other seedlings were subjected to heat stress (HS) (40 °C) for 4 h and then transferred to the
171
control conditions for another 24 h (post-stressed, PS). The 4th leaf from top of the seedlings
172
was used for the experiments.
173
To study the effect of Ca2+ on the expression of AtCBF3, some of the transgenic 8
This article is protected by copyright. All rights reserved.
rd29A::AtCBF3 lines grown for 3 weeks were pre-treated with an equal volume of distilled
175
water, 20 mM ethylene glycol bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) or
176
100 mM calcium chloride (CaCl2) (Knight, Trewavas & Knight 1996) dissolved in water for 1
177
h, followed by exposure to heat treatment（40 °C）for 0, 1, 2 h. All of the measurements of the
178
physiological and biochemical parameters were performed on the fully expanded leaves.
Accepted Article
174
179 180 181
Transcriptional analysis via quantitative PCR (Q-PCR) and semi-quantitative
RT-PCR
182 183
Fresh leaf samples (0.1 g) from wild type and transgenic plants were used for total RNA
184
extraction using Trizol reagent (TransGen Biotech, Beijing, China) according to the
185
manufacturer’s instructions. The cDNA was synthesized using a reverse transcription system
186
(TransGen Biotech, Beijing, China). Q-PCR was performed using a SYBR®PrimeScript™
187
RT-PCR Kit (TaKaRa, Dalian, China) in a 25μL volume using a CFX96TM Real–time
188
System (Bio-Rad). The Q-PCR amplification conditions were as follows: initial denaturation
189
at 95 °C for 30 s; 41 cycles at 95 °C for 5 s, 50 °C for 15 s and 72 °C for 15 s; and a single
190
melt cycle from 65 °C to 95 °C. Each condition contained at least three individual samples.
191
The linearity of the relationships and the amplification efficiency were examined and the data
192
analysis was performed using CFX Manager Software version 1.1 (Yao et al. 2013).
193
For semi-quantitative RT-PCR, the amplification conditions were as follows: 10 min at
194
94 °C, 30 s at 94 °C, 30 s at 50 °C and 30 s at 72 °C, followed by 10 min at 72 °C. Each cycle
195
was repeated 27 times. The RT-PCR products were separated using a 2.0 % (m v–1) agarose 9
This article is protected by copyright. All rights reserved.
gel. The ethidium bromide-stained gels were photographed using a Tanon-4100 Digital
197
Imaging System (Shanghai Tanon Science & Technology Co, Ltd, Shanghai, China). The
198
specific primers for each gene are presented in Table 1.
Accepted Article
196
199 200
Protein extraction and western blot analysis
201 202
Leaf sections collected from plants were placed in liquid nitrogen, and the protein
203
concentrations were quantified (Harrison et al. 1998). The amount of proteins was measured
204
using the dye-binding assay described by Bradford (1976). For immunoblotting, equal
205
amounts of the protein samples (0.418 mg) per well were loaded on an equal protein basis,
206
separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
207
transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Molsheim, France)
208
and incubated using antibodies raised against heat-shock protein 70 (HSP70) or D1 (Li et al.
209
2011). The immunoreactive protein bands were detected using peroxidase-conjugated goat
210
antibodies against rabbit IgG. Quantitative image analysis was performed using a Tanon
211
Digital Gel Imaging Analysis System (Tanon-4100, Shanghai Tanon Science & Technology
212
Co, Ltd).
213 214
Measurements of chlorophyll a fluorescence transients (OJIP)
215 216
The polyphasic chlorophyll a fluorescence transients (OJIP) were measured using a plant
217
efficiency analyzer (PEA, Hansatech Instruments Ltd., UK) according to Strasser et al. (1995). 10
This article is protected by copyright. All rights reserved.
The transients were induced by red light of approximately 3000 μmol m-2 s-1 provided by an
219
array of six light-emitting diodes (peak 650 nm). The fluorescence signals were recorded
220
within a time scan from 10 μs to 1 s at a data acquisition rate of 105 points s-1 for the first 2 ms
221
and 1000 points s-1 after 2 ms. (Tan et al. 2011)
Accepted Article
218
222
The fluorescence data derived from each sample were imported into Microsoft Office Excel
223
software; then, data analysis and processing were performed. (Gao & Strasser 2005; Pan et al.
224
2008)
225 226 227
Measurements of the net photosynthetic rate (Pn) and the maximal photochemical
efficiency of PSII (Fv/Fm)
228 229
Measurements of the Pn were performed on a fully expanded attached leaf of potato
230
seedlings using an open system (Ciras-2, PP Systems, Norfolk, UK). The measurements of
231
these photosynthetic parameters lasted approximately 10 min, during which no significant
232
recovery was detected based on these parameters. These measurements were performed in
233
parallel with the chlorophyll fluorescence recordings.
234
The Fv/Fm was measured using a portable fluorometer (FMS2, Hansatech, King’s Lynn,
235
UK). The leaf samples were dark-acclimated for 20 min before Fv/Fm measurement. The
236
minimal fluorescence level (Fo) with all PSII reaction centers open was measured by the
237
measuring modulated light, which was sufficiently low (
Accepted Article
1
AtCBF3 enhances heat tolerance of potato plants
2 3
Corresponding author:
4
Xinghong Yang
5
College of Life Science, State Key Laboratory of Crop Biology,
6
Shandong Key Laboratory of Crop Biology, Shandong Agricultural University,
7
Taian 271018,
8
People’s Republic of China
9
E-mail: [email protected]
10
Phone: +86 538 8246167
11
Fax:
+86 538 8246167
12 13 14
Title：Potato plants ectopically expressing Arabidopsis thaliana CBF3 exhibit enhanced
15
tolerance to high-temperature stress1
16
Haiou Dou1, Kunpeng Xv1, Qingwei Meng1, Gang Li1, Xinghong Yang1*
17 18
1
19
Shandong Agricultural University, Taian 271018, People’s Republic of China
State Key Laboratory of Crop Biology，Shandong Key Laboratory of Crop Biology,
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/pce.12366 1
This article is protected by copyright. All rights reserved.
Accepted Article
20 21
*Corresponding author:
22
Xinghong Yang
23
Tel: +86 538 8246167;
24
Fax: +86 538 8246167;
25
E–mail address: [email protected]
26 27 28 29 30 31 32 33 34 35
Abstract
36
CBF3, a known cold-inducible gene that encodes a transcription factor, was isolated from
37
Arabidopsis thaliana and introduced into the potato (S. tuberosum cv. ‘luyin NO.1’) under the
38
control of the CaMV35S promoter or the rd29A promoter. Our results revealed that high
39
temperatures of 40 °C or above can significantly induce AtCBF3 expression. After heat stress,
40
the net photosynthetic rate (Pn), the maximal photochemical efficiency of PSII (Fv/Fm) and
41
the accumulation of the D1 protein were higher in the transgenic lines than in the wild-type 2
This article is protected by copyright. All rights reserved.
(WT) line. Moreover, compared to the WT line, O2·– and H2O2 accumulation in the transgenic
43
lines were reduced. A Q-PCR assay of a subset of the genes involved in photosynthesis and
44
antioxidant defense further verified the above results. Interestingly, under heat stress
45
conditions, the accumulation of HSP70 increased in the WT line, but decreased in the
46
transgenic lines. These results suggest that potato plants ectopically expressing AtCBF3
47
exhibited enhanced tolerance to high temperature, which is associated with improved
48
photosynthesis and antioxidant defense via induction of the expression of many
49
stress-inducible genes. However, this mechanism may not depend on the regulatory pathways
50
in which HSP70 is involved.
51
Key words
52
AtCBF3; high temperature; photosynthesis; antioxidant defense; potato
Accepted Article
42
53 54 55 56 57 58
Introduction High temperature is strongly detrimental to plant growth and development. Heat stress is
59
one of the primary abiotic stresses that limit plant biomass production and productivity
60
(Boyer 1982). The early changes caused by high-temperature stress involve the
61
reprogramming of signal transduction components, transcription factors and proteins
62
associated with the metabolism of reactive oxygen species (ROS) under stressful conditions.
63
Photosynthetic organisms are directly exposed to acute temperature alterations and are 3
This article is protected by copyright. All rights reserved.
considered the most heat sensitive. In the temperature region of 20-40 °C, inhibition of
65
photosynthesis in spinach (Spinacia oleracea L.) has been discovered and it is completely
66
reversible by lowering the temperature (Weis 1981). However, prolonged exposures at even
67
higher temperatures (> 40 °C) generally result in an irreversible inhibition of photosynthesis,
68
due to a disruption in thylakoid membrane lipid integrity and concomitant damage to
69
photosystem II (PSII) (Berry & Björkman 1980; Seemann et al. 1984; Bilger, Schreiber &
70
Lange 1987). In other word, a certain degree of heat stress can directly damages the
71
photosynthetic apparatus and decreases the photosynthetic rate, the photochemical efficiency
72
of PSII and the duration of the assimilate supply (Prasad et al. 2008; Prasad et al. 2009).
Accepted Article
64
73
Meanwhile, high-temperature stress can promote the accumulation of ROS in the plant,
74
particularly when the antioxidant capacity to remove ROS is low (Djanaguiraman, Prasad &
75
Seppanen 2010). Under stress conditions, ROS can damage cellular components, including
76
DNA, proteins and membrane lipids, leading to loss of function and cell death (Mittler 2002).
77
The potato (Solanum tuberosum L.) is planted in two-thirds of the countries in the world
78
and is the fourth most important food crop after rice, wheat and corn (Ortiz & Wa-tanabe
79
2004). The potato prefers a cool environment, and the major potato-producing regions are
80
located in the relatively cool climates of the northern temperate zone and Andean tropical
81
highlands. In addition, the potato is a frost-sensitive species, and its normal growth and
82
development are inhibited by high temperature. When the temperature rises above 25 °C,
83
tubers growth is arrested, and when exceeding 39 °C, growth of stems and leaves is arrested.
84
Krauss & Marschner (1984) assumed that high temperature during tuber development might
85
affect tuber yield and starch content via carbohydrate metabolism within the tuber. High 4
This article is protected by copyright. All rights reserved.
temperature not only inhibits tuber growth rate but also alters carbohydrate partitioning in
87
potato plants from tubers to shoots, which reduces overall plant yield (Borah & Milthorpe
88
1962; Ewing 1981; Wolf, Marani & Rudich 1990). Thus, high temperature is one of the most
89
important environmental factors that limit the growth and yield of potatoes.
Accepted Article
86
90
The cold-regulatory C-repeat binding factor (CBF) pathway, which is widely conserved in
91
many plants (Chew & Halliday 2010), is best characterized in Arabidopsis (Hua 2009;
92
Thomashow 2010). Arabidopsis encodes three cold-inducible CBF genes, CBF1, CBF2, and
93
CBF3 (Stockinger, Gilmour & Thomashow 1997; Gilmour et al. 1998; Medina et al. 1999), also
94
referred to as DREB1b, DREB1c, and DREB1a, respectively (Liu et al. 1998). These genes
95
encode transcription factors that are members of the APETALA2 (AP2) /
96
ethylene-responsive factor (ERF) family of DNA-binding proteins (Riechmann &
97
Meyerowitz 1998). CBF proteins bind to the C-repeat (CRT) / dehydration-responsive
98
element (DRE), a regulatory element that is present in the promoters of approximately 100
99
cold-regulated (COR) genes, and induce the expression of COR genes (Maruyama et al. 2004;
100
Fowler & Thomashow 2002; Vogel et al. 2005). Expression of the CBF genes occurs within
101
approximately 15 min during low temperature (4 °C) treatment, followed by induction of the
102
CBF downstream target genes beginning at approximately 2 to 3 h. CBF/DREB1 factors
103
appear to be ubiquitously expressed in plants regardless of their freezing tolerance capacity,
104
and the ability of ectopic CBF/DREB1 transgene activity to increase freezing tolerance has
105
been demonstrated in many plants, such as wheat and barley (Morran et al. 2011), rice (Gutha
106
& Reddy 2008) and poplar (Benedict et al. 2006). In addition, in potatoes, previous
107
examination has established that the plant is responsive to ectopic CBF transgene activity 5
This article is protected by copyright. All rights reserved.
(Pino et al. 2006). Besides, additional studies showed that the Arabidopsis CBF gene
109
(AtCBF1 and AtCBF3) can function in potatoes to increase freezing tolerance (Pino et al.
110
2007; Pino et al. 2008). Moreover, direct CBF transgene expression using a stress-inducible
111
(rd29A) promoter, which induces low background expression under non-stressful conditions,
112
significantly improves freezing tolerance without negatively impacting agronomically
113
important traits in the potato. However, expression of CBF3 under the control of a constitutive
114
promoter (CaMV 35S) might not completely mimic the activation of CBF3 by low
115
temperature (Sarah J et al. 2000).
Accepted Article
108
116
Once CBF transcription factors were confirmed to improve the freezing tolerance of plants,
117
they were successively transferred to various plants in the hope of enhancing the tolerance of
118
these plants to stress. For example, AtCBF1 gene expression vectors have been transfected
119
into tomatoes, which reduced ROS accumulation and increased the proline content,
120
significantly enhancing the resistance of the plants to low temperature and drought conditions
121
(Hsieh et al. 2002). Transfection of rice with OsCBF was found to increase the content of
122
proline and soluble sugar and its resistance to cold, drought and salt stresses (Ito et al. 2006).
123
Similarly, the AtCBF3 gene has been transfected into maize, tobacco and Arabidopsis itself,
124
also improving the cold-, drought- and salt-tolerance of the plants (Gilmour et al. 2000;
125
Al-Abed et al. 2007; Liu et al. 2011).
126
Overall, the expression of CBF genes can not only enhance the resistance of plants to low
127
temperatures but also improve the drought- and salt-tolerance of plants. Based on these results,
128
previous interpretations indicate that similar DREB/CBF genes in different plants may
129
participate in a different cellular signal transduction pathway. Under stress conditions, plants 6
This article is protected by copyright. All rights reserved.
initiate various stress signal transduction pathways, and these pathways are linked together
131
via some common components, thus forming a complex signaling network (Haake et al.
132
2002). For each individual form of DREB/CBF in the same and different plants, the condition-
133
and time-induced expressions are distinct. Moreover, the CRT/DRE element to which
134
DREB/CBF transcription factors bind is located in the promoters of some stress-responsive
135
genes, indicating that DREB/CBF genes can be involved in various stress reactions
136
(Stockinger, Gilmour & Thomashow 1997; Liu et al. 1998).
Accepted Article
130
137
Despite studies on low temperature and other stresses, to our knowledge, no report is
138
available regarding whether the CBF transcription factor functions in response to high
139
temperature to enhance plant thermo-tolerance. To examine whether CBF can improve the
140
heat resistance of plants, we performed the following study.
141
In the present study, we utilized AtCBF3-transgenic potato plants to investigate whether
142
overexpression of AtCBF3 could enhance the tolerance of the potato to high temperature. In
143
addition, we also performed a preliminary examination of the regulatory pathway that plants
144
use to resist high temperature in the presence of AtCBF3 and the mechanism involved in
145
high-temperature-induced expression of AtCBF3.
146 147
Materials and methods
148 149
Plant materials
150 151
The 35S::AtCBF3 and rd29A::AtCBF3 (rd29A, the stress-inducible promoter) constructs 7
This article is protected by copyright. All rights reserved.
were introduced into Agrobacterium tumefaciens strain EHA105 using a previously described
153
transformation method (Pino et al. 2007). Young potato leaf explants (S. tuberosum cv. ‘luyin
154
NO.1’) were utilized for transgenic plant generation. The procedures of potato transformation
155
and tissue culture were performed as previously described (Pino et al. 2007). The
156
pre-cultivation medium consisted of MS-3 % sucrose (pH 5.8) containing 1 mg L–1 of the
157
auxin indole-3-acetic acid (IAA). The co-cultivation medium consisted of MS-3 % sucrose
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(pH 5.7) containing 1 mg L–1 IAA and 2 mg L–1 trans-Zeatin (ZT). Finally, the callus
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induction medium consisted of MS-3 % sucrose (pH 5.8) containing 2 mg L–1 ZT, 1 mg L–1
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IAA, 250 mg L–1 cefotaxime and 50 mg L–1 kanamycin.
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Plants and growth conditions
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T0 potato plants were grown at 25 °C under a 16 h/8 h day/night photoperiod (300-400
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μmol m−2 s−1) in a greenhouse (Pino et al. 2007). In this study, the potato plants included a
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wild type (WT) line and two transgenic lines that expressed the A. thaliana CBF3 gene under
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the control of the CaMV35S promoter (referred to as S1, S2) or the rd29A promoter (referred
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to as R1, R6). After 6 weeks, a portion of the seedlings divided into five groups were exposed
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directly to various temperatures (25, 30, 35, 40 or 45 °C) for 2 h in a growth chamber. The
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other seedlings were subjected to heat stress (HS) (40 °C) for 4 h and then transferred to the
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control conditions for another 24 h (post-stressed, PS). The 4th leaf from top of the seedlings
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was used for the experiments.
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To study the effect of Ca2+ on the expression of AtCBF3, some of the transgenic 8
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rd29A::AtCBF3 lines grown for 3 weeks were pre-treated with an equal volume of distilled
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water, 20 mM ethylene glycol bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) or
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100 mM calcium chloride (CaCl2) (Knight, Trewavas & Knight 1996) dissolved in water for 1
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h, followed by exposure to heat treatment（40 °C）for 0, 1, 2 h. All of the measurements of the
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physiological and biochemical parameters were performed on the fully expanded leaves.
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Transcriptional analysis via quantitative PCR (Q-PCR) and semi-quantitative
RT-PCR
182 183
Fresh leaf samples (0.1 g) from wild type and transgenic plants were used for total RNA
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extraction using Trizol reagent (TransGen Biotech, Beijing, China) according to the
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manufacturer’s instructions. The cDNA was synthesized using a reverse transcription system
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(TransGen Biotech, Beijing, China). Q-PCR was performed using a SYBR®PrimeScript™
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RT-PCR Kit (TaKaRa, Dalian, China) in a 25μL volume using a CFX96TM Real–time
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System (Bio-Rad). The Q-PCR amplification conditions were as follows: initial denaturation
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at 95 °C for 30 s; 41 cycles at 95 °C for 5 s, 50 °C for 15 s and 72 °C for 15 s; and a single
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melt cycle from 65 °C to 95 °C. Each condition contained at least three individual samples.
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The linearity of the relationships and the amplification efficiency were examined and the data
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analysis was performed using CFX Manager Software version 1.1 (Yao et al. 2013).
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For semi-quantitative RT-PCR, the amplification conditions were as follows: 10 min at
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94 °C, 30 s at 94 °C, 30 s at 50 °C and 30 s at 72 °C, followed by 10 min at 72 °C. Each cycle
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was repeated 27 times. The RT-PCR products were separated using a 2.0 % (m v–1) agarose 9
This article is protected by copyright. All rights reserved.
gel. The ethidium bromide-stained gels were photographed using a Tanon-4100 Digital
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Imaging System (Shanghai Tanon Science & Technology Co, Ltd, Shanghai, China). The
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specific primers for each gene are presented in Table 1.
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199 200
Protein extraction and western blot analysis
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Leaf sections collected from plants were placed in liquid nitrogen, and the protein
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concentrations were quantified (Harrison et al. 1998). The amount of proteins was measured
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using the dye-binding assay described by Bradford (1976). For immunoblotting, equal
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amounts of the protein samples (0.418 mg) per well were loaded on an equal protein basis,
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separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
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transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Molsheim, France)
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and incubated using antibodies raised against heat-shock protein 70 (HSP70) or D1 (Li et al.
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2011). The immunoreactive protein bands were detected using peroxidase-conjugated goat
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antibodies against rabbit IgG. Quantitative image analysis was performed using a Tanon
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Digital Gel Imaging Analysis System (Tanon-4100, Shanghai Tanon Science & Technology
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Co, Ltd).
213 214
Measurements of chlorophyll a fluorescence transients (OJIP)
215 216
The polyphasic chlorophyll a fluorescence transients (OJIP) were measured using a plant
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efficiency analyzer (PEA, Hansatech Instruments Ltd., UK) according to Strasser et al. (1995). 10
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The transients were induced by red light of approximately 3000 μmol m-2 s-1 provided by an
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array of six light-emitting diodes (peak 650 nm). The fluorescence signals were recorded
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within a time scan from 10 μs to 1 s at a data acquisition rate of 105 points s-1 for the first 2 ms
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and 1000 points s-1 after 2 ms. (Tan et al. 2011)
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The fluorescence data derived from each sample were imported into Microsoft Office Excel
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software; then, data analysis and processing were performed. (Gao & Strasser 2005; Pan et al.
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2008)
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Measurements of the net photosynthetic rate (Pn) and the maximal photochemical
efficiency of PSII (Fv/Fm)
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Measurements of the Pn were performed on a fully expanded attached leaf of potato
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seedlings using an open system (Ciras-2, PP Systems, Norfolk, UK). The measurements of
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these photosynthetic parameters lasted approximately 10 min, during which no significant
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recovery was detected based on these parameters. These measurements were performed in
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parallel with the chlorophyll fluorescence recordings.
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The Fv/Fm was measured using a portable fluorometer (FMS2, Hansatech, King’s Lynn,
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UK). The leaf samples were dark-acclimated for 20 min before Fv/Fm measurement. The
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minimal fluorescence level (Fo) with all PSII reaction centers open was measured by the
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measuring modulated light, which was sufficiently low (
