Botanical Studies

An International Journal

Impact Factor 1.159

Botanical Studies Cover Image
Open Access

Enhanced tolerance of transgenic potato plants expressing choline oxidase in chloroplasts against water stress

  • Yu-Jie Cheng1, 2,
  • Xi-Ping Deng1, 3Email author,
  • Sang-Soo Kwak1, 3,
  • Wei Chen1 and
  • Anthony E Eneji1, 4
Botanical StudiesAn International Journal201354:30

DOI: 10.1186/1999-3110-54-30

Received: 25 April 2012

Accepted: 9 November 2012

Published: 3 September 2013

Abstract

Background

Glycinebetaine, whose biosynthesis could be catalyzed by choline oxidase (COD), is an extremely efficient compatible solute for scavenging oxidative stress-inducing molecules and protecting the photosynthetic system in plants. To study the effects of the codA transgene for choline oxidase on the drought resistance and recovery, a transgenic potato cultivar (SC) bearing codA gene and a non-transgenic (NT) control cultivar were raised in pots under moderate and severe drought stress. The experiment was constituted by a two-day-pretreatment with 20% PEG and a four-day-water stress combined with two-day-recovery treatment.

Results

Under the four-day-water stress, plants were provided with normal water condition, 10% or 20% polyethylene glycol. The results of pretreatment showed an expression of codA gene in transgenic potato and an accumulation of glycine betaine (GB); leaf water potential was higher in SC than in NT. In the stress-recovery-treatment, SC showed stronger antioxidant ability, more efficient photosynthetic system, higher chlorophyll content, lower malondialdehyde content and better recovery from water deficit stress than NT.

Conclusion

Although this work concentrated on the short-term water stress and recover treatments on transgenic potato plants with the over-expression of CodA gene and its control line. The datas shows that the exogenous codA gene provided potato a stronger drought resistance and recovery ability.

Keywords

Choline oxidase Glycine betaine Re-watering Transgenic potato Water stress

Background

Glycinebetaine (GB, N,N,N-trimetrimethylglycine; hereafter betain) is a quaternary ammonium compound that occurs naturally in a wide variety of plants, animals and microorganisms. The accumulation of GB is induced and synthesized in the chloroplasts of higher plants under various abiotic stress, such as high salt, drought and cold (Jagendorf and Takabe 2001; Rontein et al. 2002), and the exogenous GB could enhance the resistance ability to drought (Mahouachi et al. 2012). Glycinebetain affords osmoprotection for plants and protects cell components from harsh conditions by functioning as a molecular chaperone (Sakamoto and Murata 2002).

Furthermore, it could stabilize the higher-order structure of protein and protect the activities of intracellular protein and metabolic enzymes (Demiral and Turkan 2004). In photosynthetic systems, GB efficiently protects various components of the photosynthetic machinery, such as rubulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the oxygen-evolving photosystemII (PSII) complex from stress (Murata et al. 2007). It preserves the normal cellular turgor pressure, playing an important role in respiration and photosynthesis. An exogenous application of GB improves the growth and survival of a wide variety of plants under various stress conditions (Ashraf and Foolad 2007; Hoque et al. 2007; Park et al. 2006; Chen and Murata 2008). The fact that many agronomically important crops, such as rice and potato, are betain-deficient has inevitably led to proposals that it might be possible to increase stress tolerance by genetic manipulation that would allow non-accumulators or low-level accumulators to accumulate betain at protective levels (McCue and Hanson 1990).

Glycinebetaine has three main synthetic pathways in different organisms (Sakamoto and Murata 2000). Therefore, different methods could be used to introduce a GB synthetic system into non-GB-accumulating plants to improve their stress tolerance. One of the methods was the introduction of the BADH (betaine aldehyde dehydrogenase) gene, which has been frequently introduced into a variety plants including tomato (Jia et al. 2002), tobacco (Yang et al. 2005; Ci et al. 2007; Zhou et al. 2008), wheat (Guo et al. 2000) and potato (Zhang et al. 2009) for enhanced tolerance of salt, drought or extreme temperatures. The other was COD (choline oxidase), which itself does not exist in higher plants at all. Previous reports showed that the COD gene was also introduced into Arabidopsis (Sulpice et al. 2003; Waditee et al. 2005), tobacco (Huang et al. 2000), rice (Konstantinova et al. 2002; Mohanty et al. 2002; Kathuria et al. 2009), tomato (Goel et al. 2011; PARK et al. 2007; Park et al. 2004; Li et al. 2011), maize (Quan et al. 2004), potato (Ahmad et al. 2008) and Eucalyptus globulus (Matsunaga et al. 2012) to improve their stress tolerance.

Potato (Solanum tuberosum) is one of the leading crops throughout the world. It is cultivated in more than one hundred countries and regions, with total yield and cultivated area ranked fourth among crops, only after wheat, rice and maize (Jackson 1999). With the rapid economic development in recent years, potato is increasingly becoming an important cash crop and the potato industry has seen strong development recently. Hence, an excellent potato variety with resistance or tolerance of abiotic stress is required for the steady development of the potato industry (Jiang et al. 2008).

Different types of promoters have been used in plant transformation research; these promoters can be divided into three classes: constitutive promoter, organ-specific promoter and inducible promoter (Potenza et al. 2004). The most representative constitutive promoter is CaMV 35S (Odell et al. 1985), which is one of the most widely used promoters. However, the constitutive promoter might result in the over-expression of exogenous gene and break the regular growth process of plants (Scheid et al. 2002). The use of organ-specific promoters, such as potato tuber-specific patatin promoter, can make up for this weakness. SWPA2 (Oxidative stress-inducible peroxidase promoter) is inducible promoter cloned by Kim in 2003 from sweet potato. This was confirmed by transforming the glucuronidase gene with SWPA2 and CaMV35S promoters respectively into tobacco. Under water stress, the gene expression of SWPA2-GUS was 30-fold that of CaMV35S-GUS, suggesting that SWPA2 has a very strong stress-inducible ability (Kim et al. 2003).

In this experiment, we aimed to determine the influence of an introduced codA gene on transgenic potato under water stress and rewatering treatment, and provide a basis for research on new potato varieties and glycinebetain.

Methods

Materials

We used potted transgenic potato plants (SC) expressing codA gene (from A. globiformis) in chloroplasts under the control of an oxidative stress-inducible SWPA2 promoter (Kim et al. 2003) and non-transgenic (NT) control plants (Solanum tuberosum L. cv. Superior) (Ahmad et al.2008). The vector structure with codA gene is shown in (Figure 1). This experiment was divided into pretreatment and stress-rehydration-treatment.
https://static-content.springer.com/image/art%3A10.1186%2F1999-3110-54-30/MediaObjects/40529_2012_Article_30_Fig1_HTML.jpg
Figure 1

Vector structure of pCAMBIA2300 with choline oxidase gene. LB: left border; T35S: CaMV35S terminator; P35S: CaMV35S promoter; PSWPA2: SWPA2 promoter; TEV: tobacco etch virus 5′-UTR; TP: chloroplast transit peptide; TNOS: nopaline synthesis terminator; RB: right border.

Pretreatment

Five four-week-old plants respectively from SC and NT were transferred to buckets filled with daily-aerated Hoagland nutrient solution. One week later, all plants were subjected to drought stress simulated with PEG6000 (polyethylene glycol 20%) treatment for 48 h. The fourth and fifth leaves were sampled from each plant at 0 h and 48 h after stress to determine the GB content, leaf water potential and expression of codA gene.

Stress-rehydration-treatment

A total of 90 pots (45 for SC and 45 for NT) were used for this treatment. The two potato types were allocated to three drought treatments of no stress - normal water condition, PEG 10% (moderate stress) and PEG 20% (severe stress); each treatment has 15 replications arranged into a completely randomized design. Nutrient solution (with or without PEG) was changed at 9 a.m. each day just before the determination of photosynthetic activity. The stress continued for four days and then in the following two days all plants were provided with normal water condition. Five leaves (the fourth or the fifth leaf from each plant) from five plants were chosen randomly from each treatment everyday at 10 a.m. All the samples were immediately frozen in liquid nitrogen and stored at -80°C until required for analysis.

Growth conditions

Each pot contained 1 kg disinfected dry vermiculite which was then watered to 70% of maximum field moisture capacity with Hoagland nutrient solution, with only one seedling cultivated in each pot. Pots were placed in a growth chamber under a 16 h photoperiod with light intensity of 300 μmol photons m-2s-1, 60% (w/v) relative humidity at day 25°C / night 20°C. Soil moisture was controlled by weighing each pot during the growth period.

PCR analysis

Total genomic DNA was extracted from transgenic potato and control plant with DNA kits bought from Beijing TaiKe Biotechnology Limited. First-strand cDNA synthesis was performed in a 20 ul reaction mixture containing 1 ul of total plasmid DNA. The PCR was conducted with 0.5 ul first-strand cDNA with the primers of 5′-GCT GCT GGA ATC GGG ATA-3′(forward) and 5′-TGG GCT TAT CGC GGA AGT-3′(reverse). The amplification reactions occurred at 94°C for 5 min, followed by 30 cycles (94°C 30 s, 62°C 30 s and 72°C 1 min) and finally an extension cycle of 10 min at 72°C. The PCR products were separated on 1% agarose gel, stained with ethidium bromide, and visualized under UV. The expected size of the PCR fragment was 450 bp. The UV transilluminator was obtained from Thermo Company (USA), dNTP from Roche Company (Sweden) and Taq polymerase from Fermentas Company (USA).

Glycinebetain content

Glycinebetain was measured via UV–VIS spectrophotometry (Huang et al. 2009). In brief, GB reacts with Reinecke’s salt under acidic condition to produce sediment of Reinecke’s salt, which was then dissolved in 700 ml/L acetone until the color turned pink. Acetone was used as blank control to produce an absorbance standard curve under 525 nm and the standard curve was used to determine the GB contents in samples.

Photosynthetic system and leaf water potential

From 9 a.m.—10 a.m. each day, the fifth mature and well-exposed leaves from top in five randomly tagged plants in each treatment group were sampled for measurement with a portable photosynthetic meter, LI-6400; leaf water potential was determined according to the method of Turner using a pressure chamber (Turner 1988).

Chlorophyll content

Mature and well-exposed leaves from plant (0.5 g fresh weight) were homogenized in a mortar and pestle using 10 ml of chilled 80% acetone. The homogenate was centrifuged at 10,000 rpm at 4°C for 10 min. The absorbance of the supernatant was measured at 646, 663 and 750 nm, respectively, and chlorophyll content was calculated as per the method of Arnon et al. (Arnon et al. 1974).

Extraction and assays of the activities of reactive oxygen-scavenging enzymes

The determination of the activities of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) followed those reported in Lee et al. (Lee and Lee 2002). Leaf samples (0.5 g) were homogenized in 8 ml of 50 mM potassium phosphate buffer (pH 7.0) that contained 1 mM EDTA, 1 mM ascorbic acid (ASA), 1 mM dithiothreitol (DTT), 1 mM L-glutathione (GSH) and 5 mM MgCl2. After sufficient grinding with little quartz sand, the homogenate was centrifuged at 20,000 rmp for 15 min at 4°C. The resultant supernatant was deep-freezed under -80°C and used for assays of enzymatic activity. Total protein concentration was determined according to the Bradford method (Bradford 1976) using the Bio-Rad protein assay reagent.

The activity of superoxide dismutase (SOD) was measured according to McCord and Fridovich (1969) with slight modification, by immediately monitoring the absorbance at 560 nm due to the reduction of cytochrome c. The reaction mixture contained 50 mM phosphate buffer (pH 7.8), 0.1 mM Nitrotetrazolium Blue chloride (NBT), 0.1 mM EDTA and 13.37 mM methionine.

POD activities were determined specifically at 420 nm. The reaction mixture contained 0.4 ml of 100 mM potassium phosphate buffer (pH 6), 0.16 ml of 147 mM H2O2, 0.32 ml of 5% Pyrogallol and 2.1 ml of DW. The reaction was initiated by adding 20 ul plant extract and after 10 min. The POD activity was determined by following the consumption of H2O2 (extinction coefficient 39.4 mM-1 cm-1) at 420 nm for 20 s.

The activity of catalase (CAT) was assayed by monitoring decreases in absorbance at 240 nm due to the decomposition of H2O2. The reaction mixture contained 670 ul potassium phosphate buffer (pH 7.0), 330 ul H2O2 and 30 ul of the extract. The CAT activity was determined by following the consumption of H2O2 (extinction coefficient 39.4 mM-1 cm-1) at 240 nm for 1 min.

Lipid peroxidation

Lipid peroxidation was determined as the amount of malondialdehyde (MDA, e = 155 mM ± 1 cm ± 1), a product of lipid peroxidation. 1 ml of saved supernatant (which has also been used to determinate antioxidant enzyme) was mixed with 3 ml reaction buffer including 5% Trichloroacetic acid (TCA) and 0.5% Thiobarbituric acid (TBA) was heated in 100°C water for 15 min, then cooed immediately and centrifuged. The absorbance was monitored at 450, 532 and 600 nm.

Results

Confirmation of codA DNA in transgenic potato

As shown in (Figure 2), codA does not exist in NT, while the exogenous codA gene introduced into transgenic SC plants could be observed. The total length of codA was 450 bp as we expected.
https://static-content.springer.com/image/art%3A10.1186%2F1999-3110-54-30/MediaObjects/40529_2012_Article_30_Fig2_HTML.jpg
Figure 2

Development of transgenic potato plants expressing the codA in chloroplasts. Genomic DNA PCR analysis of the codA from transgenic plants. Numbers (3–6) represent independent transgenic lines, M: size marker, number 1–2: non-transgenic plants, N: negative control (water).

Effect of dehydration pretreatment on GB accumulation and leaf water potential in transgenic and non-transgenic potato

To determine whether the expression of codA induced the synthesis of GB in the transgenic plants, the GB was analyzed after 0 h and 48 h of 20% PEG stress (Figure 3A). Also, the leaf water potential (LWP) (Figure 3B) of transgenic and non-transgenic potato was determined. At 0 h after stress, no GB accumulated in NT but a slight accumulation was noted in SC. Even at 48 h of stress, no GB was observed in NT, but the amount of GB increased significantly in SC. Leaf water potential of both potato types was significantly reduced from 0 h to 48 h after stress but the LWP of SC remained higher than that of NT.
https://static-content.springer.com/image/art%3A10.1186%2F1999-3110-54-30/MediaObjects/40529_2012_Article_30_Fig3_HTML.jpg
Figure 3

The determinations of Glycinebetaine contents and leave water potential in potatoes. GB levels (A) and leaf water potential (B) in non-transgenic (NT) and transgenic (SC) plants under water stress simulated with 20% PEG. GB was extracted from non-treated and 20% PEG-treated plants. Samples were collected after 48 h of PEG treatment. Data are expressed as the mean ± standard deviation (SD) of five replicates.

Effect of introduced codA gene on enzyme system in transgenic and non-transgenic potato following stress-rehydration treatment

The changes in antioxidant enzyme system (SOD, CAT and POD) of the two potato types under stress-rehydration-treatment are shown in (Table 1, Table 2 and Table 3). Activities of SOD and CAT varied similarly and without significant changes under normal water condition. However, under stress conditions, the SOD and CAT activities in the two potato types increased from DAY 0 to DAY 2; although at DAY 3 the values declined, they were still higher than those on DAY 0 (as an exception, the CAT activities in NT under 20% PEG stress started dropping from DAY 2). From DAY 4 to DAY 5 (rehydration period), SOD and CAT activities in both SC and NT rose again. In general, the SOD and CAT activities of the two potato types were higher under 10% PEG stress than under 20% PEG stress; under the same stress condition, SC showed higher antioxidant enzyme activities than NT.
Table 1

The activities of SOD enzyme in non-transgenic (NT) and transgenic (SC) potatoes during and after stress

PEG treatments

Potato type

SOD activity (units / mg protein)

Stress period

Recovery period

DAY 0

DAY 1

DAY 2

DAY 3

DAY 4

DAY5

Control (0% PEG)

SC

16.86±2.05

18.55±3.22

17.1±2.65

17.88±1.85

18.65±1.96

17.3±2.88

NT

17.07±2.72

18.12±1.75

18.9±3.11

17.6±1.68

16.7±2.84

17.83±2.03

Moderate stress (10% PEG)

SC

18.2±2.21

25.82±4.55

30.15±6.48

23.84±5.14

31.44*±4.97

37.28*±7.13

NT

18.21±3.48

21.75±6.54

28.26±5.84

19.52±5.78

20.89±4.22

28.44±7.61

Severe stress (20% PEG)

SC

17.92±1.57

23.18±4.21

26.77*±4.33

22.46*±6.14

25.06*±3.35

29.34±6.92

NT

16.55±2.41

18.25±3.26

17.25±5.11

11.77±4.68

17.38±3.57

22.21±4.85

Data are expressed as the mean of five replicates. Asterisks (*) represent significantly different (P < 0.05) with the NT.

Table 2

The activities of CAT enzyme in non-transgenic (NT) and transgenic (SC) potatoes during and after stress

PEG treatments

Potato type

CAT activity (units / mg protein)

Stress period

Recovery period

DAY 0

DAY 1

DAY 2

DAY 3

DAY 4

DAY5

Control (0% PEG)

SC

0.463±0.22

0.472±0.16

0.459±0.19

0.466±0.24

0.461±0.26

0.457±0.22

NT

0.465±0.20

0.469±0.17

0.463±0.23

0.454±0.19

0.456±0.18

0.465±0.11

Moderate stress (10% PEG)

SC

0.451±0.14

0.814±0.17

0.854±0.26

0.722*±0.23

0.907*±0.29

1.214*±0.42

NT

0.47±0.15

0.717±0.53

0.653±0.35

0.533±0.24

0.565±0.22

0.896±0.36

Severe stress (20% PEG)

SC

0.459±0.17

0.667±0.26

0.839*±0.44

0.676*±0.23

0.734*±0.47

0.917*±0.29

NT

0.462±0.11

0.508±0.27

0.362±0.24

0.216±0.12

0.369±0.13

0.448±0.18

Data are expressed as the mean of five replicates. Asterisks (*) represent significantly different (P < 0.05) with the NT.

Table 3

The activities of POD enzyme in non-transgenic (NT) and transgenic (SC) potatoes during and after stress

PEG treatments

Potato type

POD activity (units / mg protein)

Stress period

Recovery period

DAY 0

DAY 1

DAY 2

DAY 3

DAY 4

DAY5

Control (0% PEG)

SC

9.18±0.672

9.22±0.55

8.81±0.84

9.21±0.73

9.11±0.71

8.79±0.57

NT

8.57±0.98

9.03±0.95

9.52±0.65

9.38±0.42

9.0±0.73

9.25±0.66

Moderate stress (10% PEG)

SC

8.37±0.52

12.86±2.21

18.76±4.47

24.84*±5.44

18.6±4.75

9.67*±2.48

NT

9.2±0.86

16.44±3.86

21.3±5.33

28.16±4.16

20.78±4.22

12.07±5.61

Severe stress (20% PEG)

SC

8.84±0.69

15.51±3.26

24.07*±4.27

30.38*±5.48

25.76±4.85

13.2±3.15

NT

9.25±1.25

19.83±4.36

28.72±4.21

35.81±6.88

27.39±4.62

15.56±2.48

Data are expressed as the mean of five replicates. Asterisks (*) represent significantly different (P < 0.05) with the NT.

Variations in POD activities differed entirely from those for SOD and CAT during the drought stress and rehydration period. Under the four PEG stress conditions, from DAY 0 to DAY 3 POD activities increased progressively before dropping from DAY 4 to DAY 5 in the rehydration period. The POD activities in both potato types were significantly higher under severe than moderate stress; under the same water condition, NT showed a higher POD activity than SC.

Effect of exogenous codA gene on MDA and chlorophyll contents in transgenic and non-transgenic potato under stress-rehydration treatment

Data for chlorophyll and MDA contents in transgenic and non-transgenic potato are shown in (Tables 45). Under normal water condition, both chlorophyll and MDA contents had no significant variation and were within the regular range. Under drought stress, the MDA contents in both potato types increased from DAY 0 to DAY 3 (the period of PEG stress) but reduced in DAY 4 and DAY 5 (the period of hydration).
Table 4

Contents of MDA in non-transgenic (NT) and transgenic (SC) potatoes during and after water stress

PEG treatments

Potato type

MDA content (mmol.g.FW-1)

Stress period

Recovery period

DAY 0

DAY 1

DAY 2

DAY 3

DAY 4

DAY5

Control (0% PEG)

SC

4.38±0.58

4.21±0.34

4.25±0.56

4.36±0.34

4.31±0.46

4.28±0.53

NT

4.35±0.33

4.15±0.26

4.11±0.24

4.43±0.37

4.27±0.41

4.32±0.31

Moderate stress (10% PEG)

SC

4.16±0.61

5.88±1.67

6.92±1.88

8.98±2.17

5.74*±1.42

4.81±1.14

NT

4.24±0.31

6.17±2.54

7.25±1.57

9.02±2.64

7.18±2.47

5.92±1.13

Severe stress (20% PEG)

SC

4.33±0.26

6.36*±1.83

9.78*±2.46

11.52±2.84

6.29*±1.45

5.63*±1.57

NT

4.27±0.27

9.47±2.32

14.24±1.69

13.32±3.68

9.46±2.55

8.38±2.84

Data are expressed as the mean of five replicates. Asterisks (*) represent significantly different (P < 0.05) with the NT.

Table 5

Chlorophyll contents in non-transgenic (NT) and transgenic (SC) potatoes during and after water stress

PEG treatments

Potato type

Chlorophyll content (mg.gFW-1)

Stress period

Recovery period

DAY 0

DAY 1

DAY 2

DAY 3

DAY 4

DAY5

Control (0% PEG)

SC

1.91±0.26

1.83±0.18

1.86±0.24

1.79±0.23

1.93±0.17

1.87±0.18

NT

1.84±0.22

1.89±0.17

1.77±0.21

1.9±0.15

1.87±0.24

1.92±0.25

Moderate stress (10% PEG)

SC

1.92±0.28

1.67±0.17

1.45±0.26

1.26*±0.34

1.55±0.41

1.83±0.42

NT

1.85±0.23

1.53±0.35

1.21±0.42

0.97±0.31

1.46±0.39

1.77±0.54

Severe stress (20% PEG)

SC

1.82±0.34

1.49±0.33

1.19±0.56

0.78±0.29

1.32*±0.29

1.73*±0.38

NT

1.88±0.19

1.24±0.36

0.95±0.21

0.62±0.18

0.81±0.23

1.37±0.36

Data are expressed as the mean of five replicates. Asterisks (*) represent significantly different (P < 0.05) with the NT.

However, the MDA content in NT under 20% PEG stress started dropping earlier at DAY 3 instead of DAY 4. Chlorophyll contents in both plant types decreased from DAY 0 to DAY 3 and leveled off from DAY 4 to DAY 5. In general, the MDA contents in both SC and NT were significantly higher under 20% PEG stress than under 10% PEG stress; under the same stress condition, NT exhibited a higher MDA content than SC. On the contrary, under the four stress conditions, both plant types much higher chlorophyll under 10% PEG stress than under 20% PEG stress; for each stress level, SC showed a significantly higher chlorophyll content than NT.

Effect of exogenous codA gene on photosynthetic activities in transgenic and non-transgenic potato under stress-rehydration treatment

Data for changes in photosynthetic parameters, including photosynthetic rate (Figure 4A), stomatal conductance (Figure 4B), intercellular carbon dioxide concentration (Figure 4C) and transpiration rate (Figure 4D) of the two potato types due to the introduced gene are showed. There was no apparent variation among photosynthetic indexes of SC and NT under normal water condition. However, all photosynthetic parameters gradually and significantly decreased in both potato types during DAY 0 to DAY 3 under PEG stress, but increased markedly during the period of rehydration from DAY 4 to DAY 5. Throughout the observation period, SC showed higher photosynthetic parameters than NT and 10% PEG was less stressful to plants than 20% PEG.
https://static-content.springer.com/image/art%3A10.1186%2F1999-3110-54-30/MediaObjects/40529_2012_Article_30_Fig4_HTML.jpg
Figure 4

Changes in photosynthetic parameters of non-transgenic (NT) and transgenic (SC) potatoes during and after water stress. A: photosynthetic rate (Pn), B: Stomatal conductance (Gs), C: Intercellular carbon dioxide concentration (Ci), D: Transpiration rate. Data are expressed as the mean ± standard deviation (SD) of five replicates.

Discussion

Glycinebetain is an extremely efficient compatible solute and its presence was strongly associated with enhanced tolerance of plants in stress environments (Rhodes and Hanson 1993). Since some major crops do not produce GB by themselves, studies have been carried out to determine if exogenous application of GB could improve the growth and survival of a wide variety of plants under various stress conditions (Allard et al. 1998), the biosynthesis of GB, and its mechanisms to enhance the tolerate abiotic stress have been deeply reported (Chen and Muata, 20082011).

The codA gene in this experiment, which was obtained from Arthrobacter globiformis, directly converts choline into GB and H2O2 (Deshnium et al. 1995), and H2O2, is known to be not only a ROS but also plays many crucial roles as a signalling molecule to induces tolerance to stress (Xiong et al. 2002; Jiang et al. 2012). Kathuria et al. (2009) reported after the transformation of codA into rice, the produced H2O2 could simultaneously activate the response of transgenic plants to stresses. Therefore, GB might not be the only factor that is responsible for the tolerance improvement but the increase of H2O2 also makes contributions.

From our results, water stress simulated with 20% PEG under the control of SWPA2 promoter, the transgenic potato produced the codA gene and accumulated GB while the control plants had neither the codA gene nor GB accumulation since potato is a non-GB-accumulating plant. This demonstrated that the gene was successfully transformed and expressed in transgenic potato. When plants were subjected to water stress, the cells consequently lost water. Since one of the key functions of GB is osmoregulation, it could help to maintain the osmotic equilibrium of cells. During pretreatment, the overwhelming advantage of leaf water potential in SC relative to NT was based on the accumulation of GB.

When plants were subjected to water stress, reactive oxygen species (ROS) accumulated; at the same time the antioxidant system, especially SOD, the most important antioxidant enzyme was induced to scavenge the newly produced ROS. To determine the influence of accumulated GB on the antioxidant enzyme system and photosynthetic system of transgenic potato during water stress and recovery stage, we measured the daily activities of SOD, POD and CAT, as well as photosynthetic parameters of two plant types.

The changes in SOD and CAT activities in the two plant types followed a similar trend under water stress. The activities of both enzymes increased across stress treatments from DAY 0 to DAY 2. At DAY 3, the activities slightly reduced compared to DAY 2 but were still higher than DAY 0 because the amount of accumulated ROS exceeded the scavenging ability of antioxidant enzymes, hence, plant cells might been hurt. Ultimately from DAY 4 to DAY 5 (rehydration period) when plants were provided with normal water condition, the injured plant cells regained their structure, function and the enzyme activities rose again. Also, the activities of SOD and CAT increased more significantly in SC than NT during rehydration stage. Therefore, after stress, SC was more able to eliminate ROS and protect plants.

The POD activities of two plant types changed in a quite different way compared with SOD and CAT during the whole treatment. They gradually increased from DAY 0 to DAY 3 under water stress and reduced from DAY 4 to DAY 5 in the recovery stage. This result can be explained by the dual functional role of POD in plants. On the one hand, it can express a protective effect as a member of the scavenging enzyme system that removes H2O2 at the earlier stage of stress or aging; on the other hand, it can be also express injurious effects at the later stage of stress or aging, prompting the generation of reactive oxygen species, degradation of chlorophyll and peroxidation of membrane lipids which are products as well as indices of aging or stress. The main role of POD is generally considered to be the later one (Zhang and Kirkham 1994). Thus, due to the water stress, especially under the severe drought treatment (20% PEG), POD activities increased markedly in both potato types. During the recovery period, the antioxidant system and other physiological reactions regained their abilities resulting to removal of ROS and reduction of POD activities. In contrast to SOD and CAT, the POD activity was higher in NT than in SC, possibly because the promoter used in this experiment was derived from moderating clips of POD enzyme (SWPA2) in sweet potato. Exogenous promoters can cause transcriptional gene silencing of endogenous unlinked homologous promoters (Mette et al. 1999). Therefore, the expression of endogenous POD in SC was effected by the introduced CodA expressing construct which were regulated by the SWPA2 promoter. The expression of the endogenous promoter of POD enzyme might be suppressed by the inserted exogenous promoter and the activity of POD was inhibited to some level in SC. The SOD and CAT activities in SC were markedly superior to NT during the whole experiment. Hence, it could be suggested that the GB produced by the introduced codA gene in potato could protect its antioxidant system and enhance its drought resistance.

The protection of photosynthetic system against photodamage Because GB could internally stabilize the photosynthetic apparatus, improve the practical efficiency of photosynthetic system II and cell osmotic equilibrium (Papageorgiou and Murata 1995), the transgenic potato with extra GB exhibited much higher photosynthetic parameters than NT. For example, at DAY 1 of water stress with 10% PEG, the photosynthetic rate (Pn) and stomatal conductance (Gs) in SC dropped only slightly compared with control treatment. In addition, the intercellular carbon dioxide concentration (Ci) and transpiration rate (Tr) were higher in SC than in NT indicating that these parameters were directly related to Pn and Gs. During the recovery stage, as GB could protect the repair machinery of PS (Ohnishi and Murata, 2006; Murata et al. 2007), the photosynthetic parameters of SC recovered more efficiently than NT after stress. Moreover, the higher leaf water potential in SC (which has been proved in pretreatment) sustained a higher Tr. The MDA content was higher in NT than in SC while chlorophyll content showed an opposite trend in this experiment. This demonstrated that the GB produced by introduced codA gene in SC could prevent membrane lipid peroxidation and degradation of chlorophyll caused by stress.

Conclusion

In conclusion, the exposure of transgenic potato to four-day-stress (with 10% and 20% PEG) and two-day-recovery periods favoured the accumulation of GB and H2O2 by codA transgene; consequently the transgenic potato showed stronger antioxidant enzyme ability, more efficient photosynthetic system, higher leaf water potential and chlorophyll content and lower MDA content. It also showed better recovery from stress than the non-transgenic potato. The exogenous codA gene provided potato a stronger drought resistance and recovery ability.

Abbreviations

COD: 

Choline oxidase

CAT: 

Catalase

GB: 

Glycine betaine

MDA: 

Malondialdehyde

PEG: 

Polyethylene glycol

POD: 

Peroxidase

ROS: 

Reactive oxygen species

SOD: 

Superoxide dismutase.

Declarations

Acknowledgments

This work was conducted in the State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, China 111 Project (No.B12007) Chinese Academy of Sciences and supported by National Basic Research Program of China (2009CB118604), International Science & Technology Cooperation Program of China (2010DFA91930) and National Center for GM Crops (PJ008097), Biogreen21 Project for Next Generation, RDA, Korea.

Authors’ Affiliations

(1)
State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Northwest A&F University
(2)
Department of Forest, Northwest A&F University
(3)
Enviromental Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology
(4)
Department of Soil Science, University of Calabar

References

  1. Ahmad R, Kim M, Back K, Kim H, Lee H, Kwon S, Murata N, Chung W, Kwak S: Stress-induced expression of choline oxidase in potato plant chloroplasts confers enhanced tolerance to oxidative, salt, and drought stresses. Plant Cell Rep 2008, 27: 687–698. 10.1007/s00299-007-0479-4View ArticlePubMedGoogle Scholar
  2. Allard F, Houde M, Krol M, Ivanov A, Huner N, Sarhan F: Betaine improves freezing tolerance in wheat. Plant Cell Physiol 1998, 39: 1194. 10.1093/oxfordjournals.pcp.a029320View ArticleGoogle Scholar
  3. Arnon D, McSwain B, Tsujimoto H, Wada K: Photochemical activity and components of membrane preparations from blue-green algae. I. Coexistence of two photosystems in relation to chlorophyll a and removal of phycocyanin. Biochimica et Biophysica Acta (BBA)-Bioenergetics 1974, 357: 231–245. 10.1016/0005-2728(74)90063-2View ArticleGoogle Scholar
  4. Ashraf M, Foolad MR: Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ. Exp. Bot. 2007, 59: 206–216. 10.1016/j.envexpbot.2005.12.006View ArticleGoogle Scholar
  5. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72: 248–254. 10.1016/0003-2697(76)90527-3View ArticlePubMedGoogle Scholar
  6. Chen THH, Murata N: Glycinebetaine: an effective protectant against abiotic stress in plants. Trends Plant Sci 2008, 13: 499–505. 10.1016/j.tplants.2008.06.007View ArticlePubMedGoogle Scholar
  7. Chen THH, Murata N: Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant Cell Environ 2011, 34: 1–12. 10.1111/j.1365-3040.2010.02232.xView ArticlePubMedGoogle Scholar
  8. Ci H, Zhang N, Wang D: Enhancement of tobacco drought and salt tolerance with introduced BADH gene. Acta Agronomica Sin. 2007, 33: 1335–1339.Google Scholar
  9. Demiral T, Turkan I: Does exogenous glycinebetaine affect antioxidative system of rice seedlings under NaCl treatment? J Plant Physiol 2004, 161: 1089–1100. 10.1016/j.jplph.2004.03.009View ArticlePubMedGoogle Scholar
  10. Deshnium P, Los DA, Hayashi H, Mustardy L, Murata N: Transformation of Synechococcus with a gene for choline oxidase enhances tolerance to salt stress. Plant Mol Biol 1995, 29: 897–907. 10.1007/BF00014964View ArticlePubMedGoogle Scholar
  11. Goel D, Singh AK, Yadav V, Babbar SB, Murata N, Bansal KC: Transformation of tomato with a bacterial codA gene enhances tolerance to salt and water stresses. J Plant Physiol 2011, 168: 1286–1294. 10.1016/j.jplph.2011.01.010View ArticlePubMedGoogle Scholar
  12. Guo B, Zhang Y, Lee H, Du L, Lee Y, Zhang J, Chen S, Zhu Z: The transformation and expression of wheat with Betaine aldehyde dehydrogenase (BADH) gene. Chinese Bull. Bot. 2000, 42: 279–283.Google Scholar
  13. Hoque A, Okuma E, Banu NA, Nakamura Y, Shimoishi Y, Murata Y: Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities. J Plant Physiol 2007, 164: 553–561. 10.1016/j.jplph.2006.03.010View ArticlePubMedGoogle Scholar
  14. Huang J, Hirji R, Adam L, Rozwadowski K, Hammerlindl J, Keller W, Selvaraj G: Genetic engineering of glycinebetaine production toward enhancing stress tolerance in plants: metabolic limitations. Plant Physiol 2000, 122: 747–756. 10.1104/pp.122.3.747PubMed CentralView ArticlePubMedGoogle Scholar
  15. Huang H, Zhang D, Zheng X, Xue G, Lee C, Zhang L: The research progress of extraction, separation and determination of betaine. Medical Recapitulate 2009, 15: 3492–3494.Google Scholar
  16. Jackson S: Multiple signaling pathways control tuber induction in potato. Plant Physiol 1999, 119: 1–8. 10.1104/pp.119.1.1PubMed CentralView ArticlePubMedGoogle Scholar
  17. Jagendorf A, Takabe T: Inducers of glycinebetaine synthesis in barley. Plant Physiol 2001, 127: 1827–1835. 10.1104/pp.010392PubMed CentralView ArticlePubMedGoogle Scholar
  18. Jia G, Zhu Z, Chang F, Li Y: Transformation of tomato with the BADH gene from Atriplex improves salt tolerance. Plant Cell Rep 2002, 21: 141–146. 10.1007/s00299-002-0489-1View ArticleGoogle Scholar
  19. Jiang J, Wang L, Zhan L, Wang Y, Liu G, Yang C: Expression of Choline Oxidase Gene in Transformed Populus simonii × P. nigra Improves Salt Stress Tolerance. Chinese Bull Bot 2008, 80–84.Google Scholar
  20. Jiang JL, Miao S, Wang LY, Jiao CJ, Sun ZX, Cheng W, Li FM: Exogenous hydrogen peroxide reversibly inhibits root gravitropism and induces horizontal curvature of primary root during grass pea germination. Plant Physiol Biochem 2012, 53: 84–93.View ArticlePubMedGoogle Scholar
  21. Kathuria H, Giri J, Nataraja KN, Murata N, Udayakumar M, Tyagi AK: Glycinebetaineinduced water-stress tolerance in codA-expressing transgenic indica rice is associated with up-regulation of several stress responsive genes. Plant Biotechnol J 2009, 7: 512–526. 10.1111/j.1467-7652.2009.00420.xView ArticlePubMedGoogle Scholar
  22. Kim K, Kwon S, Lee H, Hur Y, Bang J, Kwak S: A novel oxidative stress-inducible peroxidase promoter from sweetpotato: molecular cloning and characterization in transgenic tobacco plants and cultured cells. Plant Mol Biol 2003, 51: 831–838. 10.1023/A:1023045218815View ArticlePubMedGoogle Scholar
  23. Konstantinova T, Parvanova D, Atanassov A, Djilianov D: Freezing tolerant tobacco, transformed to accumulate osmoprotectants. Plant Sci 2002, 163: 157–164. 10.1016/S0168-9452(02)00090-0View ArticleGoogle Scholar
  24. Lee Z, Lee J: Simultaneously determination of five antioxidant enzymes in a single extraction system of plants. J. Yunnan Normal University 2002, 22: 44–48.Google Scholar
  25. Li SF, et al.: Glycinebetaine enhances the tolerance of tomato plants to high temperature during germination of seeds and growth of seedlings. Plant Cell Environ 2011, 34: 1931–1943. 10.1111/j.1365-3040.2011.02389.xView ArticlePubMedGoogle Scholar
  26. Mahouachi J, Argamasilla R, Gomez-Cadenas A: Influence of exogenous glycine betaine and abscisic acid on papaya in responses to water-deficit stress. J. Plant Growth Regul. 2012, 31: 1–10. 10.1007/s00344-011-9214-zView ArticleGoogle Scholar
  27. Matsunaga E, Nanto K, Oishi M, Ebinuma H, Morishita Y, Sakurai N, Suzuki H, Shibata D: Agrobacterium-mediated transformation of Eucalyptus globulus using explants with shoot apex with introduction of bacterial choline oxidase gene to enhance salt tolerance. Plant Cell Rep 2012, 31: 225–235. 10.1007/s00299-011-1159-yView ArticlePubMedGoogle Scholar
  28. McCue K, Hanson A: Drought and salt tolerance: towards understanding and application. Trends Biotechnol 1990, 8: 358–362.View ArticleGoogle Scholar
  29. Mette M, Van der Winden J, Matzke M, Matzke A: Production of aberrant promoter transcripts contributes to methylation and silencing of unlinked homologous promoters in trans. The EMBO J. 1999, 18: 241–248. 10.1093/emboj/18.1.241View ArticlePubMedGoogle Scholar
  30. Mohanty A, Kathuria H, Ferjani A, Sakamoto A, Mohanty P, Murata N, Tyagi A: Transgenics of an elite indica rice variety Pusa Basmati 1 harbouring the codA gene are highly tolerant to salt stress. Theor Appl Genet 2002, 106: 51–57.PubMedGoogle Scholar
  31. Murata N, Takahashi S, Nishiyama Y, Allakhverdiev SI: Photoinhibition of photosystem II under environmental stress. Biochimica et Biophysica Acta (BBA)-Bioenergetics 2007, 1767: 414–421. 10.1016/j.bbabio.2006.11.019View ArticleGoogle Scholar
  32. Odell J, Nagy F, Chua N: Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 1985, 313: 810–812. 10.1038/313810a0View ArticlePubMedGoogle Scholar
  33. Ohnishi N, Murata N: Glycinebetaine counteracts the inhibitory effects of salt stress on the degradation and synthesis of D1 protein during photoinhibition in Synechococcus sp. PCC 7942. Plant Physiol 2006, 141: 758–765. 10.1104/pp.106.076976PubMed CentralView ArticlePubMedGoogle Scholar
  34. Papageorgiou G, Murata N: The unusually strong stabilizing effects of glycine betaine on the structure and function of the oxygen-evolving photosystem II complex. Photosynth Res 1995, 44: 243–252. 10.1007/BF00048597View ArticlePubMedGoogle Scholar
  35. Park E, Jekni Z, Sakamoto A, DeNoma J, Yuwansiri R, Murata N, Chen T: Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage. Plant J 2004, 40: 474–487. 10.1111/j.1365-313X.2004.02237.xView ArticlePubMedGoogle Scholar
  36. Park E, Jeknic JZ, Chen THH: Exogenous application of glycinebetaine increases chilling tolerance in tomato plants. Plant Cell Physiol 2006, 47: 706–714. 10.1093/pcp/pcj041View ArticlePubMedGoogle Scholar
  37. Park E, Jekni Z, Pino M, Murata N, Chen T: Glycinebetaine accumulation is more effective in chloroplasts than in the cytosol for protecting transgenic tomato plants against abiotic stress. Plant Cell Environ 2007, 30: 994–1005. 10.1111/j.1365-3040.2007.01694.xView ArticlePubMedGoogle Scholar
  38. Potenza C, Aleman L, Sengupta-Gopalan C: Targeting transgene expression in research, agricultural, and environmental applications: promoters used in plant transformation. In Vitro Cellular & Developmental Biology-Plant 2004, 40: 1–22.View ArticleGoogle Scholar
  39. Quan R, Shang M, Zhang H, Zhao Y, Zhang J: Engineering of enhanced glycine betaine synthesis improves drought tolerance in maize. Plant Biotechnol J 2004, 2: 477–486. 10.1111/j.1467-7652.2004.00093.xView ArticlePubMedGoogle Scholar
  40. Rhodes D, Hanson A: Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu Rev Plant Physiol Plant Mol Biol 1993, 44: 357–384. 10.1146/annurev.pp.44.060193.002041View ArticleGoogle Scholar
  41. Rontein D, Basset G, Hanson A: Metabolic engineering of osmoprotectant accumulation in plants. Metab Eng 2002, 4: 49–56. 10.1006/mben.2001.0208View ArticlePubMedGoogle Scholar
  42. Sakamoto A, Murata N: Genetic engineering of glycinebetaine synthesis in plants: current status and implications for enhancement of stress tolerance. J Exp Bot 2000, 51: 81. 10.1093/jexbot/51.342.81View ArticlePubMedGoogle Scholar
  43. Sakamoto A, Murata N: The role of glycine betaine in the protection of plants from stress: clues from transgenic plants. Plant Cell Environ 2002, 25: 163–171. 10.1046/j.0016-8025.2001.00790.xView ArticlePubMedGoogle Scholar
  44. Scheid O, Probst A, Afsar K, Paszkowski J: Two regulatory levels of transcriptional gene silencing in Arabidopsis. PNAS 2002, 99: 13659–13662. 10.1073/pnas.202380499PubMed CentralView ArticleGoogle Scholar
  45. Sulpice R, Tsukaya H, Nonaka H, Mustardy L, Chen T, Murata N: Enhanced formation of flowers in salt stressed Arabidopsis after genetic engineering of the synthesis of glycine betaine. Plant J 2003, 36: 165–176. 10.1046/j.1365-313X.2003.01873.xView ArticlePubMedGoogle Scholar
  46. Turner N: Measurement of plant water status by the pressure chamber technique. Irrigation Sci. 1988, 9: 289–308. 10.1007/BF00296704View ArticleGoogle Scholar
  47. Waditee R, Bhuiyan M, Nazmul H, Rai V, Aoki K, Tanaka Y, Hibino T, Suzuki S, Takano J, Jagendorf A: Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in Synechococcus and Arabidopsis. PNAS 2005, 102: 1318. 10.1073/pnas.0409017102PubMed CentralView ArticlePubMedGoogle Scholar
  48. Xiong L, Schumaker KS, Zhu JK: Cell signaling during cold, drought, and salt stress. Plant Cell 2002, 14: 165–183. 10.1105/tpc.010278View ArticleGoogle Scholar
  49. Yang XH, Liang Z, Lu CM: Genetic engineering of the biosynthesis of glycinebetaine enhances photosynthesis against high temperature stress in transgenic tobacco plants. Plant Physiol 2005, 138: 2299–2309. 10.1104/pp.105.063164PubMed CentralView ArticlePubMedGoogle Scholar
  50. Zhang J, Kirkham M: Drought-stress-induced changes in activities of superoxide dismutase, catalase, and peroxidase in wheat species. Plant Cell Physiol 1994, 35: 785.Google Scholar
  51. Zhang N, Ci H, Li L, Yang T, Zhang C, Wang D: Enhanced drought tolerance of potato with introduced BADH gene. Acta Agronomica Sin. 2009, 35: 1146–1150. 10.3724/SP.J.1006.2009.01146View ArticleGoogle Scholar
  52. Zhou S, Chen X, Zhang X, Li Y: Improved salt tolerance in tobacco plants by co-transformation of a betaine synthesis gene BADH and a vacuolar Na + /H + antiporter gene SeNHX1. Biotechnol Lett 2008, 30: 369–376. 10.1007/s10529-007-9548-6View ArticlePubMedGoogle Scholar

Copyright

© Cheng et al.; licensee Springer. 2013

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.