Acta Physiologiae Plantarum

, Volume 35, Issue 3, pp 919–929

Comparative physiological responses of the yeast halotolerance genes expressed in transgenic lines of tomato cv Rio Grande under saline conditions

Authors

  • Naila Safdar
    • Department of BiochemistryQuaid-i-Azam University
    • Department of BiochemistryQuaid-i-Azam University
  • Nazif Ullah
    • Department of BiochemistryQuaid-i-Azam University
Original Paper

DOI: 10.1007/s11738-012-1135-3

Cite this article as:
Safdar, N., Mirza, B. & Ullah, N. Acta Physiol Plant (2013) 35: 919. doi:10.1007/s11738-012-1135-3
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Abstract

We previously analyzed the transgenic lines of tomato cv Rio Grande over-expressing the yeast HAL I and HAL II genes for their response to salt stress under in vitro conditions. In this study, six homozygous tomato lines harbouring the yeast HAL I or HAL II genes with highest expression level were selected for exploring their physiological responses against different salt stresses in the field. These transgenic plants showed significant growth and improved water content in comparison with control under 100 and 150 mM salt stress conditions. The HAL I and HAL II lines showed better Ca2+ content than their control counterparts. Furthermore, the transgenic lines exhibited lower values of relative electrical conductivity and improved resistance against the fungal pathogens Fusarium oxysporum and Alternaria solani when tested by detached leaf and agar tube dilution assays. Physiological analyses carried out in this study suggest an involvement of multiple mechanisms in transgenic tomato plants harbouring yeast genes to confer biotic and abiotic tolerance under stress conditions.

Keywords

Antifungal assayCalcium contentFusarium oxysporumRelative electrolyte conductanceSolanaceaeWater content

Introduction

Plants growing on land experience a multitude of environmental stresses. These stresses include drought, salinity, water logging and extremes of temperature, radiation, deficiency or excess of minerals. Although appraisal of the effects of all different stresses on plants is important, in the existing literature more emphasis of researchers can be observed on salinity than on other stresses. This is due to the fact that vast areas of available land on the globe comprising a large number of countries are affected by salinity. Ashraf and Foolad (2007) have evoked a number of adverse effects of salinity on plants such as ionic effect, osmotic effect, nutrient imbalance, hormonal imbalance and production of reactive oxygen species (ROS). These adverse effects result in plant growth inhibition and even plant death.

A plant responds to salt stress at three different levels, i.e. cellular, tissue and whole plant level. Results from metabolic engineering experiments have confirmed the functional roles of compatible osmolytes such as mannitol, proline and betaine in salt adaptation of higher plants (Hu et al. 2005; Park et al. 2007; Teixeira and Pereira 2007). These organic compounds are thought to mediate osmotic adjustment, protecting sub-cellular structures and oxidative damage by their free radical scavenging capacity. The overexpression of antioxidative enzymes such as catalase, ascorbate peroxidase and superoxide dismutase has also been reported to improve the tolerance of plants to environmental stresses (Lu et al. 2007; Moriwaki et al. 2008; Chatzidimitriadou et al. 2009).

Another appealing approach is the introduction of genes that maintain the ion homeostasis in plants. Several yeast genes have been identified such as HAL 1 gene from Saccharomyces cerevisiae, which modulates the cation transport system and maintain a high internal K+ concentration and decreased intracellular Na+ ions when expressed in tomato or Arabidopsis (Gisbert et al. 2000; Yang et al. 2001). In yeast, SOD 2 gene was identified from Schizosaccharomyces pombe as a Na+/H+ antiporter on the plasma membrane and was effective for reducing Na+ toxicity in transgenic Arabidopsis and rice under salt stress conditions in greenhouse (Gao et al. 2003; Zhao et al. 2006). Another identified yeast HAL II gene does not affect ion homeostasis but is involved in the pathway of methionine biosynthesis and confer lithium and sodium tolerance (Glase et al. 1993).

We have previously reported the transformation of tomato cultivar “Rio Grande” with yeast HAL I and HAL II gene and the characterization of the inter-transformant expression variability of the homozygous lines harbouring the respective genes under in vitro salt stress conditions (Safdar et al. 2011). To further characterize the effect of these yeast genes in homozygous tomato progenies, here we report the growth responses of HAL I/HAL II overexpressing tomato lines at higher salt stress levels (100, 150 and 200 mM) under physiological green house conditions. Besides the calcium level determination in different parts of the plant, the water content was also assessed and antifungal potential and cell membrane stability tests under stressed and non-stressed conditions were also carried out in comparison with the control plants.

Materials and methods

Plant material and salt stress treatments

Six independent transgenic lines of tomato cv “Rio Grande” containing HAL I (THI-2, THI-5, THI-6) or HAL II (THII-2, THII-4, THII-5) gene and showing appropriate expression level of the transgene were selected for this study. Homozygous seeds from the third generation were germinated in a small pot containing a mixture of peat and vermiculite (2:1) and grown in a growth chamber at 25 ± 2 °C with 16 h of photoperiod, illumination of 45 μE m−2 s−1 and 60 % relative humidity. The wild type cv “Rio Grande” served as a control in the experiment. Once hardened, the plants were shifted to the green house and allowed to grow for 4 weeks before salt stress treatments. The plants were exposed to different concentrations of NaCl 50, 100, 150 and 200 mM for salt stress treatments. Control and transgenic lines were grown in three replicates for each salt treatment. Different concentrations of NaCl were mixed with Hoagland’s nutrient solution and were given on every third day to the field grown transgenic HAL I and HAL II lines along with the control plants. This salt treatment regime was repeated for a period of 6 weeks. The growth parameters of the tomato HAL I and HAL II transformants in comparison with control lines subjected to different salt stress treatments were observed after 6 weeks of treatment and recorded. Fully expanded leaves from the transgenic and control lines were then tested for cell membrane stability and antifungal assays.

Measurement of fresh weight, dry weight and water content

After 6 weeks of salt stress treatment, the transgenic and control plants were allowed to grow fruits. After harvesting the fruits, the whole plant growth was determined by measuring fresh weight, dry weight and water content. The fresh weight of each individual HAL I and HAL II transformants along with the control was measured immediately after the harvest. Dry weight was measured after drying the plants at 70 °C for 2 days. Water content of plants was calculated by using the formula “fresh weight − dry weight/dry weight”.

Polymerase chain reaction

A simple and efficient plant DNA extraction procedure for isolation of high quality DNA was carried out according to Ahmed et al. (2009). PCR was performed in 25 μl total reaction mixture. The reported sequences of NPT II, HAL I and HAL II primers were used (Safdar et al. 2011). During PCR, the DNA was denatured at 94 °C for 5 min followed by 35 amplification cycles of denaturation at 94 °C for 30 s, 30 s of annealing at 53 °C for NPT II gene, 56 °C for HAL I gene and 55 °C for HAL II gene with an extension of 72 °C for 60 s.

Cell membrane stability test

The cell membrane permeability of the transgenic and wild tomato plants was determined according to the method defined by Wang et al. (2006) based on the relative electrolyte exudation ratio. The leaves of the transgenic and control samples subjected to salt stress were thoroughly rinsed with deionized water. Six equal round sections were cut from the leaves of each sample, put in a clean beaker with 30 ml deionized water under vacuum for 15 min. The electrical conductivity was measured in micro Siemens (μS) with a digital conductivity meter and was denoted as E1. The leaves were then heated at 90 °C for 20 min and cooled at room temperature. Electrical conductivity was measured again as E2. Relative electrical conductance was calculated using the formula E1/E2 × 100.

Measurement of cation concentrations

Calcium content was determined in independent homozygous HAL I and HAL II tomato transformants in comparison with control. Plant material subjected to salt stress was separated into leaves, stems (including petiole) and roots and was dried at 70 °C for 3 days. The dried samples were weighed and digested with the nitric-perchloric acid mixture (2:1 v/v). Solutes were analyzed in the digested material by atomic absorption spectrophotometry.

Fungal resistance assays

Detached leaf assay

The tomato fungal pathogens Fusarium oxysporum and Alternaria solani were cultured in Sabouraud dextrose agar (SDA) medium for 4 days. Intact leaves from the HAL I and HAL II transformants as well as the control plants were inoculated on petri plates containing growing fungus and incubated at 28 °C for 6 days. Three leaves were inoculated on each petri plate and each treatment was performed in triplicate. After 6 days, the percentage of infection was calculated by the formula.
$$ {\text{Percentage of infection}} = \frac{\text{Number of leaves infected}}{\text{Total number of leaves inoculated}} \times \, 100 $$

Agar tube dilution assay

Antifungal activity of leaf extracts was determined using the agar tube dilution method (Hanif et al. 2007) with some modifications. Leaf tissue from the transgenic and control lines were ground in a sodium phosphate buffer (pH 7.0) and incubated at room temperature for 30 min. The crude leaf extract was centrifuged at 14,000 rpm for 20 min and the supernatant was filter-sterilized. Five hundred microlitre of the leaf extract prepared was added to the autoclaved non-solidified SDA medium cooled to about 46 °C. Tubes containing SDA and the leaf extracts of the respective transformants were allowed to solidify in a slanting position at room temperature. The mycelial discs of F. oxysporum and A. solani were inoculated in the centre of the leaf-extract amended medium, placed in incubator at 28 °C and the linear growth of the fungus was measured after 6 days of incubation. The percentage of fungal inhibition was calculated by the formula.
$$ {\text{Percentage}}\;{\text{of}}\;{\text{inhibition}} = \frac{{{\text{Linear}}\;{\text{growth}}\;{\text{of}}\;{\text{C2}}\;\left( {\text{mm}} \right) - {\text{Linear}}\;{\text{growth}}\;{\text{of}}\;{\text{sample}}\;\left( {\text{mm}} \right) \times 100}}{{{\text{Linear}}\;{\text{growth}}\;{\text{of}}\;{\text{C1}}\;\left( {\text{mm}} \right)}} $$
where C1 represents the mycelial fungal growth on medium lacking leaf extract and C2 indicates the mycelial fungal growth on medium containing leaf extract from the control plants.

Statistical analysis

The data were subjected to analysis of variance and mean comparisons among the transgenic and control lines were made using the least significant difference at 0.05 probability level. The statistical software program MSTATC version 2.00 (East Lansing, MI, USA) was used for the experimental analysis.

Results

This study is follow-up of our on-going work on genetic modification of tomato for improvement of salt tolerance (Safdar et al. 2011). Three homozygous lines of the HAL I (THI-2, THI-5, THI-6) or HAL II (THII-2, THII-4, THII-5) tomato transformants showing relatively high salt tolerance indices based on their respective gene expression analysis were selected. Transformation of these plants was confirmed by PCR which detected the presence of NPT II, HAL I and HAL II gene in a total of six independent tomato transgenic lines while the control plants showed no positive PCR results (Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs11738-012-1135-3/MediaObjects/11738_2012_1135_Fig1_HTML.gif
Fig. 1

The PCR products of: HAL 1 gene (a), HAL II gene (b) and NPT II gene (c). Lanes 1–9 PCR product of independent transgenic lines, P plasmid, C control, M 1 kb marker (Fermentas)

Growth parameters of HAL I and HAL II transformants in green house

To investigate the response of HAL I and HAL II tomato transgenic lines, 4-week-old homozygous seedlings were shifted to the green house and were treated with NaCl at 50, 100, 150 and 200 mM every 3 days for a period of 6 weeks until the plants reached the late reproductive stage. Table 1 shows that the transgenic lines had a markedly enhanced salt tolerance physiologically and their behaviour varied with the salt stress imposed. No significant difference was observed among the transgenic lines and control plants at 0 and 50 mM NaCl in any of the parameters studied. The HAL I and HAL II transformants showed improved plant height, number of nodes, leaves and stem diameter at high salt concentrations of 100 and 150 mM (Fig. 2). Control plants showed symptoms of injury at these salt concentrations by showing stunted height and necrotic leaves, whereas the transgenic lines continued to grow normally and produced flowers and fruits under these salt concentrations. As reported previously, the HAL I and HAL II transformants produced small-sized fruits in comparison with control; however, the fruit yield of the transformants was not affected by salt stresses (Safdar et al. 2011).
Table 1

Growth parameters of homozygous HAL I and HAL II tomato transformants along with the wild type plants after six week treatment of salt stress

Parameters measured

NaCl (mM)

Control

THI-2

THI-5

THI-6

THII-2

THII-4

THII-5

Plant height (cm)

0

40.0 ± 1.9

41.3 ± 1.4

38.9 ± 2.5

42.8 ± 0.6

40.1 ± 1.9

40.5 ± 2.0

42.8 ± 1.5

50

38.5 ± 1.3

40.0 ± 0.6

39.7 ± 0.4

38.7 ± 0.2

39.9 ± 0.8

40.5 ± 0.7

37.9 ± 0.4

100

27.2 ± 0.8

37.8 ± 1.7**

32.5 ± 1.0**

35.3 ± 2.6**

31.9 ± 1.5**

34.6 ± 1.4**

32.8 ± 0.7**

150

12.9 ± 1.7

29.8 ± 1.0**

22.9 ± 0.7**

25.7 ± 1.2**

23.3 ± 0.5**

25.9 ± 0.9**

23.6 ± 1.1**

200

9.6 ± 0.9

12.1 ± 0.4

9.5 ± 0.5

10.0 ± 0.1

9.0 ± 0.6

9.7 ± 1.3

9.4 ± 0.7

Stem diameter (cm)

0

1.11 ± 0.1

1.14 ± 0.2

1.11 ± 0.2

1.12 ± 0.04

1.1 ± 0.2

1.11 ± 0.2

1.02 ± 0.1

50

1.02 ± 0.04

1.11 ± 0.1

1.08 ± 0.1

1.02 ± 0.1

1.08 ± 0.1

1.11 ± 0.5

1.08 ± 0.2

100

0.95 ± 0.2

0.98 ± 0.1

0.98 ± 0.1

1.05 ± 0.2

1.02 ± 0.1

1.08 ± 0.1

1.02 ± 0.1

150

0.73 ± 0.3

1.02 ± 0.4**

1.02 ± 0.1**

1.0 ± 0.01**

0.95 ± 0.2**

1.02 ± 0.3**

1.0 ± 0.2**

200

0.63 ± 0.03

0.66 ± 0.03

0.57 ± 0.2

0.70 ± 0.1

0.63 ± 0.1

0.66 ± 0.1

0.61 ± 0.1

Number of nodes

0

10.1 ± 0.7

10.6 ± 1.5

10.4 ± 0.8

10.9 ± 1.0

10.2 ± 0.5

10.6 ± 0.9

10.2 ± 0.7

50

9.7 ± 0.8

10.4 ± 0.6

9.3 ± 1.1

9.6 ± 0.6

9.1 ± 0.3

9.4 ± 1.2

9.7 ± 1.0

100

5.6 ± 1.0

8.2 ± 0.6**

7.7 ± 0.9**

7.8 ± 1.1**

6.9 ± 0.9

7.4 ± 0.6**

7.0 ± 0.8**

150

3.4 ± 0.4

6.7 ± 0.3**

6.4 ± 0.5**

6.4 ± 0.4**

6.6 ± 0.1**

6.0 ± 0.4**

5.9 ± 0.7**

200

2.2 ± 0.1

3.3 ± 0.1

3.1 ± 0.1

3.1 ± 0.2

3.2 ± 0.1

2.9 ± 0.04

2.7 ± 0.7

Leaves/plant

0

60.3 ± 2.0

59.0 ± 2.1

58.2 ± 2.0

55.1 ± 1.7

50.3 ± 1.4

56.0 ± 1.6

51.9 ± 1.6

50

58.2 ± 1.9

57.0 ± 2.0

56.2 ± 1.1

56.8 ± 1.2

54.1 ± 1.4

53.4 ± 1.2

49.2 ± 0.4

100

29.9 ± 1.5

36.9 ± 1.2**

36.5 ± 0.8**

33.3 ± 0.7**

34.0 ± 0.6**

36.6 ± 1.4**

34.5 ± 0.9**

150

9.3 ± 1.2

30.5 ± 0.9**

33.5 ± 0.3**

31.9 ± 0.4**

25.1 ± 0.4**

28.1 ± 0.7**

24.9 ± 1.0**

200

8.1 ± 0.5

9.3 ± 0.1

10.3 ± 0.3

9.5 ± 0.1

10.0 ± 0.1

10.3 ± 0.4

9.1 ± 0.3

Degrees of injury

0

+

+

+

+

+

+

+

50

+

+

+

+

+

+

+

100

++

+

+

+

+

+

+

150

+++

++

++

++

++

++

++

200

++++

++++

++++

++++

++++

++++

++++

For each line and treatment, values are given as a mean of three replicates ± SE

** Represents the highly significant difference in data compared with control plants (P < 0.01)

The bold values indicate the most significant results obtained among HAL I and HAL II transformants in comparison with control plants. +, no injury; ++, moderate symptoms of injury/the plant grew relatively slowly and some of the leaves tip turned necrotic; +++, serious symptom of injury/the plant grew slowly and the majority of leaves start dying; ++++, nearly to death

https://static-content.springer.com/image/art%3A10.1007%2Fs11738-012-1135-3/MediaObjects/11738_2012_1135_Fig2_HTML.jpg
Fig. 2

Representative phenotypes of HAL I (1) and HAL II (2) transformants in comparison with control (C) subjected to salt treatment of 100 mM (a) and 150 mM (b). The picture was taken 6 weeks after the salt treatment

Highly significant growth difference (P < 0.01) was observed among the halotolerant transgenic lines at high salt stress, thus, plants growing under high 150 mM salt stress conditions were characterized in our project for further study to explore the possible function of yeast genes in tomato plants under highly significant salt stress. However, none of the transgenic plants carrying HAL I or HAL II genes survived at 200 mM salt stress nor did the control plants. Thus, all lines showed progressive chlorosis, reduced leaf number, growth inhibition and were severely damaged and died at the sixth week of 200 mM salt stress treatment.

Effect of constitutive HAL I and HAL II expression on the fresh weight, dry weight and water content

Progeny of HAL I and HAL II homozygous plants showed improved physiological and growth responses when cultivated under a salt stress of 100 and 150 mM compared to control. As no significant difference was observed between transgenic and wild-type plants at 50 and 200 mM salt stress concentrations, the growth behaviour of the transgenic lines was corroborated with their fresh, dry weight and water content at 100 and 150 mM salt concentrations.

As shown in Fig. 3, the fresh weight of the HAL I and HAL II transformants under selected salt concentrations improved significantly (P < 0.05) as compared to control plants. Among all the lines tested, the HAL I line THI-2 showed increased fresh weight with or without salt stress. At 150 mM NaCl, the fresh weight of control plants was severely affected but only slightly for the transgenic plants that even showed increased fresh weight. The HAL II lines also showed better performance than control plants but slightly less than HAL I lines. In general, HAL I lines continued to show improved fresh weight, dry weight and water content as compared with the HAL II lines and control plants.
https://static-content.springer.com/image/art%3A10.1007%2Fs11738-012-1135-3/MediaObjects/11738_2012_1135_Fig3_HTML.gif
Fig. 3

Fresh weight (a), dry weight (b) and water content (c) of the whole plant tested in HAL I (THI-2, THI-5, THI-6) and HAL II (THII-2, THII-4, THII-5) tomato transformants along with control type. Control plants and transgenic plants were grown on the soil for 3 months and watered every third day until the plants were subjected to salt stress. Asterisk indicates statistically significant data from the control plants at P < 0.05. Values represents mean of three replicates ± SE

Taken together, these results indicate that the HAL I overexpressing tomato transgenic plants are more tolerant to salt stress under green house conditions than the HAL II plants. The HAL I and HAL II lines showed improved physiological and growth behaviour in comparison with control plants but at relatively different rates.

Calcium level in HAL I and HAL II tomato transformants

It is known that re-establishing ion homeostasis is of critical importance for plant adaptation to salt stress. Our previous study (Safdar et al. 2011) suggested K+/Na+ ratio to be one of the key determinants of salt tolerance in HAL I transgenic lines only. To examine whether the calcium ions perform any role for increased salt tolerance in HAL I and HAL II lines, the homozygous tomato transformants subjected to 150 mM salt stress until late reproductive stage were selected and their leaves, shoots and roots along with the control plants were analyzed before and after exposure to salt stress (Table 2). Without NaCl stress, Ca2+ contents in both the HAL I and HAL II transgenic and control plants showed parallel values with little variations. On the other hand, Ca2+ concentration increased in all the experimental HAL I and HAL II lines when tested upon salt treatment compared with the control. This suggests a positive correlation between the increased Ca2+ and salt tolerance level in both transgenic HAL I and HAL II tomato plants.
Table 2

Cation concentration (mg/100 mg DW) in the leaves, shoots and roots of the 6-week old HAL I and HAL II tomato transformants grown with and without salt stress in comparison with control under green house conditions

Salt stress

Lines

Calcium ions (Ca2+) (mg/100 mg DW)

Leaf

Shoot

Root

0 mM

Control

4.7 ± 1.3

4.0 ± 0.9

2.9 ± 1.3

THI-2

5.5 ± 0.6*

5.7 ± 1.3*

3.4 ± 1.1*

THI-5

5.3 ± 1.9*

4.6 ± 1.1*

2.9 ± 1.0

THI-6

5.0 ± 1.2*

4.5 ± 1.0*

3.2 ± 2.0*

THII-2

5.3 ± 0.4*

3.9 ± 1.1

3.2 ± 1.6*

THII-4

5.6 ± 2.0*

4.5 ± 1.4*

3.0 ± 1.0

THII-5

5.7 ± 0.5*

4.9 ± 0.8*

3.3 ± 1.4*

150 mM

Control

1.8 ± 1.5

1.7 ± 1.0

0.9 ± 1.1

THI-2

4.8 ± 1.1**

3.4 ± 1.1**

2.5 ± 0.7**

THI-5

3.2 ± 1.4**

2.9 ± 0.8**

2.0 ± 1.1**

THI-6

3.1 ± 1.1**

3.3 ± 0.7**

1.9 ± 1.7**

THII-2

2.9 ± 1.1**

4.0 ± 0.8**

2.5 ± 0.9**

THII-4

3.4 ± 0.9**

3.1 ± 1.8**

2.3 ± 0.8**

THII-5

2.9 ± 1.1**

3.1 ± 1.7**

1.9 ± 1.2**

Asterisk represents the significant difference between control and transgenic lines at * P < 0.05 and ** P < 0.01, respectively

Values represent the means of three replicates ± SE

Relative electrical conductivity in transgenic and non-transgenic tomato

The relative electrical conductivity of HAL I and HAL II transgenic lines grown in a green house with and without salt treatment were tested (Fig. 4). The results showed that no significant difference was found between control and transgenic lines under non-stressed conditions. However, under 150 mM salt stress treatment, the relative electrical conductivities were distinctly lower in transgenic lines with the lowest value (16.1 %) being observed in HAL II line (THII-4) followed by the HAL I line (THI-2) which showed 21.7 % value. Interestingly, HAL II lines suffered the least membrane damage among all the transformants implying the functional role of the HAL II gene in protecting the cell membrane more efficiently compared to HAL I lines. Overall, salt stress caused more severe membrane damage to control plants than to the transgenic lines.
https://static-content.springer.com/image/art%3A10.1007%2Fs11738-012-1135-3/MediaObjects/11738_2012_1135_Fig4_HTML.gif
Fig. 4

Relative electrical conductivity of transgenic tomato and control plants before and after 150 mM salt stress. Values represent means of three replicates ± SE. Asterisk indicates significantly different from control at * P < 0.05 and ** P < 0.01, respectively

Antifungal potential of leaf/extracts prepared from HAL I and HAL II transformants

To test the possible antifungal role of the yeast genes in tomato plants, two different antifungal assays were carried out. In detached leaf assay, the leaves of the control plants infected with the Alternaria and Fusarium specie showed visible symptoms of infection after 2 days of inoculation. On the other hand, transgenic HAL I lines were able to resist the infection after 2 days of post inoculation and the necrotic symptoms were observable after 4 days of infection (Table 3). After 6 days, the control leaves showed 100 % infection and were completely colonized by fungal hyphae resulting in their death. By contrast, disease severity was relatively reduced in transgenic HAL I lines as compared to the HAL II lines. The transgenic lines showed delayed appearance of disease symptoms and smaller lesions compared to the controls as evidenced by Fig. 5a, b.
Table 3

Detached leaf and agar tube dilution method for testing transgenic HAL I and HAL II tomato lines for resistance against fungal pathogens

Lines

Detached leaf assay

Agar tube dilution assay

Detached leaf assay

Agar tube dilution assay

Alternaria solani

Fusarium oxysporum

Days after inoculation

Inhibition percentage (%) ± SE

Days after inoculation

Inhibition percentage (%) ± SE

2

4

6

2

4

6

Control

+++

++++

+++++

2.1h ± 0.0

+++

++++

+++++

0i

THI-2

++

+++

34.8a ± 0.2

++

+++

30.0b ± 1.1

THI-5

++

+++

30.0b ± 1.2

++

+++

22.7d ± 0.7

THI-6

++

+++

30.5b ± 0.7

++

+++

26.9c ± 1.1

THII-2

+

+++

++++

6.8f ± 0.4

+

+++

+++++

5.5 g ± 1.0

THII-4

+

+++

++++

7.2f ± 1.0

+

+++

++++

11.8e ± 0.4

THII-5

+

+++

++++

6.9f ± 0.3

+

+++

++++

10.9e ± 1.3

Results are based on three independent experiments and each experiment contained three replicates ± SE. Severity of infection was scored on a comparative scale as compared to the response in wild type plant: −, no infection; +, 20 % infection; ++, 40 % infection; +++, 60 % infection; ++++, 80 % infection; +++++, 100 % infection

The superscript alphabets are LSD rank orders

https://static-content.springer.com/image/art%3A10.1007%2Fs11738-012-1135-3/MediaObjects/11738_2012_1135_Fig5_HTML.jpg
Fig. 5

Fungal resistance assay in transgenic tomato plants. Response of the HAL I (1) and HAL II (2) transformants along with the control (c) plants analyzed by leaf detached method using two fungal strains Alternaria solani (a) and Fusarium oxysporum (b). Effect of leaf extract from transgenic plants on the growth of Alternaria solani (c) and Fusarium oxysporum (d). (C) Mycelial growth on medium lacking leaf extract; (C1) mycelial growth on medium containing the leaf extract from control plant

The leaf extracts from the transgenic plants also showed antifungal potential but at a lower rate. As shown in Table 3, only the transgenic HAL I lines THI-2 showed the maximum inhibition percentage (34.8 % and 30 %) against both fungal species tested. Moreover, maximum percentage of inhibition in HAL II transgenic line was found to be 7.2 % against A. solani and 11.8 % against F. oxysporum. The degree of fungal resistance varied between different HAL Iand HAL II transgenic lines with the improved resistance shown by the HAL I lines. The leaf extracts from the control plant had no significant effect on the inhibition of the fungal growth (Fig. 5c, d).

Discussion

In vitro salt response studies on the HAL I and HAL II genes have been carried out when expressed in tomato and citrus (Cervera et al. 2000; Gisbert et al. 2000; Arrillaga et al. 1998). Here, we studied transgenic tomato plants harbouring yeast genes in the field and we assessed the physiological and growth parameters of selected transformants. Furthermore, the field plants were also tested for the water content, ionic levels, relative electrolyte conductance and fungal resistance assays.

Low salt concentration of 50 mM NaCl did not affect any of the morphological parameters tested in the HAL I and HAL II transformants. Their number of leaves and plant height were comparable to the control plants. Conversely, the HAL I and HAL II transformants showed significantly better growth compared to control plants lacking the gene of interest under 100 and 150 Mm NaCl. The control plants under this salt concentration were damaged, showed necrotic symptoms and reduced chlorophyll content. The tomato transformants survived healthily at these moderate levels of salt concentrations. However, a higher salt concentration of 200 mM severely affected both the transgenic and control plants under green house conditions. Zhou et al. (2007) also studied the growth parameters of green house grown transgenic tomato plants harbouring betaine aldehyde dehydrogenase gene (BADH) under different NaCl treatments. These transgenic tomato plants showed better performance at high salt concentrations than the control plants. They observed non-significant differences between transgenic BADH tomatoes and their isogenic counterparts at low salt concentrations. Another green house study of transgenic plants was reported by Saad et al. (2010), who evaluated the green house performance of transgenic tobacco plants carrying “stress associated protein” gene (AlSAP) isolated from Aeluropus littoralis. His research group reported a slight increase in transgenic plant parameters when studied under non-stressed conditions such as whole plant weight, root and shoot length, leaf number, plant yield compared with the control. However, under salt stress, the transgenic tobacco plants continued to grow better and showed higher level of salt tolerance. Various other studies reported improved performances of different salt tolerant transgenic plants under salt stress (Rus et al. 2001; Ellul et al. 2003; Zhao et al. 2006; Khoudi et al. 2009; Zhou et al. 2009; Choi et al. 2011).

According to the two-phase salt response hypothesis, plant growth is first inhibited by cellular response to the osmotic effects and later by the toxic effects due to excessive salt accumulation in plant cells (Serrano 1996). These responses were also observed in control and HALI/HAL II tomato plants when tested under different salt concentrations. Since osmotic stress causes immediate reduction in cell expansion of roots and leaves (Vijayan 2009), fresh weight and dry weight of the whole plants under stress were used as indicators to demonstrate osmotic effects in transgenic plants. Increased fresh weight, dry weight and water content in the HAL I and the HAL II transgenic lines determined after the salt stress imposition as compared with control plants indicated decreased osmotic shock and beneficial effect of the expression of yeast genes. Number of studies has shown the direct association between the increased biomass (fresh weight, dry weight, water content) and different salt tolerant transgenic plants produced. e.g. Hasthanasombut et al. (2010) produced salt tolerant transgenic tobacco plants carrying betaine aldehyde dehydrogenase 1 gene (OsBADH1) and reported improved fresh and dry weight of the tobacco transformants under salt stress. Similarly, Gao et al. (2003) also reported increased salt tolerance of transgenic Arabidopsis plants overexpressing yeast SOD2 genes with higher fresh and dry weight than that of the control plants. Other reports mentioning the correlation of increased fresh and dry weight and water content with the salt tolerant transformants include Aswath et al. (2005), Sergeeva et al. (2006), Hussaiani and Abdin (2008), Zhou et al. (2009), Khare et al. (2010).

The level of calcium ion toxicity in transgenic and control plants exposed to a concentration of 150 mM NaCl was determined. Interestingly, Ca2+ content in the HAL II lines was almost similar to that found in HAL I lines both in the shoots and the roots. Zhao et al. (2006) expressed the plasma membrane Na+/H+ antiporter SOD2 gene from yeast in rice and reported increased salt tolerance in transgenic rice plants. They observed higher levels of K+/Na+ ratio, Ca2+ and Mg2+ ionic contents in transgenic rice plants and correlated the elevated levels of Ca2+ ions with the uptake of K+ ions. Zhou et al. (2007) also demonstrated increased K+ and Ca2+ ions in transgenic tomato plants harbouring BADH genes and showing resistance against salt stresses. Our results also suggest that Ca2+ ions play a physiologically relevant role in plant responses to salinity.

Ca2+ is well known for its essentiality in plant growth and plays important roles in stabilizing cell walls and membranes as well as a second messenger (Hepler 2005). The increased Ca2+ level in HAL I and HAL II plants supports our finding of the cell membrane stability test, where the HAL I and HAL II transgenic lines showed relatively less electrolyte exudation ratio suggesting less damage to the halotolerant transgenic plants membrane when exposed to salt stress. Zhao et al. (2009) and Xue et al. (2009) introduced the transcription factor gene YAP1 and heat shock protein gene HSP26, respectively in Arabidopsis plants and communicated increased abiotic stress tolerances in the transformants. They both detected a lower electrolyte conductance in transgenic Arabidopsis plants compared to wild type plants when exposed to salt or freezing stresses, thus, implying less membrane injury in transformants. Similar studies also reported lower electrical conductivities in transgenic tomato, potato and tobacco plants showing resistance against abiotic stresses (Wang et al. 2006; Zhou et al. 2007; Roy et al. 2006; Kim et al. 2009; Khare et al. 2010). So, it is reasonable to hypothesize that besides its known functions, the HAL I and HAL II gene also confers salt tolerance by playing an important role in protecting the membranes against salt damage. Moreover, Ca2+ ions in the HAL II transformants are suspected to play a direct or indirect role in improving salt tolerance.

In the present in vitro antifungal study, the transgenic HAL I and HAL II tomato plants were tested against F. oxysporum and A. solani which are responsible for the fusarium wilt and early blight disease in tomato. These transformants showed a certain degree of resistance against above-mentioned fungal pathogens at a relatively different level compared with the control. The effect of HAL genes on plant disease resistance has not been reported yet; however, previous studies have shown the function of certain genes that confer both biotic and abiotic stress tolerance when expressed in transgenic plants. e.g. Pathogenesis-related PR-5 osmotin like gene expressed in transgenic potato plants conferred resistance against late blight fungus P. infestans and abiotic stresses (Zhu et al. 1995). Similarly, Rajam et al. (2007) expressed thaumatin gene isolated from Thaumatococcus danielli in transgenic tobacco plants which showed enhanced survival and germination rate under salt and PEG-mediated drought stress. These transgenic plants also exhibited enhanced resistance against fungal infections caused by Pythium aphanidermatum and Rhizoctonia solani. Moreover, Prabhavathi and Rajam (2007) also reported the expression of E. coli mannitol-1-phosphate dehydrogenase gene (mtlD) in transgenic egg plants which showed tolerance to both biotic and abiotic stresses. They found that mtlD gene along with acting as an osmoprotectant and conferring salt and drought tolerance in transgenic egg plants also functions to increase the resistance against fungal infections caused by Fusarium oxysporium, Verticillium dahliae and Rhizoctonia solani under both in vitro and in vivo growth conditions. Thus, HAL I and HAL II genes along with functioning under abiotic stresses also show improved resistance against fungal infections. This can be explained by the involvement of yeast genes in the alleviation of osmotic shock which in turn could affect the fungal infection process as the osmotic stress resulting in wilting is a critical early requirement for infection. Yeast genes seem to be involved in protecting the host plants from fungal infection by reducing the osmotic stress. Our findings also support the role of yeast genes in the protection of cell membrane and this might be one of the factors contributing to the improved resistance of the HAL I and HAL II transformants against fungal infections in comparison with control plants.

In conclusion, this study has demonstrated the multiple effects of the yeast genes when introduced into the crop tomato cv “Rio Grande” which showed tolerance against both biotic and abiotic stresses at positive level. Enhancement of multiple stress tolerances in practical varieties of tomato grown as food crops would be of great benefit to agriculture local areas. In short, the introgression of genes that are known to be involved in stress response and putative tolerance might prove to be a faster track towards improving crop varieties.

Author’s contribution

Naila Safdar: Carried out the experimental work and wrote the manuscript. Bushra Mirza: Designed the project and proof read the manuscript. Nazif Ullah: Improved the English of the manuscript.

Acknowledgments

The authors sincerely thank Dr. Ramon Serrano (Instituto de Biologia Molecular y Celular de plantas, Valencia, Spain) for the kind provision of HAL I and HAL II constructs. We are also thankful to the Higher Education Commission of Pakistan for the financial support of this research project.

Conflict of interest

The authors declare that they have no conflict of interest.

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2012