Molecular Responses of Bt Transgenic Corn (Zea mays L.) to Salt (NaCl) stress

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Author: Mohamed Salah Beltagi

Molecular Responses of Bt Transgenic Corn (Zea mays L.) Plans to Salt (NaCl) Stress

Mohamed S. Beltagi Botany Department, Faculty of Science, Suez Canal University, Ismailia, Egypt.

ABSTRACT

Non-transgenic and Bt transgenic crop lines of a hybrid (YieldGard 2) corn (Zea mays L.) plants in the 4th leaf stage were subjected to 0, 50, 100 and 150 mM NaCl. Significant (P ≥ 0.05) reduction in growth of non-transgenic plants was recorded under almost all levels (50, 100 and 150 mM) of salt (NaCl) stress; while, the growth of the Bt transgenic corn plants showed no significant changes under the same levels of salt stress. Chlorophyll a contents was reduced at 150 mM NaCl only; but did not change in the Bt transgenic corn plants. Chlorophyll b was not responsive to NaCl treatments in both non-transgenic and Bt transgenic corn plants. Chlorophyll stability index (CSI) were always higher in the Bt transgenic than in the non-transgenic corn plants under all salinity levels. The analyses of SDS-PAGE revealed relative stability in the patterns of protein bands in the Bt transgenic corn plants under salinity stress. The sum of optical densities of protein bands was higher in the Bt transgenic corn plants. In response to salinity stress, a group of polypeptide (76.96, 59.38, 41.56, 33.5 and 31.26 KDa) were newly synthesized in both non-transgenic and Bt transgenic plants. Salt-susceptible polypeptides of molecular weights 325.47, 32.64 and 24.17 KDa were found only in non-transgenic corn plants and completely disappeared under all level of salt stress; while two polypeptides of molecular weights 38.59 and 30.61 KDa were totally inhibited in all salt-stressed corn plants. The synthesis of another four polypeptides (298.81, 99.82, 20.79 and 19.43 KDa) was solely specific to the Bt transgenic corn plants. Stability of chlorophyll pigments and molecular weights of salt stress responsive proteins are key genetic determinants of salt stress in Bt transgenic corn plants.

Key words: Bt Transgenic corn, salt stress, SDS-PAGE proteins, CSI .

INTRODUCTION

Soil salinity, one of the major abiotic stresses reducing agricultural productivity, affects large terrestrial areas of the world. The damaging effects of salt accumulation in agricultural soils have influenced ancient and modern civilizations. It is estimated that 20% of the irrigated land in the world is presently affected by salinity (Yeo, 1999). Reduction in growth and yield are surely the most conspicuous physiological responses of the crop to excessive salts in the media.

One of the two main approaches that have been adopted to improve salt tolerance of crops is the generation of transgenic plants to introduce novel genes or to alter expression levels of the existing genes to affect the degree of salt stress tolerance (Yamaguchi and Blumwald, 2005). For verifying the molecular basis of salinity tolerance in genetically modified (transgenic) plants, proteomic changes should be identified and detected. In contrast to the genome, the proteome is not static but rather responsive to many internal and external factors (Zhu et al., 1995). Thus, identifying differentially regulated proteins would assist in the analysis of gene expression altered under salinity stress and involved in the mechanism of salt resistance (Zörb et al., 2004).

Corn (Zea mays L.) is considered as a moderately salt-sensitive plant (Mass and Hoffman, 1977). Efforts to improve the crop performance under environmental stresses have not been that fruitful because the fundamental mechanisms for stress tolerance in plants remain to be completely understood. Bt transgenic corn is normal corn that contains one or more genes from the soil bacterium Bacillus thuringiensis, or Bt as in commonly used. The gene(s) allow the bacterium to produce one or more toxins that are toxic to certain insects, but are not toxic to mammals, including humans. The present investigation was conducted to verify and identify key genetic determinants of salt stress tolerance in Bt transgenic corn plants with a special emphasis on the role of SDS-PAGE protein profile in the mechanism of salt tolerance in corn plans.

MATERIALS AND METHODS

Plant Material Seeds of both non-transgenic and Bt transgenic crop lines of a hybrid (YieldGard 2) corn (Zea mayes L.) were kindly provided by Pioneer Hi-Bred International, Inc., USA.

Plantation Seeds of both non-transgenic (control) and B transgenic corn (Zea mays L.) crop lines were soaked (3 seeds/pot) in plastic pots (10 cm) filled with pre-sieved homogeneous garden soil (sandy loam). All planted pots were kept in the open garden in about 32/21 ºC day/night temperature and average relative humidity of 68% and irrigated regularly up to field capacity with pure water. After one week from soaking (4th leaf stage), pots were subdivided into two separate groups (non-transgenic and Bt transgenic) and each group (3 replicates/treatment) was subjected to four salinity (NaCl) levels: 0, 50, 100 and 150 mM with a 120 ml doze/pot every other day for three more weeks.

Growth Parameters By the end of the 4th week, corn plants were gently uprooted, cleaned from soil residues and prepared for measurements. Plant height, root length, shoot and root fresh and dry weights, leaf length and width and total number of leaves per plant were recorded.

Chlorophyll Pigments The contents of chlorophyll a; chlorophyll b and total chlorophyll were estimated in the 4th expanded corn leaf in 80% acetone according to the procedure described by Sadasivam and Manickam (1991). The absorbance of the extract was read at 645, 663 and 652 nm against the solvent as blank. Pigments were determined as mg/ g fresh weight residue. Chlorophyll stability index (CSI) was calculated by combining chlorophyll a+b contents in corn leaf before and after salinity stress following the formula noted by Kumari et al. (2004): CSI % = Chlorophyll before stress - chlorophyll under stress / chlorophyll under stress × 100

Protein Electrophoresis Preparation of total protein Total protein extracts were prepared by extracting appropriate portion of the frozen plant material with 0.125 M tris/borate (pH = 8.9). All extracts were kept for 24 h at 4 ºC and then centrifuged at 10,000 rpm for 20 min. The supernatants were used for electrophoresis. Gel electrophoresis SDS Polyacrylamide Gel Electrophoresis (PAGE) was carried out with gel slabs according to the method of Laemmili (1970). Protein subunit bands were stained with Coomassie blue R-250 by standard techniques. The gel was scanned using Gel-Pro-Analyzer. Statistical Analysis All parameters were statistically analyzed by multiple comparison procedure at p≤ 0.05 using t-test and mean separation by least significant difference (LSD) (Steel and Torrie, 1980).

RESULTS The Statistical analysis (P ≥ 0.05) of the growth parameters (Table 1) of corn plants revealed significant reduction in plant growth of non-transgenic plants under almost all levels (50, 100 and 150 mM) of salt (NaCl) stress; while, the growth parameters of the Bt transgenic corn plants showed no significant changes under the same levels of salinity stress. However, the growth parameters of controlled (0.0 NaCl) plants of Bt transgenic corn were always lower than those of non-transgenic plants.

The contents of chlorophyll a in the expanded leaf of non-transgenic corn plants was significantly reduced by the highest NaCl treatment (150 mM) only; but did not significantly change in the Bt transgenic corn plants. However, chlorophyll b was not responsive to NaCl treatments in both non-transgenic and Bt transgenic corn plants. On the other hand, the values of chlorophyll stability index (CSI) were always higher in the Bt transgenic than in the non-transgenic corn plants under all salinity levels (Figure 3).

The analyses of SDS-PAGE protein profiles of either non-transgenic or Bt transgenic corn plants revealed both qualitative and quantitative changes in the patterns of protein bands in response to salinity stress (Table 3; figures 2 &3). The total number of protein bands showed relative stability in the Bt transgenic corn plants under salinity stress, but showed fluctuations in the non-transgenic plants. The sum of optical density of protein bands was higher in the Bt transgenic corn plants. In response to salinity stress, a group of polypeptide (76.96, 59.38, 41.56, 33.5 and 31.26 KDa) were newly synthesized in both non-transgenic and Bt transgenic plants. Salt-susceptible polypeptides of molecular weights 325.47, 32.64 and 24.17 KDa were found only in non-transgenic corn plants and completely disappeared under all level of salt stress; while two polypeptides of molecular weights 38.59 and 30.61 KDa were totally inhibited in all salt-stressed corn plants. The synthesis of another four polypeptides (298.81, 99.82, 20.79 and 19.43 KDa) was solely specific to the Bt transgenic corn plants DISCUSSION Twenty five years ago Emanuel Epstein (1980) described the technical and biological constraints to solving the problem of salinity. Although there has been some success with technical solutions to the problem, the biological solutions have more difficult to develop because a pre-requisite for the development of salt-tolerant crops is the identification of key genetic determinants of stress tolerance (Yamaguchi and Blumwald, 2005).

The non-significant (P ≥ 0.05) growth responses of Bt transgenic corn plants to salt stress proved the magnitude of salt tolerance in those plants. Earlier consistent works (Munns, 1993) reported that growth parameters were found to be perfect indicators for screening salt tolerance in plants.

Compared to the lower chlorophyll a/b ratio in non-transgenic corn plants, the high ratio of chlorophyll a/b as well as the stability of chlorophyll b content in Bt transgenic corn plants can be ranged as an index of salt tolerance, which might produce higher photosynthetic rate and consequently high yield (Raja Babo et al., 2005). Moreover, the chlorophyll stability index (CSI) is an important index for screening plant tolerance to abiotic stresses ( Michael Gomaz and Rangasamy, 2002; Yagameena, 2004). In this investigation, the high values of chlorophyll satiability index verified the tolerance of Bt transgenic corn plants to salinity stress, results were supported by Raja Babo et al. (2005).

Over the past few years, much attention has been concentrated on resolving the identity of salt stress proteins, in order to identify and understand the role of proteins in salt tolerance. Several workers have detected a number of proteins induced by salt, reflecting the complexity of biochemical and physiological responses accompanied with the biological changes of the adaptation process of salt tolerance. In this report, corn plants, non-transgenic and Bt transgenic, responded to salt stress by both induction and repression in the synthesis of many polypeptides. In response to salinity stress, the newly synthesized proteins in common were 76.96, 59.83, 41.46, 33.5, and 31.26 KDa; while the suppressed proteins were 38.59 and 30.61 KDa. Quantitatively, the protein content in each band (band optical density) was always higher in the Bt transgenic corn plants. Other proteins (298.81, 99.82, 20.79, 19.43 KDa) were found only in Bt transgenic corn plants. Relevant salt stress proteins of 56.1, 70.8 and 93.8 KDa were reported in rice roots (Salekdeh, et al., 2002; de Souza Filho et al., 2003) and 18.0, 19.5, 21.0, 26.0, 34.0, 35.5, 37.0 and 58.0 KDa in tomato roots (Chen and Plant, 1999). In conclusion, the data presented here revealed some of the key genetic determinants of salt stress tolerance in Bt transgenic corn plants, which could be partially attributed to the stability of photosynthetic pigments under salt stress. Moreover, this investigation reported molecular weights of some salt tress responsive proteins in Bt transgenic corn plants. It is necessary to further investigate the patterns of protein expression of these salt-induced protein bands as well as the specific polypeptides of Bt transgenic corn plants to enhance our understanding of salt tolerance in transgenic corn.

REFERENCES

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Laemmili, U. K.: Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature. 227: 680-685, 1970.

Mass, E. V. and Hoffman, G. J.: Crop salt-current assessment ASCE, Irrig. Drain. Div. ASCE. 103 115-137, 1977.

Michael Gomaz, S. and Rangasamy, P.: Correlation and path analysis of yield and physiological changes in drought resistance in rice. Indian J. Mental. 19(1-2): 33-34, 2002.

Munns, R.: Physiological process limiting plant growth in saline soil: some dogmas and hypotheses. Plant Cell Environ. 16: 15-24, 1993.

Raja Babo, C., Vigayalakshini, C. and Mohandass, S.: Evaluation of rice (Oriza sativa L.) genotypes for salt tolerance. J. of Food Agric. and Envionment. 3(1): 190-195, 2005.

Sadasivam, S. and Amanickam, A.: Biochemical Methods for Agriculture Science. Wiley Eastern Ltd., New Delhi, 1991.

Steel, R.G.D and Torrie, J. H.: Principe and procedures of statistics, 2 nd ed. McGrath-Hill Inc., USA, 1980.

Yagameena Kisha: Genetic analysis for drought tolerance in rice (Oriza sativa L.). M Sc. Thesis, Tami NaduAgricultural Univ., Coimbator, Srelanka, 2004.

Yamaguchi, T. and Blumwald, E.: Developing salt-tolerant crop plants: challenges and opportunities. TRENDS in Plant Science. 10(12): 615-620, 2005.

Yeo, A. R.: Predicting the interaction between the effects of salinity and climate change on crop plants. Sci. Hort. (Amsterdam). 78: 159-174, 1999. Zhu, B., Chen, T. H. H. and Li, H.: Activation of two osmotin-like protein genes by a biotic stimuli and fungal pathogen in transgenic potato plant. Plant Physiol. 108: 929-937, 1995.

Zörb, C., Schubert, S., Neeb, A., Carl, D., Linder, M. and Schubert, S.: The biochemical reaction of maize (Zea mays L.) to salt stress is characterized by a mitigation of symptoms and not by a specific adaptation. Plant Sci. 167: 91-10, 2004.

Table 1. Mean vegetative and reproductive growth parameters of non-transgenic and Bt transgenic corn (Zea mayes L.) plants under salt (NaCl) stress.

Corn crop line Treatment Growth parameters Plant height (cm) Root length (cm) No. of leaves/plant Leaf length (cm) Leaf width (cm) Shoot F. Wt. (g) Root F. Wt. (g) Shoot D. Wt. (g) Root D. Wt. (g)

Non-transgenic C 50 100 150 69.6 55.5 48.0 41.3 38.3 19.7 20.2 11.9 7.0 6.0 5.0 4.0 50.7 41.7 35.3 31.5 3.8 1.9 2.0 1.8 11.78 6.03 5.51 3.58 2.290 0.651 0.629 0.658 1.209 0.696 0.716 0.535 0.429 0.179 0.144 0.090

Bt transgenic C 50 100 150 58.4 56.5 54.0 48.0 21.8 19.3 18.8 17.0 6.0 5.0 5.0 5.0 45.3 37.6 36.8 34.4 3.2 2.8 2.3 2.2 8.22 6.73 6.26 5.88 1.240 1.899 1.890 1.517 0.816 0.846 0.755 0.814 0.150 0.101 0.207 0.155

LSD (P ≥ 0.05) 15.25 13.90 1.65 11.41 1.27 4.32 1.177 0.346 0.192 C, 50, 100, 150 = Plants treated with 0, 50, 100 and 150 mM NaCl, respectively.

Table 2. Chlorophyll contents (mg/g) in foliage leaves of non-transgenic and Bt stransgenic corn (Zea mayes L.) plants under salt (NaCl) stress. Pigment Non-transgenic Bt transgenic LSD (P ≥ 0.05) C 50 100 150 C 50 100 150 Chlorophyll a Chlorophyll b Total Chlorophyll 0.5712 0.2094 0.7768 0.5671 0.2205 0.7873 0.5232 0.1796 0.6949 0.4560 0.1518 0.6077 0.4486 0.1327 0.5811 0.4131 0.0663 0.4763 0.3738 0.0857 0.4595 0.3669 0.0620 0.4288 0.0782 0.1126 0.2541

C, 50, 100, 150 = Plants treated with 0, 50, 100, 150 mM NaCl

Table 3. Comparative analysis of average optical density (O. D.), molecular weight (M.Wt.) and relative front (Rf) of SDS-PAE protein profile of non-transgenic and Bt transgenic corn (Zea mays L.) plants under salt (NaCl) stress. Band number Treatment & O. D. Rf Mol. Wt. (KDa) 1 2 3 4 5 6 7 8 1 201.75 - - - - - - - 0.02 325.47 2 - - - - - - - 179.89 0.04 298.81 3 127.5 84.49 - 151.83 141.54 - - 144.23 0.10 187.87 4 - - - - - 103.50 93.16 - 0.12 184.49 5 - - - 149.44 148.45 - - - 0.13 149.39 6 - 102.16 - - 152.78 131.15 117.10 - 0.18 104.81 7 153.66 - 96.85 147.28 - - - - 0.19 101.46 8 - - - - - - - 208.46 0.22 99.82 9 - - - 143.50 137.22 - - - 0.24 88.89 10 171.96 113.59 99.72 136.94 135.27 120.98 116.44 235.31 0.25 81.95 11 - 105.17 96.92 133.00 125.06 109.21 121.54 - 0.26 76.96 12 - 125.53 136.15 167.99 150.74 128.92 167.04 - 0.35 59.38 13 208.19 172.96 193.35 198.27 161.54 161.10 193.02 244.73 0.38 50.73 14 - 116.97 105.46 120.31 108.67 102.63 124.89 - 0.45 41.56 15 207.17 - - - - - - 237.61 0.48 38.59 16 157.21 90.24 76.54 84.38 79.84 74.91 86.56 188.99 0.53 34.85 17 - 96.16 76.89 86.13 78.15 78.04 86.57 - 0.56 33.50 18 146.92 - - - - - - - 0.59 32.64 19 - 89.80 73.03 95.07 84.60 80.49 91.10 - 0.62 31.26 20 116.28 - - - - - - 147.97 0.65 30.61 21 92.64 67.39 - 74.59 64.07 - - 0.72 27.36 22 - - - - - 60.91 75.30 119.15 0.76 26.63 23 70.14 - - - - - - - 0.82 24.17 24 - 53.55 38.74 - - - - 88.89 0.86 22.85 25 - - - - - 54.32 - - 0.89 22.11 26 - - - - - - 67.13 - 0.90 21.97 27 - - - - - - - 73.92 0.94 20.79 28 - - - - - - - 89.52 0.99 19.43 Total O. D. 1653.4 1218.0 0993.7 1687.9 1567.9 1206.2 1339.9 1958.7 Band/ Lane 11 12 10 13 13 12 12 12 1 and 8= untreated (control) non-transgenic and Bt transgenic plants, respectively 2, 3, 4 and 5,6,7 = non-transgenic and Bt transgenic plants treated with 50, 100, 150 mM NaCl, respectively. .

Fig.1. Growth responses of non-transgenic (left) and Bt transgenic (right) corn (Zea mays L.) plants to salt (NaCl) stress: 0, control plants; 50, 100, 150, plants treated with 50, 100, and 150 mM NaCl, respectively.

M 8 7 6 5 4 3 2 1 KDa

116►

97.4►

66.2►

37.6►

28.5►

Fig. 2. Electrophotograph of SDS-PAGE of total proteins of non-transgenic (1, 2, 3, 4) and Bt transgenic (8, 5, 6, 7) corn (Zea mays L.) plants subjected to 0, 50, 100 and 150 mM salt (NaCl) stress. M, molecular weight markers used on polyacrylamide gel.

Fig. 3. Chlorophyll stability index (CSI%) of non-transgenic and Bt transgenic corn (Zea mays L.) plants in response to salt (NaCl) stress.

Figure 4. Scan of the tracks in the Electrophotograph (Fig. 2) of SDS-PAGE of total proteins of non-transgenic (1, 2, 3, 4) and Bt transgenic (8, 5, 6, 7) corn (Zea mays L.) plants subjected to 0, 50, 100 and 150 mM salt (NaCl) stress.