- Email: [email protected]
Overexpression of a Foreign Bt Gene in Cotton Affects the Low-Molecular-Weight Components in Root Exudates1
Pedosphere 17(3): 324-330, 2007 ISSN 1002-0160/CN 32-1315/P @ 2007 Soil Science Society of China Published by Elsevier Limited and Science Press
PEDOSPHERE w,elsevier.com/locate/pedosphere
Overexpression of a Foreign Bt Gene in Cotton Affects the Low-Molecular-Weight Components in Root Exudates*' YAN Wei-Dong, SHI Wei-Ming*', LI Bao-Hai and ZHANG Min State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China). E-mail: [email protected] (Received July 12, 2006; revised January 22, 2007)
ABSTRACT Most research in the past using genetically modified crops (GM crops) has focused on the ecological safety of foreign gene (i.e., the gene flow), gene products (for example, Bt (Bacillus thuringiensis) protein), and the safety of transgenic food for humans. In this study, changes in both the species and amounts of low-molecular-weight components in cotton (Gossypium hirsutum L.) root exudates after foreign Bt gene overexpression were investigated under different nutritional conditions. Transgenic cotton containing Bt (Bt-cotton), supplemented with all the mineral nutrients, secreted more organic acids than the wild-type cotton (WT). When nitrogen was removed from the full-nutrient solution, the amount of organic acids secretion of Bt-cotton was lesser than that of WT. The roots of the transgenic cotton secreted lesser amounts of amino acids and soluble sugars than the W T roots in the full-nutrient solution. Deficiencies of P and K caused a large increase in the total amino acid and soluble sugar secretions of both Bt-cotton and WT, with larger increases observed in Bt-cotton. Because transferring the foreign Bt gene into cotton can result in alterations in the components of the root exudates, with the effect varying depending on the nutritional status, the cultivation of genetically modified crops, such as Bt-cotton, in soil environments should be more carefully assessed, and the possible effects as a result of the alterations in the root exudate components should be considered. Key words:
gene overexpression, low-molecular-weight components, nutritional status, root exudates, transgenic cotton
Citation: Yan, W. D., Shi, W. M., Li, B. H. and Zhang, M. 2007. Overexpression of a foreign Bt gene in cotton affects the low-molecular-weight components in root exudates. Pedosphere. 17(3): 324-330.
INTRODUCTION Cotton (Gossypium hirsutum L.) is one of the major economic crops in China. In the past two decades, the total acreage under cultivation increased from 4.04 (in 2000) t o 6.835 (in 1992) million hectares. In 2001, 4.8 million hectares of arable land were used for cotton production. During the same period, the total production of cotton ranged from 3.54 (in 1976) to 5.68 (in 1991) million tons. In 2001, total production of cotton reached 5.32 million tons, with an average yield of 1.1 t ha-'. Of the total, about 0.3 to 0.6 million hectares were cultivated with genetically modified (GM) cotton, expressing the modified Bt (Bacillus thuringiensis) toxin (CrylA or CpTI). Although the total cultivated area of genetically modified varieties was comparatively smaller than that of the genetically regular varieties, the area cultivated with the genetically modified variety increased largely. The release of transgenic cotton to field experiments first began in 1997, with the field area being 667 hectares. In 2002, China ranked fourth in the world in terms of GM cultivation acreage. The possible effect of the cultivated GM crops on the environment is currently a strongly debated topic in China (Meng, 1996; Fan et al., 2001) as well as in the rest of the world (Donegan et al., 1995; Escher et al., 2000; Wolfenbarger and Phifer, 2000). Furthermore, the actual cultivation area in China is expected to increase rapidly in the coming * lProject supported by the Knowledge Innovation Program of the Institute of Soil Science, Chinese Academy of Sciences, and the National Natural Science Foundation of China (No. 30270789). *2Corresponding author. E-mail: [email protected].
GENE OVEREXPRESSION AND ROOT EXUDATE COMPONENTS
325
years. Regulatory laws established in China require reporting of assessments of the effect of GM crops on the ecology before their release t o the open environment and agricultural fields. However, the current regulations are not very strictly followed. More careful environmental assessment is necessary, particularly concerning the possible interactions of each GM crop with the soil through the root-soil interface. Therefore, as a first step, low-molecular-weight components in root exudates in transgenic Bt cotton (Bt-cotton) were compared with those in the root exudates of wild-type cotton (WT). These root exudates are the predominant components affecting soil-plant interactions. Some reports have shown that root exudates influence the activities of soil microorganisms (Heuer et al., 2002; Saxena et al., 1999; Wang et al., 2002). In their studies, a product of the introduced gene, namely, Bt toxin, was also secreted from transgenic plant roots as one of the components in the root exudates; thus, effects of the root exudates on the activities of soil microorganisms can be partially attributed to the toxin itiself. Changes in the low-molecular-weight components in the root exudates of individual GM crops have not been studied in detail. Because these low-molecular-weight components play active roles in the soil-root interaction processes, it is worthwhile to observe whether the introduction of the transgene causes any alterations in their nature or quantity (Chen et al., 2002; Fan and Fan, 1995). In the present investigation, changes in both the species and amounts of low-molecular-weight components in cotton root exudates after foreign Bt gene overexpression were investigated and the effect of the nutritional status of the grqwth medium on the components in plant root exudates was also analyzed. MATERIALS AND METHODS The variety of the wild-type cotton used in the present study was Sumian 12, and the transgenic cotton with modified CrylA from Bacillus thuringiensis used was Sukang 103 variety. The two varieties were purchased from the same seed company and had identical genetic backgrounds ( i e . , Sumian 12). Both wild-type (WT) and transgenic cotton (Bt-cotton) seeds were allowed to germinate in sand quartz watered with tap water. After the appearance of cotyledons, the full-nutrient solution was supplied and preculture was carried out in a growth chamber with a light period of 14 h at 25 "C at 100 pE m-2 s -1 and a dark period of 10 h at 23 "C and 70% humidity. The full-nutrient solution consisted of 1 mmol L-lNH4HzP04, 5.75 mmol L-' Ca(N03)2, 1.3 mmol L-' CaC12, 2 mmol L-' MgS04, 6 mmol L-' KN03, 4 pmol L-' Fe-EDTA (ethylenediamine tetraacetic acid), 2 pmol L-' HsB03, 1 pmol L-' MnC12, 0.3 pmol L-' ZnCl:!, 0.1 pmol L-l CuC12, and 0.05 pmol L-l HzM004. The solution pH was
adjusted to 6.0 using a pH meter on a daily basis. Two uniform seedlings that were 10-day old and precultured as mentioned above were transferred into a 500-mL pot supplemented with the full-nutrient solution as mentioned above, or the nutrient solution lacking either nitrogen or phosphorus or potassium. In the nutrient solution lacking N, 7 mmol L-' of CaC12 was used to replace NH4H2P04 and Ca(N03)2, with KzS04 being used instead of KN03. For the nutrient solution lacking P, 1 mmol L-' of NH4HZP04 was replaced with 0.5 mmol L-' of NH4N03. In the nutrient solution lacking K , KN03 was replaced with 3 mmol L-l of NH4N03. The other nutrients were of the same concentrations as in the full-nutrient solution. After 4 days, the total root proton secretion in the medium was calculated by titration to pH 6.0 with dilute NaOH. For collecting root exudates, ten-day-old seedlings were transferred into the full-nutrient solution or into the nutrient solutions lacking N, P, or K, and kept for one week. The roots from six plants were placed in 900 mL of deionized water for 3 h (from 9:00 am to 12:OO am), and the root washings were then collected and freeze-dried. The dried residues were dissolved in ultrapure water (Millipore Simplicity, USA), adjusted to a final volume of 0.5 mL, filtered through a 0.45-pm membrane, and kept at -80 "C before analysis. The organic acid concentrations were measured using a Shimadzu HPLC LC-6A. For oxalic acid detection, a 10-pL sample was injected into the Shim-pack IC-A3 column with a mobile phase containing 10 mmol L-' of NaH2P04 and 10 mmol L-' of H3P04 (pH 2.51). The UV detector was set at 210
W. D. YAN et al.
326
nm, and the column was kept at room temperature. For citric acid and other organic acids, a 25 pLsample was injected into an SCR-102H column containing a mobile phase of ultrapure water, which was adjusted to pH 2.1 with dilute perchloric acid. The derivative phase was composed of 0.2 mmol L-l of bromothymol blue, 5% (v/v) methanol, 15 mmol L-l of Na2HP04, and 2 mmol L-l of NaOH. The UV detector was set at 425 nm, and the column was kept at room temperature (Shen et al., 2004). For determining the total soluble sugar content, 100 pL of the concentrated root exudates were sampled. The anthrone reagent (9,l0-dihydro-9-oxoanthracene1 CI4Hl0O, GFS Chemicals, USA) was used to determine the sugar content using colorimetry at 620 nm, with glucose as the standard (Lu, 2000). After reaction with ninhydrin (l12,3-indanetrione monohydrate, CgH604, Sigma, USA), colorimetry of a 100-pL sample of root exudates at 580 nm (Wang, 1998), with leucine as the standard, was used to measure the total amino acid concentration. All the treatments were performed in at least three replicates. The data obtained were calculated as the means of three replicates k standard deviations using Microsoft Excel 2000. The analysis of variance was performed using the software program SPSS 10.0 for windows. Duncan's multiple range test was carried out according to the least significance difference (LSD) values. RESULTS AND DISCUSSION
Acidification of the root medium during plant growth The changes in the medium pH and amounts of secreted protons were observed following prolonged growth of young cotton seedlings (Fig. 1; Table I). The medium pH rapidly decreased from 6.0 to about 4.0 within 3-4 days. No obvious difference was found between W T and Bt-cotton, regardless of the nutrient supply conditions, after 4 days of growth. However, during the first 3 days, with Bt-cotton 6
+ BT-FN
0 BT-NP
I
4 WT-FN
0 WT-NP
n
A BT-NN W BT-NK
5
A WT-NN
4
WT-NK
3
0
2
1
3
4
Time (days) Fig. 1 Alterations in medium pH (means of three replicates) in the 10-day old seedlings of the wild-type cotton (WT) and the transgenic cotton containing Bt (Bt-cotton) under the treatments of various nutrient supply conditions: BT-Btcotton, FN-full-nutrient solution, "-no N in the nutrient solution, NP-no P in the nutrient solution, NK-no K in the nutrient solution. Vertical bars represent standard deviations. TABLE I Total amounts (means of three replicates f standard deviations) of protons secreted by the roots (fresh weight) of the wild-type cotton (WT) and the transgenic cotton containing Bt (Bt-cotton) under the treatments of various nutrient supply conditions Treatmenta) ~
BT-FN 50.2f6.4bcb)
WT-FN
BT-NN
WT-NN
BT-NP
42.5&9.2cd
mol g-' h-l 5 0 . 7 z t 0 . 5 ~ 40.4It5.3d 5 6 . 2 f 3 . 4 a b
~
~
WT-NP
BT-NK
WT-NK
59.9f6.6a
60.6k6.8a
56.6zt8.7ab
BT-Bt-cotton, FN-full-nutrient solution, "-no N in the nutrient solution, NP-no P in the nutrient solution, NK-no K in the nutrient solution. ')Means followed by the same letter(s) are not significantly different ( P 5 0.05) using Duncan's multiple range test.
GENE OVEREXPRESSION AND ROOT EXUDATE COMPONENTS
327
being supplemented with all the nutrients, the medium pH was higher than for the other treatments. For Bt-cotton and W T with full-nutrient supply and with the treatment lacking K or P, there were no significant differences ( P 5 0.05) in the total amounts of protons secreted per unit fresh weight of roots. However, significant differences were found between Bt-cotton and W T for the treatment lacking N (Table I). To determine whether there was a difference in local proton secretion between W T and Bt-cotton, localization of acid in the region around the roots was observed in the agar medium. The pattern showed that stronger acidification occurred around mature root regions. Acidification was relatively weak around the root tips (data not shown).
Alterations in the organic acid composition in the root exudates The organic acid fractions including oxalic, citric, and acetic acids were detected in the root exudates from both W T and Bt-cotton (Figs.2 and 3). Citric acid was usually the predominant species. In addition, a peak corresponding to an unknown organic acid substance was detected in the HPLC chromatograph chart, When both Bt-cotton and W T were grown in the full-nutrient solution, it was seen that the total organic acids in the root exudates of Bt-cotton were about 35% more than that of W T (Fig. 3), whereas in the absence of P and K, the difference was only 5% to 10%. However, when the plants were subjected to nitrogen starvation, lesser amounts of total organic acids were secreted from the roots of Bt-cotton than W T (Fig.3). For both cotton types, lesser amounts of organic acids were secreted when the nutrient solutions lacked N, P, or K, compared with the full-nutrient solution.
y"
2.0
-
u)
-.E
-0
1.5 -
0
.-
1.0
-
0.5
-
c L
O
0.0
Treat rn e nt Fig. 2 Organic acids (means of three replicates) detected in the root (fresh weight) exudates of the 10-day old seedlings of the wild-type cotton (WT) and the transgenic cotton containing Bt (Bt-cotton) after 7 days of treatment under various nutrient supply conditions: BT-Bt-cotton, FN-full nutrient solution, "-no N in the nutrient solution, NP-no P in the nutrient solution, NK-no K in the nutrient solution. Vertical bars represent standard deviations. Fig. 3 Alterations in the total organic acid contents (means of three replicates) in the root (fresh weight) exudates of the 10-day old seedlings of the wild-type cotton (WT) and the transgenic cotton containing Bt (Bt-cotton) after 7 days of treatment under various nutrient supply conditions: BT-Bt-cotton, N in the FN-full-nutrient solution, "-no nutrient solution, NP-no P in the nutrient solution, NK-no K in the nutrient solution. Vertical bars represent standard deviations.
The organic acid species also differed with the nutrient supply conditions and showed different patterns between W T and Bt-cotton (Fig. 2). When complete nutrients were provided, oxalic acid, citric acid, and acetic acid were secreted more from the roots of Bt.-cotton than WT. When compared with the full-nutrient treatment, in the absence of nitrogen supply, oxalic acid secretion was measurably lower from the roots of Bt-cotton, whereas in WT, oxalic acid could not be detected. When potassium was not supplied, the oxalic acid content in the root exudates of both Bt-cotton and W T decreased below the detection limits of HPLC. However, the secretion of citric acid into the medium was enhanced
328
W. D. YAN et al.
during potassium starvation when compared with the full-nutrition supply (Fig. 2).
Alterations in total soluble sugars and amino acids in the root exudates Sugars are the main component of root exudates. It has been reported that more than 65% of the organic matter secreted into the rhixosphere by plants are sugars (Curl and Truelove, 1986). In this study, the soluble sugar level (Fig. 4) was ten-fold higher than the total organic acid (Fig. 3) or amino acid contents (Fig. 5). With full-nutrient supply, the WT root medium contained about 32 pg of sugar per gram root fresh weight (Fig. 4), whereas Bt-cotton contained only 22 pg g-l. When compared with the full-nutrient supply, phosphorus and potassium deficiencies caused considerably greater secretion of soluble sugar compounds, with increment being 3-3.5 folds for Bt-cotton and 1.5-1.7 folds for WT. Sugar secretion in W T in the case of nitrogen deficiency was 60% lower than that in the case of full nutrients, whereas in Bt-cotton it was only about 17% lower. h
90 r 80 -
1
70
5
50
3.0 r
.,-'02 5I
-
z
z;' 60 -
5 L
20
40 30 -
T
-
v
1 5 -
m 0
t
2g) 20 10-
1 0 -
E 056
Treatment
m, Treatment
Fig. 4 Alterations in the total soluble sugar contents (means of three replicates) in the root (fresh weight) exudates of the 10-day old seedlings of the wild-type cotton (WT) and the transgenic cotton containing Bt (Bt-cotton) after 7 days of treatment under various nutrient supply conditions: BT-Bt-cotton, FN-full-nutrient solution, NN-no N in the nutrient solution, NP-no P in the nutrient solution, NK-no K in the nutrient solution. Vertical bars represent standard deviations. Fig. 5 Alterations in the total amino acid contents (means of three replicates) in the root (fresh weight) exudates of the 10-day old seedlings of the wild-type cotton (WT) and the transgenic cotton containing Bt (Bt-cotton) after 7 days of treatment under various nutrient supply conditions: BT-Bt-cotton, FN-full-nutrient solution, NN-no N in the nutrient solution, NP-no P in the nutrient solution, NK-no K in the nutrient solution. Vertical bars represent standard deviations.
With full-nutrient supply, the total amino acid contents were lower in the root exudates of Bt-cotton than those in WT (Fig. 5). The effects of the nutrient deficiencies were very similar in the two types of cotton. When compared with the full-nutrient supply, deficiencies of P and K caused increases in the secretion of amino acids, being 5.5 to 6.8 folds for Bt-cotton and 2.5 to 3.2 folds for WT. Again, N deficiency led to considerably lower amino acid secretion in WT, but only a slight decrease was observed in Bt-cotton. In WT, N-deficiency treatment led to about 49% drop in amino acid secretion, whereas in Bt-cotton, this drop was only about 4%.
DISCUSSION Although Bt-cotton shows resistance to damage by cotton insects, required only lower dosages of pesticides, and has been cultivated worldwide, there has been concern about the biosafety of transgenic plants. In the past decade, considerable attention has been paid to possible ecological risks from GM crops, such as gene flow from one species to another and food safety for humans. Also, Bt, as an insecticidal toxin released from transgenic plant roots into the rhizosphere, can exist for rather long
GENE OVEREXPRESSION AND ROOT EXUDATE COMPONENTS
329
periods (Shelton et al., 2002; Ding et aZ., 2001). Besides the transgenic insecticidal toxin (Bt), all plant roots secreted large amounts of inorganic ions and organic matter into the rhizosphere. A total of 25%-40% of the photosynthetic carbon can be transferred into the soil through this root-soil interface pathway (Curl and Truelove, 1986). Among the organic fractions of root exudates, 65% of the compounds belong to sugars, 20% to organic acids, and the remaining to amino acids and others (Curl and Thelove, 1986). These compounds, particularly low-molecular-weight compounds, are the key factors involved in soil microflora chemotaxis. They have both beneficial and harmful effects on plant growth, such as improving the link between roots and soils for water and nutrient uptake, mobilizing soil insoluble nutrients, immobilizing toxic elements, and acting as elicitors of a pathogen attack or as an energy source for pathogen growth. For example, sugars and amino acids secreted by a plant are positively correlated with plant infection (Yuan et al., 2002). The results of this study showed that when compared with WT, the Bt-cotton roots would secrete less amino acids and sugars into the soil when the soil fertility was high. However, when soil P or K supply was limited, the Bt-cotton roots would exude more amino acids and sugars into the rhizosphere. Thus, Bt-cotton may have lower yield when grown in the soils with higher probability of disease-causing infection. This may be one of the reasons for the requirement of larger dosages of P and K for transgenic Bt-cotton cultivation in comparison t o W T cultivation, for better yield and quality. On the other hand, root exudates, particularly organic acids, play a very important role in improving the soil nutrient bioavailability and in stabilizing harmful elements like aluminum and heavy metals (Shen et aZ., 2001; Zhang et al., 2005). Several mineral nutrient stresses can induce enhancement of root exudation (Liu e t al., 1997; Li e t al., 2005). The present investigation also indicated that the introduction of a foreign gene could lead to changes in the amounts of low-molecular-weight components in root exudates. At present, an explanation for the mechanism of the changes of organic acid species in root exudates of Bt-cotton cannot be provided. However, plants have a very complicated structure, and as the Bt protein is constitutively produced in various parts of the transgenic plant, the metabolic processes can be considerably affected. In conclusion, the results of this study indicated that root exudate components between transgenic Bt cotton and W T were, t o some extent, different and that the soil nutrient levels further affected these components. Because transferring the foreign Bt gene into cotton can result in alterations in the components of the root exudates, with the effect varying depending on the nutritional status, the cultivation of genetically modified crops, such as Bt-cotton, in soil environments should be more carefully assessed, and the possible effects as a result of the alterations in the root exudate components should be studied in the future. ACKNOWLEDGMENTS We thank Dr. Dong Cai-Xia and Miss Chen Rong-Fu of the Institute of Soil Science, Chinese Academy of Sciences, for their assistance with the HPLC analysis. We are very grateful to Professor Andre Jagendorf of Cornell University for his critical reading of this manuscript and his valuable suggestions. REFERENCES Chen, Y. L., Guo, Y. Q. and Han, S. L. 2002. Effect of root derived organic acid on the activation of nutrients in the rhizosphere soil. Journal of Forestry Research (in Chinese). 13: 115-118. Curl, E. A. and Truelove, B. 1986. The Rhizosphere. Springer-Verlag, New York. pp. 43-57. Ding, Z. Y., Xu, C. R. and Wang, R. J. 2001. Comparison of several important isoenzymes between Bt cotton and regular cotton. Acta Ecologzca Sznzca (in Chinese). 21: 332-336. Donegan, K. K., Palm, C. J. and Fieland, V. J. 1995. Changes in levels, species, and DNA fingerprints of soil microorganisms associated with cotton expressing the Baczllus thurzngaenszs var kurstakz endotoxin. Applaed Sod Ecology. 2: 111-124. Escher, N., Kach, B. and Nentwig, W. 2000. Decomposition of transgenic Baczllus thunngzenszs maize by microorganisms
330
W. D. YAN et al.
and woodlice Porcellio scaber (Crustacea: Zsopoda). Basic Applied Ecology. 1: 161-169. Fan, J. G. and Fan, G. R. 1995. Plant root exudation and its significance in forestry. Bulletin of Botanical Research. 15: 246-251. Fan, L., Zhou, X., Hu, B., Shi, C. and Wu, J. 2001. Gene dispersal risk of transgenic plants. Chinese Journal of Applied Ecology (in Chinese). 12: 630-632. Heuer, H., Kroppenstedt, R. M., Lottmann, J., Berg, G. and Srnalla, K. 2002. Effects of T4 lysozyme release from transgenic potato roots on bacterial rhizosphere communities are negligible relative to natural factors. Applied and Environmental Microbiology. 88: 1325-1 335. Li, J. Y., Xu, R. K. and Ji, G. L. 2005. Dissolution of aluminum in variably charged soils as affected by low-molecularweight organic acids. Pedosphere. 15: 484-490. Liu, Z. Y., Li, L. M. and Shi, W. M. (eds.). 1997. Research Methods in Rhizosphere (in Chinese). Jiangsu Science and Technology Publishing House, Nanjing. pp. 13-127. Lu, R. K. (ed.). 2000. Analytical Methods for Soils and Plants (in Chinese). China Agricultural Science Press, Beijing. pp. 121-133. Meng, K. Q. 1996. Debating for biosafety of genetically engineered plants. Progress in Biotechnology (in Chinese). 16: 2-6. Saxena, D., Florest, S. and Stotzky, G. 1999. Insecticidal toxin in root exudates from Bt corn. Nature. 415: 402-480. Shelton, A. M., Zhao, J , Z. and Roush, R. J . 2002. Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants. Ann. Rev. Entomol. 47: 845-851. Shen, H., Wang, X. C. and Shi, W. M. 2001. Isolation and identification of specific root exudates in elephantgrass in response to mobilization of iron- and aluminum-phosphates. J . Plant Nutrition. 24: 1117-1 130. Shen, R. F., Iwashita, T. and Ma, J. F. 2004. Form of A1 changes with A1 concentration in leaves of buckwheat. Journal of Ezperzmental Botany. 55(394): 131-136. Wang, W. P. 1998. Improving the method for determining the total dissociative amino acid in fresh plant tissue. Journal of Bezjing Agricultural College (in Chinese). 13(3): 9-12. Wang, Z. H., Ye, Q. F., Shu, Q. Y., Cui, H. R., Xia, Y. W. and Zhou, M. Y. 2002. Impact of root exudates from transgenic plants on soil micro-ecosystems. Chinese Journal of Applied Ecology (in Chinese). 13: 373-375. Wolfenbarger, L. L. and Phifer, P. R. 2000. The ecological risks and benefits of genetically engineered plants. Science. 290: 2 088-2 093. Yuan, H. X., Li, H. L. and Wang, Y. 2002. The root exudates of cotton cultivars with the different resistance and their effects on Verticillium dahliae. Acta Phytopathologica Sinzca (in Chinese). 32: 127-131. Zhang, Y. S., Shao, X. H., Lin, X. Y. and Scherer, H. W. 2005. Effect of organic acids and protons on release of non-exchangeable NH; in flooded paddy soils. Pedosphere. 15(3): 273-280.
PEDOSPHERE w,elsevier.com/locate/pedosphere
Overexpression of a Foreign Bt Gene in Cotton Affects the Low-Molecular-Weight Components in Root Exudates*' YAN Wei-Dong, SHI Wei-Ming*', LI Bao-Hai and ZHANG Min State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China). E-mail: [email protected] (Received July 12, 2006; revised January 22, 2007)
ABSTRACT Most research in the past using genetically modified crops (GM crops) has focused on the ecological safety of foreign gene (i.e., the gene flow), gene products (for example, Bt (Bacillus thuringiensis) protein), and the safety of transgenic food for humans. In this study, changes in both the species and amounts of low-molecular-weight components in cotton (Gossypium hirsutum L.) root exudates after foreign Bt gene overexpression were investigated under different nutritional conditions. Transgenic cotton containing Bt (Bt-cotton), supplemented with all the mineral nutrients, secreted more organic acids than the wild-type cotton (WT). When nitrogen was removed from the full-nutrient solution, the amount of organic acids secretion of Bt-cotton was lesser than that of WT. The roots of the transgenic cotton secreted lesser amounts of amino acids and soluble sugars than the W T roots in the full-nutrient solution. Deficiencies of P and K caused a large increase in the total amino acid and soluble sugar secretions of both Bt-cotton and WT, with larger increases observed in Bt-cotton. Because transferring the foreign Bt gene into cotton can result in alterations in the components of the root exudates, with the effect varying depending on the nutritional status, the cultivation of genetically modified crops, such as Bt-cotton, in soil environments should be more carefully assessed, and the possible effects as a result of the alterations in the root exudate components should be considered. Key words:
gene overexpression, low-molecular-weight components, nutritional status, root exudates, transgenic cotton
Citation: Yan, W. D., Shi, W. M., Li, B. H. and Zhang, M. 2007. Overexpression of a foreign Bt gene in cotton affects the low-molecular-weight components in root exudates. Pedosphere. 17(3): 324-330.
INTRODUCTION Cotton (Gossypium hirsutum L.) is one of the major economic crops in China. In the past two decades, the total acreage under cultivation increased from 4.04 (in 2000) t o 6.835 (in 1992) million hectares. In 2001, 4.8 million hectares of arable land were used for cotton production. During the same period, the total production of cotton ranged from 3.54 (in 1976) to 5.68 (in 1991) million tons. In 2001, total production of cotton reached 5.32 million tons, with an average yield of 1.1 t ha-'. Of the total, about 0.3 to 0.6 million hectares were cultivated with genetically modified (GM) cotton, expressing the modified Bt (Bacillus thuringiensis) toxin (CrylA or CpTI). Although the total cultivated area of genetically modified varieties was comparatively smaller than that of the genetically regular varieties, the area cultivated with the genetically modified variety increased largely. The release of transgenic cotton to field experiments first began in 1997, with the field area being 667 hectares. In 2002, China ranked fourth in the world in terms of GM cultivation acreage. The possible effect of the cultivated GM crops on the environment is currently a strongly debated topic in China (Meng, 1996; Fan et al., 2001) as well as in the rest of the world (Donegan et al., 1995; Escher et al., 2000; Wolfenbarger and Phifer, 2000). Furthermore, the actual cultivation area in China is expected to increase rapidly in the coming * lProject supported by the Knowledge Innovation Program of the Institute of Soil Science, Chinese Academy of Sciences, and the National Natural Science Foundation of China (No. 30270789). *2Corresponding author. E-mail: [email protected].
GENE OVEREXPRESSION AND ROOT EXUDATE COMPONENTS
325
years. Regulatory laws established in China require reporting of assessments of the effect of GM crops on the ecology before their release t o the open environment and agricultural fields. However, the current regulations are not very strictly followed. More careful environmental assessment is necessary, particularly concerning the possible interactions of each GM crop with the soil through the root-soil interface. Therefore, as a first step, low-molecular-weight components in root exudates in transgenic Bt cotton (Bt-cotton) were compared with those in the root exudates of wild-type cotton (WT). These root exudates are the predominant components affecting soil-plant interactions. Some reports have shown that root exudates influence the activities of soil microorganisms (Heuer et al., 2002; Saxena et al., 1999; Wang et al., 2002). In their studies, a product of the introduced gene, namely, Bt toxin, was also secreted from transgenic plant roots as one of the components in the root exudates; thus, effects of the root exudates on the activities of soil microorganisms can be partially attributed to the toxin itiself. Changes in the low-molecular-weight components in the root exudates of individual GM crops have not been studied in detail. Because these low-molecular-weight components play active roles in the soil-root interaction processes, it is worthwhile to observe whether the introduction of the transgene causes any alterations in their nature or quantity (Chen et al., 2002; Fan and Fan, 1995). In the present investigation, changes in both the species and amounts of low-molecular-weight components in cotton root exudates after foreign Bt gene overexpression were investigated and the effect of the nutritional status of the grqwth medium on the components in plant root exudates was also analyzed. MATERIALS AND METHODS The variety of the wild-type cotton used in the present study was Sumian 12, and the transgenic cotton with modified CrylA from Bacillus thuringiensis used was Sukang 103 variety. The two varieties were purchased from the same seed company and had identical genetic backgrounds ( i e . , Sumian 12). Both wild-type (WT) and transgenic cotton (Bt-cotton) seeds were allowed to germinate in sand quartz watered with tap water. After the appearance of cotyledons, the full-nutrient solution was supplied and preculture was carried out in a growth chamber with a light period of 14 h at 25 "C at 100 pE m-2 s -1 and a dark period of 10 h at 23 "C and 70% humidity. The full-nutrient solution consisted of 1 mmol L-lNH4HzP04, 5.75 mmol L-' Ca(N03)2, 1.3 mmol L-' CaC12, 2 mmol L-' MgS04, 6 mmol L-' KN03, 4 pmol L-' Fe-EDTA (ethylenediamine tetraacetic acid), 2 pmol L-' HsB03, 1 pmol L-' MnC12, 0.3 pmol L-' ZnCl:!, 0.1 pmol L-l CuC12, and 0.05 pmol L-l HzM004. The solution pH was
adjusted to 6.0 using a pH meter on a daily basis. Two uniform seedlings that were 10-day old and precultured as mentioned above were transferred into a 500-mL pot supplemented with the full-nutrient solution as mentioned above, or the nutrient solution lacking either nitrogen or phosphorus or potassium. In the nutrient solution lacking N, 7 mmol L-' of CaC12 was used to replace NH4H2P04 and Ca(N03)2, with KzS04 being used instead of KN03. For the nutrient solution lacking P, 1 mmol L-' of NH4HZP04 was replaced with 0.5 mmol L-' of NH4N03. In the nutrient solution lacking K , KN03 was replaced with 3 mmol L-l of NH4N03. The other nutrients were of the same concentrations as in the full-nutrient solution. After 4 days, the total root proton secretion in the medium was calculated by titration to pH 6.0 with dilute NaOH. For collecting root exudates, ten-day-old seedlings were transferred into the full-nutrient solution or into the nutrient solutions lacking N, P, or K, and kept for one week. The roots from six plants were placed in 900 mL of deionized water for 3 h (from 9:00 am to 12:OO am), and the root washings were then collected and freeze-dried. The dried residues were dissolved in ultrapure water (Millipore Simplicity, USA), adjusted to a final volume of 0.5 mL, filtered through a 0.45-pm membrane, and kept at -80 "C before analysis. The organic acid concentrations were measured using a Shimadzu HPLC LC-6A. For oxalic acid detection, a 10-pL sample was injected into the Shim-pack IC-A3 column with a mobile phase containing 10 mmol L-' of NaH2P04 and 10 mmol L-' of H3P04 (pH 2.51). The UV detector was set at 210
W. D. YAN et al.
326
nm, and the column was kept at room temperature. For citric acid and other organic acids, a 25 pLsample was injected into an SCR-102H column containing a mobile phase of ultrapure water, which was adjusted to pH 2.1 with dilute perchloric acid. The derivative phase was composed of 0.2 mmol L-l of bromothymol blue, 5% (v/v) methanol, 15 mmol L-l of Na2HP04, and 2 mmol L-l of NaOH. The UV detector was set at 425 nm, and the column was kept at room temperature (Shen et al., 2004). For determining the total soluble sugar content, 100 pL of the concentrated root exudates were sampled. The anthrone reagent (9,l0-dihydro-9-oxoanthracene1 CI4Hl0O, GFS Chemicals, USA) was used to determine the sugar content using colorimetry at 620 nm, with glucose as the standard (Lu, 2000). After reaction with ninhydrin (l12,3-indanetrione monohydrate, CgH604, Sigma, USA), colorimetry of a 100-pL sample of root exudates at 580 nm (Wang, 1998), with leucine as the standard, was used to measure the total amino acid concentration. All the treatments were performed in at least three replicates. The data obtained were calculated as the means of three replicates k standard deviations using Microsoft Excel 2000. The analysis of variance was performed using the software program SPSS 10.0 for windows. Duncan's multiple range test was carried out according to the least significance difference (LSD) values. RESULTS AND DISCUSSION
Acidification of the root medium during plant growth The changes in the medium pH and amounts of secreted protons were observed following prolonged growth of young cotton seedlings (Fig. 1; Table I). The medium pH rapidly decreased from 6.0 to about 4.0 within 3-4 days. No obvious difference was found between W T and Bt-cotton, regardless of the nutrient supply conditions, after 4 days of growth. However, during the first 3 days, with Bt-cotton 6
+ BT-FN
0 BT-NP
I
4 WT-FN
0 WT-NP
n
A BT-NN W BT-NK
5
A WT-NN
4
WT-NK
3
0
2
1
3
4
Time (days) Fig. 1 Alterations in medium pH (means of three replicates) in the 10-day old seedlings of the wild-type cotton (WT) and the transgenic cotton containing Bt (Bt-cotton) under the treatments of various nutrient supply conditions: BT-Btcotton, FN-full-nutrient solution, "-no N in the nutrient solution, NP-no P in the nutrient solution, NK-no K in the nutrient solution. Vertical bars represent standard deviations. TABLE I Total amounts (means of three replicates f standard deviations) of protons secreted by the roots (fresh weight) of the wild-type cotton (WT) and the transgenic cotton containing Bt (Bt-cotton) under the treatments of various nutrient supply conditions Treatmenta) ~
BT-FN 50.2f6.4bcb)
WT-FN
BT-NN
WT-NN
BT-NP
42.5&9.2cd
mol g-' h-l 5 0 . 7 z t 0 . 5 ~ 40.4It5.3d 5 6 . 2 f 3 . 4 a b
~
~
WT-NP
BT-NK
WT-NK
59.9f6.6a
60.6k6.8a
56.6zt8.7ab
BT-Bt-cotton, FN-full-nutrient solution, "-no N in the nutrient solution, NP-no P in the nutrient solution, NK-no K in the nutrient solution. ')Means followed by the same letter(s) are not significantly different ( P 5 0.05) using Duncan's multiple range test.
GENE OVEREXPRESSION AND ROOT EXUDATE COMPONENTS
327
being supplemented with all the nutrients, the medium pH was higher than for the other treatments. For Bt-cotton and W T with full-nutrient supply and with the treatment lacking K or P, there were no significant differences ( P 5 0.05) in the total amounts of protons secreted per unit fresh weight of roots. However, significant differences were found between Bt-cotton and W T for the treatment lacking N (Table I). To determine whether there was a difference in local proton secretion between W T and Bt-cotton, localization of acid in the region around the roots was observed in the agar medium. The pattern showed that stronger acidification occurred around mature root regions. Acidification was relatively weak around the root tips (data not shown).
Alterations in the organic acid composition in the root exudates The organic acid fractions including oxalic, citric, and acetic acids were detected in the root exudates from both W T and Bt-cotton (Figs.2 and 3). Citric acid was usually the predominant species. In addition, a peak corresponding to an unknown organic acid substance was detected in the HPLC chromatograph chart, When both Bt-cotton and W T were grown in the full-nutrient solution, it was seen that the total organic acids in the root exudates of Bt-cotton were about 35% more than that of W T (Fig. 3), whereas in the absence of P and K, the difference was only 5% to 10%. However, when the plants were subjected to nitrogen starvation, lesser amounts of total organic acids were secreted from the roots of Bt-cotton than W T (Fig.3). For both cotton types, lesser amounts of organic acids were secreted when the nutrient solutions lacked N, P, or K, compared with the full-nutrient solution.
y"
2.0
-
u)
-.E
-0
1.5 -
0
.-
1.0
-
0.5
-
c L
O
0.0
Treat rn e nt Fig. 2 Organic acids (means of three replicates) detected in the root (fresh weight) exudates of the 10-day old seedlings of the wild-type cotton (WT) and the transgenic cotton containing Bt (Bt-cotton) after 7 days of treatment under various nutrient supply conditions: BT-Bt-cotton, FN-full nutrient solution, "-no N in the nutrient solution, NP-no P in the nutrient solution, NK-no K in the nutrient solution. Vertical bars represent standard deviations. Fig. 3 Alterations in the total organic acid contents (means of three replicates) in the root (fresh weight) exudates of the 10-day old seedlings of the wild-type cotton (WT) and the transgenic cotton containing Bt (Bt-cotton) after 7 days of treatment under various nutrient supply conditions: BT-Bt-cotton, N in the FN-full-nutrient solution, "-no nutrient solution, NP-no P in the nutrient solution, NK-no K in the nutrient solution. Vertical bars represent standard deviations.
The organic acid species also differed with the nutrient supply conditions and showed different patterns between W T and Bt-cotton (Fig. 2). When complete nutrients were provided, oxalic acid, citric acid, and acetic acid were secreted more from the roots of Bt.-cotton than WT. When compared with the full-nutrient treatment, in the absence of nitrogen supply, oxalic acid secretion was measurably lower from the roots of Bt-cotton, whereas in WT, oxalic acid could not be detected. When potassium was not supplied, the oxalic acid content in the root exudates of both Bt-cotton and W T decreased below the detection limits of HPLC. However, the secretion of citric acid into the medium was enhanced
328
W. D. YAN et al.
during potassium starvation when compared with the full-nutrition supply (Fig. 2).
Alterations in total soluble sugars and amino acids in the root exudates Sugars are the main component of root exudates. It has been reported that more than 65% of the organic matter secreted into the rhixosphere by plants are sugars (Curl and Truelove, 1986). In this study, the soluble sugar level (Fig. 4) was ten-fold higher than the total organic acid (Fig. 3) or amino acid contents (Fig. 5). With full-nutrient supply, the WT root medium contained about 32 pg of sugar per gram root fresh weight (Fig. 4), whereas Bt-cotton contained only 22 pg g-l. When compared with the full-nutrient supply, phosphorus and potassium deficiencies caused considerably greater secretion of soluble sugar compounds, with increment being 3-3.5 folds for Bt-cotton and 1.5-1.7 folds for WT. Sugar secretion in W T in the case of nitrogen deficiency was 60% lower than that in the case of full nutrients, whereas in Bt-cotton it was only about 17% lower. h
90 r 80 -
1
70
5
50
3.0 r
.,-'02 5I
-
z
z;' 60 -
5 L
20
40 30 -
T
-
v
1 5 -
m 0
t
2g) 20 10-
1 0 -
E 056
Treatment
m, Treatment
Fig. 4 Alterations in the total soluble sugar contents (means of three replicates) in the root (fresh weight) exudates of the 10-day old seedlings of the wild-type cotton (WT) and the transgenic cotton containing Bt (Bt-cotton) after 7 days of treatment under various nutrient supply conditions: BT-Bt-cotton, FN-full-nutrient solution, NN-no N in the nutrient solution, NP-no P in the nutrient solution, NK-no K in the nutrient solution. Vertical bars represent standard deviations. Fig. 5 Alterations in the total amino acid contents (means of three replicates) in the root (fresh weight) exudates of the 10-day old seedlings of the wild-type cotton (WT) and the transgenic cotton containing Bt (Bt-cotton) after 7 days of treatment under various nutrient supply conditions: BT-Bt-cotton, FN-full-nutrient solution, NN-no N in the nutrient solution, NP-no P in the nutrient solution, NK-no K in the nutrient solution. Vertical bars represent standard deviations.
With full-nutrient supply, the total amino acid contents were lower in the root exudates of Bt-cotton than those in WT (Fig. 5). The effects of the nutrient deficiencies were very similar in the two types of cotton. When compared with the full-nutrient supply, deficiencies of P and K caused increases in the secretion of amino acids, being 5.5 to 6.8 folds for Bt-cotton and 2.5 to 3.2 folds for WT. Again, N deficiency led to considerably lower amino acid secretion in WT, but only a slight decrease was observed in Bt-cotton. In WT, N-deficiency treatment led to about 49% drop in amino acid secretion, whereas in Bt-cotton, this drop was only about 4%.
DISCUSSION Although Bt-cotton shows resistance to damage by cotton insects, required only lower dosages of pesticides, and has been cultivated worldwide, there has been concern about the biosafety of transgenic plants. In the past decade, considerable attention has been paid to possible ecological risks from GM crops, such as gene flow from one species to another and food safety for humans. Also, Bt, as an insecticidal toxin released from transgenic plant roots into the rhizosphere, can exist for rather long
GENE OVEREXPRESSION AND ROOT EXUDATE COMPONENTS
329
periods (Shelton et al., 2002; Ding et aZ., 2001). Besides the transgenic insecticidal toxin (Bt), all plant roots secreted large amounts of inorganic ions and organic matter into the rhizosphere. A total of 25%-40% of the photosynthetic carbon can be transferred into the soil through this root-soil interface pathway (Curl and Truelove, 1986). Among the organic fractions of root exudates, 65% of the compounds belong to sugars, 20% to organic acids, and the remaining to amino acids and others (Curl and Thelove, 1986). These compounds, particularly low-molecular-weight compounds, are the key factors involved in soil microflora chemotaxis. They have both beneficial and harmful effects on plant growth, such as improving the link between roots and soils for water and nutrient uptake, mobilizing soil insoluble nutrients, immobilizing toxic elements, and acting as elicitors of a pathogen attack or as an energy source for pathogen growth. For example, sugars and amino acids secreted by a plant are positively correlated with plant infection (Yuan et al., 2002). The results of this study showed that when compared with WT, the Bt-cotton roots would secrete less amino acids and sugars into the soil when the soil fertility was high. However, when soil P or K supply was limited, the Bt-cotton roots would exude more amino acids and sugars into the rhizosphere. Thus, Bt-cotton may have lower yield when grown in the soils with higher probability of disease-causing infection. This may be one of the reasons for the requirement of larger dosages of P and K for transgenic Bt-cotton cultivation in comparison t o W T cultivation, for better yield and quality. On the other hand, root exudates, particularly organic acids, play a very important role in improving the soil nutrient bioavailability and in stabilizing harmful elements like aluminum and heavy metals (Shen et aZ., 2001; Zhang et al., 2005). Several mineral nutrient stresses can induce enhancement of root exudation (Liu e t al., 1997; Li e t al., 2005). The present investigation also indicated that the introduction of a foreign gene could lead to changes in the amounts of low-molecular-weight components in root exudates. At present, an explanation for the mechanism of the changes of organic acid species in root exudates of Bt-cotton cannot be provided. However, plants have a very complicated structure, and as the Bt protein is constitutively produced in various parts of the transgenic plant, the metabolic processes can be considerably affected. In conclusion, the results of this study indicated that root exudate components between transgenic Bt cotton and W T were, t o some extent, different and that the soil nutrient levels further affected these components. Because transferring the foreign Bt gene into cotton can result in alterations in the components of the root exudates, with the effect varying depending on the nutritional status, the cultivation of genetically modified crops, such as Bt-cotton, in soil environments should be more carefully assessed, and the possible effects as a result of the alterations in the root exudate components should be studied in the future. ACKNOWLEDGMENTS We thank Dr. Dong Cai-Xia and Miss Chen Rong-Fu of the Institute of Soil Science, Chinese Academy of Sciences, for their assistance with the HPLC analysis. We are very grateful to Professor Andre Jagendorf of Cornell University for his critical reading of this manuscript and his valuable suggestions. REFERENCES Chen, Y. L., Guo, Y. Q. and Han, S. L. 2002. Effect of root derived organic acid on the activation of nutrients in the rhizosphere soil. Journal of Forestry Research (in Chinese). 13: 115-118. Curl, E. A. and Truelove, B. 1986. The Rhizosphere. Springer-Verlag, New York. pp. 43-57. Ding, Z. Y., Xu, C. R. and Wang, R. J. 2001. Comparison of several important isoenzymes between Bt cotton and regular cotton. Acta Ecologzca Sznzca (in Chinese). 21: 332-336. Donegan, K. K., Palm, C. J. and Fieland, V. J. 1995. Changes in levels, species, and DNA fingerprints of soil microorganisms associated with cotton expressing the Baczllus thurzngaenszs var kurstakz endotoxin. Applaed Sod Ecology. 2: 111-124. Escher, N., Kach, B. and Nentwig, W. 2000. Decomposition of transgenic Baczllus thunngzenszs maize by microorganisms
330
W. D. YAN et al.
and woodlice Porcellio scaber (Crustacea: Zsopoda). Basic Applied Ecology. 1: 161-169. Fan, J. G. and Fan, G. R. 1995. Plant root exudation and its significance in forestry. Bulletin of Botanical Research. 15: 246-251. Fan, L., Zhou, X., Hu, B., Shi, C. and Wu, J. 2001. Gene dispersal risk of transgenic plants. Chinese Journal of Applied Ecology (in Chinese). 12: 630-632. Heuer, H., Kroppenstedt, R. M., Lottmann, J., Berg, G. and Srnalla, K. 2002. Effects of T4 lysozyme release from transgenic potato roots on bacterial rhizosphere communities are negligible relative to natural factors. Applied and Environmental Microbiology. 88: 1325-1 335. Li, J. Y., Xu, R. K. and Ji, G. L. 2005. Dissolution of aluminum in variably charged soils as affected by low-molecularweight organic acids. Pedosphere. 15: 484-490. Liu, Z. Y., Li, L. M. and Shi, W. M. (eds.). 1997. Research Methods in Rhizosphere (in Chinese). Jiangsu Science and Technology Publishing House, Nanjing. pp. 13-127. Lu, R. K. (ed.). 2000. Analytical Methods for Soils and Plants (in Chinese). China Agricultural Science Press, Beijing. pp. 121-133. Meng, K. Q. 1996. Debating for biosafety of genetically engineered plants. Progress in Biotechnology (in Chinese). 16: 2-6. Saxena, D., Florest, S. and Stotzky, G. 1999. Insecticidal toxin in root exudates from Bt corn. Nature. 415: 402-480. Shelton, A. M., Zhao, J , Z. and Roush, R. J . 2002. Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants. Ann. Rev. Entomol. 47: 845-851. Shen, H., Wang, X. C. and Shi, W. M. 2001. Isolation and identification of specific root exudates in elephantgrass in response to mobilization of iron- and aluminum-phosphates. J . Plant Nutrition. 24: 1117-1 130. Shen, R. F., Iwashita, T. and Ma, J. F. 2004. Form of A1 changes with A1 concentration in leaves of buckwheat. Journal of Ezperzmental Botany. 55(394): 131-136. Wang, W. P. 1998. Improving the method for determining the total dissociative amino acid in fresh plant tissue. Journal of Bezjing Agricultural College (in Chinese). 13(3): 9-12. Wang, Z. H., Ye, Q. F., Shu, Q. Y., Cui, H. R., Xia, Y. W. and Zhou, M. Y. 2002. Impact of root exudates from transgenic plants on soil micro-ecosystems. Chinese Journal of Applied Ecology (in Chinese). 13: 373-375. Wolfenbarger, L. L. and Phifer, P. R. 2000. The ecological risks and benefits of genetically engineered plants. Science. 290: 2 088-2 093. Yuan, H. X., Li, H. L. and Wang, Y. 2002. The root exudates of cotton cultivars with the different resistance and their effects on Verticillium dahliae. Acta Phytopathologica Sinzca (in Chinese). 32: 127-131. Zhang, Y. S., Shao, X. H., Lin, X. Y. and Scherer, H. W. 2005. Effect of organic acids and protons on release of non-exchangeable NH; in flooded paddy soils. Pedosphere. 15(3): 273-280.