Impact of salinity-tolerant MCM6 transgenic tobacco on soil enzymatic activities and the functional diversity of rhizosphere microbial communities

Research in Microbiology 163 (2012) 511e517 www.elsevier.com/locate/resmic

Impact of salinity-tolerant MCM6 transgenic tobacco on soil enzymatic activities and the functional diversity of rhizosphere microbial communities Vasvi Chaudhry a, Hung Quang Dang b, Ngoc Quang Tran b, Aradhana Mishra a, Puneet Singh Chauhan a, Sarvajeet Singh Gill b, Chandra Shekhar Nautiyal a,*, Narendra Tuteja b,** b

a CSIR - National Botanical Research Institute, Rana Pratap Marg, Lucknow, UP 226001, India Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India

Received 15 May 2012; accepted 8 August 2012 Available online 7 September 2012

Abstract The development of genetically modified plants for agriculture has provided numerous economic benefits, but has also raised concern over the potential impact of transgenic plants upon the environment. The rhizosphere is the soil compartment that is directly under the influence of living roots; it constitutes a complex niche likely to be exploited by a wide variety of bacteria potentially influenced by the introduction of transgenes in genetically modified plants. In the present study, the impact of overexpression of the salinity stress-tolerant minichromosome maintenance complex subunit 6 (MCM6) gene upon functional diversity and soil enzymatic activity in the rhizosphere of transgenic tobacco in the presence and absence of salt stress was examined. The diversity of culturable bacterial communities and soil enzymes, namely, dehydrogenases and acid phosphatases, was assessed and revealed no significant (or only minor) alterations due to transgenes in the rhizosphere soil of tobacco plants. Patterns in principal components analysis showed clustering of transgenic and non-transgenic tobacco plants according to the fingerprint of their associated bacterial communities. However, the presence of MCM6 tobacco did not cause changes in microbial populations, soil enzymatic activities or the functional diversity of the rhizosphere soil microbial community. Ó 2012 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Keywords: Bacterial diversity; CLPP; Pea MCM; Rhizosphere; Soil dehydrogenase; Transgenic plants

1. Introduction Genetically modified (GM) plants are being grown on an increasingly wide scale throughout the world, and their increasing use has been accompanied by major concern about their potential ecological and environmental impact (Wolt and Peterson, 2010). Incorporation of antibiotic resistance genes in GM plants as selection markers has raised questions about the possible transfer of these genes to indigenous microbes in the soil. For this reason, environmental risk assessment of GM plants has focused mainly on possible horizontal gene transfer (HGT) to related plants or soil- and plant-associated microbial * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (C.S. Nautiyal), [email protected] (N. Tuteja).

communities (Lee et al., 2011). The possible impact of transgenes on rhizosphere microbial communities is of interest, since little is known of the diverse functional relationships of microorganisms with transgenic plants in the soil environment (Bruinsma et al., 2003). It has been suggested that rhizospheres are altered in response to plant genetic transformation through HGT from GM plants to indigenous soil microbes (Lynch et al., 2004). Comparative studies assessing differences between microbial communities residing in the rhizospheres of GM and non-GM plants represent a first step in determining whether the presence of transgenic material produces changes in the environment. It is evident that, when GM plants are grown at a single site for a long period, they change the rhizospheric microbial metabolism, causing negative effects upon soil quality, structure and function and affecting enzyme synthesis and activity as well as soil processes such as litter decomposition and mineralization

0923-2508/$ - see front matter Ó 2012 Published by Elsevier Masson SAS on behalf of Institut Pasteur. http://dx.doi.org/10.1016/j.resmic.2012.08.004

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(Saxena and Stotzky, 2001). Wu et al. (2004) found that transient differences existed in the rhizospheric microbial community between soils amended with transgenic plants and non-transgenic plants. In contrast, there was no apparent effect of GM alfalfa on the microbial population. Studies of soil microbial and microfaunal communities also revealed differences in bacterial and fungal community physiological profiling (CLPP) (Blackwood and Buyer, 2004) and in the nematode population (Griffiths et al., 2005) under Bt maize cultivation. Donegan et al. (1995) observed effects of transgenic Bt cotton on both the abundance and diversity of indigenous soil bacteria and fungi. Several studies on long- and short-term effects of GM crops on soil microbial communities were carried out under field conditions. Oliveira et al. (2008) performed a two-year experiment under field conditions with two hybrids of transgenic Bt maize; their near-isogenic lines suggested that there were no cumulative effects of Bt maize over the short term. Another study was carried out for 2 years to assess the impact of Bt corn on rhizospheric and soil eubacterial communities and on beneficial mycorrhizal symbiosis in experimental microcosms. A polyphasic approach using denaturing gradient gel electrophoresis analyses of 16S rRNA genes showed differences in rhizospheric eubacterial communities associated with the three corn lines and a significantly lower level of mycorrhizal colonization in Bt 176 corn roots (Castaldini et al., 2005). Hannula et al. (2012), over a period of three years, investigated the impact of different potato cultivars, including a GM amylopectin-accumulating potato line, on rhizosphere fungal communities under field conditions using molecular microbiological methods; they revealed occasional differences between a transgenic line and its parental variety, indicating that differences were mainly transient in nature and could be detected either only in one soil, at one growth stage or over a one-year period. Current research on transgenic approaches have been used to determine the function of a gene vis-a-vis the response of plants under salinity stress conditions. Salinity stress is one of the major negative factors affecting the growth, development and yield of crops (Mahajan and Tuteja, 2005; Tuteja, 2007). Model plants such as tobacco have been extensively used to obtain transgenic plants with the pea minichromosome maintenance complex subunit 6 (MCM6) gene, where their role in stress tolerance has been established. The heterohexameric complex of MCM proteins (MCM2-7) unwinds the duplex chromosomal DNA ahead of the replication fork and thereby plays an important role in initiation and elongation steps in eukaryotic DNA replication, which occurs only once during the S-phase of the cell cycle (Tuteja et al., 2011). Helicase and ATPase activities were reported to be associated with complexes of MCM4/7 (Kanter et al., 2008), MCM4/6/7 (Ishimi, 1997) and MCM2-7 (Bochman and Schwacha, 2008). Recently, first direct evidence showed that a single subunit MCM6 from the pea forms a homohexamer and contains DNA helicase activity (Tran et al., 2010). Since MCM proteins play an essential role in cell division and are most likely affected during stress conditions, their overexpression in plants may help in stress

tolerance. Dang et al. (2011) reported for the first time the novel function of the pea MCM6 single subunit (PsMCM6) in conferring salinity stress tolerance in transgenic tobacco plants without affecting yield, elucidating a previously undescribed pathway for manipulating stress tolerance in crop plants. Biological and biochemical properties of soil have often been proposed as early sensitive indicators of soil ecological stress and other environmental changes. Generally, dehydrogenase (DHA) and phosphatase enzyme activities in the soil are closely related to building up of organic matter, and provide sensitive information on the microbial activity of soil. Moreover, DHA is thought to reflect the total range of oxidative activity of soil microflora and consequently may be a good indicator of microbiological activity (Visser and Parkinson, 1992; Dick, 1994; Nannipieri et al., 2003; Oliveira and Pampulha, 2006). In the present study, using the pea-MCM6-gene-overexpressing tobacco plant as our experimental system, we evaluated changes in microbial communities colonizing the rhizosphere of the MCM6 transgenic tobacco plant and its nontransgenic counterpart in the presence and absence of salt. In addition, the impact of the MCM6-transgene on rhizospheric soil DHA and phosphatase activities was also evaluated. 2. Material and methods 2.1. Cloning of pea MCM6 cDNA and tobacco transformation Pea MCM6 cDNA was cloned by screening the pea cDNA library as described earlier (Tran et al., 2010). Briefly, the cDNA library was first constructed from 5 mg poly (Aþ) RNA (isolated from pea leaves) in an UniZAP XR vector (Stratagene) following the manufacturer’s protocol. A heterologous probe of the yeast cdc21 gene (a member of the MCM protein family; a kind gift of Dr. Stephen Kearsey, University of Oxford, UK) was used to screen the library. The positive clone was sequenced and analyzed as described by Tran et al. (2010). For tobacco transformation, the complete ORF of MCM6 cDNA (2.48 kb) was PCR-amplified using forward primerc (50 -GAGGATCCATGGAAGCTTTCGGCGGTTA-30 ) starting from the translation initiation site and reverse primer (50 CCTGAGGATCCTCAGTCAACAACATAATGAG-30 ) designed to create BamHI sites at the 50 end before the start codon (underlined) and 30 ends next to the translation termination codon (underlined). The amplified fragment (2.5 kb) was cloned into plant transformation vector pBI121 (Clontech) to create pBI121-MCM6 as described earlier (Dang et al., 2011). This construct contains MCM6 and GUS (uidA) under a single cauliflower mosaic virus-35S (CaMV35S) promoter and the NPTII (kanamycin) gene as a selectable marker. The schematic representation of the final construct is shown in Fig. 1A. Tobacco (Nicotiana tabacum cv. Xanthi) leaf disks were transformed via a specific procedure (Horsch et al., 1985) with Agrobacterium tumefaciens (LBA4404 strain) containing the pBI121-MCM6 construct. Putative T0 transgenic plants were

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Fig. 1. (A) Schematic structure of the pBI121-MCM6 construct used for tobacco plant transformation. (B) MCM6-overexpressing T1 transgenic and WT tobacco plants under salt-stressed (200 mM NaCl) and non-stressed conditions after 4 months. Treatments: WT þ salt; T1-MCM6 þ salt; WT control; T1-MCM6 control (Fig. adapted from Dang et al., 2011).

regenerated from independent calli in the presence of kanamycin and further screened using PCR and Southern blot as described (Dang et al., 2011). The seeds from these T0 plants were germinated on kanamycin-containing medium to select all transgenic T1 seedlings. The positive transgenic plants were used for further study. 2.2. Experimental site and rhizosphere soil sampling Transgenic and wild type (WT) tobacco plants were grown under a 16 h light photoperiod at 25  C in the greenhouse of the International Centre for Genetic Engineering & Biotechnology, New Delhi. The experiment was conducted in an earthen pot where T1-transgenic MCM6 and non-transgenic (WT) tobacco were planted (5 months) in three replicates in the presence and absence of 200 mM NaCl salt. Harvesting of both transgenic and WT non-transgenic plants was performed four months after sowing. Rhizosphere soil samples were collected from each replicate of pots. To obtain rhizosphere soil, root material with adhering soil was removed and placed in a plastic bag. All visible plant debris was removed manually. Each soil sample was then sieved (2 mm) and stored at 4  C in the dark before analysis.

and absence of salt was determined by the culture enrichment technique (Nautiyal et al., 2008). Total rhizospheric soil aerobic bacteria were determined by the dilution plate technique. Rhizospheric soil (1 g) samples were suspended in 10 mL 0.85% saline Milli-Q water (MQW) and 10-fold serially diluted. Colony-forming units (CFU) of culturable heterotrophic bacteria were determined by spreading 100 mL of diluted sample onto nutrient agar medium. Three replicates of the inoculated agar plates were incubated at 28  C for 3 days, after which colonies were counted. 2.4. Determination of soil DHA activity Soil DHA was measured by a reduction in triphenyl tetrazolium chloride (TTC) to triphenyl formazan (TPF) (Alef, 1995), homogenized to 1 g rhizospheric soil and mixed with TTC solution (8 mg TTC per mL of 100 mM Tris buffer, pH 7.6), and then incubated at 25  C for 20 h. TPF produced was extracted from the reaction mixture with acetone and measured at 540 nm by a Shimadzu UVeVIS spectrophotometer. The activity of DHA was expressed as mg TPF g1 soil h1. 2.5. Determination of acid phosphatase activity

2.3. Quantification of heterotrophic bacteria determination Microflora associated with MCM6-transgenic and nontransgenic tobacco rhizospheric soil samples in the presence

Acid phosphatase activity was measured by the method of Tabatabai (1994). The method used p-nitrophenyl phosphate (pNP) as substrate and enzyme activity was determined from colorimetric measurements of pNP released when soil is

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2.6. Rhizospheric soil community level physiological profiling (CLPP) analysis Rhizospheric soil extracts were prepared from root-adhered soil of WT and MCM6 transgenic tobacco plants and soil was placed on clean sheets of plastic wrap. Individual rhizosphere soil samples of comparable weight (1.0 g) were shaken in 9.0 mL of sterile 0.85% saline MQW for 60 min and then made up to a final dilution 103. After incubation, 150 ml of sample were inoculated into each well of Biolog GN and Eco microplates (Biolog, Inc., Hayward, CA, USA) which contained a total of 103 different carbon sources along with a negative control well, and incubated at 28  C (Campbell et al., 1997). Each well also contained the redox indicator dye tetrazolium violet, which turns from colorless to purple in the presence of respiration, indicating the rate of carbon source utilization. Data were recorded for days 1e7 at 590 nm described earlier (Mishra and Nautiyal, 2009). Microbial activity in each microplate, expressed as the average well color development (AWCD), was determined as described by Garland (2006). Diversity and evenness indexes were calculated as described by Mishra and Nautiyal (2009). Principal component analysis (PCA) was performed on data divided by AWCD as described by Garland and Mills (1991). PCA was performed on the 5th day (120 h) with data divided by the AWCD, as described by Garland and Mills (1991). Using the method of Biolog Eco and MT plates (Biolog, Inc., Hayward, CA, USA), a comparative study was made to determine the carbon source utilization pattern of rhizosphere soil samples of transgenic and nontransgenic plants as described earlier (Campbell et al., 1997). Biolog MT plates were prepared using the manufacturer’s instructions (Biolog Inc., Hayward, CA 94545, USA). 2.7. Statistical analyses Statistical analyses were performed using MS-Excel, SPSS 16.0 and Statistica 7.0 software. Heterotrophic bacterial counts (Log10CFU) and enzyme activities were expressed as means of three replicates. 3. Results and discussion 3.1. Isolation of MCM6 cDNA and transgenic plant analysis Sequence analysis of pea MCM6 cDNA (Accession number AY169793) showed that it encodes a full-length cDNA

2.895 kp in size, with an open reading frame (ORF) of 2.484 kp, a 50 untranslated region (UTR) of 99 bp and a 30 UTR of 312 bp, including a 13 bp poly(A) tail. Analysis of transgenic plants was described earlier (Dang et al., 2011). Briefly, MCM6-overexpressing T1 transgenic tobacco plants were able to grow to maturity and set normal viable seeds under continuous salinity stress (200 mM NaCl) and in water, without yield loss (Fig. 1B). Wild type plants can grow only in water and could not survive in 200 mM NaCl (Fig. 1B). Naþ ions were found to accumulate in mature leaves and not in seeds of T1 transgenic lines as compared with WT plants. T1 transgenic plants exhibited better growth status under salinity stress conditions in comparison to WT plants. Furthermore, T1 transgenic plants maintained significantly higher levels of leaf chlorophyll content, net photosynthetic rates and therefore higher dry matter accumulation and yield with 200 mM NaCl compared to WT plants (Dang et al., 2011). 3.2. Culturable bacterial populations in rhizosphere soil of parent and MCM6 transgenic tobacco plants Plants of each line were collected and evaluated after 4 months of cultivation. Bacterial densities from the rhizospheric soil of MCM6 transgenic tobacco plants and WT plants in the presence and absence of salt were determined and it was observed that rhizospheric soil extracts varied very little in terms of total heterotrophic bacterial counts. The total

A DHA activity µg TPF g−1 soil h −1

incubated in a buffered (modified buffer pH 6.5) substrate solution; 1 g of soil was treated with 0.25 mL of toluene, 4 mL of modified buffer (pH 6.5), 1 mL of pNP made in the same buffer, mixed and incubated for 1 h at 37  C. After incubation, 1 mL of 0.5 M CaCl2 and 4 mL of 0.5 M NaOH were added, and contents were mixed and filtered through a filter paper. The absorbance in the filtrate was then measured at 400 nm using a Shimadzu UVeVIS spectrophotometer and reported as mg pNP g1 soil h1.

400

WT contro control r l WT+salt

T1-MCM6 contro control r l T1-MCM6+ salt

350 300 250 200 150 100 50 0

WT contro control r l WT+salt

B

T1-MCM6 contro control r T1-MCM6+ salt

12

Phosphatase activity µg pNP g-1 soil h -1

514

10 8 6 4 2 0

Fig. 2. Variations in DHA (A) and acid phosphatase (B) activities in WT control; T1-MCM6 control; WT þ salt (200 mM NaCl); T1-MCM6 þ salt (200 mM NaCl). Different letters indicate significant differences ( p < 0.05, n ¼ 3).

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respectively (Fig. 2). The response of DHA and acid phosphatase activity in soil amended with salt was quite different from that in soil with no salt amendment. In the presence of salt, values of DHA rose to 296.69  14.2 and 334.91  10.4 mg TPF g1 soil h1, whereas the acid phosphatase level was 9.68  0.57 and 10.9  0.28 mg pNP g1 soil h1 in non-transgenic and MCM6 transgenic tobacco plants, respectively (Fig. 2). The differing behavior of the soil enzymes was found in the presence of salt, indicating that despite this, no differences were detected when MCM6 transgenic plants were compared to their nontransgenic isolines.

1.0

WT+salt T1-MCM6+salt

Factor 2 : 24.12%

0.5

515

0.0

T1-MCM6 control WT control

-0.5

3.4. CLPP of microbial communities Analysis of rhizosphere bacterial community metabolic fingerprint structure in the rhizosphere of transgenic and nontransgenic plants was assessed using Biolog Eco and GN2 plates to monitor changes in microbial diversity. PCA was applied to the overall set of results obtained from carbon substrate utilization on Biolog Eco and GN2 plates to obtain a general view of the differences within the rhizosphere microbial community of transgenic and non-transgenic lines in the presence and absence of salt. In PCA, differences were observed by plotting scatter plot in two dimensions on the basis of their scores for the first two components. When the two factors were plotted, there appeared some overlap in cluster composition between the rhizosphere bacterial community metabolic fingerprints of transgenic-MCM6 and non-transgenic lines, as they were grouped together at 65.25 and 24.12% variance on the factor 1 and 2 axis in the presence and absence of salt. Salt caused a shift in the microbial diversity of transgenic and non-transgenic lines; however, no significant differences were found between transgenic and non-transgenic lines in the presence and absence of salt (Fig. 3). Concerning substrate diversity, there were no significant differences between transgenic and non-transgenic lines in the presence and absence of salt based on Shannon, McIntosh or Simpson diversity or the McIntosh evenness indices, except for the Shannon evenness index where the difference was not considerable (Table 1). GN and Eco plate data suggest that polymers were the most frequently utilized carbon sources by transgenic and non-transgenic lines in the presence and absence of salt. No apparent differences were observed between categorized carbon substrates among the samples. However, carbohydrate utilization differed and was

-1.0 -1.0

-0.5

0.0

0.5

1.0

Factor 1 : 65.25%

Fig. 3. Principal components analysis based on carbon source utilization by the rhizospheric microbial community of parent and transgenic plants in the presence and absence of salt. Treatments: WT control; T1-MCM6 control; WT þ salt (200 mM NaCl); T1-MCM6 þ salt (200 mM NaCl).

bacterial count observed was 5.05 and 5.13 Log10 CFU/mL in the presence of salt and 5.52 and 5.54 Log10 CFU/mL in the absence of salt in WT and MCM6 transgenic tobacco plants, respectively. 3.3. DHA and acid phosphatase enzyme activity in rhizosphere soil To test the impact of transgenic tobacco on rhizospheric soil enzymes, DHA and acid phosphatase enzyme analyses were carried out with MCM6 transgenic and non-transgenic tobacco plants. On the basis of DHA and acid phosphatase, the MCM6 transgenic tobacco plant did not appear to show significant differences when compared with its non-transgenic cultivar either in the presence or absence of salt. In the absence of salt, MCM6 transgenic and non-transgenic tobacco showed DHA activity values of 241.15  6.5 and 250.15  9.4 mg TPF g1 soil h1 and acid phosphatase activity of 4.90  0.54 and 5.04  0.55 mg pNP g1 soil h1,

Table 1 Functional diversity of microbial communities of parent and transgenic tobacco plant rhizosphere soils in the presence and absence of salt, as reflected by the Shannon index, Simpson index and McIntosh index. T1-MCM6 control

WT control Shannon diversity index Shannon evenness index McIntosh diversity index McIntosh evenness index Simpson diversity index

4.55 0.99 0.99 0.99 1.00

    

a

0.01 0.00a 0.00a 0.00a 0.00a

4.54 0.98 0.99 0.99 1.00

    

a

0.01 0.00a 0.00a 0.00a 0.00a

Different letters (a, b) in a row indicate a significant difference at P < 0.05 using the WallereDuncan test.

WT þ salt 4.57 0.99 0.99 0.99 1.00

    

T1-MCM6 þ salt b

0.01 0.00a 0.00a 0.00a 0.00a

4.56 0.98 0.99 0.99 1.00

    

0.01b 0.00a 0.00a 0.00a 0.00a

516

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Fig. 4. Categorized carbon substrate utilization pattern by the rhizospheric microbial community of parent and transgenic plants in the presence and absence of salt. Symbol represents treatments: WT control; T1-MCM6 control; WT þ salt (200 mM NaCl); T1-MCM6 þ salt (200 mM NaCl). Different letters indicate significant differences ( p < 0.05, n ¼ 3).

decreased in transgenic line MCM6 and WT in the presence of salt, whereas carboxylic acid utilization was lower in both MCM6 and WT in the presence of salt when compared with a salt-amended non-transgenic control (Fig. 4). Our results on MCM6 transgenic tobacco showed consistency with reports from earlier workers, where the introduction of transgenic plants did not alter the rhizospheric soil or soil microflora. Oliveira et al. (2008) indicated that the presence of Bt maize did not cause changes in microbial populations in soil nor in the activity of the microbial community. Likewise, some studies showed that the diversity of the soil microbial community was not affected or only slightly affected by long-term cultivation of transgenic plants (Kapur et al., 2010; Li et al., 2011). In contrast, some studies reported adverse effects of GM plants on native soil and the rhizosphere microbial population. Some workers indicated that Bt corn affects microbial communities, the activities of some enzymes and microbe-mediated processes and functions in soil (e.g., Griffiths et al., 2005, 2006; Icoz et al., 2008). Aira et al. (2010) studied microbial communities in the maize rhizosphere and found that the plant genotype (su1 and sh2 genes) strongly influenced the structure and growth of rhizosphere microbial communities. The structure and diversity of bacterial communities in rhizosphere soils of GM and non-GM Zoysia grasses were investigated by constructing 16S rDNA clone libraries. They revealed that the two clone libraries significantly differed, suggesting alterations in the composition of the microbial community associated with GM Zoysia grass (Lee et al., 2011). From the ample amount of data available, it is clear that the effect of transgenic plants on the soil microbial community, and consequently on the evolution of potential adverse effects, depends strongly on the particular plants, techniques, proteins and environmental conditions (Brusetti et al., 2004). Therefore, whatever the approach chosen, evaluation of the effects on rhizosphere processes and species composition can improve our understanding of the effects of modified genes. Moreover, an evaluation of these

interactions and use of modern molecular techniques are frequently a requirement for risk assessment (European Food Safety Authority, 2010) and should be initiated at some stage during the application for approval of GM crops. In conclusion, it was found that the transgenic MCM6, regardless of genetic modifications, did not significantly affect the microbial community structure of the rhizosphere in the absence or presence of salt amendment, based on results of the Biolog system, which was used to reflect the diversity of microbial communities by comparing metabolic fingerprints of different microbes according to their utilization of different carbon sources. Rhizospheric soil bacterial populations and enzyme activities revealed minor results indicate no evidence of adverse effects of transgenic MCM6 upon the native soil microflora in this study. Acknowledgments Work on DNA replication and plant abiotic stress tolerance in NT’s laboratory is partially supported by the Department of Biotechnology (DBT) and the Department of Science and Technology (DST), Government of India. We thank Dr. Meenu Madan for her initial help in transgenic work. References Aira, M., Brando´n, M.G., Lazcano, C., Baath, E., Domı´nguez, J., 2010. Plant genotype strongly modifies the structure and growth of maize rhizosphere microbial communities. Soil Biol. Biochem. 42, 2276e2281. Alef, K., 1995. Dehydrogenase activity. In: Alef, K., Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology and Biochemistry. Academic Press, London, pp. 228e231. Blackwood, C.B., Buyer, J.S., 2004. Soil microbial communities associated with Bt and non-Bt corn in three soils. J. Environ. Qual. 33, 832e836. Bochman, M.L., Schwacha, A., 2008. The MCM2-7 complex has in vitro helicase activity. Mol. Cell. 31, 287e293. Bruinsma, M., Kowalchuk, G.A., van Veen, J.A., 2003. Effects of genetically modified plants on microbial communities and processes in soil. Biol. Fertil. Soils 37, 329e337.

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