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Kinetics of mass and DNA decomposition in tomato leaves
Chemosphere 61 (2005) 677–684 www.elsevier.com/locate/chemosphere
Kinetics of mass and DNA decomposition in tomato leaves John Pote´ b
a,*
, Patrick Rosse´ a, Walter Rosselli b, Van Tran Van c, Walter Wildi
a
a Institut F.-A. Forel, University of Geneva, 10 route de Suisse, 1290 Versoix, Switzerland WSL Swiss Federal Research Institute, Antenne romande, P.O. Box 96, 1015 Lausanne, Switzerland c Ecologie Microbienne UMR CNRS 5557 UFR of Biology, Universite´ Claude Bernard, Lyon I, 43 boulevard 11 novembre 1918, 69622 Villeurbanne, France
Received 1 October 2004; received in revised form 11 March 2005; accepted 14 March 2005 Available online 5 May 2005
Abstract This laboratory study investigated the kinetics of leaf and DNA content decomposition in two varieties of tomato (Palmiro and Admiro) after incubation in soil for 35 days. Results revealed that the decrease of dry matter in leaves in both varieties did not follow a single exponential function and was better described by a double exponential model. Composite half-decrease times were 3.4 and 2.4 days for Palmiro and Admiro respectively. The same pattern was observed for DNA mass loss, although this was closer to a single exponential model with composite half-decrease times of 1.5 and 1.4 days. Genomic analysis showed that DNA in dried leaves at room temperature (not inoculated in the soil), remains intact or presents a weak degradation, and DNA extracted from leaves inoculated in non-sterile soil showed degradation after two days. These results indicate that before release an important quantity of DNA may be degraded inside plant tissues during decomposition in soil. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Decomposition kinetics; Tomato leaves; Exponential model; DNA release
1. Introduction Soil is a heterogeneous environmental compartmentalised into physical, chemical and biological points of view. It contains a great diversity of micro-organisms and is endowed with both biological and physico-chemical reactivity. Many studies have been made of decomposition rates in different types of plant material, especially leaf litter (Olson, 1963; Howard and Howard, 1974). Decomposition of leaves in field conditions is mainly microbial
* Corresponding author. Tel.: +41 22 950 97 24; fax: +41 22 755 13 82. E-mail address: [email protected] (J. Pote´).
(Howard and Howard, 1974; Berg et al., 1984). Decomposition of plant materials in soil and nutrient release are significantly influenced by such factors as microbial biomass, litter source, amount of litter input, the relative proportion of the different plant tissues, as well as their chemical composition (Monties, 1991; Guo and Sims, 2002; Ko¨gel-Knabner, 2002). Soil factors such as texture (Schimel, 1986; Amato and Ladd, 1992; Saggar et al., 1999; Stemmer et al., 1999), temperature, and humidity are also critical. The Earth is virtually entirely covered in nucleic acid (Trevors, 1996). Nucleic acids (i.e. DNA and RNA) are ubiquitous in many environments, including fresh water, marine water column and sediment, soil, and terrestrial subsurface. They play a key role in the biogeochemical cycles of the biosphere and represent an
0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.03.030
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enormous reservoir of novel valuable molecules for health or industry (Pillai and Ganguly, 1970; DeFlaun et al., 1986; Paul et al., 1987; Trevors, 1996; Robe et al., 2003). Soil is a complex environment and appears to be a major reservoir of nucleic acids, which seem to be concentrated in upper soil horizons and represent up to 10% of the soil organic phosphate (Baker, 1977). Several studies discuss the presence of large quantities of extracellular DNA in soil (Torsvik and Gorsksoyr, 1978; Reanney et al., 1982; Ogram et al., 1987; Steffan et al., 1988; Spring et al., 1992; Blum et al., 1997; Paget et al., 1998). However, it has been estimated that, depending on soil composition, extracellular DNA concentrations from 5 to more than 35 lg g1 (dry soil) can be extracted from soil (Frostega¨rd et al., 1999). DNA from plants has been released into the environment naturally (Soltis et al., 1992; Paget et al., 1998). By studying transgenic plants, many recent studies have focused on what happens to transgenes in the environment. The majority of these studies have monitored the presence and stability of transgene in various systems using PCR amplification. Most of these studies were carried out on the presence of extracellular plant DNA in soil. This DNA (from transgenic or non-transgenic plants) is released during the process of plant tissue decomposition or from enzymatic degradation of cell structures by pathogens (Ceccherini et al., 2003). However, little information is available on the process of DNA release and neither is there much information to be found regarding quantitative and qualitative aspects of DNA in plant material decomposing in soil. Potential ecological effects caused by deliberate or accidental release of genetically engineered micro-organisms mean survival, multiplication and dispersal of the inoculated cells as well as transfer of recombinant DNA to the indigenous microflora need monitoring (Paget and Simonet, 1994). One of the main risks of transgenic plants is the spread of their transgenes through gene flow into wild populations causing genetic pollution and giving rise to potential ecosystem disruption. The following issues associated with genes from transgenic plants must be dealt with: (i) emission, dispersal, and deposition of transgenic pollen, (ii) introgression of the transgene into wild species, (iii) stabilisation and spread of the transgene in wild species, (iv) ecological effects of the transgene in the new host population (Evenhuis and Zadok, 1991). Description of DNA dispersion from engineered plants in the environment, in particular its kinetic aspects, requires the use of models capable of assessing the characteristics of the initial source, the release process, and the means of transport (Stocco, 1994). The transport of DNA in saturated soil has recently been studied by Pote´ et al. (2003). DNA, as the signature of life, has been extensively studied in a wide range of environments. While DNA analysis has become central to work on natural gene
exchange, forensic analyses, soil bioremediation, genetically modified organisms, exobiology, and palaeontology, fundamental questions concerning DNA release from diverse organisms, there remains resistance to degradation (quantity and quality) all remain. The objective of this study was to examine: (i) the decomposition kinetics of the tomato leaf (dry matter) in soil and (ii) qualitative and quantitative aspects of DNA in tomato leaves during decomposition in soil. This study is oriented towards a conceptual approach to modelling the plant DNA release process during dry matter decomposition in soil.
2. Materials and methods 2.1. Conceptual approach 2.1.1. Decomposition of dry matter The most frequently used model to describe decomposition is the single exponential decay function (Wieder and Lang, 1982), which was first proposed by Jenny et al. (1949) and is discussed in detail by Olson (1963). Other models (double exponential, asymptotic, linear, quadratic, power) can also be used to examine decomposition data (Wieder and Lang, 1982). Thus, one can assume that the dry weight loss of tomato leaves in soil can be modelled by using an exponential law (Jenny et al., 1949; Olson, 1963). M DRY ðtÞ ¼ M DRY0 ekDRY t
ð1Þ
In which MDRY(t): dry matter weight at time t, MDRY0: initial dry matter weight, kDRY: loss rate constant (decomposition rate). Eq. (1) indicates a first order decrease and appears in many other processes such as: degradation of pesticides on the ground—natural decrease of radioactive isotopes—sedimentation of the not coagulated solids— breathing death rate of bacteria and algae. For the purposes of the study presented in this paper, kDRY will be determined by fitting an exponential model to dry weight measurements of tomato leaves. Half-decomposition time of the dry matter is given by T DRY1=2 ¼
1 ln 2 k DRY
ð2Þ
2.1.2. DNA decrease in leaves As in the dry matter case, a first order relaxation model was used to describe the decrease of DNA in dry matter: M DNA ðtÞ ¼ M DNA0 ekDNA t
ð3Þ
In which MDNA(t): DNA weight at time t, MDNA0: initial DNA weight, kDNA: release rate constant.
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However, in this case DNA weight within the samples cannot be directly measurable for instrumental reasons linked to the DNA extraction method used. Therefore, DNA weight is determined from the measurement of the DNA mass concentration in leaves, CDNA(t), and the previous dry matter weight of samples MDRY(t), using the direct relation M DNA ðtÞ ¼ C DNA ðtÞ M DRY ðtÞ
ð4Þ
This equation (Eq. (4)), an exponential model fitting, allows the determination of both kDNA and the associated DNA half-decrease time given by T DNA1=2 ¼
1 ln 2 k DNA
ð5Þ
2.2. Plants Two varieties of tomato were used in this study. Firstly, Palmiro (PAL), which has a water content of 89.3 ± 0.3%, and a concentration of DNA in dry leaves samples of 117 ± 7 lg g1. Secondly, Admiro (ADM), with water content: 88.4 ± 1.4%, and concentration of DNA in dry leaves samples of 112 ± 6 lg g1. These tomatoes were cultivated in greenhouse on hydroponic medium. After being collected, leaves were rapidly dried at 35 °C for 3 days in order for any decomposition to be avoided. The leaves were re-hydrated in sterile water at 20 °C for 5 min, after which they were incorporated into soil microcosms. 2.3. Soil The surface soil (between 0 and 15 cm depth) used in this study was collected from a cropland field in the locality of Ecogia, near the city of Geneva, Switzerland. Some soil characteristics are given in Table 1. The soil was sieved through a 5 mm sieve (W.S. Styler company mentor, USA). To determine the aggregates size distribution of the soil microcosm, a soil aliquot was dried (at 35 °C for 24 h) and sieved through four successive sieves (2/1/0.5/0.125 mm). Each fraction was then weighed and reported to the total aliquot mass. Results
Table 1 Some soil characteristics
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Table 2 Aggregates size in soil microcosms Size class (mm)
Mass (%)
<0.125 0.125–0.5 0.5–1.0 1.0–2.0 >2.0
1.2 18.7 35.4 40.1 4.5
are shown in Table 2. Sterile soil for control microcosms was dried in the oven at 150 °C overnight and autoclaved (121 °C, 50 min. Autoklav, Typ 23, MELAG, Germany) twice with a 24 h period of incubation between treatments and re-hydrated with sterile water (22 wt.%). 2.4. Microcosm description and handling PET plastic containers 21 cm in height and 8 cm by 8 cm in section were used for the microcosms. Each container was filled with a 15 cm layer of soil. Tomato leaves were placed in a non-overlapping manner between two supple polyester nets (1.4 mm mesh, thickness 130 lm) which in turn was placed on top of the soil. Leaves were then covered with a 3 cm layer of soil, this configuration mimics the burying of green manure in the field. The net was needed to make the removal of the sample easier. It was supple enough to maintain good contact between leaves and soil (Couˆteaux et al., 2002). Natural soil (without leaves) was also used for control microcosms. Microcosms were kept in a dark room at 21.5 °C. The soil humidity was stabilised (at 22 wt.%) by water addition based on container weighting. The experiment was conducted for a total of 35 days. Leaves were taken from microcosms after 2, 4, 10, 19 and 35 days, and at each time of these sampling points, three containers of each tomato variety (PAL & ADM) were sampled. Tomato leaves were removed from microcosms, dried for 3 days at 35 °C, and weighed. Care was taken to remove all pieces of leaves attached to the net. For pieces of leaves completely attached on the soil, leaves and soil were placed in water and separated by gravity. 2.5. DNA extraction from soil and tomato leaves
Soil type
Loamy sand
Sand (%) Silt (%) Clay (%) Organic matter (%) pH (H2O) Volumetric weight Water-holding capacity Bacteria biomass
78 14 8 4.3 6.5 1.32 g ml1 38.6% 108 CFU g1 dry soil
After the leaves had been removed, soil located 50 mm below the top of microcosms was taken. This was mixed and used for extracellular DNA extraction using buffer TENP (50 mM Tris, 20 mM EDTA, 100 mM NaCl, 1% [wt/vol] polyvinylpolypyrrolidone)—the protocol included no lysis treatment, as described by Frostega¨rd et al. (1999). The concentration of extracted DNA was measured spectrophotometrically (OD260) and aliquots were used for PCR amplification.
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DNA from tomato leaves was extracted using DNeasy plant DNA miniextraction kit (Qiagen, Helden Germany) and following the manufacturerÕs recommendations. The concentration of extracted DNA was measured spectrophotometrically (OD260) and aliquots were used to estimate DNA quality using electrophoresis on 0.8% agarose gel stained with ethidium bromide (10 lg ml1, Sigma, USA). All DNA extracted was passed through a purification Microspin S-400 (Pharmacia Biotech) to remove trace humic acids.
Normalised leaves dry weight (-)
680
1.0 0.8 0.6 0.4 0.2 0.0 0
2.6. PCR amplification The tomato DNA sequences in extracted soil DNA were amplified using PCR amplification based on 18S primers, F474 (5 0 -ATGAAAGACGAACAACTG-3 0 ) and F475 (5 0 -TACCTGGTTGATCCTGCC-3 0 ), which were complementary to part of tomato 18S gene. PCR was carried out in 50 ll reaction volume containing 1X PCR buffer (Invitrogen), 1.5 mM MgCl2, 200 lm of each dNTP, 200 nM of each primer, 0.5 units Taq DNA polymerase (Invitrogen), and a template DNA (100 ng). PCR was performed in a Biometra thermocycler (BIOLABO) in the following conditions: an initial denaturation at 94 °C for 5 min; 35 cycles of denaturation (94 °C for 45 s), annealing (55 °C for 45 s), extension (72 °C for 45 s); and a final extension for 5 min at 72 °C. PCR products were checked by electrophoresis on 0.8% agarose gel stained with ethidium bromide (10 lg ml1, Sigma, USA).
Palmiro Admiro Palmiro fitting Admiro fitting
10
20
30
Time (day) Fig. 1. Decrease of leaves dry matter (normalised by the initial value).
A single exponential model appeared to be inappropriate to describe the fast weight loss observed at the early stage. Consequently, a two-compartment (double exponential), first order model was chosen to fit the results (Couˆteaux et al., 2002). M DRY ðtÞ ¼ Aekat þ Bekbt In which MDNA(t): DNA weight at time t (in days); A and B: initial dry matter proportions of the fast and the slow decomposing compartments respectively (MDRY0 = A + B); ka and kb: decay rate constants for A and B fractions respectively. Each fraction decays exponentially. The models fitted are ðtÞ ¼ 0.361e1.85t þ 0.497e0.0436t M DRY
3. Results 3.1. Decomposition of leaves All the data were normalised before comparison (i.e. relative to their initial value). An initial and instantaneous loss of 15% occurred during the leaf re-hydration process preceding incorporation into the soil. This loss of hydrosoluble substances may imitate the rain leaching process of the dried tomato plant occurring in the field. Consequently, the posthydration dry weight is hereafter considered as being the initial value. No significant differences in leaf decomposition were found between the two varieties of tomato leaves (Fig. 1). The maximum difference between their normalised weights is 7% during the first 10 days. At this time, PAL shows a dry weight 49% and 22% smaller than ADM after 19 and 35 days, respectively. The decomposition was faster in the first few days of experimentation (decrease reaching 46% and 47% respectively for PAL and ADM after two days in soil). After this, more resistant parts of leaves seem to slow the decomposition down.
ðR2 ¼ 0.998Þ for Palmiro ðtÞ ¼ 0.423e1.09t þ 0.431e0.0339t M DRY
ðR2 ¼ 0.999Þ for Admiro The Palmiro and Admiro values of R2 fitted by single exponential function were 0.823 and 0.693 respectively. 3.2. DNA mass concentration The DNA mass concentration is not constant in the dry matter of leaves showing decrease through time (Fig. 2). In the first two days of experimentation, the DNA concentration for both varieties did not appreciably vary. In the meantime, the dry matter lost about half of its weight (see Section 4.1.). After the second day, the DNA concentration starts to decrease more or less linearly in parallel with the dry weight until the 19th day. 3.3. Amount of DNA For similar reasons as in the dry matter case, a double exponential model was chosen to fit DNA data (Fig. 3).
DNA concentration in leaves (µg/g)
J. Pote´ et al. / Chemosphere 61 (2005) 677–684 140 120
Initial time
100
PAL : 117 ± 7 (µg/g) ADM : 112 ± 6 (µg/g)
2ndday
4thday
80 60 10thday
40 20 0 1.0
Palmiro Admiro 0.8
35thday
19th
0.6
0.4
0.2
0.0
Normalised leaves dry weight (-) Fig. 2. DNA concentration pattern, CDNA(t) as a function of the normalised dry weight loss.
Normalised DNA amount in leaves (-)
681
1.0
Palmiro Admiro Palmiro fitting Admiro fitting
0.8 0.6 0.4 0.2 0.0 0
10
20
30
Time (day) Fig. 3. Decrease of DNA weight in leaves dry matter (normalised by the initial value).
The models fitted are M DNA ðtÞ ¼ 0.544e0.992t þ 0.456e0.133t ðR2 ¼ 0.999Þ for Palmiro ðtÞ ¼ 0.591e0.935t þ 0.409e0.131t M DNA
Fig. 4. Gel signature of DNA extracted from tomato leaves at different time to estimate potential of DNA degradation. Lane 1: Molecular Weight Standard (smart Ladder). Lane 2: PAL DNA from fresh matter. Lane 3: PAL DNA from dry matter no introduced in soil. Lane 4: ADM DNA from fresh matter. Lane 5: ADM DNA from dry matter no introduced in soil. Lane 6: PAL DNA, 2 days in soil. Lane 7: ADM DNA, 2 days in soil. Lane 8: PAL DNA, 35 days in soil. Lane 9: ADM DNA, 35 days in soil. Lane 10: PAL DNA, control microcosms, 35 days in soil. Lane 11: ADM DNA, control microcosms, 35 days in soil.
degraded after two days. It can be seen that DNA extracted from leaf samples (PAL and ADM), dried and stored in dry conditions for 40 days at 22 °C (room temperature) does not present a smear, indicating that no degradation has occurred. The amounts of extracellular DNA estimated by OD260 ranged from 24 to 31 lg g1 dry soil. No difference was observed between amounts of extracellular DNA extracted from both natural soil and from soil microcosms. The expected 935 pb long 18S DNA fragment was detected in all the DNA extracted from soil microcosms. No signal was observed for DNA extracted from natural soil and sterilised control microcosms.
4. Discussion 4.1. Leaf decomposition
2
ðR ¼ 0.999Þ for Admiro The Palmiro and Admiro values of R2 fitted by single exponential function were 0.978 and 0.980 respectively. 3.4. Quality of DNA in leaves, amount of extracellular DNA, and PCR amplification DNA degradation in the tomato leaves was qualitatively estimated by DNA size distribution on the agarose gel by comparing it with DNA standard (Romanowski et al., 1993; Widmer et al., 1996; Ceccherini et al., 2003; Pote´ et al., 2003). The analysis of agarose gel electrophoresis (Fig. 4) indicates that DNA extracted from leaves inoculated in non-sterile soil was significantly
Tomato leaf dry matter from control microcosms (sterile soil) showed only limited weight loss (Fig. 5). The dry weight loss in control microcosms after 35 days reached 33.8% and 29.4% for PAL and ADM respectively. These values must be compared with the 87.9 and 85.2% weight loss obtained with the non-sterile soil. This limited loss may be explained by microbial activity in leaves which were not sterilised (Ceccherini et al., 2003). However, this activity did not affect the DNA quality in leaves, as shown in Fig. 4. The exponential model with one single rate constant does not fit dry weight measurements well. The fitting may be improved by using a two-compartment first order model (i.e. two decomposition processes in parallel with two distinct
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researcher to understand what happens to transgenic plant DNA released into the environment (Stocco, 1994). 4.2. DNA release from leaf dry matter
Fig. 5. Quality of leaves from soil microcosms (PAL) after 35 days.
kinetics) (Couˆteaux et al., 2002). The half-decomposition time of Palmiro leaves is 0.375 ± 0.280 days for the fast fraction (42% of initial dry weight) and 15.9 ± 1.5 days for the slow fraction (58% of initial dry weight). The composite half-decomposition time is 3.40 days (numerical solution). For Admiro leaves, the halfdecomposition time is 0.638 ± 0.156 days for the fast fraction (50% of initial dry weight) and 20.5 ± 2.7 days for the slow fraction (50% of initial dry weight). The composite half-decomposition time is 2.43 days. The double exponential decay model assumes that dry matter can be partitioned into two components: firstly, a fraction relatively easily decomposed, and secondly one which is recalcitrant to decomposition (Wieder and Lang, 1982). The biodegradation of the fractions (such as simple sugars, amino acids, polyphenols, hemicellulose, cellulose and lignin) in plant residues occurs simultaneously but with different kinetics (Kumar and Goh, 2000). The amount of DNA released by decomposition of plant materials in soil can be estimated by the amount of plant material present. The rates of loss from all reservoirs (e.g. plant materials) can be expressed conveniently by parameter k (the decomposition rate), which equals the fraction of the stored quantity that is lost (short) unit time, but without implying whether the fractions are approximately constant or not (Olson, 1963; Wieder and Lang, 1982; Berg et al., 1984; Couˆteaux et al., 2002). The data concerning leaf decomposition largely contributes to predicting (modelling) the amount of tomato extracellular DNA released through time while tomato leaves are decomposing in soil. Many studies have been performed on the persistence of plant DNA in soil. However, little information is available on the release of DNA from plant materials. Modelling DNA release from plant materials may help the field
The half-decrease time of Palmiro DNA is 0.698 ± 0.070 days for the fast fraction (54% of initial DNA amount) and 5.20 ± 0.31 days for the slow fraction (46% of initial DNA amount). The composite halfdecrease time is 1.48 days (numerical solution). For Admiro DNA, the half-decrease time is 0.742 ± 0.046 days for the fast fraction (59% of initial DNA amount) and 5.31 ± 0.25 days for the slow fraction (41% of initial DNA amount). The composite half-decrease time is 1.38 days. However, the DNA amount is inferred from the dry weight of leaves. The decreases in amount of DNA agree better with the single exponential model rather than with the dry weight (i.e. the fast and slow parameters are closer). This fact suggests that the DNA and dry matter decrease processes are not closely coupled at the beginning of the experiment. The DNA released from dry matter in decomposition may or may not be in degraded state. Many studies have shown that extracellular DNA in soil is quickly degraded according to the biotic and abiotic parameters of soil, but a significant fraction of the molecules escapes from the enzymatic or chemical destruction and persist for a long time as a substance with high molecular weight (Paget et al., 1998; Frostega¨rd et al., 1999). Numerous experiments have demonstrated the mechanisms and importance of DNA adsorption onto soil components such as sand particles, clays minerals, or humic compounds (Greaves and Wilson, 1969; Lorenz and Wakernagel, 1987; Romanowski et al., 1991; Khanna and Stotzky, 1992; Blum et al., 1997; Poly et al., 2000; Demaneche et al., 2000). These results suggest that free DNA is rapidly degraded in the environment, but the adsorption of DNA onto soil components retards DNA degradation and constitutes a major mechanism of DNA molecule persistence in soil. However, a noticeable aspect of the majority of these studies is that the quality and quantity of DNA in plant materials before release into soil is not stated. Therefore, this study provides important knowledge. DNA extracted from leaf samples (PAL and ADM), dried and stored in dry conditions for 40 days at 22 °C does not present a smear on agarose gel 0.8%. The same result was observed for DNA extracted from fresh leaves and from leaves placed in the control microcosms. This indicates that DNA was not degraded in control microcosms. On the contrary, a smear was observed on all DNA samples extracted from leaves introduced into standard microcosms, indicating that DNA was degraded (Fig. 4).
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The plant decay process can affect DNA stability and persistence in leaves. It has recently been shown that the kinetics of DNA degradation in plant cells, submitted to decay processes could vary depending on various biotic and abiotic factors (Ceccherini et al., 2003). During plant decay, plant DNA can also be degraded by nucleases of microbial cells that degrade plant residues. Other microbial enzymes (cellulases, proteases, ligninases) involved in degradation of plant polymers are active during plant decay, and their activity might facilitate the access of microbial nucleases to plant DNA (Ceccherini et al., 2003). As long as the cells are not yet in decomposition, DNA in the dry matter can persist for a long time without being degraded. Thus, the DNA degradation rate present in decomposing plant cells may depend on the conditions for plant cell death and the prevailing environmental conditions. Moreover, the high degradability of DNA could also be linked to the sandy texture of the soil that does not favour soil organic matter stabilisation (Pote´ et al., 2003). The presence of smear on agarose gel does not mean that all DNA molecules are degraded in the dry matter undergoing decomposition, but it does mean that DNA molecules had been partially degraded. Soil microorganisms are responsible for many processes such as organic matter decomposition and release of organic macromolecular and nutrient (Ko¨gel-Knabner, 2002), and plant tissue decomposition and DNA release (Frostega¨rd et al., 1999; Widmer et al., 1996). The results presented in this paper show that the contact of plant material with soil leads to plant material degradation, including degradation of DNA. This degradation is perhaps predominantly due to the enzymatic activities of the soil micro-organisms. Important quantities of DNA molecules released by the decomposition of plant tissues in soil seem to be degraded. PCR reaction showed the presence of tomato gene in soil microcosms. Absence of signal in control microcosms (sterilised soil) indicate the absence of tomato DNA fragments in soil. This can be explained by the absence of soil microorganism enzymatic activity to decompose the plant tissues. This present study has discussed the kinetics of decomposition in dry matter and consequent DNA release in soil, important aspects for modelling DNA dispersion from plants into the environment. Further application and extension of this work could consider the persistence of DNA in transgenic plants leaves and the effect of microbial components during dry matter decomposition in different soils. Studies concerning the possible release into soil of exogenous extracellular DNA have, for the most part, concentrated on the detection and possible horizontal gene transfer (HGT) to soil bacteria (Paget et al., 1998; Gebhard and Smalla, 1999). It is important to consider what happens to plant DNA in dry matter decomposing process so as to formulate
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a conceptual approach model of the plant DNA (transgenic or non-transgenic) dispersion into the environment.
Acknowledgements This research was funded by financial support from F.-A. Forel Institute, University of Geneva, Switzerland. We would particularly like to thank Professor William Broughton of the Botanical Garden in Geneva for his precious help in providing access to his laboratory for various analyses. We would also like to thank Andreas Wigger from the Horticulture School of Lullier, Geneva, Switzerland for the supply of tomato samples and Dr Franck Bertolla, UMR CNRS Universite´ Claude Bernard Lyon 1 for tomato primers. Clive Prestt also kindly checked the English of this manuscript.
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Gebhard, F., Smalla, K., 1999. Monitoring field releases of transgenic modified sugar beets for persistence of transgenic plant DNA and horizontal gene transfer. FEMS Microbiol. Ecol. 28, 261–272. Greaves, M.P., Wilson, M.J., 1969. The adsorption of nucleic acids by montmorillonite. Soil Biol. Biochem. 1, 317–323. Guo, L.B., Sims, R.E.H., 2002. Eucalypt litter decomposition and nutrient release under a short rotation forest regime and effluent irrigation treatments in New Zealand. Soil Biol. Biochem. 34, 913–922. Howard, P.J.A., Howard, D.M., 1974. Microbial decomposition of tree and shrub leaf litter. OIKOS 25, 341–352. Jenny, H., Gell, S.P., Bingham, F.T., 1949. Comparative study of decomposition rates of organic matter in temperate and tropical regions. Soil Sci. 68, 419–432. Khanna, M., Stotzky, G., 1992. Transformation of Bacillus subtilis by DNA bound on montmorillonite and effect of DNase on the transforming ability of bound DNA. Appl. Environ. Microbiol. 58, 1930–1939. Ko¨gel-Knabner, I., 2002. The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter. Soil Biol. Biochem. 34, 139–162. Kumar, K., Goh, K.M., 2000. Crop residues and management practices: effects on soil quality, soil nitrogen dynamics, crop yield, and nitrogen recovery. Adv. Agronomy 68, 197– 319. Lorenz, M.G., Wakernagel, W., 1987. Adsorption of DNA to sand and variable degradation rates of adsorbed DNA. Appl. Environ. Microbiol. 53, 2948–2952. Monties, B., 1991. Plant cell walls as fibrous lignocellulosic composites: relations with lignin structure and function. Animal Feed Sci. Technol. 32, 159–175. Ogram, A., Sayler, G.S., Barkay, T., 1987. The extraction and purification of microbial DNA from sediments. J. Microbiol. Meth. 7, 57–66. Olson, J.S., 1963. Energy storage and the balance of producers and decomposers in ecological syst. Ecology 44, 322–331. Paget, E., Lebrun, M., Freyssinet, G., Simonet, P., 1998. The fate of recombinant plant DNA in soil. Eur. J. Soil Biol. 34, 81–84. Paget, E., Simonet, P., 1994. On the track of natural transformation in soil. FEMS Microbiol. Ecol. 15, 109–118. Paul, J.H., Jeffrey, W.H., DeFlaun, M.F., 1987. Dynamics of extracellular DNA in marine environment. Appl. Environ. Microbiol. 53, 170–179. Pillai, T.V.N., Ganguly, A.K., 1970. Nucleic acids in the dissolved constituents of seawater. Curr. Sci. 22, 501–504. Poly, F., Chenu, C., Simonet, P., Rouiller, J., Monrozier, L.J., 2000. Difference between linear chromosomal and supercoiled plasmid DNA in their mechanisms and extent of adsorption on clay minerals. Langmuir 16 (3), 1233–1238.
Pote´, J., Ceccherini, M.T., Tran Van, V., Rosselli, W., Wildi, W., Simonet, P., Vogel, T.M., 2003. Fate and transport of antibiotic resistance genes in saturated soil columns. Eur. J. Soil Biol. 39, 65–71. Reanney, D.C., Roberts, W.P., Kelly, W.J., 1982. Genetic interactions among microbial communities. In: Bull, A.T.S., Slater, J.H. (Eds.), Microbial Interactions and Communities. London, Academic press, pp. 287–322. Robe, P., Nalin, R., Capellano, C., Vogel, T.M., Simonet, P., 2003. Extraction of DNA from soil. Eur. J. Soil Biol. 39, 183–190. Romanowski, G., Lorenz, M.G., Wackernagel, W., 1991. Adsorption of plasmid DNA to mineral surfaces and protection against DnaseI. Appl. Environ. Microbiol. 57, 1062–1069. Romanowski, G., Lorenz, M.G., Wackernagel, W., 1993. Plasmid DNA in a groundwater aquifer microcosm-adsorption, Dnase resistance and natural genetic transformation of Bacillus subtilis. Mol. Ecol. 2, 171–181. Saggar, S., Parshotam, A., Hedley, C., Salt, G., 1999. 14Clabelled glucose turnover in New Zealand soils. Soil Biol. Biochem. 31, 2025–2037. Schimel, D.S., 1986. Carbon and nitrogen turnover in adjacent grassland and cropland ecosystems. Biogeochemistry 2, 345–357. Soltis, P.S., Soltis, D.E., Smiley, C.J., 1992. An rbcl sequence from a Miocene Taxodium (bald cypresse). Proc. Natl. Acad. Sci. USA 89, 449–451. Spring, S., Amann, R., Ludwig, W., Schleifer, K.H., Peter-Sen, N., 1992. Phylogenic diversity and identification of nonculturable magnetotactic bacteria. Syst. Appl. Microbiol. 15, 116–122. Steffan, R.J., Goksoyr, J., Bej, A.K., Atlas, R.M., 1988. Recovery of DNA from soils and sediments. Appl. Environ. Microbiol. 54, 2908–2915. Stemmer, M., Gerzabek, M.H., Kandeler, E., 1999. Inverstase and xylanase activity of bulk soil and particle-size fractions during maize straw decomposition. Soil Biol., 9–18. Stocco, P., 1994. Genie ge´ne´tique et environnement: principes fondamentaux et introduction a` la proble´matique. Georg Ed. SA, Gene`ve. 207 p. Torsvik, V.L., Gorsksoyr, 1978. Determination of bacteria DNA in soil. Soil. Biol. Biochem. 10, 7–12. Trevors, J.T., 1996. Nucleic acids in the environment. Current Opin. Biotech. 7, 331–336. Widmer, F., Seidler, R.J., Watrud, L.S., 1996. Sensitive of transgenic plant marker gene persistence in soil microcosms. Mol. Ecol. 5, 603–613. Wieder, R.K., Lang, G.E., 1982. A critique of the analytical methods used in examining decomposition data obtained from litter bags. Ecology 63, 1636–1642.
Kinetics of mass and DNA decomposition in tomato leaves John Pote´ b
a,*
, Patrick Rosse´ a, Walter Rosselli b, Van Tran Van c, Walter Wildi
a
a Institut F.-A. Forel, University of Geneva, 10 route de Suisse, 1290 Versoix, Switzerland WSL Swiss Federal Research Institute, Antenne romande, P.O. Box 96, 1015 Lausanne, Switzerland c Ecologie Microbienne UMR CNRS 5557 UFR of Biology, Universite´ Claude Bernard, Lyon I, 43 boulevard 11 novembre 1918, 69622 Villeurbanne, France
Received 1 October 2004; received in revised form 11 March 2005; accepted 14 March 2005 Available online 5 May 2005
Abstract This laboratory study investigated the kinetics of leaf and DNA content decomposition in two varieties of tomato (Palmiro and Admiro) after incubation in soil for 35 days. Results revealed that the decrease of dry matter in leaves in both varieties did not follow a single exponential function and was better described by a double exponential model. Composite half-decrease times were 3.4 and 2.4 days for Palmiro and Admiro respectively. The same pattern was observed for DNA mass loss, although this was closer to a single exponential model with composite half-decrease times of 1.5 and 1.4 days. Genomic analysis showed that DNA in dried leaves at room temperature (not inoculated in the soil), remains intact or presents a weak degradation, and DNA extracted from leaves inoculated in non-sterile soil showed degradation after two days. These results indicate that before release an important quantity of DNA may be degraded inside plant tissues during decomposition in soil. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Decomposition kinetics; Tomato leaves; Exponential model; DNA release
1. Introduction Soil is a heterogeneous environmental compartmentalised into physical, chemical and biological points of view. It contains a great diversity of micro-organisms and is endowed with both biological and physico-chemical reactivity. Many studies have been made of decomposition rates in different types of plant material, especially leaf litter (Olson, 1963; Howard and Howard, 1974). Decomposition of leaves in field conditions is mainly microbial
* Corresponding author. Tel.: +41 22 950 97 24; fax: +41 22 755 13 82. E-mail address: [email protected] (J. Pote´).
(Howard and Howard, 1974; Berg et al., 1984). Decomposition of plant materials in soil and nutrient release are significantly influenced by such factors as microbial biomass, litter source, amount of litter input, the relative proportion of the different plant tissues, as well as their chemical composition (Monties, 1991; Guo and Sims, 2002; Ko¨gel-Knabner, 2002). Soil factors such as texture (Schimel, 1986; Amato and Ladd, 1992; Saggar et al., 1999; Stemmer et al., 1999), temperature, and humidity are also critical. The Earth is virtually entirely covered in nucleic acid (Trevors, 1996). Nucleic acids (i.e. DNA and RNA) are ubiquitous in many environments, including fresh water, marine water column and sediment, soil, and terrestrial subsurface. They play a key role in the biogeochemical cycles of the biosphere and represent an
0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.03.030
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enormous reservoir of novel valuable molecules for health or industry (Pillai and Ganguly, 1970; DeFlaun et al., 1986; Paul et al., 1987; Trevors, 1996; Robe et al., 2003). Soil is a complex environment and appears to be a major reservoir of nucleic acids, which seem to be concentrated in upper soil horizons and represent up to 10% of the soil organic phosphate (Baker, 1977). Several studies discuss the presence of large quantities of extracellular DNA in soil (Torsvik and Gorsksoyr, 1978; Reanney et al., 1982; Ogram et al., 1987; Steffan et al., 1988; Spring et al., 1992; Blum et al., 1997; Paget et al., 1998). However, it has been estimated that, depending on soil composition, extracellular DNA concentrations from 5 to more than 35 lg g1 (dry soil) can be extracted from soil (Frostega¨rd et al., 1999). DNA from plants has been released into the environment naturally (Soltis et al., 1992; Paget et al., 1998). By studying transgenic plants, many recent studies have focused on what happens to transgenes in the environment. The majority of these studies have monitored the presence and stability of transgene in various systems using PCR amplification. Most of these studies were carried out on the presence of extracellular plant DNA in soil. This DNA (from transgenic or non-transgenic plants) is released during the process of plant tissue decomposition or from enzymatic degradation of cell structures by pathogens (Ceccherini et al., 2003). However, little information is available on the process of DNA release and neither is there much information to be found regarding quantitative and qualitative aspects of DNA in plant material decomposing in soil. Potential ecological effects caused by deliberate or accidental release of genetically engineered micro-organisms mean survival, multiplication and dispersal of the inoculated cells as well as transfer of recombinant DNA to the indigenous microflora need monitoring (Paget and Simonet, 1994). One of the main risks of transgenic plants is the spread of their transgenes through gene flow into wild populations causing genetic pollution and giving rise to potential ecosystem disruption. The following issues associated with genes from transgenic plants must be dealt with: (i) emission, dispersal, and deposition of transgenic pollen, (ii) introgression of the transgene into wild species, (iii) stabilisation and spread of the transgene in wild species, (iv) ecological effects of the transgene in the new host population (Evenhuis and Zadok, 1991). Description of DNA dispersion from engineered plants in the environment, in particular its kinetic aspects, requires the use of models capable of assessing the characteristics of the initial source, the release process, and the means of transport (Stocco, 1994). The transport of DNA in saturated soil has recently been studied by Pote´ et al. (2003). DNA, as the signature of life, has been extensively studied in a wide range of environments. While DNA analysis has become central to work on natural gene
exchange, forensic analyses, soil bioremediation, genetically modified organisms, exobiology, and palaeontology, fundamental questions concerning DNA release from diverse organisms, there remains resistance to degradation (quantity and quality) all remain. The objective of this study was to examine: (i) the decomposition kinetics of the tomato leaf (dry matter) in soil and (ii) qualitative and quantitative aspects of DNA in tomato leaves during decomposition in soil. This study is oriented towards a conceptual approach to modelling the plant DNA release process during dry matter decomposition in soil.
2. Materials and methods 2.1. Conceptual approach 2.1.1. Decomposition of dry matter The most frequently used model to describe decomposition is the single exponential decay function (Wieder and Lang, 1982), which was first proposed by Jenny et al. (1949) and is discussed in detail by Olson (1963). Other models (double exponential, asymptotic, linear, quadratic, power) can also be used to examine decomposition data (Wieder and Lang, 1982). Thus, one can assume that the dry weight loss of tomato leaves in soil can be modelled by using an exponential law (Jenny et al., 1949; Olson, 1963). M DRY ðtÞ ¼ M DRY0 ekDRY t
ð1Þ
In which MDRY(t): dry matter weight at time t, MDRY0: initial dry matter weight, kDRY: loss rate constant (decomposition rate). Eq. (1) indicates a first order decrease and appears in many other processes such as: degradation of pesticides on the ground—natural decrease of radioactive isotopes—sedimentation of the not coagulated solids— breathing death rate of bacteria and algae. For the purposes of the study presented in this paper, kDRY will be determined by fitting an exponential model to dry weight measurements of tomato leaves. Half-decomposition time of the dry matter is given by T DRY1=2 ¼
1 ln 2 k DRY
ð2Þ
2.1.2. DNA decrease in leaves As in the dry matter case, a first order relaxation model was used to describe the decrease of DNA in dry matter: M DNA ðtÞ ¼ M DNA0 ekDNA t
ð3Þ
In which MDNA(t): DNA weight at time t, MDNA0: initial DNA weight, kDNA: release rate constant.
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However, in this case DNA weight within the samples cannot be directly measurable for instrumental reasons linked to the DNA extraction method used. Therefore, DNA weight is determined from the measurement of the DNA mass concentration in leaves, CDNA(t), and the previous dry matter weight of samples MDRY(t), using the direct relation M DNA ðtÞ ¼ C DNA ðtÞ M DRY ðtÞ
ð4Þ
This equation (Eq. (4)), an exponential model fitting, allows the determination of both kDNA and the associated DNA half-decrease time given by T DNA1=2 ¼
1 ln 2 k DNA
ð5Þ
2.2. Plants Two varieties of tomato were used in this study. Firstly, Palmiro (PAL), which has a water content of 89.3 ± 0.3%, and a concentration of DNA in dry leaves samples of 117 ± 7 lg g1. Secondly, Admiro (ADM), with water content: 88.4 ± 1.4%, and concentration of DNA in dry leaves samples of 112 ± 6 lg g1. These tomatoes were cultivated in greenhouse on hydroponic medium. After being collected, leaves were rapidly dried at 35 °C for 3 days in order for any decomposition to be avoided. The leaves were re-hydrated in sterile water at 20 °C for 5 min, after which they were incorporated into soil microcosms. 2.3. Soil The surface soil (between 0 and 15 cm depth) used in this study was collected from a cropland field in the locality of Ecogia, near the city of Geneva, Switzerland. Some soil characteristics are given in Table 1. The soil was sieved through a 5 mm sieve (W.S. Styler company mentor, USA). To determine the aggregates size distribution of the soil microcosm, a soil aliquot was dried (at 35 °C for 24 h) and sieved through four successive sieves (2/1/0.5/0.125 mm). Each fraction was then weighed and reported to the total aliquot mass. Results
Table 1 Some soil characteristics
679
Table 2 Aggregates size in soil microcosms Size class (mm)
Mass (%)
<0.125 0.125–0.5 0.5–1.0 1.0–2.0 >2.0
1.2 18.7 35.4 40.1 4.5
are shown in Table 2. Sterile soil for control microcosms was dried in the oven at 150 °C overnight and autoclaved (121 °C, 50 min. Autoklav, Typ 23, MELAG, Germany) twice with a 24 h period of incubation between treatments and re-hydrated with sterile water (22 wt.%). 2.4. Microcosm description and handling PET plastic containers 21 cm in height and 8 cm by 8 cm in section were used for the microcosms. Each container was filled with a 15 cm layer of soil. Tomato leaves were placed in a non-overlapping manner between two supple polyester nets (1.4 mm mesh, thickness 130 lm) which in turn was placed on top of the soil. Leaves were then covered with a 3 cm layer of soil, this configuration mimics the burying of green manure in the field. The net was needed to make the removal of the sample easier. It was supple enough to maintain good contact between leaves and soil (Couˆteaux et al., 2002). Natural soil (without leaves) was also used for control microcosms. Microcosms were kept in a dark room at 21.5 °C. The soil humidity was stabilised (at 22 wt.%) by water addition based on container weighting. The experiment was conducted for a total of 35 days. Leaves were taken from microcosms after 2, 4, 10, 19 and 35 days, and at each time of these sampling points, three containers of each tomato variety (PAL & ADM) were sampled. Tomato leaves were removed from microcosms, dried for 3 days at 35 °C, and weighed. Care was taken to remove all pieces of leaves attached to the net. For pieces of leaves completely attached on the soil, leaves and soil were placed in water and separated by gravity. 2.5. DNA extraction from soil and tomato leaves
Soil type
Loamy sand
Sand (%) Silt (%) Clay (%) Organic matter (%) pH (H2O) Volumetric weight Water-holding capacity Bacteria biomass
78 14 8 4.3 6.5 1.32 g ml1 38.6% 108 CFU g1 dry soil
After the leaves had been removed, soil located 50 mm below the top of microcosms was taken. This was mixed and used for extracellular DNA extraction using buffer TENP (50 mM Tris, 20 mM EDTA, 100 mM NaCl, 1% [wt/vol] polyvinylpolypyrrolidone)—the protocol included no lysis treatment, as described by Frostega¨rd et al. (1999). The concentration of extracted DNA was measured spectrophotometrically (OD260) and aliquots were used for PCR amplification.
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DNA from tomato leaves was extracted using DNeasy plant DNA miniextraction kit (Qiagen, Helden Germany) and following the manufacturerÕs recommendations. The concentration of extracted DNA was measured spectrophotometrically (OD260) and aliquots were used to estimate DNA quality using electrophoresis on 0.8% agarose gel stained with ethidium bromide (10 lg ml1, Sigma, USA). All DNA extracted was passed through a purification Microspin S-400 (Pharmacia Biotech) to remove trace humic acids.
Normalised leaves dry weight (-)
680
1.0 0.8 0.6 0.4 0.2 0.0 0
2.6. PCR amplification The tomato DNA sequences in extracted soil DNA were amplified using PCR amplification based on 18S primers, F474 (5 0 -ATGAAAGACGAACAACTG-3 0 ) and F475 (5 0 -TACCTGGTTGATCCTGCC-3 0 ), which were complementary to part of tomato 18S gene. PCR was carried out in 50 ll reaction volume containing 1X PCR buffer (Invitrogen), 1.5 mM MgCl2, 200 lm of each dNTP, 200 nM of each primer, 0.5 units Taq DNA polymerase (Invitrogen), and a template DNA (100 ng). PCR was performed in a Biometra thermocycler (BIOLABO) in the following conditions: an initial denaturation at 94 °C for 5 min; 35 cycles of denaturation (94 °C for 45 s), annealing (55 °C for 45 s), extension (72 °C for 45 s); and a final extension for 5 min at 72 °C. PCR products were checked by electrophoresis on 0.8% agarose gel stained with ethidium bromide (10 lg ml1, Sigma, USA).
Palmiro Admiro Palmiro fitting Admiro fitting
10
20
30
Time (day) Fig. 1. Decrease of leaves dry matter (normalised by the initial value).
A single exponential model appeared to be inappropriate to describe the fast weight loss observed at the early stage. Consequently, a two-compartment (double exponential), first order model was chosen to fit the results (Couˆteaux et al., 2002). M DRY ðtÞ ¼ Aekat þ Bekbt In which MDNA(t): DNA weight at time t (in days); A and B: initial dry matter proportions of the fast and the slow decomposing compartments respectively (MDRY0 = A + B); ka and kb: decay rate constants for A and B fractions respectively. Each fraction decays exponentially. The models fitted are ðtÞ ¼ 0.361e1.85t þ 0.497e0.0436t M DRY
3. Results 3.1. Decomposition of leaves All the data were normalised before comparison (i.e. relative to their initial value). An initial and instantaneous loss of 15% occurred during the leaf re-hydration process preceding incorporation into the soil. This loss of hydrosoluble substances may imitate the rain leaching process of the dried tomato plant occurring in the field. Consequently, the posthydration dry weight is hereafter considered as being the initial value. No significant differences in leaf decomposition were found between the two varieties of tomato leaves (Fig. 1). The maximum difference between their normalised weights is 7% during the first 10 days. At this time, PAL shows a dry weight 49% and 22% smaller than ADM after 19 and 35 days, respectively. The decomposition was faster in the first few days of experimentation (decrease reaching 46% and 47% respectively for PAL and ADM after two days in soil). After this, more resistant parts of leaves seem to slow the decomposition down.
ðR2 ¼ 0.998Þ for Palmiro ðtÞ ¼ 0.423e1.09t þ 0.431e0.0339t M DRY
ðR2 ¼ 0.999Þ for Admiro The Palmiro and Admiro values of R2 fitted by single exponential function were 0.823 and 0.693 respectively. 3.2. DNA mass concentration The DNA mass concentration is not constant in the dry matter of leaves showing decrease through time (Fig. 2). In the first two days of experimentation, the DNA concentration for both varieties did not appreciably vary. In the meantime, the dry matter lost about half of its weight (see Section 4.1.). After the second day, the DNA concentration starts to decrease more or less linearly in parallel with the dry weight until the 19th day. 3.3. Amount of DNA For similar reasons as in the dry matter case, a double exponential model was chosen to fit DNA data (Fig. 3).
DNA concentration in leaves (µg/g)
J. Pote´ et al. / Chemosphere 61 (2005) 677–684 140 120
Initial time
100
PAL : 117 ± 7 (µg/g) ADM : 112 ± 6 (µg/g)
2ndday
4thday
80 60 10thday
40 20 0 1.0
Palmiro Admiro 0.8
35thday
19th
0.6
0.4
0.2
0.0
Normalised leaves dry weight (-) Fig. 2. DNA concentration pattern, CDNA(t) as a function of the normalised dry weight loss.
Normalised DNA amount in leaves (-)
681
1.0
Palmiro Admiro Palmiro fitting Admiro fitting
0.8 0.6 0.4 0.2 0.0 0
10
20
30
Time (day) Fig. 3. Decrease of DNA weight in leaves dry matter (normalised by the initial value).
The models fitted are M DNA ðtÞ ¼ 0.544e0.992t þ 0.456e0.133t ðR2 ¼ 0.999Þ for Palmiro ðtÞ ¼ 0.591e0.935t þ 0.409e0.131t M DNA
Fig. 4. Gel signature of DNA extracted from tomato leaves at different time to estimate potential of DNA degradation. Lane 1: Molecular Weight Standard (smart Ladder). Lane 2: PAL DNA from fresh matter. Lane 3: PAL DNA from dry matter no introduced in soil. Lane 4: ADM DNA from fresh matter. Lane 5: ADM DNA from dry matter no introduced in soil. Lane 6: PAL DNA, 2 days in soil. Lane 7: ADM DNA, 2 days in soil. Lane 8: PAL DNA, 35 days in soil. Lane 9: ADM DNA, 35 days in soil. Lane 10: PAL DNA, control microcosms, 35 days in soil. Lane 11: ADM DNA, control microcosms, 35 days in soil.
degraded after two days. It can be seen that DNA extracted from leaf samples (PAL and ADM), dried and stored in dry conditions for 40 days at 22 °C (room temperature) does not present a smear, indicating that no degradation has occurred. The amounts of extracellular DNA estimated by OD260 ranged from 24 to 31 lg g1 dry soil. No difference was observed between amounts of extracellular DNA extracted from both natural soil and from soil microcosms. The expected 935 pb long 18S DNA fragment was detected in all the DNA extracted from soil microcosms. No signal was observed for DNA extracted from natural soil and sterilised control microcosms.
4. Discussion 4.1. Leaf decomposition
2
ðR ¼ 0.999Þ for Admiro The Palmiro and Admiro values of R2 fitted by single exponential function were 0.978 and 0.980 respectively. 3.4. Quality of DNA in leaves, amount of extracellular DNA, and PCR amplification DNA degradation in the tomato leaves was qualitatively estimated by DNA size distribution on the agarose gel by comparing it with DNA standard (Romanowski et al., 1993; Widmer et al., 1996; Ceccherini et al., 2003; Pote´ et al., 2003). The analysis of agarose gel electrophoresis (Fig. 4) indicates that DNA extracted from leaves inoculated in non-sterile soil was significantly
Tomato leaf dry matter from control microcosms (sterile soil) showed only limited weight loss (Fig. 5). The dry weight loss in control microcosms after 35 days reached 33.8% and 29.4% for PAL and ADM respectively. These values must be compared with the 87.9 and 85.2% weight loss obtained with the non-sterile soil. This limited loss may be explained by microbial activity in leaves which were not sterilised (Ceccherini et al., 2003). However, this activity did not affect the DNA quality in leaves, as shown in Fig. 4. The exponential model with one single rate constant does not fit dry weight measurements well. The fitting may be improved by using a two-compartment first order model (i.e. two decomposition processes in parallel with two distinct
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researcher to understand what happens to transgenic plant DNA released into the environment (Stocco, 1994). 4.2. DNA release from leaf dry matter
Fig. 5. Quality of leaves from soil microcosms (PAL) after 35 days.
kinetics) (Couˆteaux et al., 2002). The half-decomposition time of Palmiro leaves is 0.375 ± 0.280 days for the fast fraction (42% of initial dry weight) and 15.9 ± 1.5 days for the slow fraction (58% of initial dry weight). The composite half-decomposition time is 3.40 days (numerical solution). For Admiro leaves, the halfdecomposition time is 0.638 ± 0.156 days for the fast fraction (50% of initial dry weight) and 20.5 ± 2.7 days for the slow fraction (50% of initial dry weight). The composite half-decomposition time is 2.43 days. The double exponential decay model assumes that dry matter can be partitioned into two components: firstly, a fraction relatively easily decomposed, and secondly one which is recalcitrant to decomposition (Wieder and Lang, 1982). The biodegradation of the fractions (such as simple sugars, amino acids, polyphenols, hemicellulose, cellulose and lignin) in plant residues occurs simultaneously but with different kinetics (Kumar and Goh, 2000). The amount of DNA released by decomposition of plant materials in soil can be estimated by the amount of plant material present. The rates of loss from all reservoirs (e.g. plant materials) can be expressed conveniently by parameter k (the decomposition rate), which equals the fraction of the stored quantity that is lost (short) unit time, but without implying whether the fractions are approximately constant or not (Olson, 1963; Wieder and Lang, 1982; Berg et al., 1984; Couˆteaux et al., 2002). The data concerning leaf decomposition largely contributes to predicting (modelling) the amount of tomato extracellular DNA released through time while tomato leaves are decomposing in soil. Many studies have been performed on the persistence of plant DNA in soil. However, little information is available on the release of DNA from plant materials. Modelling DNA release from plant materials may help the field
The half-decrease time of Palmiro DNA is 0.698 ± 0.070 days for the fast fraction (54% of initial DNA amount) and 5.20 ± 0.31 days for the slow fraction (46% of initial DNA amount). The composite halfdecrease time is 1.48 days (numerical solution). For Admiro DNA, the half-decrease time is 0.742 ± 0.046 days for the fast fraction (59% of initial DNA amount) and 5.31 ± 0.25 days for the slow fraction (41% of initial DNA amount). The composite half-decrease time is 1.38 days. However, the DNA amount is inferred from the dry weight of leaves. The decreases in amount of DNA agree better with the single exponential model rather than with the dry weight (i.e. the fast and slow parameters are closer). This fact suggests that the DNA and dry matter decrease processes are not closely coupled at the beginning of the experiment. The DNA released from dry matter in decomposition may or may not be in degraded state. Many studies have shown that extracellular DNA in soil is quickly degraded according to the biotic and abiotic parameters of soil, but a significant fraction of the molecules escapes from the enzymatic or chemical destruction and persist for a long time as a substance with high molecular weight (Paget et al., 1998; Frostega¨rd et al., 1999). Numerous experiments have demonstrated the mechanisms and importance of DNA adsorption onto soil components such as sand particles, clays minerals, or humic compounds (Greaves and Wilson, 1969; Lorenz and Wakernagel, 1987; Romanowski et al., 1991; Khanna and Stotzky, 1992; Blum et al., 1997; Poly et al., 2000; Demaneche et al., 2000). These results suggest that free DNA is rapidly degraded in the environment, but the adsorption of DNA onto soil components retards DNA degradation and constitutes a major mechanism of DNA molecule persistence in soil. However, a noticeable aspect of the majority of these studies is that the quality and quantity of DNA in plant materials before release into soil is not stated. Therefore, this study provides important knowledge. DNA extracted from leaf samples (PAL and ADM), dried and stored in dry conditions for 40 days at 22 °C does not present a smear on agarose gel 0.8%. The same result was observed for DNA extracted from fresh leaves and from leaves placed in the control microcosms. This indicates that DNA was not degraded in control microcosms. On the contrary, a smear was observed on all DNA samples extracted from leaves introduced into standard microcosms, indicating that DNA was degraded (Fig. 4).
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The plant decay process can affect DNA stability and persistence in leaves. It has recently been shown that the kinetics of DNA degradation in plant cells, submitted to decay processes could vary depending on various biotic and abiotic factors (Ceccherini et al., 2003). During plant decay, plant DNA can also be degraded by nucleases of microbial cells that degrade plant residues. Other microbial enzymes (cellulases, proteases, ligninases) involved in degradation of plant polymers are active during plant decay, and their activity might facilitate the access of microbial nucleases to plant DNA (Ceccherini et al., 2003). As long as the cells are not yet in decomposition, DNA in the dry matter can persist for a long time without being degraded. Thus, the DNA degradation rate present in decomposing plant cells may depend on the conditions for plant cell death and the prevailing environmental conditions. Moreover, the high degradability of DNA could also be linked to the sandy texture of the soil that does not favour soil organic matter stabilisation (Pote´ et al., 2003). The presence of smear on agarose gel does not mean that all DNA molecules are degraded in the dry matter undergoing decomposition, but it does mean that DNA molecules had been partially degraded. Soil microorganisms are responsible for many processes such as organic matter decomposition and release of organic macromolecular and nutrient (Ko¨gel-Knabner, 2002), and plant tissue decomposition and DNA release (Frostega¨rd et al., 1999; Widmer et al., 1996). The results presented in this paper show that the contact of plant material with soil leads to plant material degradation, including degradation of DNA. This degradation is perhaps predominantly due to the enzymatic activities of the soil micro-organisms. Important quantities of DNA molecules released by the decomposition of plant tissues in soil seem to be degraded. PCR reaction showed the presence of tomato gene in soil microcosms. Absence of signal in control microcosms (sterilised soil) indicate the absence of tomato DNA fragments in soil. This can be explained by the absence of soil microorganism enzymatic activity to decompose the plant tissues. This present study has discussed the kinetics of decomposition in dry matter and consequent DNA release in soil, important aspects for modelling DNA dispersion from plants into the environment. Further application and extension of this work could consider the persistence of DNA in transgenic plants leaves and the effect of microbial components during dry matter decomposition in different soils. Studies concerning the possible release into soil of exogenous extracellular DNA have, for the most part, concentrated on the detection and possible horizontal gene transfer (HGT) to soil bacteria (Paget et al., 1998; Gebhard and Smalla, 1999). It is important to consider what happens to plant DNA in dry matter decomposing process so as to formulate
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a conceptual approach model of the plant DNA (transgenic or non-transgenic) dispersion into the environment.
Acknowledgements This research was funded by financial support from F.-A. Forel Institute, University of Geneva, Switzerland. We would particularly like to thank Professor William Broughton of the Botanical Garden in Geneva for his precious help in providing access to his laboratory for various analyses. We would also like to thank Andreas Wigger from the Horticulture School of Lullier, Geneva, Switzerland for the supply of tomato samples and Dr Franck Bertolla, UMR CNRS Universite´ Claude Bernard Lyon 1 for tomato primers. Clive Prestt also kindly checked the English of this manuscript.
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