Proteome analysis of greenhouse-cultured lettuce with the natural soil mineral conditioner illite

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Soil Biology & Biochemistry 40 (2008) 1370–1378 www.elsevier.com/locate/soilbio

Proteome analysis of greenhouse-cultured lettuce with the natural soil mineral conditioner illite Jong-Soon Choia, Seong-Woo Chob, Tae-Seon Kimb, Kun Choa, Seok-Soon Hanc, Hong-Ki Kimc, Sun-Hee Woob, Keun-Yook Chungc, a Proteomics Team, Korea Basic Science Institute, Daejeon 305-333, Republic of Korea Department of Crop Science, Chungbuk National University, Cheongju 361-763, Republic of Korea c Department of Agricultural Chemistry, Chungbuk National University, Cheongju 361-763, Republic of Korea b

Received 16 November 2006; received in revised form 24 April 2007; accepted 27 April 2007 Available online 14 June 2007

Abstract The natural soil mineral conditioner illite effectively improved the germination and growth of lettuce when applied in particulate or powdered form. When illite was given in particulate and powdered forms, the germination rate of lettuce seeds improved remarkably up to 93% and 133%, respectively. Contrary to the developmental effects, the growth rate of lettuce treated with particulate illite improved slightly by 23%; powdered illite had no significant effects on lettuce growth rate. Thus, illite primarily affects seed germination rather than the growth of lettuce. To examine illite-induced proteins related to lettuce growth, differentially expressed proteins in lettuce leaves were analyzed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS) followed by Mascot search. From the proteomic analysis, five down-regulated proteins were identified related to storage protein, carbon metabolism and energy conversion. Three up-regulated proteins were related to energy production/conversion and carbon fixation. These results demonstrate that illite treatment as a soil conditioner helps lettuce seed germination and lettuce growth by regulating carbon metabolic flux. r 2007 Published by Elsevier Ltd. Keywords: Soil conditioner; Illite; 2-Dimensional gel electrophoresis; Matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry

1. Introduction Many soil conditioners such as polyvinyl polymer, minerals (i.e., bentonite, zeolite, perlite, and vermiculite), humus chemically derived from peat, wood coal, and compost derived from manure and straw can be used to improve soil quality (Brady, 1990). When maize was cultured with co-composted bentonite, the biomass productivity increased significantly (Soda et al., 2006). In addition, the natural mineral illite has been used as a soil conditioner to improve soil quality (Cho et al., 2005). The potassiumbearing illite positively enhanced the growth of Bacillus sp., by which the illite in turn helped to increase potassium Corresponding author. Tel.: +82 43 261 3383; fax: +82 43 271 5921.

E-mail address: [email protected] (K.-Y. Chung). 0038-0717/$ - see front matter r 2007 Published by Elsevier Ltd. doi:10.1016/j.soilbio.2007.04.032

uptake by wheat (Sheng and He, 2006). Using illite in particulate form has been reported to conserve soil nutrients against leaching and volatilization, resulting in better fertile status of the soil (Tisdale et al., 1985). Illite also prevented plant growth retardation in calcium ion-deficient soil (Tisdale et al., 1985). In particular, when its powdered form is properly used for foliar application and through soil water, the beneficial effects are proposed to enhance water holding capacity and nutrient conservation in soil. Furthermore, it can improve resistance to damage by disease, pests and continuous cropping. Agriculturally, illite enhances crop productivity by preventing the growth of unnecessary leaf and root and by degrading pesticide residues, resulting in fresher quality plants (Cho et al., 2005). Potassium deficiency for long periods in corn allows new leaves to be continuously produced, but the old leaves die (Nason and

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2.3. Protein extraction

McElroy, 1963), as potassium ions translocate from older to younger tissue when deficiency develops. Little information is known about the effects of illite as a soil conditioner on the growth of other crops, vegetables and fruits. In the present study, we evaluated and compared the effects of illite on the development and growth of greenhouse-cultured lettuce. Recently, proteomics is becoming a powerful tool to investigate genomewide protein expression involved in the specific function and metabolism of plants (Pandey and Mann, 2000). Therefore, we further analyzed the expressed protein profiles using 2-DE and MALDI-TOF/TOF MS in order to discover proteins responsive to illite that affect the growth of lettuce.

Proteins were extracted from 2-week-old seedling lettuce leaves using a previously described protocol with modifications (Salekdeh et al., 2002). Five grams of lettuce leaves were flash frozen with liquid nitrogen, ground into powder in a mortar and homogenized in extraction buffer containing 0.3% SDS, 50 mM Tris–HCl, pH 8.0, and 200 mM DTT. The resulting mixture was incubated with sample buffer (DNase, RNaseA, 50 mM Tris–HCl pH 8.0, MgCl2) for 10 min on ice. Flours were then transferred to Falcon tubes to remove insoluble particles by centrifugation at 15,000g for 15 min at 4 1C. The supernatants containing soluble proteins were transferred to new Eppendorf tubes and 10% final concentration of cold trichloroacetic acid and acetone was subsequently added to precipitate the proteins on ice for 1 h. The non-proteinous portion was discarded after centrifugation at 15,000g for 15 min at 4 1C. The pellet was washed three times with acetone and lyophilized prior to 2-DE analysis.

2. Materials and methods 2.1. Plant culture and illite treatment Red wrinkled lettuce produced by HeungNong Company in Korea was used in this study. The pots used were 25 cm in height and 25 cm in diameter; a total of 36 pots were used in the experiment. The sieved soils were added and packed in the pots and then sprayed with the proper amount of water. Lettuce seeds purchased from HeungNong Company were immersed in water for 24 h in the dark prior to sowing. Two or three seed kernels were directly sowed in pots with or without illite in a greenhouse with a 16 h photoperiod. After germination, the expanded leaves of the most actively growing 2-week-old lettuce plants were used for proteomic analysis. The percent composition of compounds (weight to weight) in illite is presented in Table 1. The analysis of components was performed by ICP-AES, X-ray diffraction (XRD), and XRF provided by the Korea Institute of Geoscience and Mineral Resources (KIGAM). Standard 1  concentration of the particulate and powdered forms of illite was defined as 3 kg and 500 g per 2.4793  102 ha, corresponding to 75 pyong (Korean floorage unit), respectively. The final concentration of illite applied is shown in Table 2.

2.4. Two-dimensional gel electrophoresis Soluble proteins of lettuce leaves were resolved on 2-D gels as described previously (O‘Farrell, 1975). The isoelectric-focusing (IEF) gel solution (per 1 l) contained 48.6 g urea, 2% (v/v) ampholytes (pH 3.5–10), 11.8 ml acrylamide/bis-acrylamide solution (29.2:0.8% (w/w), acrylamide:N,N0 -bis-methyleneacrylamide), 20.3 ml of 10% (v/v) Triton X-100, 4.5 ml of Bio-Lyte 5/7, 0.5 ml of BioLyte 3/10 and 28.8 ml of distilled water. After degassing 1 ml of IEF gel solution, 1 ml of N,N,N0 ,N0 -tetramethylethylenediamine (TEMED) and 1.3 ml of freshly prepared ammonium persulfate (10%, w/v) were added to the IEF solution. Then, the gel solution was loaded into the gel tube (3.5 mm  13–33.5 cm). The 5 ml sample was added to 1 ml of IEF solution containing 0.1 ml of 10% (w/v) SDS, 0.02 ml of Bio-Lyte 3/10, 0.1 ml of 2-mercaptoethanol, and 0.2 ml of Triton X-100. The upper and lower reservoirs Table 2 Field concentration of illite applied in particulate and powdered forms

2.2. Measurement of lettuce development and growth after illite treatment The number of germinated seeds and the plant’s length from the top shoot to the tip of root after 2 weeks of growth were used as parameters of plant development and growth, respectively.

Ratio of illite to soil

Illite particle (kg/ha)

Illite powder (kg/ha)

0 0.5 1.0 1.5 2.0

0.00 0.605  102 1.210  102 1.815  102 2.420  102

0.00 1.008  101 2.017  101 3.025  101 4.033  101

Table 1 Major components of illite in this study SiO2

Al2O3

Fe2O3

CaO

MgO

K2O

Na2O

TiO2

MnO

P2O5

Igloss

63.82

17.16

4.26

2.16

1.11

5.27

0.66

0.58

0.06

0.08

4.66

Relative quantity of each compound is expressed as percentage composition (w/w).

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were filled with 0.1 N NaOH and 0.06% phosphoric acid, respectively. After the sample solutions were loaded, the gels were run at 400, 600 and 800 V for 30 min each and then at 1200 V for 12–14 h at 9 1C. Gels were subsequently removed from the gel tubes and equilibrated in 3 ml of reducing SDS buffer [62.5 mM Tris–HCl, pH 6.8, 10% (v/v) of glycerol, 5% (v/v) of 2-mercaptoethanol, 2% (w/v) of SDS, and 0.0125% (w/v) of bromophenol blue] by mild shaking for 30 min at room temperature. The second dimension of gel electrophoresis was carried out using a 15% acrylamide separation gel and a 5% acrylamide stacking gel as previously described (Laemmli, 1970). The post-electrophoretic gels were stained with silver nitrate using the Pharmacia Biotech staining kit as reported previously (Yan et al, 2000). 2.5. Gel image analysis After silver staining, the gels were scanned using flatbed scanner and analyzed using Progenesis workstation version 2005 (Non-linear Dynamics, Newcastle-upon-Tyne, UK). By the serial comparison of illite-treated and control group gels, the protein spots were normalized by background subtraction, gel matching, and warping. Subsequently, the intensity of detected spots were calculated from the spot volume and evaluated by statistical analysis tools. The numerical spot data obtained from the independent triplicate experiments in each group were statistically assessed using the Student’s t-test.

standard peptide mixture of des-Arg bradykinin, angiotensin I, Glu-fibrinopeptide B, adrenocorticotropic hormone (ACTH) clip 1-17, ACTH clip 18-39, and ACTH clip 7-38. Internal calibration was also performed using two autolysis peaks of trypsin ([M+H]+ ¼ 842.5099 and 2211.1046). Since the lettuce genomic database is not currently available, the peptide mass fingerprinting search based on MS data from MALDI-TOF MS can not provide sufficient information for proteins with low coverage in the protein sequence (Kim et al., 2003). Thus, the internal sequences of proteins-of-interest were determined using MS/MS fragmentation analysis of selected tryptic peptide molecular ions ([M+H]+) to identify proteins by homology searches against the non-redundant BLAST database. As an example, the whole procedure of peptide fragmentation by MS/MS and the Mascot database search for the protein identification of ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) is schematized in Fig. 1. All MALDI plates containing sample peptides were irradiated with UV light (355 nm) from an Nd:YAG laser with a repetition rate of 200 Hz; 1000 and 3000 laser shots were averaged for normal mass spectra and MS/MS spectra, respectively. The samples were analyzed at a source acceleration voltage of 25 kV with two-stage reflection in MS mode. In the MS/MS experiment, the collision energy that was defined by the potential difference between the source acceleration voltage (8 kV) and the floating collision cell (7 kV) was constant at 1 kV. 2.8. Protein identification by MASCOT database search

2.6. In-gel digestion of protein spots Spots with increased or decreased intensity with more than 30% difference were manually excised with clean tips. The gel spots were cut into fine slices with a razor blade, then transferred to microcentrifuge tubes, and subjected to in-gel trypsin digestion as described with minor modification (Shevchenko et al., 1996). After dehydration with acetonitrile, the gel pieces were dried under vacuum in a Thermo Savant SpeedVac Plus (Savant, Holbrook, NY). Samples were digested with sequencing-grade trypsin (enzyme to substrate ratio (w/w), 41:20) (Promega, Madison, WI) at 37 1C, overnight. After tryptic digestion, the resulting tryptic peptides were dissolved in 0.5% (v/v) trifluoroacetic acid in 50% (v/v) acetonitrile, and then desalted using ZipTip C18 pipette tip (Millipore, Bedford, MA). The peptides were directly eluted onto MALDI plates by a-cyano-4-hydroxy-cinnamic acid (CHCA) matrix solution (10 mg/ml CHCA in 0.5% TFA/50% acetonitrile, 1:1, v/v).

The proteins were identified by searching NCBI nonredundant database using the MASCOT program (http:// www.matrixscience.com, Matrixscience, London, UK). The search parameters allowed for modifications of N-terminal Gln to pyroGlu, oxidation of methionine, acetylation of protein N-terminus, carbamido-methylation of cysteine, and acrylamide-modified cysteine. For MS/MS searches, the fragmentation of a selected peptide molecular ion peak was used to identify the protein by matching the observed peptide sequences with a probability of less than 5%. Thus, MS/MS spectra with a MASCOT score higher than the significant score (Po0.05) were assumed to be correct. When more than one peptide sequence was assigned to a spectrum with a significant score, the spectra were manually examined. Cases where the top-scoring peptide sequences had equal scores were discarded. 3. Results 3.1. The effects of illite treatment on plant development

2.7. MALDI-TOF-MS and MALDI-TOF-TOF MS analysis All mass spectra were acquired at a reflection mode by a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA). External calibration was performed with a

In order to examine the effects of illite treatment on development in lettuce seeds, we compared germination rates in the presence and absence of illite. Fig. 2 shows the number of germinated lettuce seeds when various concentrations of particulate illite were applied to pots in the

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pI

Protein proteolyze exract peptides

spot picking

MW

Peptides

2D Gel Separation

Mass Analysis

MS/MS

100 90 80 70 60 50 40 30 20 10 0 69.0

100 90 y10 80 y6 70 60 b5 b8 50 y8 b12 40 b10 b7 30 20 y4 y5 y11 b9 b11 y7 10 y12 0 828.0 660.2 955.8 1251.4 1547.0 Mass (m/z)

1464.74 m/z

y9

y1 b4 b3 y2 y3

364.6

% Intensity

% Intensity

Select Parent Ion

Protein Identification

1346.8

1865.6 2384.4 2903.2 Mass (m/z)

3422.0

Database Search

Ribulose-1,5-bisphophate carboxylase oxygenase large subunit (Monochoriavaginalis) GFKAGVKDYK EAGAAVAAES GEDSQYIAYV ALRLEDLRIP CTIKPKLGLS MRWRDRFLFC KRAQFARELG HIHRAMHAVI VGKLEGEREM PGVIPVASGG GNAPGAVANR SPELAAA

LTYYTPEYET STGTWTTVWT AYPLDLFEEG PAYSKTFQGP AKNYGRAVYE AEAIYKAQAE APIVMHDYLT DRQKNHGMHF TLGFVDLLRD IHVWHMPALT VALEACVQAR

KDTDILAAFR DGLTSLDRYK SVTNMFTSIV PHGIQVERXR CLRGGLDFTK TGEIKGHYLN GGFTANTSLA RVLAKALRMS DFIEKDRSRG EIFGDDSVLQ NEGRDLAREG

VTPQPGVPPE GRCYHIEAVP GNVFGFKALR LNKYGRPLLG DDENVNSQPF ATAGTCEEMM HYCRDNGLLL GGDHIHSGTV IFFTQDWVSM FGGGTLGHPW VEIIREASKW

Fig. 1. Flow chart of MS-based proteomic analysis. The protein spot to be analyzed was cut out of the gel and trypsinized (A). The tryptic digests were ionized on MALDI plate and scanned by MS. One parent peak (m/z 1464.74) was allowed to further fragment to obtain MS/MS spectra (B). Fragmented sequence TFQGPPHGIGQVER showing complete b and y ions series was identified as ribulose 1,5-bisphosphate carboxylase oxygenase large subunit with statistical significance (Po0.05) in Mascot search. The corresponding sequences were highlighted on the queue of amino acid sequences of the identified protein.

greenhouse. The number of germinated seeds in soil treated with illite was higher than that in untreated soil. In accordance with the increasing concentration of illite in powdered form, the germination rates concomitantly increased with statistical significance at Po0.05. When illite was applied in particulate form, 1  - and 2  -concentrated particulate illite increased the seed germination rate by 64% and 93%, respectively. When illite was applied in powdered form, 1  - and 2  -concentrated powdered illite increased

the seed germination rate remarkably by 75% and 133%, respectively. Thus, the powdered form of illite acted as a more effective soil conditioner than the particulate form. 3.2. The effects of illite treatment on plant growth In order to examine the effects of illite on the growth rate of lettuce, the vertical length of the lettuce was measured from the top shoot to the tip of root of lettuce grown in

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40

8.0

35

7.0 Whole lenght of lettuce

30 25 20 15 10 5 0

Number of germinated lettuce seeds

*

6.0 5.0 4.0 3.0 2.0 1.0

0

0.5 1.0 1.5 Concentration of particulate illite applied (x)

2.0 0.0 0

0.5 1.0 1.5 2.0 Concentration of particulate illite applied (X)

0

0.5 1.0 1.5 Concentration of powdered illite applied (X)

40 *

35

* 8.0

30

7.0

25 20 15 10 5 0 0

0.5 1.0 1.5 Concentration of powdered illite applied (x)

2.0

Fig. 2. The effects of illite treatment on seed germination of lettuce. The numbers of germinated lettuce seeds in the pots were counted according to the concentrations of particulate (A) and powdered (B) illite applied. Data are expressed as average7standard deviation from three independent experiments. The asterisk * represents statistical significance at Po0.05 by Student’s t-test.

either the presence or absence of illite after 2 weeks of growth. This 2-week period constitutes the most dynamic changes in lettuce growth after seed germination. As shown in Fig. 3, the growth of greenhouse-cultured lettuce plants in the presence of illite was compared to those grown in the absence of illite. With increasing concentrations of illite in either particulate or powdered form, the whole length of lettuce was exclusively enhanced by the particulate form. Treatment with 1  - and 2  -concentrated particulate illite slightly increased the length of the stem by 8% and 23%, respectively. In particular, 2  -particulate illiteinduced growth of the whole length of lettuce with statistical significance at Po0.05. When powdered illite was used, 1  -concentrated illite had no effect and 2  -concentrated illite increased the stem length by 11%; however, this was not statistically significant. 3.3. Differential proteome profiles by illite treatment To better understand the physiological role of illite on plant growth, we used a proteomics approach to compare the proteome profiles in the presence and absence of illite

Whole lenght of lettuce

Number of germinated lettuce seeds

1374

6.0 5.0 4.0 3.0 2.0 1.0 0.0 2.0

Fig. 3. The effects of illite treatment on the vertical growth of lettuce. The whole length of lettuce grown in pots was measured 2 weeks after seed germination according to the concentrations of particulate (A) and powdered (B) illite applied. Data are expressed as average 7 standard deviation from five to six independent experiments. The asterisk * represents statistical significance at Po0.05 by Student’s t-test.

as a soil conditioner. In order to study changes in protein patterns in lettuce caused by illite treatment, matured lettuce leaves were chosen as the source for proteomic study since growing lettuce leaves undergo photosynthesis and distribute the carbon source dynamically to sink organs such as the stem and root. The proteome profile of 2-week-old lettuce leaves treated with illite was compared to the untreated control. We found that the majority of proteins were distributed in the acidic region of pH 3.5–5 (Fig. 4A and B). We compared patterns of soluble proteins extracted from lettuce leaves of control, 1  - and 2  -illitetreated groups in either particulate or powdered form. Approximately 150–200 proteins were obviously visible by silver staining of 2D gels. Based on proteomic analysis, five proteins were down-regulated by illite and three proteins were up-regulated (Fig. 4C, Table 3). The proteins downregulated by illite treatment were shown as glycinin subunit G3 (1.5 fold, Po0.05), enolase (1.9 fold, Po0.05), hypothetical protein (4.6 fold), fructose-bisphosphate

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pH

pH

3.5 97.0 66.0

10 1

pH 10

3.5

10

2

3

45.0

3.5

1375

4

30.0

5

6

7

8 20.1 14.4

Control

Particle 1x

pH

pH

3.5 97.0 66.0

10 1

3.5

pH 10

3.5

10

2

3

45.0

Particle 2x

4

30.0

5

6

7

8 20.1 14.4

Control

Control

1 3

2 4

Powder 1x

6

5

Powder 2x

8

7

Particular Illite

Powder Illite

Fig. 4. Two-dimensional gel images of soluble protein from control, particulate illite- (1  , 2  ) (A), and powdered illite- (1  , 2  ) (B) treated lettuce leaves. Target spots were magnified and compared in control and particulate or powdered illite at 2  concentration (C).

aldolase (1.4 fold), and T protein (1.5 fold). Among them, enolase (gi|1041245, spot 3) and fructose-bisphosphate aldolase (gi|1168411, spot 6) belong to carbon metabolic enzymes. Up-regulated proteins by the treatment of illite were shown as unnamed protein product (gi|18536, spot 2, +1.7 fold), F12M16.14 (gi|7769871, spot 5, +1.3 fold, Po0.01), and Rubisco (gi|1173990, spot 8, +2.1 fold, Po0.05). 4. Discussion The soil conditioner illite has been used to enhance soil quality. However, little has been reported about the physiological effects of illite on plant development and

growth. Thus, we examined the physiological effects of illite on seed germination and the plant growth rate using a typical crop plant, lettuce. In general, seed germination is controlled by a myriad of external factors such as light (Butler et al., 1959; Hendricks, 1980), hormones (Mayer and Poljakoff-Mayver, 1982), and minerals (Obendorf and Wettlaufer, 1984). Besides these factors, the water imbibition capability plays a key role in seed germination (Kauffmann, 1969). The imbibing seed initiates the metabolic enzyme activity required for post-germination and development of shooting seeds. Mineral components in the soil also contribute to development in the seed. Potassium deficiency in plants has severe effects on the plant’s constituent materials such as carbohydrates, amino

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Table 3 Differentially expressed proteins identified by 2-DE and MALDI-TOF/TOF-MS analysis Spot No.

Calculated pI/ Mr

Annotated protein (organism)

Folda

Expression pattern

Mascot score

Gi Accession number

1 2 3 4 5 6 7 8

5.73/54208 5.07/70250 5.41/47567 10.06/8214 8.54/36968 6.85/42442 8.91/44325 6.46/49433

Glycinin subunit G3 (Glycine max) Unnamed protein product (Glycine max) Enolase (Alnus glutinosa) Hypothetical protein (Oryza sativa japonica) F12M16.14 (Arabidopsis thaliana) Fructose-bisphosphate aldolase, chloroplast precursor T protein (Flaveria pringlei) Ribulose-1,5-bisphophate carboxylase oxygenase large subunit (Monochoria vaginalis)

1.5* +1.7 1.9* 4.6 +1.3** 1.4 1.5 +2.1*

decrease increase decrease decrease increase decrease decrease increase

88 53 73 46 264 94 62 380

18639 18536 1041245 51535795 7769871 1168411 438005 1173990

*Po0.05, ** Po0.01. a Relative protein expression ratio in the illite-treated group was expressed as fold versus control. Positive and negative sign means up- and downregulation, respectively. Statistical significance in Student t-test.

acids, and proteins (Steinberg, 1951). Furthermore, potassium is an essential element for photosynthesis and protein synthesis. Plants lacking potassium experience retarded and stunted growth, resulting in low crop productivity. To overcome potassium deficiency, clay minerals such as illite and vermiculite can complement the soil quality by providing a fixed form of potassium. As shown in Fig. 2, illite positively affected the seed germination rate, depending on the concentration of illite given. In particular, the powdered form of illite had more effect than the particulate form. The powdered illite, rather than particulate illite, may be taken up more easily by lettuce seeds, resulting in the efficient germination. Thus, the difference in seed germination between the particulate and powdered forms of illite is probably associated with the ability of the minerals required for seed germination to diffuse in the soil and to be taken up by the lettuce seed. As observed in seed germination, plant growth can also be controlled by the coordination of internal and external factors (Gustafson, 1946). In particular, the contents of ingredients and trace elements are major contributors to plant growth, depending on the soil composition. The ability for uptake, distribution and accumulation of required elements in the appropriate place and time is directly related to crop productivity (Tinker, 1981). Contrary to the seed germination data, illite had little effect on whole plant growth as shown in Fig. 3. Thus, we concluded that the supplement of key elements like potassium in illite is required for the seed germination stage, but not at the plant growth stage. Detailed examination of the effects of illite at different stages of plant development, growth, and senescence are needed to further explore efficient crop productivity with this supplementary soil conditioner. To better understand the effects of illite on lettuce growth, the proteome profiles were compared in the presence and absence of illite. The soluble proteins in lettuce leaves were resolved on 2-D gels according to their isoelectric point and molecular mass by isoelectrofocusing

(IEF) and SDS-PAGE, respectively. Protein application to IEF and SDS-PAGE provides low-cost performance and flexible analysis to identify the protein by Edman sequencing and MALDI-TOF MS (Choi et al., 2000; Woo et al., 2002). Eight proteins were identified as differentially expressed proteins by the treatment of illite given as powder and particle form, in which the interest-of-protein was doubly confirmed at least from the independent triplicate experiments. Down-regulated proteins included glycinin subunit G3, enolase, hypothetical protein, fructose-bisphosphate aldolase, and T-protein. In particular, glycinin subunit G3 and enolase were shown with statistical significance, Po0.05 in our designed study. Glycinin (gi|18639, spot 1) is a predominant seed storage proteins found in many plant seeds (Cho et al., 1989), in which the illite may promote the glycinin degradation for plant growth, resulting in the decrease of glycinin spot. T-protein (gi|438005, spot 7) as one of components consisting of glycine decarboxylase multienzyme complex that converts glycine to serine is known to be higher levels in leaves in the light and closely related to Rubisco responsible for carbon fixation (Oliver and Raman, 1995). Enolase is an enzyme of gluconeogenesis to catalyze the reversible dehydration of 2-phosphoglycerate to phosphoenolpyruvate. In the previous study, carbon metabolic enzymes including enolase of Populus euphratica were down-regulated upon heat stress (Ferreira et al., 2006). Under the normal growth condition, steady-state mRNA and enzyme activities were significantly higher in roots than in green tissue such as leaves (van der Straeten et al., 1991). During the development of rice seedlings, enolase isoforms with different post-translational modification were identified as up- and down-regulated patterns (Tanaka et al., 2005). Fructose bisphosphate aldolase (FBPA) is an enzyme to catalyze the aldol cleavage of fructose 1,6-bisphophate to glyceraldehyde to glyceralgehyde-3-phosphate and dihydroxyacetone phosphate, in which FBPA is involved in degradative reactions closely linked to energy production. Furthermore, this enzyme is known to be widely

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distributed and rapidly developed during germination (Green and Baisted, 1972; Nakamura et al., 1997). Upregulated proteins were unnamed protein, F12M16.14, and Rubisco. In particular, F12M16.14 and Rubisco were shown with the statistical significance at Po0.01 and 0.05, respectively. Unnamed protein (gi|18536) assigned as spot 2 was further analyzed by homology search, resulting in the a-subunit of b-conglycinin as a storage protein. During the seed development of soybean embyro, the b-conglycinin synthesis is known to be increased and decreased as seeds neared maturity (Allen et al., 1991). Local sequence of F12M16.14 (gi|7769871, spot 5) has similarity to malate/lactate dehydrogenase involved in energy production and conversion, of which gene expression is known to be increased during the growth immediately following cucumber seed imbibition (Kim and Smith, 1994). Thus, the soil conditioner illite promoted the expression of malate/lactate dehydrogenase to produce energy for the growth of plant. One of drastic change in protein expression is Rubisco (gi|1173990, spot 8) that is the primary enzyme in photosynthetic carbon fixation and the rate-limiting factor for photosynthesis (Makino et al., 1985). Proteomic changes of Rubisco are known in the growth of rice leaves (Rakwal and Komatsu, 2004) and seed maturation in barley (Finnie et al., 2002). 5. Conclusion Illite has been used as a soil conditioner and belongs to the representative 2:1 type of soil minerals. From our experimental results, illite appears to be effective in the germination and growth of lettuce to some extent. Illite treatment primarily increased the germination rate of lettuce seeds by a maximum of 133% when applied in powdered form. Since the specific surface area of the powdered illite was greater than that of the particulate, the reaction rate of powdered illite with lettuce in the pot soil was postulated to be faster than that of the particulate. In the growth rate of lettuce, illite applied in particulate form enhanced the vertical whole length of 2-week-old lettuce by 23%. This may be due to the fact that it was used as a soil conditioner to improve soil quality rather than as a nutrient for uptake by the lettuce. We also conducted a proteomic approach to identify the proteins affected by illite treatment. The proteins identified by MALDI-TOF/ TOF MS are specifically involved in photosynthesis, energy conversion, and carbon metabolism. Thus, illite improves the quality of soil suitable for germination and plant growth in powdered and particulate form, respectively. This suggests that illite promote the stimulatory growth of lettuce by down-regulating storage protein, glycolytic and gluconeogenic enzymes to switch carbon metabolic flux to carbon fixation by up-regulating Rubisco. However, detailed proteomic profiling of lettuce is still needed to determine the effects of illite as a soil conditioner on development, growth, and senescence of crop plants.

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Acknowledgement This work was supported by a research grant from the Chungbuk National University in 2006.

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