ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 5, с. 757-760
КРАТКИЕ ^^^^^^^^^^^^^^ СООБЩЕНИЯ
УДК 581.1
CHARACTERIZATION OF PROTEASES SECRETED BY LEEK ROOTS1
© 2014 B. Adamczyk
Department of Plant Cytology and Cytochemistry, Institute of Plant Physiology, Cytology and Cytogenetic, University of iodz, iodz, Poland Finnish Forest Research Institute, Vantaa Unit, Vantaa, Finland Received November 8, 2013
Some characteristics of root-secreted proteases were studied. We measured their molecular weights and mechanism of BSA digestion in comparison to endogenous root proteases. We related these studies to culture medium N composition. The seedlings of Allium porrum L. (cv. Bartek) were cultivated on MS medium, MS without inorganic nitrogen (MS-IN), and MS without IN, but with 0.1% casein (MS-IN + 0.1% casein). Electrophoretic study showed that root-secreted proteases had one isoform with a mol wt of 45 kD, regardless of medium N composition. Difference in molecular weights of root-secreted proteases and endogenous root proteases active under used conditions (>66 kD) provide us another strong evidence that root-secreted proteases were not just leaking from the roots, but they were secreted. Proteases exuded by roots degraded BSA in a similar way as endogenous proteases, with only one SDS-PAGE-detectable product of degradation. Our results may be a powerful tool in the extraction and purification of these enzymes and also in proteomic studies.
Keywords: Allium porrum — organic nitrogen — plant nitrogen nutrition — protease secretion — proteolytic activity
DOI: 10.7868/S0015330314050029
INTRODUCTION
Nitrogen is one of the key factors regulating plant development. Plants can utilize wide array of N forms, including inorganic N compounds, simple organic forms, but also proteins [1, 2]. Plant ability to use organic nitrogen is of special importance as soil organic matter contains nitrogen predominantly in organic form, mainly as proteins [3]. Soil proteins are digested by microbial proteases; moreover, it was proven that plants can also secrete proteases by roots [4—6]. However, the knowledge about root-secreted proteases is very limited. Some of the few results available indicate that roots of numerous plants are able to secrete proteases [4, 5]. It was shown that roots of wheat seedlings can use root-secreted proteases to create a pool of accessible nitrogen [5]. Proteases secreted by the roots of Allium porrum may cleave proteins, releasing low-molecular-weight peptides that can probably be taken up by roots [7].
Molecular weight characterization of root-secreted proteases is needed. On the basis of such knowledge, it would be possible to effectively extract and purify root-
1 This text was submitted by the author in English.
Abbreviation: MS-IN — MS without inorganic nitrogen. Corresponding author: Bartosz Adamczyk. Finnish Forest Research Institute, Vantaa Unit, P.O. Box 18, FIN-01301 Vantaa, Finland; fax: + 358 10-211-2206; e-mail: Bartosz.Adamczyk@ metla.fi
secreted proteases and, in the future, perform protein sequencing and characterization of genes encoding these proteins. In addition, a comparison of molecular weights of root-secreted proteases with endogenous proteases is highly needed because it can prove finally that these enzymes are actively exuded, not just leaked from the roots.
In this paper we studied molecular weights of proteases secreted by roots of A. porrum cv. Bartek cultivated on different media and degradation products of BSA after digestion by root-secreted proteases in parallel to endogenous proteases active under used pH conditions.
MATERIALS AND METHODS
Seeds of Allium porrum L. cv. Bartek were obtained from a commercial plant distributor ("Torseed", Poland), and reagents — from "Sigma-Aldrich" (United States).
Seeds were sterilized with 70% ethanol (2 min) and later with 10% NaOCl (15 min), rinsed five times with sterile, deionized water, and germinated at 23°C in Petri dishes with two layers of filter paper moistened with sterile, deionized water (for 7 days) and later separately in tubes (for 2 weeks; whole experiment took 3 weeks) with 15 mL of autoclaved, liquid MS or MS medium without inorganic nitrogen (MS-IN), or MS medium without IN, but with 0.1% casein (MS-IN +
758 ADAMCZYK
Proteolytic activity in the culture medium and shoot and root fresh weights of seedlings cultivated on different media
Parameter MS MS-IN MS-IN + 0.1% casein
Shoot fresh weight, mg 24.50 ± 0.62a 9.80 ± 0.60b 30.20 ± 1.05c
Root fresh weight, mg 5.20 ± 0.48a 2.70 ± 0.21b 6.00 ± 0.37a
Proteolytic activity, U/mm root surface 2.60 ± 0.20a 1.10 ± 0.71a 5.20 ± 0.36b
Results are shown as mean value of six replicates (n = 6) ± standard errors. Statistically significant differences (P < 0.05) are marked with different letters.
0.1% casein) like in previous study [5]. All operations were done under aseptic conditions in a laminar airflow cabinet. The seedlings were cultivated at 23 ± 1°C air temperature, 70% relative humidity, and 16-h pho-toperiod with 380 ^mol/(m2 s) light intensity. Sterility of the culture medium was verified by microbiological tests (Microcount combi, "Schulke-Mayr", Germany). These tests are agar dip-slides having media suitable for detection of bacterial and fungal contaminations. Root surface was measured using a stereological method [8], on the basis of root length and root fresh weight.
Root endogenous proteases were extracted with 50 mM Tris—HCl buffer (pH 6.8) by grounding the plant root material in a cooled mortar. After centrifu-gation (10000 g, 5 min), the supernatant and also liquid culture media (containing secreted proteases) were purified and concentrated with a Vivapore 5 solvent absorption concentrator with a pore size of 7500 MWCO ("Sartorius Stedim Biotech", France). This concentrator lets not only to concentrate the sample but also
kD
66.0-
45.0 —
36.029.024.020.1 ■
14.2 —
Fig. 1. Molecular weights of root-secreted and endogenous proteases of seedlings cultivated on different media. M — mol wt markers (14—66 kD); 1, 2 — root-secreted and endogenous proteases of seedlings growing on MS; 3, 4 — root-secreted and endogenous proteases of seedlings growing on MS-IN; 5, 6 — root-secreted and endogenous proteases of seedlings growing on MS-IN + 0.1% casein. Gel was stained with Coomassie Brilliant Blue-250.
to purify it from compounds smaller than 7.5 kD because of the presence of membrane.
Proteolytic activity was determined using Tomarel-li method [9], in which the time-dependent release of azo-dye-coupled TCA-soluble peptide fragments from the substrate, azocasein was monitored. Briefly, to 200 ^L of partially purified culture medium (as described above), 100 ^L 0.5% (w/v) azocasein (substrate) dissolved in 0.9% NaCl in 50 mM phosphate buffer (pH 6.8) was added, and after 4 h incubation at 28°C, the reaction was arrested by adding 200 ^L of 20% (w/v) trichloroacetic acid (TCA). After centrifu-gation (10000 g, 5 min) 150 ^L of the supernatant was mixed with 50 ^L of 1 M NaOH, and after 30 min ab-sorbance at 440 nm was read (Hitachi U-2000 spectrophotometer, Japan). In the control, TCA was added prior to azocasein. One unit of protease activity was defined as the amount of enzyme that increased the absorbance by 0.1 at 440 nm per 1 h. Results are presented per 1 mm2 of the root surface.
Zymogram was performed as described by Leber and Balkwill [10]. Proteolytic enzyme aliquots were mixed with loading buffer (1 : 2) consisted of 60 mM Tris-HCl buffer (pH 6.8), 10% glycerol, 4% SDS, and 0.01% bromophenol blue. We used markers with molwts of 14-66 kD ("Sigma-Aldrich") and Mini Protean III Cell ("BioRad", United States). Our aim was to resolve with high precision only root-secreted proteases, and because of that we did not use here markers with the higher molecular weights. Running gel (10%) containing 0.1% casein (substrate) was overlaid with 4% stacking gel, and samples (20 ^L) were loaded and run at 90 V. Later, gel was soaked for 1 h in 2.5% Triton X-100 (to remove SDS) with gentle stirring followed by three washes in deionized water and later incubated overnight in a buffer (50 mM Tris-HCl (pH 6.8) with 0.02% Brij-35 (polyethylene glycol dodecyl ether), 1.17% NaCl, and 0.05% CaCl2). On the next day, gel was briefly rinsed with deionized water and stained with 0.5% Coomassie Brilliant Blue R-250 in a mixture of methanol : acetic acid : deionized water (4 : 1 : 5) for 2 h with gentle stirring, and destained with the same mixture overnight.
To study degradation products of BSA, proteolytic enzyme aliquots (as described above) were incubated with 0.5% BSA (in 50 mM Tris-HCl, pH 6.8) for 12 h in sterile conditions in proportion of1 : 1 at room tem-
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CHARACTERIZATION OF PROTEASES SECRETED BY LEEK ROOTS
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perature. Control probe was prepared in the same way, but with heat-denatured enzyme (100°C, 20 min). Such control was prepared to exclude the influence of other compounds present in probes on BSA degradation. The SDS-PAGE was performed according to manufacturer's instructions ("BioRad"). Briefly, probes of incubation mixture (proteases with BSA) were mixed with loading buffer (1 : 2) consisting of 60 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 0.01% bromophenol blue, and 5% 0-mercaptoethanol and incubated for 4 min at 95°C. We used markers with mol wts of14—66 kD. Running gel (10%) was overlaid with 4% stacking gel, and samples (20 ^L) were loaded and run at 125 V. Gel was stained as described for the zymogram.
Each experiment consisted of 6 replicates (n = 6). Proteolytic activity and fresh weight means were compared among different media, using one-way ANOVA, followed by Tukey's test. We used Statistica software ("Statsoft", United States).
RESULTS
The highest shoot and root weights and proteolytic activity in the culture medium were obtained for seedlings growing on MS-IN + 0.1% casein, followed by seedlings growing
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