ISSN 1021-4437, Russian Journal of Plant Physiology, 2006, Vol. 53, No. 6, pp. 746–750. © MAIK “Nauka /Interperiodica” (Russia), 2006. Original Russian Text © L.D. Garaeva, S.A. Pozdeeva, O.A. Timofeeva, L.P. Khokhlova, 2006, published in Fiziologiya Rastenii, 2006, Vol. 53, No. 6, pp. 845–850.
Cell-Wall Lectins during Winter Wheat Cold Hardening L. D. Garaeva, S. A. Pozdeeva, O. A. Timofeeva, and L. P. Khokhlova Faculty of Biology and Soil Sciences, Kazan State University, Kremlevskaya ul. 18, Kazan, Tatarstan, 420008 Russia; fax: 7 (843) 238-7121; e-mail:
[email protected] Received February 15, 2006
Abstract—The polypeptide composition and functional activity of cell-wall lectins from roots of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) seedlings during cold hardening were studied. Several phases of lectin activity changes were observed, which indicates their involvement in the development of general adaptation syndrome of the cell. After 0.5-h low-temperature treatment, marked alterations occurred in the profile of protein elution: lectins with mol wts of 78 and 42.5 kD disappeared and new ones with mol wts of 72, 69, 37, and 34.5 kD appeared. It was established that 17.5- and 69-kD lectins and most lectins eluted with glucose were arabinogalactan proteins (AGP), which permitted a supposition that these lectins were involved in the interaction between the cell wall and cytoskeleton. After 7-day-long hardening, total protein content reduced and lectins with mol wts of 69 and 37 kD disappeared, which corresponded to reduced lectin activity by the end of hardening. A transient appearance of 37- and 69-kD lectins, which are AGP, might indicate their involvement in the triggering the development of plant-cell defense responses. DOI: 10.1134/S1021443706060033 Key words: Triticum aestivum - cell wall - lectins - arabinogalactan proteins - low-temperature hardening
INTRODUCTION The investigation of the mechanisms of plant adaptation to variable environment is tightly related to the solving the problems of plant resistance and introduction. Plant adequate responses to various stressors depend on cell signaling systems providing for coordinated functioning of defense and adaptation systems. Cell-wall lectins could by an important component of such systems. Due to their high specificity toward carbohydrate determinates of complex glycoconjugates, lectins could be involved in the interaction between the cell wall and plasma membrane. The extracellular matrix in combination with the plasma membrane and cytoskeleton represents a structurally and functionally coupled system responsible for recognizing and transduction of environmental stress signals [1, 2]. The plant cell wall, being a metabolically active and dynamic compartment [3, 4], is involved in the development of winter plant frost resistance [5]. In the cell wall subjected to the action of low temperature, the content of extensions increases, some enzyme activities enhance, and protein composition changes [6, 7]. Although lectins are present in the cell wall [8] and their behavior depends on the structural integrity of the cytoskeleton [9], essentially nothing is known about the regulatory mechanisms of their activity and changes in their composition under unfavorable environmental conditions, low temperature in particular. In this conAbbreviations: AGP—arabinogalactan proteins; PMSF—phenylmethylsulfonyl fluoride; WGA—wheat germ agglutinin.
nection, the objective of this work was to study changes in the polypeptide composition and functional activity of cell-wall lectins under conditions of winter wheat acclimation to low temperature. MATERIALS AND METHODS Experiments were performed with roots of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) seedlings. Plants were grown in trays on tap water at an irradiance of 100 W/m2 and a 12-h photoperiod. Hardening was performed in a special chamber at 3°ë for 7 days. During this period, we took samples for determination of cell-wall lectin activity and fractional composition. Cell-wall lectins were isolated as described in [10]. After extraction of soluble lectins, the residue was homogenized in the medium containing 20 mM Kphosphate buffer, pH 7.4, 10 mM EDTA, 0.05 mM dithiotreithol, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was filtered through two layers of the cotton cloth. The residue on the cloth, containing cell walls, was washed many times with the homogenization medium and acetone and then extracted with the initial medium supplemented with 0.05% Triton X-100 and 0.9% NaCl for 3 h at constant stirring at 2°ë. After subsequent centrifugation at 10 000 g for 10 min, the pellet was discarded, whereas the supernatant (enriched in cell-wall lectins) used for lectin activity assay and further purification.
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Protein concentration, µg/ml
Lectin activity, (mg/ml) –1
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78 kD
54 kD
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42.5 kD 17.5 kD
13 17 21 25 29 33 37 41 45 Fraction number
Time of hardening Fig. 1. Changes in cell-wall lectin activity during cold hardening of winter wheat seedlings.
Lectins contained in the supernatant were precipitated with ammonium sulfate at 20–30% saturation. The pellet was recovered by centrifugation at 4000 g for 30 min, dissolved in medium containing 0.9% NaCl, 10 mM K-phosphate buffer, pH 7.4, and 0.5 mM PMSF, and dialyzed against 0.9% NaCl in 10 mM K-phosphate buffer, pH 7.4, for 24 h at 5°C. To remove thermolabile nonlectin proteins, the solution was kept for 10 min in the ice bath and centrifuged at 15000 g for 10 min. The pellet (denatured proteins) was discarded, and lectins were precipitated from the supernatant by five volumes of cooled acetone at –20°ë. Preciptated proteins were sedimented by centrifugation at 15000 g for 30 min and dried. The powder was dissolved in medium containing 0.9% NaCl, 10 mM K-phosphate buffer, pH 7.4, and 0.5 mM PMSF and clarified by centrifugation at 1500 g for 30 min. Cell-wall lectins were fractionated on the column (55 × 0.8 cm) packed with Sephadex G-75 (Sigma, United States). To this end, the supernatant was loaded onto the column preliminarily equilibrated with the 50 mM K-phosphate buffer, pH 7.4, containing 0.9% NaCl. Proteins were eluted with this buffer at the flow rate of 0.5 ml/min. Tightly absorbed lectins were eluted with the same buffer supplemented with 0.1 M D-glucose. In the 0.5-ml fractions collected, lectin activity and protein content were measured. The column was calibrated for subsequent calculation of protein molecular weights using standard proteins: BSA (66 kD), ovalbumin (45 kD), and myoglobin (17 kD). The presence of lectins and ARP in fractions obtained after chromatography was detected qualitatively from erythrocyte agglutination [11] and with Yarviv reagent [12], respectively. Lectin activity was evaluated from the lowest protein concentration inducing agglutination of trypsinized erythrocytes of the first blood group [11]. To this end, protein extracts were serially twofold diluted in the wells of U-bottomed microtiter plates. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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Fig. 2. Elution profile during gel chromatography of cell-wall proteins of unhardened winter wheat seedlings.
The results of reaction were recorded in 2 h after titration. Lectin activity was expressed in the values reciprocal to the lowest protein concentration in 1/(mg/ml). Protein content in extracts was estimated by the method of Bradford [13]. Experiments were performed in three replicates. The mean values and their standard errors are presented. RESULTS We observed several phases of lectin activity in roots during wheat seedling hardening (Fig. 1). It increased after 30 min of cold treatment, decreased by 3 h, increased again by 6 h, and decreased again, so that the activity on the 7th day was lower than the initial one. However, not only protein quantity but also their qualitative composition and functional activity are of importance for plant frost resistance. Therefore, in the next series of experiments, we separated cell-wall proteins from Mironovskaya 808 roots by gel filtration with subsequent lectin activity assay in the fractions obtained. As evident from Fig. 2, nine cell-wall protein fractions were eluted from the column. Four of them containing proteins with mol wts of 78, 54, 42.5, and 17.5 kD displayed lectin activity. Since Sephadex is based on cross-linked dextrane, which is a starch derivative, it might be that some lectins specific for glucose were absorbed on Sephadex. These lectins were eluted with 0.1 M D-glucose. Most fractions obtained did not agglutinate erythrocytes. They might be monovalent lectins with a single binding center specific for glucose. Nevertheless, some fractions displayed a capability of erythrocyte agglutination. Arabinogalactan proteins might be among monovalent β-lectins. In our experiments, only one protein fraction obtained by gel filtration (17.5-kD protein) reacted with Yarviv reagent, i.e., it was AGP. Among No. 6
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GARAEVA et al. 16 69 kD 14 12 72 kD 10 8 6 4 2 0 1 5 9
37 kD
14.5 kD 51 kD
34.5 kD
13 17 21 25 29 33 37 41 45 Fraction number
Protein concentration, µg/ml
Protein concentration, µg/ml
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9 8 7 6 5 4 3 2 1 0
72 kD
51 kD
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Fig. 3. Elution profile during gel chromatography of cellwall proteins of winter wheat seedlings hardened during 30 min.
Fig. 4. Elution profile during gel chromatography of cellwall proteins of winter wheat seedlings hardened during 7 days.
lectins isolated by affinity chromatography, those, which agglutinated erythrocytes, did not react with Yarviv reagent, whereas other proteins did react with Yarviv reagent and thus were AGP. In general, at least four lectin groups could be distinguished among cellwall proteins: (1) lectins agglutinating erythrocytes, unspecific for glucose, which are not AGP; (2) lectins agglutinating erythrocytes, specific for glucose, which are not AGP; (3) so-called classical β-lectins very specific for glucose, which do not agglutinate erythrocytes and are AGP; and (4) lectins agglutinating erythrocytes, unspecific for glucose, which are AGP.
DISCUSSION
Low-temperature treatment during 30 min increased markedly total content of cell-wall protein and changed the profile of their elution (Fig. 3). Lectins with mol wts of 78 and 42.5 kD disappeared, and new ones with mol wts of 72, 69, 37, and 34.5 kD appeared. It should be noted that, under these conditions, the content of protein eluted with glucose increased as well (table). In addition, the number of peaks in this fraction increased to ten vs. seven in control. AGP prevailed in this fraction (data not shown). After 7 days of hardening, the content of protein declined substantially (table), which corresponded to the drop in lectin activity by the end of hardening (Fig. 1). Lectins with mol wts of 69 and 37 kD disappeared from the elution profile (Fig. 4). Changes in the content of cell-wall proteins during cold hardening of winter wheat seedlings, µg/g fr wt Treatment
Unhard- 30 min of hy- 7 days of hyened pothermia pothermia
Proteins obtained 12.80 ± 0.3 13.69 ± 0.4 by gel filtration Proteins eluted 8.87 ± 0.2 14.33 ± 0.4 with glucose
2.86 ± 0.6 3.88 ± 0.6
In the perception and transduction of the hormonal or low-temperature signal, the cell surface plays a key role; it includes the cell wall, plasma membrane, and the cytoskeleton. The cell wall takes an especial place because just its capability of responsiveness determines perception of signals reporting about unfavorable environment. The occurrence of several phases of lectin activity changes during plant hardening, we observed in this study, indicates the involvement of lectins in the development of a plant stress state or unspecific adaptation syndrome of the cell. In particular, an increase in the lectin activity during first minutes of hypothermia corresponds to the alarm phase characterized by a rapid and sharp deviation of many indices from normal values with subsequent gradual recovery (Fig. 1). The second peak of lectin activity corresponds to the phase of adaptation (resistance). After completion of this phase, the plant grows normally under extreme conditions, although metabolic processes might be somewhat suppressed. For example, in our experiments, lectin activity became lower than in unhardened plants. It should be noted that the alarm phase (30 min of hardening) was accompanied not only by the increase in the total protein content but also by marked changes in the profile of their elution (Fig. 3). 37-kD lectin, we detected in plants hardened for 30 min, might be a classical wheat lectin, wheat germ agglutinin (WGA). WGA is known to occur not only in the cytoplasm but also in the space between the cell wall and plasma membrane [14]. WGA accumulates sharply under unfavorable conditions, and its possible excretion might protect the plant weakened under stress conditions against soil infection [15]. In addition, in connection with its excretion, we can suppose that WGA is involved in the recovery of the meristem, which mitotic activity is extremely responsive to environmental conditions [15].
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CELL-WALL LECTINS DURING WINTER WHEAT COLD HARDENING
As for the lectin activity in 14.5-kD fraction instead of 17.5-kD fraction in control plants, we incline to believe that they are one and the same protein. Some AGP are known to contain carboxyl-terminal glycosylphosphatidylinositol structure used to anchor them into the plasma membrane [16]. A drop in temperature induces activation of phospholipases C and D [17], which remove this anchor and release AGP from the plasma membrane into the cell wall and ambient medium; this might result in the change of their functions [18]. On the other hand, AGP modification can occur at the posttranscriptional level due to changes in the degree of glycosylation (the addition or loss of some sugar residues [19]). It seems likely that 17.5-kD protein, we detected in the cell walls of unhardened plants, is subjected to such modification under the action of low temperature. During deglycosylation, the protein moieties of AGP molecules become available for further modifications; as a result, they can be involved in the external signal transduction. On the other hand, oligosaccharides released due to degradation of AGP carbohydrate chains can function as regulatory molecules affecting gene expression and, as a sequence, plant resistance, growth, and development. After 7 days of cold hardening, the content of protein decreased substantially, which is in agreement with published data about some decrease in the activity of metabolic processes after adaptation completion [20]. A disappearance of 69- and 37-kD lectins from protein profile (Fig. 4) should be noted. Hardened plants are characterized by improved resistance. Under these condition WGA secretion into the ambient medium was evidently reduced, and therefore, it disappeared from the protein set of the cell wall. The 69-kD lectin, which appeared after 0.5-h hypothermia, is of especial interest. It belongs to AGP that can be adhesive proteins or function as signal molecules. The possibility of interaction between cell-wall proteins, including AGP, and the plasma membrane is widely discussed in the literature [21, 22]. The molecular nature of this interaction between the cell wall and plasma membrane is of especial importance during tissue freezing/thawing, when strong dehydration occurs during freezing and a rapid water uptake, during thawing [8]. The involvement of AGP in the stabilization of structural biopolymer conformation, which could be essential for functioning of both the plasma membrane and cell wall during adaptation to low temperature. The interaction between cell-wall proteins, plasma membrane, and cytoskeleton plays a critical role in the perception and transduction of external signals to various cell compartments. It is commonly accepted that cell-wall AGP contact with the receptors of the plasma membrane [18, 22]. AGP are evidently accessory proteins not altering signals, but they have specific domains for binding signaling proteins. Due to the presence of such domains, accessory proteins produce RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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a complex apparatus providing for the interaction between chains of the signaling pathways, such as receptors, protein kinases, transcription factors, protein phosphatase, and cytoskeletal proteins [4]. It was supposed that AGP bridge the plant cytoskeleton and cell wall [18, 24]. Thus, actin filament depolymerization by cytochalasin D resulted in the changes of AFP conformation and, as a sequence, in distorted contacts between the cell wall and plasma membrane [24]. The cytoskeleton is known to be one of the primary targets of low temperature action. Thus, transient depolymerization of tubulin [25] and actin [26] filaments is required for plant adaptation and improvement of their thermoresistance. The appearance or modification of cell-wall lectins, including AGP, under the effect of low temperatures evidently changes transmembrane interactions in the system cell wall–plasma membrane–cytoskeleton. This results in dynamic instability of microtubules and microfilaments, which triggers a cascade of responses providing for coordinated functioning of defense and adaptation systems in the cells and development of frost resistance. ACKNOWLEDGMENTS This work was supported by the Russian Foundation for Basic Research, project no. 04-04-49318. REFERENCES 1. Nick, P., Signals, Motors, Morphogenesis – the Cytoskeleton in Plant Development, Plant Biol., 1999, vol. 1, pp. 169–179. 2. Turkina, M.V. and Sokolov, O.I., Myosins Are Motor Proteins of the Actomyosin Motility System: Association with Membranes and with the Signal Pathways, Fiziol. Rast. (Moscow), 2001, vol. 48, pp. 788-800 (Russ. J. Plant Physiol., Engl. Transl., pp. 681–692). 3. Gorshkova, T.A., Karpita, N.S., Chemikosova, S.B., Kuz’mina G.G., Kozhevnikov, A.A., and Lozovaya, V.A., Galactans Are a Dynamic Component of Flax Cell Walls, Fiziol. Rast. (Moscow), 1998, vol. 45, pp. 275282 (Russ. J. Plant Physiol., Engl. Transl., pp. 234–239). 4. Tarchevsky, I.A., Metabolizm rastenii pri stresse (Plant Metabolism under Stress Conditions), Kazan: FEN, 2001. 5. Zabotin, A.I., Barysheva, T.S., and Zabotina, O.A., Plant Cell Wall and Hypothermic Syndrome Generation, Dokl. Akad. Nauk, 1995, vol. 343, pp. 567–570. 6. Bozart, C.E., Mullet, J.E., and Bouyer, J.S., Protein Water Potentials, Plant Physiol., 1987, vol. 85, pp. 258– 267. 7. Weisner, R.L., Wallner, S.J., and Waddel, J.M., Cell Extensin mRNA Changes during Cold Acclimation of Pea Seedlings, Plant Physiol., 1990, vol. 93, pp. 1026– 1031. 8. Komarova, E.N., Trunova, T.I., and Vyskrebentseva, E.I., Lectin Activity in Cell Walls of Winter Wheat Apical Meristems during the First Day of Hardening, Fiziol. No. 6
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