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Biologia, Bratislava, 61/1: 97—102, 2006 97 Protein reutilisation in corms of Colchicum autumnale Lenka Franková*1, Katarína Cibírová2, Károly Bóka3, Otília Gašparíková1 & Mikuláš Pšenák2 1 Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 14, SK–84523 Bratislava, Slovakia; tel.: ++421-259426119; *e-mail: [email protected] 2 Comenius University, Faculty of Pharmacy, Department of Cell and Molecular Biology of Drugs, Kalinčiakova 8, SK–83232 Bratislava, Slovakia; tel.: ++421-2-50117308 3 Department of Anatomy, ELTE, Pázmany Péter Sétany 1/C, H–1117 Budapest, Hungary Abstract: Colchicum autumnale L. is a monocotyledonous geophyte with hysteranthous leaves, i.e. flowering and leaf growth occur in different time periods. Because after the starch, the second prominent storage compound of corm is represented by proteins, we were interested in nitrogen remobilisation during the annual life cycle of C. autumnale. In this context the content of soluble and insoluble proteins were measured in parallel with determination of some exo- and endopeptidase activities. Our results indicate that the continual proteolysis occurs in both mother and new daughter corms during the whole life-cycle of the plant. L-Ala-aminopeptidase and trypsin-like endopeptidase were the most active peptidases in both mother and daughter corms. As the protein level of mother corm did not change significantly during the development of the future above-ground part under the soil surface (the first, autumnal developmental stage), the developmental profile of nitrate reductase activity was estimated followed by evaluation of total nitrogen and amino acid contents. Significant activity of root nitrate reductase was detected in the roots only in the second, vernal stage. Our results showed that the stored proteins constituted a relevant nitrogen source partly required by the growth processes of the late autumnal stage, but mainly by the intensive growth of leaves and reproductive structures during the second, photosynthetically active stage of the life-cycle. Key words: Colchicum, corm, geophyte, peptidases, proteolysis, nitrogen storage Abbreviations: d.m. – dry mass; L-Ala-AP – L-alanine-aminopeptidase; BAPA-ase – (N-benzoyl-arginine-hydrolase) trypsin-like endopeptidase; Z-Glu-Phe-CP – Z-(Z-benzyloxycarbonyl)-glutamyl-phenylalanine-carboxypeptidase; Z-GluTyr-CP – Z-glutamyl-tyrosine-carboxypeptidase; Gly-L-Pro-DPP – glycyl-L-proline-dipeptidylpeptidase; L-Leu-AP – Lleucine-aminopeptidase; PMSF – phenylmethylsulfonyl fluoride; Suc-Phe-EP – (N-succinyl-phenylalanine-hydrolase) – chymotrypsin-like endopeptidase; TDG – thiodiglycol Introduction Colchicum autumnale L. is monocotyledonous geophyte with underground basal storage stem, called corm. Because this perennial medicinal plant flowers without leaves, it belongs to the group of hysteranthous geophytes (Franková, 2005). Geophyte species are often considered as a source of carbohydrates. However, they do contain proteins the content of which is substantially lower than that of starch (Monte-Neshich et al., 1995; Shewry, 2003). Usually the underground storage organs, such as tubers, storage roots and corms, accumulate less than 10% of the stored proteins (Pate & Dixon, 1982). Unlike seed storage proteins, very little is known about the reserve proteins of the underground storage organs of geophytes (Guimarães et al., 2001). The importance and some characteristics of proteins of plant storage tubers, bulbs and corms have been reported by Shewry (2003). Vegetative storage organs contain a large amount of storage proteins, which are mobilized during seed filling, foliar (Rossato et al., 2002) and floral (Wagstaff et al., 2002) senescence, regrowth (Gomez & Faurobert, 2002) or sprouting (Shewry, 2003). In addition to their storage function, they can play additional roles in the plant, e.g. enzymatic (Strickland et al., 1995; Tonón et al., 2001; Rosahl et al., 1987), inhibitory (Yeh et al., 1997; Hou et al., 1999), antioxidant (Hou et al., 2001), antibacterial (Flores et al., 2002) or antifungal ones (Flores et al., 2002, Terras et al., 1993). The storage proteins can be defined as proteins whose major role is to act as stores of nitrogen, sulphur and carbon (Shewry, 2003). In plant cells protein breakdown is mediated by different proteolytic systems: vacuolar proteolysis, selective nuclear and cytosolic proteolysis, and organellar proteolysis. Because each cellular compartment is af- 98 fected by sugar starvation, these different proteolytic systems might be involved in starvation-induced proteolysis (Brouquisse et al., 1998). Proteins are hydrolysed to their amino acids by the sequential action of three types of enzymes: endo-, carboxy-, and aminopeptidases (Callis, 1995). Although a few papers aimed at storage proteins in tuberous geophytes have already been published (Pate & Dixon, 1982; Shewry 2003), nothing is known about the proteins and their major role in life strategy of hysteranthous geophytes generally and that of cormous ones especially. In case of C. autumnale, as a typical representative of hysteranthous cormous geophytes, neither protein content, nor protein breakdown have been studied so far. In the present study, the changes in the protein amount in the mother and daughter corms during the annual life-cycle of C. autumnale are reported and correlated with some proteolytic activities of these tissues. Potential contribution of nitrate assimilation (taken up by roots) to the protein synthesis in the daughter corm is also evaluated. To better understand the protein changes during the first part of the life cycle, the content of total nitrogen and that of total amino acids have also been determined. The results obtained were interpreted in compliance with the individual developmental stages of the life cycle of autumn crocus. Material and methods Plant material Colchicum autumnale L. was collected in a natural population (Malacky, Slovak Republic – 48◦ 26 N, 17◦ 02 E). Protein extraction and content Soluble proteins were extracted from fresh corms by 0.05 M citrate buffer pH 6.6 in presence of 0.02 M KCl, 0.002 M MgCl2 , 0.001 M dithiotreithol, 0.002 M Na2 S2 O3 and 0.5% Polyclar AT (MOHAN-KUMAR et al., 1999) for 1 h. The extract was centrifuged (10 000 g) and the sediment two times re-extracted. After centrifugation the supernatants were pooled and used for determination of soluble protein content. The sediment was washed and used for extraction of cell wall-bound proteins. To determine insoluble proteins Triton X 100 was added to extraction medium (final concentration of 0.05%) and extracted as described before. The protein content was estimated according to BRADFORD (1976) using bovine serum albumin as a standard. Enzyme preparation Corms were pulverised in presence of liquid nitrogen, washed with excess of cooled acetone (–50 ◦C, delipidization) and dried at laboratory temperature. 1 g of acetone-powder was extracted with 0.1 M Na-phosphate buffer, pH 7.5 (proteolytic enzymes) in presence of Polyclar AT. To extract nitrate reductase 0.1 M Na-phosphate buffer, pH 8.0 containing 5 mM MgCl2 , 0.5 mM EDTA, 0.5 mM PMSF, 1% glycerol and 0.05% Triton X 100 were used (FRANKOVÁ et al., 2005). After extraction (3 × 30 min) and centrifugation (10 000 g, 30 min) the proteins in supernatants were pooled and precipitated by ammonium sulphate (70% saturation). L. Franková et al. After centrifugation (10 000 g, 45 min) the sediment was dissolved in buffer used and dialysed. All operations were carried out at 4 ◦C. Determination of proteolytic activities To determine aminopeptidase activity the L-alanine pnitroanilide and L-leucine p-nitroanilide (Sigma, Richmond, USA) were used as substrates. Standard reaction mixture at final volume of 1.5 mL contained 0.1 mL of enzyme solution and 1.4 mL of substrate solution (1.4 µmol Lalanine p-nitroanilide or L-leucine p-nitroanilide) in 0.01 M Tris-HCl buffer, pH 8.0. After incubation at 30 ◦C for 30 min the reaction was stopped by adding 0.25 mL of 30% acetic acid and absorbance of released p-nitroaniline was measured at 400 nm (KOVÁCS et al., 1985). For calculation of enzyme activity the molar absorption coefficient of 9950 M−1 cm−1 was taken. In case of trypsin-like endopeptidase, chymotrypsin-like endopeptidase and dipeptidylpeptidase assay 1.4 µmol of BAPA (α-N-benzoyl-D,L-arginine p-nitroanilide), 1.4 µmol of N-succinyl-phenylalanine pnitroanilide or 1.29 µmol of glycyl-L-proline p-nitroanilide were added to the reaction mixture and assayed as described before. Carboxypeptidase activities were estimated using Zglutamine-thyrosine and Z-glutamine-phenylalanine as substrates. Reaction mixture (1 mL) contained 1.2 µmol of substrate in 0.02 M Tris-HCl, pH 7.2 and enzyme solution. After incubation (37 ◦C, 30 min) the rate of liberated Phe or Tyr was evaluated by ninhydrine method [formation of Schiffbase and detection of blueviolette dye at 570 nm, ROSEN (1957)] using Phe and Tyr as standards. All enzyme activities were expressed as µmol s−1 g−1 d.m. Nitrate reductase assay Reaction mixture at final volume of 0.5 mL included enzyme solution and 0.05 M Na-phosphate buffer pH 8.0 containing 10 mM KNO3 , 1 mM EDTA, 0.2 mM NADH and 0.01 mM FAD. After 5 min incubation at 30 ◦C the reaction was stopped by adding 50 µL of 0.5 mM Zn(CH3 COO)2 and 50 µL of 10 mM phenazine methosulphate [oxidation of NADH excess; DRUART et al. (2000)]. NO− 2 produced was determined by pipetting 1 mL of sulfanillamide (1% in 1.5 M HCl) and 1 mL of N-(1-Naphthyl)-ethylendiamine dihydrochloride (0.02% in distilled water). The absorbance of azo dye was measured at 540 nm. Total nitrogen and total amino acid content Total nitrogen assay was estimated using Kjeldahl method (FERENČÍK et al., 1981). The spectrum and level of total amino acids (free and protein bound) were analysed by HPLC. The proteins present in 0.5 g of fresh corms were suspended in 30 mL of 6 M HCl followed by nitrogen bubbling. After hydrolyzation (110 ◦C, 23 h), the sample was neutralized with 30 mL of 12% NaOH and diluted up to 100 mL with dilution buffer pH 2.2 containing 1.4% citric acid, 1.25% NaCl, 0.01% (w/w) caprylic acid and 0.5% TDG. Amino components were identified using an AAA 400 amino acid analyzer (Ingos, Prague) equipped with Ostion LG AN B cation-exchange column (0.37 × 45 cm) and with two channel photometer 440 and 570 nm. In the assay the following phosphate buffer systems were used: 0.2 M (pH 2.7), 0.4 M (pH 4.25), 1.12 M (pH 7.9), 0.2 M (pH 3.0) and 0.2 M NaOH. Protein reutilisation in corms of Colchicum autumnale 99 B A Insoluble proteins Soluble proteins 200 80 150 60 100 40 50 0 0 1.IX 15.IX 1.X 8.X 22.X 29.X 6.XI 13.XI 20.XI 27.XI 4.XII 15.I 1.II 28.II 10.III 24.III 31.III 7.IV 14.IV 21.IV 28.IV 12.V 19.V 26.V 2.V 9.VI 22.VI 20 Protein content (mg.g-1 d.m.) 100 250 Insoluble proteins Soluble proteins 15.VIII 1.IX 15.IX 1.X 8.X 22.X 29.X 6.XI 13.XI 20.XI 27.XI 4.XII 15.I 1.II 28.II 10.III 24.III 31.III 7.IV 14.IV 21.IV 28.IV 12.V 19.V 26.V 2.V 9.VI 22.VI Protein content (mg.g-1 d.m.) 120 Fig. 1. Total protein content in old mother corm (A) and new daughter corm (B). (Means ± SE, SE ≤ 7%, n = 6). 13 4 8 11 10 6 5 4 1 2 12 7 3 1 2 9 5 6 3 7 8 A B Statistics The data are means of three independent experiments performed each in two replicates. Results and discussion Generally, the polysaccharides are the most common storage materials in perennial plants (Buckeridge et al., 2000; Pate & Dixon, 1982). Only a limited group of geophytes accumulates proteins as a main storage material. Starch appeared to be the main storage component of corms of C. autumnale (Franková et al., 2003, 2004). The results in this paper revealed that the proteins formed the second major component of corm’s storages. In the mother corm the protein fraction represents only 6–11% of dry mass while that in new daughter corm forms at least 10–22% of dry mass. Fig. 2. The first developmental (autumnal) stage of C. autumnale: A – Formation of root system from the root disk after flowering: 1 – new daughter corm, 2 – root disk, 3 – root system, 4 – stem, 5 – peduncles, 6 – ovaries, 7 – sieve-like structure (connection between old and new corm), 8 – style lobes. B – Development of new shoot (regular) under the soil surface – the whole future aboveground part of the plant: 1 – old mother corm, 2 – protuberance, 3 – non-developing irregular bud, 4 – new shoot, 5 – new daughter corm, 6 – root disk, 7 – next, new regular bud for future generation, 8 – roots, 9 – stem, 10 – peduncles, 11 – young capsules with unmature seeds, 12 – rest of perianth tubes, 13 – leaves. The arrows indicate the position of soil surface. The amount of total proteins varies in dependence on the individual developmental phases of the life cycle of perennial autumn crocus. The continuity of autumn crocus is provided by the annual corm replacement, i.e. the new daughter corm replaces old mother one each year. The annual life cycle consists of two developmental stages: the first – autumnal one (September-December) followed by winter season, and the second – photosynthetically active one (March–May), which tends to senescence (June) and summer dormancy (July–August, Franková et al., 2003, 2004). The developmental profile of total proteins in mother corm (Fig. 1A) shows that their level does not change significantly during the autumnal stage. For this period the differentiation of new shoot (from regular bud ensuring the plant continuity) is characteristic (Franková et al., 2004). The appearance of ir- 100 L. Franková et al. 2 0,35 A mother corm daughter corm C 1,6 0,28 1,2 0,21 0,8 0,14 0,4 0,07 0 0 0,5 B mother corm daughter corm 0,4 0,5 mother corm daughter corm D 0,4 0,3 0,3 0,2 0,2 0,1 0,1 Jun Apr May Mar Jan Feb Dec Oct Nov Sep Jul Aug Jun Apr May Mar Jan Feb Dec Oct Nov Sep Jul 0 Aug 0 Activity (µkat.g-1 d.m.) Activity ( µkat.g-1 d.m.) mother corm daughter corm Fig. 3. Developmental profile of some exo- and endopeptidases in old mother and new daughter corm: L-Ala-AP (A), L-Leu-AP (B), N-Suc-Phe-EP (C) and BAPA-ase (D). (Means ± SE, n = 6). regular shoot (from irregular bud serving for vegetative propagation, Fig. 2B) depends on both environmental conditions and level of storage reserves in the mother corm. The regular bud gives rise to new daughter corm and its shoot regularly at the end of August. The new shoot (yet without roots) is in flower in the middle of September. After flowering the differentiation of roots and the complete future above-ground part (stem, leaves and capsules) takes place in the soil (Figs 2A,B). These events proceed without any photosynthesis and are energetically covered up by intensive reutilisation of starch reserves of mother corm (Franková et al., 2003, 2004). In the course of autumnal stage the amount of total proteins (soluble and insoluble) in the new developing corm increases (Fig. 1B), but the corresponding decrease of proteinous nitrogen was not observed in the mother corm (Fig. 1A). Thus, it seems that the proteins in new corm are formed independently from the proteins stored in the mother corm. Looking for details what happens in the new corm at the time of root (middle of October, Fig. 2A) and shoot (future above-ground part, October-November, Fig. 2B) development, we obtained following results: the levels of total nitrogen and that of total amino acids in the new corm slowly decreased during shoot development, but the amount of total proteins increased by the end of November. The opposite phenomenon was observed after complete shoot development, i.e. an increase of amino acid amount, and decrease of protein content and that of total nitrogen (Figs 1B, 4). Total protein content in mother corm remains almost constant (Fig. 4). It seems that during the first part of the autumnal stage the proteins in the new developing daughter corm (Fig 2B) are synthesised from the pool of free amino acids. This pool may be maintained by a) free amino acids from the mother corm, b) proteolytic activities in both corms, and c) nitrate assimilation in root system directly connected with new corm. Because the protein content of the mother corm almost did not change, it is difficult to find the origin of nitrogen used for metabolic processes connected with root and new shoot formation (Figs 2A,B). On the other hand, the remarkable proteolytic process was observed in both mother and daughter corms. Of the proteolytic enzymes five exopeptidases and two endopeptidases were detected having maximum activity during winter season. There were L-Ala-AP (Fig. 3A), L-Leu-AP (Fig. 3B), Z-Glu-TyrCP, Z-Glu-Phe-CP, Gly-L-Pro-DPP (data not shown), Suc-Phe-EP (Fig. 3C) and BAPA-ase (Fig. 3D). LAla-AP and BAPA-ase appeared to be the most active peptidases in both mother and daughter corms. The importance of L-Ala-AP and L-Leu-AP in protein turnover has been reported by Mikola & Mikola (1986). The highest proteolytic activities were detected in the mother corm during late autumn and winter period and at the end of the life cycle of the plant. These findings indicate that an active process of protein turnover takes place during autumnal stage in both corms. The level of free amino acids in the new daughter corm might be renewed exactly at the cost of protein degradation during the late autumnal developmental stage (end of November-December, Fig. 4). The results of amino acid analyses in the new corm presented in Fig. 5 show that N-rich amino acids Protein reutilisation in corms of Colchicum autumnale content (mg g-1 d.m.) 350 total nitrogen NC total proteins NC f ree amino acids NC total proteins OC 280 210 140 70 14.XII 7.XII 21.XI 14.XI 7.XI 31.X 0 Fig. 4. Changes in the different nitrogen fractions in new daughter corm during root and shoot formation of C. autumnale. OC – old, mother corm, NC – new, daughter corm. (Means ± SE, n = 6). 2N Content (mg g-1 d.m.) 30 4N 2N 24 18 2N 12 3N 6 Asn Arg Gln Tyr Leu Ala Ser Lys Val Pro Gly Thr Ile His Phe 0 Fig. 5. The spectrum and content of amino acids in the new daughter corm (the early autumnal stage); N – nitrogen rich amino acids. Activity (nkat g -1 d.m.) 30 25 20 15 10 5 30.IX 15.X 30.X 15.XI 30.XI 15.I 30.I 15.II 28.II 15.III 30.III 15.IV 30.IV 15.V 30.V 15.VI 0 Fig. 6. Seasonal variation of nitrate reductase activity in the root system of C. autumnale. (Means ± SE, n = 6). Asn, Gln, Arg are present at the highest levels. It means that not only proteinous nitrogen but also Nrich units can serve as immediately accessible form of reduced nitrogen. Low activity of nitrate reductase (3– 4 nkat g−1 d.m.) at the first, autumnal stage indicates that free amino acids are de novo formed only 101 at limited amounts. Paradoxically the nitrate reductase reached maximum activity during the second developmental stage (March-June, Fig. 6). For the second, photosynthetically active period following developmental processes are typical: a) the intensive growth of leaves and reproductive structures (capsules with seeds), b) formation of reserves in daughter corm (the new corm ripening), c) physical disappearance of old mother corm, d) dying back of the above-ground part. The total reutilization of the rest of protein content in the mother corm went in parallel with its physical destruction. A certain correlation between the decline in protein content (Fig. 1A) and relatively high activities of N-Suc-Phe-EP, BAPA-ase (Figs 3C,D) and Z-GluPhe-CP confirms this. The amino acids released in the new and old corms as well as those formed de novo may saturate their demands for growth and differentiation of vegetative tissues, as well as the development of reproductive structures during the second developmental stage. In addition, the level of proteinous nitrogen in the new corm also decreased to the end of spring period. So at the end of May the amount of total proteins in the new ripe corm was almost the same as it was in the old mother one at the beginning of the life-cycle (August). To maintain the critical level of storage reserves in the ripe corm at the end of the annual bioprogram seems to be the specific feature of C. autumnale. We have found that proteolytic activities are present in both corms during the whole life cycle of the plant. In addition, the activities are varying significantly depending on the individual developmental stages. These findings evoke the question what was the reason for the presence of proteolytic activities in both corms of C. autumnale during the whole annual lifecycle. It is known that one of the crucial functions of proteolysis is to help regulate metabolism. Therefore, the presence of proteolytic activities in corm tissues might be combined with the immediate degradation of enzymes and the others, less essential proteins, which are not required by the basic metabolism at a given developmental stage. This phenomenon was observed in case of developmental profile of polyphenoloxidase (Bilecová et al., 1996) and β-glucosidase (unpublished results) during the annual life-cycle of mother and daughter corms of autumn crocus. Together with protein synthesis, degradation is essential to maintain appropriate enzyme levels (Scheurwater et al., 2000). For C. autumnale it means that the levels of the most abundant enzymes with a high specific activity (such as amylases, glucosidases, polyphenoloxidases and peptidases) could also be modulated through their selective breakdown. The main significance of remarkable proteolytic activities is probably to ensure the turnover of proteins and their trade-off in distribution between the mother corm, new developing plant and its new corm. Although starch represents 50% of corm’s dry mass, the storage proteins still remain the required component of storage material of C. autumnale. Our 102 results show that the stored proteins constituted a relevant nitrogen source being required by the growth processes of the late autumnal stage, but mainly by the metabolic events taking place during the second, photosynthetically active stage, when the individual organs of vegetative part are intensively growing and the storage compounds are formed in the ripening daughter corm. 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