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Alterations in endogenous polyamines in bulbs of tuberose (Polianthes tuberosa L.) during dormancy
Scientia Horticulturae 105 (2005) 483–490 www.elsevier.com/locate/scihorti
Alterations in endogenous polyamines in bulbs of tuberose (Polianthes tuberosa L.) during dormancy§ Shweta Sood, P.K. Nagar * Division of Biotechnology, Institute of Himalayan Bioresource Technology, Palampur 176061, Himachal Pradesh, India Received 17 August 2004; received in revised form 8 February 2005; accepted 9 February 2005
Abstract Changes in the levels of endogenous polyamines were determined in tuberose (Polianthes tuberosa L.) bulbs during their dormant periods. High free putrescine and low spermine and spermidine levels were associated during initial stages of dormancy. In contrast, high spermine and spermidine levels were related with dormancy release. The conjugated putrescine level increased during the period with an increase in conjugated spermine and spermidine levels. An inverse relationship between free and conjugated polyamines was noticed only for putrescine. The possible role of polyamines is discussed in relation to dormancy. # 2005 Elsevier B.V. All rights reserved. Keywords: Dormancy; Polyamines; Tuberose; Polianthes tuberosa L.
1. Introduction Dormancy is an important adaptive mechanism which benefits survival of a species under unfavorable conditions (Juntilla, 1988). It is considered as a phase in the plants life cycle during which the usual processes contributing to orderly growth and development are inhibited. Seeds and vegetative propagules like corms, tubers, bulbs and rhizomes, etc., of § IHBT communication no. 0456. * Corresponding author. Tel.: +91 1894 230992; fax: +91 1894 230433. E-mail address: [email protected] (P.K. Nagar).
0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2005.02.010
484
S. Sood, P.K. Nagar / Scientia Horticulturae 105 (2005) 483–490
most plant species even under highly favorable conditions do not germinate/sprout immediately after maturity. This period of suspended growth has been defined as innate dormancy (Wareing, 1977) while in imposed dormancy the growth is suspended before the onset of adverse climatic conditions (Bradbeer, 1988). Because of its basic and applied importance (Lang, 1996) the regulation of dormancy has attracted considerable interest. Dormancy has been well characterized in apical buds of grapevines (Or et al., 2002), peach (Faye and Floch, 1999) and tuberous organs (Suttle, 1996). Polyamines (PAs) are ubiquitous nitrogenous compounds appear to be present in all organisms. The diamine putrescine (Put), triamine-spermidine (Spd) and tetramine-spermine (Spm), recognized as plant growth regulators are present in all plant species examined sofar (Kumar et al., 1997). There are many observations correlating alterations in polyamine titers to many physiological and developmental processes like normal and abnormal growth, cell division, differentiation, embryogenesis, fruit ripening, flower development, and normal or stress induced senescence (Galston and Kaur-Sawhney, 1995; Kakkar et al., 2000). They also regulate the rigidity and stability of cellular membranes (Ficker et al., 1994). In recent years, by using inhibitors of PA deficient, PA resistant mutants and transgenic approaches, new insights into the roles of PAs in plant developmental processes have become available (Malmberg et al., 1998; Kakkar and Sawhney, 2002). In addition to commonly occurring free polyamines, the importance of polyamines conjugated to low molecular weight compounds like hydroxycinnamic acid or bound to high molecular weight substances like specific proteins or nucleic acid is now being realized (Martin-Tanguy, 2001). Among ornamental bulbous plants valued for their beauty and fragrance of the flowers, the tuberose (Polianthes tuberosa L.) occupies a very special and selective place. Moreover, its flower is a very good source of essential oils (Hussain, 1986) which is used in the production of cosmetic and perfumery products. The tuberose bulbs remain in a rest of period for about 3 months in places where the temperature is low (5–6 8C) and an early planting is desirable for early crop. Pre-treatment of tuberose bulbs with gibberellic acid (GA3) had no effect in breaking the dormancy (Pathak et al., 1980). The mechanism controlling dormancy in tuberose bulbs is not yet been elucidated. In a previous communication (Nagar, 1995) a high level of free ABA was detected in tuberose bulbs during their dormant period which decreased with release of dormancy with concomitant increase in free auxin (IAA) levels. In a recent study (Huang et al., 2004) changes in polyamine patterns were studied during floral initiation and development in bulbs of P. tuberosa. However, there have been very few investigations about the possible role of polyamines in control of dormancy (Wang and Faust, 1994; Kakkar and Nagar, 1997) and the importance of specific polyamines or different polyamine fractions has not been demonstrated. In the present paper we evaluate the alterations in the polyamine titers during imposition of dormancy and its release in tuberose bulbs in order to understand whether their levels or the relationships between different polyamines could be involved in dormancy control.
2. Materials and methods Tuberose bulbs grown in the Institute Floriculture Farm at Palampur (altitude 1290 m, 32.68N and 78.188E) were used for the purpose. Freshly harvested (mid November) bulbs,
S. Sood, P.K. Nagar / Scientia Horticulturae 105 (2005) 483–490
485
15–18 g fresh weight (FW) was kept in pots at 15–18 8C temperature. From these, three bulbs were randomly selected at 15 days intervals till sprouting and used for determination of polyamines. 2.1. Polyamine analysis Each sample of whole bulb (1 g FW) was homogenised in chilled 10% perchloric acid (PCA). Extraction and benzyolation of PAs were performed using a modification of procedure described earlier (Flores and Galston, 1982; Sood and Nagar, 2004). In brief, the homogenised sample was vortexed, stored for overnight at 4 8C. and centrifuged at 8000 g for 30 min. The supernatant was the source of free PAs and PCA soluble polyamines conjugated to hydroxycinnamic acid while the pellet contained PCA insoluble polyamines. The latter two fractions were released by hydrolysis as described earlier (Sood and Nagar, 2004). The original supernatant, HCl hyrolysed supernatant and HCl hydrolysed pellet were separately treated with insoluble PVP (50 mg ml 1) and stirred for 45 min to remove phenolics and interfering compounds. These were then filtered and HCl was used to clean up the vials. PAs recovered from the hyrolysed and non-hydrolysed supernatants and from pellet suspension were then benzoylated as described earlier (Sood and Nagar, 2004). Benzoylamines were extracted with chloroform (1:1, v/v); the chloroform phase was dried under the stream of air and dissolved in 0.25 ml of methanol (HPLC grade) for further analysis. Reverse phase HPLC was carried out essentially as described previously (Sood and Nagar, 2004) using Lichrosorb RP-18 (5 mm) column (250 mm 4 mm i.d.) protected by a guard column. Elution was carried out using a linear gradient of methanol:water (64–75% methanol for 15 min, 75% methanol for 5 min and 75–64% methanol for 5 min) at 22 8C at a flow rate of 1 ml/min. The solvents were filtered through 0.45 mm pore size membranes and degassed. The column effluents were passed through UV detector (996 PDA detector) at 254 nm. Under these conditions, retention time for Put, Spd and Spm were 4.23 0.032, 6.257 0.038 and 8.745 0.83 min, respectively. The concentrations of PAs in the eluates were calculated from standard curves responses of the known polyamines (Put, Spd and Spm, obtained from Sigma Chemical Co., USA) which were also benzyolated as described above. Standard deviations of the means of three replicates were assessed for all the data.
3. Results and discussion Polyamine levels (free, bound and conjugated) changed appreciably during the dormancy period (Fig. 1). The highest free putrescine level (182 nmol/g dry weights) was noticed during the first 30 days after harvest while free spermine and spermidine reached their highest level at the time of bulb sprouting and spermidine was the most abundant at this time. Free putrescine content in the dormant bulbs was higher than spermine and spermidine up to 45 days (Fig. 1A). The conjugated Put level increased during the period with an increase in both conjugated Spm and Spd (Fig. 1B). The bound polyamine levels appeared to increase especially for Spm and Spd from dormancy initiation to dormancy
486
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Fig. 1. Changes in the levels of free (A) conjugated (B) and bound (C) polyamines in tuberose bulbs during dormancy. The standard error for each sampling (n = 3) is given as a vertical bar.
release (Fig. 1C). At all the stages bound and conjugated Spm and Spd levels were higher than bound and conjugated Put. The Put to Spm plus Spd ratio in the free fraction was highest during the early stages of dormancy and lowest subsequent to dormancy release (Table 1). In conjugated fraction, the ratio increased from 45 days onwards, perhaps due to an increase in all forms of conjugated polyamines. However, in the bound fraction, the ratio increased from 45 days onwards, perhaps due to an increase in all forms of conjugated polyamines.
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Table 1 Seasonal variations in putrescine to spermidine plus spermine (Put/[Spd + Spm]) ratio in the tuberose bulbs Days after harvest
Put/[Spd + Spm] Free
Conjugated
Bound
15 30 45 60 75 90
1.29 1.28 1.04 0.35 0.22 0.17
0.14 0.13 0.2 0.23 0.25 0.34
0.24 0.21 0.17 0.12 0.1 0.07
Variations in the levels of growth regulators, especially ABA and IAA have been observed in tuberose bulbs during dormancy and subsequent to its release (Nagar, 1995). These variations correlate well with those observed in the present study in relation to PA levels relating to dormancy in tuberose bulbs. In general high levels of free Put and low levels of free Spm and Spd seem to accumulate at the beginning of dormancy and opposite situation was noticed during dormancy release. The highest relative levels of Spm and Spd in tuberose bulbs were observed subsequent to dormancy release. The ratio of Put to Spd plus Spm was also very low during this period. Thus, the role of Spm and Spd could be more important subsequent to dormancy release, i.e. during active growth. In normal and dwarf apple shoots higher levels of Spd and Spm were noticed during spring than autumn (Wang and Faust, 1994). The relative high accumulation of Put during early phase of dormancy (Fig. 1A) indicates that it may play an important role in this process. It has been shown that Put is primarily responsible for the chilling tolerance of bean (Guye et al., 1986), wheat (Nadeau et al., 1987) and rice (Lee et al., 1997) and during the early phase of winter dormancy in tea (Kakkar and Nagar, 1997) relative concentration of Put is much more than other PAs. However, other work shows an involvement of Spd or Spm and not only Put in chilling tolerance of plants. For example, it has been suggested (Shen et al., 2000) that Spd plays an important roles in chilling tolerance of cucumber probably through prevention of chill-induced activation of superoxide-generating NADPH oxidases in microsomes. Spd also plays a role in the adaptation of Pringlea, a cold-adapted subantartic crucifer, to cold condition (Dufeu et al., 2003). It has been emphasized that PA level is regulated to a large extent by conjugation, compartmentation and by oxidative enzymes (Martin-Tanguy, 2001). In tuberose bulbs, an inverse relationship between the concentration of free and conjugated PA forms can be observed only for Put whereas conjugated and bound Spd and Spm show the same increase pattern with the start and release of dormancy. This indicates that the bound form of PAs have at least to some extent physioregulatory functions. Certain studies (Torrigiani et al., 1986) have suggested that conjugated PAs appear to function solely as storage pools of PAs which upon hydrolysis could supply the cell with additional PAs and could influence cell division and/or expansion and other developmental processes (Protacio and Flores, 1992; Bonneau et al., 1994). It has also been proposed that the conjugates may act as a means for PA traslocation (Havelange et al., 1996) and that they could be the preferred substrates for amine oxidases (Martin-Tanguy, 1997). There may be interconversion between free and
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conjugated PAs and that in various physiological processes this interchange is required to ensure high levels of free polyamines (Martin-Tanguy, 2001). A good positive correlation was found between free ABA levels and degree of dormancy in tuberose bulbs which decreased with sprouting of bulbs with concomitant increase of free IAA (Nagar, 1995). In a recent study, Huang et al. (2004) suggested that a significant reduction in Put and Spm is involved in the early floral initiation in P. tuberosa. These data along with the observed decrease in the ratio of Put to the rest of PAs during dormancy release of tuberose bulbs and vice-versa supports the hypothesis that the process relating to release of dormancy involves an interaction or balance between endogenous growth promoters and inhibitors as well as changes in the tissue sensitivity (Trewavas, 1991) could also be an operative mechanism during dormancy phenomenon. Further, in view of the interaction of PAs with other growth regulators (Lin, 1984; Mader and Hanke, 1997; Cvikrova´ et al., 1999) and their role in a number of growth and developmental processes in plants (Kumar et al., 1997; Kakkar et al., 2000), the existence of variations in endogenous PA levels during the annual growth cycle of many species could be considered to be involved in the control of many developmental processes. This is in agreement with the hypothesis that it is not the presence of ‘high’ but ‘adequate’ level of PAs that is important for many physiological functions (Altamura et al., 1991).
Acknowledgements The authors wish to thank Dr. P.S. Ahuja, Director of the Institute for the necessary facilities, and Department of Science and Technology, New Delhi for financial support in the form of a research project.
References Altamura, M.M., Torrigiani, P., Capitani, F., Scaramagli, S., Bagni, N., 1991. De novo root formation in tobacco thin layers is affected by inhibition of polyamine biosynthesis. J. Exp. Bot. 42, 1575–1582. Bonneau, L., Carre, M., Martin-Tanguy, J., 1994. Polyamines and related enzymes in rice differing in germination potential. Plant Growth Regul. 15, 75–82. Bradbeer, J.W., 1988. Seed Dormancy and Germination. Chapman & Hall, New York. Cvikrova´ , M., Binarova´ , P., Eder, J., Va´ gner, M., Hrubcova´ , M., Zon´ , J., Macha´ ckova´ , I., 1999. Effect of inhibition of phenylalanine ammonia-lyase activity on growth of alfalfa cell suspension culture: Alterations in mitotic index, ethylene production, and contents of phenolics, cytokinins and polyamines. Physiol. Plant. 107 (3), 329–337. Dufeu, M., Martin-Tanguy, J., Hennion, F., 2003. Temperature dependent changes of amine levels during early seedling development of the cold adapted subantarctic crucifer Pringlea antiscorbutica. Physiol. Plant. 118, 164–172. Faye, F., Floch, F.L., 1999. Changes in the activity of peach bud demosine kinase from the dormancy period to bud break. J. Plant Physiol. 154, 471–476. Ficker, E., Taglialatela, M., Wible, B.A., Henly, C.M., Brown, A.M., 1994. Spermine and spermidine as gating molecules for inward rectifier K+ channels. Science 266, 1068–1072. Flores, H.E., Galston, A.W., 1982. Analysis of polyamines in higher plants by high performance liquid chromatography. Plant Physiol. 69, 701–706.
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Galston, A.W., Kaur-Sawhney, R., 1995. Polyamines as endogenous growth regulators. In: Davis, P.J. (Ed.), Plant hormones: Physiology, Biochemistry and Molecular Biology. 2nd ed. Kluwer Academic Publishers, Dordrecht, pp. 158–178. Guye, M.G., Vigh, L., Wilson, J.M., 1986. Polyamine titre in relation to chilling-sensitivity in Phaseolus sp. J. Exp. Bot. 37, 1036–1043. Havelange, A., Lejeune, P., Bernier, A., Kaur-Sawhney, R., Galston, A.W., 1996. Putrescine export from leaves in relation to floral trasition in Sinapsis alba. Physiol. Plant. 96, 59–65. Huang, C.-K., Chang, B.-S., Wang, K.-C., Her, S.-J., Chen, T.-W., Chen, Y.-A., Cho, C.-L., Liao, L.-J., Huang, K.-L., Chen, W.-S., Liu, Z.-H., 2004. Changes in polyamine pattern are involved in floral initiation and development in Polianthes tuberosa. J. Plant Physiol. 161 (6), 709–713. Hussain, A., 1986. Ornamental Horticulture. ICAR, New Delhi, India, p. 136. Juntilla, O., 1988. To be or not to be bud dormant: some comments on the new dormancy nomenclature. Hort. Sci. 23, 805–806. Kakkar, R.K., Nagar, P.K., 1997. Distribution and changes in endogenous polyamines during winter dormancy in tea [Camellia sinensis L. (O) Kuntze]. J. Plant Physiol. 151, 63–67. Kakkar, R.K., Sawhney, V.K., 2002. Polyamine research in plants—a changing perspective. Physiol. Plant. 116, 281–292. Kakkar, R.K., Nagar, P.K., Ahuja, P.S., Rai, V.K., 2000. Polyamines and plant morphogenesis. Biol. Plant. 43, 1–11. Kumar, A., Altabella, T., Taylor, M.A., Tiburcio, A.F., 1997. Recent advances in polyamine research. Trends Plant Sci. 2, 124–130. Lang, 1996. Plant Dormancy, Physiology, Biochemistry and Molecular Biology. CAB International, Wallington, UK, 386 pp. Lee, T.M., Lur, H.S., Chu, C., 1997. Role of abscisic acid in chilling tolerance of rice (Oryza sativa L.) seedlings. II. Modulation of free polyamine levels. Plant Sci. 126, 1–10. Lin, P., 1984. Polyamine metabolism and its relation to response of the alerone layers of barley seeds to gibberellic acid. Plant Physiol. 74, 975–983. Mader, J.C., Hanke, D.E., 1997. Polyamine sparing may be involved in the inhibition of phenylpropanoid synthesis in cytokinin-starved soyabean cells. J. Plant Growth Regul. 16, 89–93. Malmberg, R.L., Watson, M.B., Galloway, G.L., Yu, W., 1998. Molecular genetic analysis of plant polyamines. Crit. Rev. Plant Sci. 17, 199–224. Martin-Tanguy, J., 2001. Metabolism and function of polyamines in plants: recent development (new approaches). Plant Growth Regul. 34, 135–148. Martin-Tanguy, J., 1997. Conjugated polyamines and reproductive development: biochemical, molecular and physiological approaches. Physiol. Plant. 100, 675–688. Nadeau, P., Delaney, S., Chouinard, L., 1987. Effect of cold hardening on the regulation of polyamine levels in wheat (Triticum aestivum L.) and alfalfa (Medicago sativa L.). Plant Physiol. 84, 73–77. Nagar, P.K., 1995. Changes in abscisic acid, phenols and indolacetic acid in bulbs of tuberose (Polianthes tuberosa L.) during dormancy and sprouting. Sci. Hort. 63, 77–82. Or, E., Vilozny, I., Eyal, Y., Ogrodovitch, A., 2002. The transduction of the signal for grape bud dormancy breaking induced by hydrogen cyanamide may involve the SNF-like protein kinase GDBRPK. Plant Mol. Biol. 43, 483–494. Pathak, S., Chaudhary, M.A., Chaudhary, S.K., 1980. Germination and flowering in different sized bulbs of tuberose (Polianthes tuberosa L.). Indian J. Plant Physiol. 23, 47–54. Protacio, C.M., Flores, H.E., 1992. The role of polyamines in potato tuber formation. In vitro Cell Dev. Biol. 28P, 81–86. Shen, W., Nada, K., Nachibana, S., 2000. Involvement of polyamines in the chilling tolerance of cucumber cultivars. Plant Physiol. 124, 431–439. Sood, S., Nagar, P.K., 2004. Changes in endogenous polyamines during flower development in two diverse species of rose. Plant Growth Regul. 44, 117–123. Suttle, J.C., 1996. Dormancy in tuberous organs: problems and perspectives. In: Lang, G.A. (Ed.), Plant Dormancy: Physiology, Biochemistry and Molecular Biology. CAB International, Wallington, UK, pp. 133–143.
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Trewavas, A.J., 1991. How do plant growth substances work? Plant Cell Environ. 14, 1–12. Torrigiani, P., Serafini-Fracassini, D., Biondi, S., Bagni, N., 1986. Evidence for the subcellular localization of polyamines and their biosynthetic enzymes in plant cells. J. Plant Physiol. 124, 23–29. Wang, S.Y., Faust, M., 1994. Changes in polyamine content during dormancy in flower buds of ‘Anna’ apples. J. Am. Soc. Hort. Sci. 119, 70–73. Wareing, P.F., 1977. Growth substances integration in the whole plant. In: Jennings, D.H. (Ed.), Proceedings of the Symposium XXXI on Integration of Activity in Higher Plants, Society of Experimental Biology, pp. 337–365.
Alterations in endogenous polyamines in bulbs of tuberose (Polianthes tuberosa L.) during dormancy§ Shweta Sood, P.K. Nagar * Division of Biotechnology, Institute of Himalayan Bioresource Technology, Palampur 176061, Himachal Pradesh, India Received 17 August 2004; received in revised form 8 February 2005; accepted 9 February 2005
Abstract Changes in the levels of endogenous polyamines were determined in tuberose (Polianthes tuberosa L.) bulbs during their dormant periods. High free putrescine and low spermine and spermidine levels were associated during initial stages of dormancy. In contrast, high spermine and spermidine levels were related with dormancy release. The conjugated putrescine level increased during the period with an increase in conjugated spermine and spermidine levels. An inverse relationship between free and conjugated polyamines was noticed only for putrescine. The possible role of polyamines is discussed in relation to dormancy. # 2005 Elsevier B.V. All rights reserved. Keywords: Dormancy; Polyamines; Tuberose; Polianthes tuberosa L.
1. Introduction Dormancy is an important adaptive mechanism which benefits survival of a species under unfavorable conditions (Juntilla, 1988). It is considered as a phase in the plants life cycle during which the usual processes contributing to orderly growth and development are inhibited. Seeds and vegetative propagules like corms, tubers, bulbs and rhizomes, etc., of § IHBT communication no. 0456. * Corresponding author. Tel.: +91 1894 230992; fax: +91 1894 230433. E-mail address: [email protected] (P.K. Nagar).
0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2005.02.010
484
S. Sood, P.K. Nagar / Scientia Horticulturae 105 (2005) 483–490
most plant species even under highly favorable conditions do not germinate/sprout immediately after maturity. This period of suspended growth has been defined as innate dormancy (Wareing, 1977) while in imposed dormancy the growth is suspended before the onset of adverse climatic conditions (Bradbeer, 1988). Because of its basic and applied importance (Lang, 1996) the regulation of dormancy has attracted considerable interest. Dormancy has been well characterized in apical buds of grapevines (Or et al., 2002), peach (Faye and Floch, 1999) and tuberous organs (Suttle, 1996). Polyamines (PAs) are ubiquitous nitrogenous compounds appear to be present in all organisms. The diamine putrescine (Put), triamine-spermidine (Spd) and tetramine-spermine (Spm), recognized as plant growth regulators are present in all plant species examined sofar (Kumar et al., 1997). There are many observations correlating alterations in polyamine titers to many physiological and developmental processes like normal and abnormal growth, cell division, differentiation, embryogenesis, fruit ripening, flower development, and normal or stress induced senescence (Galston and Kaur-Sawhney, 1995; Kakkar et al., 2000). They also regulate the rigidity and stability of cellular membranes (Ficker et al., 1994). In recent years, by using inhibitors of PA deficient, PA resistant mutants and transgenic approaches, new insights into the roles of PAs in plant developmental processes have become available (Malmberg et al., 1998; Kakkar and Sawhney, 2002). In addition to commonly occurring free polyamines, the importance of polyamines conjugated to low molecular weight compounds like hydroxycinnamic acid or bound to high molecular weight substances like specific proteins or nucleic acid is now being realized (Martin-Tanguy, 2001). Among ornamental bulbous plants valued for their beauty and fragrance of the flowers, the tuberose (Polianthes tuberosa L.) occupies a very special and selective place. Moreover, its flower is a very good source of essential oils (Hussain, 1986) which is used in the production of cosmetic and perfumery products. The tuberose bulbs remain in a rest of period for about 3 months in places where the temperature is low (5–6 8C) and an early planting is desirable for early crop. Pre-treatment of tuberose bulbs with gibberellic acid (GA3) had no effect in breaking the dormancy (Pathak et al., 1980). The mechanism controlling dormancy in tuberose bulbs is not yet been elucidated. In a previous communication (Nagar, 1995) a high level of free ABA was detected in tuberose bulbs during their dormant period which decreased with release of dormancy with concomitant increase in free auxin (IAA) levels. In a recent study (Huang et al., 2004) changes in polyamine patterns were studied during floral initiation and development in bulbs of P. tuberosa. However, there have been very few investigations about the possible role of polyamines in control of dormancy (Wang and Faust, 1994; Kakkar and Nagar, 1997) and the importance of specific polyamines or different polyamine fractions has not been demonstrated. In the present paper we evaluate the alterations in the polyamine titers during imposition of dormancy and its release in tuberose bulbs in order to understand whether their levels or the relationships between different polyamines could be involved in dormancy control.
2. Materials and methods Tuberose bulbs grown in the Institute Floriculture Farm at Palampur (altitude 1290 m, 32.68N and 78.188E) were used for the purpose. Freshly harvested (mid November) bulbs,
S. Sood, P.K. Nagar / Scientia Horticulturae 105 (2005) 483–490
485
15–18 g fresh weight (FW) was kept in pots at 15–18 8C temperature. From these, three bulbs were randomly selected at 15 days intervals till sprouting and used for determination of polyamines. 2.1. Polyamine analysis Each sample of whole bulb (1 g FW) was homogenised in chilled 10% perchloric acid (PCA). Extraction and benzyolation of PAs were performed using a modification of procedure described earlier (Flores and Galston, 1982; Sood and Nagar, 2004). In brief, the homogenised sample was vortexed, stored for overnight at 4 8C. and centrifuged at 8000 g for 30 min. The supernatant was the source of free PAs and PCA soluble polyamines conjugated to hydroxycinnamic acid while the pellet contained PCA insoluble polyamines. The latter two fractions were released by hydrolysis as described earlier (Sood and Nagar, 2004). The original supernatant, HCl hyrolysed supernatant and HCl hydrolysed pellet were separately treated with insoluble PVP (50 mg ml 1) and stirred for 45 min to remove phenolics and interfering compounds. These were then filtered and HCl was used to clean up the vials. PAs recovered from the hyrolysed and non-hydrolysed supernatants and from pellet suspension were then benzoylated as described earlier (Sood and Nagar, 2004). Benzoylamines were extracted with chloroform (1:1, v/v); the chloroform phase was dried under the stream of air and dissolved in 0.25 ml of methanol (HPLC grade) for further analysis. Reverse phase HPLC was carried out essentially as described previously (Sood and Nagar, 2004) using Lichrosorb RP-18 (5 mm) column (250 mm 4 mm i.d.) protected by a guard column. Elution was carried out using a linear gradient of methanol:water (64–75% methanol for 15 min, 75% methanol for 5 min and 75–64% methanol for 5 min) at 22 8C at a flow rate of 1 ml/min. The solvents were filtered through 0.45 mm pore size membranes and degassed. The column effluents were passed through UV detector (996 PDA detector) at 254 nm. Under these conditions, retention time for Put, Spd and Spm were 4.23 0.032, 6.257 0.038 and 8.745 0.83 min, respectively. The concentrations of PAs in the eluates were calculated from standard curves responses of the known polyamines (Put, Spd and Spm, obtained from Sigma Chemical Co., USA) which were also benzyolated as described above. Standard deviations of the means of three replicates were assessed for all the data.
3. Results and discussion Polyamine levels (free, bound and conjugated) changed appreciably during the dormancy period (Fig. 1). The highest free putrescine level (182 nmol/g dry weights) was noticed during the first 30 days after harvest while free spermine and spermidine reached their highest level at the time of bulb sprouting and spermidine was the most abundant at this time. Free putrescine content in the dormant bulbs was higher than spermine and spermidine up to 45 days (Fig. 1A). The conjugated Put level increased during the period with an increase in both conjugated Spm and Spd (Fig. 1B). The bound polyamine levels appeared to increase especially for Spm and Spd from dormancy initiation to dormancy
486
S. Sood, P.K. Nagar / Scientia Horticulturae 105 (2005) 483–490
Fig. 1. Changes in the levels of free (A) conjugated (B) and bound (C) polyamines in tuberose bulbs during dormancy. The standard error for each sampling (n = 3) is given as a vertical bar.
release (Fig. 1C). At all the stages bound and conjugated Spm and Spd levels were higher than bound and conjugated Put. The Put to Spm plus Spd ratio in the free fraction was highest during the early stages of dormancy and lowest subsequent to dormancy release (Table 1). In conjugated fraction, the ratio increased from 45 days onwards, perhaps due to an increase in all forms of conjugated polyamines. However, in the bound fraction, the ratio increased from 45 days onwards, perhaps due to an increase in all forms of conjugated polyamines.
S. Sood, P.K. Nagar / Scientia Horticulturae 105 (2005) 483–490
487
Table 1 Seasonal variations in putrescine to spermidine plus spermine (Put/[Spd + Spm]) ratio in the tuberose bulbs Days after harvest
Put/[Spd + Spm] Free
Conjugated
Bound
15 30 45 60 75 90
1.29 1.28 1.04 0.35 0.22 0.17
0.14 0.13 0.2 0.23 0.25 0.34
0.24 0.21 0.17 0.12 0.1 0.07
Variations in the levels of growth regulators, especially ABA and IAA have been observed in tuberose bulbs during dormancy and subsequent to its release (Nagar, 1995). These variations correlate well with those observed in the present study in relation to PA levels relating to dormancy in tuberose bulbs. In general high levels of free Put and low levels of free Spm and Spd seem to accumulate at the beginning of dormancy and opposite situation was noticed during dormancy release. The highest relative levels of Spm and Spd in tuberose bulbs were observed subsequent to dormancy release. The ratio of Put to Spd plus Spm was also very low during this period. Thus, the role of Spm and Spd could be more important subsequent to dormancy release, i.e. during active growth. In normal and dwarf apple shoots higher levels of Spd and Spm were noticed during spring than autumn (Wang and Faust, 1994). The relative high accumulation of Put during early phase of dormancy (Fig. 1A) indicates that it may play an important role in this process. It has been shown that Put is primarily responsible for the chilling tolerance of bean (Guye et al., 1986), wheat (Nadeau et al., 1987) and rice (Lee et al., 1997) and during the early phase of winter dormancy in tea (Kakkar and Nagar, 1997) relative concentration of Put is much more than other PAs. However, other work shows an involvement of Spd or Spm and not only Put in chilling tolerance of plants. For example, it has been suggested (Shen et al., 2000) that Spd plays an important roles in chilling tolerance of cucumber probably through prevention of chill-induced activation of superoxide-generating NADPH oxidases in microsomes. Spd also plays a role in the adaptation of Pringlea, a cold-adapted subantartic crucifer, to cold condition (Dufeu et al., 2003). It has been emphasized that PA level is regulated to a large extent by conjugation, compartmentation and by oxidative enzymes (Martin-Tanguy, 2001). In tuberose bulbs, an inverse relationship between the concentration of free and conjugated PA forms can be observed only for Put whereas conjugated and bound Spd and Spm show the same increase pattern with the start and release of dormancy. This indicates that the bound form of PAs have at least to some extent physioregulatory functions. Certain studies (Torrigiani et al., 1986) have suggested that conjugated PAs appear to function solely as storage pools of PAs which upon hydrolysis could supply the cell with additional PAs and could influence cell division and/or expansion and other developmental processes (Protacio and Flores, 1992; Bonneau et al., 1994). It has also been proposed that the conjugates may act as a means for PA traslocation (Havelange et al., 1996) and that they could be the preferred substrates for amine oxidases (Martin-Tanguy, 1997). There may be interconversion between free and
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conjugated PAs and that in various physiological processes this interchange is required to ensure high levels of free polyamines (Martin-Tanguy, 2001). A good positive correlation was found between free ABA levels and degree of dormancy in tuberose bulbs which decreased with sprouting of bulbs with concomitant increase of free IAA (Nagar, 1995). In a recent study, Huang et al. (2004) suggested that a significant reduction in Put and Spm is involved in the early floral initiation in P. tuberosa. These data along with the observed decrease in the ratio of Put to the rest of PAs during dormancy release of tuberose bulbs and vice-versa supports the hypothesis that the process relating to release of dormancy involves an interaction or balance between endogenous growth promoters and inhibitors as well as changes in the tissue sensitivity (Trewavas, 1991) could also be an operative mechanism during dormancy phenomenon. Further, in view of the interaction of PAs with other growth regulators (Lin, 1984; Mader and Hanke, 1997; Cvikrova´ et al., 1999) and their role in a number of growth and developmental processes in plants (Kumar et al., 1997; Kakkar et al., 2000), the existence of variations in endogenous PA levels during the annual growth cycle of many species could be considered to be involved in the control of many developmental processes. This is in agreement with the hypothesis that it is not the presence of ‘high’ but ‘adequate’ level of PAs that is important for many physiological functions (Altamura et al., 1991).
Acknowledgements The authors wish to thank Dr. P.S. Ahuja, Director of the Institute for the necessary facilities, and Department of Science and Technology, New Delhi for financial support in the form of a research project.
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