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Variations of the Patterns of Abscisic Acid and Proline during Maturation of Nicotiana tabacum Pollen Grains
J. Plant Physiol.
Vol. 147. pp. 355 -358 (1995)
Variations of the Patterns of Abscisic Acid and Proline during Maturation of Nicotiana tabacum Pollen Grains FATIHA CHIBI, TRINIDAD ANGOSTOt,
and ANGEL MATILLA*
Departamento de Biologfa Vegetal, Universidad de Granada, 18001-Granada I E. Politecnica Superior, Universidad de Almeda, 0412C-Almena, Spain .. For correspondence Received March 8,1995' Accepted July 20,1995
Summary
Abscisic acid (ABA) and proline were evaluated during in situ development of anthers and pollen grains of Nicotiana tabaeum. No conjugated forms of ABA were detected. The maximum accumulation of free ABA in the whole anther occurred at the mid-binucleate stage (MB), almost 95 % of the ABA being located in the pollen; this ABA distribution was not observed in the microspore (Mi) or mature (Ma) stages of the anther, in which the sporophytic tissue provided more ABA than did the pollen. A maximum in proline accumulation was also found at the MB stage of the anther; however, at the Ma pollen stage (when the ABA was scarcely detectable) the proline accumulation was maximum. The germination of the pollen grain was inhibited by exogenous ABA and stimulated by fluridone, a compound that inhibits carotenoid biosynthesis. The results of the present work suggest an important role for ABA in maturation and germination, as well as in the elongation of the pollen tube of N. tabacum.
Key words: Abscisic acid, anther, germination, maturation, Nicotiana tabacum, pollen, proline. Abbreviations: ABA - abscisic acid; AMGLu - maturation medium; GK Ma - matured; Mi - microspore; MB = mid-binucleate; T z tetrade.
Introduction
Abscisic acid (ABA), like other plant hormones, has multiple roles during the life cycle of a plant (Zeevaart and Creelman, 1988). So, stomatic control (Mansfield et al., 1990), water relations (Davies and Zhang, 1991), proline biosynthesis and osmoregulation (Skiver and Mundy, 1990; Delauney and Verma, 1993), and embryogenesis, dormancy and germination of seeds (Quatrano, 1987), among the physiological processes, are under ABA control. In most fruits, seeds or embryos, changes in ABA levels during development show a similar pattern: a fairly steep rise during development, followed by a sharp drop as the seeds mature (Quatrano, 1987). However, even though ABA has been detected in the reproductive apparatus (v.e. pollen, © 1995 by Gustav Fischer Verlag, Stuttgart
=
germination medium;
styles and ovary) of various species of Angiosperms and Conifers (Shibuya et al., 1978; Webber, 1988), nothing until now was known about its alteration during the development of pollen grain, perhaps because of the difficulty of obtaining sufficient amounts for ABA quantification. Recently, we have demonstrated that ethylene and polyamines are involved in «in vivo. and «in vitro. development of Nicotiana tabaeum pollen (Chibi et al., 1993; 1994; Chibi and Matilla, 1994), and also during its embryogenesis (Garrido et al., 1995). As a preliminary approach, we quantified endogenous levels of ABA and free-proline in intact anthers as well as in isolated pollen grains of N. tabacum during different stages of development in order to gain further information about the role that ABA could play in anther and pollen development.
356
FATIHA CHffiI, TRINIDAD ANGOSTO, and ANGEL MATILLA
Materials and Methods
Plant material Nicotiana tabacum plants were grown in a climate chamber under a 16 h light period at 25°C. Anthers and pollen were collected at different developmental stages: microspore (Mi), mid-binucleate (MB) and mature (Ma) (Fig. 1). For determining the stage of pollen development, one anther was squashed and the released pollen was stained with a droplet of 4 % carmine in 45 % propionic acid and viewed under a light microscope. Two min later, the precise stage of development was determined (Garrido et al., 1991).
~
.
FW
-6
The pollen grains matured in situ were cultured for 2 h in GK medium (Brewbaker and Kwack, 1963) modified to contain a double concentration of boric acid (Benito-Moreno et al., 1988). The number of pollen grains with growing tubes were counted under an invened microscope. More than 1000 pollen grains were counted for each run. ABA (1 to 20 11M) or fluridone (1 to 50 11M) solutions were added to GK in order to examine the effect of these substances on the germination process.
''-
~
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~ -o
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.c. '\-,
; -ECll
-
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Pollen germination
0
1,5
0.5
E~
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MB
Me
Fig. 1: Variations in fresh weight of whole anther (.) and its pollen content (D) during the microspore, mid-binucleate and mature stages of N. tabacum. Data are the mean of 5-6 experiments ± SD. to room temperature and the absorbance read at 520 nm using toluene as a blank.
Results and discussion
Quantification ofendogenous ABA Abscisic acid was extracted from intact anthers or isolated pollen at different stages of development according to Vaughan and Milborrow (1984) with several modifications (Angosto and Matilla, 1993). All steps were carried out under green light to minimize isomerization. To anthers or isolated pollen samples, the following were added: 10 mL acetone: acetic acid (99: 1), v/v) containing 2-6di-t-butyl-4-methyl phenol (BHT, 10 I1gmL -1); 1ilL HC-ABA (422 GBqmol-t, 740KBqmL-t, Amersham) and indolbutyric acid (10 mg) as an internal standard. The mixture was ground in a mortar and centrifuged at 27,000go for 15min at 4°C. The pellet was extracted twice more with acetone: acetic (99: 1, v/v). The combined supernatants were evaporated to dryness in a rotavapor at 35°C and the dried residue was dissolved in 30 mL ethanol: acetic acid (0.2 %; 1: 1, v/v), filtered through four C l8 Sep-Pack (Waters) filters and then concentrated in a rotavapor to 5 mL. This volume was extracted three times with cold ethyl ether, the free ABA being in the ether phase and the bound ABA in the aqueous phase. The aqueous phase was hydrolyzed for 30 min at 60°C, pH 11.0 (adjusted with 1 mol/L NaOH), and when cold, was adjusted to pH 3.0 with 1mol/L HCI and fractionated once more with ethyl ether as described above. ABA was methylated with ice-cold etheric diazomethane (Schlong and Guellerman, 1960) and analysed by gas chromatography combined to mass spectrometry (GC-MS). The GC capillary column (12 m x 0.33 mm) was packed with methyl-silicone (panicle size 0.25I1mol/L) and operated at 200 °C and 250°C in the injector and detector, respectively; hydrogen was used as the GC-carrier gas. The gas chromatography was combined with mass spectrometry, and atomic-absorption spectrometry (GC-M5-AAS) gave spectra similar to those of commercial ABA (Sigma, Chern. Co).
Free.proline determination Isolated pollen or whole anthers from different stages were homogenized in 1 mL 3 % aqueous sulphosalicylic acid and centrifuged at 1,000 x g. One mL of supernatant was reacted with 1 mL acid ninhydrin and 1 mL glacial acetic acid in a test tube for 60 min at 100 0C. The reaction was stopped in an ice bath. The mixture was extracted with 4 mL toluene and mixed with a test-tube stirrer for 15 sec. The chromophore containing toluene was separated, warmed
As the anther of N. tabacum was about to reach maturity, its FW decreased slowly, while that of its pollen content increased 4-fold from the Mi to Ma stages (Fig. 1). In recent publications, we demonstrated that the processes related to the development and maturation provoked: (a) a decrease in both arginine-decarboxylase (ADC) and ornithine-decarboxylase (ODC) activities (Chibi et al., 1994), (b) important alterations in the levels of free and bound-polyamines in the anthers (Chibi et al., 1994) as well as in the isolated pollen grains (Chibi et al., 1993), and (c) an increase in ethylene production (Chibi and Matilla, 1994). In the present work, we demonstrate that in the three (Mi, MB and Ma) developmental stages of N. tabacum anthers studied, ABA exists in its free form (Fig. 2). Conjugated forms of ABA have not been detected (data not shown). In the whole anther, the level of free ABA was similar at both Mi and Ma stages; however, at the MB stage, the free ABA value was 4.5-fold greater. In the case of isolated pollen, the maximum obtained at the MB stage was 8 and 40-fold greater with respect to Mi and Ma stages, respectively (Fig. 2). After the first mitosis of the N. tabacum microspore, a bicellular pollen grain is formed (Mascarenhas, 1989). The increase of endogenous ABA coincided with this cellular process. Kyo and Harada (1985) found that the addition of ABA enhanced pollen division in a pollen culture of Nicotiana tustica, and Saini and Aspinall (1982) reported that exogenous ABA or water stress decreased the rate of meiosis in wheat pollen mother cells. Isolated pollen and whole anther presented, as in the case of ethylene production (Chibi and Matilla, 1994), the same pattern with respect to ABA content in the three developmental stages studied; however, the minimum in ethylene production (in the MB stage) corresponded to a maximum in ABA content (Fig. 2). ABA inhibited embryogenesis in anther culture of Brassica oleraceae concomitantly with an increase in ethylene production (Biddington et al., 1993). In N. tabacum, the strong decrease of ABA from the MB to Ma stage (Fig. 2), together with other gene processes (Mas-
ABA and proline during pollen maturation
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Fig. 2: Variations in ABA content of whole anther (.) and its pollen content (0) during the microspore, mid-binucleate and mature stages of N. tabacum. Data are the mean of 3-4 experiments ± SD.
carenhas, 1990, 1993), could allow germination upon inhibition. The accumulation of proline is related to the mechanism of cell survival under stress conditions (Delauney and Verma, 1993). Dessication (e.g. pollen dessication) may be included in this kind of stress (Aspinall, 1980). However, little is known about proline levels and their possible functions during the reproductive process under stress conditions. In N tabacum pollen, it has been established that during the maturation phase, the pollen grain undergoes a progressive water loss, a decrease in its volume and an increase in the sucrose content resulting from starch degradation (Hoekstra et al., 1988; Mascarenhas, 1989; Tupy et al., 1992). In addition, we quantified endogenous proline in anthers as well as in isolated pollen (Fig. 3). In the whole anther, proline was scarcely detected at the stage of tetrate (T); then, a burst of proline concentration occurred in the transition from Mi to MB stages, decreasing 50 % in the Ma stage. By contrast, proline levels in the pollen of the anther increased progressively with development, being at MB and Ma 5 and ll-fold greater, respectively, than at the Mi stage (Fig. 3). The relationship between ABA and proline accumulation has previously been described in other plant systems (Pesci and Reggianni, 1992; Delauney and Verma, 1993; Angosto and Matilla, 1993). In the present work: (a) similar patterns of proline and ABA accumulation were found in the whole anthers, (b) ABA was preferentially accumulated in MB pollen, whereas the maximum accumulation of proline (Ma stage) coincided with the minimum level of ABA, and (c) the sporophytic tissue of the anther contained higher amounts of proline at the MB stage than at the Mi and Ma stages, while ABA reached its minimum at the MB stage for the entire developmental period studied. The germination of pollen grains of N tabacum matured in situ was inhibited by exogenous ABA and stimulated by fluridone (Fig. 4). On the other hand, we observed that the length of pollen tubes of those germinated pollen was increased by ABA (data not shown). These results are in keeping with the data obtained by Sindhu et al. (1986), who reported a stimulation of Hippeastrum vivatum pollen tube elongation at concentrations of ABA between 1-10 mg L -1, and indicated that ABA is involved in the regulation of emergence and elongation of
MB
Mo Fig. 3: Variations in proline content during the tetrade, microspore, mid-binucleate and mature stages of whole anthers (.) and its pollen content (0) of N. tabtU:um. Data are the mean of three different experiments ± SD. T
Mi
60
+
40
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+Fluridone
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.~
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Fig. 4: Effect of ABA and fluridone on germination percentage of in situ matured pollen of N. tabacum. ABA or fluridone were added in GK medium and the emergence of pollen tube measured. Data are the mean of three different experiments ± SD.
the pollen tube in the same way as putrescine (Chibi et al., 1994). Unfortunately, the role of the sporophytic tissue in controlling the source-sink relationship between the sporophytic tissue itself and the pollen grain during the in situ maturation process is still unknown. The in vitro maturation of the Mi (Stauffer et al., 1991; Tupy et al., 1992) or MB pollen (Chibi et al., 1993) presents an appropriate system to study the influence of the sporophytic tissue on the pollen grain development. We used this approach to examine the role of polyamines and ethylene in the maturation process of N tabacum pollen (Chibi et al., 1993; Chibi and Matilla, 1994; Chibi et al., 1994), but it was not possible to measure ABA due to the difficulty of obtaining sufficient amounts of pollen. The addition of ABA or fluridone to the maturation or germination media showed no effect on both processes (data not shown). On the basis of these results, we can hypothesize that in N tabacum the pool of ABA at the MB stage was sufficient to carry out the pollen maturation process without the necessity of ongoing ABA biosynthesis, and that, similar to «in situ», ABA should be synthesized during the germination process, confering to ABA an important role in development, maturation and germination of N tabacum pollen.
358
FATIHA CHIBI, TRINIDAD ANGOSTO, and ANGEL MATILLA
Acknowledgements
This research was supported by a Grant from La Direcci6n General de Investigaci6n Cientffica y Desarrollo Tecnol6gico (DGICYT), Spain, PB 93/1112.
References ANGOSTO, T. and A. J. MATILLA: Variations in seeds of three endemic leguminous species at different altitudes. Physiol. Plant. 87,329-334 (1993). ASPINALL, D.: Role of abscisic acid and other hormones in adaptation to water stress. In: TURNER, N. C. and P. J. KRAMER (eds.): Adaptation of plants to water and high temperature stress. Wiley & Sons, New York, pp. 155-172 (1980). BENITo-MORENO, R. M., F. MACKE, A. ALWEN, and E. HEBERLE-BoRS: In situ seed production after pollination with in vitro matured, isolated pollen. Planta 176,145-148 (1988). BIDDINGTON, N. 1., H. T. ROBINSON, and J. R. LYNN: ABA promotion of ethylene production in anther culture of Brussels sprouts (Brassica oleracea var. gemmifera) and its relevance to embryogenesis. Physiol. Plant. 88, 577-582 (1993). BREWBAKER, J. 1. and B. H. KWACK: The essential role of calcium ion in pollen germination and pollen tube growth. Am. J. Bot. 50, 859-865 (1963). CHIBI, F., T. ANGOSTO, D. GARRIDO, and A J. MATILLA: Requirement of polyamines for in vitro maturation of the mid-binucleate pollen of Nicotiana tabacum. J. Plant Physiol. 142, 452-456 (1993). CHIBI, F. and A. J. MATILLA: The involvement of ethylene in the in vitro maturation of mid-binucleate pollen of Nicotiana tabacum. J. Exp. Bot. 45, 529-532 (1994). CHIBI, F., A. J. MATILLA, T. ANGOSTO, and D. GARRIDO: Changes in polyamine synthesis during anther development and pollen germination in tobacco (Nicotiana tabacum). Physiol. Plant. 92, 61-68 (1994). DAVIES, W. J. and J. ZHANG: Root signals and the regulation of growth and development of plants in drying soil. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 55-76 (1991). DELAUNEY, A J. and D. P. S. VERMA: Proline biosynthesis and osmoregulation in plants. The Plant Journal 4, 215-223 (1993). GARRIDO, D., B. CHARVAT, R. M. BENITo-MORENO, A. ALWEN, O. VICENTE, and E. HEBERLE-BoRS: Pollen culture for haploid plant production in tobacco. In: NEGRUTIU, I. and G. GHARTI-CHHETIlI (eds.): A laboratory guide for cellular and molecular plant biology. Birkhauser, Basel, pp. 59-69 (1991). GARRIDO, D., F. CHIBI, and A. MATILLA: Polyamines in the induction of Nicotiana tabacum pollen embryogenesis by starvation. J. Plant Physiol. 145,731-735 (1995). HOEKSTRA, F. A, T. VON ROEKEL, N. TEN PAS: Pollen maturation and desiccation tolerance. In: CIlESTI, M., GoIll, P., and E. PACINI (eds.): Sexual Reproduction in Higher Plants. Springer, Berlin, pp. 291-296 (1988).
Kyo, M. and H. HARADA: Studies on conditions for cell division and embryogenesis in isolated pollen culture of Nicotiana rustica. Plant Physiol. 79, 89-94 (1985). MANSFIELD, T. A., A. M. HETHERINGTON, and C. J. ATKINSON: Some current aspects of stomatal physiology. Ann. Rev. Plant Physio!. Plant Mol. Biol. 41, 55-75 (1990). MASCAIlENHAS, J. P.: The male gametophyte of flowering plants. The Plant Cell 1, 657 -664 (1989). - Gene activity during pollen development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 41, 317 -338 (1990). - Molecular mechanisms of pollen tube growth and differentiation. The Plant Cell 5, 1303-1314 (1993). PESCI, P. and R. REGGIANI: The process of abscisic acid-induced proline accumulation and the levels of polyamines and quaternary ammonium compounds in hydrated barley leaves. Physiol. Plant. 84, 134-139 (1992). QUATRANO, R. S.: The role of hormones during seed development. In: DAVIES, P. J. (ed.): Plant Hormones and their role in plant growth and development. Martinus Nijhoff Publishers, The Netherlands, pp. 494-514 (1987). SAINI, H. S. and D. ASPINALL: Sterility in wheat (Triticum aestivum 1.) induced by water deficit or high temperature, possible mediation by abscisic acid. Aust. J. Plant Physiol. 9, 529 - 537 (1982). SCHLONG, H. and J. 1. GELLERMAN: Sterification of fatty acids with diazomethane on a small scale. Anal. Chem. 32, 1412-1414 (1960). SHIBUYA, T., M. FUNAMIZU, and Y. KrrAHARA: Abscisic acid from Pi· nus densiflora pollen. Phytochem. 17, 322-323 (1978). SINDHU, R. K., A. S. BASRA, and C. P. MALIK: Hormonal effects on tube elongation, 14COZ fixation and phosphoenolpyruvate carboxylase activity in amaryllis pollen: promotion by abscisic acid. Plant Growth Regul. 4, 293-298 (1986). SKIVER, K. and J. MUNDY: Gene expression in response to ABA and osmotic stress. Plant Cell 2, 503-512 (1990). STAUFFER, c., R. M. BENITO-MORENO, and E. HEBERLE-BoRS: Seed set after pollination with in vitro matured, isolated pollen of Triti· cum aestivum. Theor. Appl. Genet. 81, 576-580 (1991). Tupy, J., 1. RIHovA, V. CAPKOVA, and V. ZARSKY: Differentiation and maturation of tobacco pollen in situ and in suspension culture. In: OTTAVIANO, E., D. 1. MULCAHY, M. SAIlI GOIlLA, and G. BERGAMINI MULCAHY (eds.): Angiosperm pollen and ovules. Springer, New York, pp. 309-314 (1992). VAUGHAN, G. T. and B. V. MILBOIlIlOW: The resolution HPLC of RS-{2.14C)Me-1' ,4'-eis-diol of abscisic acid on the metabolism of (- )-R- and (+ )-S-abscisic acid. J. Exp. Bot. 35, 110-120 (1984). WEBBER, J. E.: The role of plant growth regulators in the development and germination of conifer pollen. Plant Growth Regul. 6, 217 -236 (1988). ZEEVAIlT, J. A. D. and R. A. CIlEEMAN: Metabolism and physiology of abscisic acid. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39, 439-473 (1988).
Vol. 147. pp. 355 -358 (1995)
Variations of the Patterns of Abscisic Acid and Proline during Maturation of Nicotiana tabacum Pollen Grains FATIHA CHIBI, TRINIDAD ANGOSTOt,
and ANGEL MATILLA*
Departamento de Biologfa Vegetal, Universidad de Granada, 18001-Granada I E. Politecnica Superior, Universidad de Almeda, 0412C-Almena, Spain .. For correspondence Received March 8,1995' Accepted July 20,1995
Summary
Abscisic acid (ABA) and proline were evaluated during in situ development of anthers and pollen grains of Nicotiana tabaeum. No conjugated forms of ABA were detected. The maximum accumulation of free ABA in the whole anther occurred at the mid-binucleate stage (MB), almost 95 % of the ABA being located in the pollen; this ABA distribution was not observed in the microspore (Mi) or mature (Ma) stages of the anther, in which the sporophytic tissue provided more ABA than did the pollen. A maximum in proline accumulation was also found at the MB stage of the anther; however, at the Ma pollen stage (when the ABA was scarcely detectable) the proline accumulation was maximum. The germination of the pollen grain was inhibited by exogenous ABA and stimulated by fluridone, a compound that inhibits carotenoid biosynthesis. The results of the present work suggest an important role for ABA in maturation and germination, as well as in the elongation of the pollen tube of N. tabacum.
Key words: Abscisic acid, anther, germination, maturation, Nicotiana tabacum, pollen, proline. Abbreviations: ABA - abscisic acid; AMGLu - maturation medium; GK Ma - matured; Mi - microspore; MB = mid-binucleate; T z tetrade.
Introduction
Abscisic acid (ABA), like other plant hormones, has multiple roles during the life cycle of a plant (Zeevaart and Creelman, 1988). So, stomatic control (Mansfield et al., 1990), water relations (Davies and Zhang, 1991), proline biosynthesis and osmoregulation (Skiver and Mundy, 1990; Delauney and Verma, 1993), and embryogenesis, dormancy and germination of seeds (Quatrano, 1987), among the physiological processes, are under ABA control. In most fruits, seeds or embryos, changes in ABA levels during development show a similar pattern: a fairly steep rise during development, followed by a sharp drop as the seeds mature (Quatrano, 1987). However, even though ABA has been detected in the reproductive apparatus (v.e. pollen, © 1995 by Gustav Fischer Verlag, Stuttgart
=
germination medium;
styles and ovary) of various species of Angiosperms and Conifers (Shibuya et al., 1978; Webber, 1988), nothing until now was known about its alteration during the development of pollen grain, perhaps because of the difficulty of obtaining sufficient amounts for ABA quantification. Recently, we have demonstrated that ethylene and polyamines are involved in «in vivo. and «in vitro. development of Nicotiana tabaeum pollen (Chibi et al., 1993; 1994; Chibi and Matilla, 1994), and also during its embryogenesis (Garrido et al., 1995). As a preliminary approach, we quantified endogenous levels of ABA and free-proline in intact anthers as well as in isolated pollen grains of N. tabacum during different stages of development in order to gain further information about the role that ABA could play in anther and pollen development.
356
FATIHA CHffiI, TRINIDAD ANGOSTO, and ANGEL MATILLA
Materials and Methods
Plant material Nicotiana tabacum plants were grown in a climate chamber under a 16 h light period at 25°C. Anthers and pollen were collected at different developmental stages: microspore (Mi), mid-binucleate (MB) and mature (Ma) (Fig. 1). For determining the stage of pollen development, one anther was squashed and the released pollen was stained with a droplet of 4 % carmine in 45 % propionic acid and viewed under a light microscope. Two min later, the precise stage of development was determined (Garrido et al., 1991).
~
.
FW
-6
The pollen grains matured in situ were cultured for 2 h in GK medium (Brewbaker and Kwack, 1963) modified to contain a double concentration of boric acid (Benito-Moreno et al., 1988). The number of pollen grains with growing tubes were counted under an invened microscope. More than 1000 pollen grains were counted for each run. ABA (1 to 20 11M) or fluridone (1 to 50 11M) solutions were added to GK in order to examine the effect of these substances on the germination process.
''-
~
I,D ~ o
~ -o
~c
C>I~
.c. '\-,
; -ECll
-
c
C>I
4
o
h Mi
Pollen germination
0
1,5
0.5
E~
'-'--
MB
Me
Fig. 1: Variations in fresh weight of whole anther (.) and its pollen content (D) during the microspore, mid-binucleate and mature stages of N. tabacum. Data are the mean of 5-6 experiments ± SD. to room temperature and the absorbance read at 520 nm using toluene as a blank.
Results and discussion
Quantification ofendogenous ABA Abscisic acid was extracted from intact anthers or isolated pollen at different stages of development according to Vaughan and Milborrow (1984) with several modifications (Angosto and Matilla, 1993). All steps were carried out under green light to minimize isomerization. To anthers or isolated pollen samples, the following were added: 10 mL acetone: acetic acid (99: 1), v/v) containing 2-6di-t-butyl-4-methyl phenol (BHT, 10 I1gmL -1); 1ilL HC-ABA (422 GBqmol-t, 740KBqmL-t, Amersham) and indolbutyric acid (10 mg) as an internal standard. The mixture was ground in a mortar and centrifuged at 27,000go for 15min at 4°C. The pellet was extracted twice more with acetone: acetic (99: 1, v/v). The combined supernatants were evaporated to dryness in a rotavapor at 35°C and the dried residue was dissolved in 30 mL ethanol: acetic acid (0.2 %; 1: 1, v/v), filtered through four C l8 Sep-Pack (Waters) filters and then concentrated in a rotavapor to 5 mL. This volume was extracted three times with cold ethyl ether, the free ABA being in the ether phase and the bound ABA in the aqueous phase. The aqueous phase was hydrolyzed for 30 min at 60°C, pH 11.0 (adjusted with 1 mol/L NaOH), and when cold, was adjusted to pH 3.0 with 1mol/L HCI and fractionated once more with ethyl ether as described above. ABA was methylated with ice-cold etheric diazomethane (Schlong and Guellerman, 1960) and analysed by gas chromatography combined to mass spectrometry (GC-MS). The GC capillary column (12 m x 0.33 mm) was packed with methyl-silicone (panicle size 0.25I1mol/L) and operated at 200 °C and 250°C in the injector and detector, respectively; hydrogen was used as the GC-carrier gas. The gas chromatography was combined with mass spectrometry, and atomic-absorption spectrometry (GC-M5-AAS) gave spectra similar to those of commercial ABA (Sigma, Chern. Co).
Free.proline determination Isolated pollen or whole anthers from different stages were homogenized in 1 mL 3 % aqueous sulphosalicylic acid and centrifuged at 1,000 x g. One mL of supernatant was reacted with 1 mL acid ninhydrin and 1 mL glacial acetic acid in a test tube for 60 min at 100 0C. The reaction was stopped in an ice bath. The mixture was extracted with 4 mL toluene and mixed with a test-tube stirrer for 15 sec. The chromophore containing toluene was separated, warmed
As the anther of N. tabacum was about to reach maturity, its FW decreased slowly, while that of its pollen content increased 4-fold from the Mi to Ma stages (Fig. 1). In recent publications, we demonstrated that the processes related to the development and maturation provoked: (a) a decrease in both arginine-decarboxylase (ADC) and ornithine-decarboxylase (ODC) activities (Chibi et al., 1994), (b) important alterations in the levels of free and bound-polyamines in the anthers (Chibi et al., 1994) as well as in the isolated pollen grains (Chibi et al., 1993), and (c) an increase in ethylene production (Chibi and Matilla, 1994). In the present work, we demonstrate that in the three (Mi, MB and Ma) developmental stages of N. tabacum anthers studied, ABA exists in its free form (Fig. 2). Conjugated forms of ABA have not been detected (data not shown). In the whole anther, the level of free ABA was similar at both Mi and Ma stages; however, at the MB stage, the free ABA value was 4.5-fold greater. In the case of isolated pollen, the maximum obtained at the MB stage was 8 and 40-fold greater with respect to Mi and Ma stages, respectively (Fig. 2). After the first mitosis of the N. tabacum microspore, a bicellular pollen grain is formed (Mascarenhas, 1989). The increase of endogenous ABA coincided with this cellular process. Kyo and Harada (1985) found that the addition of ABA enhanced pollen division in a pollen culture of Nicotiana tustica, and Saini and Aspinall (1982) reported that exogenous ABA or water stress decreased the rate of meiosis in wheat pollen mother cells. Isolated pollen and whole anther presented, as in the case of ethylene production (Chibi and Matilla, 1994), the same pattern with respect to ABA content in the three developmental stages studied; however, the minimum in ethylene production (in the MB stage) corresponded to a maximum in ABA content (Fig. 2). ABA inhibited embryogenesis in anther culture of Brassica oleraceae concomitantly with an increase in ethylene production (Biddington et al., 1993). In N. tabacum, the strong decrease of ABA from the MB to Ma stage (Fig. 2), together with other gene processes (Mas-
ABA and proline during pollen maturation
o
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Mo
Fig. 2: Variations in ABA content of whole anther (.) and its pollen content (0) during the microspore, mid-binucleate and mature stages of N. tabacum. Data are the mean of 3-4 experiments ± SD.
carenhas, 1990, 1993), could allow germination upon inhibition. The accumulation of proline is related to the mechanism of cell survival under stress conditions (Delauney and Verma, 1993). Dessication (e.g. pollen dessication) may be included in this kind of stress (Aspinall, 1980). However, little is known about proline levels and their possible functions during the reproductive process under stress conditions. In N tabacum pollen, it has been established that during the maturation phase, the pollen grain undergoes a progressive water loss, a decrease in its volume and an increase in the sucrose content resulting from starch degradation (Hoekstra et al., 1988; Mascarenhas, 1989; Tupy et al., 1992). In addition, we quantified endogenous proline in anthers as well as in isolated pollen (Fig. 3). In the whole anther, proline was scarcely detected at the stage of tetrate (T); then, a burst of proline concentration occurred in the transition from Mi to MB stages, decreasing 50 % in the Ma stage. By contrast, proline levels in the pollen of the anther increased progressively with development, being at MB and Ma 5 and ll-fold greater, respectively, than at the Mi stage (Fig. 3). The relationship between ABA and proline accumulation has previously been described in other plant systems (Pesci and Reggianni, 1992; Delauney and Verma, 1993; Angosto and Matilla, 1993). In the present work: (a) similar patterns of proline and ABA accumulation were found in the whole anthers, (b) ABA was preferentially accumulated in MB pollen, whereas the maximum accumulation of proline (Ma stage) coincided with the minimum level of ABA, and (c) the sporophytic tissue of the anther contained higher amounts of proline at the MB stage than at the Mi and Ma stages, while ABA reached its minimum at the MB stage for the entire developmental period studied. The germination of pollen grains of N tabacum matured in situ was inhibited by exogenous ABA and stimulated by fluridone (Fig. 4). On the other hand, we observed that the length of pollen tubes of those germinated pollen was increased by ABA (data not shown). These results are in keeping with the data obtained by Sindhu et al. (1986), who reported a stimulation of Hippeastrum vivatum pollen tube elongation at concentrations of ABA between 1-10 mg L -1, and indicated that ABA is involved in the regulation of emergence and elongation of
MB
Mo Fig. 3: Variations in proline content during the tetrade, microspore, mid-binucleate and mature stages of whole anthers (.) and its pollen content (0) of N. tabtU:um. Data are the mean of three different experiments ± SD. T
Mi
60
+
40
r+
+Fluridone
ABA
~ o
.§ o
+
c:
.~
rt
20
t:» CI
o
.... 20
I
10
L..
50
cone ent rotionlpM)
Fig. 4: Effect of ABA and fluridone on germination percentage of in situ matured pollen of N. tabacum. ABA or fluridone were added in GK medium and the emergence of pollen tube measured. Data are the mean of three different experiments ± SD.
the pollen tube in the same way as putrescine (Chibi et al., 1994). Unfortunately, the role of the sporophytic tissue in controlling the source-sink relationship between the sporophytic tissue itself and the pollen grain during the in situ maturation process is still unknown. The in vitro maturation of the Mi (Stauffer et al., 1991; Tupy et al., 1992) or MB pollen (Chibi et al., 1993) presents an appropriate system to study the influence of the sporophytic tissue on the pollen grain development. We used this approach to examine the role of polyamines and ethylene in the maturation process of N tabacum pollen (Chibi et al., 1993; Chibi and Matilla, 1994; Chibi et al., 1994), but it was not possible to measure ABA due to the difficulty of obtaining sufficient amounts of pollen. The addition of ABA or fluridone to the maturation or germination media showed no effect on both processes (data not shown). On the basis of these results, we can hypothesize that in N tabacum the pool of ABA at the MB stage was sufficient to carry out the pollen maturation process without the necessity of ongoing ABA biosynthesis, and that, similar to «in situ», ABA should be synthesized during the germination process, confering to ABA an important role in development, maturation and germination of N tabacum pollen.
358
FATIHA CHIBI, TRINIDAD ANGOSTO, and ANGEL MATILLA
Acknowledgements
This research was supported by a Grant from La Direcci6n General de Investigaci6n Cientffica y Desarrollo Tecnol6gico (DGICYT), Spain, PB 93/1112.
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