Flavonols Stimulate Development, Germination, and Tube Growth of ...

Received for publication February 11, 1992 Accepted February 21, 1992

Plant Physiol. (1992) 100, 902-907 0032-0889/92/100/0902/06/$01 .00/0

Flavonols Stimulate Development, Germination, and Tube Growth of Tobacco Pollen' Bauke Ylstra2, Alisher Touraev, Rosa Maria Benito Moreno, Eva Stoger, Arjen J. van Tunen, Oscar Vicente, Joseph N. M. Mol, and Erwin Heberle-Bors* Institute of Microbiology and Genetics, University of Vienna, Vienna Biocenter, Dr. Bohrgasse 9, 1030 Vienna, Austria (B.Y., A.T., R.M.B.M., E.S., O.V., E.H.-B.); and Department of Genetics, Free University Amsterdam, de Boelelaan 1087, 1081 HV Amsterdam, The Netherlands (A.J.v.T., J.N.M.M.)

ABSTRACT

expressed in the anther, with a peak of expression in the mid-binucleate stage of pollen development (8, 23-25). A similar expression pattern was also observed in transgenic tobacco plants (24; B. Ylstra, E. Stoger, unpublished observation). Petunia plants lacking chs gene expression are male sterile (19, 21), indicating that anther-derived flavonoids may play a role in male gametogenesis. In vitro culture of isolated microspores has shown that an important function of the anther for the development of the microspores and pollen grains is the stepwise provision of low mol wt nutrients and anabolic precursors (2, 4, 18, 20). Germination frequency and seed set of this pollen are, however, lower compared with mature pollen taken directly from the plant (2, 20). Obviously, in vitro pollen lacks factors that are provided in vivo by the sporophyte. The in vitro pollen can thus be considered as 'minimal pollen' that fulfills the minimal requirements for pollination and fertilization but lacks factors for optimal reproductive success (28). It should, therefore, react very sensitively to added compounds that in vivo are provided by the anther wall. Here, we show that flavonols but not other flavonoids produced by the anther are present in diffusates of mature tobacco (Nicotiana tabacum L.) pollen and have a strong stimulatory effect on in vitro pollen development, pollen germination, and pollen tube growth.

The effect of anther-derived substances on pollen function was studied using pollen produced by in vitro culture of immature pollen of tobacco (Nicotiana tabacum L.) and petunia (Petunia hybrida). Addition of conditioned medium consisting of diffusates from in situ matured pollen strongly increased pollen germination frequency and pollen tube growth, as well as seed set after in situ pollination. Thin-layer chromatography and depletion of phenolic substances by Dowex treatment indicated that flavonols are present in the diffusate and may be the active compounds. When added to the germination medium, flavonols (quercetin, kaempferol, myricetin) but not other flavonoids strongly promoted pollen germination frequency and pollen tube growth in vitro. The best results were obtained at very low concentrations of the flavonols (0.151.5 Mm), indicating a signaling function. The same compounds were also effective when added during pollen development in vitro.

Male gametophyte and gamete formation in plants occurs by a close interaction with the surrounding sporophytic tissues, particularly the tapetum (1, 14, 28). A variety of factors have been suggested to play a role in this interaction. However, their function is still largely unknown. Flavonoids are secondary plant products that include pigments (chalcones, anthocyanins) and colorless compounds (flavanones, flavones, and flavonols) that are involved in pollination, seed dispersal, UV light protection, and plant/ pathogen interaction (9, 11, 23). Flavonoids are present in pollen of many species of angiosperms and gymnosperms, as well as in spores of ferns and mosses (29). Flavonoid biosynthesis is initiated by chalcone synthase, followed by the synthesis of flavanones by chalcone isomerase. Recently, it was shown that the chs and chi genes are coordinately

MATERIALS AND METHODS In Vitro Culture of Immature Pollen

Microspores or young binucleate pollen grains were isolated from tobacco (Nicotiana tabacum L. cv Petit Havana SR1) and petunia (Petunia hybrida cv Wi 15) flowers. Microspores were cultured in a MR26 medium consisting of the Murashige-Skoog (16) minerals, 0.5 M sucrose, 3 mm glutamine, 100 mg. L` of inositol, 2% coconut water, and 1 g* L` of lactalbumin hydrolysate (pH 7) in wells (330 ,L of pollen suspension at a density of 105 grains/mL) of tissue clusters 24 (Costar, Cambridge, MA) at 280C in darkness. Young binucleate pollen grains were cultured in a AMGLU medium consisting of Murashige-Skoog minerals, 0.5 M sucrose, and 3 mm glutamine (pH 7) to maturity following the procedure of Benito Moreno et al. (2). Maturity was judged by microscopical analysis (33 ,m in diameter, fully packed with starch

l This work was supported by the Austrian 'Fonds zur Forderung der wissenschaftlichen Forschung', the 'Forschungsforderungsfonds fur die gewerbliche Wirtschaft, and 'btF-biotechnologische Forschungsges. m.b.H.,' Linz, Austria. B.Y. was partly financed by a Stimuleringsprogramma voor Intemaltionalisering van het Hoger Onderurijs TIR grant of the Dutch Ministry of Education and Science. 2 Present address: Center for Plant Breeding and Reproduction Research (CPRO-DLO), P.O. Box 16, 6700 AA Wageningen, The Netherlands.

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FLAVONOLS AND POLLEN

grains) and by germination ability in a germination medium (Brewbaker-Kwack [3] minerals with a double amount of boric acid, 10% sucrose, GK medium). In situ pollination with the in vitro matured pollen was done as described by Benito Moreno et al. (2). To obtain a homogeneous population of mid-binucleate tobacco pollen (no starch grains, nucleolus in vegetative nucleus present, generative cell detached from intine), pollen was isolated from flower buds of 18 to 19 mm in length into a solution of 0.4 M mannitol. After two washings, the pollen suspension was layered on top of a 60% Percoll in 0.4 M mannitol solution and was centrifuged at 300g for 15 min. The remaining pollen on top of the Percoll solution was removed, washed three times, and cultured in AMGLU medium with or without flavonoids.

903

medium and was then gently shaken for 10 min. The resin removed by centrifugation, and the medium was filter sterilized. The conditioned medium was dialyzed (Spectra POR 1, mol wt cutoff 6000-8000) twice for 8 h against 100 volumes of fresh GK medium at 0°C. For heat inactivation, the samples were heated for 10 min at 800C. was

Flavonoids Naringenin chalcone (4,2',4',6'-tetrahydroxychalcone [21]), naringin, and naringenin were isolated from petunia flowers (8). Quercetin (3,3',4',5,7-pentahydroxyflavone dihydrate), kaempferol [3,5,7-trihydroxy(4-hydroxyphenyl)-4H 1-benzopyran-4-one] and myricetin (3,3',4',5,5',7-hexahydroxyflavone) were purchased from Sigma. All flavonoids were dissolved in DMSO immediately before addition to the media. In control experiments, no effect of DMSO (.

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To identify more precisely the type of compounds involved in growth stimulation of tobacco pollen tubes, a TLC analysis was performed. Flavonoids are known to be present in pollen (29), and in fact, TLC revealed the presence in the conditioned medium of compounds with the mobility and color of the flavonols quercetin and kaempferol used as standards (Fig. 3). To show directly the effect of flavonols on pollen tube growth, various flavonols and other flavonoids were added to minimal pollen produced by in vitro culture of isolated microspores. All of the flavonols tested (quercetin, kaempferol, myricetin) had a strong stimulatory effect on germina-

tion frequency and pollen tube length (Figs. 1, top and bottom, and 4) at concentrations ranging from 0.15 to 1.5 A-M. Maximum restoration of the effect of the conditioned medium was 80%. The other flavonoids tested (naringenin chalcone, naringin, naringenin) had no effect (data not shown). Addition of the tobacco pollen diffusate and of flavonols to in vitro matured petunia pollen also had a stimulatory effect on pollen germination (data not shown). Petunia pollen, however, usually gave a less efficient response. The poor synchronism of in vivo pollen development in this species did not allow homogeneous starting populations. Also, the in vitro culture conditions established for tobacco pollen may not have been adequate for petunia pollen.

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Figure 1. Minimal pollen germinating overnight in control GK medium (top), in conditioned GK medium made from pollen isolated from closed anthers by 5-min incubation (middle), and in GK medium containing 1.5 AM quercetin (bottom).

served, indicating that heat-labile substances (including most proteins) were not directly involved in growth control of tobacco pollen tubes (Fig. 2A). Dialysis produced a reduction of the effect of the conditioned medium, suggesting that low mol wt substances are involved in growth control. The effect of dialysis was, however, small. Depletion of compounds from the conditioned medium by incubation in the presence of a Dowex resin that can bind phenolic substances reduced pollen germination frequency and pollen tube growth to the levels of the nonconditioned germination medium serving as a control (Fig. 2B).

Flavonols Stimulate in Vitro Pollen Development Flavonols were also tested for their effect on tobacco pollen development in vitro. A highly homogeneous population of mid-binucleate pollen produced by density centrifugation in Percoll was cultured in AMGLU medium, and aliquots of pollen suspension were transferred to germination medium at different time intervals. Germination frequency was determined 10 h after transfer to germination medium. After 48 h of development, no pollen tubes were seen, indicating that the pollen was not yet mature (Table II). After 60 h of pollen development in control medium, some pollen (6.2%) germinated, whereas pollen developed in quercetin-containing medium germinated at a frequency of 40.9%. Pollen tubes of quercetin-treated pollen were longer than in the control (Fig. 5). After 72 h, 25.5% of the pollen developed in control medium germinated, whereas 59.9% germinated when pollen development took place in quercetin-containing medium. Kaempferol and myricetin also stimulated pollen development but to a lower extent. These results indicate that midbinucleate pollen reach maturity (maximum germination frequency) within 72 h of in vitro culture and that flavonols reduce the time required to reach maturity. DISCUSSION The data presented in this article strongly suggest that the effect of pollen diffusates on pollen germination and tube growth is largely due to the presence of flavonols in mature pollen. Other flavonoids such as chalcones, flavanones, and flavones had no such effect. We have shown that the low mol wt flavonoid compounds stimulate pollen germination and tube growth. On the other hand, removal of small molecules by dialysis reduced the germination-promoting

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Figure 2. A, Effect of dialyzed or heat-treated conditioned media (made from pollen from closed anthers by 5-min incubation) on germination of minimal pollen. Germination frequency, average; pollen tube length, average ± SD. B, Effect of Dowex-treated conditioned medium (made from pollen from closed anthers by 5-min incubation) on germination of minimal pollen. Germination frequency, average; pollen tube length, average ± SD. preco., Preconditioned medium.

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effect of the diffusate only a little. This may indicate that high mol wt compounds, i.e. lipids or proteins (6), may be additional factors. Possibly, their effect is by binding the flavonols. An interesting finding was that the effective concentrations were very low, i.e. in the micromolar range. This fact, together with the chemical specificity, suggests that the flavonols act as signal molecules, similar to plant hormones. Even the third classical definition of a hormone, apart from effectiveness at low concentration and chemical specificity, applies to the flavonols: synthesis at one location and action at another. The genes for flavonoid biosynthesis in petunia and tobacco

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Figure 4. Effects of flavonols on germination of minimal pollen when added to the germination medium. Germination frequency, average; pollen tube length, average ± SD.

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YLSTRA ET AL.

Plant Physiol. Vol. 100, 1992

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Figure 5. Germination of minimal pollen in GK medium (after 6 h) that had developed for 60 h in AMGLU medium (left) and for 60 h in AMGLU medium plus 10 AM quercetin (right).

anthers are expressed in the sporophyte, i.e. in the anther wall tissues (22; B. Ylstra, E. Stbger, unpublished observation), and the flavonoids synthesized by the tapetal flavonoid enzymes are incorporated in the exine cavities of the pollen (30). Flavonoid action, however, is in the pollen grains after they have landed on a stigma, as suggested by their stimulatory effect on pollen germination. For these reasons, we suggest that during the development of the male gametophyte, flavonols act as plant growth regulators or plant hormones. It is interesting that the flavonols also had an effect on pregermination pollen development, indicating a second control point for flavonols. This coincides with the finding that chs-antisense mutants (with a chimeric anther-specific promoter) are male sterile caused by an arrest in pollen development (21). In chs-cosuppressor mutants (introduction of a chs transgene into wild-type petunia), seed set was prevented, whereas pollen development was apparently unaffected (19). Male fertility was restored when mutant pollen was used to pollinate wild-type stigmas, probably by uptake of flavonols synthesized by the stigma (conditional male fertility). We predict that restoration can also be achieved by using the socalled mentor effect (7), i.e. by mixing the mutant pollen with

wild-type pollen to pollinate mutant stigmas. Flavonols produced by the wild-type pollen should promote pollen tube growth of the mutant pollen, resulting in fertilization and seed set. Little is known about the mechanism of action of flavonols as signal molecules (11). Recently, Rubery (17) proposed that quercetin and other flavonoids bind as natural ligands to the phytotropin or N-1-naphthylphthalamic acid receptor (5, confirmed in ref. 26), which interacts with and controls the auxin efflux carrier in the plasma membrane involved in polar auxin transport (10, 15). We imagine that in pollen a block of the auxin efflux carrier by flavonols increases the intracellular concentration of auxin, which, in turn, promotes the polar tube growth. This view is corroborated by Vesper and Kuss (27) who found in the presence of N-1-naphthylphthalamic acid a stronger stimulation by IAA of elongation growth of maize coleoptiles. Alternatively, pollen tube growth promotion may result from the inhibition by flavonoids of oxidative destruction of IAA (12, 13), resulting again in an increased endogenous level of auxin. Further experiments are required to unravel the precise mode of action of flavonols in pollen development.

Table II. Germination Frequencya of in Vitro Pollenb after Three Time Intervals in the Presence of Flavonols (all 10 lsM) The data are means ± SE of three experiments; six Petri dishes were used for each treatment.

suggestions.

ACKNOWLEDGMENT We are thankful to Bernd Zbell, Heidelberg, Germany, for valuable

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LITERATURE CITED 1. Albertini L, Souvre A, Audran JC (1987) Le tapis de l'anthere et ses relations avec les microsporocytes et les grains de pollen. Rev Cytol Biol Veg Bot 10: 211-242 2. Benito Moreno RM, Macke F, Alwen A, Heberle-Bors E (1988) In situ seed production after pollination with in vitro matured isolated pollen. Planta 176: 145-148 3. Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50: 859-865 4. Heberle-Bors E (1989) Isolated pollen culture in tobacco: plant reproductive development in a nutshell. Sex Plant Reprod 2: 1-10

FLAVONOLS AND POLLEN 5. Jacobs M, Rubery PH (1988) Naturally occurring auxin transport regulators. Science 241: 346-349 6. Kirby EG, Vasil IK (1979) Effect of pollen-protein diffusates on germination of eluted pollen samples of Petunia hybrida in vitro. Ann Bot 44: 361-367 7. Knox RB, Gaget M, Dumas C (1987) Mentor pollen techniques. Int Rev Cytol 107: 315-332 8. Koes RE, van Blokland R, Quattrochio F, van Tunen AJ, Mol JNM (1990) Chalcone synthase promoters in Petunia hybrida are active in pigmented and unpigmented cell types. Plant Cell 2: 379-392 9. Kuhn DN, Chapall J, Boudet A, Hahlbrock K (1984) Induction of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase mRNAs in cultured plant cells by UV induction or fungal elicitor. Proc Natl Acad Sci USA 81: 1102-1106 10. Lembi CA, Morre DJ, Thomson K-S, Hertel R (1971) N-1naphthylphthalamic-acid-binding activity of a plasma membrane-rich fraction from maize coleoptiles. Planta 99: 37-45 11. Long S (1989) Rhizobium-legume nodulation: life together in the underground. Cell 56: 203-214 12. Marigo G, Boudet AM (1977) Relations polyphenols-croissance: mise an evidence d'un effect inhibiteur des composes phenoliques sur le transport polarise de l'auxine. Physiol Plant 41: 197-202 13. Marigo G, Boudet AM (1979) Effects of an increase in levels of phenolic compounds on the auxin content and growth of Lycopersicum esculentum. Z Pflanzenphysiol 92: 33-38 14. Mascarenhas JP (1990) Gene activity during pollen development. Annu Rev Plant Physiol Plant Mol Biol 41: 317-338 15. Michalke W, Schmieder B (1979) Fractionation of particulate material from maize coleoptile homogenates with polyethylene glycol. Planta 145: 129-135 16. Murashige T, Skoog E (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497 17. Rubery PH (1990) Phytotropins: receptors and endogenous ligands. Soc Exp Biol Symp 44: 119-146 18. Stauffer C, Benito Moreno RM, Heberle-Bors E (1991) Seed set after pollination with in-vitro-matured, isolated pollen of Triticum aestivum. Theor Appl Genet 81: 576-580

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19. Taylor LP, Jorgensen R (1992) Conditional male fertility in chalcone synthase-deficient petunia. J Hered 83: 11-17 20. Tupy J, Rihova L, Zarsky V (1991) Production of fertile tobacco pollen from microspores in suspension culture and its storage for in situ pollination. Sex Plant Reprod 4: 284-287 21. van der Meer IM, Stam ME, van Tunen AJ, Mol JNM, Stuitje AR (1992) Inhibition of flavonoid biosynthesis in petunia anthers by antisense approach results in male sterility. Plant Cell 4: 253-262 22. van Tunen AJ, Mol JNM (1987) A novel purification procedure for chalcone flavanone isomerase from Petunia hybrida and the use of its antibodies to characterize the Po mutation. Arch Biochem Biophys 257: 85-91 23. van Tunen AJ, Mol JNM (1990) Control of flavonoid synthesis and manipulation of flower colour. In D Grierson, ed, Plant Biotechnology Series. Blacky and Son, Glasgow, Scotland, pp 94-130 24. van Tunen AJ, Mur LA, Brouns GS, Rienstra J-D, Koes RE, Mol JNM (1990) Pollen and anther-specific chiA and B promoters from Petunia hybrida: tandem promoter regulation of chiA gene expression. Plant Cell 2: 393-401 25. van Tunen AJ, Mur LA, Recourt K, Gerats AGM, Mol JNM (1991) Regulation and manipulation of flavonoid gene expression in anthers of Petunia: the molecular basis of the Pomutation. Plant Cell 3: 39-48 26. Venis MA (1991) Recent progress with auxin and phytotropin receptors. In M Kutacek, MC Elliot, I Machackova, eds, Molecular Aspects of Hormonal Regulation of Plant Development. SPB Academic Publishing bv, The Hague, The Netherlands, pp 177-184 27. Vesper MJ, Kuss CL (1990) Physiological evidence that the primary site of auxin action in maize coleoptiles is an intracellular site. Planta 182: 486-491 28. Vicente 0, Benito Moreno RM, Heberle-Bors E (1991) Pollen cultures as a tool to study plant development. Cell Biol Rev 25: 295-305 29. Wiermann R (1968) Untersuchungen zum Phenylpropanstoffwechsel des Pollens. Ber Dtsch Bot Ges 81: 232-238 30. Wiermann R, Vieth K (1983) Outer pollen wall, an important accumulation site for flavonoids. Protoplasma 118: 230-233