Plant Physiol. (1 995) 107: 639-643

Rapid Communication

Steroid Hormones Stimulate Germination and Tube Growth of in Vitro Matured Tobacco Pollen'

Bauke Ylstra, Alisher Touraev, Albert O. Brinkmann, Erwin Heberle-Bors, and Arjen J. van Tunen*

Department of CelI Biology, DLO-Centre for Plant Breeding and Reproduction Research, P.O. Box 16, 6700 AA Wageningen, The Netherlands (B.Y., A.J.v.T.); lnstitute for Microbiology and Genetics,

University of Vienna, Dr. Bohrgasse 9, A-1 030 Vienna, Austria (A.T., E.H.-B.); and Department of Endocrinology and Reproduction, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam,

The Netherlands (A.O.B.)

A study of the effects of different steroids on germination and tube growth of tobacco pollen (Nicofiana fabacum 1. cv Petit Havana SRI) matured in vitro is presented. Application of the mammalian steroid sex hormones (testosterone, progesterone, and estradiol) resulted in a stimulation of pollen germination and tube elongation. The presence of both steroids and flavonols in the germination medium strongly enhanced the growth of tobacco male gametophytes.

During the process of plant fertilization, a pollen grain lands on the stigma of a receptive pistil, germinates, and forms a tube. This pollen tube grows through the transmit- ting tissue of the style until it reaches an ovule. Subse- quently, the tube unloads at its apex, and the two sperm cells fuse with the female reproductive cells in a double- fertilization event. Much is known about the cytology and histology of female and male gametophyte development, whereas only limited information is available about phys- iological, biochemical, and molecular aspects of these pro- cesses (for reviews, see Mascarenhas, 1993; Russell, 1993).

It has been shown that a specific class of small phenolic compounds present in the pollen exine, the flavonols, plays an important role during plant fertilization and that fla- vonols stimulate in vitro germination and tube growth of tobacco (Nicotiana fabacum) pollen matured in vitro (Ylstra et al., 1992). Genetic suppression of flavonoid biosynthesis in the reproductive organs of transgenic petunia plants resulted in the formation of (self-Isterile pollen (Taylor and Jorgensen, 1992; van der Meer et al., 1992; Ylstra et al., 1994). Fertility of these flavonoid-depleted pollen was restored by the addition of flavonols (Mo et al., 1992; Ylstra et al., 1994).

In addition to being essential for the plant, flavonols may also be important for animals and humans since they form

' This work was supported by the Technology Foundation (STW), grant No. VB100.2356, and the Austrian Research Founda- tion for Basic Science, grant No. 56004.

* Corresponding author; e-mail a.j.van.tunen@cpro.agro.nl; fax 31- 8370 -1 8094.

a large component of the diet of vertebrates and may have a vitamin-like function. It was suggested that they might serve a role as anticarcinogenic compounds (Hertog et al., 1992) and/or reduce the risk of coronary heart disease (Hertog et al., 1993). In addition, flavonols have also been reported to mimic the action of certain estrogens in mam- mals and might induce estrogenic activity (for a review, see Miksicek, 1993). Furthermore, it was shown that the flavo- no1 quercetin can interact with the estrogen type 11-binding site in rats (Markaverich et al., 1988). Finally, homology was observed between a flavonoid biosynthesis enzyme (dihydroflavonol-4-reductase) and a steroid biosynthesis enzyme (3P-hydroxysteroid dehydrogenase; Baker, 1991). Although flavonols and steroids are chemically unrelated (Fig. l) , these results suggest a relationship between ste- roids and flavonols in animals.

The brassinosteroids represent an interesting group of plant steroids (Mitchell et al., 1970). These compounds were isolated from mature Brassica pollen (Grove et al., 1979), and they showed strong growth-promoting effects in severa1 plant species (Yokota and Takahashi, 1985). Love and Love (1945) reported that other steroids, the mamma- lian androgens and estrogens, might be involved in the process of sex determination and development in higher plants. Grunwald (1980) reviewed the current understand- ing of the biosynthesis of steroids in plants. Despite the studies performed, the existence and potential role of ste- roid hormones in plants remains unclear (Leighton-Jones and Roddick, 1988).

In this article, we address the question of whether ste- roids have a function during plant reproduction and whether their action might be flavonol related. Therefore, we used a tobacco in vitro pollen maturation and germi- nation assay, which is a useful tool to investigate factors influencing pollen development, germination, and tube growth (Benito-Moreno et al., 1988; Heberle-Bors et al., 1991). In a previous study, this system was used to demonstrate the important role for flavonols in the growth of the pollen tube (Ylstra et al., 1992). In this paper, we show that certain mammalian sex hormones stimulate pollen germination and tube growth either alone or in combination with flavonols.

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640 Ylstra et al. Plant Physiol. Vol. 107, 1995

OH O

B

D I ç=o

O &

C OH

H & ONHCk$COOH

Figure 1. Chemical structures of a flavonol (quercetin, A) and the steroids testosterone (B), R1881 (C), progesterone (D), estradiol (E), 4-androstene-3,I 7-dione (F), glycocholic acid (G), p-sitosterol (H), and brassinolide ( I ) . The asterisk in B indicates the C-17 position.

MATERIALS AND METHODS

Plant Material

Nicotiana tabacum L. cv Petit Havana SR1 plants were grown in climate chambers at 25°C with a 16-h daylength under mercury vapor light (15,000 lux at pot level).

Microspore lsolation and Culture

Microspores were isolated essentially as described by Benito-Moreno et al. (1988) and cultured for 3 d at 25°C in the dark at a density of 4 X 105 microspores/mL in an M1 medium containing 10 mM KNO,, 1 mM Ca(NO,),, 1 mM MgSO,, 0.16 mM H,BO,, 1 mM uridine, 0.5 mM cytidine, and 1 mM sodium phosphate buffer at pH 7 (Tupy et al., 1991). To mature tobacco pollen in vitro, the cultures were diluted 1:l in a second medium containing 1.17 M SUC and 100 mM L-Pro (Tupy et al., 1991) and were incubated for 2 d. The in vitro matured tobacco pollen was collected by

centrifugation and resuspended directly in 400 pL of Brew- baker and Kwack (1963) germination medium with 10% Suc, in 12-well plates, with or without steroids and/or flavonols, at a density of 0.5 X 105 pollen/mL and cultured another 8 to 10 h. To determine germination frequency, 500 to 1000 pollen were counted, and pollen with tubes longer than 30 pm were judged to be germinated. To determine the pollen tube length, at least 60 (control) or 300 (quercetin plus testosterone) tubes were measured. To do this, slides were projected on a digitizer and the tube length was determined using the Sigma-Scan, version 3.90, software program (Jandel Scientific, Corte Madera, CA). A11 exper- iments were repeated at least three times. Quantitative data are presented from one representative experiment.

Flavonols and Steroids

Quercetin (3,3',4',5,7-pentahydroxyflavone dihydrate) was purchased from Sigma and a stock solution of 0.1 M

was made in DMSO. Progesterone (4-pregnene-3,20-dione) was purchased from Fluka Chemie AG; glycocholic acid (N-[3a,7a,l2a-trihydroxy-24-oxocholan-24yl]Gly), 4 andro- stene-3,17-dione, p-sitosterol ([3p]-stigmast-5-en-3-01), es- tradiol (1,3,5[1O]-estratriene-3,17~-diol), and testosterone (17P-hydroxy-4-androsten-3one) were purchased from Sigma. The synthetic androgen R1881 (17P-hydroxy-17a- methyl-estra-4, 9,11-trien-3one) was purchased from Du- Pont NEN, and 28-homo-brassinolide and 24-epi-brassino- lide were a kind gift of Michael E. Baker (University of San Diego, La Jolla, CA). Stock solutions (0.1 M) of the steroids and steroid hormones were made in ethanol. Immediately before the initiation of the cultures the compounds were added to the media. No effects of DMSO and/or ethanol (<0.1%) on pollen germination were observed in control experiments.

RESULTS

Testosterone Strongly Stimulates Male Gametophyte Development

Using in vitro matured tobacco pollen, we showed pre- viously that quercetin and other flavonols have a strong stimulatory effect on the growth of the pollen tube when compared with a control culture (Ylstra et al., 1992). The stimulatory effect of unglycosylated flavonols was measur- able at 0.3 FM and reached a plateau at 1 p ~ . Increasing the concentration or combining quercetin with other flavonols did not give a further stimulatory effect on germination or pollen tube growth.

For severa1 reasons explained earlier, we suspected that steroids could influence the germination and tube growth of pollen, and we added those compounds to in vitro matured tobacco pollen. The presence of 10 p~ testoster- one, the androgenic steroid hormone, in the germination medium resulted in an increased germination frequency and longer pollen tubes (Fig. 2). Detailed quantitative anal- ysis revealed that this stimulatory effect was detectable at 2 p~ and leveled off between 5 and 20 p ~ .

In a second set of experiments we observed that the presence of 1 p~ quercetin in combination with 10 FM

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Steroid Hormones and Pollen Tube Growth 641

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Figure 2. Average tube length (hatched bar) and percentage of ger-mination (filled bar) of in vitro matured tobacco pollen in the pres-ence of different concentrations of testosterone.

testosterone resulted in the formation of extremely longpollen tubes within several hours after initiation of germi-nation (Fig. 3). Tube growth was significantly faster thanobserved in any concentration of flavonols or steroids sep-arately. Quantitative analysis of these experiments re-vealed that the combination of steroids and flavonols alsoincreased the frequency of pollen germinating and that theeffects observed were additive (Fig. 4). Figure 4B alsoindicates that addition of quercetin alone gives rise topollen with longer tubes than the addition of testosteronealone.

Only Specific Steroids Stimulate Pollen Germination andTube Growth

To determine the specificity of testosterone, differentclasses of steroids were tested for their pollen tube growth-stimulating effect on pollen tubes. First, a number of mam-

malian steroids were tested (Table I). Glycocholic acid, themost important bile acid of herbivores, which is known forits emulsifying capacity, did not effect pollen tube growth.4-Androstene-3,17-dione, the immediate precursor for tes-tosterone, and estradiol did not show an effect either. Theknown steroid hormones testosterone, progesterone, andestradiol were able to stimulate pollen tube growth both inthe absence or presence of the flavonol quercetin. Differ-ences between male and female steroid hormones were notobserved in these experiments. Since the hormone prepa-rations may not have been totally pure and may havecontained traces of other (steroid) compounds, the syn-thetic androgen R1881 was also tested. Beyond this test,R1881 cannot be further metabolized (Bonne and Raynaud,1976), thus excluding the possibility that metabolized ste-roid compounds were influencing the assay. As is shown inTable I, R1881 stimulated germination and tube growth ina similar way as testosterone.

Second, a set of phytosteroids were assayed: jS-sitosterol(together with stigmasterol, the most common and widelydistributed sterol in plants), 28-homo-brassinolide, and 24-epi-brassinolide. Separately or in combination with fla-vonols, these plant steroids could not mimic the effectcaused by the mammalian steroids (Table I). 28-Homo-brassinolide completely inhibited pollen germination, evenin the presence of quercetin.

Considered together, these experiments show that cer-tain mammalian steroid hormones specifically stimulatepollen tube growth and germination.

DISCUSSION

In this report we describe our observations that mam-malian steroid sex hormones strongly and specifically stim-ulate the germination and growth of plant male gameto-phytes. Addition of steroids to the germination medium ofin vitro matured tobacco pollen resulted in a significantincrease in the percentage of germinating pollen and thelength of the tubes. Our experiments also showed that a

B D

.* *

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Q*..:Figure 3. Light microscope photographs showing the effect of testosterone and/or quercetin on germination and tube growthof in vitro matured pollen. A, Control, no additions; B, with 10 /J.M testosterone; C, with 1 /*M quercetin; D, with 10 /J.Mtestosterone and 1 fiM quercetin. www.plantphysiol.orgon July 14, 2018 - Published by Downloaded from

Copyright © 1995 American Society of Plant Biologists. All rights reserved.

642 Ylstra et al. Plant Physiol. Vol. 107, 1995

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8 40

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t31 Testosterone (1 O pM) Quercetin (1 pM) Testa& Quercetin

30200 2W-400 400-800 800-800 800-1OM) 10001200 12001400 14001800 >16W

Tube length, pm

Figure 4. Graphic representation showing the effect of testosterone and/or quercetin on germination and tube growth of in vitro matured pollen. A, Percentage of germination; 6, length of pollen tubes distributed in nine different classes. Each class contains the number of pollen with a given tube length as a percentage of the total number of pollen. The percentage of ungerminated pollen is not given but can be deduced from A. T (or Test.), Testosterone; Q, quercetin.

combination of steroids and flavonols stimulated male ga- metophyte development significantly more than these two compounds could do separately. The quantitative data re- veal that steroids function independently of flavonols and most likely have a different site of action. It can be con- cluded, therefore, that the plant flavonols not only exhibit certain steroid-related functions in mammalian systems (Markaverich et al., 1988; Miksicek, 1993) but mammalian steroid hormones can have flavonol-related effects during plant male gametophyte development.

In contrast to the mammalian steroid sex hormones, sterols such as glycocholic acid and fl-sitosterol or plant steroids such as 28-homo- and 24-epi-brassinolides did not stimulate male gametophyte development. Likewise, 4-an- drostene-3,17-dione, although closely related to the ste-

Table 1. The influence o f steroids on tube growth of an in vitro matured tobacco pollen in the presence or absence of flavonols

These data compare with the photographs presented in Figure 3. --, No germination; -, as in Figure 3A; +, as in Figure 3, B or C; and + +, as in Figure 3D. nt, Not tested.

~

Compound No Flavonols Flavonols

+ Control -

Mammalian steroids (10 p ~ ) Testosterone + + + Progesterone + + + Estradiol + + + 4-Androstene-3,17-dione -

Clycocholic acid - + +

Synthetic steroids (1 O I*.M

Plant steroids (1 O p ~ )

R1881 + + + nt

+ p-Si tosterol - 28-Homo-brassinol ide _ _ _ _ 24-Epi-brassinol ide -

roids testosterone, progesterone, and estradiol (Fig. l), also did not enhance pollen germination and development. We hypothesize that the type of rest group at the C-17 position (in Fig. 1, compare F with B, C, and D), like in mammalian hormone responses, might be important for activity of steroid compounds in pollen tubes. In summary, we con- clude that only specific steroids affect pollen development.

The levels of the steroid sex hormones that had to be applied to the tobacco germination medium were relatively high, compared to the active concentration in mammalian assay conditions. Possibly poor uptake of the steroids by pollen requires high concentrations in the medium. Alter- natively, mammalian steroids may only partly mimic the signal function of an endogenous plant steroid compound or steroid hormones exhibit a nonsignaling structural or biochemical role in the process of plant male gametophyte development. Although the stimulation of the in vitro de- velopment of pollen by steroids is clear, their biological function during plant development and pollen tube growth remains to be solved. Also, the presence of estrogen and/or testosterone compounds in the plant kingdom is still equiv- oca1 (Saden-Krehula et al., 1979; Grunwald, 1980; Leighton- Jones and Roddick, 1988; Zhang et al., 1991).

During the last few decades, many research groups have tried to develop in vitro pollen germination systems for various commercially important plant species. But such systems were developed for only a limited number of species (Brewbaker and Kwack, 1963; Benito-Moreno et al., 1988; Tupy et al., 1991; Mascarenhas, 1993). Flavonols have been proven to be successful enhancers of pollen tube growth in tobacco, petunia, tomato, and Brassica (Sedgley, 1975; Ylstra et al., 1992; Groot and de Ruiter, 1993). Fur- thermore, preliminary results indicated that inclusion of certain flavonols in germination media improved in vitro germination of wheat pollen (Dr. J.S. Heslop-Harrison, per-

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Steroid Hormones and Pollen Tube Growth 643

sonal communication). W e suggest that steroids i n combi- nation with flavonols be applied to further improve pollen germination systems for these and other plant species.

ACKNOWLEDCMENTS

The authors thank Dr. J.S. Heslop-Harrison for sharing re- sults prior to publication. Furthermore, we are indebted to Dr. M.E. Baker for his gift of the brassinosteroids. We also thank Dr. J.J.M. Dons and Prof. Dr. J.N.M. Mo1 for critically reading the manuscript.

Received September 19, 1994; accepted November 17, 1994. Copyright Clearance Center: 0032-0889/95/107/0639/05. .-

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