Scientia Horticulturae

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1 Scientia Horticulturae 122 (2009) Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: Fructose inhibits pear pollen germination on agar medium without loss of viability Keiko Okusaka, Shin Hiratsuka * Graduate School of Bioresources, Mie University, Tsu, Mie , Japan ARTICLE INFO ABSTRACT Article history: Received 19 March 2008 Received in revised form 10 March 2009 Accepted 23 March 2009 Keywords: Acetocarmine Fructose Japanese pear Pollen germination Respiration Pollen of Japanese pear (Pyrus pyrifolia Nakai) germinated well on agar medium containing 10% sucrose or glucose, but not on agar containing fructose. The inhibitory effect of fructose was dose-dependent. Sucrose enhanced pollen tube growth much more effectively than glucose. Addition of 5% fructose to 5% or 10% sucrose or glucose media suppressed germination completely. Ungerminated pollen, however, showed similar respiration rate and stainability against acetocarmine dye as germinated pollen. When pollen was transferred onto fructose medium after culturing it on glucose or sucrose medium for 1 2 h, germination was completely impeded. Reversely, pollen transferred to sucrose or glucose medium from fructose medium germinated at almost the same ratio as pollen on sucrose or glucose medium without transfer. Thus, pollen inhibition by fructose is reversible. Compared with uncultured pollen, cultured pollen contained less than half amount of total sugars, even if failed in germination on fructose medium. Germinated pollen on sucrose and glucose media contained sucrose and glucose, but ungerminated pollen on fructose medium contained only trace levels of these sugars, suggests that pollen on fructose medium predominantly uses sucrose and glucose as respiration substrates and cannot maintain the constant levels of these sugars. However, as pollen germination occurred on agar medium without any sugar, fructose may impede a physiological factor that triggers germination, and once the trigger is impeded, many physiological pathways including sugar biosynthesis may be blocked. ß 2009 Elsevier B.V. All rights reserved. 1. Introduction Control of pollination and fertilization is extremely important not only in plant breeding but also in fruit production of many types of fruit trees. The total number of pollen grains on a stigma often exceeds the number necessary to fertilize all the ovules, leading to competition among growing pollen tubes in the style (Howden et al., 1998). Consequently, rapid growth of the pollen tube is essential for male reproductive success, and, indeed, the pollen tube is the fastest growing plant cell known. In maize, for example, the rate of pollen tube growth can reach 1 cm h 1, and pollen respires 10 times faster than vegetative tissue (Dickinson, 1965; Tadege and Kuhlemeier, 1997; Taylor and Hepler, 1997). To enable the pollen tube to grow at this rate, rapid synthesis of cell wall material (Derksen et al., 1995) and a high energy supply are necessary. During pollen tube elongation, sugars are utilized as an energy source for synthesis of cell wall materials such as pectins, cellulose and callose (Mascarenhas, 1993; Derksen et al., 1995). In pollen germination assay in vitro, sucrose is generally used as an energy source in many plant species, because it usually * Corresponding author. Fax: address: hiratuka@bio.mie-u.ac.jp (S. Hiratsuka). stimulates pollen germination and subsequent tube growth. Pollen germination tests of Primula obconica (Higuchi, 1969) and Diospyros kaki (Nakagawa, 1974) showed that germination was promoted most effectively by sucrose but was greatly suppressed in a solid fructose medium. However, in Japanese pear Yakumo, pollen germination rate was highest on glucose medium, followed by sucrose medium, while germination was inhibited on fructose medium (Nakagawa, 1974). Thus, the effect of sugars on pollen germination differs among plant species, and little information is available on sugar metabolism in pear. To obtain basic information for breeding and fruit production of pear, we investigated the germination response of pollen to several sugars. In this paper, we refer to ungerminated pollen on the medium as ungerminated pollen and pollen before culture as uncultured pollen. 2. Materials and methods 2.1. Plant materials Adult Japanese pear trees ( Kousui, Pyrus pyrifolia Nakai) planted in the orchard of Mie University, Tsu, Japan, were used. Anthers were collected from flowers just before anthesis, /$ see front matter ß 2009 Elsevier B.V. All rights reserved. doi: /j.scienta

2 52 K. Okusaka, S. Hiratsuka / Scientia Horticulturae 122 (2009) dehisced and dried in a bottle with desiccant and stored at 30 8C until use Pollen germination assay in vitro Pollen grains were cultured at 25 8C for 24 h in the dark on solid medium containing 10% sugar (sucrose, glucose, or fructose), 1% agar and 0.01% H 3 BO 3. The cultured pollen was stained with 0.1% cotton blue dye (0.1% cotton blue/phenol:glycerol:lactic acid:h 2 O = 1:1:1:1), and percentage germination and tube length were determined under a light microscope. More than 100 pollen grains and 30 pollen tubes were counted to determine pollen germination and tube length, respectively. The experiment was repeated three times Viability test of cultured pollen To check the viability of ungerminated pollen on fructose medium, pollen grains were cultured for 1, 3 and 6 h as described above and stained with acetocarmine solution (1% carmine/45% acetic acid containing 2% iron alum). Then they were observed under a light microscope. Pollen grains cultured on sucrose medium served as a control Respiration of cultured pollen Respiration rate of ungerminated pollen on fructose medium was measured 0, 1, 3 and 6 h after culture using oxygen electrode apparatus (Rank brother s Co. Ltd., Cambridge, UK). To collect all pollen grains from the agar medium, a piece of filter paper (10 mm 10 mm) was put onto the medium on which 10 mg of pollen grains had been scattered. Fructose inhibition of pollen germination was occurred in the same manner in this system. After culture, the pollen was removed from the filter paper by rinsing in 3 ml of 0.05 M phosphate buffer (ph 7.4) contained in an electrode tank maintained at 25 8C, and the oxygen decrease in the buffer was recorded for 15 min Pollen transfer experiment To know whether the pollen inhibition by fructose is reversible, pollen transfer experiments were conducted. First, pollen grains were cultured on 10% sucrose or glucose medium for 1 or 2 h at 25 8C in the dark, and transferred onto 10% fructose medium and then cultured for additional 23 or 22 h, respectively. Reversely, pollen cultured on fructose medium was transferred to sucrose or glucose medium. Pollen culture on first medium was conducted on filter paper put onto the agar medium as described above, and pollen was transferred by contacting the paper with the surface of second agar medium. Sum of culture period on 1st and 2nd media was 24 h. After incubation, pollen was stained with cotton blue dye and observed under a light microscope. (Showadenko Co. Ltd., Tokyo, Japan). The running conditions were as follows: column temperature, 70 8C; eluent, H 2 O; flow rate, 1 ml min 1 ; detector, L-7490 RI (Hitachi Co. Ltd., Tokyo, Japan). Sucrose, glucose and fructose were identified and quantified by comparison with standard sugars. Because pollen contained not only sucrose, glucose and fructose but also several unidentified sugars, total sugar was calculated by quantifying unidentified sugars using sucrose as a standard. 3. Results 3.1. Pollen germination and pollen tube growth Pollen germination and tube growth were compared among the agar media with different carbon sources. As shown in Fig. 1, pollen germinated on glucose and sucrose media, while germination was completely inhibited on fructose medium. Germination rate was almost equal between glucose and sucrose media, but pollen tubes grew significantly better on sucrose medium. Inhibition by fructose was dose-dependent; pollen germinated on media containing less than 1.0% fructose but germination was completely inhibited at more than 3% (Fig. 2). Furthermore, fructose inhibited germination in the presence of sucrose or glucose; pollen did not germinate on glucose + fructose or on sucrose + fructose medium (Fig. 3) Viability of ungerminated pollen To examine the viability of ungerminated pollen on fructose medium, acetocarmine stainability and respiration rate were checked after 1-, 3- and 6-h culture. Pollen on fructose medium was stained by acetocarmine dye similarly to that on sucrose medium (Fig. 4). In addition, rapid respiration occurred within 1 h of culture and decreased thereafter on both media, although pollen on sucrose medium respired more actively (Fig. 5). These results indicate that ungerminated pollen on fructose medium still has the potential to germinate Pollen transfer experiment To know whether pollen inhibition by fructose is reversible, pollen transfer experiments were conducted at 1 or 2 h after 2.6. Sugar analysis in cultured pollen Ten milligrams of cultured pollen on filter paper were suspended in 0.05 M phosphate buffer (ph 7.4) as described above and gathered by centrifugation at 2,000 g for 5 min. The sedimented pollen was washed twice with the same buffer and resuspended in 70% ethanol in 0.05 M phosphate buffer (ph 7.4). Then it was homogenized with a motor-driven glass homogenizer and the homogenate was boiled for 30 min and centrifuged at 35,000 g for 20 min. The supernatant was gathered, concentrated in vacuo at 40 8C, and filtered through a 0.45-mm membrane (Nihon Millipore K.K., Tokyo, Japan). Sugars in the filtrate were separated by HPLC equipped with a SUGAR SC1011 column Fig. 1. Pollen germination (A) and pollen tube growth (B) on agar media containing different sugars. Concentration of each sugar = 10%. Culture period = 24 h. Vertical bars indicate SE. Values with different letters are significant by Duncan s multiple range test at 5% level.

3 K. Okusaka, S. Hiratsuka / Scientia Horticulturae 122 (2009) Fig. 2. Dose-dependent inhibition of pollen germination by fructose at five concentrations (%) (values are mean SE). Culture period = 24 h. Fig. 3. Combined effects of fructose with glucose or sucrose on pollen germination (values are mean SE). Note the complete inhibition on glucose or sucrose medium by adding 5% fructose. Culture period = 24 h. culture. At 1 h after culture, pollen grains imbibed and swelled but did not germinate on sucrose and glucose media. Although a few pollen grains began to germinate at 2 h, many grains were just before germination (data not shown). When pollen was carried from sucrose medium to fructose medium at 1 or 2 h after culture and incubated for additional 23 or 22 h, respectively, almost no germination was observed (Table 1). Transfer from glucose to fructose medium also showed similar results. On the other hand, pollen transferred from fructose to sucrose medium showed 17.4% germination at 1-h culture on 1st medium and 20.3% at 2-h culture, respectively. Similarly, transfer from fructose to glucose medium showed 13.9% at 1-h and 20% at 2-h culture, respectively. These germination ratios were almost the same as that on sucrose or glucose medium without transfer (Table 1). The tube length of these pollen was similar to that of nontransferred pollen on sucrose and glucose media (data not shown) Sugar content in cultured pollen Since pear pollen contained not only sucrose, glucose and fructose but also several unidentified sugars, we expressed total sugar as the sum of all sugars, including unidentified ones. Germinated pollen on sucrose and glucose media contained less Fig. 4. Acetocarmine stainability of pollen cultured on medium containing sucrose or fructose. Each sugar concentration = 10%. Pollen was stained 1, 3 and 6 h after culture. Note the similar stainability of pollen grains between sucrose and fructose media. Fig. 5. Comparison of respiration rate between pollen cultured on sucrose and fructose media. Each sugar concentration = 10%. Vertical bars indicate SE. Values with different letters at each culture period are significant by Duncan s multiple range test at 5% level.

4 54 K. Okusaka, S. Hiratsuka / Scientia Horticulturae 122 (2009) Table 1 Pollen germination on second medium after transfer from first medium. 1st medium 2nd medium Culture period (h) on 1st medium Pollen germination (%) G F 1 0 G F S F 1 0 S F F S F S F G F G G S F 24 0 Sum of culture period on 1st medium and 2nd medium = 24 h. G, glucose medium; S, sucrose medium; F, fructose medium. Values in the table are mean SE. than half amount of total soluble sugars in uncultured pollen, and considerably less sucrose, glucose and fructose (Fig. 6). However, ungerminated pollen on fructose medium contained much less sugar than germinated pollen (Fig. 6). Comparing the content of each sugar in the pollen showed that glucose content was almost equal in sucrose and glucose media, but that sucrose and fructose contents were significantly greater in sucrose medium than glucose medium, fructose content being quite low in glucose medium. Pollen on fructose medium contained trace levels of each sugar (Fig. 6). 4. Discussion The results show that sucrose, a disaccharide, stimulates pollen germination and tube growth of Japanese pear much more effectively than a monosaccharide, glucose, suggesting that sucrose is more readily available for pollen growth than glucose (Fig. 1). The germination results did not match the results of previous research on Japanese pear Yakumo, in which glucose stimulated pollen germination and tube growth more effectively than sucrose (Nakagawa, 1974). Nakagawa (1974) reported that media containing 10% sucrose or glucose caused 20.4% or 34.2% germination, whereas media containing 15% sucrose or glucose caused 29.4% or 6.7% germination, respectively. Therefore, experimental conditions, including sugar concentration, seem to greatly affect the germination results, and the contradiction between Nakagawa s data and ours may be due to the differences in purity of chemicals, cultivars, pollen viability and so on. Nevertheless, sucrose appears to be generally superior to glucose for pear pollen germination and pollen tube growth. However, fructose, a monosaccharide, inhibited pollen germination dose-dependently (Fig. 2), even if mixed with sucrose or glucose (Fig. 3). Thus, fructose seems to play an inhibitory role in pear pollen germination, but the pollen was stained positively by acetocarmine dye and showed adequate respiration rate (Figs. 4 and 5). Accordingly, some physiological pathway for germination is likely to be impeded in pollen on fructose medium. One possibility is that pollen cannot take up fructose from agar medium; a high level of sugar uptake would be required to enable the fast growth of the pollen tube (Mascarenhas, 1993; Schlüpmann et al., 1994; Derksen et al., 1995). This hypothesis, however, may not apply to fructose, because pear pollen could germinate without any sugar and inhibition occurred when sucrose was added to fructose medium (Figs. 2 and 3). Because the effect of fructose is known to be conditiondependent (Portnoi and Horovitz, 1977), we examined the effect of medium conditions on the inhibitory action of fructose as follows: (1) the effects of agar concentrations in the medium ranging from 0.25% to 1.0%; (2) the use of agarose instead of agar or agar washed Fig. 6. Comparison of sugar content in pollen cultured on agar media containing different sugars. Culture period = 24 h. Each sugar concentration in the medium = 10%. Vertical bars indicate SE. Values with different letters are significant by Duncan s multiple range test at 5% level. Data are compared between uncultured, glucose, sucrose and fructose media for respective sugar contents. by water; and (3) adding to medium 0.07% Ca(NO 3 ) 2, 0.02% MgSO 4 7H 2 O, 0.01% KNO 3, 5% polyethylene glycol. However, no germination was observed in any of the trials on fructose medium (data not shown). Meanwhile, pollen inhibition by fructose is reversible; ungerminated pollen on fructose medium was able to germinate on the medium without fructose, and cultured pollen on sucrose medium could not germinate on fructose medium (Table 1). Thus, fructose seems to impede germination at any stages of germination process only when it is present there, but does not act as a lasting cytotoxin on germination. This idea is supported by the results in Figs. 4 and 5, which pollen on fructose medium was stained by acetocarmine dye and respired similarly to that on sucrose medium. Because stored carbohydrates in the grain enable pollen germination and early stages of tube elongation, as described below, it is likely that something triggers pollen germination and that fructose in the agar medium impedes this trigger.

5 K. Okusaka, S. Hiratsuka / Scientia Horticulturae 122 (2009) Itis supposed that pollen tubegrowthis divided into two phases in the pistil. During phase I, pollen germinates by using stored nutrients in the grain and tube elongation occurs very slowly for several hours after germination. During phase II, the pollen tube absorbs nutrients from the style and the rate of tube elongation becomes 2 5 times faster than duringphase I(HerreroandDickinson, 1980; Mulcahy and Mulcahy, 1982; Cruzan, 1986; Singh et al., 1992; Herrero and Hormaza, 1996). The present study supports this idea; compared with germinated pollen on sucrose or glucose medium, total sugar content of uncultured pollen was about three times as high, and sucrose, glucose and fructose contents were 5 20 times higher (Fig. 6). Large quantities of stored sugars in the grain may be consumed for respiration and synthesis of cell wall materials during germination and early pollen tube development; thereafter, the growing pollen tubes maintain constant levels of sugar by absorbing and synthesizing them in the pollen. However, total sugar content was also less than one-third in the pollen on fructose medium compared to uncultured pollen, though the pollen did not germinate. The stored sugars may be used for respiration, because the ungerminated pollen on fructose medium respired actively (Fig. 5). In addition, almost no sucrose, glucose and fructose were detected in the pollen on fructose medium (Fig. 6), suggests that pollen predominantly uses sucrose, glucose and fructose as respiration substrates, and that normally germinating pollen maintains these sugars at constant level. Thus, not only sucrose and glucose but also fructose seems to be essential for normal pollen germination and subsequent tube growth. Nevertheless, we cannot yet explain why only exogenous fructose inhibits pollen germination. Our results will contribute to a better understanding of basic physiology of pollen germination and pollen tube growth and lead to improvements in controlling pollination and fertilization in fruit trees. Based on the present results, we examined the cultivar differences in sucrose content in pear pollen and found that pollen grains with higher sucrose content show better elongation of pollen tube in the style, even if the pollen grows in an incompatible style (data not shown). In summary, sucrose greatly stimulates pear pollen germination and pollen tube growth, while fructose strongly inhibits germination on agar medium where fructose may impede a physiological trigger of germination. Most likely, once the trigger is impeded, many physiological pathways, including sugar biosynthesis, are blocked, resulting in germination failure. References Cruzan, M.B., Pollen tube distributions in Nicotiana glauca: evidence for density dependent growth. Am. J. Bot. 73, Derksen, J., Rutten, T., van Amstel, T., de Win, A., Doris, F., Steer, M., Regulation of pollen tube growth. Acta Bot. Neerl. 44, Dickinson, D.B., Germination of lily pollen: respiration and tube growth. Science 150, Herrero, M., Dickinson, H.G., Pollen tube growth following compatible and incompatible intraspecific pollination in Petunia hybrida. Planta 148, Herrero, M., Hormaza, J.I., Pistil strategies controlling pollen tube growth. Sex. Plant Reprod. 9, Higuchi, H., The physiology and regulation of heteromorphic and gametophytic self-incompatibility in plants. PhD thesis. Nagoya University, Nagoya, Japan (in Japanese). Howden, R., Park, S.K., Moore, J.M., Orme, J., Grossniklaus, U., Twell, D., Selection of T-DNA-tagged male and female gametophytic mutants by segregation distortion in Arabidopsis. Genetics 149, Mascarenhas, J.P., Molecular mechanisms of pollen tube growth and differentiation. Plant Cell 5, Mulcahy, G.B., Mulcahy, D.L., The two phases of growth of Petunia hybrida (Hort. Vilm-Andz. ) pollen tubes through compatible styles. Acta Bot. Neerl. 18, Nakagawa, S., Problems on fruit setting in fruit trees. Agric. Hortic. 49, (in Japanese). Portnoi, L., Horovitz, A., Sugars in natural and artificial pollen germination substrates. Ann. Bot. 41, Schlüpmann, H., Bacic, A., Read, S.M., Uridine diphosphate glucose metabolism and callose synthesis in cultured pollen tubes of Nicotiana alata Link et Otto. Plant Physiol. 105, Singh, A., Evensen, K.B., Kao, Th., Ethylene synthesis and floral senescence following compatible and incompatible pollinations in Petunia inflata. Plant Physiol. 99, Tadege, M., Kuhlemeier, C., Aerobic fermentation during tobacco pollen development. Plant Mol. Biol. 35, Taylor, L.P., Hepler, P.K., Pollen germination and tube growth. Ann. Rev. Plant Physiol. Plant Mol. Biol. 48,