Comparison of Reproductive Biology Including Morphology of Reproductive Organs,Viability of Pollens,Flower Visitors,and Flavonols Accumulation Between Fertile and Sterile Cultivars in Bougainvillea

XU Su-Xia HUANG Qing-Yun LIN Chun-Song XU Yi-Ding LI Zi-Yang LIU Yu-Mei HUANG Yi-Jin

(1.Key Laboratory of Fujian Provincial Subtropical Plant Physiology & Biochemistry,Fujian Institute of Subtropical Botany,Xiamen 361006； 2.School of Landscape Architecture,Beijing Forestry University,Beijing 100083； 3.Department of Agriculture,Northeast Agricultural University,Harbin 150030； 4.Plant Introduction & Quarantine and Plant Product Key Laboratory of Xiamen City,Xiamen Overseas Chinese Subtropical Plant Introduction Garden,Xiamen 361002； 5.The First Affiliated Hospital of Xiamen University,Xiamen 361003)

Abstract Plants in Bougainvillea are characterized with high sterility. In this study, morphology of reproductive organs, viability of pollens, flower visitors, and flavonols accumulation were compared between two sterile and four fertile cultivars. The calyx in four fertile cultivars was oval and bigger than that in other sterile cultivars. Pollens were size-consistent in fertile cultivars. Viability of pollens with 11.1 to 46.5 germination rate in fertile cultivars was much higher than that in the sterile. Seven kinds of insect visitors were found in six cultivars, which displayed no discrimination among various cultivars. The results indicated that kaempferol/ and quercetin/glycosylated derivatives were the major flavonols accumulating in pollens using the florescence dye, diphenylboric acid 2-aminoethyl ester(DPBA). Florescence strength in tips of pollen tubes was much robuster than that in other parts. Accumulation of flavonols resulted in sharp difference between fertile cultivars and sterile cultivars in the study, which suggested that flavonols especially quercetin might play positive roles in fertility of plants in Bougainvillea.

Key words Bougainvillea;fertility;pollen;viability;flavonol

Bougainvillea, a member of the Nyctaginaceae family, comprises 18 cultivars of spiny trees, shrubs and vines[1]. Bougainvilleas are among the most floriferous shrubs of the tropics, which can hardly be excelled by any other plant. Among 18 cultivars only three cultivars,BougainvilleaspectabilisWilld.,B.glabraChoisy. andB.peruviana, are most commonly used in horticulture. Recently, some researches indicated thatB.spectabilisshowed great potential in virus-resistance[2～5]and pollution-tolerance[6]. Specially, a kind of like-insulin compound, pinitol, was first isolated from leaves ofB.spectabilis[7]and evaluated[8]. The compound can be developed into dietary supplements as insulin substitutes in curing diabetes[9].

Although these cultivars have much potential, the important cultivars inBougainvilleaare almost sterile which causes difficulty in sexual reproduction. The high level of sterility makes the cross breeding very difficult, which greatly narrows the gene pool of the cultivars[10～11]. The fact of poor seeds set discouraged the passion for the utilization. Since the last century, researchers worked hard at the uncovering the secret of high sterility. Oval viability partly contributed the low fertility[11～12]. Low pollen viability was possibly one of the reasons of the sterility inBougainvillea[13]. Pollen fertility is flavonoid-dependent in a wide range of cultivars(angiosperms, gymnosperms, and mono- and dicotyledonous plants)[14～16]. Flavonoid-less pollen fails to produce a functional pollen tube, and this defect can be complemented rapidly by the external addition of specific flavonols, which restores pollen tube growth[15]. Then, what is the true key to the confusing question?

Our study tried to partly discover the factors causing sterility of these cultivars based on comparison of morphology of pollens, anthers and perianth, flower visitors, pollen germination rate, flavonols in pollen, between fertile and sterile cultivars.

1 Materials and Methods1.1 Plant materials and growth conditions

In the study, cultivars were planted in Fujian Institute of Subtropical Botany of Xiamen, China(118°04′04″E, 24°26′46″ N, annual average temperature of 22℃). Four fertile cultivars(B.glabra‘Brazil’(XZ),B.glabra‘Alba’(XB),B.glabra‘Dream’(FZ) and F1 cultivars hybridized by XB × XZ) and two sterile cultivars(B. ‘Zakiriana’(R) andB. ‘Imperial Delight’(RW)), were chosen for investigation. Fresh pollens were collected for culture and observation from booming flowers(two little flowers were in bloom and the third was not). Field observation was carried out from October to July during two flowering seasons(the years from 2016 to 2017).

1.2 Methods Comparison of pollens and calyx among six cultivars

Each axillary shoot develops one to four inflorescences. Each inflorescence is represented by a four-flowered umbel. Plants inBougainvilleashowed asynchronous flowering and within the inflorescence, flower opening is consecutive. Study materials were collected as two flowers opening within an inflorescence. Pollens and each calyx of these flowers were collected and observed.

Fig.1 Six varieties R, RW. The sterile cultivars; XZ, F1, XB and FZ. The fertile cultivars The parents of F1 were XZ and XB.

1.3 Flower visitors

Flower visitors were investigated for two months. Diurnal observations were carried out from July to August throughout the morning, for a total of 100 h. We sampled each plant for 2-2.5 h. We observed each plant during flower lifetime and recorded the insect kinds visiting the flowers, only counting those insects that contacted anthers or stigma at least during part of their visit to the flowers(i.e. they can act as pollinators). Insects were identified in the field, and some specimens were photographed for further identification.

1.4 Observation of Pollens and pollen tubes

Fresh pollens were shed on the B&K medium containing 0.35% agar and cultured for 2 hours at 25℃ in dark. The medium contained sugar 200 g·L-1, H3PO30.2 g·L-1, MgSO4·7H2O 0.2 g·L-1, Ca(NO3)2·4H2O 0.3 g·L-1, KNO30.1 g·L-1, pH6.0. Pollen tubes were counted under an optical microscope after culturing for more than 2 h. Pollen viability was evaluated by germination rate. Observations were conducted using a Leica TCS SP5 photomicroscope.

1.5 Flavonol Accumulation in pollens and pollen tubes Using DPBA

To determine whether flavonols accumulate in pollens, we used diphenylboric acid 2-aminoethyl ester(DPBA) dye to detect flavonols and their derivatives[17]. DPBA fluoresces with distinct spectral properties in complex with kaempferol and quercetin[18～19], with Q-DPBA having a longer wavelength fluorescence emission than K-DPBA.

Pollens fell on the BK medium with 0.35% agar and germinated for 1.5 h. Then, a drop of DPBA solution containing 0.01%(v/v) Triton X-100 and 2.52 mg·mL-1DPBA was added on pollens and cultured for 1.5 h[20]. Then, using distilled water washed off DPBA solution twice.

A Leica TCS SP5 was used to excite the pollens with 40% maximum laser power at 458 nm. These settings collect fluorescence at 475-504 nm for kaempferol and 577-619 nm for quercetin[17]. Thus, quercetin was indicated by yellow inflorescence and kaempferol by green fluorescence. All micrographs within each figure were acquired using identical offset, gain, and pinhole settings using the same detectors.

1.6 Quantification of Flavonols in flowers of the sterile and fertile cultivars by UPLC

Fresh flowers were collected in Sep 2017 and rinsed in ddH2O. Hydrolysis of the samples followed the method as described by Hertog et al.[21]. Briefly, each sample was repeated four times. First, 0.5 g of ground freeze-dried sample in a flask was mixed with ascorbic acid solution(80 mg in 7.5 mL water). Then the sample was imbibed with the liquid, and 12.5 mL of methanol and 5 mL of 6 mol·L-1HCl were added. Samples were boiled in a water bath under reflux for 2 h at 90℃. Finally, the samples were cooled at room temperature, and then filtered through a 0.22 μm RC filter(SRI, Roth, Germany) prior to injection of 10 μL to HPLC. Triplicates were performed for each sample.

All chemicals of chromatographic grade were used in the study. Quercetin and kaempferol(Sigma-Aldrich, Buchs, Germany) and isorhamnetin(Sigma-Aldrich, Buchs, Germany) were used as standards. All solution was prepared using ddH2O(18.2 MΩ·cm). HPLC-grade water was obtained from a Milli-Q System(Millipore, Billerica, MA, USA). All standards were dissolved into methanol to a final concentration of 1.0 mg·mL-1and stored at 4℃.

All measurements were performed on a Waters AcquityTMUltra Performance LC Spectrometry(Quattro Premier XE) equipped with an autosampler, and a Masslynx 4.1 data handling system. The analytical column was BEH C18(50 mm×2.1 mm I.D., 1.7 μm, Waters, USA). An aliquot of 10 μL solution was injected for HPLC analysis. Chromatograms were acquired at 350 nm for flavonols. Formic acid-acetonitrile(0.1:99.9, v/v/v) was selected as phase A and TFA-formic acid-acetonitrile-water(0.1:2:35:62.9, v/v/v/v) as phase B[22]. Additional chromatographic conditions were as follows: 0 min, B 30%; 12 min, B 48%; 22 min, B 55%; 35 min, B 60%; 40 min, B 30%; the flow rate, 0.3 mL/min; the injection volume, 10 μL; temperature, 35℃.

2 Results

2.1 Comparison of anthers, pollen and calyx tube between six cultivars

Quite inconspicuous flowers are tubular, constricted in the middle, and radically symmetric. Four small flowers units consist of an inflorescence with four colorful bracts together. Each flower has tubular calyx. Obvious differences were observed between the fertile and the sterile(Fig.2). The calyx tube was fat, bulging in the fertile cultivars, while, slim and long in the sterile cultivars. In the study, the fertile cultivars were XZ, XB, F1(XB x XZ) and FZ. Also, XB can set more fruits than the other three. If the inflorescence set fruits, usually no all three flowers produce fruits. The fruit is covered by the perianth tube. If the flower would not set fruit the entire inflorescence falls off after blooming.

Fig.2 Comparison of calyxes among six materials Up. Blooming; Down. Wilting flowers

The mature flower is composed of five sepals, eight stamens, and a gynoecium containing a single ovule. InBougainvillea, eight stamens arise sequentially at divergence angles of a 3/8 spiral[13]. When pollens are mature they would shed. Much difference could be found in morphology of pollens between two types of materials. For instance, fertile pollens were light-colored sphere with visible rough-surfaced reticulate pattern, but sterile pollens had dark-colored, sticky outer wall. Sterile pollens were also partly malformed but fertile pollens were uniform in size and shape. Thus, when pollens were shed in water, fertile pollens were loosely dispersed in water and sterile pollens were sticky, two to four or more stayed in water(Fig.3).

Fig.3 Comparison of pollens between the fertile and the sterile cultivars A. Pollens from XZ; B. Pollens from R(Bar=50 μm)

2.2 Flower visitors

Seven kinds of insects(Moths, two kinds of butterflies, flower flies, bamboo bees, bees and ants) were found in the flowers of six cultivars during 100 h observation. Six kinds of insects(moths, one kind of butterflies, flower flies, bamboo bees and ants) were found to visit XZ, two types of insects(one kind of butterflies, moths) visited XB, one type of insects(butterflies) visited FZ, one type of insects(butterflies) visited RW, and four types of insects(two types of butterflies, bees) visited R. Flowers were visited by insects including butterflies, moths, flies, bees and ants(Fig.4-5). The insects belong to Lepidoptera, Diptera and Hymenoptera, respectively. Long proboscis of insects was introduced to the perianth tube. The constriction of the perianth probably facilitates pollination, since it enforces contact between the proboscis and the stigma(Fig.4-5). Insects without long proboscis, i.e. ants, were also both pollinators and nectar thieves(Fig.4). Insects often visited all four flowers of an inflorescence and several inflorescences in each plant. In fact, among seven types of insects, ants were orthodox nectar and pollens thieves who always ate nectar and also ate up anthers visited. After ants-visiting, the flowers only had blank perianth(Fig.4:B-D).

Fig.4 Flowers visitors A. Moth(Macroglossum stellatarum) in F1; B. Butterfly(Pelopidas mathias) in XB; C. Flower fly(Eristalis arvorum) in R; D. Bee(Apis cerana) in XZ; E. Butterfly(Papilio polytes) in R; F. Bamboo bee(Xylocopa dissimilis Lep.) in XZ

Fig.5 Ants acted as thieves A. An inflorescence in blooming; B. An ant was climbing into the calyx and the other was doing the same(In the outer surface of the calyx, there were lots of pollens which were carried out by ants); C. In the calyx, two ants were sharing pollens; D. One of them was climbing backward off the calyx and carrying lots of pollens on the backs(Pollens were carried around by ants)

MaterialsVisual field 1*Visual field 2*Visual field 3*Visual field 4*Visual field 5*Pollens countedGermination rate(%)Annual germination rate(%)XZ134/1007/237/266/1014/3319235.4219/5329/6818/696/1813/2122925.331.335/1020/554/144/2529/8318733.1XB14/613/293/207/647/6123510.222/542/245/844/565/532711011.136/308/409/714/64—20513.2F1(XB/XZ)14/613/293/207/647/6123510210/2714/755/283/4712/4522219.814.934/329/546/6212/989/2126715.0FZ112/2724/950/10019/3324/4229755.0258/12131/8848/14221/4518/3543140.846.5313/3118/3429/6632/6254/14333643.5

Note:*data mean germinated pollens/total pollens in the visual field.

2.3 Pollen germination

Pollens were investigated in two sterile and four fertile cultivars. Obvious difference was observed in germination of pollens among them. Comparison of pollen germination rate was shown in Table 1. It indicated that sterile pollens almost not germinate pollen tubes. Pollens in four fertile cultivars presented different germination ability(Table 1). Germination rate of pollens ranked from 11.1% to 46.5% viability of pollens in FZ was highest(46.5%) and that in XB was lowest(11.1%). Viability of pollens in XZ performed well with 31.3% of germination rate. Sterile pollens burst during culturing or no tubes occurred.

2.4 Flavonols distribution in pollens

To analyze the functional significance of flavonol accumulation in pollens, we used a dye, diphenylboric acid 2-aminoethyl ester(DPBA), to visualize flavonols and their glycosylated derivatives in live pollens. DPBA fluorescence with distinct spectral properties in complex with kaempferol and quercetin, with Q-DPBA having a longer wavelength fluorescence emission than K-DPBA. Thus, quercetin/glycosylated derivatives was indicated by yellow florescence and kaempferol/glycosylated derivatives by green fluorescence. Under the settings of 40% maximum excitation laser power at 458 nm, yellow Q-DPBA fluorescence and green K-DPBA fluorescence were captured at 475-504 nm and 577-619 nm. The absence of the DPBA signal in control pollens confirmed the specificity of this staining in pollen and tube. The levels of DPBA fluorescence was at background levels in control. Except for control, pollens of all materials(Xb, XZ, F1, FZ and RW, R), were stained with DPBA. It suggested that kaempferol/ and quercetin/ glycosylated derivatives were the major flavonols giving fluorescing signals when the macrographs of free-DPBA control was dark(Fig.6). Further, florescence of pollen tubes was acquired in the four fertile cultivars(Fig.6). Obvious difference of florescence strength was not found between the fertile and the sterile cultivars, or among six cultivars. Strength of yellow Q-DPBA fluorescence did not make obvious difference from green K-DPBA florescence(Fig.6).

The adding time of DPBA was set during pollens culture. Four treatments included simultaneously, 0.5, 1 and 1.5 h after culturing. Of them, the time set, 1.5 h after culturing was appropriate, under which pollen tubes grew well and florescence signals was strong. Pollens did not germinate in the fertile cultivars when DPBA was added simultaneously. With the time going florescence signals was detected both in wall of pollen and nuclear area in the fertile cultivars. Florescence strength of tips of pollen tubes was much robuster than that in other parts, which indicated flavonols/glycosylated derivatives would move with pollen tubes growth.

Fig.6 Confocal micrographs of DPBA-stained pollens & pollen tubes Confocal micrograph showed Q-DPBA fluorescence and K-DPBA fluorescence in pollens and pollen tubes of six cultivars. DPBA bound to quercetin is shown in yellow and to kaempferol is shown in green. The materials were R, RW, FZ, XZ, XB and F1(XB x XZ). The last column was control without adding DPBA named as C(Bar=75 μm)

2.5 Quantification of flavonols in flowers of the sterile and fertile cultivars by UPLC

Amounts of four flavonols(quercetin, rutin, kaempferol and Isorhamnetin) in floral organs were analyzed using UPLC. Rutin is the glycosylated derivatives of quercetin and isohemnetin is the metabolite of quercetin, which could help estimate amounts of quercetin. The results showed that quercetin was main component of flavonols in six cultivars(Table 2). As far as rutin was concerned, quercetin / derivatives ranged from 246-1 879 μg·g-1dry weight. Amounts of kaemferol was much lower than quercetin, undetectable or traceable(0.8-3.5 μg·g-1dry weight). Contents of quercetin in fertile cultivars were much higher than that in sterile cultivars(Table 2). For instance, 365-1 879 μg·g-1rutin, 11.4-27.9 μg·g-1quercetin, 4.4-35.4 μg·g-1isohemnetin were detected in four fertile cultivars. 246-310 μg·g-1rutin, 4.3-4.6 μg·g-1isohemnetin in two sterile cultivars(Table 2).

Table2Contentsofflavonols/glycosylatedderivatives(μg·g-1dry weight)

CultivarsRutinQuercetinKaempferolIsohemnetinXB1879.8±109.327.9±6.23.5±1.835.4±15.7XZ365.9±4.215.2±0.62.3±0.44.4±0.2F1823.4±27.911.4±0.40.8±0.14.6±0.3FZ491.9±13.516.2±0.81.7±0.113.2±0.2RW246.9±13.8004.3±0.1R310.1±9.8004.6±0.1

3 Discussions

3.1 The roles of flower visitors in Bougainvillea

Many insects visited these plants in this study. Also, visitors could be seen continuously during blooming. Obviously, fragrance or nectar was the drive force of continuous visiting. Plants inBougainvilleacould emit fragrance or secrete nectae or both. Fragrance is emitted by the papillae located on the lobe margins of the perianth. The nectary is located at the base of the stamina tube. It secretes fructose-dominant nectar with amino acids, phenols and reduced acids[13]. Nectae was secretted in continuous way during blooming because insects visited flowers during whole blooming period.

Main visitors were bees, moths, butterflies and ants. López and Galetto[13]thought only insects of the order Lepidoptera visited flowers, however, insects of the order Hymenoptera, i.e. ants, also visit the flowers inBougainvillea(Fig.3). Different cultivars ofBougainvilleapresent a suit of different traits to attract pollinators, such asB.spectabilisandB.glabrashowing coloured flowers and bracts whileB.stipitatashowing fragrance emission[13]. Nectar is the reward offered to pollinators. Given different visitors, composition of nectar should be not related to the pollinator’s preferences. Insects visited flowers inBougainvilleain open flowers and wilting flowers(Fig.4). It was suspected these flowers secreted nectar throughout the flower lifetime.

3.2 Morphology of pollen and fertility

It was speculated that patterns of the pollen tubes were related to fruit set rate, i.e. fast germination and growth and slim and long pollen tube increased fruit set. The slimmer and longer was the pollen tube, the faster germinate pollen. Pollen tubes in XZ were slimmer and longer than that in other cultivars and XZ had higher fruit set rate than that in others.

FZ presented 46.5% germination rate of pollens and has obvious oval calyx. It showed such clues in other four fertile cultivars(Table 1 and Fig.1). The cultivar has oval calyx and then it could germinate high-vatality pollens. It seemed closely relevant between diameter of calyx and viability of pollens. Obviously, plants inBougainvilleaare self-incompatible and pollinators bring good quality pollens to the neighbor, like XB and XZ.

3.3 Potential function of quercetin and kaemferol in Bougainvillea

We chose DPBA to indicate the accumulation of kaempferol and quercetin in pollens. Under the settings of 40% maximum excitation laser power at 458 nm, yellow Q-DPBA fluorescence and green K-DPBA fluorescence were captured at 475-504 nm and 577-619 nm[17-19]. It suggested that kaempferol and quercetin were accumulated in pollens of all cultivars(Fig.6). Strength of yellow Q-DPBA fluorescence did not make obvious difference from green K-DPBA florescence under the same microscope settings. Also, obvious difference of florescence strength was not found among six cultivars. However, together with the results of pollen germination test in vitro and flavonol by UPLC detection in floral organs, we did not think that contents of quercetin was similar to that of kaemferol in pollens, or flavonols in fertile cultivars were similar to that in sterile cultivars. Conclusively, the method did not present an efficient tool to quantify the content of flavonols and their glycosylated derivatives in the study. In addition, the fact that pollens did not germinate in fertile cultivars if DPBA was added simultaneously into medium suggested that DPBA probably inhibite the germination switch. In the study, quercetin was the majority of flavonols in six cultivars. Amounts of kaemferol was much lower than quercetin, even undetectable or traceable. Contents of quercetin in fertile cultivars were much higher than that in sterile cultivars. Several researches reported the positive function of flavonols on pollen reproductive function[23]. Pollens devoid of flavonols were unable to germinate or produce a functional pollen tube on self-pollinations in petunia[15,24], maize[25]and tobacco[23].

In conclusion, four clues of universal sterility inBougainvilleawere provided: Low pollen viability universally is one of the main reasons of sterility in these cultivars; The role of pollinators is of importance in successful fertilization in self-incompatible plants inBougainvillea; Differences in structure morphology provides clues between the fertile and the sterile; Accumulation of flavonols made sharp difference between fertile cultivars and sterile plants. Thus, flavonols maybe play important roles in fertility of plants inBougainvillea.