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The effect of putrescine on pollen performance in hazelnut (Corylus avellana L.)
Scientia Horticulturae xxx (xxxx) xxxx
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The effect of putrescine on pollen performance in hazelnut (Corylus avellana L.) Aslıhan Çetinbaş-Gença,*, Giampiero Caib, Stefano Del Ducac, Filiz Vardara, Meral Ünala a
Department of Biology, Marmara University, Kadıköy, 34722, Istanbul, Turkey Department of Life Sciences, University of Siena, via Mattioli 4, 53100, Siena, Italy c Department of Biological, Geological and Environmental Sciences, University of Bologna, via Irnerio 42, 40126, Bologna, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hazelnut Pollen germination Pollen elongation Polyamine Putrescine
One of the most substantial molecules for pollen germination and tube growth are polyamines. Putrescine is the most abundant polyamine and the molecule from which spermine and spermidine originate. In this study, the effects of different exogenous putrescine concentrations (0.05, 0.25, 0.5 and 2.5 mM) on pollen performance of hazelnut (Corylus avellana) were investigated. Germination ratio and tube length were induced by 0.05 and 0.25 mM putrescine treatment. Putrescine concentration above 0.25 mM inhibited the pollen germination, tube elongation and, caused morphological alterations such as apex swelling. While the 0.05 and 0.25 mM putrescine treatment did not cause significant change in callose accumulation and actin filament distribution at tube apex, concentrations above 0.25 mM caused dense callose accumulation and increased the actin filament anisotropy at tube apex. Moreover, putrescine treatment caused an increase of reactive oxygen species level at the apex, especially in swollen tube apex, at the concentration of 2.5 mM. Reactive oxygen species detoxification mechanisms, which can be examined by changes in the amount of hydrogen peroxide and enzyme activities, were not disrupted by 0.05 and 0.25 mM putrescine treatment, while they are affected by 0.5 and 2.5 mM putrescine treatment. Eventually, low doses (0.05 and 0.25 mM) of putrescine can be used as a performance enhancing agent for hazelnut pollen tube growth, while the higher concentrations (0.5 and 2.5 mM) cause adverse effects reducing fertilization success.
1. Intoduction
and night temperature differences or seasonal temperature changes (Heslop-Harrison et al., 1986). During this waiting period, the sustainability of pollen performance is important for the success of fertilization and it also affects yield. Therefore, the increase in pollen performance also results in an increase in reproduction efficiency and thus in production yield. So, to improve the pollen performance, exogenous treatment to pollen or pollen tubes can be recommended during the pollination period (including the waiting process). Researchers have generally considered the pollen germination rate, tube length and tube abnormality rate to evaluate pollen performance (Williams and Reese, 2019). Moreover, callose accumulation at tube apex have been used as an evaluation criterion because it affects pollen performance adversely and reduce the fertilization performance (Sawidis et al., 2018; Çetinbaş-Genç, 2019). Callose is a fundamental constituent of the tube wall, but it is absent at the tube apex (Williams et al., 2016; Aloisi et al., 2017). Existence of callose at the tube apex is an abnormal situation and it causes the reduction of reproductive
Hazelnut (Corylus avellana L.) is a member of Betulaceae family. It is globally the most significant nut crop and a significant resource in almost all areas of industry (Novara et al., 2017; Armstrong and Turner, 2018; Candellone et al., 2019). Although it is mainly cultivated in Turkey and Italy, its cultivation is becoming widespread all over the world because of its commercial value (Novara et al., 2017; ÇetinbaşGenç et al., 2019). Since the hazelnut’s reproductive biology is very unusual, understanding the development of some stages in reproductive biology and pollination are the main focus for increase the cultivation yield (Çetinbaş-Genç et al., 2019). In hazelnut, the pollen grains or pollen tubes wait for 2–3 months in different parts of the pistil for the maturation of ovary (Liu et al., 2014). Also, since pollination starts in winter and ends in spring, during this long waiting period, pollen and pollen tubes are exposed to low or high-temperature stresses due to day
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Corresponding author at: Department of Biology, Marmara University, Göztepe Campus, 34722, Istanbul, Turkey. E-mail addresses: [email protected] (A. Çetinbaş-Genç), [email protected] (G. Cai), [email protected] (S.D. Duca), fi[email protected] (F. Vardar), [email protected] (M. Ünal). https://doi.org/10.1016/j.scienta.2019.108971 Received 5 September 2019; Received in revised form 18 October 2019; Accepted 21 October 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Aslıhan Çetinbaş-Genç, et al., Scientia Horticulturae, https://doi.org/10.1016/j.scienta.2019.108971
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oxygen species (ROS) that led to the oxidative stress in pollen tube (You and Chan, 2015). Superoxide dismutase (SOD) promotes the reduction of superoxide to hydrogen peroxide (H2O2). Catalase (CAT) catalyzes the deterioration of H2O2 thereby overcoming the oxidative stress (Wang et al., 2010). Consequently, alterations in these compounds can be used as indicators to determine the positive or negative effect of PAs. The purpose of this manuscript is to assess the effect of exogenously applied Put on hazelnut pollen performance, by focusing on callose accumulation, actin cytoskeleton dynamics, and ROS detoxification system. The application of Put in proper doses can be assessed to ensure the maintenance of pollen performance during the pollen's waiting period and can provide a new perspective to pollination and fertilization in hazelnut.
success by preventing the transfer of sperm nuclei to embryo sac (Del Duca et al., 2010). Callose accumulation is likely strictly associated with the actin cytoskeleton dynamics because insertion of callose synthase in the plasma membrane is supervised via actin cytoskeleton (Cai et al., 2011). In accordance with the composition of actin filaments, pollen tube is divided into three area; the apex, the sub-apex and the shank. Actin filaments exist as parallel bundles in the shank and are joined with the actin fringe in the sub-apex. The characteristic structure of actin filaments at the apex, as curved and dynamic filaments, is required for the elongation of pollen tube (Cai et al., 2015). Accordingly, callose distribution and actin organization are critical parameters used in the indication of pollen performance (Kang et al., 2010). Many substance are reported to improve pollen germination and tube growth, such as flavonols (Ylstra et al., 1992), boron (Wang et al., 2003), gibberellic acid (Voyiatzsis and Paraskevopoulou-Paroussi, 2005) and polyamines. One of the most important class is polyamines (PA), because PA homeostasis is responsible for events that occur throughout the whole pollen lifetime (Aloisi et al., 2016). So, whether PAs affect pollen germination and tube growth was examined in many species. Putrescine (Put) is the most abundant PA and also the molecule from which spermine and spermidine originate (Sequera-Mutiozabal et al., 2017). The promoting effect of Put on pollen efficiency has been shown in pear (Dixin and Shaoling, 2002) and almond (Sorkheh et al., 2011). Despite the evidences, more data is needed to understand the effects of Put on pollen performance. In general, PAs homeostasis must be delicately regulated and exogenous treatment of PAs has a different effect during pollen germination. Low concentrations of exogenous PAs induce pollen tube emergence while high doses strongly block this process and alter tube form (Aloisi et al., 2017; Çetinbaş-Genç, 2019). PAs might also organize the enzymatic activity of pectin-methyl esterase and they might lead to decreased grades of unesterified pectins and therefore to softer cell walls (Charnay et al., 1992). In Arabidopsis thaliana pollen tubes, exogenous spermidine increased the cytosolic free Ca2+ concentration; spermidine oxidation by PAO generates H2O2, which acts as a second messenger to activate Ca2+ channels, thus inducing Ca2+ ion influx beyond optimum grades and causing the prevention of tube elongation. Activation of the Ca2+ currents by spermidine is considerably disrupted in KO-PAO mutants, but the Ca2+ channels can still be activated following treatment of H2O2 (Wu et al., 2010a, 2010b). In Malus domestica, during pollen tube growth, both RNA and protein biosynthesis were stimulated by addition of spermidine. In the same system, PA-conjugation to actin and tubulin catalyzed by TGase affects their capability to mediatize and their coaction with motor proteins both in vivo and in vitro, as demonstrated by the amorphous structures of microtubules when pollen tubulin is incubated with high concentrations of Put, while tubular-like structures are formed at lower concentrations (Del Duca et al., 2009). Also, PAs may regulate the installation and structures of cell wall polysaccharides, such as pectins. In soybean’s cell walls, positively-charged PAs might compete by unesterified (acidic) pectins in binding Ca2+ ions (Charnay et al., 1992). PAs usually exist in the cells in free and bound form and the effect of exogenous PAs is multifactorial. The molecular mechanism of action of PAs has generally related with their polycationic backbone capable of set-up electrostatic interplays with anion groups of different biological molecules as proteins, nucleic acids, and membrane phospholipids. Furthermore, PAs strongly bind in vitro to pectic substances and to isolated cell wall polysaccharides. Beside the electrostatic interactions, the covalent binding to glutamyl residues of specific proteins, catalyzed by transglutaminase could affect protein functionality. Other covalent bonds to phenylpropanoids, abundant in some plant families, as hydroxyl-cinnamic acids give rise to phenolamides, involved in the structure of the cell wall and concerned to fertility (Aloisi et al., 2016). It is accepted that PA affect the pollen germination and tube growth according to the dosage (Aloisi et al., 2015; Çetinbaş-Genç, 2019). Excessive dose of PAs causes an excessive accumulation of reactive
2. Material and methods 2.1. In vitro pollen germination, tube elongation and tube abnormality Pollen materials were collected from Akçakoca/Düzce (Turkey) in February 2019 and were stored at −20 °C after dehydration process. Pollen germination was carried out at 20 °C with 50% RH for 24 h, in parallel with former works (Heslop-Harrison et al., 1986; Novara et al., 2017; Carniel et al., 2018; Çetinbaş-Genç et al., 2019). Brewbaker& Kwack pollen germination medium (BK medium) with 12 % sucrose was used for the germination medium and the solution was supplemented with 0.05 mM, 0.25 mM, 0.5 mM and 2.5 mM Put (Brewbaker and Kwack, 1963; Çetinbaş-Genç et al., 2019). BK medium without Put was used for control group. Pollen grains whose tubes were at least twice as long as the pollen diameter were considered as germinated. For each individual treatment, 3 slides were prepared and 500 pollen grains were investigated for each slide. Eventually pollen germination percentages were scored by considering 1500 pollen grains for each individual treatment. In addition, 5 slides were prepared for each individual treatment and 100 pollen tube were investigated. Afterwards, tube length and abnormality rate were scored considering almost 500 germinated pollen grains for each group. Pollen tube abnormalities were investigated using 2% aceto-orcein which facilitate the examination of tube morphology by staining cytoplasm and nucleus (ÇetinbaşGenç et al., 2019).
2.2. Determination of callose accumulation Pollen tubes were incubated by 0.1 % decolorized Aniline Blue to visualize callose (Chen et al., 2007). Almost 150 tubes were monitored at 455 nm using an Olympus BX-51 fluorescence microscope with 40X objective and KAMERAM software. To examine the quantitative accumulation at the tube apex, fluorescence intensity (FI) was scored in an area of 400 μm2 at the apex of 50 pollen tubes for each group. FI measurement was performed by handling the ‘Rectangle Selection’ tool of ImageJ (https://imagej.nih.gov/ij/).
2.3. Determination of actin filament anisotropy Pollen tubes were fixed for 30 min in pH 6.9 buffer containing 100 mM PIPES, 5 mM MgSO4, 0.5 mM CaCl2, 0.05% (v/v) Triton X-100, 1.5% (m/v), formaldehyde, and 0.05% (m/v) glutaraldehyde, and washed two times in same buffer also containing 10 mM EGTA and 6.6 μM Alexa 488-phalloidin (Lovy-Wheeler et al., 2005). 150 pollen tubes were monitored at 488 nm using a fluorescence microscope. To examine the quantitative difference of actin filaments at tube apex, actin filament anisotropy was measured on 20 pollen tubes for each group. Anisotropy measurement was evaluated using the ‘Fibril Tool’ plugin of ImageJ (Boudaoud et al., 2014). 2
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it did not exist at the apex of control tubes, as well as in 0.05 mM and 0.25 mM Put treated tubes (Fig. 2A). Conversely, callose was present at the apex of both 0.5 mM and 2.5 mM Put treated pollen tubes, in addition to existing throughout the shank (Fig. 2A, arrows). To examine the variations of callose at the tube apex, FI of callose was measured in 400 μm2 areas of the apex. FI decreased by 12.20 % at 0.25 mM and increased by 15.86 % at 0.05 mM, insignificantly. However, FI significantly increased by 78.40 % at 0.5 mM, 113.40 % at 2.5 mM in compare to control (Fig. 2B). To understand whether the actin cytoskeleton could impact on changes in the pollen performance, actin filaments were visualized and analyzed. Distribution of actin filaments did not show remarkable difference between the control and 0.05 mM, 0.25 mM Put. Actin filaments were lined up as irregular at the apex while filaments arranged linearly in the shank (Fig. 2C). On the other hand, actin arrangement exhibited critical changes at 0.5 mM and 2.5 mM Put treated pollen tubes, especially in the swollen apex (Fig. 2C, arrows). Disorganizations of actin filaments were quite obvious in the swollen tube apex, while there were no changes in the tube shank. In order to determine the subtler differences at the apex, anisotropy analysis was performed. Anisotropy of actin filaments increased by 5.93 % at 0.05 mM, 8.47% at 0.25 mM, insignificantly. However, anisotropy of actin filaments significantly increased by 23.44% at 0.5 mM and 43.50% at 2.5 mM in compare to control (Fig. 2D). In order to determine whether Put treatment effected the ROS localization, pollen tubes were stained by CM-H2DCFDA. Tubes showed a uniform ROS fluorescence signal in control tubes as well as after 0.05 mM and 0.25 mM Put application (Fig. 3A). On the contrary, ROS fluorescence signal was more intense at the apex after treatment with 0.5 mM and 2.5 mM Put, especially at the swollen tube apex (Fig. 3A, arrow). To examine the variations of ROS accumulation at the apex, the FI of apex localized ROS was measured. To allow us to focus on the apex localized ROS even if pollen tubes are different lengths, a region starting from the pollen tube tip with width and length of about 20 μm were determined and FI was measured in this 400 μm2 area of apex. Quantification analysis showed that FI of ROS accumulated at the apex and insignificantly increased by 18.98 % at 0.05 mM and, significantly increased by 91.03 % at 0.25 mM, 155 % at 0.5 mM and 362.48 % at 2.5 mM (Fig. 3B). To analyze the impact of Put on ROS detoxification system, SOD, CAT enzyme activity and H2O2 content were determined. The activity of SOD insignificantly increased by 68.39 % at 0.05 mM, 82.38 % at 0.5 mM and 12.17 % at 2.5 mM while significantly increased by 112.17 % at 0.25 mM (Fig. 3C). H2O2 content insignificantly increased by 5.80 % at 0.05 mM and significantly increased by 65.66 % at 2.5 mM while insignificantly decreased by 39.81 % at 0.25 mM and 46.85 % at 0.5 mM (Fig. 3D). The activity of CAT enzyme insignificantly increased by 24.44 % at 0.05 mM, 22.22 % at 0.25 mM, and insignificantly decreased by 8.88 % at 0.5 mM and 4.44 % at 2.5 mM (Fig. 3E).
2.4. Determination of ROS Germinated pollen grains were stained by 20 μM CM-H2DCFDA. Almost 150 pollen tubes were observed at 500 nm using a fluorescence microscope. To examine the ROS quantity at the apex, FI of ROS was scored in areas of 400 μm2 at the apex of 50 pollen tubes for each group. Measurement was performed with the ‘Rectangle Selection’ tool of ImageJ. 2.5. Determination of SOD, CAT enzyme activity and H2O2 content To analyze the effect of Put on ROS detoxification system in hazelnut pollen, SOD and CAT enzyme activity and H2O2 content were determined. To separate the germinated pollen grains from non-germinated pollen grains, filter mesh with suitable pore size for hazelnut (30–40 μm) was used according to literature with some modification (Chen et al., 2009). Almost 0.03 g of germinated pollen grains were homogenized in 1.5 ml of 50 mM PBS (pH 7.8). After centrifugation at 12,000 g for 15 min at 4 °C, the supernatants were used as enzyme sources for SOD and CAT experiments. To analyze the SOD activity, 2.4 ml of sample buffer (1.5 ml of 50 mM PBS pH 7.8, 300 μl of 130 mM L-methionine, 300 μl of 750 μM nitro blue tetrazolium, 300 μl of 100 μM EDTA-Na2) and 300 μl enzyme sources were mixed and incubated under light (50 μmol m−2s-1) for 3 min. The mixture was measured at 560 nm using a spectrophotometer (Li et al., 2000). To detect CAT activity, 1.5 ml of 0.2 M PBS containing 1% (m/v) PVP (pH 7), 1 ml of 72 mM H2O2 and 0.2 ml enzyme source were mixed. The mixture was spectrophotometrically evaluated by reduce in absorbance for 2 min at 240 nm (Prochazkova et al., 2001). To determine the H2O2 content, almost 0.03 g germinated pollen grains were homogenized with 2 ml of the extraction buffer including 0.1% TCA, 1 M KI, 10 mM PBS. After centrifugation at 12,000 g for 15 min at 4 °C, supernatants were incubated in dark chamber for 20 min and then measured the absorbance at 390 nm (Junglee et al., 2014). All spectrophotometrically analyses were conducted using IMPLEN nanophotometer. 2.6. Data analysis Statistical analyses were carried out by one-way analysis of variance (SPSS 16.0 software). All data presented are means ± SD with P < 0.05 followed by the least significant difference test (LSD). 3. Results To reveal the Put effect on hazelnut pollen performance, germination percentage and tube lengths were evaluated. Germination rate insignificantly increased by 10.6 % at 0.05 mM and 21.64 % at 0.25 mM. Conversely, Put concentration above 0.25 mM restricted germination. Germination reduced by 0.31 % at 0.5 mM and 8.73 % at 2.5 mM in compare to control, insignificantly (Fig. 1A). Pollen tube lengths presented a similar trend as the germination percentages. The tube length increased by 20.02 % at 0.05 mM and 10.64 % at 0.25 mM, significantly. Conversely, germination rate significantly decreased by 8.39 % at 0.5 mM and 22.54 % at 2.5 mM in compare to control (Fig. 1B). The abnormity ratio was also analyzed to evaluate the Put effect on hazelnut pollen performance. The control pollen tubes were generally regular with intact apex. Tube abnormalities decreased by 16.92 % at 0.05 mM, insignificantly. On the other hand, abnormality rate insignificantly increased by 95.66 % at 0.25 mM, 75.59 % at 0.5 mM and 285.03 % at 2.5 mM (Fig. 1C). The most widespread abnormalities were a swollen tube apex in all cases (Fig. 1D). Also, it is remarkable that the swelling is not only at the apex but also in the subapex in the 2.5 mM Put treatment. To examine the impact of different Put concentration on callose accumulation at the tube apex, pollen tubes were incubated with aniline blue. Callose was deposited equally along the tube shank although
4. Discussion In this study, the effect of Put on the performance of C. avellana pollen was detected as a dose-dependent mode. Put dosages up to 0.25 mM are capable of improving or at least not altering pollen germination and tube elongation. In addition, it was found that concentrations above 0.25 mM decrease both pollen germination and tube elongation. Similarly, Dixin and Shaoling (2002) have shown an excitation potency of Put on pollen germination and tube elongation in Pyrus serotine. It is likely that low doses of PAs can be most efficient in enhancing the pollen germination and tube elongation (Wolukau et al., 2004; Sorkheh et al., 2011). Song et al. (1999) stated that low concentrations of spermine and spermidine also have ameliorative effect on tomato pollen performance while high concentrations have not. It has been indicated that PAs organize the enzymatic activity of pectin-methyl esterase and they might lead to decreased grades of unesterified 3
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Fig. 1. Effect of Put on pollen germination, tube growth and tube morphology in hazelnut. A. Pollen germination rates, B. Pollen tube lengths, C. Pollen tube abnormality rates, D. Sample images of normal and abnormal pollen tubes. Bar: 20 μm. Means within each bar followed by the same letters are not significantly different by LSD test at P < 0.05 level (n = 4).
Fig. 2. Effect of Put on callose accumulation and actin filament organization in hazelnut pollen tubes. A. Representative images of callose accumulation pattern; arrows indicate the dense callose accumulation at apex, B. FI of callose in 400 μm2 area of apex, C. Representative images of actin filament distribution; arrows remark the disorganization (superimposed filaments at 0.5 mM and collapse of filament at apex at 2.5 mM) of actin filaments at apex, D. Actin filament anisotropy in 400 μm2 area of apex. Bar: 10 μm. Means within each bar followed by the same letters are not significantly different by LSD test at P < 0.05 level (n = 4).
morphology, cell wall or cell cytoskeleton are considered as a significant hallmark in appraising pollen performance (Sawidis et al., 2018; Çetinbaş-Genç, 2019). Any difference in these parameters can induce problems on the reproductive performance (Jia et al., 2017) and thus on pollination and fertilization success (Wang et al., 2013; Deveci
pectins and therefore to softer cell walls (Charnay et al., 1992). Also, researchers have indicated that PAs can affect organelle and vesicle transport by modifying actin filament organization and altering cytosolic free Ca2+ concentration (Wu et al., 2010a, 2010b). Differentiation or anomaly of pollen tube properties such as 4
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Fig. 3. Effect of Put on ROS accumulation and ROS detoxification system in hazelnut pollen. A. Representative images of ROS accumulation pattern; arrow indicate dense FI of ROS, B. FI of ROS in 400 μm2 area of apex, C. Changes in SOD activity, D. Changes in H2O2 content, E. Changes in CAT activity. Bar: 10 μm. Means within each bar followed by the same letters are not significantly different by LSD test at P < 0.05 level (n = 4).
accumulation at the tube tips is generally observed in engorged tubes due to the incompatibility or environmental stress (Ünal et al., 2013; Laggoun et al., 2019). Disturbance of the actin cytoskeleton is one of the main reasons for the generation of tube anomaly (Ketelaar et al., 2012). In C. avellana, disorganization of actin filaments was detected after 0.5 mM and 2.5 mM Put applications, at swollen tube apex in particular. Similarly, Aloisi et al. (2017) showed that 0,1 mM spermine treatment excited actin filament disruptions in abnormal tube apex of Pyrus communis. The dynamics of the actin cytoskeleton is critical for tube growth because a proper organized actin cytoskeleton is necessary to transfer organelles and vesicle and to direct tube growth (Qu et al., 2013). Dynamics of actin filaments can be evaluated by measuring the anisotropy rate. Increase in anisotropy refers to less actin dynamics (Parrotta et al., 2016). In C. avellana, all Put treatment increased the anisotropy value of actin filaments at the apex. However, the increment was significant only after 0.5 mM and 2.5 mM Put treatments. These higher anisotropy values indicated the decrease in dynamics of apical actin filaments that consequently affects adversely pollen tube elongation. Comparably, higher anisotropy was shown in heat-treated pollen tubes of Nicotiana tabacum (Parrotta et al., 2016). ROS are important signaling molecules for plants and play multiple roles during whole pollen lifetime (Jiang et al., 2014). Apex-positioned ROS are essential to sustain tube elongation (Potocky et al., 2007; Swanson and Gilroy, 2010). Disturbance of tip-positioned ROS was shown in diphenyleneiodonium chloride-excited stunted tubes of Pyrus pyrifolia (Jiang et al., 2014) and Cupressus arizonica (Pasqualini et al., 2015), respectively. In a conformable manner with these results, control and 0.05 mM Put-treated pollen tubes demonstrated an obvious apexpositioned ROS fluorescence signal. However, apex-localized ROS signal increased with increasing Put concentrations. Swollen pollen tube apex showed remarkable ROS fluorescence signal, especially after 0.5 and 2.5 mM Put treatment. This extreme fluorescence signal of ROS at high Put concentrations may explain the block of pollen tube growth and the high rate of morphological abnormality. Extreme ROS accumulation in pollen tubes are detoxified by enzymatic antioxidants (Wang et al., 2016). SOD supports the reduction of superoxide, a type of toxic ROS, to H2O2. CAT accelerates the degradation of H2O2 (Wang et al., 2010), thus scavenging the oxidative stress. The increase in SOD
et al., 2017). Wang et al. (2016) have shown the expanded pollen tube apex of Camellia sinensis after cold treatment. Moreover, herbicide-induced expansion of tube apex was detected in Hyacinthus orientalis (Deveci et al., 2017). An et al. (2018) showed abnormal and ruptured pollen tubes after 5-Aminolevulic acid treatment in Pyrus pyrifolia. Here, we show that Put treatments increased the tube abnormalities in C. avellana. More anomaly was monitored as the concentration of Put increases, suggesting that the Put-induced tube anomaly is concentration-dependent. These results indicate that Put-induced morphological abnormalities are parallel to Put-induced tube growth inhibition. Almost all of the abnormalities were the swelling of the tube tip. Similarly, Aloisi et al. (2015) has shown swollen tips of Pyrus communis pollen tubes after exposion to spermine. In addition, we have shown swollen tips in both low and high temperature stressed pollen tubes of C. avellana (Çetinbaş-Genç et al., 2019). In C. avellana' s 2.5 mM Put treated pollen tubes; swelling is not only at the apex but also in the subapex. This recalls what Aloisi et al. (2017) have already observed in the pear pollen tubes treated with spermine. Callose is a basic element of the tube wall and it is not present at the apex of control pollen tubes (Aloisi et al., 2017). Because of its structure, callose acts as an isolation barrier or molecular filter (Ünal et al., 2013). Therefore, the existence of callose at the tube apex decreases or block tube growth and prevents the transfer of sperm nuclei to the embryo sac. Thus, the presence of callose at the tube apex is an important parameter that can be used to evaluate pollen performance. In addition, the relative abundance of cell wall polymers can be affected by various treatments and changes at the level of cell wall components are considered as tools to analyze the stress-induced effects in pollen performance (Laggoun et al., 2019). Boron deficiency is known to induce changes in callose accumulation at the tube apex of Nicotiana tabacum (Yang et al., 1999) and Picea meyeri (Wang et al., 2003). Here, in C. avellana, dense callose deposition was visualized at the tube apex after 0.5 mM and 2.5 mM Put treatment. Considering that Put concentration above 0.25 mM adversely affects both pollen germination and tube elongation, our results were coherent with prior works documented that callose localized at the apex of aberrant or inefficient pollen tubes (Del Duca et al., 2010; Hao et al., 2013). Del Duca et al. (2010) has reported callose plugs in the apical region that concerned to the interception of the tube elongation in Pyrus communis. Callose 5
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activity after 0.05 and 0.25 Put treatment showed that the toxic superoxide radical in the pollen tubes is successfully converted to H2O2. In addition, reduced H2O2 amount and increased CAT activity indicated that toxic H2O2 has been successfully converted to harmless water. These findings showed that the ROS detoxification mechanisms in pollen tubes were not disturbed by 0.05 and 0.25 mM Put application. The uptrend in SOD activity after 0.5 and 2.5 Put treatment showed that toxic superoxide radical is converted to H2O2, especially proved by the high content of H2O2 at 2.5 mM. However, decreased CAT activity showed that toxic H2O2 could not be successfully converted to water after 0.5 and 2.5 mM Put treatment. These findings showed that the ROS detoxification mechanisms in pollen tubes were disturbed by the 0.5 and 2.5 mM Put treatment. All this data suggests that low Put concentration are not effective in improving pollen performance. However, high concentrations of Put prevent effective scavenging of ROS. As a result, a high concentration of ROS could alter the organization of actin filaments (Wilkins et al., 2011). Indirectly, alterations in the actin organization could affect the deposition of callose at the pollen tube apex because callose synthase is joint into the apical membrane by transport and fusion of secretory vesicles carried throughout the actin filaments (Cai et al., 2011). Hypothetically, an altered dynamics of actin filaments, as induced by high levels of ROS, could lead to the accumulation of enzyme at the pollen tube apex or a lesser removal of the same enzyme via endocytosis, thus altering callose level.
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5. Conclusion The application of Put in proper doses can be assessed to ensure the maintenance of pollen performance during the pollen's waiting period and can provide a new perspective to pollination and fertilization in hazelnut. The results open the way to expand hazelnut production by enhancing the pollen performance with polyamines and bring a new perspective to pollination and fertilization of hazelnut. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgement This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Aloisi, I., Cai, G., Faleri, C., Navazio, L., Serafini-Fracassini, D., Del Duca, S., 2017. Spermine regulates pollen tube growth by modulating Ca2+−dependent actin organization and cell wall structure. Front. Plant Sci. 8, 1701. https://doi.org/10.3389/ fpls.2017.01701. Aloisi, I., Cai, G., Serafini-Fracassini, D., Del Duca, S., 2016. Polyamines in pollen: from microsporogenesis to fertilization. Front. Plant Sci. 7, 155. https://doi.org/10.3389/ fpls.2016.00155. Aloisi, I., Cai, G., Tumiatti, V., Minarini, A., Del Duca, S., 2015. Natural polyamines and synthetic analogs modify the growth and the morphology of Pyrus communis pollen tubes affecting ROS levels and causing cell death. Plant Sci. 239, 92–105. https://doi. org/10.1016/j.plantsci.2015.07.008. An, Y.Y., Lu, W.Y., Li, J., Wang, L.J., 2018. ALA inhibits pear pollen tube growth through regulation of vesicle trafficking. Sci. Hortic. 241, 41–50. https://doi.org/10.1016/j. scienta.2018.06.081. Armstrong, C.G., Turner, N.J., 2018. Management and traditional production of beaked hazelnut (k’áp’xw-az’, Corylus cornuta; Betulaceae) in British Columbia. Hum. Ecol. 46 (4), 547–559. https://doi.org/10.1007/s10745-018-0015-x. Boudaoud, A., Burian, A., Borowska-Wykręt, D., Uyttewaal, M., Wrzalik, R., Kwiatkowska, D., Hamant, O., 2014. FibrilTool, an ImageJ plug-in to quantify fibrillar structures in raw microscopy images. Nat. Protoc. 9 (2), 457–463. https://doi. org/10.1038/nprot.2014.024. Brewbaker, J.L., Kwack, B.H., 1963. The essential role of calcium ion in pollen germination and pollen tube growth. Am. J. Bot. 50 (9), 859–865. https://doi.org/10. 1002/j.1537-2197.1963.tb06564.x. Carniel, F.C., Gorelli, D., Flahaut, E., Fortuna, L., Del Casino, C., Cai, G., Nepi, M., Prato,
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The effect of putrescine on pollen performance in hazelnut (Corylus avellana L.) Aslıhan Çetinbaş-Gença,*, Giampiero Caib, Stefano Del Ducac, Filiz Vardara, Meral Ünala a
Department of Biology, Marmara University, Kadıköy, 34722, Istanbul, Turkey Department of Life Sciences, University of Siena, via Mattioli 4, 53100, Siena, Italy c Department of Biological, Geological and Environmental Sciences, University of Bologna, via Irnerio 42, 40126, Bologna, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hazelnut Pollen germination Pollen elongation Polyamine Putrescine
One of the most substantial molecules for pollen germination and tube growth are polyamines. Putrescine is the most abundant polyamine and the molecule from which spermine and spermidine originate. In this study, the effects of different exogenous putrescine concentrations (0.05, 0.25, 0.5 and 2.5 mM) on pollen performance of hazelnut (Corylus avellana) were investigated. Germination ratio and tube length were induced by 0.05 and 0.25 mM putrescine treatment. Putrescine concentration above 0.25 mM inhibited the pollen germination, tube elongation and, caused morphological alterations such as apex swelling. While the 0.05 and 0.25 mM putrescine treatment did not cause significant change in callose accumulation and actin filament distribution at tube apex, concentrations above 0.25 mM caused dense callose accumulation and increased the actin filament anisotropy at tube apex. Moreover, putrescine treatment caused an increase of reactive oxygen species level at the apex, especially in swollen tube apex, at the concentration of 2.5 mM. Reactive oxygen species detoxification mechanisms, which can be examined by changes in the amount of hydrogen peroxide and enzyme activities, were not disrupted by 0.05 and 0.25 mM putrescine treatment, while they are affected by 0.5 and 2.5 mM putrescine treatment. Eventually, low doses (0.05 and 0.25 mM) of putrescine can be used as a performance enhancing agent for hazelnut pollen tube growth, while the higher concentrations (0.5 and 2.5 mM) cause adverse effects reducing fertilization success.
1. Intoduction
and night temperature differences or seasonal temperature changes (Heslop-Harrison et al., 1986). During this waiting period, the sustainability of pollen performance is important for the success of fertilization and it also affects yield. Therefore, the increase in pollen performance also results in an increase in reproduction efficiency and thus in production yield. So, to improve the pollen performance, exogenous treatment to pollen or pollen tubes can be recommended during the pollination period (including the waiting process). Researchers have generally considered the pollen germination rate, tube length and tube abnormality rate to evaluate pollen performance (Williams and Reese, 2019). Moreover, callose accumulation at tube apex have been used as an evaluation criterion because it affects pollen performance adversely and reduce the fertilization performance (Sawidis et al., 2018; Çetinbaş-Genç, 2019). Callose is a fundamental constituent of the tube wall, but it is absent at the tube apex (Williams et al., 2016; Aloisi et al., 2017). Existence of callose at the tube apex is an abnormal situation and it causes the reduction of reproductive
Hazelnut (Corylus avellana L.) is a member of Betulaceae family. It is globally the most significant nut crop and a significant resource in almost all areas of industry (Novara et al., 2017; Armstrong and Turner, 2018; Candellone et al., 2019). Although it is mainly cultivated in Turkey and Italy, its cultivation is becoming widespread all over the world because of its commercial value (Novara et al., 2017; ÇetinbaşGenç et al., 2019). Since the hazelnut’s reproductive biology is very unusual, understanding the development of some stages in reproductive biology and pollination are the main focus for increase the cultivation yield (Çetinbaş-Genç et al., 2019). In hazelnut, the pollen grains or pollen tubes wait for 2–3 months in different parts of the pistil for the maturation of ovary (Liu et al., 2014). Also, since pollination starts in winter and ends in spring, during this long waiting period, pollen and pollen tubes are exposed to low or high-temperature stresses due to day
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Corresponding author at: Department of Biology, Marmara University, Göztepe Campus, 34722, Istanbul, Turkey. E-mail addresses: [email protected] (A. Çetinbaş-Genç), [email protected] (G. Cai), [email protected] (S.D. Duca), fi[email protected] (F. Vardar), [email protected] (M. Ünal). https://doi.org/10.1016/j.scienta.2019.108971 Received 5 September 2019; Received in revised form 18 October 2019; Accepted 21 October 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Aslıhan Çetinbaş-Genç, et al., Scientia Horticulturae, https://doi.org/10.1016/j.scienta.2019.108971
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oxygen species (ROS) that led to the oxidative stress in pollen tube (You and Chan, 2015). Superoxide dismutase (SOD) promotes the reduction of superoxide to hydrogen peroxide (H2O2). Catalase (CAT) catalyzes the deterioration of H2O2 thereby overcoming the oxidative stress (Wang et al., 2010). Consequently, alterations in these compounds can be used as indicators to determine the positive or negative effect of PAs. The purpose of this manuscript is to assess the effect of exogenously applied Put on hazelnut pollen performance, by focusing on callose accumulation, actin cytoskeleton dynamics, and ROS detoxification system. The application of Put in proper doses can be assessed to ensure the maintenance of pollen performance during the pollen's waiting period and can provide a new perspective to pollination and fertilization in hazelnut.
success by preventing the transfer of sperm nuclei to embryo sac (Del Duca et al., 2010). Callose accumulation is likely strictly associated with the actin cytoskeleton dynamics because insertion of callose synthase in the plasma membrane is supervised via actin cytoskeleton (Cai et al., 2011). In accordance with the composition of actin filaments, pollen tube is divided into three area; the apex, the sub-apex and the shank. Actin filaments exist as parallel bundles in the shank and are joined with the actin fringe in the sub-apex. The characteristic structure of actin filaments at the apex, as curved and dynamic filaments, is required for the elongation of pollen tube (Cai et al., 2015). Accordingly, callose distribution and actin organization are critical parameters used in the indication of pollen performance (Kang et al., 2010). Many substance are reported to improve pollen germination and tube growth, such as flavonols (Ylstra et al., 1992), boron (Wang et al., 2003), gibberellic acid (Voyiatzsis and Paraskevopoulou-Paroussi, 2005) and polyamines. One of the most important class is polyamines (PA), because PA homeostasis is responsible for events that occur throughout the whole pollen lifetime (Aloisi et al., 2016). So, whether PAs affect pollen germination and tube growth was examined in many species. Putrescine (Put) is the most abundant PA and also the molecule from which spermine and spermidine originate (Sequera-Mutiozabal et al., 2017). The promoting effect of Put on pollen efficiency has been shown in pear (Dixin and Shaoling, 2002) and almond (Sorkheh et al., 2011). Despite the evidences, more data is needed to understand the effects of Put on pollen performance. In general, PAs homeostasis must be delicately regulated and exogenous treatment of PAs has a different effect during pollen germination. Low concentrations of exogenous PAs induce pollen tube emergence while high doses strongly block this process and alter tube form (Aloisi et al., 2017; Çetinbaş-Genç, 2019). PAs might also organize the enzymatic activity of pectin-methyl esterase and they might lead to decreased grades of unesterified pectins and therefore to softer cell walls (Charnay et al., 1992). In Arabidopsis thaliana pollen tubes, exogenous spermidine increased the cytosolic free Ca2+ concentration; spermidine oxidation by PAO generates H2O2, which acts as a second messenger to activate Ca2+ channels, thus inducing Ca2+ ion influx beyond optimum grades and causing the prevention of tube elongation. Activation of the Ca2+ currents by spermidine is considerably disrupted in KO-PAO mutants, but the Ca2+ channels can still be activated following treatment of H2O2 (Wu et al., 2010a, 2010b). In Malus domestica, during pollen tube growth, both RNA and protein biosynthesis were stimulated by addition of spermidine. In the same system, PA-conjugation to actin and tubulin catalyzed by TGase affects their capability to mediatize and their coaction with motor proteins both in vivo and in vitro, as demonstrated by the amorphous structures of microtubules when pollen tubulin is incubated with high concentrations of Put, while tubular-like structures are formed at lower concentrations (Del Duca et al., 2009). Also, PAs may regulate the installation and structures of cell wall polysaccharides, such as pectins. In soybean’s cell walls, positively-charged PAs might compete by unesterified (acidic) pectins in binding Ca2+ ions (Charnay et al., 1992). PAs usually exist in the cells in free and bound form and the effect of exogenous PAs is multifactorial. The molecular mechanism of action of PAs has generally related with their polycationic backbone capable of set-up electrostatic interplays with anion groups of different biological molecules as proteins, nucleic acids, and membrane phospholipids. Furthermore, PAs strongly bind in vitro to pectic substances and to isolated cell wall polysaccharides. Beside the electrostatic interactions, the covalent binding to glutamyl residues of specific proteins, catalyzed by transglutaminase could affect protein functionality. Other covalent bonds to phenylpropanoids, abundant in some plant families, as hydroxyl-cinnamic acids give rise to phenolamides, involved in the structure of the cell wall and concerned to fertility (Aloisi et al., 2016). It is accepted that PA affect the pollen germination and tube growth according to the dosage (Aloisi et al., 2015; Çetinbaş-Genç, 2019). Excessive dose of PAs causes an excessive accumulation of reactive
2. Material and methods 2.1. In vitro pollen germination, tube elongation and tube abnormality Pollen materials were collected from Akçakoca/Düzce (Turkey) in February 2019 and were stored at −20 °C after dehydration process. Pollen germination was carried out at 20 °C with 50% RH for 24 h, in parallel with former works (Heslop-Harrison et al., 1986; Novara et al., 2017; Carniel et al., 2018; Çetinbaş-Genç et al., 2019). Brewbaker& Kwack pollen germination medium (BK medium) with 12 % sucrose was used for the germination medium and the solution was supplemented with 0.05 mM, 0.25 mM, 0.5 mM and 2.5 mM Put (Brewbaker and Kwack, 1963; Çetinbaş-Genç et al., 2019). BK medium without Put was used for control group. Pollen grains whose tubes were at least twice as long as the pollen diameter were considered as germinated. For each individual treatment, 3 slides were prepared and 500 pollen grains were investigated for each slide. Eventually pollen germination percentages were scored by considering 1500 pollen grains for each individual treatment. In addition, 5 slides were prepared for each individual treatment and 100 pollen tube were investigated. Afterwards, tube length and abnormality rate were scored considering almost 500 germinated pollen grains for each group. Pollen tube abnormalities were investigated using 2% aceto-orcein which facilitate the examination of tube morphology by staining cytoplasm and nucleus (ÇetinbaşGenç et al., 2019).
2.2. Determination of callose accumulation Pollen tubes were incubated by 0.1 % decolorized Aniline Blue to visualize callose (Chen et al., 2007). Almost 150 tubes were monitored at 455 nm using an Olympus BX-51 fluorescence microscope with 40X objective and KAMERAM software. To examine the quantitative accumulation at the tube apex, fluorescence intensity (FI) was scored in an area of 400 μm2 at the apex of 50 pollen tubes for each group. FI measurement was performed by handling the ‘Rectangle Selection’ tool of ImageJ (https://imagej.nih.gov/ij/).
2.3. Determination of actin filament anisotropy Pollen tubes were fixed for 30 min in pH 6.9 buffer containing 100 mM PIPES, 5 mM MgSO4, 0.5 mM CaCl2, 0.05% (v/v) Triton X-100, 1.5% (m/v), formaldehyde, and 0.05% (m/v) glutaraldehyde, and washed two times in same buffer also containing 10 mM EGTA and 6.6 μM Alexa 488-phalloidin (Lovy-Wheeler et al., 2005). 150 pollen tubes were monitored at 488 nm using a fluorescence microscope. To examine the quantitative difference of actin filaments at tube apex, actin filament anisotropy was measured on 20 pollen tubes for each group. Anisotropy measurement was evaluated using the ‘Fibril Tool’ plugin of ImageJ (Boudaoud et al., 2014). 2
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it did not exist at the apex of control tubes, as well as in 0.05 mM and 0.25 mM Put treated tubes (Fig. 2A). Conversely, callose was present at the apex of both 0.5 mM and 2.5 mM Put treated pollen tubes, in addition to existing throughout the shank (Fig. 2A, arrows). To examine the variations of callose at the tube apex, FI of callose was measured in 400 μm2 areas of the apex. FI decreased by 12.20 % at 0.25 mM and increased by 15.86 % at 0.05 mM, insignificantly. However, FI significantly increased by 78.40 % at 0.5 mM, 113.40 % at 2.5 mM in compare to control (Fig. 2B). To understand whether the actin cytoskeleton could impact on changes in the pollen performance, actin filaments were visualized and analyzed. Distribution of actin filaments did not show remarkable difference between the control and 0.05 mM, 0.25 mM Put. Actin filaments were lined up as irregular at the apex while filaments arranged linearly in the shank (Fig. 2C). On the other hand, actin arrangement exhibited critical changes at 0.5 mM and 2.5 mM Put treated pollen tubes, especially in the swollen apex (Fig. 2C, arrows). Disorganizations of actin filaments were quite obvious in the swollen tube apex, while there were no changes in the tube shank. In order to determine the subtler differences at the apex, anisotropy analysis was performed. Anisotropy of actin filaments increased by 5.93 % at 0.05 mM, 8.47% at 0.25 mM, insignificantly. However, anisotropy of actin filaments significantly increased by 23.44% at 0.5 mM and 43.50% at 2.5 mM in compare to control (Fig. 2D). In order to determine whether Put treatment effected the ROS localization, pollen tubes were stained by CM-H2DCFDA. Tubes showed a uniform ROS fluorescence signal in control tubes as well as after 0.05 mM and 0.25 mM Put application (Fig. 3A). On the contrary, ROS fluorescence signal was more intense at the apex after treatment with 0.5 mM and 2.5 mM Put, especially at the swollen tube apex (Fig. 3A, arrow). To examine the variations of ROS accumulation at the apex, the FI of apex localized ROS was measured. To allow us to focus on the apex localized ROS even if pollen tubes are different lengths, a region starting from the pollen tube tip with width and length of about 20 μm were determined and FI was measured in this 400 μm2 area of apex. Quantification analysis showed that FI of ROS accumulated at the apex and insignificantly increased by 18.98 % at 0.05 mM and, significantly increased by 91.03 % at 0.25 mM, 155 % at 0.5 mM and 362.48 % at 2.5 mM (Fig. 3B). To analyze the impact of Put on ROS detoxification system, SOD, CAT enzyme activity and H2O2 content were determined. The activity of SOD insignificantly increased by 68.39 % at 0.05 mM, 82.38 % at 0.5 mM and 12.17 % at 2.5 mM while significantly increased by 112.17 % at 0.25 mM (Fig. 3C). H2O2 content insignificantly increased by 5.80 % at 0.05 mM and significantly increased by 65.66 % at 2.5 mM while insignificantly decreased by 39.81 % at 0.25 mM and 46.85 % at 0.5 mM (Fig. 3D). The activity of CAT enzyme insignificantly increased by 24.44 % at 0.05 mM, 22.22 % at 0.25 mM, and insignificantly decreased by 8.88 % at 0.5 mM and 4.44 % at 2.5 mM (Fig. 3E).
2.4. Determination of ROS Germinated pollen grains were stained by 20 μM CM-H2DCFDA. Almost 150 pollen tubes were observed at 500 nm using a fluorescence microscope. To examine the ROS quantity at the apex, FI of ROS was scored in areas of 400 μm2 at the apex of 50 pollen tubes for each group. Measurement was performed with the ‘Rectangle Selection’ tool of ImageJ. 2.5. Determination of SOD, CAT enzyme activity and H2O2 content To analyze the effect of Put on ROS detoxification system in hazelnut pollen, SOD and CAT enzyme activity and H2O2 content were determined. To separate the germinated pollen grains from non-germinated pollen grains, filter mesh with suitable pore size for hazelnut (30–40 μm) was used according to literature with some modification (Chen et al., 2009). Almost 0.03 g of germinated pollen grains were homogenized in 1.5 ml of 50 mM PBS (pH 7.8). After centrifugation at 12,000 g for 15 min at 4 °C, the supernatants were used as enzyme sources for SOD and CAT experiments. To analyze the SOD activity, 2.4 ml of sample buffer (1.5 ml of 50 mM PBS pH 7.8, 300 μl of 130 mM L-methionine, 300 μl of 750 μM nitro blue tetrazolium, 300 μl of 100 μM EDTA-Na2) and 300 μl enzyme sources were mixed and incubated under light (50 μmol m−2s-1) for 3 min. The mixture was measured at 560 nm using a spectrophotometer (Li et al., 2000). To detect CAT activity, 1.5 ml of 0.2 M PBS containing 1% (m/v) PVP (pH 7), 1 ml of 72 mM H2O2 and 0.2 ml enzyme source were mixed. The mixture was spectrophotometrically evaluated by reduce in absorbance for 2 min at 240 nm (Prochazkova et al., 2001). To determine the H2O2 content, almost 0.03 g germinated pollen grains were homogenized with 2 ml of the extraction buffer including 0.1% TCA, 1 M KI, 10 mM PBS. After centrifugation at 12,000 g for 15 min at 4 °C, supernatants were incubated in dark chamber for 20 min and then measured the absorbance at 390 nm (Junglee et al., 2014). All spectrophotometrically analyses were conducted using IMPLEN nanophotometer. 2.6. Data analysis Statistical analyses were carried out by one-way analysis of variance (SPSS 16.0 software). All data presented are means ± SD with P < 0.05 followed by the least significant difference test (LSD). 3. Results To reveal the Put effect on hazelnut pollen performance, germination percentage and tube lengths were evaluated. Germination rate insignificantly increased by 10.6 % at 0.05 mM and 21.64 % at 0.25 mM. Conversely, Put concentration above 0.25 mM restricted germination. Germination reduced by 0.31 % at 0.5 mM and 8.73 % at 2.5 mM in compare to control, insignificantly (Fig. 1A). Pollen tube lengths presented a similar trend as the germination percentages. The tube length increased by 20.02 % at 0.05 mM and 10.64 % at 0.25 mM, significantly. Conversely, germination rate significantly decreased by 8.39 % at 0.5 mM and 22.54 % at 2.5 mM in compare to control (Fig. 1B). The abnormity ratio was also analyzed to evaluate the Put effect on hazelnut pollen performance. The control pollen tubes were generally regular with intact apex. Tube abnormalities decreased by 16.92 % at 0.05 mM, insignificantly. On the other hand, abnormality rate insignificantly increased by 95.66 % at 0.25 mM, 75.59 % at 0.5 mM and 285.03 % at 2.5 mM (Fig. 1C). The most widespread abnormalities were a swollen tube apex in all cases (Fig. 1D). Also, it is remarkable that the swelling is not only at the apex but also in the subapex in the 2.5 mM Put treatment. To examine the impact of different Put concentration on callose accumulation at the tube apex, pollen tubes were incubated with aniline blue. Callose was deposited equally along the tube shank although
4. Discussion In this study, the effect of Put on the performance of C. avellana pollen was detected as a dose-dependent mode. Put dosages up to 0.25 mM are capable of improving or at least not altering pollen germination and tube elongation. In addition, it was found that concentrations above 0.25 mM decrease both pollen germination and tube elongation. Similarly, Dixin and Shaoling (2002) have shown an excitation potency of Put on pollen germination and tube elongation in Pyrus serotine. It is likely that low doses of PAs can be most efficient in enhancing the pollen germination and tube elongation (Wolukau et al., 2004; Sorkheh et al., 2011). Song et al. (1999) stated that low concentrations of spermine and spermidine also have ameliorative effect on tomato pollen performance while high concentrations have not. It has been indicated that PAs organize the enzymatic activity of pectin-methyl esterase and they might lead to decreased grades of unesterified 3
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Fig. 1. Effect of Put on pollen germination, tube growth and tube morphology in hazelnut. A. Pollen germination rates, B. Pollen tube lengths, C. Pollen tube abnormality rates, D. Sample images of normal and abnormal pollen tubes. Bar: 20 μm. Means within each bar followed by the same letters are not significantly different by LSD test at P < 0.05 level (n = 4).
Fig. 2. Effect of Put on callose accumulation and actin filament organization in hazelnut pollen tubes. A. Representative images of callose accumulation pattern; arrows indicate the dense callose accumulation at apex, B. FI of callose in 400 μm2 area of apex, C. Representative images of actin filament distribution; arrows remark the disorganization (superimposed filaments at 0.5 mM and collapse of filament at apex at 2.5 mM) of actin filaments at apex, D. Actin filament anisotropy in 400 μm2 area of apex. Bar: 10 μm. Means within each bar followed by the same letters are not significantly different by LSD test at P < 0.05 level (n = 4).
morphology, cell wall or cell cytoskeleton are considered as a significant hallmark in appraising pollen performance (Sawidis et al., 2018; Çetinbaş-Genç, 2019). Any difference in these parameters can induce problems on the reproductive performance (Jia et al., 2017) and thus on pollination and fertilization success (Wang et al., 2013; Deveci
pectins and therefore to softer cell walls (Charnay et al., 1992). Also, researchers have indicated that PAs can affect organelle and vesicle transport by modifying actin filament organization and altering cytosolic free Ca2+ concentration (Wu et al., 2010a, 2010b). Differentiation or anomaly of pollen tube properties such as 4
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Fig. 3. Effect of Put on ROS accumulation and ROS detoxification system in hazelnut pollen. A. Representative images of ROS accumulation pattern; arrow indicate dense FI of ROS, B. FI of ROS in 400 μm2 area of apex, C. Changes in SOD activity, D. Changes in H2O2 content, E. Changes in CAT activity. Bar: 10 μm. Means within each bar followed by the same letters are not significantly different by LSD test at P < 0.05 level (n = 4).
accumulation at the tube tips is generally observed in engorged tubes due to the incompatibility or environmental stress (Ünal et al., 2013; Laggoun et al., 2019). Disturbance of the actin cytoskeleton is one of the main reasons for the generation of tube anomaly (Ketelaar et al., 2012). In C. avellana, disorganization of actin filaments was detected after 0.5 mM and 2.5 mM Put applications, at swollen tube apex in particular. Similarly, Aloisi et al. (2017) showed that 0,1 mM spermine treatment excited actin filament disruptions in abnormal tube apex of Pyrus communis. The dynamics of the actin cytoskeleton is critical for tube growth because a proper organized actin cytoskeleton is necessary to transfer organelles and vesicle and to direct tube growth (Qu et al., 2013). Dynamics of actin filaments can be evaluated by measuring the anisotropy rate. Increase in anisotropy refers to less actin dynamics (Parrotta et al., 2016). In C. avellana, all Put treatment increased the anisotropy value of actin filaments at the apex. However, the increment was significant only after 0.5 mM and 2.5 mM Put treatments. These higher anisotropy values indicated the decrease in dynamics of apical actin filaments that consequently affects adversely pollen tube elongation. Comparably, higher anisotropy was shown in heat-treated pollen tubes of Nicotiana tabacum (Parrotta et al., 2016). ROS are important signaling molecules for plants and play multiple roles during whole pollen lifetime (Jiang et al., 2014). Apex-positioned ROS are essential to sustain tube elongation (Potocky et al., 2007; Swanson and Gilroy, 2010). Disturbance of tip-positioned ROS was shown in diphenyleneiodonium chloride-excited stunted tubes of Pyrus pyrifolia (Jiang et al., 2014) and Cupressus arizonica (Pasqualini et al., 2015), respectively. In a conformable manner with these results, control and 0.05 mM Put-treated pollen tubes demonstrated an obvious apexpositioned ROS fluorescence signal. However, apex-localized ROS signal increased with increasing Put concentrations. Swollen pollen tube apex showed remarkable ROS fluorescence signal, especially after 0.5 and 2.5 mM Put treatment. This extreme fluorescence signal of ROS at high Put concentrations may explain the block of pollen tube growth and the high rate of morphological abnormality. Extreme ROS accumulation in pollen tubes are detoxified by enzymatic antioxidants (Wang et al., 2016). SOD supports the reduction of superoxide, a type of toxic ROS, to H2O2. CAT accelerates the degradation of H2O2 (Wang et al., 2010), thus scavenging the oxidative stress. The increase in SOD
et al., 2017). Wang et al. (2016) have shown the expanded pollen tube apex of Camellia sinensis after cold treatment. Moreover, herbicide-induced expansion of tube apex was detected in Hyacinthus orientalis (Deveci et al., 2017). An et al. (2018) showed abnormal and ruptured pollen tubes after 5-Aminolevulic acid treatment in Pyrus pyrifolia. Here, we show that Put treatments increased the tube abnormalities in C. avellana. More anomaly was monitored as the concentration of Put increases, suggesting that the Put-induced tube anomaly is concentration-dependent. These results indicate that Put-induced morphological abnormalities are parallel to Put-induced tube growth inhibition. Almost all of the abnormalities were the swelling of the tube tip. Similarly, Aloisi et al. (2015) has shown swollen tips of Pyrus communis pollen tubes after exposion to spermine. In addition, we have shown swollen tips in both low and high temperature stressed pollen tubes of C. avellana (Çetinbaş-Genç et al., 2019). In C. avellana' s 2.5 mM Put treated pollen tubes; swelling is not only at the apex but also in the subapex. This recalls what Aloisi et al. (2017) have already observed in the pear pollen tubes treated with spermine. Callose is a basic element of the tube wall and it is not present at the apex of control pollen tubes (Aloisi et al., 2017). Because of its structure, callose acts as an isolation barrier or molecular filter (Ünal et al., 2013). Therefore, the existence of callose at the tube apex decreases or block tube growth and prevents the transfer of sperm nuclei to the embryo sac. Thus, the presence of callose at the tube apex is an important parameter that can be used to evaluate pollen performance. In addition, the relative abundance of cell wall polymers can be affected by various treatments and changes at the level of cell wall components are considered as tools to analyze the stress-induced effects in pollen performance (Laggoun et al., 2019). Boron deficiency is known to induce changes in callose accumulation at the tube apex of Nicotiana tabacum (Yang et al., 1999) and Picea meyeri (Wang et al., 2003). Here, in C. avellana, dense callose deposition was visualized at the tube apex after 0.5 mM and 2.5 mM Put treatment. Considering that Put concentration above 0.25 mM adversely affects both pollen germination and tube elongation, our results were coherent with prior works documented that callose localized at the apex of aberrant or inefficient pollen tubes (Del Duca et al., 2010; Hao et al., 2013). Del Duca et al. (2010) has reported callose plugs in the apical region that concerned to the interception of the tube elongation in Pyrus communis. Callose 5
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activity after 0.05 and 0.25 Put treatment showed that the toxic superoxide radical in the pollen tubes is successfully converted to H2O2. In addition, reduced H2O2 amount and increased CAT activity indicated that toxic H2O2 has been successfully converted to harmless water. These findings showed that the ROS detoxification mechanisms in pollen tubes were not disturbed by 0.05 and 0.25 mM Put application. The uptrend in SOD activity after 0.5 and 2.5 Put treatment showed that toxic superoxide radical is converted to H2O2, especially proved by the high content of H2O2 at 2.5 mM. However, decreased CAT activity showed that toxic H2O2 could not be successfully converted to water after 0.5 and 2.5 mM Put treatment. These findings showed that the ROS detoxification mechanisms in pollen tubes were disturbed by the 0.5 and 2.5 mM Put treatment. All this data suggests that low Put concentration are not effective in improving pollen performance. However, high concentrations of Put prevent effective scavenging of ROS. As a result, a high concentration of ROS could alter the organization of actin filaments (Wilkins et al., 2011). Indirectly, alterations in the actin organization could affect the deposition of callose at the pollen tube apex because callose synthase is joint into the apical membrane by transport and fusion of secretory vesicles carried throughout the actin filaments (Cai et al., 2011). Hypothetically, an altered dynamics of actin filaments, as induced by high levels of ROS, could lead to the accumulation of enzyme at the pollen tube apex or a lesser removal of the same enzyme via endocytosis, thus altering callose level.
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5. Conclusion The application of Put in proper doses can be assessed to ensure the maintenance of pollen performance during the pollen's waiting period and can provide a new perspective to pollination and fertilization in hazelnut. The results open the way to expand hazelnut production by enhancing the pollen performance with polyamines and bring a new perspective to pollination and fertilization of hazelnut. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgement This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Aloisi, I., Cai, G., Faleri, C., Navazio, L., Serafini-Fracassini, D., Del Duca, S., 2017. Spermine regulates pollen tube growth by modulating Ca2+−dependent actin organization and cell wall structure. Front. Plant Sci. 8, 1701. https://doi.org/10.3389/ fpls.2017.01701. Aloisi, I., Cai, G., Serafini-Fracassini, D., Del Duca, S., 2016. Polyamines in pollen: from microsporogenesis to fertilization. Front. Plant Sci. 7, 155. https://doi.org/10.3389/ fpls.2016.00155. Aloisi, I., Cai, G., Tumiatti, V., Minarini, A., Del Duca, S., 2015. Natural polyamines and synthetic analogs modify the growth and the morphology of Pyrus communis pollen tubes affecting ROS levels and causing cell death. Plant Sci. 239, 92–105. https://doi. org/10.1016/j.plantsci.2015.07.008. An, Y.Y., Lu, W.Y., Li, J., Wang, L.J., 2018. ALA inhibits pear pollen tube growth through regulation of vesicle trafficking. Sci. Hortic. 241, 41–50. https://doi.org/10.1016/j. scienta.2018.06.081. Armstrong, C.G., Turner, N.J., 2018. Management and traditional production of beaked hazelnut (k’áp’xw-az’, Corylus cornuta; Betulaceae) in British Columbia. Hum. Ecol. 46 (4), 547–559. https://doi.org/10.1007/s10745-018-0015-x. Boudaoud, A., Burian, A., Borowska-Wykręt, D., Uyttewaal, M., Wrzalik, R., Kwiatkowska, D., Hamant, O., 2014. FibrilTool, an ImageJ plug-in to quantify fibrillar structures in raw microscopy images. Nat. Protoc. 9 (2), 457–463. https://doi. org/10.1038/nprot.2014.024. Brewbaker, J.L., Kwack, B.H., 1963. The essential role of calcium ion in pollen germination and pollen tube growth. Am. J. Bot. 50 (9), 859–865. https://doi.org/10. 1002/j.1537-2197.1963.tb06564.x. Carniel, F.C., Gorelli, D., Flahaut, E., Fortuna, L., Del Casino, C., Cai, G., Nepi, M., Prato,
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