不同重金属对‘黄花’梨花粉萌发及花粉管生长的影响

Effects of different types of heavy metals on pollen germination and tube growth of the 'Huanghua' pear

【目的】检测几种常见重金属对梨花粉萌发及花粉管生长的作用特点。【方法】采用液体培养法研究镍(Ni)、镉(Cd)、铜(Cu)、铅(Pb)和锌(Zn)等5种常见重金属对‘黄花’梨花粉萌发以及花粉管长度的影响,以电子显微镜检测重金属处理后花粉管尖端形态变化,并用NBT结合荧光探针标记分析花粉管尖端ROS分布。【结果】随着Ni^(2+)、Cd^(2+)、Cu^(2+)、Pb^(2+)和Zn^(2+)浓度增加,花粉萌发率越低且对花粉管伸长的抑制作用越明显。在以上5种重金属中,Pb对梨花粉萌发及花粉管伸长的影响最小,而Cu的影响最大。经过重金属处理后,花粉管尖端会出现不同程度的肿胀现象,并且维持花粉管极性生长的尖端活性氧(reactive oxygen species,ROS)浓度梯度受到破坏。Cu处理后花粉管尖端肿胀率最高,且显著高于其他4种重金属处理。【结论】以上5种重金属对‘黄花’梨花粉萌发和花粉管伸长的抑制作用呈现浓度依赖性,并表现出Pb<Ni<Cd<Zn<Cu的趋势。经重金属处理后,花粉管尖端发生肿胀,尖端ROS浓度梯度被破坏。不同处理下花粉管尖端肿胀率与其花粉萌发及花粉管伸长所受抑制程度相一致。

[Objective] Heavy metal pollution has become one of the serious environment problems along with social and economy development. These excessive heavy metals often exhibit negative effects on plant cells or organ growth, such as pollen germination and tube growth. During the pear reproductive period, pollen grains are exposed in air and are prone to pollution by some heavy metals. However, the effects of heavy metals on pear pollen germination and tube growth remain unknown. This present study evaluates the properties of Ni, Cd, Pb, Cu and Zn treatments on pear pollen germination and tube growth, respectively. [Methods] Pollen grains from the cultivar ＇Huanghua＇ （Pyrus pyrifolia） are collected from the Zhejiang A ＆ F University Fruit Experimental Yard, preserved by drying in air at ambient temperature （25 ℃） for 12 h, and then stored in silica gel at -20℃. Mature pear pollen grains were cultured in a culture medium in the dark at 25 ℃ for 3 h with a constant temperature shaker （100 r· min-1）. The culture medium con- tained 0.03% CaCl2,0.01% H3BO3, 10% sucrose, 15% polyethylene glycol 4 000 and 30 mmol·L-1 MES, pH 6.0 （adjusted with Trizma base）. Ni〉, Cd2＋, Cu〉, Pb2＋ and Zn2＋ were added to the culture medium before the pollen grains culture. The final treatment concentrations of Ni2＋, Cd〉, Cu〉, pb2＋or Zn〉 were 25, 50 and 100 μmol· L-1, respectively. When the germinated tube length was more than the diameter of the pollen grain, this pollen grain was considered as pollen germination. The germination rate is equal to the number of germinated pollen/total pollen number; 100 pollen grains （or tubes） were counted in each repeat. Pollen tube length and shape changes of the pear pollen tube tip were captured and analyzed by a microscope with a CCD camera and Image-Pro plus6.0 software, respectively. Data were analyzed with both Excel and SPSS 22.0 software. The tip-localized reactive oxygen species （ROS） were stained by Nitroblue tetrazolium （NBT; 1 g. L-1） and 5-（and-6）-chloromethyl-2＇, 7＇-dichlorodihydrofluorescein diacetate （CM-H2DCF-DA; 20 μmol· L-1） for 5 min, respectively. The stained samples were visualized by using a OLYMPUS BX51 microscope equipped with a CCD camera Sensys 1401E （Olympus Optical, Tokyo, Japan）. [Results ] 25, 50 or 100 μmol· L-1 Ni2＋, Cd2＋, Cu2＋, Pb2＋ and Zn2＋ showed inhibition of the ＇Huanghua＇ pear pollen germination rate and tube growth. The inhibition degree increased along with the concen- tration of the above heavy metal, and exhibited a dose-dependent correlation. The pollen germination rate showed no statistical significance between 25 and 0 μmol· L-1 Pb2＋ treatments, whereas the 25 μmol· L-1 Pb2＋ treatments significantly （P〈0.05） inhibited pollen tube length. Moreover, Ni displayed a similar nega- tive manner to Pb treatments. This result indicated that 25 μmol· L-1 Pb2＋ or Ni2＋ showed more negative effects on tube elongation than pollen germination. In contrast to Pb and Ni, the 25 μmol· L-1 Cd2＋, Zn2＋ or Cu2＋ treatments significantly （P〈0.05） inhibited pollen germination and tube growth. At the same concentration in our present work, Cd showed less negative effects on pollen germination and tube growth than Zn and Cu. Furthermore, Cu treatments showed the highest toxicity on the pear pollen germination rate and tube growth. The pollen germination rate was 10.0% after treatment with 25 μmol· L-1 Cu2＋, which extremely significantly （P〈0.01） inhibited the pollen germination rate in contrast to the control （52.6%）. The inhibition of the pollen tube average length was consistent with the pollen germination rate after Cu treat- ments. Among the above five different heavy metals, Pb treatments displayed the least effects on the pollen germination rate and tube growth. Additionally, the pollen tube tip shape is essential for rapid tube growth. Features of different heavy metals on the shape of the pear pollen tube tip were investigated after different heavy metal treatments. Three main forms of swollen tip could be observed in the pear pollen tube after different heavy metal treatments. The swollen tip rate was the lowest after Pb treatment, whereas the Cu treatment showed the highest effect. The swollen tip rate by the Cu treatment was significantly （P〈 0.05） more than the Pb, Ni, Cd and Zn treatments, respectively. Furthermore, the tip-localized ROS gradi- ent plays an essential role for pear pollen tube growth. Interestingly, the tip-localized ROS gradient was disturbed by the above heavy mental treatments, which suggested that the tip-localized ROS gradient disturbance could be involved in the inhibition processes of pear pollen tube growth after heavy metal treat- ments. [ Conclusion ] Different concentrations of Ni2＋, Cd2＋, Cu2＋, Pb2＋ and Zn2＋ showed various negative effects on pear pollen germination and tube growth in this work. This negative degree displayed a dose-dependent manner after one the above five heavy metal treatments. Three main types of swollen pollen tube tip were found after heavy metal treatments, and the rate of swollen pollen tube tip induced by Cu and Pb showed the highest and the lowest, respectively. Furthermore, pollen tube tip-localized ROS was disrupt- ed by heavy metal treatments. Combined with the pollen germination rate, tube length and tube swollen tip rate analysis after different heavy metal treatments, our results showed that a negative effect trend of different heavy metals was Pb 〈 Ni 〈 Cd 〈 Zn 〈 Cu on ＇ Huanghua＇ pear pollen germination and tube growth.