重楼属植物花药开裂和关闭机制研究

Anther opening is very important for pollen maturation and release and it is a complicated process. Defects in anther opening give rise to the failure of pollen release from anther locules and hence affect pollination. Anthers of different kinds of plants may have different opening mechanism..Paris L. is a temperate genus in the family of Trilliaceae.Roots of most Paris L. plants are chief raw materials of many Chinese patent drugs.We observed that anthers of some kinds of Paris L.plants open in the morning and close in the evening naturally during the flower anthesis,and close when it is raining and re_open when the rain stop. We concluded that the efficient opening and closing of P. polyphylla.var. yunnanensis anthers may be an adaptation to conserve pollen throughout the life of the flower, a mechanism that might enhance the male fitness.Such a particular phenomenon have never been reported by now except ecophysiology of anther opening and closing in P. polyphylla var. yunnanensis was studied by us. .To further this studies,and to gain a better understanding of anther opening and closing in Paris L.,based on the studies of ecophysiology of anther opening and closing in P. polyphylla var. yunnanensis we addressed the following four points: （1）In the process of microsporogenesis, male gametophyte development and pollen release,we study anther structure, ultrastructure using traditional paraffin wax section, transmission electron microscope(TEM), scanning electron microscope(SEM),endugenous hormones by Enzyme-Linked Immunosorbent Assays(ELISA), potassium by confocal laser scanning microscopy（potassium-binding benzofuran isophthalate, PBFI） and discuss the relationships with anther opening and closing; (2) By spraying different concentrations of hormones on anthers we study the influence of exogenous hormone on anther opening and closing; (3) Isolating and cloning the PIP aquaporins and analysing the expression in anther development and (4) Discussing the mechanism of anther opening and closing in Paris L..We hope the results of this research can be helpful to inheritance breeding and resource protection for Paris L.. At the same time, we hope this research can open up new areas in plant reproduction biology.

前期研究发现，多种重楼属植物的花药存在每天上午开裂、傍晚关闭，雨淋关闭、雨停开裂的特殊现象，是保护花粉、增强雄性适合度的适应机制。本项目在前期进行生理生态学研究的基础上，针对重楼属植物花药开裂-关闭的这一适应机制，进一步对滇重楼花药、小孢子、花粉形成及散粉过程与花药开裂-关闭的关系进行研究：通过常规石蜡切片、电镜观察等方法研究花药的组织细胞结构，采用酶联免疫法测定内源激素，用共聚焦激光扫描显微镜观察花粉内K+分布变化；研究不同外源激素条件下，滇重楼花药的开裂-关闭；分离克隆滇重楼水通道蛋白基因PIP同源序列，并分析其在花药不同发育时期组织器官中的表达；揭示重楼属植物花药开裂-关闭的机制。开展这一研究，不仅可丰富植物生殖生物学的研究内容，而且可为重楼资源的繁殖、保护与利用奠定理论基础。

多种重楼属植物的花药存在每天上午开裂、傍晚关闭，雨淋关闭、雨停开裂的特殊现象，本项目针对重楼属植物花药开裂-关闭的这一适应性机制，继续对其进行相关生理生态学研究，还通过常规石蜡切片、电镜观察等方法研究花药的组织细胞结构，采用酶联免疫法测定内源激素，用共聚焦激光扫描显微镜观察花粉内K+分布变化；研究不同外源激素条件下，滇重楼花药的开裂-关闭；进行滇重楼转录组测序揭示重楼属植物花药开裂-关闭的机制。主要结果如下：.1、在滇重楼花药开裂的过程中,光照强度增强、温度升高、相对湿度下降；温度是影响滇重楼花药开裂时间的主导因子,升温促进花药开裂,降温促进花药关闭；高湿度及黑暗推迟花药开裂,但并不能阻止花药开裂；低温可使滇重楼花药持续关闭,而光照强度越高,花药持续关闭所需的温度越低。 .2、滇重楼花药壁由表皮和内层组成，内层细胞壁加厚。当花药裂开时在靠近花药裂口的内层细胞壁比未开裂和关闭的明显加厚；随着花药在上午沿着花药长度方向裂开，花药壁逐渐收缩并向外弯曲致花药裂开，以之相反，下午花药壁向内弯曲致花药关闭；内层细胞中的质体的形状在裂开和关闭状态有较大区别，需要进一步研究。.3、茉莉酸、吲哚乙酸和赤霉素的含量在花药全部裂开时达到最大值，而脱落酸的含量变化不明显。.4、K+ 荧光强度与花药的状态有关，一天中随着开裂和关闭的整个过程，花粉周围和萌发孔处的荧光强度逐渐增强，到全开后花药壁和花粉萌发孔周围出现极强荧光，待半关闭、全关闭后花粉周围的荧光强度逐渐减弱。.5、茉莉酸、吲哚乙酸和赤霉素在低浓度条件下促进花药裂开、延迟关闭，浓度高则抑制了整个进程。不同浓度的脱落酸对重楼花药开裂和关闭进程的影响并不明显。.6、通过转录组比较，获得了一批与花药开裂和关闭的unigene，结果显示花药裂开是传粉的正常状态，而裂开花药的关闭是对光、温度、雨水等外界因素反应的非正常状态。对这些unigene的注释，发现一批如HLN、SNR、HD的上调表达基因。裂开的花药频繁的关闭可能上调了一系列表观遗传修饰基因，受精后这些表观遗传修饰基因最终影响到种子萌发和休眠。.7、滇重楼花药开裂-关闭与种子“二次休眠”没有直接关系。.开展这一研究，不仅可丰富植物生殖生物学的研究内容，而且可为重楼资源的繁殖、保护与利用奠定理论基础。