【作者】 胡荻；

【导师】 高素萍；

【作者基本信息】 四川农业大学 ， 风景园林学， 2019， 博士

【摘要】 蓝花丹(Plumbago auriculata Lam.)是白花丹科白花丹属植物,原产非洲南部,因其独特的淡蓝紫色花朵及较长的花期等观赏价值特性,被我国多地引种作为园林造景植物,深受消费市场追捧。由于蓝花丹有长柱头花型(Long-style,L)和短柱头花型(Short-style,S)分别于两株,是一种典型的二型花柱异长植物。花柱异长是植物在进化过程中表现的一种遗传多态性,通常伴随着自交不亲和(Self-incompatibility,SI)特性。SI是植物防止自交促进异交的策略之一,可分为同型自交不亲和(Homomorphic self-incompatibility,Hom SI)和异型自交不亲和(Heteromorphic self-incompatibility,HetSI)两类,蓝花丹即为典型的异型自交不亲和(HetSI)植物。早在达尔文时期,植物的HetSI特性就为人们所发现,但就目前研究的数量和深度都远远不及Hom SI的。尽管前人对花柱异长与HetSI反应的相关性研究取得了一些进展,但其反应机制仍需更多异型植物材料及不同层次的试验研究证据进一步补充。如,柱头-雄蕊的多态性对其受精障碍的贡献仍然是有争议的问题;直接或间接导致HetSI反应的代谢物质及相关代谢机制并不清楚,导致在代谢组学层面的研究证据极其匮乏。前人对Hom SI反应的代谢组学研究表明,能量物质的缺乏可能是造成受精失败的原因之一,那么,HetSI反应是否也与能量缺乏息息相关?为此,本研究结合形态学证据,使用UPLC-MS/MS和GC-MS两种检测手段,从代谢组学层面,分析HetSI反应发生的主要代谢物质及其与HetSI反应的相关性,试图从代谢层面补充HetSI反应机制的证据。研究主要结果及结论如下:1.蓝花丹异型花互补式雌雄异位,密集的花柱乳突细胞簇和突出的花粉纹饰是促使异交的因素之一。(1)蓝花丹花筒狭长,互补式雌雄异位,这种形态特征能有效促进花粉传递,促进花粉异交。(2)不同型花柱,花粉的微观结构均有差异。S型花柱上着生的乳突细胞簇着生在柱头的爪状结构上很密集,数量更多,更有利于对花粉的抓取,促进花粉异交。(3)蓝花丹花粉呈橄榄状,L型花粉纹饰呈密集的疣状结构,每个疣状结构上还着生着小突起。S型花粉的疣状结构相较比较圆润。2.蓝花丹S型花柱主要代谢物富集程度高于L型花柱代谢组结果显示大量的氨基酸、类黄酮、有机酸和糖及糖苷等能量物质在S型花柱中富集(基于UPLC-MS/MS检测结果),其富集程度高于L型花柱。丰富的营养物质在S型花柱中积累,为SC(亲和)花粉的萌发与花粉管的生长提供能量,促进花粉萌发与花粉管生长速率,使SC花粉在S型花柱上萌发更快,萌发率更高且花粉管的生长速率更快。3.蓝花丹HetSI反应发生的关键时间节点为授粉后2 h,SI(不亲和)授粉柱头表面未见花粉管萌发。(1)通过荧光显微镜(Fluorescence microscope,FM)观察发现,SC花粉在授粉后2 h左右观察到花粉管萌发,说明在这个时间点前花粉与柱头的识别已经完成。4 h后花粉管伸入柱头,8 h后SC花粉成功到达子房完成整个受精过程。SI花粉一直到8 h都没有观察到花粉管,甚至没有花粉粘附在柱头上。另外,SC花粉在S型花柱萌发率更高,速率更快,授粉的成功率也更高,这与S型花柱中更充足的能量物质供给息息相关。(2)通过扫描电镜(Scanning electron microscope,SEM)观察发现,在S×L授粉组合中观察到了大量的“足状”结构从萌发沟中“流”出,与柱头上的乳突状结构发生交联,萌发沟甚至没有直接接触到柱头的乳突状结构。而L×S授粉组合中,花粉与柱头连接的方式并不相同,柱头表面的乳突状结构直接与花粉的萌发沟连接。另外,SC花粉与花柱连接点周围的花柱乳突状结构呈现干瘪状态而SI花粉并没有观察到上述类似的结构,通过进一步观察发现,SI花粉并不能成功在柱头上水合与萌发,花粉甚至不能粘附在柱头上。结果表明授粉后2 h为HetSI反应发生生长形态差异的关键时间节点,且发生的空间位点位于柱头表面。形态学和生理代谢的差异部分是同时出现的,这为后续代谢组学研究取样时间的确定提供了依据。4.UPLC-MS/MS与GC-MS两种代谢物检测方法可行,各有侧重,互为补充。(1)UPLC-MS/MS共检测出代谢物142种,GC-MS检测出53种。UPLC-MS/MS检测出的代谢物数量较多,对次生代谢物检测能力强;GC-MS检测数量虽少,但对初生代谢物检测能力强,两种方法互补。试验检出化合物大致可以分为氨基酸、其他有机酸(盐)、脂肪酸及脂类、糖类及糖苷、类黄酮及其他代谢物。(2)试验数据分析结果表明,不同授粉组合代谢组存在显著差异(VIP>1且P value<0.05)。在L×S与L×L比较组中,UPLC-MS/MS检测结果中有显著差异的代谢物67种,GC-MS检测结果中共有显著差异代谢物24种。在S×L与S×S比较组中,UPLC-MS/MS检测结果中有显著差异的代谢物67种,GC-MS检测结果中共有显著差异代谢物41种。5.能量物质的缺乏并非是导致蓝花丹HetSI反应的原因,花粉-花柱初期特异性识别可能是受精失败的原因之一。SI授粉组合中氨基酸、糖类等与能量供给相关的代谢物大量富集,花粉管生长缺乏能量物质供应并不是导致HetSI反应的原因之一。三羧酸循环(TCA cycle)与糖酵解是植物的主要碳源在花粉管的生长过程中供给能量,其中间物质在SI授粉组合中含量很高。这充分说明了HetSI反应中花粉管不是因为能量供给不足而不生长,HetSI反应受精失败可能是由于花粉-花柱初期蛋白特异性识别不匹配造成的。6.花粉管的生长速率差异是造成花柱代谢物变化趋势差异的原因之一。检出大量代谢物如其他有机酸(盐)和醇类等的相对含量在L型授粉组合(L×L,L×S)与S授粉组合(S×S,S×L)中变化趋势相反。这种差异可能与L型相较S型柱头上的SC花粉萌发与花粉管生长滞后有关。萌发初期,花粉是自养的,花粉水合后,相关酶通过磷酸化作用使代谢物快速生成、富集,相对含量在L×S中升高,为随后的花粉萌发提供物质基础。花粉内部营养物质耗尽后,由花柱供给花粉管后期非自养生长。S×L中,花粉管生长速率快,花粉管到达非自养阶段,消耗花柱中的能量相关营养物质,造成代谢物含量降低。综上所述,结合蓝花丹HetSI反应在形态、结构,代谢化合物方面的研究结果,认为其不仅具有典型的花柱异长特征,且不同花型花柱能量代谢物含量差异较大。其HetSI反应发生在柱头与花粉的识别阶段,关键时间节点为授粉后2 h。花柱能量物质的缺乏可能直接影响花粉的萌发和花粉管的生长速率,但并不是导致HetSI反应的原因之一,其原因与柱头-花粉特异性蛋白识别直接相关。更多还原

【Abstract】 Plumbago auriculata Lam.native to southern Africa,it is now imported in many places in China,with good adaptability.The unique light blue flowers have excellent ornamental value and deeply loved by the consumer market.Plumbago auriculata is a typical plant with two different morphs:long-styles and short-styles.Heterostyly is a genetic polymorphism while heterostyly plants are usually associated with self-incompatibility(SI)acts as a safeguard against self-fertilization and inbreeding depression.The self-incompatibility mechanism comprises the homomorphic(Hom SI)and the heteromorphic(HetSI)self-incompatibility,discovered in Darwin’s time.Indeed,there is little evidence established the metabolic mechanism of the HetSI.Although previous studies on the HetSI mechanism have made some progress,there are still deficiencies,the HetSI mechanism still needs to be supplemented by more experimental evidence of heteromorphic plant from different levels.For instance,the contribution of stigma-stamen polymorphism to fertilization disorder in the HetSI is still controversial and the metabolites that directly or indirectly cause the incompatibility reaction are not clear which leading to a lack of evidence at the metabolomics level.Previous metabolomics studies on the Hom SI reaction have shown that the lack of energy may be one of the causes of fertilization failure.Then,is the HetSI mechanism also related to the lack of energy? To address the problem,this study combined morphological evidence with UPLC-MS/MS and GC-MS to analyze the main metabolites involved in the HetSI mechanism and their correlation with the HetSI reaction from the level of metabolomics,in order to supplement the evidence of the HetSI mechanism.The main results and conclusions of the study are as follows:1.P.auriculata has reciprocally arranged female and male reproductive organs,dense clusters of papilla cells on styles and protuberant pollen ornamentation are the factors that promote cross-pollinations.(1)P.auriculata has long and narrow flower tube and reciprocally arranged female and male reproductive organs,which can effectively promote pollen transfer and cross-pollinations.(2)Pollens and styles of different morphs showed distinct characters.The stigmatic papillaes on the S style are more numerous has a higher density on the claw structured stigmas,which are more beneficial to grasp pollens.(3)P.auriculata has olive-like pollens.The L pollens have dense verrucous structures,each verrucous structure is born with small bumps.However,the verrucous structures on the S stigma pollens are relatively rounder.2.The concentration of main metabolites in the S style have higher levels than that in the L styleMetabolic results showed that higher levels of amino acids,flavonoids,other organic acids,carbohydrates and glucosides in the S styles(based on UPLC-MS/MS).Abundant energy-related nutrients accumulate in the S style,providing enough energy for faster pollen germination and pollen tube growth.SC pollens had a higher germination rate and faster growth rate on the S style.3.The key time node of the HetSI mechanism of P.auriculata was 2 h after pollinations,and no pollen tube germination was observed on the surface of stigma after SI pollinations.(1)According to fluorescence microscope(FM)observation,pollen tube germination was observed about 2 h after cross-pollinations,indicating that identification between pollen and stigma had been completed before this time point.4 h later,the pollen tubes extended into the stigmas,8 h later,pollen tubes were successfully reached the ovarys and completed the whole fertilization process.Incompatible pollen was observed and no pollen tube was found germinated on the stigmas or even adhered on the stigmas.It is worth mentioning that the SC pollens on the S stigmas have a higher the germination rates,a faster growth rates and a higher efficiency of successful fertilizations,which is closely related to the sufficient supply of energy-related nutrients in the S styles.(2)Through scanning electron microscope(SEM)observations,a large number of "foot" structures were observed in the S×L pollinations which "flow" out of the germination groove and connected to the papillaes on the stigma,it is even not necessary for the germination groove to touch the stigmatic papillaes.However,in L×S pollinations,the connection mode between pollen and stigma was not the same,papillaes on stigma was directly connected to the germination groove on the SC pollens.Interestingly,the stigmatic papillae around the germinated pollen became less globular.While no similar structures mentioned above have been observed in the SI pollinations,the SI pollens on the stigmas never successfully rehydrated and germinated or even adhered on the stigmas.The experiment indicated that 2 h was the key time node for the HetSI mechanism and the HetSI mechanism was happened on the stigma surfaces,.This results provides an ideal sampling time for follow-up metabolomics studies.4.UPLC-MS/MS and GC-MS metabolites detection methods are feasible,with different focuses and complementary.(1)A total of 142 metabolites are detected by UPLC-MS/MS,while 53 metabolites are detected by GC-MS.The amount of metabolites detected by UPLC-MS/MS is larger.UPLC-MS/MS has a strong ability to detect secondary metabolites.Although the number of GC-MS detection is less,it has a strong ability to detect primary metabolites.The two methods complement each other.The compounds can be classified into amino acids,other organic acids,fatty acids and lipids,sugars and glycosides,flavonoids and other metabolites.(2)The statistical results show that there are significant differences between different pollinations(VIP>1 and P value<0.05).In the L×S and L×L comparison groups,there are 67 metabolites with significant differences in UPLC-MS/MS results,and 24 metabolites were significantly changed in GC-MS detection.In the S×L and S×S comparison groups,there are 67 metabolites that significantly changed that detected by UPLC-MS/MS,and 41 metabolites with significant difference in the GC-MS detection.5.The lack of energy nutrients is not the reason for the HetSI mechanism,and the specific identification of pollen-style on early stage may be one of the reasons for the fertilization failures.In SI pollination,metabolites related to energy supply were abundant,such as amino acids and carbohydrates.The lack of energy supply for pollen tube growth is not one of the reasons leading to the HetSI mechanism.The tricarboxylic acid cycle(TCA cycle)and glycolysis are the main carbon sources of plants that supply energy during the growth of pollen tubes.This fully indicates that insufficient energy supply do not leading the HetSI mechanism.6.The variation of pollen tube growth rate is one of the reasons cause the reverse trends of style-metabolites.The changes of style-metabolites of P.auriculata are complex,and the differences of germination and growth rates of the SC pollens on different types of styles may be one of the reasons for the diversity of metabolic trends.UPLC-MS/MS detect a large number of metabolites such as other organic acids and alcohols that show an opposite trend in the L×S and S×L pollinations.These changes may be closely related to the complex mechanism of plants and the rates of pollen germination and tube growth.Through the morphological study,the SC pollen germinations on the L stigmas are relatively delayed.After pollen hydration,enzymes became active through phosphorylation,enriched metabolites rapidly in to supply pollen autotrophy.However,the growth of non-autotrophic pollen tubes in S×L pollination may consume energy-related nutrients in the style which may cause the decline of metabolites.In conclusion,this paper discussed the characteristics of the HetSI mechanism of P.auriculata from the perspective of morphological structures and metabolomics.The conclusion is that P.auriculata is a typical HetSI plant,and the content of energy metabolites varies greatly with different types of styles.The HetSI mechanism of P.auriculata occurred in the stage of stigma-pollen identification,and the key time node for the occurrence was 2 h after successful pollinations.The lack of energy substances in the style may directly affect the germination and growth rate of pollen tubes,but the lack of energy-related nutrients is not one of the reasons for the HetSI mechanism.The HetSI mechanism of P.auriculata may be directly related to the stigma-pollen specific recognitions.更多还原