高等植物自交不亲和性的分子生物学

自交不亲和性是大多数高等植物防止近亲繁殖的一种遗传屏障。它涉及受精时雄配子（花粉）和雌蕊之间的相互作用。目前,已经分离获得了编码控制雌蕊自交不亲和性的Ｓ基因。在孢子体型自交不亲和的芸苔属中,雌蕊Ｓ基因编码Ｓ位点糖蛋白（ＳＬＧ）和Ｓ受体激酶（ＳＲＫ）。它们可能与磷酸化和去磷酸化参与了的某种信号传递有关,最后导致自交花粉生长的抑制。在配子分配体型自交不亲和的茄科中,雌蕊Ｓ位点糖蛋白为一种核糖核酸酶,称为Ｓ－核酸酶（Ｓ－ＲＮａｓｅ）。自交不亲和反应与Ｓ－核酸酶引起的花粉管ＲＮＡ降解有关,并且可能通过花粉管特异性地摄入Ｓ－核酸酶或者花粉管内存在的特异性的核酸酶抑制剂的作用,达到对自交花粉生长的抑制。另外,从配子体型自交不亲和的罂粟中,分离到了与芸台属和茄科不同的雌蕊Ｓ基因,其作用机理可能与Ｃａ＋＋参与的信号传递有关。     

白菜自交不亲和性的荧光测定

通过亲和指数法及荧光显微观察对白菜的自交不亲和性进行了测定。结果表明,白菜自交不亲和的反应部位在柱头,自花授粉后柱头表面产生明显的胼胝质反应。两种观测法的结果相吻合,荧光显微镜观察法准确、方便,可应用于白菜自交不亲和系的育种实践。     

高等植物自交不亲和性的分子生物学

自交不亲和性是大多数高等植物防止近亲繁殖的一种遗传屏障。它涉及受精时雄配子（花粉）和雌蕊之间的相互作用。目前,已经分离获得了编码控制雌蕊自交不亲和性的Ｓ基因。在孢子体型自交不亲和的芸苔属中,雌蕊Ｓ基因编码Ｓ位点糖蛋白（ＳＬＧ）和Ｓ受体激酶（ＳＲＫ）。它们可能与磷酸化和去磷酸化参与了的某种信号传递有关,最后导致自交花粉生长的抑制。在配子分配体型自交不亲和的茄科中,雌蕊Ｓ位点糖蛋白为一种核糖核酸酶,称     

无籽沙糖桔WUBE1基因的功能验证

为了解泛素活化酶E1基因(UBE1)在无籽沙糖桔自交不亲和反应中的作用,通过根癌农杆菌介导法将来源于自交不亲和无籽沙糖桔(Citrus reticulata ‘Wuzishatangju’) WUBE1基因转化烟草(Nicotiana tabacum)。结果表明,外源基因WUBE1已导入烟草基因组中并得到表达。转WUBE1基因的自交授粉组合花粉管在生长过程中,部分花粉管出现停止生长的现象,到达花柱基部的花粉管数量少于异交授粉和野生型自交组合。转WUBE1基因烟草的花粉生活力、发芽率、自交和异交后每个果荚中的种子数与野生型烟草无显著差异。这表明单一的WUBE1基因不能调控无籽沙糖桔自交不亲和反应,很可能是通过Ub/26S途径参与了无籽沙糖桔自交不亲和反应。     

芸薹属中自交不亲和反应的信号转导

自交不亲和现象在芸薹属(Brassica)植物中普遍存在,芸薹属中表现的是典型的孢子体型自交不亲和性.单元特异性的S位点受体激酶(SRK)基因和S位点花粉胞被蛋白(SCR/SP11)发生识别后,一系列相关蛋白-臂重复蛋白(ARC1)、M位点蛋白激酶(MLPK)等,引发了自交不亲和反应信号的传导,最终产生自交不亲和反应.文章就这方面的研究进展作介绍.     

泛素/26S蛋白酶体途径与显花植物自交不亲和反应

植物的生长和发育离不开短命调控蛋白的有选择性降解,其中一种重要的降解方式就是泛素,26S蛋白酶体途径。在这个途径中,泛素（ubiquitin）和26S蛋白酶体起着至关重要的作用,需要被降解的蛋白会通过E1-E2-E3酶接合反应由Ub进行标记,随后标记蛋白会被26s蛋白酶体识别并降解。自交不亲和反应也正是通过此途径实现的,ARC1（arm repeat containing 1）和SCFs（skp1-cul1-F-box-proteins）作为E3s分别在孢子体自交不亲和和配子体自交不亲和反应中起作用。本文综述了就泛素/26S蛋白酶体途径的组成及其在自交不亲和反应中的作用。     

泛素/26S蛋白酶体途径与显花植物自交不亲和反应

植物的生长和发育离不开短命调控蛋白的有选择性降解, 其中一种重要的降解方式就是泛素/26S蛋白酶体途径。在这个途径中, 泛素(ubiquitin)和26S蛋白酶体起着至关重要的作用, 需要被降解的蛋白会通过E1-E2-E3酶接合反应由Ub进行标记, 随后标记蛋白会被26S蛋白酶体识别并降解。自交不亲和反应也正是通过此途径实现的, ARC1(arm repeat containing 1)和SCFs (skp1-cul1-F-box-proteins)作为E3s分别在孢子体自交不亲和和配子体自交不亲和反应中起作用。本文综述了就泛素/26S蛋白酶体途径的组成及其在自交不亲和反应中的作用。     

青花菜自交不亲和特性的荧光鉴定

利用亲和指数和荧光显微测定法对青花菜高代自交材料进行自交不亲和特性的快速鉴定。利用蕾期和花期授粉进行亲和指数测定。荧光显微观察发现自交亲和材料与不亲和材料蕾期授粉花粉能够正常萌发,花粉管穿越柱头乳突细胞到达子房完成授粉受精过程;自交不亲和材料花期授粉后花粉与柱头乳突细胞表面发生强烈胼胝质反应,花粉在柱头上不能萌发,即使有少量萌发,其花粉管生长也不正常,最终不能伸入柱头。荧光显微观察法结果准确、方便,受外界环境条件影响较小,且与亲和指数法鉴定结果相一致,可有效应用于青花菜自交不亲和特性的快速鉴定,从而加速新品种选育进程。     

菊花自交不亲和性初步研究

以菊花品种杂交为对照,通过自交亲和指数、受精作用及自交有性过程荧光显微镜观察,对菊花自交不亲和性进行了研究。结果表明:菊花自交难以结实,具不亲和性;菊花自交不亲和的反应部位在雌蕊柱头,表现为花粉粒粘附少、萌发率低及诱导胼胝质生成,花粉管在柱头上出现各种异常现象而难以进入花柱。     

配子体自交不亲和植物花粉S基因研究进展

配子体自交不亲和植物的自交不亲和性是由雌蕊自交不亲和因子和花粉自交不亲和因子相互作用的结果。目前已经分离和鉴定了雌蕊自交不亲和基因及其表达产物。最近从金鱼草、Prumusdulcis、梅等植物中分离的F-box基因,它具有花粉S基因特点,即在花药、成熟的花粉和花粉管中特异表达;在基因位置上,与S-RNase基因紧密连锁;不同物种或同一物种不同品种F-box基因间核苷酸和氨基酸序列上存在高度多态性。通过分子生物学方法和杂交授粉试验证明所分离的F-box基因是花粉自交不亲和基因,但目前尚未分离出该类基因相应的表达蛋白。主要综述了配子体自交不亲和植物花粉自交不亲和基因的发现、基因的结构、雌蕊自交不亲和因子和花粉自交不亲和因子相互作用的模型。     

Turnover of diacylglycerol kinase 4 by cytoplasmic acidification induces vacuole morphological change and nuclear DNA degradation in the early stage of pear self-incompatibility response

Pear has an S-RNase-based gametophytic self-incompatibility (SI) system. Nuclear DNA degradation is a typical feature of incompatible pollen tube death, and is among the many physiological functions of vacuoles. However, the specific changes that occur in vacuoles, as well as the associated regulatory mechanism in pear SI, are currently unclear. Although research in tobacco has shown that decreased activity of diacylglycerol kinase (DGK) results in the morphological change of pollen tube vacuole, whether DGK regulates the pollen tube vacuole of tree plants and whether it occurs in SI response, is currently unclear. We found that DGK activity is essential for pear pollen tube growth, and DGK4 regulates pollen tube vacuole morphology following its high expression and deposition at the tip and shank edge of the pollen tube of pear. Specifically, incompatible S-RNase may induce cytoplasmic acidification of the pollen tube by inhibiting V-ATPase V₀ domain a1 subunit gene expression as early as 30 min after treatment, when the pollen tube is still alive. Cytoplasmic acidification induced by incompatible S-RNase results in reduced DGK4 abundance and deposition, leading to morphological change of the vacuole and fragmentation of nuclear DNA, which indicates that DGK4 is a key factor in pear SI response.     

Neither compatible nor self-incompatible pollinations of Brassica napus involve reorganization of the papillar cytoskeleton

Compatible pollination of Brassica napus necessitates pollen hydration, pollen germination and growth of the pollen tube through the loosened walls of stigmatic papillar cells, whereas self-incompatible (SI) pollinations fail at one of these stages. Analyses of the early stages of pollination show that at high (but not low) relative humidities, both compatible and SI pollen hydrates, but SI germination is reduced and the rare pollen tubes generally fail to penetrate the papillar walls, although there is some wall loosening. Inside the papillae, both compatible and SI interactions may induce the formation of callose, but there is no evidence for a major accumulation of cytoplasm or secretory vesicles in the vicinity of the pollen tubes and neither microtubule nor F-actin patterns re-arrange in this zone. These observations indicate that the source of the wall-loosening enzymes is probably the pollen tube or pollen coat, and that the common cellular responses of plants to attempted invasions have become suppressed in the papilla–pollen tube interaction.     

Self-incompatibility in passion fruit: cellular responses in incompatible pollinations

Self-incompatibility (SI) is a genetic mechanism in angiosperms that prevents selfing. The SI system in passion fruit (Passiflora edulis Sims) was investigated using hand pollinations. Pollen tube growth was inspected by microscopy, and sequence analysis of potential regulators of this process was carried out. The results revealed that the pollen tubes grew slowly and were often completely arrested in the stigma in an incompatible combination. Under these circumstances the pollen tube was rapidly and significantly rearranged, followed by the rapid deposition of callose in the stigma during the SI response. The structural changes in the pollen grain after an incompatible pollination were investigated using scanning electron microscopy. Furthermore, ultrastructural observations during incompatible interactions showed that the membrane system of the pollen tube was damaged, and fertilisation was not observed or was considerably delayed when compared to compatible interactions. The analysis presented here provides evidence that the passion fruit genome presents similar sequences to those encoding factors involved in SI in different species. These results suggest that, in the SI system of passion fruit, the rejection of an incompatible pollen grain is characterised by drastic structural changes in both pollen and pollen tube.     

The different mechanisms of gametophytic self-incompatibility

Self-incompatibility (SI) involves the recognition and rejection of self or genetically identical pollen. Gametophytic SI is probably the most widespread of the SI systems and, so far, two completely different SI mechanisms, which appear to have evolved separately, have been identified. One mechanism is the RNase system, which is found in the Solanaceae, Rosaceae and Scrophulariaceae. The other is a complex system, so far found only in the Papaveraceae, which involves the triggering of signal transduction cascade(s) that result in rapid pollen tube inhibition and cell death. Here, we present an overview of what is currently known about the mechanisms involved in controlling pollen tube inhibition in these two systems.     

Cytomechanical properties of papaver pollen tubes are altered after self-incompatibility challenge

Self-incompatibility (SI) in Papaver rhoeas triggers a ligand-mediated signal transduction cascade, resulting in the inhibition of incompatible pollen tube growth. Using a cytomechanical approach we have demonstrated that dramatic changes to the mechanical properties of incompatible pollen tubes are stimulated by SI induction. Microindentation revealed that SI resulted in a reduction of cellular stiffness and an increase in cytoplasmic viscosity. Whereas the former cellular response is likely to be the result of a drop in cellular turgor, we hypothesize that the latter is caused by as yet unidentified cross-linking events. F-actin rearrangements, a characteristic phenomenon for SI challenge in Papaver, displayed a spatiotemporal gradient along the pollen tube; this suggests that signal propagation occurs in a basipetal direction. However, unexpectedly, local application of SI inducing S-protein did not reveal any evidence for localized signal perception in the apical or subapical regions of the pollen tube. To our knowledge this represents the first mechanospatial approach to study signal propagation and cellular responses in a well-characterized plant cell system. Our data provide the first evidence for mechanical changes induced in the cytoplasm of a plant cell stimulated by a defined ligand.     

The Causes of self-sterility in natural populations of the relictual angiosperm, Illicium floridanum (Illiciaceae)

BACKGROUND AND AIMS: Illicium floridanum, a species belonging to the basal extant angiosperm taxon Illiciaceae, reportedly exhibits self-incompatibility (SI). To date, the site and timing of SI within the carpel of this species remains unidentified. Thus, the objective of this research was to determine the cellular and temporal aspects of SI in I. floridanum. METHODS: Following controlled application of cross- and self-pollen in natural populations of I. floridanum, embryo sac development and temporal aspects of stigma receptivity, as well as pollen tube growth, fertilization, and embryo and endosperm development, were investigated with the aid of light and fluorescence microscopy. KEY RESULTS: Flowers of I. floridanum exhibited complete dichogamy whereby stigmas only supported cross- and self-pollen tube growth prior to anther dehiscence. In contrast to earlier reports of SI in this species, a prezygotic SI resulting in rejection of self-pollen tube growth at the stigma was absent and there were no significant differences between cross- versus self-pollen germination and pollen tube growth within the style and ovary during the first 5 d after pollination. Structural development of the four-celled embryo sac was not differentially influenced by pollen type as noted to occur in other angiosperms with late-acting ovarian SI. The ovule micropyle and embryo sac were penetrated equally by cross- and self-pollen tubes. In addition, there were no statistically significant differences in cross- versus self-fertilization. A resting zygote and multicellular endosperm at a variety of developmental stages was present by 30 d after application of cross- or self-pollen. CONCLUSIONS: In the clear absence of a prezygotic SI that was previously reported to result in differential self-pollen tube growth at the stigma, self- sterility in I. floridanum is likely due to early-acting inbreeding depression, although late-acting post-zygotic ovarian SI cannot be ruled out.     

Investigating mechanisms involved in the self-incompatibility response in Papaver rhoeas

Sexual reproduction in flowering plants is controlled by recognition mechanisms involving the male gametophyte (the pollen) and the female sporophyte (the pistil). Self-incompatibility (SI) involves the recognition and rejection of self- or incompatible pollen by the pistil. In Papaver rhoeas, SI uses a Ca(2+)-based signalling cascade triggered by the S-protein, which is encoded by the stigmatic component of the S-locus. This results in the rapid inhibition of incompatible pollen tube growth. We have identified several targets of the SI signalling cascade, including protein kinases, the actin cytoskeleton and nuclear DNA. Here, we summarize progress made on currently funded projects in our laboratory investigating some of the components targeted by SI, comprising (i) the characterization of a pollen phosphoprotein (p26) that is rapidly phosphorylated upon an incompatible SI response; (ii) the identification and characterization of a pollen mitogen-activated protein kinase (p56), which exhibits enhanced activation during SI; (iii) characterizing components involved in the reorganization and depolymerization of the actin cytoskeleton during the SI response; and (iv) investigating whether the SI response involves a programmed cell death signalling cascade.     

A cascade signal pathway occurs in self-incompatibility of Pyrus pyrifolia

Pear (Pyrus pyrifolia L.) possesses an S-RNase-based gametophytic self-incompatibility (GSI) system and S-RNase, the self-incompatibility (SI) determinant in the pistil, has also been implicated in the rejection of self-pollen and genetically identical pollen. We have demonstrated that S-RNase depolymerises actin cytoskeleton, triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube, which indicates programmed cell death (PCD) may occur in SI response of Pyrus pyrifolia. Recently, we have identified that S-RNase specifically disrupted tip-localized reactive oxygen species (ROS) of incompatible pollen tube via arrest of ROS formation in mitochondria and cell walls in Pyrus pyrifolia. Furthermore, tip-localized ROS disruption not only decreased the Ca²⁺ current and depolymerised the actin cytoskeleton, but it also induced nuclear DNA degradation in the pollen tube. The results mentioned above indicate that a cascade signal pathway may occur in SI of Pyrus pyrifolia and PCD is used to terminate the incompatible pollen tubes growth. In this addendum, we review the cascade signal pathway of Pyrus pyrifolia SI.Key words: S-RNase, programmed cell death, reactive oxygen species, actin cytoskeleton, Ca²⁺ current, nuclear DNA     

Polyamines and transglutaminase activity are involved in compatible and self-incompatible pollination of Citrus grandis

Pollination of pummelo (Citrus grandis L. Osbeck) pistils has been studied in planta by adding compatible and self-incompatible (SI) pollen to the stigma surface. The pollen germination has been monitored inside the pistil by fluorescent microscopy showing SI altered morphologies with irregular depositions of callose in the tube walls, and heavy callose depositions in enlarged tips. The polyamine (PA) content as free, perchloric acid (PCA)-soluble and -insoluble fractions and transglutaminase (TGase) activity have been analyzed in order to deepen their possible involvement in the progamic phase of plant reproduction. The conjugated PAs in PCA-soluble fraction were definitely higher than the free and the PCA-insoluble forms, in both compatible and SI pollinated pistils. In pistils, pollination caused an early decrease of free PAs and increase of the bound forms. The SI pollination, showed highest values of PCA-soluble and -insoluble PAs with a maximum in concomitance with the pollen tube arrest. As TGase mediates some of the effects of PAs by covalently binding them to proteins, its activity, never checked before in Citrus, was examined with two different assays. In addition, the presence of glutamyl-PAs confirmed the enzyme assay data and excluded the possibility of a misinterpretation. The SI pollination caused an increase in TGase activity, whereas the compatible pollination caused its decrease. Similarly to bound PAs, the glutamyl-PAs and the enzyme activity peaked in the SI pollinated pistils in concomitance with the observed block of the pollen tube growth, suggesting an involvement of TGase in SI response.     

Involvement of extracellular calcium influx in the self-incompatibility response of Papaver rhoeas

We have previously demonstrated that increases in cytosolic free Ca2+ are triggered by the self-incompatibility (SI) response in incompatible Papaver rhoeas (the field poppy) pollen. However, one key question that has not been answered is whether extracellular Ca2+ may be involved. To address this question, we have used an ion-selective vibrating probe to measure changes in extracellular Ca2+ fluxes around poppy pollen tubes. Our data reveal several findings. First, we confirm that there is an oscillating Ca2+ influx directed at the apex of the pollen tube; we also provide evidence that Ca2+ influx also occurs at the shanks of pollen tubes. Second, upon challenge with self-incompatibility (S) proteins, there is a stimulation of Ca2+ influx along the shank of incompatible pollen tubes, approximately 50 microm behind the pollen tube tip. This demonstration of SI-induced Ca2+ influx suggests a role for influx of extracellular Ca2+ in the SI response.