Ethylene signaling is critical for synergid cell functional specification and pollen tube attraction

ETHYLENE INSENSITIVE 3 (EIN3) is a key regulator of ethylene signaling, and EIN3‐BINDING F‐BOX1 (EBF1) and EBF2 are responsible for EIN3 degradation. Previous reports have shown that the ebf1 ebf2 double homozygous mutant cannot be identified. In this study, the genetic analysis revealed that the ebf1 ebf2 female gametophyte is defective. The pollination experiment showed that ebf1 ebf2 ovules failed to attract pollen tubes. In female gametophyte/ovule, the synergid cell is responsible for pollen tube attraction. Observation of the pEIN3::EIN3‐GFP transgenic lines showed that EIN3 signal was over‐accumulated at the micropylar end of ebf1 ebf2 female gametophyte. The overexpression of stabilized EIN3 in synergid cell led to the defect of pollen tube guidance. These results suggested that the over‐accumulated EIN3 in ebf1 ebf2 synergid cell blocks its pollen tube attraction which leads to the failure of ebf1 ebf2 homozygous plant. We identified that EIN3 directly activated the expression of a sugar transporter, SENESCENCE‐ASSOCIATED GENE29 (SAG29/SWEET15). Overexpression of SAG29 in synergid cells blocked pollen tube attraction, suggesting that SAG29 might play a role in ethylene signaling to repel pollen tube entry. Taken together, our study reveals that strict control of ethylene signaling is critical for the synergid cell function during plant reproduction.     

持续激活型CIPK9在花粉管生长过程中的生物学功能及亚细胞定位分析

植物的花粉管生长是一个多因素参与的生理学过程,需要多种信号传导系统来引导植物细胞完成。钙离子作为第二信使,可以通过钙传感器CBLs激活下游的蛋白激酶CIPKs参与调控细胞的极性发育过程。该研究中 CIPK9 被确定为候选基因,其C端与绿色荧光蛋白(GFP)相融合,通过基因枪技术在烟草花粉中进行瞬时表达,观察对应的亚细胞定位及花粉管中诱导的表型。结果表明:(1)GFP标记的CIPK9定位于花粉管中高速运动的颗粒状细胞器,并可随胞质环流进行规律的运动,为进一步探究CIPK9的生物学功能,还构建了持续激活型CIPK9(CACIPK9)。(2)与全长CIPK9相比较,CACIPK9缺少C末端的调控区域,并在激酶区域的激活环中进行了点突变,从而表现出不受调控的持续高活性。(3)缺少C端调控区的CACIPK9表现出非特异性的亚细胞定位,即与GFP对照相同的胞内弥散定位,说明CIPK9的C末端调控区对于其在花粉管中的正确定位发挥重要的调控作用。另外,CACIPK9过表达可以引起花粉管的去极化生长表型。这表明CIPK9作为钙信号下游家族的一员参与了花粉管极性生长的相关过程,并对花粉管的生长具有一定的调控作用。     

Actin organization and regulation during pollen tube growth

Pollen is the male gametophyte of seed plants and its tube growth is essential for successful fertilization. Mounting evidence has demonstrated that actin organization and regulation plays a central role in the process of its germination and polarized growth. The native structures and dynamics of actin are subtly modulated by many factors among which numerous actin binding proteins (ABPs) are the most direct and significant regulators. Upstream signals such as Ca²⁺, PIP₂ (phosphatidylinositol-4,5-bis-phosphate) and GTPases can also indirectly act on actin organization through several ABPs. Under such elaborate regulation, actin structures show dynamically continuous modulation to adapt to the in vivo biologic functions to mediate secretory vesicle transportation and fusion, which lead to normal growth of the pollen tube. Many encouraging progress has been made in the connection between actin regulation and pollen tube growth in recent years. In this review, we summarize different factors that affect actin organization in pollen tube growth and highlight relative research progress.     

Pollen germination and pollen tube growth in Fraxinus pennsylvanica

With regard to adaptation of green ash (Fraxinus pennsylvanica Marshall) to ecological conditions in Croatia, pollen germination and pollen tube length after 2, 4 and 6 hours were examined in vitro at 10, 15, 20 and 25°C during two years 2001 and 2002. Narrow leaved ash (F. angustifolia Vahl) pollen served as a control in 2002. The year, time and temperature, and the interaction between time and temperature were significant for both germination percentage and pollen tube length. Interactions year × temperature and year × time were significant for pollen tube length only. The highest germination percentage (17.86% in 2001 and 19.40% in 2002) of green ash pollen was at 15°C after 6 hours. The pollen tube length was greatest at 20°C (393.46 μm) in 2001 and 25°C (899.50 μm) in 2002 after 6 hours. Narrow leaved ash pollen had the highest germination percentage (19.22%) at 20°C after 6 hours and was significantly reduced at 25°C. The pollen tube length was greatest at 25°C (518.90 μm) after 6 hours. It can be concluded that green ash pollen has satisfactory germination in ecological conditions in Croatia and that the optimum temperature for pollen germination is higher than 20°C.     

Actin is involved in pollen tube tropism through redefining the spatial targeting of secretory vesicles

In order to accurately target the embryo sac and deliver the sperm cells, the pollen tube has to find an efficient path through the pistil and respond to precise directional cues produced by the female tissues. Although many chemical and proteic signals have been identified to guide pollen tube growth, the mechanism by which the tube changes direction in response to these signals is poorly understood. We designed an experimental setup using a microscope-mounted galvanotropic chamber that allowed us to induce the redirection of in vitro pollen tube growth through a precisely timed and calibrated external signal. Actin destabilization, reduced calcium concentration in the growth medium and inhibition of calcium channel activity decreased the responsiveness of the pollen tube to a tropic trigger. An increased calcium concentration in the medium enhanced this response and was able to rescue the effect of actin depolymerization. Time-lapse imaging revealed that the motion pattern of vesicles and the dynamics of the subapical actin array undergo spatial reorientation prior to the onset of a tropic response. Together these results suggest that the precise targeting of the delivery of new wall material represents a key component in the growth machinery that determines directional elongation in pollen tubes.     

Sugar composition of pollen grain and pollen tube cell walls

The sugar composition of pollen grain and pollen tube cell walls was studied for Camellia japonica, C. sasanqua, C. sinensis, Tulipa gesneriana and Lilium longiflorum. In all species, the main components of pollen grain walls were arabinose, galactose, glucose and uronic acid. On the other hand, the pollen tube walls consisted mostly of glucose. The pollen tube wall of C. japonica was fractionated into hemicellulose, α-cellulose and pectic substance fractions in yields of 61, 19 and 3 %, respectively. The hemicellulose fraction was composed essentially of glucose. The sugar composition of the pollen tube wall was not influenced by the nature of exogenously supplied sugars. Rapid growth of the pollen tube seemed to correlate with the synthesis of hemicellulosic glucan.     

蛋白质可逆磷酸化对花粉管生长的调控作用

花粉管极性生长受多种信号与代谢过程的调控,主要包括Rop GTPase信号途径、磷脂酰肌醇信号通路、Ca²⁺信号途径、肌动蛋白动态变化、囊泡运输、细胞壁重塑等,这些过程都受到蛋白质可逆磷酸化作用的调节。如：(1) Rop调节蛋白(GEF、GDI和GAP)的可逆磷酸化可以改变其活性,从而调节Rop GTPase;同时,蛋白激酶还可能作为Rop下游的效应器分子参与Rop下游信号途径的调节;(2) 蛋白质可逆磷酸化作用既能够激活/失活质膜上的Ca²⁺通道或Ca²⁺泵,又参与调节胞内贮存Ca²⁺的释放,从而调控花粉管尖端Ca²⁺梯度的形成;此外,蛋白激酶还作为Ca²⁺信号的感受器,磷酸化相应的靶蛋白,参与Ca²⁺信号下游途径的调节;(3) 肌动蛋白结合蛋白(ADF和Profilin)的活性也受到蛋白质可逆磷酸化的调节,进而调控肌动蛋白聚合与解聚之间的动态平衡;(4) 蛋白质磷酸化作用调节胞吞/胞吐相关蛋白的活性,并调控质膜的磷脂代谢,从而参与调控囊泡运输过程;(5) 胞质丝氨酸/苏氨酸蛋白激酶和蔗糖合酶的可逆磷酸化可以调节其在花粉管中的功能与分布模式,参与花粉管细胞壁重塑;(6) 转录调节蛋白与真核生物翻译起始因子的可逆磷酸化可以改变其活性,从而调控RNA转录与蛋白质合成。文章主要综述了花粉管生长过程中重要蛋白质的可逆磷酸化作用对上述关键事件的调节。     

Electrophoretic profiling and immunocytochemical detection of pectins and arabinogalactan proteins in olive pollen during germination and pollen tube growth

Background and Aims

Cell wall pectins and arabinogalactan proteins (AGPs) are important for pollen tube growth. The aim of this work was to study the temporal and spatial dynamics of these compounds in olive pollen during germination.

Methods

Immunoblot profiling analyses combined with confocal and transmission electron microscopy immunocytochemical detection techniques were carried out using four anti-pectin (JIM7, JIM5, LM5 and LM6) and two anti-AGP (JIM13 and JIM14) monoclonal antibodies.

Key Results

Pectin and AGP levels increased during olive pollen in vitro germination. (1 → 4)-β-d-Galactans localized in the cytoplasm of the vegetative cell, the pollen wall and the apertural intine. After the pollen tube emerged, galactans localized in the pollen tube wall, particularly at the tip, and formed a collar-like structure around the germinative aperture. (1 → 5)-α-l-Arabinans were mainly present in the pollen tube cell wall, forming characteristic ring-shaped deposits at regular intervals in the sub-apical zone. As expected, the pollen tube wall was rich in highly esterified pectic compounds at the apex, while the cell wall mainly contained de-esterified pectins in the shank. The wall of the generative cell was specifically labelled with arabinans, highly methyl-esterified homogalacturonans and JIM13 epitopes. In addition, the extracellular material that coated the outer exine layer was rich in arabinans, de-esterified pectins and JIM13 epitopes.

Conclusions

Pectins and AGPs are newly synthesized in the pollen tube during pollen germination. The synthesis and secretion of these compounds are temporally and spatially regulated. Galactans might provide mechanical stability to the pollen tube, reinforcing those regions that are particularly sensitive to tension stress (the pollen tube–pollen grain joint site) and mechanical damage (the tip). Arabinans and AGPs might be important in recognition and adhesion phenomena of the pollen tube and the stylar transmitting cells, as well as the egg and sperm cells.     

山玉兰花粉离体萌发和花粉管生长特性的研究

山玉兰(Magnolia delavayi)是木兰科木兰属的常绿乔木或大型灌木,是重要的园林造景、庭院绿化素材,也是重要的育种资源。山玉兰花粉的研究对其杂交育种的成败具有重要影响,但目前尚未见其花粉活力的相关报道。该研究以新鲜的山玉兰花粉为对象,采用悬滴培养法分析了温度、光照以及培养液的蔗糖和硼酸浓度对山玉兰花粉萌发的影响。结果表明:(1)山玉兰花粉萌发时,最适宜的温度为27℃。(2)光暗条件下,山玉兰花粉以浓度为5%的蔗糖培养效果最佳,其萌发率在16%以上;而硼酸浓度则以0.001%的培养效果最佳。(3)蔗糖与硼酸共同作用可有效促进花粉萌发和花粉管生长。其中,在光照条件下,以5.0%蔗糖+0.001%硼酸为最适宜的培养液,花粉萌发率达41.27%,花粉管长达281.49μm;而在黑暗条件下,则以5.0%蔗糖+0.01%硼酸为最适宜的培养液,花粉萌发率达45.71%,花粉管长达254.00μm。该研究结果为进一步开展人工辅助授粉、发掘山玉兰的种质资源工作奠定了基础。     

Evaluation of pollen tube growth ability in Petunia species having different style lengths

Pollen tube growth is essential for the fertilization process in angiosperms. When pollen grains arrive on the stigma, they germinate, and the pollen tubes elongate through the styles of the pistils to deliver sperm cells into the ovules to produce the seeds. The relationship between the growth rate and style length remains unclear. In previous studies, we developed a liquid pollen germination medium for observing pollen tube growth. In this study, using this medium, we examined the pollen tube growth ability in Petunia axillaris subsp. axillaris, P. axillaris subsp. parodii, P. integrifolia, and P. occidentalis, which have different style lengths. Petunia occidentalis had the longest pollen tubes after 6 h of culture but had a relatively shorter style. Conversely, the pollination experiments revealed that P. axillaris subsp. parodii, which had the longest style, produced the longest pollen tubes in vivo. The results revealed no clear relationship between the style lengths and the growth rate of pollen tubes in vitro. Interspecific pollinations indicated that the styles affected pollen tube growth. We concluded that, in vitro, the pollen tubes grow without being affected by the styles, whereas, in vivo, the styles significantly affected pollen tube growth. Furthermore, interspecific pollination experiments implied that the pollen tube growth tended to be suppressed in the styles of self-incompatibility species. Finally, we discussed the pollen tube growth ability in relation to style lengths.     

G蛋白对花粉管生长的调控作用

花粉萌发和花粉管生长是花粉与雌蕊相互作用过程中受到高度调控的发育过程,它涉及花粉与雌蕊的识别作用、细胞间及细胞内信息传递等生理反应。近年的研究表明G蛋白作为一类重要的信号分子在调控花粉管生长中起重要作用。着重介绍G蛋白对花粉管生长的调控作用以及此过程中G蛋白与其它信号组分的协同作用。     

Effects of Yariv phenylglycoside on cell wall assembly in the lily pollen tube

Arabinogalactan-proteins (AGPs) are proteoglycans with a high level of galactose and arabinose. Their current functions in plant development remain speculative. In this study, (β-D-glucosyl)₃ Yariv phenylglycoside [(β-D-Glc)₃] was used to perturb AGPs at the plasmalemma-cell wall interface in order to understand their functional significance in cell wall assembly during pollen tube growth. Lily (Lilium longiflorum Thunb.) pollen tubes, in which AGPs are deposited at the tip, were used as a model. Yariv phenylglycoside destabilizes the normal intercalation of new cell wall subunits, while exocytosis of the secretory vesicles still occurs. The accumulated components at the tip are segregated between fibrillar areas of homogalacturonans and translucent domains containing callose and AGPs. We propose that the formation of AGP/(β-D-Glc)₃ complexes is responsible for the lack of proper cell wall assembly. Pectin accumulation and callose synthesis at the tip may also change the molecular architecture of the cell wall and explain the lack of proper cell wall assembly. The data confirm the importance of AGPs in pollen tube growth and emphasize their role in the deposition of cell wall subunits within the previously synthesized cell wall. Received: 14 August 1997 / Accepted: 9 September 1997     

LeSTIG1, an extracellular binding partner for the pollen receptor kinases LePRK1 and LePRK2, promotes pollen tube growth in vitro

As pollen tubes grow through the pistil they are thought to perceive and respond to diverse signals. The tomato pollen-specific receptor kinases LePRK1 and LePRK2 might participate in signaling during pollen tube growth. We previously showed that the extracellular domain of LePRK2 interacts with a pollen protein, LAT52, before but not after pollen germination. To determine whether LePRK2 might have different binding partner(s) after pollen germination, we characterized two more proteins that, like LAT52, were identified in yeast two-hybrid screens using the extracellular domains of LePRK1 and LePRK2 as baits. We show that LeSHY, a leucine-rich repeat protein from pollen, and LeSTIG1, a small cysteine-rich protein from pistil, can bind the extracellular domains of both LePRK1 and LePRK2 in vitro. In vitro binding assays with the extracellular domain of LePRK2 suggested that LeSTIG1 could displace binding of LAT52, consistent with the idea that LePRK1 and LePRK2 might interact with different ligands at different stages of pollen tube growth. Exogenous LeSTIG1 promotes pollen tube growth in vitro. The interaction of these pollen kinases with LeSTIG1 supports the notion that LePRK1 and LePRK2 are involved in mediating pollen-pistil interactions.     

植物生长调节物质对丰水梨花粉萌发和花粉管生长的影响

采用花粉液体培养法研究了植物生长调节物质对梨花粉萌发和花粉管生长的影响,结果表明：较低浓度的赤霉素、三十烷醇、吲哚乙酸及2,4-D均能促进花粉萌发和花粉管生长,而超过一定浓度时却起抑制作用,最适宜于花粉萌发和花粉管生长的赤霉素浓度为50～300mg／L,三十烷醇为3～100mg／L,吲哚乙酸为5～25mg／L,2,4-D为5～10mg／L。萘乙酸对花粉萌发和花粉管生长有抑制作用,抑制程度随培养基内其浓度的增大而加强。多效唑和脱落酸对花粉萌发有抑制作用,其抑制程度随浓度的上升而增强,但他们对花粉管生长却有促进怍用,其最适宜于花粉管生长的浓度分别为400mg／L和60mg／L,超过此浓度后,促进作用又有所下降,甚至出现抑制作用,如多效唑浓度达到1000mg／L时,能强烈地抑制花粉管生长。     

Membrane receptor-mediated mechano-transduction maintains cell integrity during pollen tube growth within the pistil

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The effect of temperature on pollen germination, pollen tube growth, and stigmatic receptivity in peach

Temperature is a major climatic factor that limits geographical distribution of plant species, and the reproductive phase has proven to be one of the most temperature-vulnerable stages. Here, we have used peach to evaluate the effect of temperature on some processes of the progamic phase, from pollination to the arrival of pollen tubes in the ovary. Within the range of temperatures studied, 20 degrees C in the laboratory and, on average, 5.7 degrees C in the field, the results show an accelerating effect of increasing temperature on pollen germination and pollen tube growth kinetics, as well as an increase in the number of pollen tubes that reach the style base. For the last two parameters, although the range of temperature registered in the field was much lower, the results obtained in the laboratory paralleled those obtained in the field. Increasing temperatures drastically reduced stigmatic receptivity. Reduction was sequential, with stigmas first losing the capacity to sustain pollen tube penetration to the transmitting tissue, then their capacity to offer support for pollen germination and, finally, their capacity to support pollen grain adhesion. Within a species-specific range of temperature, this apparent opposite effect of temperature on the male and female side could provide plants with the plasticity to withstand changing environmental effects, ensuring a good level of fertilization.     

Nt-RhoGDI2 regulates Rac/Rop signaling and polar cell growth in tobacco pollen tubes

Rac/Rop-type Rho-family small GTPases accumulate at the plasma membrane in the tip of pollen tubes and control the polar growth of these cells. Nt-RhoGDI2, a homolog of guanine nucleotide dissociation inhibitors (GDIs) regulating Rho signaling in animals and yeast, is co-expressed with the Rac/Rop GTPase Nt-Rac5 specifically in tobacco (Nicotiana tabacum) pollen tubes. The two proteins interact with each other in yeast two-hybrid assays, preferentially when Nt-Rac5 is prenylated. Transient over-expression of Nt-Rac5 and Nt-RhoGDI2 depolarized or inhibited tobacco pollen tube growth, respectively. Interestingly, pollen tubes over-expressing both proteins grew normally, demonstrating that the two proteins functionally interact in vivo. Nt-RhoGDI2 was localized to the pollen tube cytoplasm and effectively transferred co-over-expressed YFP-Nt-Rac5 fusion proteins from the plasma membrane to this compartment. A single amino acid exchange (R69A), which abolished binding to Nt-RhoGDI2, caused Nt-Rac5 to be mis-localized to the flanks of pollen tubes and strongly compromised its ability to depolarize pollen tube growth upon over-expression. Based on these observations, we propose that Nt-RhoGDI2-mediated recycling of Nt-Rac5 from the flanks of the tip to the apex has an essential function in the maintenance of polarized Rac/Rop signaling and cell expansion in pollen tubes. Similar mechanisms may generally play a role in the polarized accumulation of Rho GTPases in specific membrane domains, an important process whose regulation has not been well characterized in any cell type to date.