Tomato Pistil Factor STIG1 Promotes in Vivo Pollen Tube Growth by Binding to Phosphatidylinositol 3-Phosphate and the Extracellular Domain of the Pollen Receptor Kinase LePRK2

The speed of pollen tube growth is a major determinant of reproductive success in flowering plants. Tomato (Solanum lycopersicum) STIGMA-SPECIFIC PROTEIN1 (STIG1), a small Cys-rich protein from the pistil, was previously identified as a binding partner of the pollen receptor kinase LePRK2 and shown to promote pollen tube growth in vitro. However, the in vivo function of STIG1 and the underlying mechanism of its promotive effect were unknown. Here, we show that a 7-kD processed peptide of STIG1 is abundant in the stigmatic exudate and accumulates at the pollen tube surface, where it can bind LePRK2. Antisense LePRK2 pollen was less responsive than wild-type pollen to exogenous STIG1 in an in vitro pollen germination assay. Silencing of STIG1 reduced both the in vivo pollen tube elongation rate and seed production. Using partial deletion and point mutation analyses, two regions underlying the promotive activity of the STIG1 processed peptide were identified: amino acids 80 to 83, which interact with LePRK2; and amino acids 88 to 115, which bind specifically to phosphatidylinositol 3-phosphate [PI(3)P]. Furthermore, exogenous STIG1 elevated the overall redox potential of pollen tubes in both PI(3)P-dependent and LePRK2-dependent manners. Our results demonstrate that STIG1 conveys growth-promoting signals acting through the pollen receptor kinase LePRK2, a process that relies on the external phosphoinositide PI(3)P.     

Pollen tube localization implies a role in pollen-pistil interactions for the tomato receptor-like protein kinases LePRK1 and LePRK2.

We screened for pollen-specific kinase genes, which are potential signal transduction components of pollen-pistil interactions, and isolated two structurally related receptor-like kinases (RLKs) from tomato, LePRK1 and LePRK2. These kinases are similar to a pollen-expressed RLK from petunia, but they are expressed later during pollen development than is the petunia RLK. The abundance of LePRK2 increases when pollen germinates, but LePRK1 remains constant. Both LePRK1 and LePRK2 are localized to the plasma membrane/cell wall of growing pollen tubes. Both kinase domains have kinase activity when expressed in Escherichia coli. In phosphorylation assays with pollen membrane preparations, LePRK2, but not LePRK1, is phosphorylated, and the addition of tomato style, but not leaf, extracts to these membrane preparations results at least partially in specific dephosphorylation of LePRK2. Taken together, these results suggest that LePRK1 and LePRK2 play different roles in postpollination events and that at least LePRK2 may mediate some pistil response.     

A Calcium-Dependent Protein Kinase Interactswith and Activates A Calcium Channel toRequlate Pollen Tube Growth

ABSTRACT Calcium, as a ubiquitous second messenger, plays essential roles in tip-growing cells, such as animal neu-rons, plant pollen tubes, and root hairs. However, little is known concerning the regulatory mechanisms that code anddecode Ca2＋ signals in plants. The evidence presented here indicates that a calcium-dependent protein kinase, CPK32,controls polar growth of pollen tubes. Overexpression of CPK32 disrupted the polar growth along with excessive Ca2＋accumulation in the tip. A search of downstream effector molecules for CPK32 led to identification of a cyclic nucleotide-gated channel, CNGC18, as an interacting partner for CPK32. Co-expression of CPK32 and CNGC18 resulted in activationof CNGC18 in Xenopus oocytes where expression of CNGC18 alone did not exhibit significant calcium channel activity.Overexpression of CNGC18 produced a growth arrest phenotype coupled with accumulation of calcium in the tip, simi-lar to that induced by CPK32 overexpression. Co-expression of CPK32 and CNGC18 had a synergistic effect leading tomore severe depolarization of pollen tube growth. These results provide a potential feed-forward mechanism in whichcalcium-activated CPK32 activates CNGC18, further promoting calcium entry during the elevation phase of Ca2＋ oscilla-tions in the polar growth of pollen tubes.     

Relation of Glycosidases to Amaryllis vittata Pollen Tube Growth

The role of glycosidases activity in the regulation of pollentube extension in Amaryllis vittata during in vitro germinationwas investigated. No significant change in the enzyme activities(-glucosidase, -galactosidase, rß-glucosidase andrß-galactosidase) at different stages of tube growthwas found. No increase in patent rß-glucosidase activityassayed directly in a suspension of intact germinating pollenwas observed. The results are discussed in the light of thedifferential role of wall-bound glycosidases in cells showingoverall surface growth and tip growth i.e., pollen tubes. (Received February 4, 1981; Accepted June 5, 1981)     

The Control of Growth of Tomato Pollen

Tomato pollen grains germinate readily in a solution containingonly sugar and boric acid, although subsequent growth is ata rate much lower than that in the style. These experimentswere designed to test the hypothesis that the action of boronis to prevent the toxic effects of high auxin levels, and toinvestigate the role of auxin in germination and pollen-tubegrowth. No evidence could be found for an interaction between indol-3yl-aceticacid and boron of the kind required by the hypothesis. Severalinhibitors of growth and metabolism (maleic hydrazide, trans-cinnamicacid, iodoacetate and abscisic acid), and indol-3yl-acetic acidat high concentrations and ethylene inhibited germination tovarying extents, but promoted tube elongation. It is suggestedthat there are two distinct phases in the early growth of thepollen grain—germination and elongation—and thatthey differ in their sensitivity to chemical treatment. An activeendogenous inhibitory system appears to be established soonafter germination, which controls the rate of growth of thepollen tube. This inhibitory system can be inactivated by treatmentswhich cause the production of ethylene.     

Arabidopsis RIC1 Severs Actin Filaments at the Apex to Regulate Pollen Tube Growth

Pollen tubes deliver sperms to the ovule for fertilization via tip growth. The rapid turnover of F-actin in pollen tube tips plays an important role in this process. In this study, we demonstrate that Arabidopsis thaliana RIC1, a member of the ROP-interactive CRIB motif-containing protein family, regulates pollen tube growth via its F-actin severing activity. Knockout of RIC1 enhanced pollen tube elongation, while overexpression of RIC1 dramatically reduced tube growth. Pharmacological analysis indicated that RIC1 affected F-actin dynamics in pollen tubes. In vitro biochemical assays revealed that RIC1 directly bound and severed F-actin in the presence of Ca²⁺ in addition to interfering with F-actin turnover by capping F-actin at the barbed ends. In vivo, RIC1 localized primarily to the apical plasma membrane (PM) of pollen tubes. The level of RIC1 at the apical PM oscillated during pollen tube growth. The frequency of F-actin severing at the apex was notably decreased in ric1-1 pollen tubes but was increased in pollen tubes overexpressing RIC1. We propose that RIC1 regulates F-actin dynamics at the apical PM as well as the cytosol by severing F-actin and capping the barbed ends in the cytoplasm, establishing a novel mechanism that underlies the regulation of pollen tube growth.     

花粉管细胞结构与生长机制研究进展

花粉管的极性顶端生长是一个复杂的动力学过程,在高等植物有性生殖过程中起着重要的作用。花粉管的生长过程包括许多方面,其中最为重要的是花粉管细胞骨架动态和胞质运动。本文较全面地综述了花粉管的结构、细胞骨架、胞质运动、囊泡转运及循环、线粒体运动以及内质网和高尔基体之间囊泡运动等。     

FIMBRIN1 Is Involved in Lily Pollen Tube Growth by Stabilizing the Actin Fringe

An actin fringe structure in the subapex plays an important role in pollen tube tip growth. However, the precise mechanism by which the actin fringe is generated and maintained remains largely unknown. Here, we cloned a 2606-bp full-length cDNA encoding a deduced 77-kD fimbrin-like protein from lily (Lilium longiflorum), named FIMBRIN1 (FIM1). Ll-FIM1 was preferentially expressed in pollen and concentrated at actin fringe in the subapical region, as well as in longitudinal actin-filament bundles in the shank of pollen tubes. Microinjection of Ll-FIM1 antibody into lily pollen tubes inhibited tip growth and disrupted the actin fringe. Furthermore, we verified the function of Ll-FIM1 in the fim5 mutant of its closest relative, Arabidopsis thaliana. Pollen tubes of fim5 mutants grew with a larger diameter in early stages but could recover into normal forms in later stages, despite significantly slower growth rates. The actin fringe of the fim5 mutants, however, was impaired during both early and late stages. Impressively, stable expression of fim5pro:GFP:Ll-FIM1 rescued the actin fringe and the growth rate of Arabidopsis fim5 pollen tubes. In vitro biochemical analysis showed that Ll-FIM1 could bundle actin filaments. Thus, our study has identified a fimbrin that may stabilize the actin fringe by cross-linking actin filaments into bundles, which is important for proper tip growth of lily pollen tubes.     

花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响

以烟草为材料,通过半体内实验,就花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响进行了观察。发现用EGTA及钙调素抗血清处理柱头或花粉均可抑制花粉在柱头上的萌发;向花柱引导组织中显微注射纯化钙调素可促进花粉管束伸长,而注射钙调素抗血清可抑制花粉管束伸长;同时证实玉米花柱和花粉细胞壁中均存在钙调素及钙调素结合蛋白,而且花粉和花柱细胞壁中钙调素结合蛋白的种类有差异。结果表明存在于花粉和花柱细胞外的钙调素对花粉萌发和花粉管伸长均有促进作用。     

Inhibition of Camellia japonica Pollen Tube Growth by Maltose

Various oligosaccharides were studied with regard to their effecton the in vitro growth of Camellia japonica pollen tube. Sucrose,raffinose, melezitose, cellobiose, turanose and isomaltose,especially the first four, promoted pollen tube growth, whilemaltotriose, trehalose, gentiobiose, palatinose, melibiose,lactose and lactulose had little effect. Maltose strongly inhibitednot only the tube growth on sugar-free medium but also sugar-stimulatedgrowth, except in the case of sucrose stimulation. Glycosidaseactivities toward the growth-stimulating oligosaccharides weredetected in the extract of sucrose-grown pollen, but the activitiesof -glucosidase and -galactosidase were much lower than thoseof ß-fructosidase and ß-glucosidase. Maltosesuppressed the increase in UDP-glucose level of the glucose-grownpollen but not that of the sucrose-grown one. These resultssuggest that maltose acts, directly or indirectly, somewherein the pathway from glucose to UDP-glucose via glucose-1-phosphate,but does not interfere with the direct conversion of sucroseto UDP-glucose. (Received December 1, 1984; Accepted May 24, 1985)     

肌动蛋白在丝瓜花粉管顶端生长中的作用

用非固定荧光标记的鬼笔环肽作为肌动蛋白探针观察并证明了丝瓜未萌发的花粉粒和不同生长时期花粉管中肌动蛋白纤丝的分布及其形态变化。又用细胞松弛素B（CB）、氯两嗪（CPZ）及N-乙酰马来酰胺（NEM）证明了丝瓜花粉管伸长与肌动蛋白既有密切的关系,也受Ca2 的调节。     

GA_3对含笑花粉萌发及花粉管生长的影响

以含笑（Michelia figo）花粉为试材,采用花粉离体培养法,研究GA3对含笑花粉萌发和花粉管生长的影响。结果表明,GA3可以促进含笑花粉提早萌发,20～200 mg/L GA3对含笑花粉萌发和花粉管生长起促进作用,浓度超过200 mg/L花粉萌发和花粉管生长均受到抑制。以GA3200 mg/L的促进作用最好。     

Effect of Growth Regulators and Light on Pollen Germination and Pollen Tube Growth in Pinus roxburghii Sarg

Pine (Pinus roxburghii) pollen grown in suspension cultureswas used to study the effects of growth regulators and lightconditions on germination and pollen tube growth. Indol-3-ylacetic acid, gibberellic acid, ethylene, abscisic acid and cyclicAMP (cAMP) at low concentrations (1–10 mg 1^–1) promotedgermination and tube growth. Addition of 1 and 10 mg 1^–1cAMP to any of the growth regulators had a promotory effect.Pollen tube growth decreased in white light as compared to thedark, and was increased in red light. Far-red light counteractedthe effect of red light. The effect of growth regulators incausing the enhanced tube growth appears to be manifested throughsubstances such as cAMP, and phytochrome seems to be involved. Pinus roxburghii, pine, pollen germination, pollen tube growth, growth regulators, cyclic AMP, phytochrome     

Interference of the Histone Deacetylase Inhibits Pollen Germination and Pollen Tube Growth in Picea wilsonii Mast

Histone deacetylase (HDAC) is a crucial component in the regulation of gene expression in various cellular processes in animal and plant cells. HDAC has been reported to play a role in embryogenesis. However, the effect of HDAC on androgamete development remains unclear, especially in gymnosperms. In this study, we used the HDAC inhibitors trichostatin A (TSA) and sodium butyrate (NaB) to examine the role of HDAC in Picea wilsonii pollen germination and pollen tube elongation. Measurements of the tip-focused Ca²⁺ gradient revealed that TSA and NaB influenced this gradient. Immunofluorescence showed that actin filaments were disrupted into disorganized fragments. As a result, the vesicle trafficking was disturbed, as determined by FM4-64 labeling. Moreover, the distribution of pectins and callose in cell walls was significantly altered in response to TSA and NaB. Our results suggest that HDAC affects pollen germination and polarized pollen tube growth in Picea wilsonii by affecting the intracellular Ca²⁺ concentration gradient, actin organization patterns, vesicle trafficking, as well as the deposition and configuration of cell wall components.     

Overexpression of the Fibroblast Growth Factor Receptor 1 (FGFR1) in a Model of Spinal Cord Injury in Rats

Spinal cord injury (SCI) is a severe condition that affects many people and results in high health care costs. Therefore, it is essential to find new targets for treatment. The fibroblast growth factor receptor 1 (FGFR1) signalling pathway has a history of being explored for SCI treatment. Several groups have examined the effect of high availability of different FGFR1 ligands at the injury site and reported corticospinal tract (CST) regeneration as well as improved motor functions. In this study, we investigated overexpression of the FGFR1 in rat corticospinal neurons in vivo after injury (unilateral pyramidotomy) and in cerebellar granule neurons (CGNs) in vitro. We show that overexpression of FGFR1 using AAV1 intracortical injections did not increase sprouting of the treated corticospinal tract and did not improve dexterity or walking in a rat model of SCI. Furthermore, we show that overexpression of FGFR1 in vitro resulted in decreased neurite outgrowth compared to control. Thus, our results suggest that the FGFR1 is not a suitable therapeutic target after SCI.     

Structural Analysis of the Human Fibroblast Growth Factor Receptor 4 Kinase

The family of fibroblast growth factor receptors (FGFRs) plays an important and well-characterized role in a variety of pathological disorders. FGFR4 is involved in myogenesis and muscle regeneration. Mutations affecting the kinase domain of FGFR4 may cause cancer, for example, breast cancer or rhabdomyosarcoma. Whereas FGFR1–FGFR3 have been structurally characterized, the structure of the FGFR4 kinase domain has not yet been reported. In this study, we present four structures of the kinase domain of FGFR4, in its apo-form and in complex with different types of small-molecule inhibitors. The two apo-FGFR4 kinase domain structures show an activation segment similar in conformation to an autoinhibitory segment observed in the hepatocyte growth factor receptor kinase but different from the known structures of other FGFR kinases. The structures of FGFR4 in complex with the type I inhibitor Dovitinib and the type II inhibitor Ponatinib reveal the molecular interactions with different types of kinase inhibitors and may assist in the design and development of FGFR4 inhibitors.     

Genetic Variation in Sorghum for the Inhibition of Maize Pollen Tube Growth

Aniline blue fluorescence was used to study the growth of maizepollen tubes in the stigmas of 13 diverse sorghum accessions.In 12, only short maize pollen tubes were formed, but in thesingle exception (Sorghum nervosum Nr481) maize pollen tubesgrew at least as far as the base of the style. The S. bicolorgenotypes S9B and CMS (a cytoplasmic male sterile line) werehybridized with Nr481, and analysis of maize pollen tube growthin F₁ plants, and BC₁ plants using Nr481 as the recurrent parent,suggested that differences in inhibition of pollen tube growthwere due to variation at a single locus, which we propose todesignate lap (Inhibition of alien pollen tubes). AccessionNr481 appears to be homozygous for a recessive allele permittingmaize pollen tube growth. Attempts were made to produce sorghumx maize hybrids using Nr481 and CMS derivatives which were knownto allow maize pollen tube growth to the base of the style.A putative hybrid endosperm was obtained in one Nr481 x Seneca60 maize cross, but this was not repeatable and no hybrid plantswere produced. A fundamental problem may be the large size ofthe maize pollen tube, which could have difficulty growing throughthe sorghum ovary and in entering the micropyle. Sorghum bicolor spp. bicolor (L.) Moench, Zea mays L, sorghum, maize, pollen tube growth, hybridization barriers