Free IAA in stigmas and styles during pollen germination and pollen tube growth of Nicotiana tabacum

Although many studies have emphasized the importance of auxin in plant growth and development, the thorough understanding of its effect on pollen–pistil interactions is largely unknown. In this study, we investigated the role of free IAA in pollen–pistil interactions during pollen germination and tube growth in Nicotiana tabacum L. through using histo and subcellular immunolocalization with auxin monoclonal antibodies, quantification by HPLC and ELISA together with GUS staining in DR5::GUS -transformed plants. The results showed that free IAA in unpollinated styles was higher in the apical part and basal part than in the middle part, and it was more abundant in the transmitting tissue (TT). At the stage of pollen germination, IAA reached its highest content in the stigma and was mainly distributed in TT. After the pollen tubes entered the styles, the signal increased in the part where pollen tubes would enter and then rapidly declined in the part where pollen tubes had penetrated. Subcellular localization confirmed the presence of IAA in TT cells of stigmas and styles. Accordingly, a schematic diagram summarizes the changing pattern of free IAA level during flowering, pollination and pollen tube growth. Furthermore, we presented evidence that low concentration of exogenous IAA could, to a certain extent, facilitate in vitro pollen tube growth. These results suggest that IAA may be directly or indirectly involved in the pollen–pistil interactions. Additionally, some improvements of the IAA immunolocalization technique were made.     

Ethylene response to pollen tube growth in Nicotiana tabacum flowers

In flowers of Nicotiana tabacum L., pollination induces a transient increase in ethylene production by the pistil. The characteristic dynamics of the increase in ethylene correspond to the main steps of the pollen-tube journey into the pistil: penetration into the stigma, growth through the style, entry into the ovary and fertilization. Ethylene is synthesized de novo in the pistil, and its production is reduced in the dark. Ethylene production was monitored in tobacco flowers after pollination with incongruous pollen from three different Nicotiana species, N. rustica, N. repanda and N. trigonophylla, and with pollen from Petunia hybrida. Pollen from all of these different sources can germinate on the stigma surface but each pollen type shows a different behavior and efficiency in penetrating the pistil tissues. Thus, these different crosses provided a model with which to study the response of the pistil to pollination and fertilization. Ethylene evolution upon pollination in tobacco differed in each cross, suggesting that ethylene is correlated with the response to pollen tube growth in the tobacco flower.     

Development of membrane- and calcium-gradients during pollen germination of Lilium longiflorum

Chlorotetracyclin (10^-4M) has been used to observe the distribution of membrane-associated calcium during pollen germination of Lilium longiflorum. For comparison, the general membrane distribution has been determined with 4·10^-5 M fluorescamine. The pollen grains show a calcium gradient with either weak or strong chlorotetracycline-fluorescence intensity, but always increasing toward the germination colpus. This gradient intensifies during germination, reaching a maximum before the pollen tube emerges. The typical tip-to-base calcium gradient of the tube does not change during growth. Independent of the developmental stage, the pollen grains show a flat fluorescamine-fluorescence gradient with the highest intensity in one half of the grain. Pollen tubes reveal a tip-to-base membrane gradient, independent of their length. As an additional marker for membrane distribution, the distribution of phosphorus, measured by proton-induced X-ray emission in chemically fixed tubes, has been used. A tip-to-base phosphorus gradient, distinct from the calcium gradient measured with the same method, was detected.Abbreviation CTC chlorotetracycline     

Effect of toluidine blue on pollen germination and pollen tube growth

Toluidine blue is known to induce gynogenic haploids in significant numbersin Populus]. Because the efficacy of a chemical in inducing gynogenesis depends largely on its effeot on pollen germination, on pollen tube growth, and on male gamete formation, the effect of toluidine blue (0, 1, 10 and 100 mgl-1) on these processes was studied in treated pistils ofSolatium nigrum (4 X), as well as on cultured pollen grains ofS. nigrum andTrigonella foenumgraecum. Irrespective of the time of application, toluidine blue (1 and 10 mg I-1) had no effect on pollen germination or pollen tube growth in pistils ofS. nigrum; at 100 mg I-1 it invariably inhibited both the processes. Almost similar responses were elicited by cultured pollen grains. InT. foenum-graecum toluidine blue had no effeot on pollen germination and suppressed tube growth. Gamete formation was inhibited, to various degrees, at all the concentrations tested; at 100 ing I-1 hardly any pollen tube showed gamete formation. Based on our results, and those on other systems, the potentiality of toluidine blue as an inducer of gynogenesis has been analysed.     

Thigmotropism in the growth of pollen tubes of Lilium longiflorum

The pollen tubes of Lilium longiflorum were clearly seen togrow regularly along minute network filaments. By analyzingthe nature of this growth regularity, we found that the pollentubes showed a kind of thigmotropism in their growth. (Received October 14, 1974; )     

Effect of X-radiation on pollen tube growth and seed setting in crosses between Nicotiana tabacum and N. rustica

Summary In vitro andin vivo studies of the effect of irradiating pollen with different doses of X-rays were carried out inNicotiana rustica andN. tabacum. Dosages upto 9600 r were found to enhance the rate of growth of pollen tubers per unit time. Advantage was taken of this observation to overcome the incompatibility normally found in the crossN. tabacum x N. rustica as a result of the inadequate growth ofrustica pollen tubes intabacum style. Thus, whenrustica pollen exposed to 4800 r and 9600 r of X-rays were used to pollinatetabacum, fertilization resulting in the formation of viable hybrid seeds occurred. In the crossN. rustica×N. tabacum there is partial seed failure as a result of the hyperplastic development of the nucellar and endothelial cells. However, when X-rayedtabacum pollen were used in the cross, seed development registered a marked improvement. Since there is now evidence for suggesting that the contents of even those pollen tubes which do not participate in fertilization have some role to play in the development of the seed, it seems likely that radiation-induced biochemical changes in pollen may prove advantageous when conditions for seed development are abnormal as in instances of somatoplastic sterility. Besides such indirect biochemical effects, irradiation of pollen may also lead to genetic changes resulting in the inactivation of zygotic lethals. Thus, there is much scope for investigating the use of radiation to overcome inter-specific and inter-generic cross-incompatibility barriers.With 2 Figures in the Text     

类整合素在烟草花粉萌发和花粉管生长中的作用

The effects of antiserum against human VnR integrin and integrin antagonist GRGDSP peptides on Nicotiana tabacum pollen germination and tube growth both in in vitro and in semi-vivo conditions were studied. No obvious inhibitory effects on pollen germination and tube growth in vitro were observed when anti-VnR serum or GRGDSP peptides was added to BK culture medium, but the enhancement of pollen germination and tube growth in vitro promoted by calmodulin was depressed by adding anti-VnR serum or GRGDSP peptides to BK culture medium. In addition to that, pollen germination and tube growth on stigma, as well as tube growth in styles were also inhibited at some extent by treating stigma and microinjecting GRGDSP peptides or anti-VnR serum into styles. The role of integrin-like proteins in regulation of pollen germination and tube growth in situ was discussed.     

The effect of cycloheximide and 6-methylpurine on in vivo compatible and incompatible pollen tube growth in Lilium longiflorum

Summary Cycloheximide, a potent inhibitor of protein synthesis, placed in styles of Lilium longiflorum at 10^–4 M in stigmatic exudate before, 6, or 12 hr after compatible or incompatible pollination retarded all pollen tube growth. An inhibitor of RNA synthesis, 6-methylpurine, placed in the style at 10^–4 M in stigmatic exudate before, 6, or 12 hr after pollination restricted compatible pollen tube growth to lengths not significantly different thanincompatiblepollen tubes in treated or nontreated styles. While pollen tube growth in the style of L. longiflorum appears to require protein synthesis, only compatible pollen tube growth requires RNA synthesis. Stigmatic exudate proved to be an excellent carrier of exogenous substances into the style of L. longiflorum. Paper number 7047 of the Scientific Journal Series Minn. Agr. Exp. Sta. Research was supported in part by funds provided by the Graduate School, University of Minnesota.The authors wish to thank the United Bulb Co., Mount Clemens, Mich. for lily bulbs and L. H. Fuchigami and L. V. Gusta, Dept. of Hort. Sci., Univ. of Minn. for advice on use of the inhibitors. Mr. Drewlow is a National Science Foundation predoctoral trainee.     

A novel pollen tube growth assay utilizing a transmitting tract-ablated Nicotiana tabacum style

Sexual plant reproduction requires multiple pollen–pistil interactions from the stigma (pollen adhesion, hydration, and germination) to the ovary (fertilization). Understanding the factors that regulate pollen tube growth is critical to understanding the processes essential to sexual reproduction. Many pollen tube growth assays (PTGAs) have shorter and slower pollen tube growth when compared to pollen tube growth through the style. The identification and study of factors that regulate pollen tube growth have been impeded by a lack of an efficient and reproducible PTGA. The objective of this research is to develop a robust assay for Nicotiana tabacum pollen tube growth in an environment that supports sustained and normal growth yet is amenable to testing the effects of specific factors. In this paper, we introduce a novel PTGA, which uses pistils from N. tabacum that lack a mature transmitting tract (TT) due to tissue-specific ablation. The TT-ablated style supports normal pollen tube growth and the hollow structure of the style allows modification of the growth environment by direct injection of test material. This PTGA is robust and allows for rapid and accurate measurement of pollen tube length and pollen tube morphology, supporting pollen tube growth from 20 to 35°C and at pH ranging from 4.8 to 7.6. Use of the ablated style for a PTGA is a novel method for the culture of pollen tubes with sustained growth in vivo while permitting the application of treatments to the growing pollen tubes.     

The influence of RNA-synthesis inhibitors on in vivo pollen tube growth and the self-incompatibility reaction in Lilium longiflorum thunb

Summary Injection of 3 X 10^–4 g/ml acridine orange, 2 X 10^–5 g/ml actinomycin-D, or 1.34 X 10^–3 g/ml 6-methyl-purine in stigmatic exudate into the hollow styles of Lilium longiflorum immediately before, or 6 or 12 hr after pollination restricted compatible pollen tube growth to lengths typical of incompatible tubes. Treatment 24 hr after pollination resulted either in no effect on compatible tube growth or in a significant retardation of compatible tube growth but not to the length of tubes in incompatibly pollinated styles. Aqueous solutions of these inhibitors at the same concentrations injected into lily styles 6 or 12hr before pollination significantly stimulated incompatible pollen tube growth. These results suggest that RNA synthesis in the style is necessary for the self-incompatibility reaction and RNA synthesis in the pollen tube is required for compatible tube growth. The RNA synthesis in the pollen tube may be completed by 24 hr after pollination.Paper number 7439 of the Scientific Journal Series, Minn. Agr. Exp. Sta., St. Paul, Minn. The author gratefully acknowledges the support of the Graduate School, Univ. of Minn. and the United Bulb Co., Mount Clemens, Mich.     

Introduction and differential use of various promoters in pollen grains of Nicotiana glutinosa and Lilium longiflorum

Summary As part of our research to develop an alternative system for the transformation of recalcitrant plant species we investigated the use of the male gametophyte as a transformation vector. Therefore the activity of four different promoters (CaMV 35S, LAT52, chiA PA2 and TR2') was analyzed in pollen of a dicot (Nicotiana glutinosa) and a monocot (Lilium longiflorum) plant species. Gene constructs in which the ß-glucuronidase (GUS) gene was placed under the control of these promoters were introduced in pollen using a particle delivery system. No activity of the Cauliflower Mosaic Virus (CaMV) 35S promoter was detected in pollen of both N. glutinosa and L. longiflorum. The promoter of the tomato flower-specific LAT52 gene was highly active in N. glutinosa pollen but remained silent in L. longiflorum pollen. A similar expression pattern was observed for the pollen-specific Chalcone Flavanone Isomerase chiA PA2 promoter originally isolated from petunia. The TR2 mannopine synthase promoter of Agrobacterium tumefaciens, however, was active in pollen from Solanaceous species and also in pollen from the monocot L. longiflorum. This suggests that the TR2' promoter is active in vegetative and sporogenous tissues of dicot and monocot plant species.Abbreviations ADH1 Alcohol Dehydrogenase 1 - A. tumefaciens Agrobacterium tumefaciens - CaMV Cauliflower Mosaic Virus - ChiA Chalcone Flavanone Isomerase A - L. longiflorum Lilium longiflorum - N. glutinosa Nicotiana glutinosa - Nos Nopaline Synthase - N. tabacum Nicotiana tabacum     

Hydrodynamics and cell volume oscillations in the pollen tube apical region are integral components of the biomechanics of Nicotiana tabacum pollen tube growth

Pollen tube growth is localized at the apex and displays oscillatory dynamics. It is thought that a balance between intracellular turgor pressure (hydrostatic pressure, reflected by the cell volume) and cell wall loosening is a critical factor driving pollen tube growth. We previously demonstrated that water flows freely into and out of the pollen tube apical region dependent on the extracellular osmotic potential, that cell volume changes reflect changes in the intracellular pressure, and that cell volume changes differentially induce, increases or decreases in specific phospholipid signals. This article shows that manipulation of the extracellular osmotic potential rapidly induces modulations in pollen tube growth rate frequencies, demonstrating that changes in the intracellular pressure are sufficient to reset the pollen tube growth oscillator. This indicates a direct link between intracellular hydrostatic pressure and pollen tube growth. Altering hydrodynamic flow through the pollen tube by replacing extracellular H₂O with ²H₂O adversely affects both cell volume and growth rate oscillations and induces aberrant morphologies. Normal growth and cell morphology are rescued by replacing ²H₂O with H₂O. Further studies revealed that the cell volume oscillates in the pollen tube apical region. These cell volume oscillations were not from changes in cell shape at the tip and were detectable up to 30 μm distal to the tip (the longest length measured). Cell volume in the apical region oscillates with the same frequency as growth rate oscillations but surprisingly the cycles are phase-shifted by 180°. Raman microscopy yields evidence that hydrodynamic flow out of the apex may be part of the biomechanics that drive cellular expansion. The combined results suggest that hydrodynamic loading/unloading in the apical region induces cell volume oscillations and has a role in driving cell elongation and pollen tube growth.     

Cytoplasmic acidification and current influx follow growth pulses of Lilium longiflorum pollen tubes

We confirm that there is not a standing pH gradient in the tips of lily pollen tubes, but show that there are pulses of H⁺ that occur during pulsatile growth. The [H⁺] increases in a zone at the tips of the tubes and travels rapidly as far as 60 μm down the shaft of the tube. The tip-localized pH was found to drop to 6.0 during the largest pulses, from an average cytosolic level of 7.05 in tubes that had not yet begun to pulse. Correlation analysis indicates that the peaks of the pH pulses lag the peaks of the growth pulses by slightly more than 7.5 sec. Vibrating probe measurements reveal an influx of ionic current that peaks 7.5 sec after the peaks of the growth pulses. While this current may in part be H⁺ influx, we give evidence that K⁺ influx is also a component of the current pulses. The timing of the H⁺ and current pulses suggests that they may be involved in terminating the growth pulses.     

Signalling pathways in pollen germination and tube growth

Summary. Signalling is an integral component in the establishment and maintenance of cellular identity. In plants, tip-growing cells represent an ideal system to investigate signal transduction mechanisms, and among these, pollen tubes (PTs) are one of the favourite models. Many signalling pathways have been identified during germination and tip growth, namely, Ca²⁺, calmodulin, phosphoinositides, protein kinases, cyclic AMP, and GTPases. These constitute a large and complex web of signalling networks that intersect at various levels such as the control of vesicle targeting and fusion and the physical state of the actin cytoskeleton. Here we discuss some of the most recent advances made in PT signal transduction cascades and their implications for our future research. For reasons of space, emphasis was given to signalling mechanisms that control PT reorientation, so naturally many other relevant works have not been cited. Correspondence and reprints: Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal.     

Power spectrum,growth velocities and cross-correlations of longitudinal and transverse oscillations of individual Nicotiana tabacum pollen tube

We report on our results concerning growth rate and oscillation modes of the individual pollen tube apex. The observed volumetric growth and growth rate periodicity in the longitudinal (axial) direction are accompanied by transverse oscillations with similar frequencies but higher energies than the axial modes. Examination of the time-domain coherence between oscillations in mutually perpendicular directions revealed minimal energy dissipation in the unperturbed (isotonic) case, opposite to the two remaining cases (hypertonic, hypotonic) with notable correlations. We conjecture that the minimal energy loss is therefore optimal in the natural growth conditions. The longitudinal growth velocity is also found to be the fastest in the unperturbed case. As a result, the isolated system (pollen tube tip) is conserving energy by transforming it from elastic potential energy of extending apical wall to the kinetic energy of periodical motion. The energy dissipation is found to be about 20 % smaller in axial direction than in lateral one, indicating that the main energy consumption is dedicated to the elongation. We further observe that the hypertonic spectrum is shifted towards lower and the hypotonic towards higher frequencies with respect to the isotonic spectrum. In consequence, the turgor pressure inside the growing cell influences monotonically the frequency of both modes of oscillations. The calculated power spectrum seen as a measure of the overall energy efficiency of tip growth under hypertonic, hypotonic and isotonic conditions implies that the biochemistry has been fine tuned to be optimal under normal growth conditions, which is the developmental implication of this work. A simple theoretical extension of the Ortega equation is derived and analysed with respect to its contribution to power spectrum. We show that the plastic term, related to the effective turgor pressure, with maximum contribution at frequency f = 0 is responsible for the steady growth. In turn, the elastic contribution dependent on the time derivative of pressure fluctuations tends to move the system into oscillatory mode (f > 0). None of those mechanisms is privileged over another. The coupling mechanism is naturally generated through a convolution of those two terms and will decide about the overall character of the growth for each particular case.     

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.     

Effect of mercury on pollen germination and tube growth in <Emphasis Type="Italic">Lilium longiflorum</Emphasis>

Pollen development and germination were adversely affected by the presence of mercury, whereas low-concentrations stimulated the whole procedure. Mercury caused morphological anomalies during the tube growth, characterized by irregularly increasing diameters and swelling tips. The main effect was the anomalous cell wall formation at the tip where a substantial number of organelles were found reducing the secretory vesicles. The dense organelle concentration caused a significant reduction of cytoplasmic movement integrity, and the cytosol streaming was gradually reduced or stopped completely. Electron dense, multilamellar myelin-like structures (MMS) of membranous material were frequently present, in close contact with plasmalemma or away from it. A loose network of fibrillar material and spherical aggregates mostly at the tip region were observed which progressively were loosened into the surrounding medium. Elevated mercury concentrations can affect plant reproduction, resulting in anomalies in gamete development and consequently loss of plant biodiversity.