Temperatures during flower bud development affect pollen germination,self‐incompatibility reaction and early fruit development of clementine (Citrus clementina Hort. ex Tan.)

• One of the key environmental factors affecting plant reproductive systems is temperature. Characterising such effects is especially relevant for some commercially important genera such as Citrus. In this genus, failure of fertilisation results in parthenocarpic fruit development and seedlessness, which is a much‐prized character. Here, we characterise the effects of temperature on flower and ovary development, and on pollen–pistil interactions in ‘Comune’ clementine (Citrus clementina Hort. ex Tan.).
• We examine flower bud development, in vitro pollen germination and pollen–pistil interaction at different temperatures (15, 20, 25 or 30 °C). These temperatures span the range from ‘cold’ to ‘hot’ weather during the flowering season in many citrus‐growing regions.
• Temperature had a strong effect on flower and ovary development, pollen germination, and pollen tube growth kinetics. In particular, parthenocarpic fruit development (indicated by juice vesicle growth) was initiated early if flowers were exposed to warmer temperatures during anthesis.
• Exposure to different temperatures during flower bud development also alters expression of the self‐incompatibility reaction. This affects the point in the pistil at which pollen tube growth is arrested and confirms the role of sub‐ and supra‐optimal temperatures in determining the numbers of pollen tubes reaching the ovary.

     

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H₂DCFDA‐staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into ‘low‐ROS’ and ‘high‐ROS’ subpopulations. Pollen germination assays following fluorescence‐activated cell sorting revealed that the high‐ROS pollen germinated with a frequency that was 35‐fold higher than the low‐ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose‐dependent alterations in DCF‐fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry‐based approaches to investigate metabolic changes during stress responses in pollen.     

Pollen germination in Welwitschia mirabilis Hook. f.: differences between the polyplicate pollen producing genera of the Gnetales

Pollen grains of the seed plant genera Ephedra L. and Welwitschia Hook. f. (Gnetales) are of similar size, shape, and have a polyplicate exine with alternating thicker and thinner regions. Ephedra pollen is considered inaperturate and the exine is shed during germination, leaving the male gametophyte naked. The shed exine curls up and forms a characteristic structure with transverse striations. Such upcurled exines have been found in situ in Early Cretaceous seeds with affinities to Ephedra. The purpose of this study was to document the germination of Welwitschia pollen and investigate whether they also discard their exine during this process.

The pollen grains of Welwitschia are monoaperturate with a distinct, distal sulcus. During germination, the sulcus splits open and the gametophyte expands to a spherical form that extends out of the exine. The pollen tube starts to grow one or two hours later and as in Ephedra, it is displaced towards one side. The exine is not shed but remains as a “cap” that partly covers the male gametophyte. Thus, in this respect the germination process is distinctly different from that in Ephedra and this study demonstrates that discharging the exine during pollen germination is unique to Ephedra, among the polyplicate pollen producing genera in the Gnetales.     

STAMINATE BLOOM DATE AND TEMPERATURE RESPONSES OF POLLEN GERMINATION AND TUBE GROWTH IN TWO WALNUT (JUGLANS) SPECIES

Pollen was collected from ten cultivars of Juglans regia and three cultivars of the later-blooming species, J. nigra. Extensive phenological data were available for these cultivars. Cultivars were chosen on the basis of staminate bloom date to include the earliest and latest blooming individuals available and a representative range throughout the bloom season. Mean staminate bloom dates for the cultivars examined covered a period of 46 days over which time mean daily temperatures rose 6 C. In order to determine if adaptations to temperature were expressed by the gametophyte generation, pollen was subjected to controlled temperatures from 5 to 40 C in 2 to 4 C increments and analyzed for germination percentages and pollen tube elongation. A positive relationship was found for pollen germination percentage and mean staminate bloom date such that earlier blooming individuals showed lower minimum temperature thresholds for germination, and optimum temperature for pollen germination was positively correlated with mean staminate bloom date. Differences in pollen tube growth, determined separately from hydration and germination responses, were less clear. Most J. regia clones had lower minium temperature thresholds for growth than the J. nigra clones, but there was no clear relationship to earliness of bloom within the species. No differences were discerned in optimum temperatures for pollen tube growth either between the two species or within species.     

Optimization of conditions for germination of cold-stored <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> pollen

One of the rare weak points of the model plant Arabidopsis is the technical problem associated with the germination of its male gametophyte and the generation of the pollen tube in vitro. Arabidopsis pollen being tricellular has a notoriously low in vitro germination compared to species with bicellular pollen. This drawback strongly affects the reproducibility of experiments based on this cellular system. Together with the fact that pollen collection from this species is tedious, these are obstacles for the standard use of Arabidopsis pollen for experiments that require high numbers of pollen tubes and for which the percentage of germination needs to be highly reproducible. The possibility of freeze-storing pollen after bulk collection is a potential way to solve these problems, but necessitates methods that ensure continued viability and reproducible capacity to germinate. Our objective was the optimization of germination conditions for Arabidopsis pollen that had been freeze-stored. We optimized the concentrations of various media components conventionally used for in vitro pollen germination. We found that in general 4 mM calcium, 1.62 mM boric acid, 1 mM potassium, 1 mM magnesium, 18% sucrose at pH 7 and a temperature of 22.5°C are required for optimal pollen germination. However, different experimental setups may deviate in their requirements from this general protocol. We suggest how to optimally use these optimized methods for different practical experiments ranging from morphological observations of pollen tubes in optical and electron microscopy to their bulk use for molecular and biochemical analyses or for experimental setups for which a specific medium stiffness is critical. F. Bou Daher and Y. Chebli contributed equally to this study.     

Variation in pollen viability among Picea abies genotypes – potential for unequal paternal success

An in vitro germination method was used to study variation in pollen viability, that is pollen-tube growth rate together with germination percentage, among the Picea abies genotypes in a seed orchard. The method permits easy, rapid screening of large numbers of genotypes. Significant variation in pollen viability among the genotypes was evident, the differences among the pollen-lot means being 7–10–fold in different years. No correlation was found between the average pollen viability and the phenology, growth or growing-site characteristics of the pollen donors. However, there appeared to be pollen lots that either benefit from a higher germination temperature or else germinate faster at lower temperatures. The significant variation in pollen viability among the pollen donors indicates a potential for male gametophyte competition. This, together with the observed genotype-environment interactions in pollen performance, may contribute to the variable genetic composition of seed produced in the seed orchard. Received: 9 December 1999 / Accepted: 22 December 1999     

Ricerche Embriologiche su Senecio Vulgaris L. var. Thyrrenus Fiori

Abstract

Embryological researches on SENECIO VULGARIS L. var. THYRRENUS Fiori. — Male gametophyte, development of tapetal cells and female gametophyte have been studied in Senecio vulgaris L. var thyrrenus Fiori.

1) The development of male gametophyte is normal. Divisions of the microspore mother cells are of the simultaneous type. The division of the generative nucleus has never been observed till the pollen grain was in the anther.

2) The tapetal cells follow a very simple development. The nucleus of each cell divides only twice starting at the same time with the meiotic divisions of pollen mother cells but ending much earlier; subsequently, as usually happens with the Asteraceae, the ameboid involution of the tapetum begins. Endomitosis or any other process which leads to a polyploidy not due to nuclear fusion, has never been observed.

3) The female gametophyte is eight nucleate of the normal type (Polygonum). At maturity it shows only three antipodal cells whose nucleus undergoes at first, two or three divisions. Only later these new nuclei, always within the antipodal cell, may fuse in a polyvalent one.     

In vitro pollen germination and pollen tube growth differences among Quercus robur L. clones in response to meteorological conditions

The impact of meteorological conditions on in vitro pollen germination and pollen tube growth during the initial phases of the development of male flowers in the Pedunculate Oak, Quercus robur, is studied. Phenological observations of male flowers and pollen sampling were performed on the field trial established with grafted Pedunculate Oak clones. During the investigation, weather conditions (absolute minimum and maximum daily air temperature, minimum absolute relative humidity of air and amount of precipitation) were recorded by an automatic meteorological station installed at the field trial. Influence of meteorological conditions on pollen germination and pollen tube growth was studied in the following stages of male flower: (I) during the last ten days of flower bud dormancy, (II) during swelling of the buds, (III) during bud burst and beginning of male catkins elongation, (IV) during the final stage of male flower catkins elongation. High temperatures and low relative air humidity during the bud burst and beginning of the male catkins elongation reduced pollen germination and pollen tube growth. Weather conditions did not significantly affect pollen germination and pollen tube growth during the swelling of flower buds, or in the final stage of male catkins elongation.     

Auxin and flavonoids in the progame phase of fertilization in petunia

The contents of IAA and flavonoids (Fls) were monitored in developing anthers, in vitro growing pollen tubes, and in the in vivo pollen-pistil system of two petunia (Petunia hybrida L.) clones, self-compatible and self-incompatible. In both clones, the development of male gametophytes was accompanied by the increase in the IAA (from 10 to 60–70 ng/g fr wt) and Fls (from 2 to 20 mg/g fr wt) contents. In both clones, pollen grain germination was accompanied by a substantial (by 10–30%) increase in the IAA content during the first two hours and Fl content during the first hour. Treatments with IAA and Fls stimulated both in vitro pollen grain germination and pollen tube growth by 25–30%. Male gametophyte germination in vivo, on the pistil surface, was accompanied by the increase in the IAA content from 90 to 200 ng/g fr wt during 8 h, whereas the content of Fl increased from 2 to 3 mg/g fr wt during the first hour and was maintained later at this level. In the pollen-pistil system, IAA and Fls were distributed evenly in the tissues of stigma, style, and ovary. On the basis of data obtained, we concluded that Fls might be endogenous mediators of IAA transport, which is one of the principal regulators of male gametophyte growth and development in the progame phase of fertilization, but are not involved in the mechanism of gametophyte incompatibility.     

Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana

Despite much effort, a robust protocol for in vitro germination of Arabidopsis thaliana pollen has been elusive. Here we show that controlled temperatures, a largely disregarded factor in previous studies, and a simple optimized medium, solidified or liquid, yielded pollen germination rates above 80% and pollen tube lengths of hundreds of microns, with both Columbia and Landsberg erecta (Ler) ecotypes. We found that pollen germination and tube growth were dependent on pollen density in both liquid and solid medium. Pollen germination rates were not substantially affected by flower or plant age. The quartet1 mutation negatively affected pollen germination, especially in the Ler ecotype. This protocol will facilitate functional analyses of insertional mutants affecting male gametophyte function, and should allow detailed gene expression analyses during pollen tube growth. Arabidopsis thaliana can now be included on the list of plant species that are suitable models for physiological studies of pollen tube elongation and tip growth.     

Protection of pollen germination from adverse temperatures: a possible role for proline

Abstract After germination, pollen grains of Lilium longiflorum became very sensitive to short periods of heat stress as shown by the greatly reduced germination percentages upon subsequent incubation at the optimal temperature. Addition of proline to the incubation medium made pollen more resistant to heat. It was demonstrated that in a short time a large amount of proline was taken up by the cell. Germination and metabolic functions were completely or partially protected from heat damage by proline treatment. As well, it was shown that proline treatment at least partially protected pollen grains from cold stress. These results suggest that the high proline concentrations found in pollen of many species may confer resistance to germinating pollen grains at unfavourable temperatures thereby enhancing the chances of successful fertilization.     

DEVELOPMENT OF THE POLLEN TUBE OF ZAMIA FURFURACEA (ZAMIACEAE) AND ITS EVOLUTIONARY IMPLICATIONS

Compared with pollen tubes of conifers, gnetophytes, and angiosperms, the pollen tube of cycads is exclusively a vegetative structure, uninvolved with the siphonogamous conduction of sperm to an egg. The cycad pollen tube appears to function primarily to obtain nutrients for the extensive growth and development of the male gametophyte. Previous workers have suggested that, similar to an haustorial fungus, the cycad pollen tube penetrates the reproductive tissues of the sporophyte by enzymatically destroying nucellar cells. These earlier studies did not document the precise structural relationship between the growing male gametophyte and its “host” tissue, the nucellus. Pollen tube growth, and its relation to the nucellus, was examined in Zamia furfuracea with light and transmission electron microscopy. Following germination, the pollen tube of Zamia furfuracea grows intercellularly through the subepidermal layers of the micropylar apex of the nucellus. Electron micrographs clearly show additional localized outgrowths of the pollen tube penetrating the walls of individual nucellar cells. Intracellular haustorial growth ultimately leads to the complete destruction of each penetrated cell, and appears to induce the degeneration of proximal unpenetrated nucellar cells. This pattern of intracellular penetration of the sporophyte by the male gametophyte in Zamia furfuracea is fundamentally different from what has been described in any other major group of seed plants (where intercellular growth of the male gametophyte is the rule), and suggests that the heterotrophic and tissue-specific relationships that male gametophytes of seed plants have with their host sporophytes are substantially more diverse than had previously been known.     

长花柱型滇丁香小孢子发生及雄配子体发育

利用常规石蜡切片法和细胞学压片法,对异型花柱植物滇丁香的长花柱型植株的小孢子发生、雄配子体发育及花粉萌发进行观察。结果表明:(1)长花柱型滇丁香具5枚花药,花药4室。(2)花药壁由1层表皮、1层花药内壁、2层中层和1~3层绒毡层组成;花药壁发育方式为基本型,绒毡层类型为腺质绒毡层。(3)小孢子母细胞减数分裂的胞质分裂为同时型,四分体排列方式为四面体型,偶有左右对称型;不同药室间小孢子母细胞减数分裂不同步。(4)成熟花粉粒为二细胞型。(5)小孢子发生和雄配子体发育过程正常,表明长花柱型滇丁香属于发育正常的两性花。(6)授粉4h后,长花柱型滇丁香的花粉在长、短两种花柱柱头上的萌发率分别达(91.8±1.6)%和(93.2±1.1)%,且两者间无显著性差异(t=1.585,df=8,p=0.152),表明长花柱型滇丁香的成熟花粉粒在长、短两种花柱柱头上的萌发均正常。     

植物有性生殖对温度胁迫反应的研究进展

开花植物的有性生殖阶段对温度胁迫高度敏感,高温热害和低温冷害都会对这一过程造成严重影响。本文全面总结了温度胁迫对作物有性生殖的影响,明确花粉发育过程是有性生殖过程中对温度胁迫最敏感的时期;转录组和蛋白质组的研究结果表明,蛋白激酶、热激转录因子、热休克蛋白等可能参与花粉发育期对热胁迫的信号转导。理解植物在有性生殖发育阶段如何适应温度胁迫的机理,为遗传育种实践中筛选对温度耐受的作物品种提供指导,也为基因工程选育对温度耐受的品种提供可能。     

Role of ethylene in the control of gametophyte-sporophyte interactions in the course of the progamic phase of fertilization

We investigated dynamics of the content of 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene production in male gametophyte development and germination in fertile (self-compatible and selfincompatible) and sterile clones of petunia. Fertile male gametophyte development was accompanied by two peaks of ethylene production by anther tissues. The first peak occurred during the microspore development simultaneously with the degeneration of both the tapetal tissues and the middle layers of the anther wall. The second peak coincided with dehydration and maturation of pollen grains. In the anther tissues of the sterile line of petunia, tenfold higher ethylene production was observed at the meiosis stage compared with that in fertile male gametophytes. This fact correlated with the degeneration of both microsporocytes and tapetal tissues. Exogenously applied ethylene (1–100 ppm) induced a degradation of the gametophytic generation at the meiosis stage. According to the obtained data, ethylene synthesis in germinating male gametophyte is provided by a 100-fold ACC accumulation in mature pollen grains. The male gametophyte germination, both in vitro, on the culture medium, and in vivo, on the stigma surface, was accompanied by an increase in ethylene production. Depending on the type of pollination, germination of pollen on the stigma surface and the pollen tube growth in the tissues of style were accompanied by various levels of ACC and ethylene release. The male gametophyte germination after self-compatible pollination was accompanied by higher content of ACC as compared with the self-incompatible clone, whereas, after the self-incompatible pollination, we observed a higher level of ethylene production compared with compatible pollination. For both types of pollination, ACC and ethylene were predominantly produced in the stigma tissues. Inhibitor of ethylene action, 2,5-norbornadiene (NBN), blocked both the development and germination of the male gametophyte. These results suggest that ethylene is an important factor in male gametophyte development, germination, and growth at the progamic phase of fertilization.     

Distinct expression of members of the LHT amino acid transporter family in flowers indicates specific roles in plant reproduction

Sexual plant reproduction necessitates proper development of pollen, pollen germination and tube growth through various tissues of the pistil, the female organ of the flower. Finally, sperm cells are released to fertilize the female gametophyte. These processes require high metabolic activities of all tissues involved and rely on the delivery of nitrogen assimilates for success. However, transporters mediating nitrogen fluxes are mostly unknown. The presented work provides an expression analysis of members of the LHT amino acid transporter family in relation to pollen development and pollen–pistil interaction. Expression of Arabidopsis LHTs was analyzed during flower development and the location of LHT function resolved by transporter-GFP and promoter-GUS studies. GFP-LHT localization in onion cells indicates that all LHTs analyzed are targeted to the plasma membrane. We further showed that LHTs are expressed in anthers and male gametophytes where they are proposed to function in transport of amino acids for pollen development and maturation. Expression in germinating pollen, pollen tubes and transmitting tissue of the pistil points to a role of LHTs in support of the fertilization process. Overall, our study suggests that LHT function in flowers is cell or tissue specific, developmentally regulated and highly coordinated between male and female tissue.     

Ethylene is Involved in the Control of Male Gametophyte Development and Germination in Petunia

The time courses of 1-aminocyclopropane-1-carboxylic acid (ACC) content and ethylene production in developing anthers of petunia fertile and sterile lines and the effects of exogenously applied ethylene and an inhibitor of ethylene action, 2,5-norbornadiene (NBD), on male gametophyte development and germination were investigated. Fertile male gametophyte development was accompanied by two peaks of ethylene production by anther tissues. The first peak occurred during microspore development simultaneously with degeneration of both tapetal tissues and middle layers of the anther wall. The second peak coincided with maturation and dispersal of pollen grains. The mature pollen is characterized by a high ACC content (up to 300 nmol/g). Exogenously applied ethylene (1–100 ppm) induced degradation of gametophytic generation at the meiosis stage. NBD completely inhibited anther development at the early stages of its development and delayed anther dehiscence. In anther tissues of the petunia sterile line, tenfold higher ethylene production was observed at the meiosis stage compared to that in fertile male gametophytes and this correlated with degeneration of both microsporocytes and tapetal tissues. In vitro male gametophyte germination was accompanied by an increase of ethylene production, whereas NBD completely blocked male gametophyte germination. These results suggest that ethylene is an important factor in male gametophyte development and germination.     

Growth and development of conifer pollen tubes

Conifer pollen tubes are an important but underused experimental system in plant biology. They represent a major evolutionary step in male gametophyte development as an intermediate form between the haustorial pollen tubes of cycads and Ginkgo and the structurally reduced and faster growing pollen tubes of flowering plants. Conifer pollen grains are available in large quantities, most can be stored for several years, and they grow very well in culture. The study of pollen tube growth and development furthers our understanding of conifer reproduction and contributes towards our ability to improve on their productivity. This review covers taxonomy and morphology to cell, developmental, and molecular biology. It explores recent advances in research on conifer pollen and pollen tubes in vivo, focusing on pollen wall structure, male gametophyte development within the pollen wall, pollination mechanisms, pollen tube growth and development, and programmed cell death. It also explores recent research in vitro, including the cellular mechanisms underlying pollen tube elongation, in vitro fertilization, genetic transformation and gene expression, and pine pollen tube proteomics. With the ongoing sequencing of the Pinus taeda genome in several labs, we expect the use of conifer pollen tubes as an experimental system to increase in the next decade.